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MIAMI UNIVERSITY The Graduate School

Certificate for Approving the Dissertation

Meepa A. Lokuge

Candidate for the Degree:

Doctor of Philosophy

______Dr. Kenneth G. Wilson, Director

______Dr. David A. Francko, Co-director

______Dr. Susan R. Barnum, Reader

______Dr. Nancy Smith-Huerta, Reader

______Dr. Richard E. Lee

Graduate School Representative

ABSTRACT

TISSUE CULTURE, GENETIC TRANSFORMATION, AND COLD TOLERANCE MECHANISMS IN COLD-HARDY PALMS

By Meepa A. Lokuge

Palms are a familiar and characteristic feature of tropical landscapes. Some palm species survive temperatures below -6.70C (200 F) and few survive temperatures below -17.70C (00 F). Needle palm, cabbage palm and Chinese windmill palm are very resistant to cold under USDA Plant Hardiness Zone 6 conditions. The first part of this study was undertaken to develop a tissue culture system for the clonal propagation of cold-hardy palms with desired characters and with the ultimate goal of producing a system for genetic transformation. Windmill palm was regenerated from shoot apical meristem tissues via indirect organogenesis, giving rise to viable plants that fully acclimated to greenhouse conditions. With cabbage palm 1.5 µM dicamba was optimal for the induction of somatic embryogenesis from zygotic embryos. The second part of this study was aimed at developing a genetic transformation system for cold-hardy palms. Cabbage palm was selected because it’s widespread use throughout USDA Zone 8 and previous data suggest that with minor improvement in cold tolerance this palm could be grown in even colder areas. Cabbage palm zygotic embryos were successfully transformed with the marker genes gfp and gus using the two most common plant transformation methods, biolistic and Agrobacterium-mediated transformation. Results indicated that Agrobacterium -mediated transformation gave more promising results when compared with the biolistic method. Plants exhibit two strategies for surviving extremely cold weather: freeze avoidance and freeze tolerance. Both strategies involved supercooling mechanisms and other adaptations that have not been characterized in palms. The final part of this dissertation was aimed at studying these mechanisms using the most cold- hardy palm, the needle palm, as a model system. According to our results needle palm supercooling capacity is already pronounced even in warm-incubated foliage and does not change significantly after exposure to cold-acclimating conditions. To further investigate the molecular mechanisms underlying this cold tolerance, a proteomic approach was used to examine initial changes of the leaf proteome upon cold treatment. Protein identification was difficult due to non- availability of relevant genome sequences. Nevertheless, 2- dimensional gel electrophoresis suggested that significant changes in protein products occur in needle palm leaves when challenged with non-lethal cold.

Keywords Cold-hardy palms; regeneration; somatic embryogenesis; genetic transformation; supercooling; proteomics; Chinese windmill palm (Trachycarpus fortunei); cabbage palm, (Sabal palmetto); needle palm (Rhapidophyllum hystrix)

TISSUE CULTURE, GENETIC TRANSFORMATION, AND COLD TOLERANCE MECHANISMS IN COLD-HARDY PALMS

A DISSERTATION

Submitted to the Faculty of

Miami University in partial

fulfillment of the requirements

for the degree of

Doctor of Philosophy

Department of Botany

Meepa A. Lokuge

Miami University

Oxford, Ohio

2006

Dissertation Director: Dr. Kenneth G. Wilson

Meepa A Lokuge

2006

Table of Contents

Chapter 1: Introduction 1 Literature Cited 29

Chapter 2: Regeneration of Trachycarpus fortunei (Hook) H. Wendl. (Chinese windmill palm) plants via organogenesis 43 Abstract 43 Introduction 43 Materials and Methods 45 Results and Discussion 47 Literature Cited 54

Chapter 3: Induction of somatic embryogenesis in Sabal palmetto; Walter Schultes & Schultes F: Morphological observations and 2-D protein profile Comparison 62 Abstract 62 Introduction 62 Materialsand Methods 64 Results and Discussion 66 Literature Cited 70

Chapter 4: Genetic transformation of Sabal palmetto Walter Schultes & Schultes F. zygotic embryos using biolistic and Agrobacterium-mediated Methods 86 Abstract 86 Introduction 86 Materials and Method 87 Results and Discussion 93 Literature Cited 98

Chapter 5: Investigations on cold tolerance mechanism in needle palm (Rhapidophyllum hystrix) and identification of cold responsive Protein 106 Abstract 106 Introduction 107 Materials and Methods 109 Results and Discussion 113 Literature Cited 117

Chapter 6 Conclusions 134 Literature Cited 138

iiiv Tables

Table 2-1 Callusing frequency of Trachycarpus fortunei zygotic embryos grown on media containing differential 2, 4-D concentrations 61 Table 3-1 Effect of different auxins and cytokinins on embryogenic callus induction in S. palmetto 81 Table 3-2 Effect of different dicamba concentrations on embryogenic callus induction in S. palmetto 82 Table 3-3 Total protein content of the different stages of embryogenesis in S. palmetto 83 Table 3-4a Total number of protein spots in gels from different stages of S. palmetto tissue culture 83 Table 3-4b Qualitative analysis of spots from S. palmetto tissue culture gels 84 Table 3-4c Quantitative analysis of spots from S. palmetto tissue culture gels 85 Table 4-1a GUS activity in the tissues placed on media with different osmotica from biolistic experiment 102 Table 4-1b ANOVA for transient GUS expression for optimization of media for biolistic method 102 Table 4-2a Percentage of tissues showing GUS activity when infected with EHA105:pCAMBIA 1301 103 Table 4-2b Percentage of tissues showing GUS activity when infected with EHA105:pCAMBIA 1305.1 104 Table 4-2c Percentage of tissues showing GUS activity when infected with EHA105:pCAMBIA 1305.2 105 Table 5-1 Supercooling points of needle palm leaves treated at 4 C for 14 days 129 Table 5-2 Relative water content of needle palm leaves treated at 4 C for 14 Days 130 Table 5-3 Spot number comparison between gels obtained from needle palm leaf protein extracts treated at 4 C for 14 days 131 Table 5-4 Spot identification by MALDI-TOF-MS analysis and database search for proteins spots that have been upregulated 132, 133

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Figures

Figure 2-1 Trachycarpus fortunei callus derived from mature zygotic Embryos 58 Figure 2-2 Different stages of plant regeneration of T. fortunei via indirect Organogenesis 59 Figure 2-3 Scanning electron micrographs of leaf stomata form different stages of T. fortunei regeneration 60 Figure 3-1 Sabal palmetto seed morphology 73 Figure 3-2 Major steps of S. palmetto seedling development 74 Figure 3-3 Stereomicrographs of major stages in the development of somatic embryos from S. palmetto zygotic embryos 75 Figure 3-4 A Scanning electron micrograph of 4-week callus tissue of S. Palmetto 76 Figure 3-4B Scanning electron micrograph of 6-week old tissue culture 77 Figure 3- 4C Scanning electron micrograph of 7-week old tissue cultures 78 Figure 3-5 Two-dimensional gel electrophoresis proteome map of S. palmetto tissue cultures 79 Figure 3-6 Selected regions of Fig. 3-5 to highlight some of the differentially expressed proteins 80 Figure 4-1 pCAMBIA 1301 vector 90 Figure 4-2 pCAMBIA 1305.1 vector 91 Figure 4-3 pCAMBIA 1305.2 vector 92 Figure 4-4 Histochemical GUS assay on bombarded and Agrobacterium co-cultivated mature zygotic embryos of S. palmetto 101 Figure 5-1 Graph of average supercooling point in Rhapidophyllum hystrix leaves vs. days at 40 C 121 Figure 5-2 Supercooling points and relative water content of R. hystrix treated at 40 C for 2 weeks 122 Figure 5-3A 2DE gel image of protein extract from R. hystrix leaves of at 260C 123 Figure 5-3B 2DE gel image of protein extract from leaves R. hystrix leaves t Treated at 40 C for 2 days 123 Figure 5-3C 2D E gel image of protein extract from R. hystrix leaves treated at 40 C for 4 days 124 Figure 5-3D 2DE gel image of protein extract from R. hystrix leaves treated at 40 C for 8 days 124 Figure 5-3E 2DE gel image of protein extract from R. hystrix leaves treated at 40 C for 10 days 125 Figure 5-3F 2DE gel image of protein extract from R. hystrix leaves treated at 40 C for 14 days 125 Figure 5-4A Western blot for dehydrin detection from a day 0 (cold untreated) leaf sample from R. hystrix 126 Figure 5-4B Western blot for dehydrin detection from day 14 (cold treated) leaf sample from R. hystrix 126

v Figure 5-5A Identification of superoxide dismutases (SODs) in R. hystrix leaves treated at 40 C for 14 days 127 Figure 5-5B Identification of Cu/Zn isoform and Mn-SOD isoform in R. hystrix using potassium cyanide 127 Figure 5-5C Identification of Cu/Zn isoform and Mn-SOD isoform in R. hystrix using hydrogen peroxide 128

viv

Dedication

Dedicated in loving memory of my parents Sirisena Lokuge and Kusuma Lokuge.

viiv

Acknowledgements

I would like to acknowledge many people who helped me through this study. First, I would like to thank my major advisor Dr. Kenneth G. Wilson and my co-advisor, Dr. David A. Francko whose intellectual guidance and enthusiasm made this degree possible and for that I am tremendously grateful and deeply honored. You have been wonderful mentors, always ready to listen to my problems, showed me different ways to approach problems and inspired me to be persistent to accomplish this dissertation. I will cherish the memory of working with both of you in the years to come.

Besides my advisors, I would like to thank my committee members, Dr. Susan R. Barnum, Dr. Nancy Smith-Huerta, and Dr. Richard E. Lee. A special thank to my committee member Dr. Richard E. Lee for being encouraging and supportive during my times of hardship in research.

I am grateful to Dr. John W. Hawes from the Department of Chemistry and Biochemistry, Miami University for his help with my proteomics work. I am also grateful to Dr. Pennock, Department of Zoology, Miami University for his assistance during my visits to his lab to use the biolistic instrument. I owe debt of gratitude to Mike Elnitsky from Ecophysiology and Cryobiology lab for helping me with supercoooling measurements.

I would also like to extend my sincere thanks to Barb Wilson and Vicky Sandlin from the Botany department for their ungrudging assistance whenever I needed their help.

My sincere appreciation and deepest gratitude to my late parents who had been the greatest strength and inspiration in my life to all your sacrifices and dedication which made me what I am today. Words cannot begin to describe how grateful I am to both of you.

Last, but not least, I would like to thank my two wonderful sisters who constantly inspired me to be persistent and pursue my interests in life. Your support, encouragement and most of all your belief in me have been the greatest gifts I could ever hope to receive.

viiiv

Chapter 1

Introduction

Palm

Palms are a familiar and characteristic feature of tropical and subtropical landscapes. Although the great majority of palms are tropical to subtropical, some palm species survive temperatures below -6.70C (20 0 F) and a few tolerate temperatures below -17.70C (0 0 F) and are thus found in warm-temperate landscapes (Francko, 2003).

They occur in habitats ranging from the mesic forests of the southeastern United States, the rainforests of the Americas and Southeast Asia, to many of the tropical deserts and savannas of the world. To some cultures, such as those of the Middle East, southwestern Asia, and northern Africa, the presence of palms is equated to life since trees occur only at oases, often the sole sources of water in the region. In the other parts of the world palms are equally important as a source of food, shelter, furniture, and clothing. In the Plant Kingdom, Palms are eclipsed by only the grass family (i.e. grains) in importance to the lives of humans.

The palms are a unique group of monocotyledons, with distinctive trunks, leaves, and flowers. Approximately 300 genera with about 2650 tree and liana (vines) species are widely distributed over the tropical and warm-temperate regions of the world.

Palms are classified as angiosperms and are found in the family Arecaceae. Due to the fact that they are monocots like bamboo and grasses, there are many physiological differences between palms and other trees that require that they be treated differently when it comes to care and maintenance as well as transplanting. Palms are considered woody plants that have solid stems and trunks. However, being a monocot, this stem tissue is not differentiated into wood, bark, pith, or annual growth rings like most trees but rather have scattered bundles of vascular tissue distributed throughout the cross- section of the trunk. Without these vascular bundles, the strength of the trunks of palms would be significantly reduced. Stems of palms tend to be cylindrical in shape and usually have no-leaf bearing lateral branches. There is usually one main growing point known at the terminal bud where all leaves arise. Most tropical palms have pinnate shaped leaves while temperate palms have palmate leaves. The root system found on palms is fibrous making them very sturdy and hard to uproot once established but at the same time very easy to transplant. (http://www.flmnh.ufl.edu/fossilhall/Library/Sabal/sabal.htm).

Cold hardy palms

All palms are not deemed reliable for planting due to the fact that some are not cold tolerant. For purpose of this study, cold-hardy palms are defined as species capable

1 of surviving exposure to–17.70C (0 0 F). Whether a palm can survive a period of cold depends on many things. Indeed, one palm may survive a cold snap while an adjacent palm of same species succumbs. Some factors affecting cold hardiness include: age of the palm, health of the palm, root growth, genetic make up of the individual palm, duration of cold, relative humidity, microclimate, protection (either natural or artificial) and soil ( reviewed by Francko, 2003). The cold tolerance mechanisms in palms are poorly understood.

All experts agree that Sabal, Rhapidophyllum, and Trachycarpus are cold hardy since they commonly survive in the Southeastern US. The genus Sabal, commonly called palmetto palms, consists of roughly New World 20 species. Most of them are native to the coastal regions of the southeastern United Sates, west to northeastern Mexico. Bermuda, some Carribbean Islands, Panama and northern South America habitats include coastal dunes and tidal flats, and savannas, and shaded swamps. Some species, such as the dwarf palmetto, Sabal minor, have a subterranean trunk, leaving only the crown of the palm above ground. Sabal species can be difficult to identify as hybridization occurs frequently, particularly in cultivated species. (http://www.flmnh.ufl.edu/fossilhall/Library/Sabal/sabal.htm). Sabal mexicana is native to coastal areas of Mexico and Texas and greatly resembles Sabal palmetto.

The flowers of Sabal species are bisexual and pollination usually occurs with the aid of insects. Wind may also assist in pollination. Seeds are often dispersed by birds and small mammals that feed on the prolific numbers of fruit produced by mature trees. Germination occurs in soil within 2 to3 months. Although Sabal palmetto is not a rare or endangered species, some related species are threatened and require conservation efforts to insure their future.

Needle palm (Rhapidophyllum hystix) (Pursh) H. Wendl & Drude

Needle palm, accepted as the most cold-hardy palm on earth, is native to the southeastern US. It has reportedly survived temperatures as low as – 23 0C without significant damage to foliage (Francko, 2003). The genus Rhapidophyllum is monospecific. Extremely sharp quills surround the leaf sheaths of this palm, and both the scientific and common names of this palm have been derived based on that character. The state of Florida lists needle palm as endangered species because it is commercially exploited.

Needle palm is a large shrub that can grow to about 10 ft (3m) in height and width. It produces suckers freely. Multiple stems create a rounded clump of indeterminate width. The needle palm does not form a long trunk but instead has a slowly lengthening crown. The stems are composed of old leaf bases, fiber and long slender spines. As each stem matures, more slender spines grow from between the leaf attachments. The needles are dark brown or black and are very slender and sharp. Each stem carries about 12 erectly held leaves. The foliage is glossy deep green on top with a dull silvery white underside. Leaves are fan-shaped and slender petioles are smooth. The tip of each leaflet is blunt and notched as if trimmed.

2 The needle palm is dioecious (although hermaphroditic individuals are also reported) and has a tightly compact inflorescence. The inflorescence is about 6-12 in (15-30 cm) long, held close to the stem, and does not usually extend above the leaf bases. Obscured by foliage and fiber and protected by the sharp needles it is often not visible. Small yellow to purplish-brown flowers are held on the inflorescence. This palm flowers irregularly with blooms typically appearing in spring and early summer. Seeds are roughly spherical and red to brown in color. They have a fuzzy fleshy covering and are very well protected by the sharp needles.

Cabbage palm (Sabal palmetto)

The sabal palm, Sabal palmetto, also known as the cabbage palmetto and Carolina palm, it is the official state tree of Florida and South Carolina. This palm usually occurs near the coast from southeastern North Carolina to the Florida Keys, west along the northern Gulf of Mexico, Cuba, and various islands of Bahamas. It is considered one of the most common species of native trees in North America. It occurs along sandy shores, commonly in crowded groves, as islets in vast expanses of salt marsh grasses, and in inland hardwood hammocks.

The average height of the mature palm is 80-90 ft (25-28 m). The trunk is highly variable. Sometimes the trunks may are covered with crisscross pattern of old leaf bases. The leaves are fan shaped and may be 3 to 6 feet (1-2m) in length. The leaf stalk or petiole is unarmed and may be longer than the leaf itself. Often the petiole base is split. The flower stalk is 2 to 3 ft (0.6-1m) or more in length, producing numerous whitish flowers followed by global, shiny, black fruit, 0.3 in (0.8cm) diameter. Cabbage palms exhibit moderate growth rates (http://www.Inla.org/palms.html)

Since it occurs further north than most other New World palms, the sabal palm is relatively cold tolerant, making this species a favorite with home owners and landscapers. To meet the ever-increasing demand for this ornamental tree, the species is widely cultivated and several varieties are available through commercial outlets.

S. palmetto has been used by humans in many ways for thousands of years. The fruits have been credited with the ability to improve the digestion of native people in southeastern United States. Early human inhabitants of Florida had a variety of uses for the sabal palm. The bud of the tree was eaten as food, the fibrous trunk was used for shelter materials and the large, thick fronds were used as roofing material. The fibrous trunk is famous for the great battle during the Revolutionary war as the building of fort moultry. The tree became famous because of the ability of the trunks to with stand cannon balls. Native Americans in south Florida still use the sabal palm to build chickee huts, shelters with water tight palm-thatched roofs and constructed with hand-hewn logs without the aid of any kind of metal, including nails.

(http://www.flmnh.ufl.edu/fossilhall/Library/Sabal/sabal.htm

3 Chinese windmill palm (Trachycarpus fortunei)

Trachycarpus fortunei, commonly known as Chusan palm or Chinese windmill palm is one of the hardiest arborescent palm species in the world. Windmill palm can withstand severe freezes with little or no damage (ca -13 to -140C; 7 to 90F). This palm is native to temperate and subtropical mountainous areas of Asia including southeastern China, Taiwan and the Chusan Islands. It is commonly grown as landscape specimen in central and northern Florida, the southeastern U.S. Atlantic and Gulf Coasts, and in mild areas along the west coast. Chinese windmill palm has been cultivated in China and Japan for thousands of years, grown for its coarse but very strong leaf fiber, used for making ropes, sacks, and other coarse cloth where great strength is important.

This palm is beautifully compact and can grow to 65ft (20 m) in height on a single stem up to 8 to 12 in (20-30 cm) in width. The trunk is slender and very rough with the persistent leaf bases clasping the stem as layers of coarse fibrous material loose mat of coarse gray or brown fiber. The stem is narrower at the base than at the top.

The large fan-like leaves, with the long petiole bare except for two rows of small spines, terminating in a rounded fan of numerous leaflets with ragged drooping tips. This palm has light to dark green palmate leaves that are lighter, almost silvery (glaucous), on the underside. They are held on thin flattened stems that are finely toothed along both edges. Leaves are arranged into a symmetrical crown that is about 8-10 ft (2.5-3.1m) wide. Specimens grown in full sun and/or under poor conditions may have much smaller, more compact crowns.

This palm is dioecious so male and female flowers are borne on separate plants. Flowers are densely arranged on inflorescences. Yellow inflorescences arise from a packet like bud in late winter and or early spring and is held inside the crown. Round or oblong fruits can be seen on female flowers in late summer. Fruits are kidney-shaped drupes and are blue-black in color.

Plant Regeneration

In nature, the methods of plant propagation may be either asexual (by multiplication of vegetative parts) or sexual (through germination of seeds). Sexually propagated plants demonstrate a high amount of heterogeneity since their seed progeny are not true-to-type unless they have been derived from inbred lines. Asexual reproduction on the other hand, gives rise to plants which are genetically identical to the parent plant and thus permits perpetuation of the unique characters of the cultivars. Clonal propagation is the multiplication of genetically identical copies of cultivars by asexual reproduction.

The in vivo clonal propagation of plants is often difficult, expensive and even unsuccessful. The tissue culture methods offer an alternative means of plant vegetative propagation. Clonal propagation through tissue culture (micropropagation) serves to save both time and space. Using this technique thousands of seedlings can be regenerated in a

4 very short time. Every cell of a plant has the potential to be regenerated into a complete plant by tissue culture techniques, a property known as the totipotency.

There are clear assumptions that vegetative propagation of plants proceeds from a callus phase, through a process of somatic embryogenesis or organogenesis to the production of plantlets. Both somatic embryogenesis and organogenesis are controlled by plant hormones and other factors added to the culture medium. Some species, such as soybeans (Trick, 1997), banana (May, 1995 and Sagi et al., 1995) and sugar beet (Krens and Jamar, 1989 and Hall et al., 1996) can be regenerated via either method so the choice depends on which gives the best yield or the easiest outcome.

Somatic embryogenesis

Somatic embryogenesis is the process of a single cell or a group of cells initiating the developmental pathway which can lead to reproducible regeneration of non-zygotic embryos. This unique ability of producing morphologically and developmentally normal embryos from undifferentiated somatic cells in culture through asexual embryogenesis found only in the Plant Kingdom. Somatic embryos are capable of germinating to form complete plantlets. Under natural conditions this pathway is not normally followed, but from tissue cultures somatic embryogenesis occurs most frequently as an alternative to organogenesis of whole plants. Many factors contribute to somatic embryo development and among them are genotype, developmental stage of the explant, auxin concentrations, growing conditions of the donor plant, and media composition. Often times, more than one factor controls somatic embryogenesis. Somatic embryogenesis, which allows the mass- propagation of clonal plants, has created many hopes in the field of in vitro plant culture in recent years.

Somatic embryo production was first observed from carrot cells (Steward et al., 1958). Since then this pathway has been reported in various higher plants, angiosperms as well as in gymnosperms (Dunstan et al., 1995). This unique developmental potential of plant cells is recognized as a potential model for investigating morphology and regulation of plant embryogenesis as well as an important pathway for plant regeneration from culture systems (Dong and Dunstan, 1999). Somatic embryos are also used as target materials for plant transformation studies e.g., via particle bombardment (Ellis, 1995 and Charest et al., 1996). Low frequency, synchrony of embryo maturation and embryo conversion into plantlets still remain as main challenges associated with somatic embryogenesis.

Gene expression during somatic embryogenesis

Somatic embryo development and differentiation are either directly or indirectly regulated by changes in gene expression. The normal expression of genes during somatic embryogenesis establishes the polarity and eventual morphological pattern of the plant. Much attention has been given to the elucidation of the molecular mechanisms underlying this unique process of embryogenesis (Thomas, 1993). Identification of particular embryo-specific genes and embryo-stage-specific genes is important to understand molecular mechanisms of plant embryo development. In the last decade, seed

5 storage protein genes and late embryogenesis- abundant genes (lea) have been studied extensively (Black, 1991 and Shewry et al., 1995). LEA proteins and storage proteins were first cloned and characterized in angiosperm plants. lea genes show increased expression in the late stage of embryo development and disappear during subsequent germination of somatic embryos. It is believed that lea gene products protect the embryos from precocious germination during seed development and from damage due to desiccation.(Dong and Dunstan, 1999). The formation of somatic embryos and subsequent plant regeneration are two independent processes indicating that the conversion into plantlets is a second control point in the process of in vitro plant regeneration.

Organogenesis

Organogenesis is the generation of organs, usually shoots from a variety of tissues including leaf fragments, cotyledons, hypocotyls and scutella from embryos. These tissues generally have the potential to generate the shoots when placed in the medium containing shoot inducing hormone, usually a cytokinin, such as 6-benzylamino purine. For example bananas are generally propagated by shoot tip culture (May, 1995). The advantage of this system is that shoots can form roots easily. It was also reported that only the base of the monocot leaf explants is capable of generating shoots due to the fact that meristematic tissues are present only in the leaf base (Maheshwari et al., 1995).

Importance of micropropagation of palms

The majority of palms are propagated by seeds, except date palm, which can be propagated vegetatively by offshoots. Although the date palm (Phoenix dactylifera L.) has a limited degree of vegetative propagation, there is no natural vegetative means for either coconut (Cocos nucifera L.) or oil palm (Elaeis guineensis Jacq.).

Since most economically important members of the family Arecaceae are propagated by seed, there is a great deal of variation between seedlings. Coconut and oil palm are specially cultivated for the production of edible oil. The variation due to seed propagation affects the yield and consequently the oil production. Therefore, for the production of identical clones in large numbers from high yielding palms the technique of tissue culture offers an important advantage.

The importance of the clonal propagation of palms cannot be overestimated considering the fact that palm seeds are recalcitrant and have a short storage life. Therefore, tissue culture will be the ideal method for germplasm storage.

Micropropagation of palms

Date Palm (Phoenix dactylifera L.)

Date palm is primarily cultivated for its edible fruits, however many other parts of the palm are used for various purposes. For example, seeds are used to feed animals while mature leaves are used to make baskets, mats, screens and fans. It is one of many examples of a tree crop that can benefit immediately from the recent advances in plant tissue culture and biotechnology.

Date palm is conventionally propagated through seeds. Impossibilities of predicting adult characteristics of the seedlings as well as slow growth have severely restricted improvement of this tree crop. Seed propagation method cannot be used commercial propagation of cultivars of interest due to several reasons. The most obvious is the heterozygous characteristics of seedlings. Thus is related to the dioecious nature of the date palm. Approximately one-half of the progeny are male, which produce no fruits. Large variations in phenotype can occur within the progeny and some desirable characters may be lost. Another important drawback of seed propagation is that the growth and maturation of seedlings is extremely slow in this ancient crop. Date palm can be vegetatively propagated through offshoots. These offshoots are produced from axillary buds situated on the base of the trunk during juvenile phase of the life cycle but the number of off shoots produced from a palm can be limited (Parveek et al., 1984).

In vitro studies on date palm have been carried out in many laboratories in different countries of the world. Date palm cloning is feasible and many reports are available on successful production of plantlets through micropropagation. Methods for large scale propagation of date palm are now available and some of the commercial tissue culture laboratories have already adopted the methods for large-scale production of elite palms (Spurr, 1985). These techniques can also be used in plant breeding programmes in addition to providing large number of cloning material. However, only a few reports are available in literature on successful establishment of plantlets in soil (Branton and Blake, 1989).

Regeneration of date palm usually occurs through the somatic embryogenesis via a callus phase (Tisserat and De Mason, 1980; Sharma et al., 1984; Tisserat, 1984; Kackar et al., 1989) while there are no reports of organogenesis from callus. Somatic embryos derived from callus usually develop into normal plantlets. Continuous production of plantlets is possible through somatic embryogenesis i.e. by the multiplication of embryogenic tissue giving rise to secondary embryogenesis.

Canary Island date palm

The Canary Island date palm (Phoenix canariensis) is a prominent feature of the landscape throughout subtropical and Mediterreneon climate of the world. Huong et al. (1999) reported the successful regeneration of P. canariensis through somatic embryogenesis using zygotic embryos and shoot tips as explants. The most suitable medium for embryogenesis callus proliferation and maintenance was MS (Murashige and Skoog, 1962) medium supplemented with 2, 4-D, kinetin and abscisic acid (ABA). In addition to this they have also used ABA for somatic embryo maturation. Activated charcoal, which acts as an adsorbent/ antioxidant, has been used in some palm tissue culture systems including P. dactylifera, to stimulate embryogenesis (Ammirato, 1983; Gabr and Tisserat, 1985). In contrast, in Phoenix canariensis tissue culture the presence of activated charcoal, combined with highest auxin concentration, completely inhibited the induction of embryogenic callus (Huong et al., 1999).

Oil Palm (Elaeis guineensis Jacq.)

There are two species of oil palm i.e. E. guineensis (African oil palm) and E. oleifera (South American oil palm). Oil from this palm is widely used in food industry to manufacture products including margarine, confectionery, shortenings, and also in frying snacks.

Several commercial laboratories now use tissue culture methods for the large-scale production of clones from oil palm. However, many details related to these methods have not yet been published mainly due to its commercial applications (Rao and Ganapathy, 1993). Nevertheless, several authors (Corley et al., 1977: Jones, 1974: Noiret, 1981) have described the advantages of producing identical clones from individual selected palms using tissue culture methods.

The various groups have used different explants from young as well as mature palms to initiate tissue cultures in oil palm. Among them are seedling embryos (Rabechault, et al., 1970); stem apex and leaf bases (Rabechault et al., 1970); meristem tissue from mature- palms (Stravinsky, 1970). shoot apex and seedling roots from as germinated embryos (Jones, 1974); leaf tissues (Rabechault and Martin, 1976); young leaf sections (Loiret, 1981); young leaf sections from nursery and mature palms (Hanower and Pannetier, 1982) and roots from mature palms (Zaid and Tisserat, 1983);. In addition Sambanthamurthi et al., (1996) first reported the isolation of highly variable protoplasts from various oil palm tissues and the formation of microcallus from protoplasts of polyembryogenic cultures.

(Alberlenv-Bertossi et al., (1999) first reported that embryogenic suspension cultures of oil palm allow mass propagation of somatic embryos However, the regeneration rates were very low according to their report. Rabechault and Martin (1976) reported the first successful regeneration of plantlets from leaf tissues. Following this there were refinements in the techniques by Corley et al. (1979).

Coconut (Cocos nucifera L.)

Coconut palm is one of the most important crops in many tropical countries. It is a major oil crop on an industrial scale and an important subsistence and cash crop for small holders. It has been described as one of nature’s greatest gifts to man because of the importance not only in economically but also socially. Coconut has a long history, in both the eastern and western hemispheres, and its widespread and apparently ancient occurrence in both hemispheres has led to uncertainty as to its center of origin.

The tree has a single meristem and axillary meristems form inflorescences leaving it unsuitable for propagation by cutting and grafting. Coconut palms do not produce suckers. Seed is the only method of propagation. Coconut is effectively an out-breeding heterozygous plant and as a result, seedlings exhibit great variability in selected characters. These characters cannot be evaluated until they reach maturity after 5 to 8 years of growth due to the slow growth rate. Thus many advantages can be gained by eliminating this variability, if cloning of selected, high performance palms could be achieved.

Since early 1970s, in vitro culture of vegetative explants has been attempted at several research institutes of coconut growing countries. No protocol is still available for reproducible, in vitro propagation of clonal plants on a large scale (Buffard-Morel et al., 1995). Major problems associated with coconut tissue culture systems are difficulties in obtaining plants, microbial contamination, low somatic embryogenesis and poor germination of somatic embryos and difficulties in successful establishment of regenerated plantlets in soil.

Various groups have used different sources of explants, such as shoot tip merited and root meristematic tissue, zygotic embryos, anther, plumule, inflorescence, leaf, and endosperm tissue to induce callus or to develop organized structures. However plant regeneration was reported only on few occasions. Success in producing a limited number of plantlets through somatic embryogenesis has been reported, with successful establishment of only a few vegetatively propagated plantlets in the field (Hornung, 1995).

Early investigations on coconut tissue culture were based on the clear assumption i.e. coconut tissue response would be similar to oil palm under in vitro conditions. Oil palm produces slow growing callus which is multiplied and induced to differentiate somatic embryos and germination of somatic embryos to produce plantlets. However, it has proved difficult to obtain plantlets in coconut tissue and the production of callus and organogenesis in callus is somewhat different from oil palm. Coconut tissue cultures show a tendency towards somatic embryogenesis. They do not multiply further and generally follow an unbalanced growth resulting in occasional production of shoots and rarely a complete plantlet (Branton and Blake, 1983).

Genetic Transformation

A large proportion of the field-planted material of some of major crops has been genetically modified using genetic transformation (Birch, 1997) during last few years. In addition, these genetic transformation studies allowed us to understand some basic aspects of biochemistry, plant physiology, and plant development (Birch, 1997). Cloning genes that were known from the phenotypes they produce, testing and evaluating the hypotheses on developmental roles of those genes and understanding the regulatory aspects of key metabolic pathways were all possible through transformation technology (Koncz et al., 1989).

Genetic transformation is potentially the most powerful tool so far afforded by biotechnology to create improved varieties. The new technology involves the introduction of foreign genes into plants by methods which by-pass the conventional process of sexual seed production. This eliminates the requirements of crossability as a prerequisite to gene transfer. Genetic engineering is now widely used as a relatively fast and precise means of achieving improved stress tolerance in model plants. Many organisms have evolved characters that will allow them to survive in extreme environments (cold, drought), and the gene(s) that confer these properties can potentially be introduced into higher plants through genetic transformation.

Transgenic crops have been adopted rapidly and widely in US agriculture. They provide cost savings and convenience to farmers and environmental safety benefits to the community. Transfer, integration and expression of foreign genes into many commercially cultivated tree species has been demonstrated so far (Ellis, 1995). Although the first transgenic crops were introduced as recently as 1995, the total area covered by seven transgenic crops (maize, soybean, cotton, tomato, potato, canola, and sugar beets) reached 81 million acres in 1998. However, no large-scale plantation of genetically modified tree species has been initiated yet. Woody plants do not possess any unique feature that would make them unsuitable for genetic manipulation through transgenic technology. However, there are some serious limitations that must be overcome in order to be successful with genetic engineering of trees. The main difference between annual crops and woody tree species is the long life span of tress. Therefore, this feature requires that a transgene be expressed correctly throughout the life of the tree. The lack of large-scale regeneration systems form tissue cultures due to highly recalcitrant nature in most tree species is another important factor limiting their improvement through gene transfer.

Most current transformation protocols require a tissue culture system to ultimately recover plants. It is the totipotency of plant cells that underlies most transformation systems. The target cells used for most current transformation systems are a part of an excised explant cells grown in culture media. Following gene transfer, the transformed cells are first selected in selection media and then allowed to grow and differentiate into shoots through organogenesis or somatic embryos through somatic embryogenesis. This

10 is achieved with the correct combinations of auxins and or cytokinins Therefore, a tissue culture step seems essential for the production of transgenic plants.

However, there is a technique called ‘in planta” transformation that has been used in order to avoid tissue culture completely. For this transformation system, target tissues are usually microspores, whole plants, or plant cells since they can directly grow into shoots or somatic embryos. Advantages of this technique are, somoclonal variation which occurs frequently in tissue culture systems can be avoided and plants that are highly recalcitrant to in vitro regeneration can be transformed. Main disadvantage of this approach is that it can often result in the production of chimeric plants (Minocha and Wallace, 1999). Although, this transformation technique may yield in low frequency of transformants it is suitable in cases where a reliable regeneration system has not yet been established or hard to be established.

For a successful gene transfer, first it is necessary to have an efficient method for introducing the desired gene into cells of the receiver organisms and then a method for growing the transformed cells into fertile transgenic organism. Gene transfer into plants cells is somewhat more difficult than in bacterial and animal cells may be due to the presence of cell walls. Nevertheless it has been accomplished by several methods including Agrobacterium-mediated gene transfer, particle bombardment, electroporation and microinjection.

Agrobacterium-mediated transfer

Agrobacterium-mediated transfer is the most commonly used method of transformation and it uses the bacterium Agrobacterium tumefaciens to transfer the desired DNA into plant. When it encounters a plant, the bacterium naturally transfers part of its DNA into plant’s chromosomes and induce crown gall tumors.

This system was historically the first successful plant transformation system, in plant genetic engineering in early 1980s. The breakthrough in genetic engineering g in plants came by characterizing plasmids carried by the bacterial plant pathogens Agrobacterium tumefaciens and A. rhizogenes. These provide natural genes transfer and gene expression in plant systems. In recent years, A. tumefaciens has been treated as nature’s most effective plant genetic engineer. A. tumefaciens is the species of choice but A. rhizogenes also has been used to transfer genes into number of plant species.

Agrobacterium mediated transformation has the following advantages 1. Agrobacterium is capable of infecting intact plant cells, tissues and organs. Therefore, tissue culture limitations are much less of a problem. 2. Transformed tissues can be regenerated more rapidly. 3. It is a natural means of gene transfer. 4. Agrobacterium is capable of transferring large fragments of DNA very effectively without substantial rearrangements as in particle bombardment. 5. Integration of T-DNA is a precise process. 6. The stability of gene transfer is excellent.

However, it has the limitation of host range; some important crops cannot be infected with Agrobacterium. Progress has been achieved to overcome this limitation by the development of highly virulent strains of Agrobacterium. Sometimes, cells in a tissue that are capable of regenerating are difficult to transform. It may be due to the fact that embryogenic cells are in deep layers so cannot be reached by Agrobacterium, or simply are not targets for T-DNA transfer. In recent years wounding of tissues by sonication has been used to facilitate the entry of bacteria into the target tissue.

Nevertheless, Agrobacterium-mediated gene transfer has been used successfully for a broad range of plant taxa including a number of woody plants. For sometime, monocots, specially cereals were thought not to be susceptible to Agrobacterium-mediated T-DNA transfer (DeCleene, 1985). However, Hiei et al., 1994 have shown that the interaction of Agrobacterium with higher plants cells to deliver T-DNA is universal Different strains of Agrobacterium show variation n their effectiveness for the induction of crown gall tumours in woody plants (Ellis, 1995).

Particle bombardment (biolistic method)

Biolistics coats the desired DNA onto microscopic metal beads and then fires these at high velocity into pieces of plant tissue. The beads deliver DNA into plant cells, some of which will incorporate the DNA into their chromosomes. The transgenic cells resulting from these processes can be selected and regenerated into whole plants by tissue culture technique. Since the development by Klein et al., (1987), this method has become most commonly used method of plant transformation. This method is found to be useful for transformation of plant tissues that are recalcitrant to Agrobacterium mediated transformation.

This method has been used to create many transgenic organisms, including mammalian cells, microorganisms, and plant species. The use of particle bombardment requires careful consideration of a number of parameters including physical (particle type, size and velocity, DNA concentration, the form of the plasmid DNA), environmental and biological parameters (explant type and stage, pretreatment).

There are several advantages that make this technique a method of choice for engineering crop species.

1. This technique is universal: Transient gene expression has been demonstrated in numerous tissues from many different species including plants and animals. 2. Allows the study of basic plant developmental processes and also clarify the origin of germline in regenerated plants by utilizing chromogenic markers. 3. Transformation of recalcitrant species is possible. 4. The need for callus tissue is eliminated or can be reduced therefore minimize the time that has to be spent on tissue culture. 5. The DNA constructs to be inserted can be created on common cloning plasmids.

12 6. It is clean and safe. 7. Transformation of organized tissue: The ability to genetically engineer organized and tissues with regeneration potential permits introduction of foreign genes into elite germplasm. 8. There is no need for protoplasts. 9. The gene of interest and the selectable marker gene can be physically separate during shooting if desired.

However, there a are few disadvantages associated with this system. One of the main problems is that in plants gene transfer leads to integration of transgene in multiple copies and rearrangement (Kohli et al., 1998). This method can also lead to the emergence of chimeric plants. In addition to that, tissue damage can also result due to the lack of control over the velocity of bombardment. However, biolistics has been effectively used in woody plant to successfully transform various tissues including stems, nodules, zygotic embryos, somatic embryos, protoplasts and seedlings (McCown et al., 1991; Charest, et al. 1993; Duchesne, et al., 1993).

Electroporation

Electroporation (electroinjection) is the process where electrical impulses of high field strength are used to reversibly permeabilize cell membranes reversibly and allow substances add to medium, including DNA to enter. Electroporation was believed to be limited for protoplasts and has been used for a long time for transient and integrative transformation of protoplasts. The range of tissues that can be transformed by electroporation seems to be narrower but they include tobacco mesophyll cells (Morikawa et al., 1986) as well as cultured sugar beet cells (Lindsey and Jones, 1987). This method is simple, fast, convenient, simple, has low cell toxic and is relatively inexpensive to obtain transient and stable transformation in tissues. Cell type, type and the concentration of enzymes used to remove cell wall and duration and the voltage of the electric pulse are some factors that affect the transformation efficiency by this method (Lin et al., 1997). DNA constructs to be inserted can be created on common cloning plasmids and multiple genes present on different plasmids can be inserted by using mixtures of DNA (Minocha and Wallace, 1999) are the main advantages of this method. The main drawback of this method is the difficulty in regenerating plants from protoplasts. In addition to that many variables that have to be considered in the procedure. Nevertheless, in barley (Salmenkallio-Marttila et al., 1995), and in rice (Rao et al., 1995) stable transformation by electroporation has been obtained. In woody plants gene transfer has been successful by electroporation. For example Picea glauca (Bekkaoui et al., 1988, 1990).

Microinjection

Microinjection is the direct mechanical introduction of DNA using a fine syringe under microscopical control into specific target. Microinjection remains the method of choice for the production of transgenic animals by transferring DNA into mammalian zygotes. It is host-range independent and when compare with electroporation it does not necessarily require a protoplast regeneration system. However, it is generally successful with protoplasts. The disadvantages of microinjection process are expensive, slow, and most importantly requires highly skilled and experienced personnel. Only a part of the plant can be transformed by this technique, resulting in the production of chimeric plants. With plant cells it difficult to anchor cells as opposed to mammalian cells attached together which makes easier to introduce DNA via microinjection.

Importance of reporter genes in transformation

In plant transformation studies, reporter genes are necessary for the rapid detection of DNA introduction. The β-glucuronidase (gus) gene is the most widely used reporter gene in cereal transformation (Baruah -Wolff et al., 1999). Although it has some useful properties, one of the main disadvantages of using this as the reporter gene is that GUS histochemical staining is destructive, preventing callus proliferation and plant regeneration from transformed tissues after its identification. An alternative reporter gene, GFP (green fluorescent protein), provides opportunity to recover the putative transformed tissue after its identification but GFP visualization requires expensive microscopes. In addition non destructive GUS histochemical protocols become available in the recent past.

The use of antibiotics as selective agents to inhibit the growth of non-transformed tissues has become a controversial aspect of plant transformation studies. These antibiotics can inhibit the growth and development of transformed tissues, if the starting material is a delicate explant such as the protocorm-like bodies of orchids (Chia et al., 1994). In addition, there is also the problem of field release of transgenic plants containing antibiotic resistance genes. There are concerns about whether the use antibiotic resistant genes will increase the development of antibiotic resistance in human pathogens and make current medicines no longer active. The antibiotics used in plant transformation are not commonly used in medicine, therefore the like hood of such an occurrence is rare. Nonetheless, alternative types of marker genes without any antibiotic specific genes have been developed and will likely replace the antibiotic resistance method in future. It may also be possible that an efficient reporter gene could eliminate the use of selectable markers together (Baruah -Wolff et al., 1999).

Each method of gene transfer technique has its own unique advantages and disadvantages. Therefore, to develop an efficient transformation method for a particular

14 plant species one has to rely upon empirical information developed with own experience and work on the optimization of the selected protocol with the particular target tissue.

Monocot transformation

Particle bombardment remains as the preferred method for the genetic engineering of monocots (Christou, 1995; 1996). Transgenic monocots, including maize (Fromm et al., 1990; Gordan-Kamm et al., 1990), rice (Christou et al., 1991), wheat (Vasil et al., 1992), oat (Somers et al., 1992), sugar cane (Bower and Birch, 1992), banana (Sagi et al., 1995), and soybean (Ponnappa et al., 1999), have been successfully obtained via particle bombardment. Agrobacterium-mediated transformation is also being used with increasing frequency (Hiei et al., 1994; Ishida et al., 1996) for monocot transformation.

In general with monocot tissue culture systems, callus can be easily imitated from explant with the correct auxin in the medium and therefore normally for transformation studies tissue culture approach is chosen. Transformation of monocotyledonous plants in general is reported to have a low efficiency (Wilmink and Dons, 1993). One of the factors that can be used to increase the transformation efficiency is, an effective selection technique (Christou, 1992 and Ritala et al., 1994). Selection is important to inhibit growth of the non-transformed cells which will enable only transformants to survive and regenerate into complete transgenic plants. Without a proper selection, a majority of the non- transformants will dominate the culture and result in chimera plants. For genetic transformation of any plant, it is important to obtain a transgenic which possess the gene(s) in the plant genome. The presence of an effective selection system is especially important for plants like oil palm with a slow tissue culture and regeneration process. A poor selection system will result in the production of chimeric plants by allowing the nontransformant cells to replicate, because the selection agents can no longer active due to prolonged period in culture medium.

Oil palm transformation

Parveez, et al. (2000) describes the production of transgenic oil palm (Elaeis guinensis Jacq) using the biolistic method. Oil palm is the first member of the family Arecaceae that has been transformed. Development of an efficient gene transfer system for oil palm seemed to have remained one of the major priorities in oil palm genetic engineering. The biolistic method has initially been chosen as the method for oil palm transformation as it has been the most successful method for most monocotyledons.

Optimization of physical and biological parameters, including testing of promoters and selective agents, has been carried out as a prerequisite for stable transformation by monitoring transient gus gene expression. Physical parameters, (helium pressures, distance from rupture disc to macrocarrier, distance from macrocarrier to stopping plate, distance from stopping plate to target tissue, vacuum pressures, number of bombardments, particle types and sizes and the effect of calcium chloride and spermidine on microcarrier-DNA binding) have been optimized for DNA delivery into oil palm embryogenic callus using biolistic devices (Parveez et al. 1996b). For this investigation,

15 embryogenic callus were bombarded with gold or tungsten particles coated with a pEmuGN plasmid containing β-glucuronidase gene driven by an Emu promoter.

According to several investigations, there are number of biological parameters that can influence transformation rates with the microprojectile bombardment. Some of them include, tissue pre-culture (Vasil et al., 1991), duration between bombardment and GUS staining (Reggiardo et al. 1991), the type of concentration of osmoticum (Perl et al., 1992), the explant type (Miki et al., 1993), duration of osmoticum treatment pre-and post- bombardment (Vain et al., 1993) use of different genotypes (Moore et al., 1994),, and DNA concentration (Klein et al., 1988). Parveez et al., (1998a) described the successful optimization of biological parameters affecting transient GUS gene expression in oil palm embryogenic calli via microprojectile bombardment. Parameters optimized were explant type using gold and tungsten microcarrier, bombardment preculture, time between bombardment and GUS staining, genotype, immature embryo preculture, DNA concentration, osmoticum type and concentration, and osmoticum treatment duration before and after bombardment.

The effectiveness of different selective agents kanamycin, geneticin, G-418, hygromycin and basta for the inhibition of the immature embryo derived embryogenic callus were evaluated by Parveez et al. (1996a). According to that investigation it has been concluded that genes conferring resistance to basta and hygromycin act as suitable markers for oil palm transformation. Parveez and Christou (1998) describe the successful recovery of genetically engineered embryogenic oil palm callus tissues by particle bombardment using genes encoding β-glucuronidase, phosphinotricin acetyltransferase and hygromycin phosphotrasnferase. The stable integration of transgenes has been confirmed by molecular analysis including PCR and southern blot hybridization. Therefore, this has resulted in the successful transfer of reporter genes into oil palm and regeneration of oil palm, thus making it possible to improve oil palm through genetic engineering.

Besides application of the biolistic method, studies on transformation mediated by Agrobacterium and utilization of the gfp gene as a selectable marker gene has now been initiated in oil palm (Parveez et al., 2000). Most recently Abdullah et al., (2005) reported successful transformation of oil palm immature zygotic embryos using both the biolistic and Agrobacterium-mediated transformation methods. Their transformation efficiencies were comparable to other plant systems reported with as high as 97.4% recorded for biolistic and 64.4% for Agrobaterium-mediated transfer.

Plant responses to environmental stresses

During their life cycle, plants have to deal with various environmental stresses. Among abiotic environmental stresses, drought and low temperature affect plant growth most seriously. A common element in response to many environmental stresses is cellular dehydration. A lack of soil moisture, low relative humidity, water loss from wounding, elevated temperatures, and reduced xylem conductivity caused by pathogens are among the conditions and treatments that can affect tissue water potential in plants and cause deficiency symptoms (Shinozaki and Shinozaki, 1996; Bray, 1997). Plants have evolved many mechanisms to defend themselves against abiotic and biotic stresses. Plants respond to dehydration and low temperature with a number of biochemical, physiological and developmental changes. Molecular and cellular changes to these stresses have been analyzed extensively at the biochemical level and various kinds of proteins and smaller molecules, including sugars, proline, and glycine betaine, which accumulate under these stress conditions.

Mechanisms of plant cold tolerance

There are two main mechanisms that allow plants to withstand subfreezing temperatures; freeze avoidance and freeze tolerance (Levitt, 1980). Both survival strategies are shown by woody plants (Sakai and Larcher, 1987, Malone and Asworth, 1991). Tissues or cells that avoid freezing rely on supercooling in which cellular water remains liquid well below the freezing point. The theoretical limit of supercooling for pure water is -38.10 C (-370 F) (Rasmussen and MacKenzie, 1972, Gusta et al., 1983). Freezing of supercooled water can be observed as low temperature exotherms, i.e. heat of fusion produced by the phase change from liquid to solid. This freezing avoidance occurs in certain tissues like xylem ray cells of many hard-woods, and in organs like shoot and floral primodia of conifers and flower buds of angiosperms (Sakai and Larcher, 1987).

Freezing tolerance involves water migration into intercellular spaces and a gradual growth of extracellular ice. This happens due to the low concentration of solutes in the intercellular spaces and results in the cellular dehydration. Therefore, ensuing dehydration stress is known to be the one of the major survival mechanism in woody plants (Levitt, 1980). The hardiest species of woody plants exhibit increased freezing tolerance; in a fully acclimated state. Under these conditions plants may survive experimental freezing to -1960 C (-3210 F) (George et al., 1974).

Environmental control of cold-hardening

Weiser, (1970) suggested that there are three stages involved in the seasonal cold acclimation process of woody plants native to the temperate zone. The first stage is dependent on the photoperiod. In woody plants short days usually induce growth cessation, a pre requirement for cold acclimation. During this stage plant depends mainly on photosynthesis, stores abundant low molecular weight organic substances, and proceed at relatively warm temperatures in autumn. Cells in the first stage can survive

17 temperatures well below 00 C (320 F) though not fully hardened. In contrast, the second stage of cold acclimation is induced by cold, specially subzero temperatures where plants undergo metabolic and structural changes. This leads to a considerable increase in cold hardening. In many woody plant species, the maximum level of cold hardiness is observed first after an exposure to low freezing temperatures (-30 to -500 C; -22 to - 580F). This has been defined as the third phase of cold-hardening accordingly shortening photoperiod and low temperature are considered to be the two main factors triggering cold acclimation in woody plants.

Plant cold acclimation

During the growing season plants are generally very sensitive to freezing. However, as the year progresses, many plants in temperate regions sense changes in environment that signal the coming winter and respond by increased expression of freeze avoidance and freeze tolerance mechanisms. The main environmental factor responsible for this response is low non-freezing temperatures; the response is known as cold acclimation.

Survival of plants at sub-zero temperatures is largely determined by the ability to cold acclimate. Species capable of cold acclimation are endowed with a greater than normal tolerance of freezing temperatures after exposing to low positive temperatures previously (Levitt, 1980; Thomashow, 1994). During cold acclimation, a number of structural, biochemical and physiological changes occur including the expression of many genes, alterations in membrane lipid composition and accumulation of sugars, proline, soluble proteins, and organic acids (Thomashow, 1994; Hughes and Dunn, 1996).

Although a variety of physiological alterations are correlated with cold acclimation (Levitt, 1980, Sakai and Larcher, 1987) a clear role for any of these changes in the development of freezing tolerance in plants is yet to be determined. However, protein synthesis appears to be required for development of freezing tolerance (Chen et al., 1983), and accumulation of several distinct polypeptides has been observed during cold acclimation (Guy, 1990b). Consequently, accumulation of these novel proteins was suggested to play a role in the observed increase in freezing tolerance (Cloutier, 1984; Guy and Haskell, 1987; Mohapatra et al., 1987; Kurkela et al., 1988; Gilmour et al., 1988; Lang et al., 1989; Perras and Sarhan, 1989) in plants.

Accumulation of specific transcripts under stress conditions in plants

Accumulation of specific transcripts is observed during cold acclimation in many plant species (Hughes and Dunn, 1996; Thomoshow, 1999,). By differential screening of cDNA clones corresponding to these cold regulated transcripts have been isolated. The derived amino acid sequences from some of these cold regulated transcripts show homologies with LEA/Rab or dehydrin proteins (Hughes and Dunn, 1996; Thomoshow, 1999). . Other cold regulated transcripts encode a thiol protease, a (LTP) lipid transfer protein, or a NADPH-aldose reductase (Schaffer and Fisher; 1988. Hughes et al., 1992;

18 Lee and Cheng, 1993) and phosphophenol pyruvate carboxykinase (Vasquez et al. (2000). Vasquez et al., 2000 also demonstrated that in rapeseed corresponding protein to the transcript was induced by exposure to cold temperature.

Glycine-rich proteins (GRPs)

Glycine rich proteins consist of repetitive amino acid pattern with high glycine content. They are a class of simple-structured proteins, resemble animal keratins or silk fibronins i.e. proteins that are characterized by both strength and flexibility. GRPs and /or their corresponding genes have been identified in a variety of plants including both monocots and dicots. Their exact function is not known but most of them are considered to be structural cell wall components like HRGPs (hydroxyproline-rich glycoproteins) or PRPs (proline-rich proteins). GRPs are also expressed in response to a variety of abiotic stress factors, such as wounding (Yashuda et al., 1997), water stress (Molina et al., 1997), and cold (Horvath and Oslon, 1999). Cornels et al., (2000) have isolated and characterized cDNAs encoding two GRPs which accumulate in chickpea (Cicer arietinum L.) in response to fungal and other abiotic stress factors.

Heat Shock Proteins

Heat shock proteins are also called stress proteins and they are present in all cells. They are induced when cell undergoes various types of stresses like cold, heat and oxygen deprivation. Induction of heat shock proteins (HSPs) is one of the most universal and rapid responses of all cells to heat stress. In addition to the heat stress, expression of HSP101 mRNAs in wheat leaves was induced by a 2-h dehydration and a treatment with 5x10-5M ABA, but was not affected by chilling or wounding. This indicates that HSP101 proteins may be involved in both heat and drought response in wheat. It was demonstrated that proteins belonging to HSP 100 family are involved in the acquisition of thermotolerance in yeast (Sanchez and Lindquist, 1990; Lindquist and Kim, 1996). Their studies also reveal that intracellular function of HSP is to aid renaturation of proteins that were denatured by heat.

Protein phosphatases 2C (PP2Cs)

Protein phosphatases 2C are a class of evolutionarily conserved serine/threonine protein phosphatases. . Structurally, plant PP2Cs consists of a catalytic core domain and N-terminal extension to regulate the catalytic activity of the enzyme (Rodriguez, 1998). They are primarily involved in stress responses. Several plant genes encoding PP2Cs have been identified in the past few years (Rodrigiez, 1998. Miyazaki, et al., 1999). Vranova et al., 2000 have isolated a protein phosphatase 2C (PP2C)-homologous cDNA from Nicotina tabacum (NtPP2Cl) It has been shown that the expression of NtPP2Cl was strongly induced by drought, but repressed by oxidative stress and heat stress. It is suggested that NtPP2Cl operates at the junction of drought, heat shock and oxidative stress.

19 Plant Fibrillins

Plant fibrillins are widespread from cyanobacteria to higher plants. They are nuclear-encoded plastid proteins upregulated during chromoplast differentiation in certain fruits (Deruere et al., 1994) and in flowers (Vishnevetsky et al., 1996). They are also named as plastid lipid –associated proteins or ChrC. Fibrilins have been first purified from pepper fruit and cucumber flowers. They seem to be involved in the stabilization of lipid structures in aqueous environments. During fruit ripening in pepper, fibrillin is necessary for the assembly of the carotenoid-storing fibrils in chromoplasts. (Deruere et al., 1994). Chen et al., 1998 reported the accumulation of fibrilins in drought-stressed pepper leaves. This has been confirmed by the cloning of a cDNA encoding a potato chloroplast protein which is homologuos to pepper fibrillin (Gillet et al., 1998) which was previously characterized as a water stress protein. The protein, localized in the stoma of the thylakoids (Pruvot et al., 1996), was accumulated under low temperature or high light (Pruvot et al., 1996).

Dehydrins

Dehydrins are characterized by conserved amino acid motifs, including a DEYGNP motif and a lysine rich block (KIKEKLPG). (Dure et al., 1989; Close et al., 1993a, b; Close, 1997). The lysine-rich block can be seen as a longer but less conserved stretch of amino acids, the K-segment (EKKGIMD-KIKEKLPG) in addition to dehydrin sequence like glycine-rich repeats (Close, 1996, 1997). Close, (1996) hypothesized that dehydrin and compatible solutes act together to stabilize macromolecules such as proteins and nucleic acids, thereby stabilizing the protoplasm.

Dehydrins are found in several species of monocots and dicots and in two families of gymnosperms, the Pinaceae (Pinus edulis Engelm.) and Ginkgogaceae (Ginkgo biloba L.).Using specific antibodies that recognize a dehydrin synthetic peptide (KIKEKLPG), Close, et al. (1993a) reported the presence of dehydrins ranging from 15 to 120kDa in those plants. So far, studies related to dehydrin in tree species have focused mainly on cold acclimation, stratification, freezing and desiccation tolerance (Arora and Wisniewski, 1994; Finch-Savage et al., 1994; Jarvis et al., 1996; Wisniewski et al., 1996; Artlip et al., 1997).

Plant abiotic stress and reactive oxygen species (ROS)

Many crop plants significantly affect due to extremely cold temperatures. The mechanisms of cold-temperature injury very complex, and can cause injury (Sakai and Larcher, 1987). These mechanisms may depend on species, degree of hardiness, and temperature conditions, including cooling rates and the time of the initiation of ice formation (Guy, 1990a). The conditions of the plasma membrane has shown to be critical to cellular behavior during cold-temperature exposure. Plasma membrane is the primary site of freeze injury Several mechanisms have been proposed to be involved in membrane damage at low temperatures. They include injury to membrane bound ATPase (Iswari and Palta, 1989), structural transitions (Rajashekar et al., 1979), membrane phase transitions (Pearce and William, 1985), loss of bound water (Weiser, 1970), and phospholipids degradation by phospholipase D (Yoshida and Sakai 1974).

Oxidative stress occurs when there is an excess free radicals within the cells. It is harmful and is proposed as one of the causes of cold-temperature damage (McKesie and Bowley, - 1997; Halliwell and Gutteridge, 1999). Hydrogen peroxide (H2O2), superoxide anion (O2 ), and the hydroxyl radical (OH.), are among reactive oxygen species (ROS). They have high oxidizing potentials, are responsible for oxidative stress. ROS are generated in organisms under normal conditions and play a role in signal transduction (Fridovich, 1991; Shinosaki and Shinosaki, 1997). Both inefficient deactivation or excessive production of ROS can cause plant injury leading to cell death (Monk et al., 1989). The ROS is known to degrade of polysaccharides, induce peroxidation of cell membrane components, denaturation of enzymes, and the nicking cross linking and scission of DNA strands (Halliwell and Guteridge, 1999; Monk et al., 1989). These data suggest that ROS are produced during exposure to cold and that oxidative stress contributes to cold induced damage of plant cells (Benson and Noronhe-Dutra, 1988).

Plants have evolved very efficient antioxidant systems to scavenge ROS (Allen, 1995) to protect against oxidative stress (Allen 1995). The antioxidant systems can be easily divided into groups; antioxidant enzymes, lipid soluble, membrane associated antioxidants (e.g. alpha tocopherol, beta carotene and ubiquinone), and water-soluble antioxidants (glutathione and ascorbate) (Halliwell and Gutteridge, 1999). Antioxidant enzymes are the most active among these and have efficient protective mechanism against oxidative stress (Halliwell and Gutteridge, 1999). The antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), peroxidase, and the enzymes in the water-water cycle (Allen, 1995). CAT (EC 1.11.1.6) degrades H2O2 into H2O and O2. SOD (EC 1.15.1.1) catalyzes the dismutation of two superoxide radicals (O2-), resulting in the production of H2O2 and O2. SOD has several isozymes, which can be classified by the location of and catalytic metals (Allen, 1995). Peroxidase oxidizes an organic substrate (RH2) with H2O2 producing oxidized substrate and water. The water-water cycle, includes the activity of APX (ascorbate peroxidase), DHAR (dehydroascorbate reductase), GR (glutathione reductase) and MDAR (mono-dehydroascorbate reductase), and is a pathway involved in scavenging superoxide radicals and H2O2 (Asada, 1999).

21 In plants, antioxidant enzymes have shown to play a major role in response to low temperature stress (McKersie et al. 1997). In maize CAT is an essential enzyme to protect mitochondria against chilling stress was induced by low temperature treatment (Prasad et al., 1994). In tobacco over expression of chloroplast CuZnSOD gene increased resistance to oxidative stress (McKersie et al., 1993). In green algae Chlorella there was a high amount of MnSOD with increased chilling tolerance (Clare et al., 1984). The elevated activity level usually correlated with increased stress tolerance (Allen, 1995). Therefore, increased expression of antioxidant systems may protect plants from ROS generated as a result of cold/oxidative stress.

Many plants can become more cold tolerant of cold temperatures through cold acclimation. During cold acclimation, many different physiological changes occur which result in increased in freezing tolerance. One of these changes may be the increased expression of antioxidant enzyme genes to protect from oxidative damage.

Proteomics

Over the last few years there has been a tremendous increase in understanding of the cellular biology of higher organisms, largely as a consequence of the availability of detailed genomic information for a number of species. . Biology depends on the selective read out of individual genes from a particular genome. These are first copied into primary transcripts, processed into mRNA, translated into a protein sequence and finally, post translational modifications (e.g. glycosylation and phosphorylation) occur. Higher eukaryotes show many differences in the mechanisms used in their control of cellular function when compare with lower organisms. In higher organisms number of different proteins are often produced from a single gene and therefore one gene equals to one protein is obsolete. . In addition to that in higher eukaryotes, many different types of posttranslational modifications occur. These different transcriptional and post-translational isoforms can have very different functional roles. Cells use partitioning between different isoforms of the same protein as a central mechanism of cellular control. Although there are modern RNA measurement techniques such as transcript imaging and DNA microarrays for the analysis of transcriptome, they do not give proper indication of the quality and the quantity of the final gene product or the proteins. Studies reveal that protein amounts are not often correlated with mRNA amount (Gygi et al., 1999) due to the differences in mRNA and protein turn over/stability. Therefore, the world of proteins, which is more diverse and complex than the genomics, has been addressed in the post-genomic era. The proteome comprises all the proteins present in an organism, tissue or cell at a particular time. In contrast to the genome, the proteome is not static but highly dynamic. Proteomics is the systematic analysis and documentation of all proteins of a given organism or a specific type of tissues at a given time”. (Wasinger et al., 1995; Blackstock and Weir, 1999; Cahill et al., 2000; Anderson et al., 2001).

Proteomics analysis mainly consists of four steps. Sample preparation, protein separation, identification and functional analysis. The proteomics concept implies the use of analytical techniques mainly 2DE coupled with mass spectrometry. Proteomics analysis

22 is becoming a powerful tool in the functional characterization of plants. Interestingly, in plants, the proteome approach has been widely to study changes in cellular protein expression in response to various biotic and biotic stresses (Thiellement et al., 1999; Porubleva, 2001; van Wijk, 2001; Kersten et al., 2002; Xing et al., 2002). In plants, some examples of comparative proteomics studies are directed towards in search for specific markers involved in plant tolerance to water deficit (Riccardi et al., 1998). In biomedical research, genomic information is no longer considered to be of prime importance. Since the genomes of several organisms, including Homo sapiens, have been sequenced to completion, biomedical research seems to be going through a change. That is mainly because it is at the protein level where biological processes in health and disease can be manipulated for biomedical research (Broder and Venter, 2000). Proteins can be used as biomarkers to detect the earliest stages of diseases like cancer or diabetes. Genetic differences between people are reflected in the different mix of proteins in their cells. Proteins show how cells respond to pathogens or chemicals, and how cells change as they age. Proteins also serve as traffic cops directing complex biochemical signaling pathways in the body. Therefore, proteomics is becoming a very useful tool in the area of biomedical research today.

Proteomics technologies

Two dimensional electrophoresis (2DE) or the roots of modern proteomics dates back to 1975 (Klose, 1975; O’Farrel, 1975) although the term “proteome” was first introduced in a conference by Wilkins in 1994. Proteome refers to the systematic analysis of protein population in a subcellular compartment, cell, tissue or in an organ. Still today 2DE remains as the most resolutive technique for the separation of complex protein mixtures, but it does not involve identification of proteins. Therefore, two main analytical techniques are primarily used in current proteomic research: 2DE for separation followed by visualization of proteins in crude protein extracts coupled with mass spectrometry (Fenn et al., 1989; Karas et al., 1989) for the identification and characterization of proteins.2-DE is based on first dimension, isoelectric focusing (IEF), by which the proteins are separated according to their isoelectric pH (pI) in pH gradient polyacrylamide gels and second dimension SDS-PAGE, by which the proteins are separated according to their molecular weights. Visualization of the separated proteins is achieved by using different stains such as coomassie brilliant blue or silver stain. Protein quantification is done based on the size and the color intensity of the detected spot. The accuracy of the method, however, is restricted due to the low dynamic range of most stains. The recent development of newer stains for protein detection are more sensitive, bind proteins more reproducibly and more constantly and permit the detection of posttranslational modifications.

Although the basic separation principles of the technique have unchanged since the beginning, the recent use of immobilized gradient strips (Bjellqvist et al., 1993) has made IEF more consistent, allowing easier comparison among different gel samples. It also increases the resolution and the reproducibility of gels. (Gorg, 1991, Wildgruber, 2000).. Other areas of development of proteomics research come from newer methods for solubilization of hydrophobic proteins (Chevallet et al., 1998), higher gel resolution and

23 reproducibility (apparatus for casting, protein staining and image analysis). The most recent technical developments in proteomics are directed towards improved resolution and better quantification of proteins and include modern techniques for sample pre- fractionation, free-flow electrophoresis or with chromatofocusing device such as the multicompartment electrolyzer (Herbert and Righetti, 2000),. Difference gel electrophoresis (DIGE) uses separate Cyb dyes to prelabel different samples which then can be analyzed on one gel. This avoids shifts in gel patterns which normally occurs when samples are conventionally separated on two gels are compared (Tonge et al., 2001). This technique which is also based on 2DE is much more reliable for quantitative analysis (Alban et al., 2003). The labeling procedure appears relatively easy to use in DIGE, dedicated equipment and expensive software is necessary for evaluation of gels making it somewhat limited for use.

Identification of the large number of proteins separated by 2DE is commonly achieved by automated MALDI-MS (Matrix Assisted Laser Desorption Ionization – Mass Spectrometry), peptide matching followed by extensive database search (Henzel et al., 1993). MS technology for the protein identification consists of generating ions from the protein sample, separating these ions based on their charge or mass, and then detecting ions using analyzers. The two sources of MS that have been used widely are MALDI and ESI. MALDI-TOF allows the high-precision measurement of masses of peptides resulting from the digestion of protein by an endopeptidase usually trypsin. If it is required to obtain more structural information from the separated proteins, nano liquid chromatography (LC)-electrospray ionization (ESI)-MS/MS method has to be employed (Fenn et al, 1989). Analyzers that have been used in mass spectrometry range from time of flight (TOF) to complex and more sophisticated analyzers such as fourier transform ion cyclo resonance (FTICR). The final step is the analysis of obtained mass spectral data. Bioinformatics tools are used to analyze mass spectra the data to answer question(s) of interest. e.g. identification of protein(s) in the fraction, search for protein modifications.

Ideally one spot on 2 D gel should contain one distinct protein species. However, despite the high resolution of 2-DE gels (Klose and Kobalz, 1995), some spots contain several proteins or protein species in different concentrations. Thus, there is a possibility that one spot may consist of major and minor protein components. While it is easy to identify the major component is by database search using peptide mass finger printing (PMF), minor components are not identified unambiguously. Therefore, the detection of different protein spot components from a same spot remains as a challenge for the analysis of 2- DE gels, mass spectrometry and MS databases(Fenn et al., 1989; Karas et al., 1989) for the identification and characterization of proteins.In a recent paper, MudPIT was used to analyze rice proteome, in which the combined 2DE and MudPIT approaches identified 2528 unique proteins (Koller et al., 2002; Whitelegge, 2002).

Plant proteomics studies

Plant proteomics started in the early 1980s and all of the concepts described above have been applied to the field of plant proteomics (Thiellement et al.; van Wijk, 2001). However when compare with proteomics research in animals and yeast plant proteomics is still at its infancy (Zivy and de Vienne, 2000; van Wijk 2001; Kersten et al., 2002). 2DE (using immobilized pH gradient strips in the first dimension and SDS- PAGE in the second dimension) based proteomic approaches have made a considerable progress in plant proteomics research. A study of the complete genetic information of plants and the resulting activities in the cellular level such as transcription, post translational modifications, protein expression, and protein-protein interaction, is essential for complete understanding of plant biology.

Proteomics studies have been conducted using different plant of tissues of different plant species including Arabidopsis (Kamo et al., 1995;Tsugita et al., 1996; Komatsu et al., 1999; Rakwal and Komatsu, 2000) rice (Komatsu et al., 2003; Rakwal and Agrawal, 2003) and maize ( Chang et al., 2000).. The number of spots resolved in plant proteomic 2-D projects can be between a few hundred and below 2000 and greatly depends on the plant species as well as the tissue (Tsugita et al., 1994; Kamo et al., 1995; Porubleva et al., 2001). When compare with animal tissues, plant tissues have low protein content and the protein yield can be even lower due to the presence of compounds from the secondary metabolism. These compounds can negatively affect the protein yield during protein extraction. Because of this reason, the resolution of plant 2DE has not been improved in the past few years (Tsugita and Kamo, 1999).

In plants, research is now being directed to study the proteomes from subcellular compartments such as membranes or organelles. Complete proteome has been fractionated into sub-proteome such as organelles, subcellular compartments and multiprotein complexes. This improves the resolution and sensitivity and reduces the overall complexity (Jung et al., 2000) because the resolution of a protein spot on a 2D gel is limited by several factors including size, abundance and other electrophoretic properties. Several proteomics research groups have studied subcellular proteomes and protein complexes in plants.e.g. proteins in cell wall, the plasma membranes of nuclei, mitochondria, chloroplast, endoplasmic reticulum and golgi apparatus (Rouquie et al., 1997; Peltier et al., 2000; Prime et al., 2000; Peltier et al., 2001, Krift et al., 2001; Millar et al., 2001; Bae et al., 2003).

Application of proteomics in plant stress studies

The identification of proteins separated on 2-D gels is a useful way to examine the metabolic changes induced by environmental conditions/treatments. In recent years, proteomic approach has been widely used to study the effect abiotic stresses on different plant species. Effect of drought on protein expression in the needles of maritime pine (Costa et al., 1998) and in elongating zone of maize leaves (Riccardi et al., 1998) were studied by quantifying and analyzing the spots on 2-D gel images. In both species a larger proportion of proteins showed a different response to drought. Accordingly, in pine needles, a total of 38 spots were affected by the drought while 24 being up-regulated, and in maize out of 78 spots that have been affected, 50 were upregulated. Proteins that were up- or down-regulated by drought were further characterized/identified by microsequencing. Among the identified proteins, were enzymes involved in different cellular mechanisms, such as glycolysis and lignin synthesis, protection against oxidative stress, chaperon function (HSP), .in addition to hydrophilic proteins including LEA (late embryogenesis-abundant) proteins. In pine needles, degradation products of low molecular weight were also detected indicating the proteolysis of the ribulose bisphosphate carboxylase large subunit upon exposure to drought. Similar observations have been observed Rey et al. (1998) with potato (Solanum tuberosum).

Dehydration or water deficit induces various biochemical, physiological and morphological responses in plants. For instance stomatal closure and reduced growth of above ground parts. Dehydration stress induces different genes and many cellular functions are also affected by this stress. High salinity and water stress both increase osmotic potential and therefore some metabolic changes are common in both stresses. Several studies report the identification of proteins induced under these conditions. . In several studies, induced proteins have been identified. Moons et al. (1997) identified 3 proteins in rice roots; group 2 (also called dehydrins) and group 3 LEA proteins (late embryogenesis abundant protein) and a novel class of glycine-rich proteins by microsequencing and western blotting. Rey et al. (1998) showed the induction of a thioredoxin-like protein in potato chloroplasts under water stress.

Chang et al. (2000) first reported the systematic identification of plant proteins by mass spectrometry (Zivy and Vienne, 2000). Pre-treatment of plant roots at low oxygen levels (hypoxia) seems to improve the tolerance to anoxia and glycolytic enzyme levels is known to increase by anoxia. Chang et al. (2000) compared the protein changes in maize root tips subjected to anoxia with those induced by hypoxia. According to their findings, increased levels of glycolytic enzymes could not alone account for acclimation brought about by hypoxia treatment. Subsequently, they identified the other proteins that were synthesized during hypoxia. . In recent years there has been an increased interest in plant response to abiotic stress, mainly because of its possible applications to breeding programs of cultivated species.

26 In plants as well as in other organisms, the synthesis of heat shock proteins (HSPs) is induced under elevated temperatures. Their synthesis is correlated to the acquisition of thermal tolerance, i.e., the ability to withstand higher temperatures. Plants when compare with other organisms, synthesize a large number of low molecular weight heat shock proteins According to a study done by (Nover and Scharf 1984) there was an induction of 48HSPs in tomato cell cultures.

2 DE has been to study the protein changes associated with cold stress in plants. This technique is mainly used to identify the induced proteins whose regulation may be related with cold acclimation and to describe the protein response of various tissues to cold. Hausman et al., 2000 reported the accumulation of two families of high-molecular mass polypeptides in poplar, in response to chilling in cuttings as in vivo raised shoots. In rape seedlings, there is an induction of proteins by low temperature (Meza-Basso et al., 1986). Guy and Haskell (1987) were able to detect polypeptides associated with cold acclimation in spinach seedlings: Accordingly, the synthesis of these polypeptides was increased during the period of freezing tolerance acquisition, and reduced during re- acclimation. van Berkel et al., 1994 detected quantitative changes of 26 proteins in response to cold treatment of potato tubers. Cabane et al. (1993) identified chilling- acclimation related proteins in soybean, among them is heat shock protein (HSP70). Sabehat et al., 1996 has shown that long-term chilling tolerance of tomato fruit acquired by heat shock treatment is correlated to the persistence of HSPs. Danyluk et al. (1991) has initiated a genetic approach to study the response of three varieties of Triticum aestivum to cold treatment.

Sections of the dissertation

Based on the research published on previous literature I hypothesize that cold hardy palms can be regenerated and somatic embryogenesis can be induced with the correct combination of hormones with the suitable explant. I further hypothesize that S. palmetto zygotic embryos can be genetically transformed using marker genes by both the biolistic and Agrobacteium- mediated transformation methods. To elucidate the needle palm cold tolerance mechanism I proposed the use of proteomics technology and I hypothesize that identifiable cold responsive proteins can be isolated and characterized in needle palms using palm leaf protein profiles as the starting material. Chapter 2 describes efforts to regenerate cold hardy palm Trachycarpus fortunei via organogenesis. Chapter 3 describes efforts to induce somatic embryogenesis in Sabal palmetto, along with morphological observation and some proteomics efforts to discriminate non- embryogenic and embryogenic tissues. Chapter 4 describes the use of both biolistic and genetic transformation methods to transform S. palmetto mature zygotic embryos using marker genes gus and gfp. Chapter 5 describes the use of 2-D gel electrophoresis technique along with protein identification using MALDI-TOF-MS to search for cold responsive proteins in the most cold hardy palm Rhapidophyllum hystirx (needle palm). This chapter also describes an effort to elucidate the cold tolerance mechanism in needle palm. Chapter 6 describes the summary of the research work described in this dissertation and some future directions related to some of the projects.

References

http://www.Inla.org/palms.html

http://www.flmnh.ufl.edu/fossilhall/Library/Sabal/sabal.htm

Abdulla R, Zainal A, Heng WY, Li LC, Beng, YC, Phing LM, Sirajuddin SA, Ping WYS, Joseph JL, Jusoh SA, Muad MR, Huey YL (2005) Immature embryo; A useful tool for oil palm ( Elaeis guineensis Jacq.) genetic transformation studies. Electronic Journal of Biotechnology 8:25-34 Aberlenv-Bertossi F, Noirot M, Duval Y (1999) BA enhances the germination of oil palm somatic embryos derived from embryogenic suspension cultures. Plant Cell, Tissue and Organ culture 56:53-57 Alban A, David SO, Bjorkesten L, Andersson C, Sloge E, Lewis S, Currie I (2003) A novel experimental design for comparative two-dimensional gel analysis: Two- dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics 1:36-44 Allen RD (1995) Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol. 107:1048-1054 Ammirato PV (1983) Embryogenesis. Handbook of Plant Cell Culture. 1:82-123 Anderson NG, Matheson A, Anderson NL (2001) Back to the future: the human protein index (HPI) and the agenda for post-proteomic biology. Proteomics 1:3-12 Arora R, Wisniewski ME (1994) Cold acclimation in genetically related (sibling) deciduous and evergreen peach (Prunus persica (L.) Batsch). 11. A 60-kilodalton bark protein in cold acclimated tissues of peach in heat stable and related to the dehydrin family of proteins. Plant Physiol. 105:95-101 Artlip TS, Callahan AM, Bassett CL, Wisniewski ME (1997) Seasonal expression of a dehydrin gene in sibling deciduous and evergreen genotypes of peach (Prunus persica L.) Batsch). Plant Mol. Biol. 33:61-70 Asada K (1999) The water-water cycle in chloroplast: Scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Physiol Plant Mol Biol 50:601- 639 Bae MS, Cho EJ, Choi EY, Park OK (2003) Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J 36:652–663 Baruah-Wolff J, Harwood WA, Lonsdale DA, Harvey A, Rull R, Snape JW (1999). Luciferase as a reporter gene for genetic transformation studies in rice (Oryza sativa L.). Plant Cell Reports 18:715-720 Bekkaoui F, Pilon M, Laine E, Raju DSS, Crosby WL, Dunstan DI (1988) Transient gene expression in electroporated Picea glauca protoplasts. Plant cell Rep. 6:476- 479

Bekkaoui F, Datla RSS, Pilon M, Tautorus TE, Crosby WL, Dunstan DI (1990) the effects of promoter on transient expression in conifer cell lines. Theor. Appl. Genet. 79:353-359 Benson EE, Noronha-Dutra AA (1989) Chemiluminescence in cryopreserved plant tissue cultures: the possible involvement of singlet oxygen in cryoinjury. Cryo-Letters 9: 120-131 Birch RG (1997) Plant transformation: problems and strategies for practical application. Ann Rev Plant Physiol Plant Mol. Biol. 48:297-326 Black M (1991) Involvement of ABA in the physiology of developing and mature seeds, In: Abscisic acid: physiology and biochemistry. Davis WJ, Jones HG (Eds.) BIOS Scientific Publishers, Oxford. 99-124 Blackstock WP, Weir MP (1999) Proteomics: quantitative and physical mapping of cellular proteins. Trends Biotechnol. 17: 121-127 Bower R, Birch RG (1992) Transgenic sugarcane plants via microprojectile bombardment. Plant J. 2:409-416 Branton RL, Blake JJ (1983) Development of organized structures in callus derived from explants of Cocos nucifera L. Ann Bot 52:673-678 Branton RL, Blake J (1989) Date palm (Phoenix dactylifera L.) In: Biotechnology in Agricultural Forestry Bajaj YPS (Ed.) Trees-11 Springer-Verlag, Berlin, Heideberg.161-175 Bray EA (1997) Plant responses to water deficit. Trends Plant Sci. 2:48-54 Broder S, Venter JC (2000) Sequencing the entire genomes of free-living organisms: the foundation of pharmacology in the new millennium. Annu. Rev. Pharmacol. Toxicol. 40:97–132 | Buffard-Morel J, Verdeil JL, Dussert S, Magnaval C, Huet C, Grosdemange F (1995) Initiation of somatic embryogenesis in coconut (Cocos nucifera L.), In: Lethal Yellowing: Research and Practical Aspects. Oropeza C (Ed.), Kluwer Academic Publishers, Netherlands. 217-223 Cabane M, Calvet P, Vincens P, Boudet AM (1993) Characterization of chilling- acclimation-related proteins in soybean and identification of one as a member of the heat shock protein (HSP 70) family. Planta 190: 346 Cahill DJ (2000) Protein arrays: high throughput solution for proteomics research? In: Proteomics: A trends guide. Blackstock W, Mann M (Eds.). Amsterdam, Elsevier Science: 49-53 Chang WW, Huang L, Shen M, Webster C, Burlingame AL, Roberts JK (2000) Patterns of protein synthesis and tolerance of anoxia in root tips of maize seedlings acclimated to a low-oxygen environment, and identification of proteins by mass spectrometry. Plant Physiol 122: 295-318 Charest PJ, Calero N, Lachance D, Mitsumune M, Yoo BY (1993) The use of microprojectile DNA delivery to bypass the long life cycle of tree species in gene expression studies. In: Current Topics in Botanical Research. Council of scientific and Industrial research. Menon, J (Ed.) New Delhi 151-163

Charest PJ, Devantier Y, Lachance D (1996) Stable genetic transformation of Picea mariana (Black spruce) via particle bombardment. In Vitro cell. Dev. Biol. 32P: 91-99 Chen HH, Li PH, Brenner ML (1983) Involvement of abscisic acid in potato cold acclimation. Plant Physiol. 71:362–365 Chen HC, Klein A, Xiang M, Backhaus RA, Kuntz M (1998) Drought- and wound- induced expression in leaves of gene encoding a chromoplast carotenoid- associated protein. The Plant Journal 14: 317-326 Chevallet M, Santoni V, Poinas A, Rouquie D,Fuchs A,Kiefer S, Rossignol M, Lunardi J, Garin J, Rabilloud T (1998) New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 19: 1901-1909 Chia T, Chan Y, Chua N (1994) The firely luciferase gene as a non-invasive reporter for Dendrobium transformation. Plant J 6:441-446 Christou P, Ford TL, Kofron MP (1991) Production of transgenic rice (Oryza sativa L.) plants from agronomically important Indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/technology 9:957-962 Christou P (1992) Genetic transformation of crop plants using microprojectile bombardment. Plant J. 2:275-281 Christou P (1995) Strategies for variety independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment. Euphytica 85: 3-27 Christou P (1996) Transformation technology. Trends in Plant Science 1: 423-431 Clare DA, Rabinowitch HD, Fridovich I (1984) Superoxide dismutase and chilling injury in Chlorella ellipsoidea. Arch. Biochem. Biophys. 231: 158-163 Close TJ, Fenton RD, Moonam F (1993a) A view of plant dehydrin using antibodies specific to the carboxy terminal peptide. Plant Mol. Boil. 23:279-286 Close TJ, Fenton RD, Yang A, Asghar R, deMason DA, Crone DE, Meyer NC, Moonan F (1993b) The protein. In: Close TJ, Bray EA (Eds.). Plant Responses to cellular dehydration during Environmental Stress, American Society of Plant Physiologists, Maryland, pp. 104-117 Close TJ (1996) Dehydrins: emergence of a biochemical role of a family of plant dehydrin proteins. Physiol. Plant 97:795-803 Close TJ (1997) Dehydrins: a commonality in the response of plants to dehydration and low temperature. Physiol. Plant 100: 291-296 Cloutier Y (1984) Changes of protein patterns in winter rye following cold acclimation and desiccation stress. Can J Bot 62: 366-371 Corley RHV, Barrett JN, Jones LH (1977) Vegetative propagation of oil palm via tissue culture. Oil Palm News 22:2-7

Corley RHV, Wooi KC Wong CY (1979) Progress with vegetative propagation of oil palm. Planter 55:377-380 Cornels H, Ichinose I, Barz W (2000) Characterization of cDNAs encoding two glycine rich proteins in chickpea (Cicer arietinum L.): Accumulation in response to fungal infection and other stress factors. Plant Science 154:83-88 Costa P, Bahrman N, Frigerio JM, Kremer A, Plomion C (1998). Water-deficit- responsive proteins in maritime pine. Plant Mol Biol 38: 587-596 Danyluk J, Rassart E, Sarhan, F (1991) Gene expression during cold and heat shock in wheat. Biochem. Cell Biol. 69, 383-391 Danyluk J, Perron A, Houde M, Limin A, Fowler B, Benhamou N, Sarhan F (1998) Accumulation of an acidic dehydrin in the vicinity of the plasma membrane during cold acclimation of wheat. Plant Cell 10:623-638 DeCleene M (1985) The susceptibility of monocotyledons to Agrobacterium tumefaciens. Phytopathol. Zeitsch. 113:81-89 Deruere J, Romer S, D’Harlingue A, Backhaus RA, Kuntz M, Camara B (1994) Fibril assembly and carotenoid over accumulation in chromoplasts: a model for supramolecular lipoprotein structures. The Plant Cell 6:119-133 Dong JZ, Dunstan DI (1999). Molecular biology of somatic embryogenesis in conifers. In:Moleuclar Biology of Woody Plants. Jain SM, Minocha SC. Dordrecht, Boston, London, Kluwer Academic Publishers.1:51-87 Duchesne IC, Lelu MA, Aderkas PV, Charest PJ (1993) Microprojectile-mediated DNA delivery in haploid and diploid embryogenic cells of Larix spp. Can. J.For. Res, 23:312-316 Dunstan DI, Tautorus TE, Thorpe TA (1995) Somatic embryogenesis in woody plants. In: In vitro embryogenesis in plants. Thrope TA. Dordrecht, Kluwer. 471-541 Ellis DD (1995) Transformation of gymnosperms. In: Somatic Embryogenesis in woody Plants. Jain SM, Gupta PK, Newton RJ (Eds.), Vol.1. Kluwer Acad. Publ., Dordrecht, The Netherlands. 227-251 Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246: 64-71 Finch-Savage WE, Pramanik SK, Bewley JD (1994) The expression of dehydrin proteins in desiccation sensitive (recalcitrant) seeds of temperate trees. Planta 193:478-485 Francko DA (2003) Palms Won't Grow Here and Other Myths: Warm-Climate Plants for Cooler Regions. Portland, OR and Cambridge, Timber Press, 307pp Fridovich I (1991) Molecular oxygen: friend and foe. Active Oxygen/Oxidative Stress and Plant Metabolism. S. K. Pell EJ. Rockville, American Society of Plant Physiologists: 1-5 Fromm ME, Morrish F, Armstrong C, Williams R,Thomas J, Klein TM (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Bio/Technology 8:833-839

Gabr MF, Tisserat B (1985) Propagating palms in vitro with special emphasis on the date palm (Phoenix dactylifera L.). Scientia Horticulturae 25:255-262 George MF, Burke MJ, Pellett HM, Johnson AG (1974) Low temperature exotherms and woody plant distribution. HortScience 9:519-522 Gillet B, Beyly A, Peltier G, Rey P (1998) Molecular characterization of CDSP34, a chloroplastic protein induced by water deficit in Solanum tuberosum L. plants, and regulation of CDSP34 expression by ABA and high illumination. The Plant Journal 16:257-262 Gilmour SJ, Hajela RK, Thomoshow MF (1988) Cold acclimation in Arabidopsis thaliana. Plant Physiol 87:745-750 Gordon-Kamm WJTMS, Mangano ML, Adams TR, Daines RJ, Start WG, O'Brien JV, Chambers SA, Adams Jr WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants. Plant Cell 2: 603-618 Gorg A (1991) Two dimensional electrophoresis. Nature 349:545-546 Gusta L, Tyler NJ, Chen TH-H (1983) Deep undercooling in woody taxa growing north of the -40 0C isotherm. Plant Physiology 72: 122-128 Guy CL, Haskell D (1987) Induction of freezing tolerance of spinach is associated with the synthesis of cold acclimation induced proteins. Plant Physiol 84:733-738 Guy CL (1990a) Molecular mechanism of cold acclimation. In: Katterman F (Ed.) environmental Injury to Plants, Academic Press, London, pp. 35-61 Guy CL (1990b) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annu Rev Plant Physiol Plant Mol Biol 41:187-223 Gygi SP, Rochon Y, Franza BR, Aebersold R (1990) Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 19:1720-1730 Hall RD, Riksen BT, Weyens GT (1996) A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cell. Nat. Biotechnol. 14 1133-1138 Halliwell B, Gutteridge JMC (1999) Free Radicals in Biology and Medicine. New York, Oxford University Press Hanower J, Pannetier C (1982) In vitro vegetative propagation of the oil palm (Ealies guineensis Jacq.) In: Plant Tissue and Cell culture. Fujiwara A (Ed.) Maruzen, Tokyo.pp. 745-746 Hausman JF, Evers D, Thiellement H, Jouve L (2000) Compared responses of poplar cuttings and in vitro raised shoots to short-term chilling treatments. Plant Cell Rep. 19: 954-960 Henzel WJ, Billeci TM, Slults JT, Wong SC, Grimley C, Watanabe C (1993) Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc. Natl. Acad. Sci. USA 90: 5011- 5015 Herbert BR, Righetti, PG (2000) A turning point in proteome analysis: Sample pre- fractionation via multicompartment electrolyzers with isoelectric membranes. Electrophoresis 21:3639-3648

Hiei Y, Ohta S, Komari T, KumashooT (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens and sequence and sequence analysis of the boundaries of the T-DNA. Plant J. 6:271-282 Hornung R (1995) Micropropagation of Cocos nucifera L. from plumular tissues excised from mature zygotic embryos. Plantations, Res. Dev.2: 38-41 Horvath DP, Olson PA (1999) Cloning and characterization of cold-regulated glycine rich RNA-binding protein genes from leafy spruge (Euphorbia esula L.) and comparison to heterozygous genomic clones, Plant Mol. Biol. 38:531-538 Hughes MA, Dunn MA, Pearce RS, White AJ, Zhang L (1992) An abscisic –acid responsive, low temperature barley gene homology with a maize phospholipid transfer protein, Plant Cell. Env.15861-865 Hughes MA, Dunn MA (1996) The molecular biology of plant acclimation to low temperature. Journal of experimental botany 47:291-305 Huong LTL, Baiocco M, Huy BP, Mezzetti B, Santilocchi R, Rosati P (1999) Somatic embryogenesis in Canary Island date palm. Plant Cell, Tissue and Organ Culture 56: 1-7 Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High frequency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat. biotechnology 14:745-750 Iswari S, Palta JP (1989) Plasma membrane ATPase activity following reversible and irreversible freezing injury. Plant Physiol 90: 1088-1095 Jarvis SB, Taylor MA, MacLeod MR, Davies HV (1996) Cloning and characterization of the cDNA clones of three genes that are differentially expressed during dormancy-breakage in the seeds of Douglas fir (Pseudotsuga menziesii). J. Plant Physiol. 147:559-566 Jones LH (1974) Propagation of clonal oil palm by tissue culture. Oil Palm News 17:1-8. Planter 50:374-381 Jung E, Heller M, Sanchez JC, Hochstrasser DF (2000) Proteomics meets cell biology: the establishment of subcellular proteomes. Electrophoresis 21: 3369-3377 Kackar NL, Solanki KR, Joshi, SP (1989) Micropropagation of date palm (Phoenix dactylifera L.) cv. Khadrawy using tissue culture technique. Animals Arid Zone 28:137-141 Kamo M, Kawasaki T, Miyatake N, Tsugita A (1995) Separation and characterization of Arabidopsis thaliana proteins by two-dimensional electrophoresis. Electrophoresis 16: 423-300 Karas M, Bahr U, Ingendoh A, Hillenkamp F (1989) Laser desorption-ionization mass spectrometric of proteins with masses 100,000 to 250,000 dalton. Angew. Chem. Int. Ed. Engl 28: 760-761 Kersten B, Burkle L, Kuhn EJ, Giavalisco P, Konthur Z, Lucking A, Walter G, Eickhoff H, Schneider U (2002) Large scale plant proteomics. Plant Mol Biol 48: 133-141 Klein TM, Wolf ED, Wu R, Sanford JC (1987) High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327:70-73

Klein TM, Gradziel T, Fromm ME, Sanford JC (1988) Factors influencing gene delivery into Zea mays cells by high velocity microprojectiles. Bio/Technology 6:559-563 Klose J (1975) Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26: 231-243 Klose J, Kobalz U (1995) Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome. Electrophoresis 16: 1034-1059 Kohli A, Leech M, Vain P, Laurie DA, Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration of hot spots. Proc Natl Acad Sci USA 86:8467-8471 Koller A, Washburn MP, Lange BM, Andon NL, Deciu C, Haynes PA, Hays L, Schieltz D, Ulaszek R, Wei J, Wolters D, Yates JR (2002) Proteomic survey of metabolic pathways in rice. Proc. Natl. Acad. Sci. USA 99: 11969-11974 Koncz C, Martini N, Mayerhofer R, Koncz-Kalman Z, Korber H, Redei GP, Schell J (1989) High frequency T-DNA mediated gene tagging in plants. Proc. Natl. Acad. Sci. USA 86:8467-8471 Krens FA, Jamar D (1998) The role of explant source and culture condition on callus induction and shoot regeneration in sugar beet (Beta vulgaris L.) J. Plant Physiol. 134:651-655 Kruft V, Eubel H, Jansch L, Werhahn W, Braun HP (2001) Proteomic approach to identify novel mitochondrial proteins in Arabidopsis. Plant Physiol 127: 1694- 1710 Kurkela S, Franck M, Heino P, Lang V, Palva ET (1988) Cold induced gene expression in Arabidopsis thaliana (l.) Heynh. Theor Appl Genet 79:801-806 Lee SP, Chen THH (1993) Molecular cloning of abscisic acid responsive mRNAs expresses during the induction of freezing tolerance in bromegrass (Bromus inermis Leyss) suspension culture. Plant Physiol. 101:1089-1096 Levit J (1980) Responses of plants to environmental stress. Chilling, freezing and high temperature stresses. New York: Academic Press Lewis BD, Karlin-Neumann G, Davis RW, Spalding EP (1997) Calcium activated anion channels and membrane depolarization induced by blue light and cold in Arabidopsis seedlings. Plant Physiology 114:1327-1334 Lin CH, Xiao L, Hou BH, Ha SB, Saunders JA (1997). Optimization of electroporation conditions for expression of GUS activity in electroporated protoplasts and intact plant cells. Plant Physiol. Biochem. 35: 959-968 Lindquist S, Kim G (1996) Heat-shock protein 104 expression is sufficient for thermotolerance in yeast. Proc. Natl. Acad. Sci. USA 93:5301-5306 Lindsey K, Jones MGK (1989). Transient gene expression in electroporated protoplasts and intact cells of sugar beet. Plant Mol Biol 10: 43-52 Li R, Brawley SH, Close TJ (1998) Proteins immunologically related to dehydrins in fucoid algae. J. Phycol. 34:642-650

35 Loiret C (1981) Vegetative propagation of oil palm by somatic embryogenesis. In: Pushparaj E and Chew PS (Eds.) Oil Palm in the Eighties. Inc Soc Plant Kuala Lumpur.pp. 163-172 Maheshwari N, Rajyalakshmi K, Baweja K (1995) In vitro culture of wheat and genetic transformation-retrospect and prospect. Cri. Rev. Plant Csi. 14:149-178 Malone SR, Ashworth EN (1991) Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques. Plant Physiology 95:871-881 May GD (1995) Generation of transgenic banana (Musa acuminata) plants via Agrobacterium-mediated transformation. Biotechnology 13:486-492 McCown BH, McCabe DE, Russell DR, Robinson DJ, Barton KA, Raffa KF (1991) Stable transformation of Populus and incorporation of pest resistance by electric discharge particle acceleration. Plant Cell Rep. 9:590-594 McKersie BD, Chen YR, De Beus M, Bowler SR, Inze D, D'Halluin K, Botterman J (1993) Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa ( Medicago sativa L.). Plant Physiol 103: 1155-1163 McKersie BD, Bowley SR (1997) Active oxygen and freezing tolerance in transgenic plants. In: Plant Cold Hardiness. Li PH, Chen THH (Eds.) New York, Plenum Press: 203-214 Meza-Basso L, Alberdi M, Ragnal M, Ferrero-Cardinanos M, Delsney M (1986) Changes in protein synthesis in rapeseed (Brassica napus) seedling during a low temperature treatment. Plant Physiol 82:733-738 Miki BL, Fobert PF, Charest PJ, Iyer VN (1993) Procedures for introducing foreign genes into plants. In: Methods in Plant Molecular Biology and Biotechnology. Thompson JE, Glick BR (Eds) CRC Press, Boca Raton, FL. 67-88 Millar AH, Sweetlove LJ, Giege P, Leaver CJ (2001) Analysis of the Arabidopsis mitochondrial proteome. Plant Physiol 127:1711-1727 Minocha SC, Wallace JC (1999). Gene transfer techniques and their relevance to woody plants. In: Molecular Biology of Woody Plants. Jain SM, Minocha SC (Eds) Dordrecht, Boston, London, Kluwer Academic Publishers. 2: 1-24 Miyazaki S, Koga R, Bohnert HJ, Fukuhara T (1999) Tissue and environmental response specific expression of 10 PP2C transcripts in Mesembryanthemum crystallinum. Molecular and general genetics 261:307-316 Mohapatra SS, Poole RJ, Dhindsa RS (1987) Changes in protein patterns and translatable messenger RNA population during cold acclimation of alfalfa. Plant Physiol 82:733-738 Molina A, Mena M, Carbonero P, Garcia-Olmedo F (1997) Differential expression of pathogen-responsive genes encoding two types of glycine-rich proteins in barley, Plant Mol. Biol. 33:803-810 Monk LS, Fagerstedt KV, Crawford RMM (1989) Oxygen toxicity and superoxide dismutase as an antioxidant in physiological stress. Physiol. Plant 76:456-459 Moons A, Gielen J, Vandekerckhove J, Van der Straeten D, Gheysen G, Van Montagu M (1997) An abscisic-acid and salt-stress-responsive rice cDNA from a novel plant gene family. Planta 202: 443-454

Moore PJ, Moore AJ, Collins GB (1994) Genotype and developmental regulations of transient expression of a reporter gene in soybean zygotic cotyledons. Plant Cell Rep. 13:556-560 Morikawa H, Iida A, Matsui M, Ikegami M, Yamada Y (1986) Gene transfer into intact cells by electroinjection through cell walls and membranes. Gene 41: 121-124 Noiret JM (1981) Application of in vitro culture to improvement and production of clonal material in the oil palm. Oleagineux 36:123-126 Nover L, Scharf KD (1984) Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures. Eur J Biochem 139:303–313 Nylander M, Svensson J, Tapio Palva E, Welin BV(2001) Stress-induced accumulation and tissue specific localization of dehydrins in Arabidopsis thaliana. 263:263-279 Nyman LP, Gonzales CJ, Arditti J (1983) Reversible structural changes associated with callus formation and plantlet development form aseptically cultured shoots of taro (Colocasia esculenta var. antiquorum). Ann. Bot 51:279-286 O'Farrell (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250: 4007-4021 Palva ET (1994) gene expression under low temperature stress. In: Stress Induced gene Expression in Plants. Baasra AS (Ed.) Harwood Academic, New York, 103-130 Pareek OP. (1984) Date palm growing potential of Indian arid zone. Indian Horticulture 29:2-8 Parveez GKA, Chowdhury MKU, Saleh NM (1996a) Determination of minimal inhibitory concentration of selection agents for oil palm (Elaeis guineensis Jacq.) Transformation. Asia Pacific Journal of Molecular biology And Biotechnology. 4: 219-228 Parveez GKA, Chowdhury MKU, Saleh NM (1997) Physical parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq) using the biolistic device. Industrial Crops and Products 6:41-50 Parveez GKA, Chowdhury MKU, Saleh NM (1998a) Biological parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) embryogenic calli via microprojectile bombardment. Industrial Crops and Products 8:17-27 Parveez GKA, Christou P (1998b) Biolistic-mediated DNA delivery and isolation of transgenic oil palm (Elaeis guineensis Jacq.) embryogenic callus cultures. Journal of Oil Palm Research 10:29-38 Parveez GKA, Masri MM, Zainal A, Majid NA, Yunus AMM, Fadilah HH, Rasid O, Cheah S (2000) Transgenic oil palm: production and projection. Biochem. Soc. Trans 28:969-972 Patton WF (2000) A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics. Electrophoresis 21:1123-1144

Pearce RS, William JHM (1985) Wheat tissues freeze-etched during exposure to extracellular freezing: distribution of ice. Planta 163:295-303 Perl A, Kless H, Blumenthal A, Galili G, Galun E (1992) Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures. Mol. Gen. Genet. 235:279-284 Perras M, Sarhan F (1989) Synthesis of freezing tolerance proteins in leaves, crown and roots during cold acclimation of wheat. Plant Physiol 89:577-585 Peltier JB, Frisco G, Kalume DE, Roepstorff P, Nilsson F, Adamska I, van Wijk KJ (2000) Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins. Plant Cell 12:319-342 Peltier JB, Ytterberg J, Liberies DA, Roepstorff P, van Wijk KJ (2001) Identification of a 350 kDa CIpP protease complex with 10 different CIp isoforms in chloroplasts of Arabidopsis thaliana. J Biol Chem 276:16318-16327 Ponnappa T, Brzozowski AE, Finer JJ (1999) Transient expression and stable transformation of soybean using jellyfish green fluorescent protein (GFP). Plant Cell reports 19:6-12 Porubleva L, Vander Velden K, Kothari S, Oliver DJ, Chitnis PR (2001). The Proteome of Maize Leaves: Use of Gene Sequences and EST Data for Identification of Proteins with Peptide Mass Fingerprints. Electrophoresis 22: 1724-1738 Prasad TK, Anderson MD, Martin BA, Stewart CR (1994) Evidence for chilling-induced oxidative stress in maize seedlings and regulatory role for hydrogen peroxide. Plant Cell 6: 65-74 Prime T, Sherrier D, Mahon P, Packman L, Dupree P (2000) A proteomic analysis of organelles from Arabidopsis thaliana. Electrophoresis 21:3488-3499 Pruvot G, Massimino J, Peltier G, Rey P (1996) Effects of low temperature, high salinity and exogenous ABA on the synthesis of two chloroplastic drought-induced proteins in Solanum tuberosum. Physiologia Plantarum 97:123-131 Rabechault H, Ahee J, Guenin G (1970) Colonies cellularies et formes embryos in vitro a partier de cultures d’embryons de palmier a huile (var.dura Becc.) CR Acad Sci Paris. 270:3067-3070 Rabechault H, Martin JP (1976) Multiplication vegetative du palmier a huile (Eleais guineensis Jacq.) a l’aide de cultures de tissue foliares. CR Acad Sci Paris Ser 283: 1735-1737 Rajashekar CB, Gusta LV, Burke MJ (1979) Membrane structure transition: probable relation to frost damage in hardy herbaceous species. In: Low Temperature Stress in Crop Plants. Lyon JM, Graham D, Raison JK. (Eds.) London, Academic Press: 255-274 Rakwal R, Agrawal GK (2003) Rice proteomics: current status and future perspectives. Electrophoresis 24:3378-3389 Rao KV, Keerti Rathore S, Hodges TK (1995) Physical, chemical and physiological parameters for electroporation-mediated gene delivery in rice protoplasts. Transge. Res. 4:361-368

Rasmussen DH, MacKenzie AP (1972) Effect of solute on ice-solution interfacial free energy; calculation from measured homogeneous nucleation temperature. Water structure at the water polymer interface. J. HHG. New York, Plenum Publishing Corporation: 126-145 Reggiardo MI, Arana JL, Orsaria LM, Permingeat HR, Spitteler MA, Vallejos RH (1991) Transient transformation of maize tissues by microparticle bombardment. Plant Sci. 75:237-243 Rey P, Pruvot G, Becuwe N, Eymery F, Rumeau D, Peltier G (1998) A novel thioredoxin-like protein located in the chloroplast is induced by water deficit in Solanum tuberosum. Plant J. 13:97-107 Riccardi F, Gazeau P, de Vienne D, Zivy M (1998) Protein changes in response to progressive water deficit in maize. Plant Physiol 117: 1253-1263 Ritala A, Aspegren K, Kurten U, Salmenkallio-Marttila M, Mannonen L, Hannus R, Kauppinen V, Teeri TH, Enari T (1994) Fertile transgenic barley by particle bombardment of immature embryos. Plant Mol. Biol. 24:317-325 Rodeiguez PL (1998) Protein phosphatase 2C (PP2C) function in higher plants. Plant Mol. Biol. 38:919-927 Rouquie D, Peltier JB, MarquisMansion M, Tournaire C, Doumas P, Rossignol M (1997) Construction of a directory of tobacco plasma membrane proteins by combined two-dimensional gel electrophoresis and protein sequencing. Electrophoresis 18:654-660 Sabehat A, Weiss D, Lurie S (1996) The correlation between heat-shock protein accumulation and persistence and chilling tolerance in tomato fruit Plant Physiol. 110: 531-537 Sagi B, Panis L, Remy S, Schoofs H, Smet KD, Swennen R, Cammue BPA (1995) Genetic transformation of banana and plantain (Musa sp.) via particle bombardment. Bio/Technology 13:481-485 Sakai A, Larcher W (Eds) (1987) Frost Survival of Plants. Responses and Adaptation to freezing Stress. Springer Verag. New York Salmenkallio-Marttila M, Aspegren K, Akerman S, Kurten U, Mannonen L, Ritala R, Teeri T, Kauppinen V (1995) Transgenic barley (Hordeum vulgare) by electroporation of protoplasts. Plant Cell Rep. 15:301-304 Sambanthamurthi R, Parman SH, Noor MRM (1996) Oil palm (Elaeis guineensis) protoplasts: isolation, culture and microcallus formation. Plant Cell, Tissue and Organ Culture 46:35-41 Sanchez Y, Lindquist S (1990) Hsp104 is required for induced thermotolerance. Science 248: 1112-1115 Schaffer MA, Fisher RL (1988) Analysis of mRNAs that accumulated in response to low temperature identifies a thiol protease gene in tomato. Plant Physiol. 87:431-436 Sharma DR, Dwara S, Chowdhury JB (1984) Somatic embryogenesis and plant regeneration in date palm (Phoenix dactylifera L.) cv. Khadravi through tissue culture. Indian J Exp Biol 22:596-598

Shewry PR, Napier JA, Tatham A (1995) Seed storage proteins: Structures and biosynthesis. Plant Cell 7:945-956 Shinozaki K, Yamaguchi-Shinozaki K (1996) Molecular responses to drought and cold stress. Curr. Opin. Biotechnol. 7:161-167 Somers DA, Rines HWGUW, Kaeppler HW, Bushnell WR (1992) Fertile, transgenic oat plants. Bio/Technology 10:1589-1594 Spurr JW (1985) Commercial date palm micropropagation Twyfords. Agri Cell Rep 4:40 Staritsky G (1970) Tissue culture of oil palm (Elaeis guineensis Jacq.) as a tool for its vegetative propagation. Euphytica 19:288-292 Steward FC, Mapes MO, Mears K (1958) Growth and organized development of cultured cells. 11. Organization in cultures grown from freely suspended cells. Am. J. Bot. 45:705-708 Thiellement H, Bahrman N, Damerval C, Piomion C, Rossignol M,Santoni V, de Vienne D, Zivy M (1999) Proteomics for genetic and physiological studies in plants. Electrophoresis 20:2013-2026 Thomas TL (1993) Gene expression during plant somatic embryogenesis and germination: an overview. Plant Cell 5:1401-1410 Thomashow MF (1994) Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance. In: Arabidopsis. Meyerowitz EC Sommerville C (Eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 807-834 Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms, Annu Rev Plant Mol Biol. 50:571-599 Tisserat B (1984) Propagation of date palms by shoot tip culture. Hort Sci 19:230-231 Tisserat B, De Mason DA (1980) A histological study of development of adventitive embryos in organ cultures of Phoenix dactylifera L. Ann Bot (London) 46:465-72 Tonge R, Shaw J, Middleton B, Rowlinson R, Rayner S, Young J, Pognan F, Hawkins E, Currie I, Davison M (2001) Validation and development of fluorescence two- dimensional differential gel electrophoresis proteomics technology. Proteomics 3:377-96 Trick HN (1997) Recent advances in soybean transformation. Plant Tissue Cult. Biotechnol. 3:9-26 Tsugita A, Kawakami T, Uchiyama Y, Kamo M, Miyatake N, Nozu Y (1994) Separation and characterization of rice proteins. Electrophoresis 15:708-720 Tsugita A, Kamo M, Kawakami T, Ohki Y (1996) Two-dimensional electrophoresis of plant proteins and standardization of gel patterns. Electrophoresis 17: 855-865 Tsugita A, Kamo M (1999) 2-D electrophoresis of plant proteins. Meth. Mol. Biol. 112: 95-97 Vain P, McMullen MD, Finer JJ (1993) Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep. 12:84-88

van Berkel JSF, Gebhardt C (1994) Transcripts accumulating during cold storage of potato (Solanum tuberosum L.) tubers are sequence related to stress-responsive genes Plant Physiol 104: 445-452 van Wijk KJ. (2001) Update on plant proteomics. Challenges and Prospects of Plant Proteomics. Plant Physiol 126:501-508. Vasil V, Brown SM, Re D, Fromm ME, Vasil IK (1991) Stably transformed callus lines from microprojectile bombardment of cell suspension cultures of wheat. Bio/Technology 9:743-747 Vasil V, Castillo AM, Fromm ME, Vasil IK (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile callus. Bio/Technology 10:667-674 Vasquez JS, Gallois P, Delseny M (2000) Accumulation and nuclear targeting of Bnc24, a Brassica napus ribosomal protein corresponding to a mRNA accumulating in response to cold treatment. 156:35-46 Vishnevetsky M, Ovadis M, Itzhaki H, Levy M, Libal-Weksler Y, Adam Z, Vainstein A (1996) Molecular cloning of a carotenoid-associated protein from Cucumber sativus corollas:homologous genes involved in carotenoid sequestration in chromoplasts. The Plant Journal 10:1111-1118 Vranova E, Langebartels C, Montagu MV, Inze D, Camp WV (2000) Oxidative stress, heat shock and drought differentially affect expression of tobacco protein phosphatase 2C. J Exp Bot. 51:1763-1764 Wasinger VC, Corwell SJ, Cerpa-Poljak A, Yan JX, Gooley AA, Wilkins MR, Duncan MW, Harris R, Williams KL, Humphery-Smith I. (1995) Progress with gene- product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 16: 1090-1094 Weiser BV (1970) Cold resistance and injury in woody plants. Science 169:1269-1278 Whitelegge JP (2002) Plant proteomics: BLASTING out a MudPIT. Proc. Natl. Acad. Sci. USA 99:11564-11566 Wildgruber R, Harder A, Obermaier C, Boguth G, Weiss W, Fey SJ, Larsen PM, Gorg (2000) Towards higher resolution: two-dimensional electrophoresis of Saccharomyces cerevisae proteins using overlapping narrow immobilized pH gradients. Electrophoresis 21:2610-2616 Wilmink A, Dons JJM (1993) Selective agents and marker genes for use in transformation of monocotyledonous plants. Plant Mol. Biol. Rep. 11:165-185 Wisniewski M, Close TJ, Artlip T, Arora R (1996) Seasonal patterns of dehydrins and 70-kDa heat-shock proteins in bark tissues of eight species of woody plants. Physiol. Plant.96:496-505 Xing T, Quellet T, Miki BL (2002) Towards genomic and proteomic studies of protein phosphorylation in plant pathogen interactions. Trends Plant Sci. 7: 224-230 Yasuda E, Ebinuma H, Wabiko H (1997) A novel glycine rich/hydrophobic 16 kDa polypeptide gene from tobacco: similarity to proline rich protein genes and its wound-inducible and developmentally regulated expression. Plant Mol. Bio. 33:667-678

Yoshida S, Sakai A (1974) Phospholipid degradation in frozen plant cells associated with freezing injury. Plant Physiol 53:509-511 Zaid A, Tisserat B (1983) Morphogenic responses obtained from a variety of somatic explant tissues of date palm. The Bot Mag (Tokyo) 96:67-73 Zivy M, de Vienne D (2000) Proteomics: a link between genomics, genetics and physiology. Plant Mol Biol 44:575-580.

Chapter 2

Regeneration of Trachycarpus fortunei (Hook) H. Wendl. (Chinese

windmill palm) plants via organogenesis

Keywords Windmill palm, Trachycarpus fortunei, mature zygotic embryos, meristem, auxins, cytokinins, organogenesis

Abstract Trachycarpus fortunei (Family - Arecaceae) is an attractive palm with foliage that can withstand extremely low environmental temperatures (ca. -130 to -140 C; 70 to 90 F) without damage. Native to temperate and subtropical mountains areas of Asia, it is now a major landscape palm in warm-temperate regions worldwide, including the U.S. With modest improvements in cold resistance, this palm is a potential candidate for general horticultural use in even colder regions. The only known method of propagation for this species is through seeds, and seedlings which can exhibit great variability. Many desirable characters cannot be evaluated until the palm is several years old. If cloning of selected, high performance palms could be achieved via tissue culture techniques then significant advantages could be made by eliminating this variability. A novel procedure was developed for clonal propagation of T. fortunei using the apical meristem as the explant source. In this study we report the regeneration of plantlets from shoot apical meristem via primary callus production and it is a two-step organogenetic process. One- month-old callus induced from meristem tissues in the modified MS media was transferred into media with gradually decreasing concentrations of auxin (2, 4-D). Shoot induction occurred from organogenic callus with the introduction of low concentration of BAP without forming somatic embryos. Shoots were grown in shoot germination medium and root formation occurred without a requirement for a specific rooting medium. Plantlets were grown under controlled aseptic conditions until they were ready for acclimatization in the greenhouse. SEM studies suggest that greenhouse acclimatization is required for high plantlet survival. The late-juvenile stage palms (short trunks and mature foliage) are now successfully growing under greenhouse conditions and are ready to transfer to field conditions. Once refined for consistent plant production, this regeneration system may be helpful for biolistic or Agrobacterium-mediated transformation of this horticulturally important palm.

Abbreviations ABA- Abscisic acid; BAP - 6- Benzylaminopurine; TDZ -Thidiazuron; 2, 4-D - 2, 4 –Dichlorophenoxy acetic acid

Introduction Trachycarpus fortunei, commonly known as Chusan palm or Chinese windmill palm is one of the cold hardy palms which can withstand severe freezes with little or no damage. T. fortunei is native to temperate and subtropical mountainous areas of Asia including Taiwan, Chusan Islands and southeastern China (Jones 1995). It is commonly grown as a landscape specimen in central and northern Florida, the southeastern U.S. Atlantic and Gulf coasts, and along the west coast. This slow growing palm can grow up

43 to 33ft (10 m) in height on a single stem up to 8-10 in (20-30 cm) diameter. The trunk, narrower at the base, is very rough with the persistent leaf bases clasping the stem as layers of coarse fibrous material. With aging the fibers turn gray, and on large old trees fibers fall off. It has leaves with a long petiole terminating in a rounded fan of numerous leaflets. The waxy leaves on this palm are bright green above and silver-green on the underside. The flowers are aromatic, yellowish in color, borne in large branched panicles; the fruit is a blue-black, kidney shaped drupe.

Propagation of T. fortunei is carried out only by seeds. As it has a single apical meristem, it is unsuitable for propagation by cutting or grafting. Seed propagation leads to wide variation in the progeny due to heterozygosity. Therefore, there is a need to develop an effective protocol for plant regeneration in T. fortunei, not only to supplement nursery- derived seedling transplantation, but also for propagation of selected trees with advantageous phenotypes.

Generally, palms have been highly recalcitrant to in vitro cultivation. This is thought to be due to natural inhibitors that cannot be removed as yet by any other means. Jesty and Francis (1991) demonstrated that the regulation of cell and nuclear parameters in coconut leaves are under separate control. The disruption of this cell coordination is a key contributory factor for recalcitrant cellular behavior of coconut under in vitro conditions. Nevertheless, there has been worthwhile progress in the past few decades in achieving plant regeneration in economically important palms, mainly through somatic embryogenesis. This includes date palm (Tisserat and Mason, 1980; Sharma et al., 1984; Tisserat, 1984a; Kackar et al., 1989); Canary Island date palm (Huong et al., 1999); oil palm (Jones, 1974; Rabechault and Martin 1976; Corley et al., 1977; Pannetier et al., 1981; Paranjothy and Oathman 1982; and Wooi 1990), and coconut palm (Pannetier and Buffard-Morel. 1982; Gupta et al., 1984; Blake, 1990; Verdeil et al., 1994). Corely et al. (1977) and Tisserat (1979 and 1981) have reported the regeneration of oil palm and date palm, respectively, from embryogenesis callus.

Plant regeneration can be achieved via organogenesis or somatic embryogenesis. Each of these two processes has advantageous over the other process. Somatic embryogenesis leads to the production of bipolar structure containing shoot and root axis. This bipolar structure has a closed independent vascular system (Thorpe, 1993). In contrast, during organogenesis tissues and cells undergo changes. These changes lead to the production of a unipolar structure with either shoot or root primodium with a distinct vascular connection to the explant vasculature (Thorpe, 1993.) In organogenesis, organs (desirably shoots) are generated from variety of tissues. Leaf fragments, cotyledons, scutella from embryos and hypocotyls generally have the potential to generate shoots. During plant development, shoots and roots are differentiated at different time periods. Cytokinin- (e.g., 6-benzylamino purine) enriched media usually enhances microshoot induction on tissues and then they are rooted in an auxin-enriched media to develop plantlets. Sometimes shoots form adventitious roots easily without any external supply of auxins. Organogenesis is greatly influenced by many factors such as genotype and plant age, physiological state of the explant, the in vitro environment, light/temperature regimes, and media composition, specially hormone concentrations (Tanimoto and Harada, 1982).

44 In general, juvenile explants, such as cotyledons, hypocotyls, embryos cotyledons, or bud explants from seedlings are more responsive to in vitro regeneration than mature tissues (e.g. bud explants from mature tissues). Immature embryos have given much higher plant regeneration rate when compare with mature embryos in rye (Ward and Jordan, 2001). Micropropagation from juvenile explants, although useful for differentiation studies, has a disadvantage in that it is difficult to predict how a seedling will perform upon reaching maturity. The present investigation was carried out with the objective of developing a workable regeneration protocol using apical meristems of Chinese windmill palm. This paper reports for the first time an in vitro procedure for plantlet regeneration from apical meristem-derived callus of T. fortunei.

The physiological status of in vitro- grown plants is an important factor which determines the success rate during ex-vitro acclimatization (Derbergh, 1991). The different growth conditions during in-vitro growth, such as, artificial gas atmosphere, low light intensity, high sucrose level in culture medium, and high relative humidity can lead to alterations in physiological status of the plant (Zacchini et al., 1997) and in leaf anatomy (Rillo et al., 1988). . In vitro-grown plants taken from an aseptic environment with carefully controlled light, temperature and humidity are extremely fragile. Therefore, it is important to study various anatomical, physiological and biochemical parameters of in- vitro-grown plants during acclimatization. A part of the present study was undertaken with the objective to investigate the status of stomata of in-vitro-grown plants. Based on such information, the levels of humidity, light and temperature during acclimatization could be altered to improve growth and vigor of the plants once transferred to field conditions.

Materials and Methods

Optimization of conditions for callus induction using mature zygotic embryos as explant

Mature seeds were sterilized and seed coats were softened by soaking them in 10% commercial bleach for 6 to 7 days. Embryos can be located by the appearance of a white or bleached spot. Prior to embryo extraction, seed coats were shaved in the area where embryos are located without damaging the embryogenic axis. Embryos were then placed horizontally on various media in petri-dishes.

For preliminary media screening, media were solidified or semi-solidified with agar or agar gel. Formulations include different concentrations of 2, 4-D (100, 75, 50 and 25 µM), different sugars (sucrose and glucose – 3%, 4% and 5%) and several types of activated charcoal (Sigma, Fluka and Carbon Activado Polvo QP-QUIMICA MEYER) with different concentrations – 0.25%, 0.2%, 0.15% and 0.1%). A minimum of 20 embryos were tested for each treatment and the experiment was triplicated. Cultures were incubated in the dark at 280 C for 2 months. Results from the preliminary media screening experiments indicated that 4% sucrose, 0.25% activated charcoal (Carbon Activado Polvo QP-QUIMICA MEYER) 0.4% agar gel appeared to provide the conditions for better callus formation from T. fortunei mature

45 zygotic embryos. In order to determine the optimal 2, 4-D concentration and the basal media, experiments were conducted using a variety of media alterations with 4% sucrose, 0.25% charcoal and 0.4% Agargel™. Culturing media contained different basal media (MS with vitamins (Murashige and Skoog, 1962), N-6 (Chu et al. 1978) and CRI-72-H (Karunaratne and Periyappperuma 1989)) with 13 different concentrations of 2, 4-D (0, 2.4, 3, 7.8, 10, 24, 30, 78, 100, 150, 200, 240 and 300 µM). Cultures were incubated in the dark for 2 months and callusing frequency was recorded. They were then gradually transferred onto media containing reduced concentrations of 2, 4- D to induce embryogenesis (Fernando and Gamage, 2000). Cultures were incubated in each medium for one month before transferring into next lower concentration of 2, 4-D. The quality and the viability of the callus were recorded during each transfer. Calli that became necrotic/brown and unhealthy were recorded as low quality and the calli that did not change in color and stayed healthy without necrosis were recorded as high quality. At the end of the culturing period with the lowest 2, 4 -D concentration (usually 2 µM) calli were transferred into media with different cytokinins i.e. BAP, TDZ and ABA each with 5 µM concentration and were incubated in dark at 280 C with the purpose of inducing shoot formation.

Organogenesis of T. fortunei using shoot apical meristem as explant source

Collection of explant material from the palm was carried out by a destructive method. Three-year-old T. fortunei plants were purchased from Plant Delights Nursery, Inc. (USA). Crowns of the palms along with the apical meristem zone were cut and mature leaves were removed carefully. Then immature leaves were detached with the exception of the ones immediately covering the shoot apical meristem. Excised apical meristem tissues were thoroughly washed with running distilled water and mild liquid soap and sterilized distilled water as soon as they were taken into the laboratory. After this stage all the steps were carried out under clean environmental conditions in a laminar flow cabinet. The tissues were sterilized by soaking in a solution of commercial bleach (10 %) plus a few drops of Tween 20 with gentle shaking for about 30 minutes. The tissue was then washed thoroughly with sterile distilled water to remove any residual bleach. Using a fine scalpel and syringe needles, the remaining scale leaves and leaf sheaths covering the meristem tissue were aseptically removed, taking extra precautions not to damage the meristem until it was exposed. Whole meristem sections were cut into small pieces (approximately 2 mm) under sterile conditions and they were placed vertically on the culture medium containing MS basal media with vitamins, 24 µM 2, 4 - D, 4% commercial sucrose, 0.25% activated charcoal (Carbon Activado Polvo QP- QUIMICA MEYER) and 0.4% agargel. Media pH was adjusted to 6.0 before autoclaving.

Dissected meristem tissues were allowed to grow in callus induction medium in the dark at 280 C for about four weeks. The induced calli were subcultured into media with gradually decreasing concentrations of 2, 4-D (16, 12, 8, 4 and 2 µM) in four-week intervals. After the fourth week in media with 2 µM 2, 4-D callus were transferred onto the same media but with BAP, TDZ or ABA at 5 µM concentration to induce shoot formation.

Shoot induction, proliferation of multiple shoots and shoot development

Shoot induction occurred from organogenic calli (developed from shoot apical meristem) with the introduction of low concentration of BAP without forming somatic embryos. After shoot initiation, elongated, well developed shoots (35) were separated from proliferated clump and were grown on Y3 media (Eeuwens, 1976) containing 5µM BAP under low light intensity with a 16:8h photoperiod. Root formation occurred without a requirement for rooting hormones. Plants were transferred on to new media every four weeks and allowed to grow in the controlled environment until they had at least three leaves and a well-developed root system.

Twelve in vitro–grown plants, developed from organogenic meristem derived calli, were selected for acclimatization. The plants were removed from tissue culture vessels, rinsed with water to wash media off the roots and drenched in a dilute fungicide solution before transferring to clear polypropylene bags containing pre-sterilized potting medium, moistened with a dilute fertilizer solution. To maintain high relative humidity, the bags were sealed at the top and kept under low light intensity (120-200 µmol m -2 s-1). After three weeks, one end of the bag was cut to reduce relative humidity. Subsequently at the end of the fourth week, plants were fully exposed to ambient conditions and kept under higher light intensity (650-800 µmol m -2 s-1) for a period of three weeks. The plants were then repotted in larger polybags containing a potting mixture. After four months, the plants were fully exposed to greenhouse conditions with higher light intensity. The plantlets were watered 2 to 3 times per week and a liquid fertilizer was applied at one week intervals.

Scanning electron microscopy studies

SEM studies were used to observe stomata from different stages of plant regeneration. Leaf tissues were obtained from the plantlets just before transferring to ex- vitro conditions and after two weeks and two months of acclimatization in the greenhouse conditions. Tissue segments (ca. 3mm) were first fixed with 2% glutaraldehdye and then with 2% osmium tetroxide and dehydrated in graded series of ethanol. The samples were critical point dried with liquid CO2. They were mounted on aluminum stub with high conductivity silver paint, and coated with gold for 45 s using a sputter coater. Samples were observed in a JEOL T200 scanning electron microscope. The status of stomata (open or closed), position of stomata in the leaf surface (on the surface or sunken) and number of open stomata were recorded.

Results and Discussion

To date there are no reports of in vitro tissue -culture regeneration of Chinese windmill palm. From this study we first report plant regeneration of windmill palm through organogenesis from apical meristem derived callus tissues. Regeneration of plantlets reported here occurred via primary callus production and it is an indirect organogenesis process. Major steps of plant regeneration process are shown in Figure 2

47 (A - F). We utilized two different explant materials for plant regeneration but only apical meristem explants gave rise to plantlets.

There are several reports indicating regeneration of palms but most of them have been achieved via somatic embryogenesis (Verdeil et al., 1989, Fernando and Gamage 2000). However through somatic embryogenesis pathway, somoclonal variation may encounter as a result of callogenesis leading to embryoid production. Therefore, experiments have been targeted to seek alternative ways of in vitro propagation which could reduce culture- induced variation in tissue culture plants. One of the methods employed is direct organogenesis of plant propagation in vitro. The successful development of this technique minimizes somoclonal variation, reduces the number of steps in culture, and possibly the length of the culture time. Callus-mediated organogenesis or indirect organogenesis is also characterized by increased somoclonal variation limiting the application of callus cultures in micropropagation and breeding programmes. However, callus-mediated organogenesis and somatic embryogenesis have been the main in vitro systems for many monocot plant systems studied (Kallak et al., 1997). In addition to somatic embryogenesis, there are reports indicating regeneration of palms via both direct organogenesis (Tisserat et al., 1984) and indirect organogenesis (Kundu and Sett, 1999; Wang et al., 2003) pathways using shoot apical meristem as the explant issue. Our study also reports plant regeneration via indirect organogenesis from shoot apical meristem of windmill palm.

Callus induction and shoot initiation

In the first part of our study, optimization of the conditions for callus induction was carried out using mature zygotic embryos as the explant. Three basal media (MS, N- 6 and CRI-72-H) were tested with 13 different concentrations of 2, 4-D.; callusing frequency was recorded in each case ( Table 1) as well as the quality and the viability of the callus upon transferring into media with next lowest concentration of 2, 4-D. MS media starting with 24 µM 2, 4-D concentration gave better results than the other basal media or concentrations of 2, 4-D for callus induction. Although the callusing frequency, 17% (Table 1) was not the highest, the majority (~65%) of the callus initiated on MS media with 24 µM remained viable and healthy (Figure 1) upon transferring onto media with low concentration of auxin without showing any necrosis. However, mature zygotic embryo explants did not give rise to any embryogenic callus (upon transferring into media with lowering concentration of 2, 4-D) or organogenic callus (with the introduction of cytokinins such as BAP or TDZ – unpublished data). Therefore, this medium did not promote any regeneration, with some exceptions, when callus growth was accompanied by rhizogenesis. Callus induced with 24 µM 2, 4- D could be maintained in the same media for a long period of time. The media composition ( MS with initial 24 µM 2,4-D )was selected to proceed with shoot–tip meristem callus induction assuming that one of the factors that contributed to non-formation of organogenic callus or embryogenic callus from windmill palm mature zygotic embryos may be the limited morphogenetic potential of older tissues /explant source. The shoot- tip meristem tissues initiated callus, when cultured on MS media with 24 µM 2, 4-D (Figure 2A). In contrast to zygotic embryo explants, meristematic explants developed

48 organogenic callus (Figure 2B) and shoot induction from those organogenic callus occurred (Figure 2C) with the introduction of BAP (5 µM) with the low concentration of 2, 4-D (2 µM) in the media. Out of 50 meristem tissue pieces that were cultured, 15 gave rise to calli. These calli were cultured on media (5 each) containing 2 µM 2, 4-D and 5 µM BAP, 2 µM 2, 4-D and 5 µM TDZ and 2 µM 2, 4-D and 5 µM ABA. However, introduction of TDZ or ABA each at 5µM concentration did not induce shoot formation or organogenic callus formation with meristematic tissue. Out of 5 calli cultured in media with BAP only one developed 35 shoots. In this study calli formation frequency from meristem tissue is 30% while the formation of organogenic calli frequency is 20%. Root formation from the induced shoots occurred naturally without a requirement of any rooting hormones. The regenerated plantlets were successfully established in soil (Figure 2F). In our study, from the culturing of an apical meristem to regeneration of plantlets through primary callus formation and organogenesis required about 9 months.

To induce calli in palm tissue culture, relatively high levels of 2, 4-D or synthetic auxins have been used (Dias et al, 1984, Gabr and Tisserat, 1985). However, in our preliminary studies, zygotic embryo explants from T. fortunei showed high level of necrosis in the presence of high concentrations of 2, 4-D with MS and CRI-72-H media tested. In contrast, Bhaskaran and Smith (1992) established callus cultures from shoot tips of Phoenix dactylifera in the presence of 100mg/L 2, 4-D and reported successful regeneration of plantlets through somatic embryogenesis. Gabr and Tisserat (1985) also obtained embryogenic callus from shoot tips of Phoenix dactylifera on MS medium supplemented with 100mg/L 2,4-D but reported a failure in the formation of plantlets from embryos. Kundu and Sett (1999) reported the use of relatively low dosage of 2,4-D (9.025 µM) to induce callus formation from the shoot tip culture of another palm, rattan (Calamus flagellum) and have obtained plantlets via indirect organogenesis. Wang et al. (2003) reported the necrosis of tissue even with very low concentration of 2, 4-D and reported a successful regeneration of Areca catechu (Arecaceae) again via indirect organogenesis using different hormones, i.e. benzyladenine (BA) and TDZ (0.02 and 0.2 mg/L) instead of 2, 4-D to induce callus from shoot tips. Therefore even among the species of the same family (Arecaceae) the type of the hormone as well as concentration of hormones required to induce callus formation varied greatly. It is also well documented that regeneration in plant systems is genotype dependent (Barr et al., 1996; Kallak et al., 1997).

According to the C. flagellum regeneration reported by Kundu and Sett (1999), shoot differentiation has occurred when callus were placed media containing benzyladenine (BA; 8.88 µM) and naphthalene acetic acid (NAA; 5.38 µM). Root formation occurred in rooting media on MS medium with 0.53 µM NAA. From another study, Wang et al. (2003) reported in vitro adventitious shoot formation occurred in the same media they have used for callus induction. It consisted of equal concentrations of benzyladenine (BA-0.2mg/L) and thidiazuron (TDZ- 0.2mg/L). However, root induction required different media i.e. 0.1mg/L NAA in MS basal media.

Our study required the presence of low concentration of 2, 4-D for callus induction and the introduction of BAP for shoot induction and there was no requirement for hormones

49 for root induction. However, the time taken for our study is considerably longer (9 months) when compare with the two other similar palm regeneration systems (C. flagellum – 18 weeks and A. catechu - 20 weeks) via indirect organogenesis discussed in this paper in addition to the differences in some of the hormones used for the regeneration.

In a separate study Tisserat (1984b) reported propagation of date palms using shoot tip cultures via direct organogenesis. Shoot-tip cultures have been first established in MS media containing thiamine, HCl, myo-inosotiol, NAA (0.1mg/L) and charcoal. When transferred these established shoots in liquid or agar medium with 0.1mg/L NAA and 10mg/ L BA without charcoal and allowed further shoot differentiation and proliferation. Adventitious roots were obtained readily after culturing separated shoots in media containing 0.1mg/L NAA without charcoal. Following several weeks in culture plantlets have been successfully transplanted in soil with a survival rate close to 100%. Up to about 20 shoots have been obtained from a single shoot culture after 6 months in culture. However, not all shoots showed such proliferation in vitro suggesting the refinement of this technique is necessary to maximize offshoot production in vitro. In contrast to this study, our regeneration occurred via the callus phase and required charcoal throughout all stages. We did not need any rooting hormone to induce root formation from shoots. However, similar to our studies, this study too required benzylaminopurine for shoot proliferation but shoot proliferation has occurred in the presence of both BA and NAA as well. Although one of our calli produced 35 shoots, we also observed some calli cultured on our media did not produce shoots, also suggesting the requirement of culture media refinement for consistent shoot production.

It is generally accepted that adult palm tissues are highly recalcitrant to in vitro culture conditions (Reynolds and Murashige, 1979). Tisserat (1984a) reported that organs and older tissues of the date palm, have limited morphogenetic potential though yielding callus. Verdeil et al. (1989) obtained only callus with adult coconut tissues and no apparent embryogenesis. Since we did not obtain any embryogenic or organogenic callus with mature zygotic embryos explants with the media tested, and obtained organogenic calli with shoot-tip meristems our results agree with those of previous authors that tissues from young plants are the most embryogenic or embryogenic (Pannetier and Morel, 1982; Raju et al., 1984). In addition, meristem tip culture is extremely useful for the production of pathogen-free plant material.

Effect of cytokinins on shoot initiation

Different cytokinins seem to induce shoot formation in many plant species (Vaibhav et al., 2001). In some plant species combination of BAP and TDZ has enhanced the shoot formation (Khalafalla and Hattori, 1999). Nielsen et al. (1995) proposed a model for cytokinin action in the plant cell. Accordingly, both BAP and TDZ can bind to a receptor, a cytokinin-binding protein which has two binding sites. One site binds adenine-type cytokinins naturally, while the other binds phenylurea-type cytokinins. Therefore, the combination of both BAP and TDZ may bind actively to both binding sites and it may result in enhanced shoot formation. Conversely, it has been suggested that

50 excess of TDZ and BAP alone or in combination for binding sites can make them toxic to plants (Malik and Saxena, 1992; Lu, 1993; Nielsen et al., 1995; Tegeder et al., 1995). According to our study, BAP induced organogenic calli formation and subsequent shoot induction while TDZ did not have that effect on the callus derived from meristem tissue. If we assume that the BAP concentration of (5.0 µM) is near optimal then we would need to speculate that the TDZ concentration was too low or lethal at this level of BAP. One of the future directions of this research would be to use a range of TDZ concentrations in combination with a narrow range of BAP combinations around (5.0 µM) to check for synergistic effects on shoot initiation.

SEM studies and Acclimatization

In vitro-grown plants, taken from an aseptic environment where temperature, light and humidity were controlled are extremely fragile. Therefore, they should exhibit a progress in stomatal regulation and related anatomical characters indicating that they could adjust well to the changing environmental conditions during acclimatization and the plants can be considered fully acclimatized at the time of field planting. The percentage of open stomata in leaves of in vitro-grown plants should be higher than in the plants grown in the controlled environment and it should decrease during the course of acclimatization indicating an improvement in stomatal regulation. Our SEM studies indicated that leaves from tissue cultured plants growing in chambers (controlled conditions) had little epidermal wax, had practically no cuticle, and stomata present on the leaf surface were all (100%) in the open state (Figure 3A and B). There was no significant difference in the stomata number in the abaxial and adaxial leaf surfaces (6 to 8 in a field of observation). In contrast, leaves from the plants growing in the greenhouse conditions for two months had more wax, had fully sunken stomata on the surface of the leaves (Figure 3D and E) and the stomatal openings could not be observed due to wax deposition. The intermediate stage (i.e. two weeks after transferring into greenhouse conditions) stomata have begun to sink in the leaf surface (Figure 3C) indicating that they are adjusting to changing environmental conditions. The present study also confirmed that the percentage of open stomata (100%) in the leaves of in vitro grown plants, just before transferring to ex vitro conditions, was higher than that of the plants fully exposed to greenhouse conditions. This may be due to the unique culture vessel microclimate where high relative humidity, low light intensity and artificial gas atmosphere exists. Santamaria and Davies (1994) reported that unacclimatized leaves of in vitro cultured Delphinium had permanent stomatal opening or poor control of water loss. Stomata of in vitro grown banana leaves were also found to be completely open (Ross and Ludders, 1998). These observations are comparable with the results obtained for in vitro-grown windmill palms. Towards the later stage of acclimatization, a decrease in the percentage of open stomata in in vitro grown plants was observed, thus indicating that the in vitro- grown plants have adjusted to the external atmospheric conditions and attained a good control of stomata regulation prior to being transferred into the field. In the meanwhile, plants develop an efficient root system, build cuticle on leaves, and become photosynthetically active (unpublished data). Towards the later stage of acclimatization, a decrease in the percentage of open stomata in in vitro grown plants was observed, thus indicating that the in vitro plants have adjusted to the external atmospheric conditions and

51 attained a good control of stomatal regulation prior to being transferred into the filed. In the mean time, plants develop an efficient root system, build cuticle on leaves, and become photosynthetically active (unpublished data). Regenerated plants have a survival rate 16% after 6 months in the green house. However, 84% loss in the greenhouse cannot be explained as a problem related to acclimatization since plants had functional stomata, strongly developed root system, and the photosynthetic rate was comparable to the ones grown naturally (unpublished data).

In conclusion, a plant regeneration protocol through indirect organogenesis of Trachycarpus fortunei was developed. Mature zygotic embryos developed non- embryogenic/ non-organogenic callus under the different auxins and cytokinins tested. Somatic embryogeneis was not induced by decreasing the hormone concentrations in the media as in some other palm species (Fernando and Gamage, 2000). Out of the two explants tested, shoot apical meristem tissues seem to be most suitable in plant regeneration via indirect organogenesis process. Shoot meristems produced organogenic callus after decreasing auxin concentration (2, 4-D) and with the introduction of BAP at a very low concentration. These calli produced a large number of shoots (up to 35 per callus) that developed into plantlets which were successfully grown under greenhouse conditions (Figure 2F). Acclimatization of plants into ex-vitro conditions was required for better survival rate of plants upon transferring into field conditions. SEM observations on stomata suggested that plants had fully adapted to greenhouse conditions. Once refined for consistent plant production, protocol established in this investigation may facilitate future research in genetic transformation and in vitro propagation of T. fortunei with superior characters.

One of the future directions of this research would be to test for the somoclonal variation among regenerated plantlets. Plant regeneration through tissue culture may result in increased spontaneous phenotypic and genetic variation in regenerated plants and which is referred to as somoclonal variation (Larkin and Scowcroft, 1981) and it is now considered to be a general phenomenon. Morphological variations resulting from somoclonal variation have been extensively studied for several crop and fruit species. For instance, plants showed considerable variation for morphological traits such as flower color and shape, plant leaf morphology and color, plant height, resistance to disease, and maturity date ( reviewed by Bajaj, 1990; Hammerschlag, 1992).These variations are heritable i.e., transmitted through meiosis and are usually irreversible. Somoclonal variation can be assessed by analysis of phenotype, chromosome number and structure, proteins, or direct DNA evaluation of plants (De Klerk, 1990).

Besides somoclonal variation, another source of variations observed in plants regenerated from tissue culture concerns variation from epigenetic origin. Epigenetics refer to modifications in gene expression brought about by heritable, but potentially reversible, changes in chromatic structure and/or DNA methylation (reviewed by Henikoff and Matzke, 1997). In this case it seems worthwhile to identify molecular markers found in plants that show abnormality which is usually 5-10%. If these abnormalities do not appear to involve genomic modifications at the nucleotide sequence level, then the efforts can be concentrated on the identification of clonal conformity markers firstly at the

52 mRNA level and secondly by studying the sequence specific DNA methylation. These studies will eventually lead to the identification and characterization of marker genes, which can be assessed for their potential as clonal conformity markers.

Acknowledgements

This project was supported by grants from the Ohio Plant Biotechnology Consortium, and the Miami University, Department of Botany Academic Challenge program.

References

Bajaj YPS (1990) In. Somoclonal variation - origin, induction, cryopreservation, and implications in plant breeding. Somoclonal variation in crop improvement. Bajaj. YPS (Ed) Springer-Verlag, Berlin 3-48 Barr SN, Payne LA, Dale MFB, Wilkison MJ (1996) Positive correlates of shoot regeneration from potato protoplast culture. Plant Cell Rep 15:350-354 Bhaskaran S, Smith R (1992) Somatic embryogenesis from shoot tip and immature inflorescence of Phoenix dactylifera cv Bahree. Plant Cell Rep 12:22-25 Blake J (1990) Coconut (Cocos nucifera L.) Micropropagation. In: Biotechnology in Agriculture and Forestry. Vol.10 Legumes and oil seed crops. Bajaj YPS (Ed.) Springer Verlag, Berlin, Heidelberg. 10:538-554 Corley RHV, Barrett JN, Jones LH (1977) Vegetative propagation of oil palm via tissue culture. Oil Palm News 22:2-7 Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY (1975) Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Science Sinica. 18:659-668 David A (1982) In vitro propagation of gymnosperms. In: Tissue culture in forestry. Bonga JM, Durzan, Martinus Nijhoff (Eds.). W. Junk Publishers, The Hague: 72-108 Debergh PC (1991) Acclimatization techniques of plants from in vitro. Acta Hort 289: 291-300 De Klerk GJ (1990) How to measure somoclonal variation. Acta Botanica Neerlandica 39: 129-144 Dias AC, Guerra MP, Cordoba AS, Kemper EL (1994) Somatic embryogenesis and plant regeneration in the tissue culture of Genoma gamiova (Arecaceae). Acta Hortic. 360:167-171 Eeuwens CJ (1976) Mineral requirements for growth and callus initiation of tissue explants excised from mature coconut palms. Physiol. Plant 36:23-28 Fernando SC, Gamage CKA ((2000) Abscisic acid induced somatic embryogenesis in immature embryo explants of coconut (Cocos nucifera L.). Plant Sci. 151:193- 198 Gabr MF, Tisserat B (1985) Propagating palms in vitro with special emphasis on the date palm (Phoenix dactylifera). Sci. Hortic. 25:255-262 Gupta PK, Kendurka SV, Kulkarni VD, Shirgurkar MV, Mascarenhas AF (1984) Somatic embryogenesis and plants from zygotic embryos of coconut (Cocos nucifera L.) In vitro Plant Cell Rep 3:222-225 Hammerschlag HA (1992) In: Somoclonal variation. Biotechnology of perennial fruit crops. Hammerschlag FA, Litz RE (Eds). Cambridge, CAB International: 35-55 Henikoff S, Matzke MA (1997) Exploring and explaining epigenetic effects. Trends in Genetics 13: 293-295 Huong LTL, Baiocco M, Huy BP, Mezzetti B, Santilocchi R, Rosati P (1999) Somatic embryogenesis in canary Island date palm. Plant Cell, Tissue and Organ Culture 56:1-7 Jesty JHF, Francis D (1992) Cellular responses of leaf explants of Cocos nucifera L. In vitro Plant Cell Tissue and Organ Culture 28:235-244.

54 Jones DL (1995) Palms throughout the world. Washington DC, Smithsoniam Institution Press, 410pp Jones LH (1974) The oil palm clonal propagation by tissue culture. Biologist 30:181-188 Kackar NL, Solanki KR, Joshi SP (1989) Micropropagation of date palm ( Phoenix dactylifera L.) cv. Khadrawy using tissue culture technique. Animals Arid Zone 28:137-141 Kallak H, Reidla M, Hilpus I, Virumae K (1997) Effects of genotype, explant source and growth regulators on organogenesis in carnation callus. Plant Cell Tissue and Organ Culture 51:127-135 Karunaratne S, Periyapperuma K (1989) Culture of immature embryos of coconut. Plant Science 62:247-253 Khalafalla MM, Hattori K. (1999) A combination of thidiazuron and benzyladenine promotes multiple shoot production from cotyledonary node explants of faba bean (Vicia faba L.). Plant Growth Regulation 27:145-148 Kundu M, Sett R (1999) Regeneration through organogenesis in rattan. Plant Cell Tissue and Organ Culture. 59:219-222 Larkin PJ, Scowcroft WR (1981) Somoclonal variation - a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60: 197-214 Lu C (1993) The use of Thidiazuron in tissue culture. In Vitro Cell Dev Biol 29: 92-96 Malki KA, Saxena PK (1992) Thidiazuron induces high-frequency shoot regeneration in intact seedling of pea (Pisum sativum) chick pea (Cicer arietinum) and lentil (Lens culinaris). Aus J Plant Physiol. 19:731-740 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiology 15:473-497 Nielsen JM, Hansen J, Brand K (1995) Synergism of thidiazuron and benzyladenine in axillary shoot formation depends on sequence of application in Miscanthus x ogiformis Giganteus. Plant Cell Tissue and Organ culture 41:165-170 Pannetier C, Arhus, P, Lievoux D (1981) Neoformation of young Elaeis guineensis plants from primary callus obtained from leaf segments cultured in vitro. Oleagineux 36:119-122 Pannetier C, Buffard-Morel J (1982) First results of somatic embryo production from leaf tissue of coconut. Oleagineux 37:352-353 Paranjothy K, Oathman R (1982) In vitro propagation of oil palm. Plant Tissue Culture. A. Fujiwara, Maruzen, Tokyo: 747-748 Rabechault H, Martin JP (1976) Multiplication vegetative du palmier a huile (Eleais guineensis Jacq.) a l'aide de cultures de tissue foliares. CR Acad Sci Paris Ser 283:1735-1737 Raju CR, Prakash Kumar P, Chandramohan M, Iyer RD (1984) Coconut plantlets from leaf tissue cultures. J. Plant Crops 12:75-91 Reynolds JF, Murashige T (1979) Asexual embryogenesis in callus cultures in palms. In Vitro 5:383-387 Rillo EP, Paloma MBF, Rodriguez BJ, Ignacio MTR (1988) Screening of coconut populations for resistance to Cadang-cadang disease using in vitro cultured embryos., Philippine Coconut Authority, Albay research centre: 39-40

55 Ross K, Ebert GS, Ludders P (1998) Influence of in vitro growth condition on stomata density, index and aperture of grape, coffee and banana plantlets. Plant Tissue Culture and Biotech. 4: 21-27 Santamaria JM, Davies WJ (1994) Control of water loss by Delphininum plants cultured in vitro. In: Physiology, Growth and Development of Plants in Culture. P. J. Lumsden, J. R. Nicolas, W. J. Davies. (Eds.) Kluwer, Dordrecht: 155-164 Sharma DR, Dwara S, Chowdhury JB (1984) Somatic embryogenesis and plant regeneration in date palm (Phoenix dactylifera L.) cv. Khadravi through tissue culture. Indian J Exp Biol 22: 596-598 Tanimoto S, Harada H (1982) Physiological and hormonal factors influencing organogenesis in Rudbeckia bicolor explants cultured in vitro. Plant and Cell Physiology 23: 107-113 Tegeder M, Gebhardt D, Schieder O, Pickardt T (1995) Thidiazuron-induced plant regeneration from protoplast of Vicia faba cv. Mythos. Plant Cell Rep 15: 164- 169 Thorpe TA (1993) In vitro organogenesis and somatic embryogenesis. In: Morphogenesis in Plants. Roubelakis-Angelakis KA, Tran Thanh Van KT (Eds.) New York, Plemun Press: 19-38 Tisserat, B (1979) Propagation of date palm (Phoenix dactylifera L.). In vitro. J. Exp Bot 90:1275-1283 Tisserat B, De Mason DA (1980) A histological study of development of adventitive embryos in organ cultures of Phoenix dactylifera L. Ann Bot (London) 46: 465- 472 Tisserat B (1981) Production of free-living date palms through tissue culture. Date Palm J 1: 43-54 Tisserat B (1984a) Clonal propagation of Palms. In: Cell culture and somatic cell genetics of plants, Laboratory procedures and applications. Vol.1 Vasil IK (Ed.) Academic Press. Inc. 74-81 Tisserat B (1984b) Propagation of date palms by shoot tip culture. Hort Sci 19: 230-231 Vaibhav T, Kavindra T, Singh N, Deo B (2001) Comparative studies of cytokinins on in vitro propagation of Bacopa monniera. Plant Cell Tissue and Organ Culture 66: 9-16 Verdeil JL, Buffard-Morel J, Pannetier C (1989) Somatic embryogenesis of coconut (Cocos nucifera L.) from leaf and inflorescence tissue, research findings and prospects. Oleagineux 44:409-411 Verdeil JL, Huet C, Grosdemanger F, Buffard-Morel J (1994) Plant regeneration from cultured immature inflorescence of coconut (Cocos nucifera L.): evidence for somatic embryogenesis. Plant Cell Rep. 13:218-221 Wang HC, Chen JT, Wu SP, Lin MC, Chang WC (2003) Plant regeneration through shoot formation from callus of Areca catechu. Plant cell tissue and organ Culture. 75:95-98 Ward KA, Jordan MC (2001) Callus formation and plant regeneration from immature and mature embryos of rye (Secale cereale L.). In vitro cell Dev Biol 37:361-368 Wooi KC (1990) Oil palm (Elaeis guineensis Jacq.): Tissue culture and micropropagation. In: Biotechnology in Agriculture and Forestry. Bajai YPS, (Ed.) Springer- Verlag Berlin Heideberg. 10:569-592

56 Zacchini M, Morini S, Vitgliano (1997) Effect of photoperiod on some stomatal characteristics of in vitro cultured fruit tree shoots. Plant Cell tissue and Organ Culture 49:195-20

Figure 1. T. fortunei callus derived from mature zygotic embryos growing on MS basal media supplemented with 24 µM 2, 4-D, 4% sucrose and 0.25% charcoal.

58 A B

C D

E F

Figure 2. Different stages of plant regeneration of T. fortunei via indirect organogenesis. A. Apical meristem tissues growing in the callus induction medium. B. Organogenic callus development on the media with low concentration of 2, 4-D (2µM) and BAP (5µM). C. Shoot induction from the organogenic callus. D. Shoot development and multiple shoot proliferation. E. Plantlet development. F. Regenerated plants after fully acclimatized into greenhouse conditions.

59 A B

D E

Figure 3. SEM observation of leaf stomata from different stages of T. fortunei regeneration. A. Presence of open (100%) stomata on the surface of tissue cultured plantlets just before transferring into greenhouse conditions. B. Detailed structure of a stomata from tissue cultured plants before transferring. C. Stomata after 2 weeks acclimatization to greenhouse conditions. Stomata begin to sink in the leaf epidermis. D. Leaf surface after 2 months in the greenhouse; stomata could not clearly be observed due to wax deposition. E. Detailed structure of an area where stomata could be located after 2 months greenhouse acclimatization.

Table 1. Observed callusing frequency in media containing differential 2-4, D concentrations

% Callusing frequency (Initial)

2, 4 – D (µM)

Medium 0 2.4 3 7.8 10 24 30 78 100 150 200 240 300

MS 0 0 2 8 13 17 16 14 12 5 0 0 0

N-6 0 2 4 5 10 14 16 16 17 20 12 10 5

CRI 72- 0 0 1 7 15 18 18 10 6 3 0.5 0 0 H

Chapter 3

Induction of somatic embryogenesis in Sabal palmetto; (Walter Schultes

& Schultes F.). Morphological observations and 2- D protein profile

comparison

Keywords - Sabal palmetto, dicamba, mature zygotic embryos, embryogenic callus, somatic embryos, 2-D electrophoresis

Abstract Sabal palmetto (cabbage palm), the state tree of Florida and South Carolina, is widely cultivated in the U.S. throughout USDA Zone 8. This paper reports on induction of somatic embryogenesis from S. palmetto tissues with the ultimate goal of developing a system that can be genetically manipulated. Many different auxins/cytokinins and combinations were tested to induce embryogenesis from mature zygotic embryos of this palm. The presence of the auxin dicamba was required to induce embryogenic callus and subsequent somatic embryogenesis in S. palmetto. In terms of embryogenic callusing frequency, 1.5 µM dicamba in MS basal media (with vitamins) supplemented with 0.25% charcoal and 4% sucrose was the best medium combination. Media with TDZ and zeatin along with dicamba combination also gave rise to embryogenic callus. However, media only with 1.5 µM dicamba were optimal for the development of somatic embryos-like structures from embryogenic callus when cultures were maintained in the same media. Cultures from different stages of embryogenesis were observed under the stereo microscope and scanning electron microscope. It is clearly a pathway different from zygotic embryo germination. There were major differences in the protein profiles of tissues taken from the different developmental stages of somatic embryogenesis induction. A large number of proteins were upregulated when compare with non embryogenic callus, during the induction of embryogenic callus and subsequent somatic embryogenesis in S. palmetto.

Abbreviations ABA - Abscisic acid; BAP- Benzylaminopurine; 2, 4-D, - 2, 4 – Dicholorophenoxy acetic acid; 2iP- N6 – (2-Isopentyl) adenine; TDZ- Thidiazuron

Introduction Sabal palmetto (cabbage palmetto) is widely cultivated in the U.S throughout USDA Zone 8. It is considered one of the most common species of native tress in North America and has recognized as the official state tree of Florida and South Carolina. This palm usually occurs near the coast from southwestern North Carolina to the Florida Keys, west along northern Gulf of Mexico, Cuba, and various islands in Bahamas.

The cabbage palm occurs further north than most of the New World palms and therefore is relatively cold-tolerant and hardy. This feature has made this species a favorite with landscapers and home owners. Established wild-type palms survive exposure to temperatures below -120C (100 F) without significant foliar injury, and some individuals

62 have known to survive subzero F cold (Riffle, 1998). Juvenile, non-trunked palms in various USDA Zone 6a-7a locations regularly survive typical winter conditions with defoliation (reviewed by Francko, 2003). The collective data suggest that, with relatively minor improvements in cold tolerance and cold acclimation potential, cabbage palm could prove suitable for general horticultural use under USDA Zone 7 conditions.

In recent years, genetic transformation has become the most powerful technique to create improved plant varieties. It involves the introduction of foreign genes into plants by different methods (most common are biolistic method and Agrobacterium-mediated transformation) which by-pass the conventional process of sexual seed production. This eliminates the requirements of cross compatibility as a prerequisite to gene transfer. Many organisms have evolved characters which enable them to survive in extreme environments e.g. cold. The gene(s) that confer these properties can potentially be introduced into higher plants by genetic transformation methods. Most current transformation protocols require a tissue culture system to ultimately recover genetically engineered plants. Totipotency which is unique to plant cells underlies most transformation systems. These systems require target tissues/cells and they are usually a part or whole of an excised explant grown in culture medium. Following gene transfer, the transformants are selected and allowed to grow in culture media to differentiate into somatic embryos or shoots. Parveez and coworkers (1997) first reported on physical parameters useful in evaluating genetic transformation events in oil palm. Subsequently Abdullah et al. (2005) reported the successful transformation of oil palm immature embryos and embryogenic calli using biolistic methods. He concluded that immature embryos of oil palm were a useful tool for genetic transformation studies using both sources of material. In a separate chapter of this dissertation, we report the first successful transformation of S. palmetto mature zygotic embryos using marker genes by both biolistic and Agrobacterium-mediated methods.

Micropropagation of plants is achieved via somatic embryogenesis or organogenesis. Somatic embryogenesis involves the development of embryos from embryogenically- competent somatic cells under in vitro conditions. In contrast, organogenesis is the process by which microshoots and roots develop on different media during different time periods of plant development. Somatic embryogenesis involves the development of embryos having both a shoot and a root pole, similar to the zygotic embryo. However, somatic embryo development in vitro requires different media for the induction, development and maturation processes. The developmental program in the embryogenically-competent somatic cells proceeds in a similar way to the development of zygotic embryos, but under the influence of specific hormones. Different cell types in the same organ or tissue may follow different pathways to differentiate into somatic embryos or shoots or roots under in vitro conditions. In general cells from zygotic embryos are more likely to develop somatic embryos than are the cells from other organs, such as leaves or roots. The genetic program for the development of embryos in the cells of immature or even mature zygotic embryos is not fully repressed and can be activated relatively easily as compared to cells from other tissue like leaves or roots. There are reports indicating that in some palms plant regeneration has been achieved through somatic embryogenesis derived from zygotic embryos (coconut; Hornung, 1995;

63 Fernando and Gamage, 2000). Huong et al., (1999) reported the successful regeneration of Phoenix canariensis through somatic embryogenesis from callus derived from plumule tissues excised from mature zygotic embryos. However, there are also reports indicating that somatic embryos have been obtained successfully from organs/tissues (e.g. leaf tissue) other than embryos as well (Pannetier and Buffard-Morel, 1982).

Mordhortst and coworkers (1997) propose that after completing the induction of embryogenesis, the events leading to zygotic and somatic embryos are common. Both zygotic and somatic embryos pass through similar stages in development. In monocots the stages are globular, scutellar (transition), and coleoptilar shaped while in dicots the stages are globular, heart and torpedo-shaped (Gray et al., 1995; Toonen and de Vries, 1996

The biochemical and molecular changes associated with somatic embryogenesis have been studied in several species such as rapeseed ( Crouch, 1982),carrot ( Choi and Sung, 1984, Dodeman and Ducreux 1996 ), rice ( Chen and Luthe, 1987), pea ( Stirn and Jacobson, 1987), cotton ( Shoemaker et al., 1987), Dactylis glomerata (Hahne et al., 1988), Trifolium ( McGee et al., 1989), coffee ( Yuffa et al., 1994), Camellia japonica ( Pedrosco et al.,1995), soybean ( Stejskal and Griga, 1995), barley ( Stirn et al., 1995), birch ( Hvoslef-Eide and Corke, 1997), and sugarcane ( Blanco et al, 1997).

Different biochemical variables have been used as markers to discriminate between embryogenic and non-embryogenic tissues in plant tissue culture. Among those are mostly proteins (Sung and Okimoto, 1981; Komamine et al., 1992), isozymes, and ethylene (Feirer and Simon, 1991). Proteins are valuable indicators of differentiation and have been used in taxonomy as genetic markers. They could be useful to identify specific stages of development of somatic embryos as well (Menendez et al., 1994). It is possible that biochemical marker can be used for early identification of embryogenic callus cultures before any morphogenic changes become visible. Therefore, its use would help to optimize culture conditions necessary for somatic embryogenesis, to monitor the course of somatic embryogenesis, and to discriminate cultures to follow multiplication process.

The first part of the present investigation was aimed at inducing embryogenic callus and somatic embryogenesis from S. palmetto mature zygotic embryos with the ultimate goal of developing a system that can be genetically manipulated. The second part was aimed at comparing protein profiles from tissues from different stages of embryogenic development and non-embryogenic callus with the ultimate goal of identifying potential biomarkers for embryogenesis in S. palmetto.

Materials and Methods

Callus induction

Mature seeds of S. palmetto were sterilized and softened by soaking them in 10% commercial bleach for 6 to 7 days. Prior to embryo extraction, seed coats were shaved in

64 the area where embryos are located without damaging the embryogenic axis. Extracted embryos were placed horizontally on petri-dishes containing MS basal media (with vitamins) supplemented with different auxins/cytokinins in different concentrations (Table 1), 4% commercial sucrose, 0.25% activated charcoal (Carbon Activado Polvo QP-QUIMICA MEYER) and agar gel (0.4%). A minimum of 20 embryos were tested for each treatment and the experiment was conducted at least three times. Cultures were incubated in the dark at 290 C for 2 months and observations were recorded weekly.

Growth/development of zygotic embryos during germination

S. palmetto seeds were sterilized in the same way as described previously. Seeds were placed on the same media as above but without hormones. Seedlings were allowed to grow in the dark for about a month and then were transferred to light. The purpose of this study is to identify major stages in seedling development. Photographs from the main stages of embryogenic callus induction and zygotic embryo germination were taken using a stereomicroscope (Olympus SZX12).

Scanning electron microscopy studies (SEM)

Scanning electron microscopy was used for further morphological observations of the different stages of embryogenic callus formation and induction of somatic embryogenesis. Samples were fixed in 3% glutaraldehyde in 0.05M phosphate buffer pH (6.03) and dehydrated in graded series of ethanol. The samples were critical point dried with liquid CO2. They were mounted on aluminum stub with high conductivity silver paint, and coated with gold for 45 s using a sputter coater. Samples were observed in a JSM 840A scanning electron microscope.

2- D Gel electrophoresis

Tissue samples from different stages of embryogenic callus induction as well as non- embryogenic callus were frozen in liquid nitrogen. Total proteins were isolated according to the method described by Tsugita and Kamo (1999). The protein content of the crude extract was measured using the Bradford assay (Biorad).

A 10 mg aliquot of crude protein was resuspended in 250 µl of isoelectric focusing (IEF) rehydration buffer. Immobilized pH gradient (IPG) strips (11 cm, pH 5 to 8; Biorad) acrylamide gel strips were passively rehydrated with 185µl of the protein solution (150 µg) over a period of 16 hr. The strips were then focused on a Protean IEF Cell (Biorad) at a constant current of 25µA per gel. The running parameters were; 20 min 250V linear ramp, 2.5 hrs 8000V linear ramp, 80K V/h 8000V rapid ramp and a hold step at 500V slow slope. Runs were performed at 200 C.

Isoelectric focused IPG strips were treated with a DTT equilibration buffer for 10 min at room temperature with shaking and then with iodoacetamide equilibration buffer for 10 min at room temperature. Strips were inserted into Criterion Ready gradient gels (Tris-

65 HCl, 8 to 16% resolving, 4% stacking) and run in 25 mM Tris/192 mM Glycine/0.1% w/v SDS electrophoresis buffer, pH 8.3 at 200V for 60 minutes. Gels were stained with Bio safe Coomassie stain for 1 hr, destained in nanopure water and photographed with Alpha-imager (Alpha Innotech Corporation). Gels were analyzed using Biorad PDQuest software. Preliminary qualitative as well as quantitative analysis of data were carried out using analysis set manager tools in the PDQuest 2-D analysis software version 7.3.0 from Biorad.

Results and Discussion

Callus initiation and somatic embryogenesis induction

We used different combinations of auxins and or cytokinins to induce embryogenic callus and subsequent somatic embryogenesis using S. palmetto zygotic embryos as the explant source. The results are shown in Table 1 and 2. Zeatin alone, TDZ and ABA (each at 5 µM concentrations) with different concentrations of 2, 4-D did not induce any callogenesis. All other auxins and or cytokinins gave rise to callus formation with some of the concentrations tested. Formation of embryonic callus occurred only in the presence of dicamba (5 µM) with different concentrations of 2, 4-D. In contrast, 2, 4-D alone or with BAP, picloram, zeatin, 2iP, BAP and 2iP, kinetin gave rise to the non- embryogenic callus formation. Nabors et al. (1983) described how one can distinguish between embryogenic and non-embryogenic cereal callus by visual examination. It is clear from our results that the same applies for S. palmetto. Many other authors have also described meristematic, organized surface of embryogenic tissue versus elongated, unorganized non-embryogenic. This seems to be a universal feature whether the plants are dicots or monocots, herbaceous or woody.

A second set of experiments was carried out to determine the optimal concentration of dicamba (based on results from the first series of experiments) to induce embryogenic callus (Table 2). Dicamba alone, dicamba with the auxin 2, 4-D, or dicamba with different cytokinins (TDZ, zeatin) produced callus induction and in some cases embryogenic callus induction. Callus induction occurred with all the concentrations of zeatin tested with 5 µM dicamba, most of the concentrations of TDZ with 5 µM dicamba with some concentration of dicamba alone or dicamba with either TDZ, zeatin or 2, 4-D. The highest embryogenic callusing frequency (nearly 78%) occurred with 1.5 µM dicamba although 5 µM zeatin and 3 µM dicamba (60%) was a close second. During the 2- month time period in culture the embryogenic calli in media with 1.5 µM dicamba gave rise to somatic embryos as indicated by stereo microscopy (Figure 2) and SEM studies (Figure 3). Some of the embryogenic callus induced in the media with dicamba and TDZ developed into haustoria-like structures but most of them gave rise to somatic embryos- like structures as observed with 1.5 µM dicamba concentration. Thus, for embryogenic callus induction and subsequent somatic embryogenesis in S. palmetto tissue culture 1.5 µM dicamba concentrations seemed to be optimal. This pathway was clearly different from normal zygotic embryo germination/seedling development (Figure 1).

66 This is the first report indicating promising results for induction of embryogenic callus and somatic embryogeneis in S. palmetto and the use of dicamba to induce embryogenic callus in palms. Fernando and Gamage, (2000) reported the use 2, 4-D and ABA to induce somatic embryogenesis from coconut immature zygotic embryos. In date palm, embryogenic callus has been induced from leaf and inflorescence explants using 2, 4-D (Fki et al., 2003). Huong et al., (1999) used picloram and kinetin, 2, 4-D and 2 iP to produce embryogenic callus from zygotic embryos and shoot tip explants from canary Island date palm. In addition to palms, for many plants, 2, 4-D has been widely regarded as the most effective auxin for inducing somatic embryogenesis (Kamada and Harada, 1979; Brown et al., 1995; Halperin, 1995).

To initiate embryogenesis and embryo development in many plant species including carrot, embryogenic callus or cell cultures should be first induced on auxin supplemented medium and then transferred to an auxin-free medium (Halperin and Wetherell, 1964; Merkle et al, 1995). In S. palmetto a primary culture without such a transfer resulted in the formation of somatic embryos. This suggests that a brief lag time is needed to obtain somatic embryo formation with less callus proliferation. Accordingly, this system seems to be especially suitable for the analysis of early developmental stages of somatic embryogenesis since it allows continuous observations from the same culture. Histological knowledge concerning ontogeny of S. palmetto palm somatic embryoids can provide important information for improving the somatic embryogenesis of this palm. Thus it would be necessary to confirm the origin of embryoids by histological analysis.

Development of embryogenic callus and somatic embryos in S. palmetto

S. palmetto zygotic embryos, excised from the seed have the germinal end or petiole limb (left) separated by a slight constriction from the cotyledonary limb or haustorial end (right) (Fig 1). When these embryos were placed on the callus induction media containing 1.5 µM dicamba (Figure 3A), a swollen area developed in the basal part of the cotyledons of zygotic embryos (Figure 3B and 4A) during the first 4 weeks indicating the development of callus tissue. This stage had compact organized cells (Figure 4A) on the surface. Somatic embryos developed from these calli after 6 weeks of initial culturing. First, pre globular embryo masses developed from this callus tissue ultimately giving rise to irregular shaped somatic embryos. (Figure 3C-1, 3C -2 and 4B). These embryos continued to grow in the media (Figure 3D and 4C) without geminating into plantlets.

2- D SDS-PAGE of S. palmetto tissue cultures

Our 2-D electrophoresis study showed that the morphological changes in relation to the development of somatic embryos were associated with considerable changes in the total protein content (Table 3). The protein content was particularly high (9.8 mg/g fresh weight) when the somatic, embryos were formed from embryogenic callus. At this stage of development about a 2.8-fold increase in protein content could be observed as

67 compared to callus stage (1.8 mg/g). A possible explanation to these higher protein levels detected in 6-week and 7-week old cultures may be an activation of metabolism during somatic embryo development. Fillipecki and Przybecki (1994) reported that in Cucumis sativus L, an ornamental woody plant, embryogenic lines had a considerably higher concentration of proteins than non-embryogenic ones. It was suggested that this difference in protein content could be related to the presence of numerous small cells which are characterized by an intensive cell metabolism in embryogenic cell lines. The 2- fold difference in 6-week (9.8 mg/g) and 7-week (18.7 mg/g) stages we observed in our study may be partly due to the accumulation of storage proteins at the later developmental stage (Table 3).

There were several changes in the 2-D electrophoresis protein patterns during the embryogenesis in S. palmetto. The 2-D gel images obtained for each sample from different stages of somatic embryogenesis exhibited between 200 and 400 protein spots with different isoelectric pH and apparent molecular masses (Figure 5). The spots showed good resolution in both electrophoresis dimensions and good reproducibility was observed in the replicate gels from the same sample allowing further analysis. Under our conditions, 288 reproducible spots could be detected in the non-embryogenic callus stage and 348 spots could be detected in 4-week-old callus. In 6-week-old embryogenic callus 419 spots could be observed while in 7-week- old-cultures 365 spots were detected (Table 4a). In embryogenic cultures an increase in spot number (348) may be related to embryogenic callus induction when compare with non-embryogenic callus samples (288). The increase in the protein number in 6-week- old cultures may be due to the differentiation of somatic embryos into specialized and more complex structures. Reinbothe et al.1992 has shown that the protein number was higher in somatic embryos than in induced callus in Vitis rupestris. It is similar to what we observed in our study. This was considered as strong metabolic activity during the process of embryo morphogenesis. In contrast, 7-week-old callus exhibited a reduced number of detected spots compared to 6-week-old developmental stages although the same amount (150 µg) of proteins was loaded on each IEF gel. The lower protein spot number in 7-week-old cultures may indicate the onset of somatic embryo maturation process or it could be related to the relative abundance of proteins as compared with those that were generally accumulated in 7 -week-old cultures. Somatic embryo maturation process involves accumulation of major storage products and results in existence of a limited number of proteins as shown by Gianazza et al. (1992) in Vitis mature somatic embryos.

In addition, qualitative analyses (Table 4b) allowed us to determine the number of spots present in each gel (representing each sample) and compare these with gels from other stages. It indicated that 6-week-old cultures had considerably large number of spots (139 and 167, respectively) when compared with non-embryogenic callus and 7-week-old cultures.

The quantitative analyses of spots in each of these gels are shown in Table 4c. When compared with non embryogenic callus, there was an increase in the quantity in large number of spots in 4-week, 6-week and 7-week-old cultures. (2-fold increase in 158 spots in 6-week-old callus, 78 spots in 4-week-old cultures, and 66 spots in 7-week-old

68 cultures). Sometimes the quantity of the spots in these gels was greater by several fold when compare with non embryogenic callus (Table 4c). These proteins may possibly be candidate proteins for indicating the developmental stage of somatic embryos in S. palmetto. Figure 5 illustrates a portion of gels showing some spots that are found only in embryogenic callus and/or somatic embryo cultures. They are not detectable in non- embryogenic callus.

Formation of somatic embryos and subsequent plant regeneration are two independent processes. This indicates the conversion into plantlets is a second control point in the plant regeneration (Strin et al., 1995). However, in some cases well formed somatic embryos derived from tissue cultures cannot be regenerated into plants. It is similar to the situation we observed in our study. In grapes, somatic embryos showed genotype-specific blocks in the conversion into plantlets; some are blocked after the topedo stage while others are blocked in the globular and heart-stage. A comparison of starch and lipid metabolism in somatic and zygotic embryos of grape vine revealed a remarkable difference (Faure and Aarrouf (1994) in this aspect. Somatic embryos of some grape genotypes were unable to use their reserves resulting in their inability to germinate into plantlets. In carrot and in Citrus inhibitory proteins secreted into the medium have been identified, suggesting the accumulation of a 57-kDA glycosylated polypeptide in cultures inhibited further development of somatic embryos (Satoh and Fujii, 1988; Gavish et al., 1992). In Citrus it was revealed that this polypeptide inhibited the transition of embryo initials to globular embryos rather than embryo initiation. Normal progression of embryo development seems to require the absence of this specific glycosylated peptide in the medium.

Despite numerous attempts we were unable to germinate somatic embryos of S. palmetto (unpublished data), germination of somatic embryos still remains as a very challenging task. Embryo development stopped at the irregular-shaped stage (Figure 3B and 3C) developing many thousands of unorganized cells on the embryos rather than developing into plants. Further analysis of these protein profiles may reveal important information related to the inability of these embryos to develop into plantlets.

Acknowledgements

This project was supported by grants from the Ohio Plant Biotechnology Consortium, and the Miami University, Department of Botany Academic Challenge program.

References

Abdullah R, Zainal A, Heng WY, Li LC, Beng YC, Phing LM, Sirajuddin, SA, Ping WYS, Joseph JL, Jusoh SA, Muad MR, Huey YL (2005) Immature embryo; A useful tool for oil palm ( Elaeis guineensis Jacq.) genetic transformation studies. Electronic Journal of Biotechnology 8:25-34 Blanco MA, Nieves N, Sanchez M, Borroto CG, Hernandez Z (1997) Protein changes associated with plant regeneration in embryogenic calli of sugarcane (Saccharum sp.). Plant Cell Tissue Org Culture 51:153-158 Brown DC, Finstad KI, Watson EM (1995) Somatic embryogenesis in herbaceous dicots. In: In vitro Embryogenesis in Plants. Thorpe TA (Ed.) Dordrecht, Boston, London, Kluwer Academic Publishers: 345-415 Chen LJ, Luthe DS (1987) Analysis of proteins from embryogenic and non-embryogenic rice (Oryza sativa L.) calli. Plant Sci. 48:181-188 Choi JH, Sung ZR (1984) Two-dimensional gel analysis of carrot somatic embryogenic proteins. Plant Mol. Biol. Rep. 2:19-25 Crouch ML (1982) Non-zygotic embryos of Brassica napus L. contain embryo-specific storage proteins. Planta 156:520-524 Dodeman VL, Ducreux G (1996) Total protein pattern expression during induction and development of carrot somatic embryos. Plant Sci. 120:57-69 Faure O, Aarrouf J (1994) Metabolism of reserve products during development of somatic embryos and germination of zygotic embryos in grapevine. Plant Sci. 96: 167-178 Feirer RP, Simon PW (1991) Biochemical differences between carrot inbreds differing in plant-regeneration potential. Plant cell reports 10:152-155 Fernando SC, Gamage CKA (2000) Abscisic acid. induced somatic embryogenesis in immature embryo explants of coconut (Cocos nucifera L.). Plant Sci. 151:193- 198 Fillipecki MK, Przybecki Z (1994) Marker proteins of somatic embryogenesis in cucumber (Cucumis sativus L.) Genet. Polon. 35:1-9 Fki L, Masmoudi R, Drira N, Rival A (2003) An optimized protocol for plant regeneration from embryogenic suspension cultures of date palm, Phoenix dactylifera L., cv. Deglet Nour. Plant Cell Rep. 21:517-524 Francko DA (2003) Palms Won't Grow here and Other Myths: Warm-Climate Plants for Cooler Regions. Portland, Timber Press, 307pp Gavish H, Vardi A, Fluhr R (1992) Suppression of somatic embryogenesis in Citrus cell cultures by extracellular proteins. Planta 186:511-517 Gianazza E, De Ponti P, Scienza A, Villa P, Martinelli L (1992) Electrophoresis 13:203- 209 Gray DJ, Compton ME, Harrell RC, Cantliffe DJ (1995) Somatic embryogenesis and the technology of synthetic seed. In: Somatic Embryogenesis and Synthetic seed1. Biotechnology in Agriculture and Forestry. Bajaj YPS (Ed.). Berlin, Springer- Verlag. 30:126-151 Hahne G, Mayer JE, Lorz H (1988) Embryogenic and callus specific proteins in somatic embryogenesis of the grass, Dactylis glomerata L. Plant Sci. 55:267-279

70 Halperin W, Wetherell DF (1964) Adventive embryony in tissue cultures of the wild carrot, Daucus carota.Amer. J. Bot. 51:274-283 Halperin W (1995) Some historical issues and unresolved problems. In: In Vitro embryogenesis in Plants. Thorpe TA (Ed.) Dordrecht, Boston, London, Kluwer Academic Publishers: 345-415 Huong LTL, Baiocco M, Huy BP, Mezzetti B, Santilocchi R, Rosati P (1999) Somatic embryogenesis in canary Island date palm. Plant Cell, Tissue and Organ Culture 56:1-7 Hvoslef-Eide AK, Corke FMK (1997) Embryogenesis specific protein changes in birch suspension cultures. Plant Cell Tissue Organ Culture 51:35-41 Kamada H, Harada H (1979) Studies on the organogenesis in carrot tissue cultures. I. Effects of growth regulators on somatic embryogenesis and root formation. Z. PflPhysiol. 91:255-266 Komamine A, Kawahara R, Masumoto M, Sunabori S, Toya T, Fujiwara M, Tsukahara M, Smith J, Ito M, Fukuda K, Nomura K, Fujimura T (1992) Mechanisms of somatic embryogenesis in cell cultures: Physiology, Biochemistry and Molecular Biology. In vitro Cell Dev Biol. 28P:11-14 McGee JD, Williams EG, Collins GB, Hidebrand DF (1989) Protein profiles associated with high-and low-frequency regeneration. J Plant Physiol 135:306-312 Menendez-Yuffa A, Garcia de E, Segura-Nieto M (1994) Comparative study of protein electrophoretic patterns during embryogenesis of Coffea arabica cv. Ctimor. Plant Cell Rep. 13:197-202 Merkle SA, Parrot WA, Flinn BS (1995) Morphogenic aspects of somatic embryogenesis. In: In Vitro embryogenesis in Plants. Thorpe TA (Ed.) Dordrecht, Boston, London, Kluwer Academic Publishers Mordhorst AP, Toonen MAJ, Vries SC (1997) Plant embryogenesis. Critical Reviews in Plant Sciences 16:535-576 Nabors MW, Heyser JW, Dykes TA, DeMott KJ (1983) Long-duration, high-frequency plant regeneration from cereal tissue cultures. Planta 157:385-391 Pannetier C, Buffard-Morel J (1982) First results of somatic embryo production from leaf tissue of coconut Cocos nucifera L. Oleagineux 37:349-354 Parveez GKA, Chowdhury MKU, and Norihan MS (1997) Physical parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) using the biolistic device. Industrial Crops and Products 6:41-50 Pedroso MC, Hilbert JL, Vasseur J, Pais MS (1995) Polypeptides associated with the induction of direct somatic embryogenesis in Camellia japonica leaves I. Identification of embryo-specific polypeptides. J. Exp. Bot. 46:1579-1584 Reinbothe C, Tewes A, Reinbothe S (1992) Altered gene-expression during somatic embryogenesis in Nicotiana plumbaginifolia and Digitalis lanata. Plant Sci. 82: 47-58 Riffle RL (1998) The Tropical Look. Timber Press, 428pp Satoh S, Fujii T (1988) Purification of GP57 an auxin-regulated extracellular glycoprotein of carrots, and its immunocytochemical localization in dermal tissues. Planta 175:364-373

71 Shoemaker RC, Chridtofferson SE, Galbraith DW (1987) Storage protein accumulation patterns in somatic embryos of cotton ( Gossypium hirsutum L.). Plant Cell Rep. 6:12-15 Stejskal J, Griga M (1995) Comparative analysis of some isozymes and proteins in somatic and zygotic embryos of Soybean (Glycine max L. Merr.). J. Plant Physiol. 146:497-502 Stirn S, Jacobson HJ (1987) Marker proteins associated for embryogenic differentiation patterns in pea callus. Plant Cell Rep. 6:50-54 Stirn S, Mordhorst AP, Fuchs S, Lorz H (1995) Molecular and biochemical markers for embryogenic potential and regenerative capacity of barley ( Hordeun vulgare L.). Plant Sci. 106:195-206 Sun ZR, Okimoto R (1981) Embryogenic proteins in somatic embryos of carrot. Proc. Natl. Sci. USA. 78:3683-3687 Toonen MAJ, De Vries SC (1996) Initiation of somatic embryos from single cells. In: Embryogenesis: the Generation of a Plant. Wang TL, Cuming A (Eds.). Oxford, Bios Scientific Publishers. 173-189 Tsugita A, Kamo M (1999) 2-D electrophoresis of plant proteins. Methods Mol biol. 112: 95-97 Yuffa AM, Garcia EG, Nieto MS (1994) Comparative study of protein electrophoretic patterns during embryogenesis in Coffea arabica cv. Catimor. Plant Cell Rep. 13: 197-202

Figure 1. Sabal palmetto seed morphology

A B C

D E F

H I J

Figure 2. Major steps in normal S. palmetto seedling development. After radical emergence, further development occurs primarily as elongation of the cotyledon stalk, with its distally attached radicle and epicotyle, and the growth of the haustorium (cotyledon) within the seed. (A – B). Radicle is emerging from the seed while keeping contact with the seed through cotyledon stalk. C. Ligule can be clearly seen in this stage D. Cotyledon stalk, ligule, primary root and plumule (in green) are seen here. F. Seedling leaf develops while primary root continues to grow. F. Developing seedling H. Adventitious root development. I-K; Various in between stages described above at higher magnifications.

A B

C-1 C-2

Figure 3. Stereomicrographs showing major stages in the development of somatic embryos from S. palmetto mature zygotic embryos. A. Freshly extracted zygotic embryo B. Development of embryogenic callus (4 weeks old). C-1 and C-2. Somatic embryos begin to develop from embryogenic callus (6 weeks).D. Appearance of somatic embryos on the surface of the tissue.

Figure 4A. Scanning electron micrograph of 4-week-old callus tissue showing many thousands of cells on the surface.

Figure 4B. Scanning electron micrograph of 6-week-old tissue culture showing the development of irregular-shaped embryos on the callus tissue.

Figure 4C. Scanning electron micrograph of 7-week-old tissue culture with further development of somatic embryos.

Non-embryogenic callus 4-week-old callus

6-week-old callus 7-week-old callus

Figure 5. Two-dimensional gel electrophoresis proteome maps of S. palmetto tissue cultures. First-dimension focusing used11-cm IPG strips with a linear pH gradient 5-8 loaded with 150 µg of total proteins per each strip. In the second dimension 8-16% SDS-PAGE gels were used. Proteins were visualized by Coomassie brilliant blue stain. Samples used for proteomic analysis were collected at 4, 6 and 7 weeks and also from non-embryogenic callus.

79 Non-embryogenic callus

Embryogenic callus – 4 weeks old

Somatic embryoids – 6-week-old

Figure 6 – Selected regions in Figure 5 to high light representative example of the protein spots that changed in qunatity.

80 Table 1. Effect of different auxins/and cytokinins on embryogenic callus induction from S. palmetto mature zygotic embryos

2,4- D MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS+ MS + Concentration 2,4- 2,4- 2,4- 2,4- 2,4-D + 2,4-D + 2,4-D 2,4- 2,4-D 2,4-D + 5µM in µM D D + D + D + 5 µM 5 µM + D + + 5 5 µM zeatin 5 5 5 dicamba picloram 5 µM 5 µM µM kinetin µM µM µM zeatin 2 iP BAP+ ABA BAP TDZ 2iP 100 NEC NG NEC NG EC NG NG NG SG SG SG 75 NEC NG NEC NG EC SG NG NG SG SG SG 50 NEC NG NEC NG EC SG NG SG NEC SG SG 38 NEC SG NEC SG EC NEC SG NEG NEC NEC SG 25 NEC SG NEC SG EC NEC SG NEC NEC NEC SG 19 NEC SG NEC SG EC NEC SG NEC NEC NEC SG 12.5 NEC SG NEC SG EC NEC NEC NEC NEC NEC SG 9.5 NEC SG SG SG EC NEC NEC NEC NEC NEC SG 6.25 NEC SG SG SG EC NEC NEC NEC NEC NEC SG 4.75 NEC SG ZEG SG EC NEG NEC NEC NEC SG SG 3 NEC SG ZEG SG EC ZEG NEC ZEG ZEG SG SG 2.4 ZEG SG ZEG SG EC ZEG NEC ZEG ZEG SG SG 1.5 ZEG SG ZEG SG EC ZEG NEC ZEG ZEG SG SG 0 ZEG SG ZEG ZEG EC ZEG ZEG ZEG Z EG SG SG

ZEG- Zygotic Embryo Germination; NEC- Non Embryogenic Callus; EC – Embryogenic Callus; NG- No Growth at all; SG – Some Growth but no callus

Table 2. Effect of different dicamba concentrations on embryogenic callus induction from S. palmetto mature zygotic embryos

MS+ Concentration MS+ dicamba MS+ MS+ dicamba+ MS+ dicamba+ MS+ TDZ+ 5 µM zeatin+ in µM dicamba+5 µM 5 µM zeatin 5 µM 2,4-D dicamba 5 µM TDZ dicamba- 100 CI CI CI CI CI CI 75 CI CI CI CI ECI - 6.66% ECI -2.3% 50 CI CI CI CI ECI - 10% ECI -3.8% 38 CI CI CI CI ECI - 30.0% ECI -4.1% 25 CI CI CI CI ECI - 36.66% ECI -4.8% 19 CI CI CI CI ECI - 46.66% ECI -5.2% 12.5 CI CI ECI -5% CI ECI - 40% ECI -6.5% 9.5 CI CI ECI - 11% CI ECI - 36.66% ECI -8.4% 6.25 ECI - 3.33% ECI ECI - 18% ECI - 2.3% ECI - 33.03% ECI -11.8% 4.75 ECI - 9.99% ECI 1.665% ECI - 30% ECI - 3.4% ECI - 30.03% ECI -14% 3 ECI – 22.67% ECI 17.29% ECI - 60% ECI - 5.8% ECI - 12.39% ECI -28% 2.4 ECI -51.60% ECI 10.6%; ECI - 50% ECI - 7.2% ECI- 10.96% ECI -20% 1.5 ECI -77.96% ECI 6.13% ECI - 18% ECI - 6.5% ECI - 10% ECI -18% 0 ZEG ZEG ZEG NEC ECI - 9.02% ECI- 10%

CI – Callus Induction; ECI- Embryogenic Callus Induction; NEC – Non Embryogenic Callus; ZEG- Zygotic Embryo Germination

Table 3. Total protein content (mg/g of fresh weight) of the different stages of embryogenesis

Non embryogenic 4-week-old tissues 6- week-old tissues 7-week-old tissues callus

1.8 3.4 9.8 18.7

Table 4 - Summary of the tissue culture gel analysis data (PDQuest)

Table 4a. Total Number of Spots on each gel

Tissue Total Number of spots

Non embryogenic callus 288

4 weeks old callus 348

6 weeks old callus 419

7 weeks old callus 365

Table 4b. Qualitative Analysis of spots from tissue culture gels

Control Non 4-week-old 6-week-old 7-week-old embryogenic embryogenic culture culture callus callus

Non NA 64 139 57 embryogenic callus

4 weeks old 46 NA 121 38 callus

6 weeks old 0 0 NA 0 callus

7 weeks old 87 81 167 NA callus

NA- Not Applicable. Values indicate the number of protein spots that appear to be present on that gel when compare with the control gel.

84 Table 4c. Quantitative Analysis of spots from tissue culture gels

Non 4-week- old 6-week-old 7-week-old callus Control embryogenic callus callus callus

Increase in 2 3 4 5 62 34562 3456 2 3 456 fold Non NA 7873001588531 66 5 311 embryogenic callus 9 6 3 3 2 NA 12 3000 6 1 00 0 4 weeks old callus

17 7 3 2 1192220 NA 18 11 742 6 weeks old callus

8 5 3 3 39 3110 11 4332 NA 7 weeks old callus

NA- Not Applicable. Values indicate the number of proteins that appear to have increased quantity in specific fold when compare with the control gel.

85 Chapter 4

Genetic transformation of Sabal palmetto Walter Schultes & Schultes F.

zygotic embryos using biolistic and Agrobacterium-mediated methods

Keywords; Sabal palmetto, GUS, GFP, biolistic, Agrobacterium tumefaciens

Abstract. This report outlines the use of biolistic and Agrobacterium-mediated methods for transient expression of marker genes in mature zygotic embryos of Sabal palmetto (cabbage palm). The gus reporter gene, encoding β-glucuronidase and green florescent protein (GFP) gene were used in biolistic experiments while only the gus gene was used for Agrobacterium-mediated transformation. Different osmotica and optimizing conditions were used to enhance the transformation efficiency. According to our results mature zygotic embryos were amenable to both biolistic and Agrobacterium-mediated gene transfer. Near 100% transient GUS expression was observed with the Agrobacterium-method and the maximum frequency obtained with the biolistic method was 30% (GUS in 0.2M mannitol and 0.2 M sorbitol medium). Transformation frequencies were slightly higher with gfp (33 %) than gus (9.4%) without osmotic treatment. The, transformation frequencies from these methods were not comparable to oil palm (as high as 97.4% for biolistic and 64.4% for Agrobacterium-mediated gene transfer). There was a difference in the expression frequency when three different GUS vectors, pCAMBIA1301, pCAMBIA 1305.1 and pCAMBIA1305.2 were used for Agrobacterium-mediated transformation. When tissues were infected with strains with vectors pCAMBIA 1305.1 and 1305.2, the frequency was always higher than the strain with the vector 1301. In addition, expression was stronger and much more rapid with Agro plus GUS vectors pCAMBIA 1305.1 and 1305.2. In all cases, transformation was higher in sonicated than non sonicated tissues. Addition of pluronic acid (a surfactant) to the medium greatly increased the transformation efficiency with all three vectors.

Introduction Sabal palmetto, the state tree of Florida and South Carolina, is widely cultivated in the U.S. throughout USDA Zone 8. Established wild-type palms survive exposure to temperatures below -120C (100 F) without significant foliar injury, and some individuals have known to survive subzero F cold (Riffle, 1998). Juvenile, non-trunked palms growing in various USDA Zone 6a-7a locations regularly survive typical winter conditions with defoliation (reviewed by Francko, 2003). The collective data suggest that, with relatively minor improvements in cold tolerance and cold acclimation, cabbage palm could prove suitable for general horticultural use under USDA Zone 7 conditions. We are developing a novel tissue-culture-based cloning system for S. palmetto and have reported success in producing embryogenic callus (unpublished data – another chapter in this dissertation).

The objective of the present investigation was to develop a reliable method for DNA delivery into S. palmetto tissue cultures with potential of being incorporated into whole

86 plants, a prerequisite for creating next-generation cold-hardier palms. Agrobacterium- mediated and biolistic (particle gun) gene transfer systems have both been used successfully in monocot plant systems (Jansson et al., 2001). Agrobacterium can transfer part of its own DNA into cells while biolistics use a mechanical device to accelerate high-velocity, DNA coated microprojectiles across the cell wall into cells ( Klein, 1987) resulting in the penetration of the protoplasm by particles and the introduction of DNA into the cell. The method is rapid, simple and to a considerable degree genotype and tissue independent. The DNA is incorporated into the chromosome of the cells that are competent for DNA uptake. According to Christou (1995; 1996) particle bombardment is the preferred method of genetic engineering of monocots. Transgenic monocots including maize (Fromm et al., 1990; Gordan Kamm et al., 1990), rice (Christou et al., 1991), wheat (Vasil et al., 1992) and oat (Somers et al., 1992) have been obtained successfully via particle bombardment.

Early research conducted on oil palm underscores the potential utility of biolistics in this study (Parveez and Christou, 1998). In oil palm, the condition for delivering DNA into embryogenic calli has been optimized successfully based on transient GUS expression (Parveez et al., 1997 and Parveez et al., 1998). Use of six different constructs carrying GFP gene driven by different promoters( CaMV 35 S, ubiquitin – Elliot et al., 1999, HBT – a chimeric maize C4PPDK gene promoter with a CaMV 35 S enhancer – Sheen, 1993) to bombard oil palm embryogenic callus has been reported ( Parveez et al., 2000) with the 35 S-SGFP-TYG giving the most promising results. Most recently Abdullah et al., (2005) reported the successful transformation of immature embryos of oil palm by both biolistic and Agrobacterium-mediated method using the gus marker gene. Transformation frequencies reported were higher in biolistic method (97.4%) than in Agrobacterium-mediated transformation (64.4%), frequencies comparable to most of the other plant species studied.

Reporter genes are necessary for rapid selection of transformed cells. While GUS assay for β- glucuronidase is limited because it destroys the tissues during assay, green fluorescent protein (GFP) provides the opportunity to recover living transformed tissues after identification. On the other hand the gus reporter is more practical because it requires no special equipment such as florescence microscope and it is detectable in even in green tissues. Results presented here include preliminary genetic transformation studies of S. palmetto mature zygotic embryos using both biolistic and Agrobacterium - mediated methods.

Materials and Methods

Tissue preparation for transformation studies

Mature zygotic embryos were extracted from sterilized S. palmetto seeds and were cultured on callus induction medium (CIM – unpublished data another chapter of this dissertation) consisting of MS basal salts and vitamins (Murashige and Skoog, 1962), 1.5µM Dicamba, 4% commercial sucrose, 0.4% agar gel (Phytotechnology) and 0.25% activated charcoal (Carbon Activado Polvo QP-QUIMICA MEYER). The medium pH

87 was adjusted to 6.0 prior to autoclaving. Embryos were incubated in the dark at 280 C for five days prior to particle bombardment or Agrobacterium-mediated transformation.

Transformation by particle bombardment

Five-day-old mature zygotic embryos (N=20) were placed on osmotic media at the center of a petri dish 4 hrs prior to bombardment. Nine combinations of sorbitol and mannitol were tested in comparison to the control with sucrose. Concentrations tested were: 0.2 M sorbitol, 0.4 M sorbitol, 0.6 M sorbitol, 0.2 M mannitol, 0.4 M mannitol, 0.6 M mannitol, 0.2 M sorbitol + 0.2 M mannitol, 0.4 M sorbitol + 0.4M mannitol, 0.6 M sorbitol + 0.6 M mannitol.

An E.coli strain (AC001) containing the plasmid pEmuGN (Last et al., 1991) was used for this experiment. This 6.5-kb plasmid contains a gus reporter gene under the control of an Emu promoter. The Emu promoter is truncated and four copies of OCS promoter element from octopine synthase gene (Agrobacterium tumefaciens) and six copies of the anaerobic responsive element from maize Adh1 gene have been added in tandem.

Plasmid DNA was extracted using an alkaline lysis method (Sambrook et al., 1982). The extracted plasmid fraction was subjected to triple restriction digestion (Hind 111/Sal1/BamH1) and analyzed on 6% polyacrylamide gels to ensure that the pEmu promoter was intact. Purity and the concentration of DNA were determined by using a nanodrop ND-1000 spectrophotometer.

For transformation with GFP, the construct (35 S-SGFP-TYG –; Sheen, 1993) which has a gfp gene with a chromophore 65-SYG mutated (Tsien et al., 1995) to TYG driven by the CaMV 35 S promoter was used. This system is especially good for plant materials with high autoflorescence. Florescence is about 120-fold brighter than the original jellyfish GFP in plant cells with a single excitation peak (blue light). It was run on 1% agarose to ensure the intactness of DNA. Purity and the concentration were determined in the same way as for GUS plasmid DNA.

DNA precipitation onto gold particles (0.6 µm) was performed according to the instruction manual for the Biolistic PDS-1000/He particle delivery system (Biorad, Herculus, CA). Gold particles were prepared and stored at 40 C at 60 mg/ml in sterile 50% glycerol. The tissues were bombarded according to the protocol supplied for the Biolistic PDS-1000/He device. A 8 µl aliquot of DNA/gold mixture was pipetted onto the middle of the macrocarrier, allowed to dry in the air and bombarded onto each petri dish containing the tissue. The bombardment conditions used in this experiment were as follows: rupture pressure 1100 psi; distance form rupture disc to macrocarrier, 6mm; distance from macrocarrier to stopping plate, 11 mm; distance from stopping plate to target tissue, 90 mm and 2 µg of DNA was used per each bombardment. Tissues that were in media with osmoticum were transferred onto normal callus induction medium 19 hrs after bombardment. Plates with tissues were incubated in the dark and GUS assays were performed three days following bombardment. Lima bean cotyledons, known to be a good model system for GFP expression were bombarded with the same GFP and were

88 used as the positive control for GFP expression. Zygotic embryos that were not bombarded were used as the negative control. Experiments were run in duplicate.

GUS assays were performed according to the method of Jefferson (1987). Bombarded tissues as well as non-bombarded tissues were transferred into an empty 96-well-plate and covered with filter-sterilized GUS substrate (Klein et al., 1988a) containing 20% methanol and were incubated at 370 C for 48 hrs. Methanol was added to the reaction mixture to suppress endogenous GUS-like activity (Kosugi et al., 1990) Tissues were observed under the stereomicroscope (Olympus SZX12) and the number of embryos that had blue spots was recorded for each treatment condition Based on difference from controls in either the intensity or the size of the blue spots, if the intensity and size of the blue spots differed from the, controls they were recorded.

GFP-expressing embryos were detected using a florescence microscope (Nikon SMZ 1500). The number of GFP-positive embryos with characteristic green color spots and the number of spots per each embryo were counted and recorded from days 1-14.

Agrobacterium- mediated transformation using GUS

Agrobacterium tumefaciens EHA105, a hyper-virulent strain was transformed separately with the vectors pCAMBIA 1301, 1305.1 and 1305.2 according to the protocol described by (Vainstein et al., 1997).

89 pCAMBIA 1301

Figure 1 (taken from www.CAMBIA.org)

The vector pCAMBIA 1301 uses E.coli gusA (N358Q — to avoid N-linked glycosylation) with an intron (from the castor bean catalase gene) inside the coding sequence. This ensures the expression of glucuronidase activity is not from expression by residual A. tumefaciens cells but is derived from eukaryotic cells. It also contains the selectable marker gene hygromycin phosphotransferase (hpt 11) under the control of a CaMV 35S promoter and CaMV 35 S terminator.

The vectors pCAMBIA 1305.1 and 1305.2

The vectors pCAMBIA 1305.1 and pCAMBIA 1305.3 have reporter gene isolated from Staphylococcus species. It is a new gene with superior properties to E.coli GUS i.e. better catalytic activity for more rapid detection of activity and a version (1305.2) with the rice glycine-rich protein signal peptide for extracellular secretion providing rapid, in vivo gus assays pCAMBIA1305.1

Figure 2 (taken from CAMBIA organization)

The vector pCAMBIA 1305.1 is based on pCAMBIA1300. E. coli gusA has been replaced by GUSplus™ and is a compact binary vector with a broad host range ori for low copy, stable replication in Agrobacterium and a pBR322 ori for high copy replication in E.coli. It contains the hygromycin resistance gene (hpt II) for plant selection. The GUSplus™ gene has the intron from the castor bean catalase gene to make sure the detection of plant-specific glucuronidase expression.

91 pCAMBIA 1305.2

Figure 3 (taken from CAMBIA organization)

The final vector used, pCAMBIA 1305.2 is similar to pCAMBIA1305.1 except for the presence of the rice glycine-rich protein signal peptide sequence (GRP) inserted between the Nco I and Bgl II sites. The version in pCAMBIA1305.2 with a signal peptide is an alternative to GFP, which offers sensitive in vivo observation of gus activity without the need for UV detection. Bacteria were streaked onto plates containing YEP medium (An et al., 1988) with kanamycin as the selection agent and allowed to grow for 2 to 3 days. Loopfuls of bacteria from those plates were inoculated into YEP liquid media and allowed to grow for 18 hrs with vigorous shaking (150 rpm) at 280 C. Absorbance at 600nm of a portion of this culture was adjusted to 1.0 with YEP medium and this culture was used to inoculate culture directly. With the rest, bacteria were precipitated from the rest of the liquid culture and the OD was adjusted to 1.0 at 600nm using MS liquid media. Bacteria were allowed to stabilize in MS liquid media (MS media + acetosyringone (100µM), MS + pluronic acid F-68 (0.03%), MS + acetosyringone (100µM) + pluronic acid (0.03%) for 3 hrs in dark at room temperature. This procedure was performed with all three strains with three GUS vectors i.e EHA105:pCAMBIA1301, EHA105: pCAMBIA 1305.1 and EHA105: pCAMBIA 1305.2.

Tissue cultures for transformation were prepared in the same way as for particle bombardment and cultured on callus induction medium but without any osmoticum in

92 medium. They were incubated in the same media (callus induction media) until used for the transformation.

Several different methods were employed to inoculate bacterial suspension to target plant cells. Bacterial suspension were directly introduced onto the growing embryos and incubated in dark at 250 C for 3 days. Tissues that were not inoculated with bacterial culture were used as the control.

Second, zygotic embryos (N=20) were inoculated with the bacterial cultures stabilized as described above and half of the embryos from each culture condition were sonicated for 5 seconds (Bransonic 221) while in the same media. Inoculation was carried out for 45 min. Tissues that were not treated with bacterial suspensions were used as a negative control while mature tobacco leaf tissues treated with bacteria stabilized in MS medium alone were used as the positive control. Two independent experiments were performed.

Finally, tissues inoculated with pCAMBIA1301 were transferred (without blotting) onto callus induction medium with or without acetoryringone and the tissues inoculated with pCAMBIA 1305.1 and 1305.2 were transferred (without blotting) onto callus induction medium without acetosyringone. Tissues were allowed to grow in the dark at 250 C for 3 days before testing for GUS activity.

Tissues were tested for GUS activity as described before for biolistic method and observed under the stereomicroscope. The number of embryos positive for GUS activity (showing blue spots) was recorded and if the intensity and the size of blue spots differed significantly they were recorded.

Results and Discussion

Our results indicate that S. palmetto tissues were transformed with high efficiency ( almost 100%) using the Agrobacterium-transformation method while particle bombardment resulted in maximum of 33% transformation efficiency, which is lower than the other palm species studied so far ( Abdullah et al., 2005). However, the biolistic PDS-1000/He device was capable of delivering DNA successfully into mature zygotic embryos.

To achieve efficient transformation using biolistic technology both biological physical and biological parameters need to be evaluated carefully. Optimization of physical parameters to increase the transformation efficiency, in oil palm embryogenic calli has been carried out (Parveez et al., 1997). These optimization conditions seemed to be necessary to minimize tissue damage during bombardment (Perl et al., 1992) and the bombardment variability (Taylor and Vasil 1991). They include, size and the velocity of the macrocarrier, concentration of spermidine and CaCl2, distance between macrocarrier and the target plate (Klein et al., 1998b). Optimization conditions are also species- specific and tissue-dependent (Parveez et al., 1997). Studies have also been carried out with several plant species including oil palm to determine the best biological parameters for particle bombardment. Among those parameters that can influence transformation

93 efficiency are different explant types (Parveez et al., 1998; Miki et al., 1993) different genotypes (Moore et al., 1994) and tissue preculture (Bilang et al., 1993; Taylor et al., 1993). In addition, the concentration and the type of the osmoticum (Perl et al., 1992), duration of osmoticum treatment pre- and post-bombardment (Vain et al., 1993) can also influence the transformation efficiency. In this study we evaluated the use of different osmotica in the media to enhance the transformation efficiency and used gus reporter gene to measure efficiency. Particle bombardment causes high velocity DNA penetration into cells and can damage cell structures. Therefore, improvement of the quality of the starting material as well as reduction of stresses during bombardment can provide major advancements for plant transformation.

According to our results (Table 1a) placement of tissues in osmoticum medium containing 0.2M mannitol and 0.2 M sorbitol 4 hrs prior to and 19 h after bombardment resulted in increase (3-fold) in transient GUS expression. This is consistent with the results obtained from previous researchers using other plant species (Vain et al., 1993) where equimolar concentrations of mannitol and sorbitol gave higher transformation efficiency. In another study, equimolar sorbitol and mannitol has been reported as the best osmoticum treatment for microorganisms (Armaleo et al., 1990; Shark et al., 1991). In our study all but the combination of 0.6M mannitol and sorbitol seem to increase the transformation efficiencies. In contrast, with oil palm, the highest transformation efficiency has been reported with 0.4M mannitol and combination of mannitol and sorbitol at 0.2M concentrations gave fairly low transformation efficiency (Parveez et al., 1998). In our study 0.4M concentrations of sorbitol or mannitol or both have shown higher GUS activity than the tissues without any osmotic treatment but have not shown a significant difference among treatments. Although GUS transient transformation efficiency could be increased using equimolar concentration (0.2M) of sorbitol and mannitol, according to the ANOVA (Table 1b) there is no significant difference between treatments.

It is believed that osmotic enhancement can stabilize the cell membrane which may have damaged during the bombardment. Osmoticum treatment also can enhance the transformation efficiency by reducing the cell growth (Vain et al., 1993). Osmotic enhancement of transient expression results from plasmolysis of cells which may reduce cell damage by preventing extrusion of protoplasm from bombarded cells (Armaleo et al., 1990; Sanford et al, 1990). Incubation of tissues in the media with osmotica after the bombardment may have increased the transformation efficiency. It is reported that to be effective, plasmolysed state must be maintained few hours before and after bombardment (Vain et al., 1993).

Transformation using green fluorescent protein gfp as a selectable marker gene

In normal medium (without any added osmoticum) GFP expression was about 33% which is higher than the GUS expression from the tissue growing in the normal media (9.42%). All the embryos showed autoflorescence but characteristic green color spots due to GFP expression could be easily distinguished. Number of spots present varied from 5- 50 in embryos positive for GFP expression. It has been reported that

94 generally the number of spots and their brightness decline drastically after three days of bombardment and totally fade after two weeks. (Parveez et al., 2000). In contrast, in our observations the number of spots and brightness of the spots appearing after bombardment did not fade or decline with time during two weeks period of time. This observation is consistent with oil palm tissues bombarded with the same GFP construct where it has shown the highest number of spots and a few spots retained expression even 5 months after bombardment (Parveez et al., 2000).

Agrobacterium-mediated transformation

The type and the developmental stage of the infected plant tissues, the concentration of Agrobacterium tumefaciens, the composition of the media used for co- cultivation infection and tissue culture, co-cultivation temperature, the selectable marker genes used, light or dark culture environment, the type of vector, and the plant genotype are some factors that influence transformation efficiencies of Agrobacterium-mediated transformation (Hiet et al., 1997). Therefore, when developing an efficient transformation protocol, it is critical to find the right combination of many factors that act together during transformation. The complexity of factors that influence transformation is probably the reason why Agrobacterium- mediated transformation in monocotyledonous plant species has been difficult to achieve (Hiei et al., 1997; Ishida et al., 1996).

In this study, embryos that were inoculated directly with bacterial cultures (with three different vectors) did not show any GUS activity while all the tissues that were inoculated with bacterial cells stabilized in MS medium with or without other compounds show positive results for GUS activity. Therefore, stabilizing the bacterial cells in the MS medium seems to be critical for the transformation of S. palmetto zygotic embryos. The results obtained are summarized in Table 2 and in Figure 4.

According to the results obtained for tissues infected with three different vectors, tissues that were sonicated show higher activity than the tissues that were not sonicated (Table 2a, 2b and 2c). In Agrobacterium-mediated transformation method, physical wounding of the tissues is common as this enhances transformation efficiency (Rashid et al, 1996). Trick and Finer, 1997 reported 100- to 1400-fold increase in transient GUS expression in various tissues of Ohio buckeye, soybean, cowpea, maize, white spruce and wheat. Their scanning electron microscopy and light microscopy studies revealed that SAAT (Sonication assisted Agrobacterium-mediated transformation) treatment produces small and uniform fissures and channels through the tissue. This may provide the Agrobacterium easy way access to internal plant tissues. In addition, wounding is believed to enhance the production of certain phenolic signals in plants, which would increase the accessibility of a putative cell-wall binding factor to the bacterium (Stachel et al., 1985).

The presence of acetosyringone (AS), a phenolic compound, in the media seems to extend the host range of Agrobacterium. The presence on AS in the Agrobacterium co- cultivation medium has markedly increased T-DNA delivery in plant tissues (Wu et al., 2003). Some dicot plants are known to produce AS in contrast monocots do not (Usami et

95 al., 1987). In our study, activity was enhanced when AS was present in the inoculation medium (100µM) with all three vectors. In addition with the vector 1301 it has also enhanced the expression when the co-cultivation media contained AS (Table 2a, 2b and 2c). However, in this study co-cultivation media with or without AS were not employed for tissues infected with the strains containing pCAMBIA1305.1 and 1305.2. Agrobactearium transformation of some monocots requires AS (Hiei et al., 1997). Absence of AS has lead to complete failure of transformation in several monocots (Ishida et al., 1996, Rashid et al., 1996) but in our study tissues expressed GUS activity without the addition of AS. This is consistent with the results obtained for the monocot Typha latifolia GUS activity (Rogers et al., 2004). That indicates the possible in vivo production of AS or other phenolic substances in these plants which are necessary for Agrobacterium infection and subsequent vir gene. Addition of 100µM AS increased the percent of explants that expressed GUS activity, as reported in T. latifolia (Rogers et al., 2004). In our study too we have observed an increase in GUS activity with the addition of AS. During the co-cultivation period, phenolic inducers such as acetosyringone may work with other signaling factors such as temperature and the acid environment to enhance the expression of vir genes. Weir et al. (2001) reports that, enhance transient GFP expression could also observed in wheat cultures with AS at 400 µM in the co- cultivation media but not the inoculation media.

The addition of pluronic acid F-68 into the inoculation medium greatly enhanced GUS expression (100%) with all three vectors (Table 2a, 2b, 2c). Tissues infected with EHA105: pCAMBIA 1301 gave 100% expression frequency when tissues were sonicated, inoculation media contained pluronic acid and co-cultivation media contained AS. In contrast issues infected with EHA105:pCAMBIA1305.1 and EHA105: pCAMBIA 1305.2 gave 100% transformation efficiency when tissues were sonicated, inoculation media contained pluronic acid and co-cultivation media did not contain AS. It has been reported that the addition of 0.03% pluronic F-68 to the inoculation medium (Cheng et al., 1997) dramatically increased transient GUS expression in sorghum by up to 100-fold. However, in our study some other factors especially tissue sonication, contributed to obtain 100% transformation efficiency in the presence of pluronic acid. It is believed that pluronic acid, a surfactant, enhances T-DNA delivery either by eliminating certain substances that inhibit A. tumefaciens attachment or by aiding A. tumefaciens attachment. GUS staining in embryos varied from single spots to large patches of blue (Cheng et al., 1997). In our study we mostly observed large blue patches on the embryos (Figure 4 G).

There was a difference in the expression frequency when three different GUS vectors were used; 1305.1 and 1305.2 vectors always giving higher expression frequencies than the vector 1301. (Table 2a, 2b, 2c- tissues in MS media with sonication and without AS in co-cultivation media frequencies were 1301 – 72.5%; 1305.1-75%; 1305.2 – 79%). However, this difference in frequencies among vectors varies greatly with the conditions used. The biggest difference in the frequency was observed when tissues were non- sonicated and the media contained pluronic acid (Tables 2a, 2b and 2c -1301- 60%, 1305.1- 87% and 1305.2 – 89% In addition, the expression was stronger and much quicker with Agro-plus GUS vectors pCAMBIA 1305.1 and 1305.2. This may well be

96 explained by the fact that these vectors have reporter gene isolated from Staphylococcus sp with superior properties to E.coli GUS. They have better catalytic activity for more rapid detection of GUS activity and a version with the rice glycine-rich protein signal peptide for extracellular secretion providing rapid, in vivo GUS assays.

In conclusion, these preliminary results demonstrate that S. palmetto zygotic embryos can be successfully transformed using both methods. According to the results from this study Agrobacterium-mediated transformation show more promising results (efficiency is several-fold higher than the biolistic method). Use of equimolar concentration (0.2M) of osmotica has increased the transient gus expression by the biolistic method. With Agrobacterium- mediated transformation, incubation in the co-cultivation media, presence of pluronic acid in the co-cultivation media, presence of acetosyringone in the culturing media as well as tissue sonication are some of the factors that enhanced transformation efficiency. However, present study did not include a comprehensive study leading to the optimization of parameters (physical and biological) for transient expression of marker genes in zygotic embryos by the biolistic method.

Acknowledgements

This research has been made possible through grants from the Ohio Plant Biotechnology Consortium and additional support from the Miami University, Department of Botany Academic Challenge program. The ungrudging assistance from Dr. David Pennock and his group at the Department of Zoology, Miami University with the gene gun work is gratefully acknowledged. We would also like to acknowledge Dr. John Finer (Ohio State University) for providing us with a sample of GFP DNA, Dr. Richard Brettell from CSIRO Australia for providing the E.coli strain with GUS construct, Drs. Richard Jefferon and Osmat Jefferson from CMABIA organization Australia for providing us with the Agrobacterium strain EHA 105 and GUS vectors pCMABIA 1301, pCAMBIA 1305.1 and pCMABIA 1305.2.

References

Abdulla R, Zainal A, Heng WY, Li LC, Beng, YC, Phing LM, Sirajuddin SA, Ping WYS, Joseph JL, Jusoh SA, Muad MR, Huey YL (2005) Immature embryo; A useful tool for oil palm ( Elaeis guineensis Jacq.) genetic transformation studies. Electronic Journal of Biotechnology 8:25-34 An G, Ebert PR, Mitra A, Ha, SB (1988) Binary vectors. In: Plant Molecular Biology Manual. Gelvin SB, Schilperoort RA, Verma DPS. (Eds.) Dordrecht, Kluwer Academic Publishers: A3/1-19 Armaleo D, Ye GN, Klein TM, Shark KB, Sanford JC, Johnston SA (1990) Biolistic Nuclear Transformation of Saccharomyces cerevisiae and other fungi. Current Genetics 17:97-103 Bilang R, Zhang S, Leduc N, Iglesias VA, Gisel A, Simmonds J, Potrykus I, Sautter C (1993) Transient gene expression in vegetative shoot apical meristem of wheat after ballistic microtargeting. Plant J 4:735-744 Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115:971-980 Christou P, Ford TL, Kofron MP (1991) Production of transgenic rice (Oryza sativa L.) plants from agronomically important Indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Biotechnology 9:957-962 Christou P (1995) Strategies for variety independent genetic transformation of important cereals, legumes and woody species utilizing particle bombardment. Euphytica 85:3-27 Christou P (1996) Transformation technology. Trends in Plant Science 1:423-431 Elliott AR, Campbell JA, Dugdale B, Brettell RIS, Grof CPL (1999) Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Reports 18:707-714 Francko DA (2003) Palms Won't Grow Here and Other Myths: Warm-Climate Plants for Cooler Regions. Portland, OR and Cambridge, Timber Press, 307pp Fromm ME, Morrish F, Armstrong C, Williams R, Thomas J, Klein TM (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Biotechnology 8:833-839 Gordon-Kamm WJTMS, Mangano ML, Adams TR, Daines RJ, Start WG, O'Brien JV, Chambers SA, Adams Jr WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603-618 Hiei Y, Komari T, Kubo T (1997) Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol 35:205-218 Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize ( Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotech 14:745-750.

98 Jansoon C, Roussy I, Ahlandsberg S (2001). Transformation and regeneration capacities for five Nordic barley elite cultivars-evaluation of tissue culture response and transient expression. Hereditas 134:97-101 Jefferson RA (1987) Assaying chimeric genes in plants. Plant Mol. Biol. Rep. 5(387-405) Klein TM (1987) High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327:70-73 Klein TM, Gradziel T, Fromm ME, Sanford JC (1988a) Factors influencing gene delivery into Zea mays cells by high velocity micoprojectiles. Bio/technology. 6:559-563 Klein TM, Harper EC, Svab Z, Sanford JC, Fromm ME, Maliga P (1988b) Stable genetic transformation of intact Nicotina cells by the particle bombardment process. Proc. Natl. Acad. Sci. USA. 85: 8502-8505 Kosugi S, Ohtai Y, Nakajima K, Arai Y (1990) An improved assay of beta-glucuronidase in transformed cells; methanol almost completely suppresses a putative endogenous beta-glucuronidase activity. Plant Science 70: 133-140 Last DI, Brettel RIS, Chamberlain DA, Chaudhury AM, Larkin PJ, Marsh EL, Peacocks WJ, Dennis ES (1991) pEmu: An improved promoter for gene expression in cereal cells. Theor. Appl. Genet. 81: 581-588 Miki BL, Fobert PF, Charest PJ, Iyer VN (1993) Procedures for introducing foreign DNA into plants. Method Plant Mol. Biol. Biotechnol: 67-68 Moore PJ, Moore AJ, Collins GB (1994) Genotype and development regulation of transient expression of a reporter gene in soybean zygotic cotyledons. Plant Cell Rep. (13): 556-560 Murashige T, Skoog F (1962) A revised method for rapid growth and bioassays with tobacco leaf cultures. Physiol. Plant 15: 473-497 Parveez GKA, Chowdhury MKU, Saleh NM (1997) Physical parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) using the biolistic device. Industrial Crops and Products 6: 41-50 Parveez GKA, Christou P (1998) Biolistic-mediated DNA delivery and isolation of transgenic oil palm (Elaeis guineensis Jacq.) embryogenic callus cultures. Journal of Oil Palm Research 10: 29-38 Parveez GKA, Chowdhury MKU, Saleh NM (1998) Biological parameters affecting transient GUS gene expression in oil palm (Elaeis guineensis Jacq.) embryogenic calli via microprojectile bombardment. Industrial Crops and Products: 17-29 Parveez GKA, Masri MM, Zainal A, Majid NA, Yunus AMM, Fadilah HH, Rasid O, Cheah S (2000) Transgenic oil palm: production and projection. Biochem. Soc. Trans 28: 969-972 Perl A, Kless H, Blumenthal A, Galili G, Galun E (1992) Improvement of plant regeneration and GUS expression in scutellar wheat calli by optimization of culture conditions and DNA-microprojectile delivery procedures. Mol. Gen. Genet. 235: 279-284 Rashid H, Yokoi S, Toriyama K, Hinata K (1996) Transgenic plant production mediated by Agrobacterium in Indica rice. Plant Cell Reports 15: 727-730 Riffle RL (1988). The Tropical Look, Timber Press, 428pp Rogers SMD, Nandakumar R, Chen (2004) Factors affecting Agrobacterium-mediated transient transformation of the wetland monocot, Typha latifolia. Plant Cell, Tissue and Organ Culture 79:31-38

99 Sambrook J, Fritsch EF, Maniatis T (1982) Molecular Cloning - A Laboratory Manual. New York, Cold Spring Harbor Sanford JC, Smith FD, Russell JA (1992) Optimizing the biolistic process for different biological applications. In: Methods in Enzymology. Wu R (Ed.) 217:483-509 Shark KB, Smith FD, Harpending PR, Rasmussen JL, Sanford JC (1991) Biolistic transformation of a prokaryote, Bacillus megaterium. Applied and environmental microbiology 57:480-485 Sheen J (1993) Protein phosphatase activity is required for light-inducible gene expression in maize. EMBO Journal 12:3497-3505 Somers DA, Rines HWGUW, Kaeppler HW, Bushnell WR (1992) Fertile, transgenic oat plants. Biotechnology 10:589-1594 Stachel SE, Messens E, Van Ontagu M, Zambryski P (1985) Identification of signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624-629 Taylor MG, Vasil IK (1991) Histology and physical factors affecting, transient GUS expression in pearl millet (Pennisetum glaucum (L.) R. Br.) embryos following microprojectile bombardment. Plant Cell Rep. 10:120-125 Taylor MG, Vasil V, Vasil IK (1993) Enhanced GUS gene expression in cereal/grass cell suspension and immature embryos using the maize ubiquitin-based plasmid pAHC25. Plant Cell Rep. 12:491-495 Trick HN, Finer JJ (1997) SAAT: Sonication assisted Agrobacterium-mediated transformation. Transgenic Research 6:329-336 Tsien RY, Heim R, Cubitt AB (1995).Improved green florescence. Nature 373:663-664 Usami S, Morikawa S, Takebe, Machida (1987) Absence of monocotyledonous plants of the diffusible plant factors inducing T-DNA circulation and vir gene expression in Agrobacterium. Mol. Gen. Genet. 290: 221-226 Vain P, McMullen MD, Finer JJ (1993) Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Reports 12:84-88 Vainstein A, Tzfira T, Jensen CS, Wang W, Zuker A, Vinocur B, Altman A (1997) Transgenic Populus tremula: a step-by-step protocol for its Agrobacterium - mediated transformation. Plant Molecular Biology Reporter 15:219-235 Vasil V, Castillo AM, Fromm ME, Vasil IK (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile callus. Bio/technology 10:667-674 Weir B, Wang MB, Upadhyaya AR, Elliot AR, Brettell RIS (2001) Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker. Aus. J. Plant Physiol. 28: 807-818 Wu H, Sparks C, Amoah B, Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell rep. 21:659- 668

100 A B

C D

E F

G H

Figure 4; Histochemical GUS assay on bombarded and co-cultivated mature zygotic embryos of S. palmetto. A. non –bombarded zygotic embryo showing no GUS activity. B. freshly bombarded embryo showing the expression of GUS gene as blue spots C. Non co-cultivated embryos D. Tobacco leaf tissue co-cultivated with EHA105: pCAMBIA 1301. E. An embryo co-cultivated with EHA105: pCAMBIA 1301. F. An embryo co-cultivated with pCAMBIA 1305.1 G. An embryo co-cultivated with EHA105: pCAMBIA 1305.2 H. An embryo co-cultivated with EHA105: pCAMBIA 1301 after stabilizing the bacterial cells in MS and pluronic acid (0.03%)

101

Table 1a – Percentage of tissues showing GUS activity in media (with or without osmoticum)

Media GUS Media GUS Media GUS activity activity activity 0.2M 15% 0.4M 15.8% 0.6M 11.2% sorbitol 3+-1.41 sorbitol 3+- sorbitol 3.5+3.53 (N=40) 1.41(N=38) (N=62) 0.2M 17.3% 0.4 M 14.8% 0.6M 10.33% mannitol 4+-2.82 mannitol 2+- 1.41 mannitol 1.5+2.12 (N= 46) (N=27) (N=29) 0.2M 30% 0.4M 14.23% 0.6M 9.93% sorbitol+ 6+4.24 sorbitol+ 3.5+-0.707 sorbitol+ 3.5+0.707 0.2 M (N=40) 0.4 M (N=49) 0.6 M (N=70.5) mannitol mannitol mannitol Normal 9.42% callus 2.5+- 3.53 induction (N=53) media Minimum of 20 embryos were tested during one experiment and two independent experiments were performed. Percentage is followed by the mean value and standard deviation and the total number of embryos tested.

Table 1b – ANOVA of transient GUS expression for optimization of media for biolistic method

Sum of Degree of Mean Source of Square feedom square Variation (SS) (df) (MS) F P-value F crit Between Groups 0.058624 9 0.006514 0.384439 0.917018 3.020382 Within Groups 0.169437 10 0.016944

Total 0.228061 19

102

Table 2a: Percentage of tissues showing GUS activity after infected with EHA105: pCAMBIA 1301

SAAT SAAT NON SAAT NON SAAT

acetosyringone No acetosyringone No acetosyringone acetosyringone

MS 77.5 % 72.5% 70% 50% 15.5 +-3.53 14.5+-3.53 14+-2.82 10+- 1.41 (N=40) (N=40) (N=40) (N=40) MS+ 82.5% 73% 72% 50% acetosyringone 16.5+- 4.94 15+-1.41 14+- 10+-2.82 (N=40) (N=41) 4.24(N=39) (N=40) MS+ pluronic 100% 87.5 % 66% 60% acid 20+-0 17.5+- 10.60 13.5+- 3.53 12+- 4.24 (N=40) (N=40) (N=41) (N=40) MS+ pluronic 84% 79% 75% 70% acid + 13.5+- 2.12 15.5+- 3.53 15+- 0 14+- 8.48 acetosyringone (N=32) (N=39) (N=40) (N=40)

SAAT, Sonication Assisted Agrobacterium-mediated Transformation. Minimum of 20 embryos were tested during one experiment and two independent replicates were performed. Percentage is followed by the mean value and standard deviation and the total number of embryos tested.

103

Table 2b: Percentage of tissues showing GUS activity after infected with EHA105: pCAMBIA 1305.1

SAAT NON SAAT

MS 75% 54% 15+- 1.41 (N=40) 10.5+- 0.707 (N=39) MS+ acetosyringone 83.5% 64% 17.5+-3.53 (N=35) 12.5+- 3.53(N=39)

MS+ pluronic acid 100% 87% 22+- 1.41 (N=44) 17+- 5.65 (N=39)

MS+ pluronic acid + 83% 72% acetosyringone 17+- 4.24 (N=41) 14+- 2.83(N=39)

SAAT, Sonication Assisted Agrobacterium-mediated Transformation; Minimum of 20 embryos were tested during one experiment and two independent replications were performed. Percentage is followed by the mean value and standard deviation and the total number of embryos tested.

104 Table 2c: Percentage of tissues showing GUS activity after infected with EHA105: pCAMBIA 1305.2

SAAT NON SAAT

MS 79% 62% 15+-1.41 (N=38) 12+-2.82 (N= 39) MS+ acetosyringone 83% 68% 17+-4.24 (N=41) 13.5 +- 4.94 (N=40) MS+ pluronic acid 100% 89% 19+-2.82 (N=38) 17+- 1.41(N=38)

MS+ pluronic acid + 87% 74% acetosyringone 17+-7.07(N=39) 15.5+- 7.77 (N=42)

SAAT, Sonication Assisted Agrobacterium-mediated Transformation; Minimum of 20 embryos were tested during one experiment and two independent experiments were performed. Percentage is followed by the mean value and standard deviation and the total number of embryos tested.

105 Chapter 5

Investigations on cold tolerance mechanism in Rhapidophyllum hystrix

(Pursh) H. Wendl & Drude (needle palm) and identification of cold- responsive

proteins

Key words; Rhapidophyllum hystrix, cold acclimation, cold tolerance, supercooling point, superoxide dismutase, dehydrin, cold responsive proteins

Abstract Needle palm (Rhapidophyllum hystrix, Family – Arecaceae), accepted as the most cold hardy palm on earth, is native to the southeastern US. It has reportedly survived temperatures as low as – 230 C (-90 F) without significant damage to foliage. Plants have evolved two efficient strategies to tolerate extreme winter conditions i.e. freeze avoidance and freeze tolerance. Both are represented by woody plants and involve supercooling mechanisms. Tissues depending on freezing avoidance show deep supercooling where all cellular water remains liquid below the freezing point. In theory the supercooling point of pure water is –38.10 C (-370F). The other survival mechanism is freezing tolerance through exposure to increasingly low, yet nonfreezing temperatures. Adaptations of freezing tolerance in plants are associated with numerous physiological and genetic alterations that are necessary to protect vital physiological processes and critical cell structures. They are regulated by a complex system that is programmed at the gene expression level. Little is known about needle palm survival mechanism(s) at extremely cold temperatures. Proteomics is one of the most recent molecular tools used to analyze low resistance in plants. To elucidate the underlying molecular mechanism/s in needle palm cold tolerance, I initially focused on the quantitative changes in the leaf protein profiles with the changes in supercooling point (SCP) during treatments. Our SCP data indicate that this palm supercools extensively even without acclimation. Proteomic analyses, by 2D electrophoresis revealed that a number of proteins have a higher expression while some proteins disappeared or had a lower abundance along with the treatment at 40 C. The majority of proteins did not show significant changes in the quantity. MALDI -TOF-MS analyses enabled us to putatively identify some of these proteins. Among the accumulating or de novo proteins expressed are heat shock proteins, thioredoxins, peroxidases, nudix hydrolases, ATP synthase, oxygen evolving complex and RUBISCO activase. However, according to our native gel electrophoresis, superoxide dismutase activity did not seem to be markedly upregulated by exposure to cold.

List of abbreviations used: 2DE – 2 Dimensional electrophoresis; IPG – Immobilized pH gradient strips; MALDI-TOF-MS – Matrix Assisted Laser Desorption/ionization time-of-flight mass spectroscopy

106 Introduction Needle palm (Rhapidophyllum hystrix, Family – Arecaceae), accepted as the most cold hardy palm, is native to central and northern Florida as well as coastal areas of Mississippi, Georgia, Alabama and the Carolinas. It has reportedly survived temperatures as low as – 23 0C (-90 F) without significant damage to foliage (Francko, 2003). Leaf sheaths are surrounded by very sharp spines and common and scientific names reflect that property. Rhapidophyllum hystrix leaves are palmate with glossy green upper surface and silvery undersides. Flowers are unisexual and plants are dioecious, requiring more than one plant to produce seeds. The seeds are brown and are covered with whitish hairs. These palms are not usually very tall; old needle palms can produce trunks up to 1 meter in height. As the plant grows stems produce offshoots and the plant grows in width, rather than the height, ultimately reaching ca. 3m tall and broad.

Plants have evolved two efficient strategies to tolerate extreme winter conditions i.e. freeze avoidance and freeze tolerance (Levitt, 1980). Woody plants exhibit both these survival mechanisms. (Sakai and Larcher, 1987, Malone and Asworth, 1991). Tissues depending on freezing avoidance show supercooling where cellular water remains liquid below the freezing point. In theory the supercooling point of pure water is –38.10 C (- 370F) (Rasmussen and MacKenzie, 1972, Gusta et al, 1983). Freezing of deep supercooled water can be observed as low temperature exotherms, i.e. heat of fusion produced by the change from liquid to solid phase. Freezing avoidance is known to occur in certain tissues and organs in temperate tress: flower buds of angiosperms, shoot and floral primodia of conifers (Sakai and Larcher, 1987). Compounds, such as sugars, amino acids, and other solutes, induce supercooling in plant tissues.

The other survival mechanism is freezing tolerance through exposure to increasingly low, yet nonfreezing temperatures which is also called cold acclimation. During freezing water migrates into intercellular spaces resulting in gradual growth of extracellular ice. This happens due to the initial low solute concentration in the intercellular space. At slow cooling rates, cold-acclimated tissues undergo equilibrium freezing; i.e. diffusion of cellular water is rapid enough to maintain the chemical equilibrium between the protoplasm and the ice. Water continues to migrate from the cell into the intercellular spaces. Therefore, ensuing dehydration stress is the major survival mechanism in woody plants (Levitt, 1980). Some of the hardiest species of woody plants exhibit freezing tolerance; in a fully acclimated state they may survive experimental freezing to – 196 0C (-3210 F) (George et al., 1974).

Adaptations of freezing tolerance in plants are associated with numerous physiological and genetic alterations that are necessary to protect vital physiological processes and critical cell structures. They are regulated by a complex system that is programmed at the gene expression level. Cold acclimation is accompanied by reduced water content of tissues and accumulation of putative cryoprotectant compounds such as soluble carbohydrates and proteins (Levitt, 1980; Guy, 1990). The biochemical changes in plasma membrane include qualitative changes in proteins concomitantly with an increase in membrane fluidity, phospholipids enrichment and fatty acid unsaturation (Yoshida and Uemura, 1990). Cell structural changes involve an argumentation of cytoplasm and

107 reduction of vacuole size (Pomeroy and Siminovitch, 1971; Wisniewski and Asworth, 1986).

In recent years Arabidopsis has become one of the most widely used plant species for studies on cold acclimation and freezing injury. This plant cold-acclimates in a very short time period and shows various changes in cells, tissues, and organs (Thomashow, 1998, 2001). Although there have been several studies to identify proteins that change in quantity during cold acclimation in woody plants (Renaut et al., 2004) there are no reports or attempts to identify proteins associated with palm cold tolerance. Recent advances in technology along with the availability of genome sequence database have enabled researchers to identify plant proteins of relatively small amounts separated by 2D-PAGE (Rossignol, 2001). Peptide mass finger printing, coupled with MALDI-TOF- MS, seems to be a powerful tool for protein identification in plant system. However, it depends considerably on the availability of genome sequence database with which mass information of whole or fragmented peptides is referred to. In fact, because the genome sequence project has completed with Arabidopsis, several studies on protein identification of Arabidopsis have been published with plasma membrane, endoplasmic reticulum (ER; Prime et al., 2000), mitochondria ( Kruft et al., 2001), and chloroplasts (Peltier et al., 2002).

The reactive oxygen species (ROS): superoxide, hydrogen peroxide, the hydroxyl radical and singlet oxygen are produced in plants to various degrees in a number of metabolic pathways. Plants normally possess scavenging systems that keep ROS species below damaging levels (Larson, 1988). It is well documented that the production of ROS can exceed the capacity of the scavenging systems under environmental stresses such as high light intensities, chilling, drought, etc., resulting in oxidative damage (Bowler et al, 1992). Plants react to oxidative stimuli by synthesizing antioxidant enzymes including superoxide dismutases, glutathione reductase, ascorbate peroxidases and catalases (Scandalios 1990, Bowler et al, 1994, Foyer et al., 1994, Anderson et al., 1995, Foyer et al., 1997). Superoxide dismutases play an essential role in the protection of cellular components against oxidative damage (Malan et al., 1990, Bowler et al., 1992). In higher plant, SODs are classified according to their respective metal cofactors: copper/zinc (Cu/Zn-SOD), iron (Fe-SOD) and manganese (Mn-SOD). There is increasing evidence that chilling causes elevated levels of active oxygen species (Omran, 1980; Wise and Naylor, 1987; Prasad et al., 1994) which likely contribute significantly to chilling damage. Thus the ability of a plant to improve its active-oxygen-scavenging capacity may be a key element in stress tolerance.

The main objective of the present investigation was to begin elucidating the underlying molecular mechanism/s responsible for needle palm cold tolerance using a proteomic approach coupled with supercooling point determinations in the plant leaves. Since palm genome databases are yet to be established we extended our investigation to identify dehydrins and superoxide dismutases which are known to be induced upon exposure to cold temperatures in plants.

108 Materials and Methods

Plant treatment conditions

A total of five needle palms (3 to 4 year old) were pre-acclimated in a growth chamber with a 16:8 photoperiod at 26 0C (790F) for one month period.Plants were watered every other day and maxicrop nutrient solution was applied once per week. Afterwards, plants were cold treated at 4 0 C (390 F) with 16: 8 photoperiod for a period of two weeks in an incubator. Two leaf pieces were removed from each plant from the third leaf closest to the apex one for supercooling point measurements and the other for protein analysis on days 0, 2, 4, 8, 10 and 14 around mid-day in the light cycle. For protein analysis leaves were immediately frozen in liquid nitrogen and stored in a – 80 0 C freezer until further use. Supercooling measurements were conducted just after harvesting the leaves. Supercooling point (SCP) measurements

Freezing analysis was carried out according to the procedure developed at the Institute of Low Temperature Sciences, Sapporo (Ishikawa and Sakai 1981, Ishikawa, 1984) with modifications. Mature fan leaves from needle palm were used in supercooling experiments. Leaf segments (1.5cm x 1.5cm) were folded in half at the midrib to enclose Cu/CuNi thermocouples, then wrapped with a tape to ensure that thermocouple was in contact with the tissue and then wrapped in aluminum foil. Tissues were cooled in a refrigerated ethanol bath at a cooling rate of 10 C/min starting from 5 0C (410F) to a final temperature of –25 0C (-130F). The temperature of the thermocouple was recorded on a chart recorder. Upon freezing, a rise (the exotherm) in temperature was observed as the latent heat of fusion was released. This chart was used to determine the temperature of the sample just prior to freezing and that was taken as the supercooling point or temperature of crystallization.

The fresh weight of the each tissue segment was recorded just before supercooling measurements. After measuring the supercooling point, tissues were dried in 65 0C oven until the weight becomes constant and then the dry weight was recorded for each tissue sample.

For each group (0, 2, 4, 6, 10 and 14 day) SCP data and water content data from all the plants were pooled. Graphs of SCP vs duration of cold treatment and SCP vs relative water content were plotted from the average data from each group.

Preparation of soluble protein extracts from plant leaves

Total protein was isolated from approximately 0.4–0.5 g of the harvested leaf tissues according to Tsugita and Kamo (1999). These protein extracts were used for 2- D gel electrophoresis and Western blotting experiments (both 1- D and 2-D). A soluble protein fraction used for superoxide dismutase activity determinations were extracted according to the method of Mauro et al. (2005). Protein quantifications were performed using standard Bio-Rad Bradford assay (Bradford, 1976). 2- D Electrophoresis

109

Isoelectric focusing and second dimension of SDS-PAGE gel electrophoresis

10 mg (dry weight) protein samples were dissolved in rehydration/sample buffer (8M urea, 50 mM dithiothreitol (DTT), 4% 3-[(3-cholamidopropyl)dimethylammonio]-2- hydroxy-1-propanesulfonate (CHAPS), 0.2% carrier ampholytes, and 0.002% bromophenol blue (ProteomeWorksSystem.com – Biorad). Isoelectric focusing was performed on 11 cm IPG strips (pH 4-7) using a Protean IEF cell (Biorad). About 100 μg of protein was loaded onto each IPG strip. These samples required 80,000-100,000 volt hours for optimum focusing. After focusing IPG strips were immediately used to run gels or were stored at –80 0C until further use. Reduction and alkylation of cysteines were conducted by treating the strips in equilibration buffer 1 (6M urea, 2% SDS, 0.05M Tris- HCl, pH 8.8, 20% glycerol and 2% DTT) and equilibration buffer 11 (6M urea, 2% SDS, 0.05M Tris-HCl, pH 8.8, 20% glycerol and 2.5 % idoacetamide) respectively. The second dimension of SDS-PAGE was run by using Criterion precast gels (Tris-HCl, 8-16% resolving, 4% stacking), and the gels were stained with biosafe coomassie brilliant blue solution according to the instructions given by the manufacturer. Altogether, 30 samples were isolated from 5 plants under 6 different treatment conditions. Three gels were run from each protein sample extracted. Accordingly the control group consisted of 15 gels and 5 experimental groups also consisted of 15 gels each.

Imaging and image analysis

Images of 2-D gels (90) were collected using Alpha Imager and 85 of them were analyzed by using the PDQuest 2-D analysis software version 7.3.0 (Biorad). The number of protein spots were ascertained using the spot detection wizard tool in the PDQuest software. Matchsets were generated from replicate gels and replicate groups were prepared using the gels that belong to each group. Group consensus was achieved using the analysis set manager tool from the PDQuest 7.30 version of the software for each replicate group (representing 15 gels) before comparing the spots. In order to make gels quantitatively comparable, the images were normalized to account for differences in staining intensities, imaging and sample loading variable.

110 In-gel trypsin digestion

In-gel trypsin digestion was carried out according to the method of University of Cincinnati Mass Spectrometry Facility. Protein spots of interest were excised from gels and 100 μl of 25mM ammonium bicarbonate (NH4HCO3) 50% acetonitrile (ACN) solution was added to each spot and vortexed for 10 minutes. This step was repeated twice and gel pieces were then dried completely by using a speed vac. The dried gel pieces were then treated with 25 μl 10mM DTT in 25 mM NH4HCO3. They were incubated at 56 0C for one hour. The supernatant was removed, 25 μl of 55mM iodoacetamide was added onto each tube, and the mixture was incubated at room temperature for 45 minutes in the dark. Supernatants were again discarded. Gel pieces were washed with 100 µl NH4HCO3 and vortexed for 10 minutes. Supernatants were discarded and gel pieces were dehydrated with ~ 100 μl of 25 mM NH4HCO3 in 50% ACN, vortexed for 5 minutes. This step was repeated one time. The gel pieces were then dried completely using the speed vac. They were then treated with 25 µl of 12.5 ng/µl trypsin (Sigma) in 25mM NH4HCO3. Gel pieces were rehydrated at 4 0C for 10 minutes. The mixtures were incubated overnight at 37 0C. The supernatants from trypsin-digested mixtures were collected in separate tubes, and peptides were extracted twice by treating the gel pieces with 30 μl 50% acetonitrile, 5% formic acid and vortexing for 30 minutes and sonicating for 5 minutes. The peptide extracts were collected, vortexed and dried to a 10 μl volume using a speed vac. Samples were concentrated and cleaned up by using ZTC1 8S0 08 Zip Tips (Fisher Scientific).

MALDI-MS analyses and database searching

Some of the samples were analyzed using a Bruker Reflex III MALDI MS in the Department of Chemistry and Biochemistry at Miami University. The balance of samples was analyzed by the Mass Spectrometry Facility at the University of Cincinnati. Peptide masses obtained from the MALDI-TOF analysis were used to search Viripindiatae databases in NCBI, SWISS-PROT (http://us.expas.org), and Genebank (www.ncbi.nlm.nih.gov) and assigned using and MASCOT (http://www.matrixscience.com), ProFound (http://prowl.rockefeller.edu, and ProteinProspector (http://prospector.ucsf.edu). The parameters used for the searches were as follows: variable modifications were considered (cysteine as the S-carboamidomethyl derivative and methionine in oxidized form), up to two-missed cleavage sites were allowed, and the restriction was placed in pH range 4 – 7.

111 Western blotting experiments for the detection of dehydrins

Isolated proteins (as described above) from day 0 and day 14 separated via 2-D electrophoresis were transferred from gels on to nitrocellulose membranes (0.45µM) using a Bio-Rad semi dry Trans blot apparatus. Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween 20 for 18 hrs at 4 0 C. Immunoblots were obtained by incubating the membranes with antidehydrin antibodies (1:1000 dilution) 2 hr at room temperature. Reactive spots were detected with anti-rabbit IgG secondary antibody (1:3000 dilution) conjugated to alkaline phosphatase (Biorad) by a color development substrate. Color development reagent consisted of NBT (nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3'-indolyphosphate p-toluidine salt)). Spots that correspond in the original gels were subjected to MALDI analysis. Isolated leaf proteins from all the plants (as mentioned above) were subjected to 1-D PAGE using 8 to 16% Tris-HCl gels (Biorad) and immunoblots were prepared using the previously described method for 2-D gels.

1- D Native gel electrophoresis and detection of superoxide dismutase (SOD) activity

1-D Native electrophoresis was carried out using 8 to 16% linear Tris-HCl gels (Biorad). All operations were carried out at 4 0C. 100 µg of the soluble proteins extracted under native conditions (from plants with different cold treatment durations) were mixed with 20 μl of sample loading buffer (prepared without SDS and β- mercaptoethanol). 5 μl of commercial SOD (Sigma) was mixed with 5 μl of sample loading buffer was used as the control. Gels were run using the following voltage conditions; 80V for 15 minutes and 145 V for one hour. After running, all gels were first equilibrated in running buffer for 15 minutes. Secondly the treatment was different i.e. some gels were equilibrated in 0.1M KPO4, pH 7.8, and 1mM EDTA solution for 30 minutes. For the determination of different SOD isozymes, rest of the gels were incubated either with 2 mM KCN; 0.1M KPO4 (pH7.8) and 1 mM EDTA solution for 30 minutes or with 5 mM H2O2; 0.1M KPO4 pH7.8 and 1 mM EDTA solution for 30 minutes. All the gels were then incubated each in 80 ml of gel incubation solution (100 mM KPO4 pH 7.8; 1 mM riboflavin; 16 mg NBT, 200 µl TEMED) and dark incubated for 45 minutes. Gels were washed twice in washing solution (100mM KPO4, pH 7.5; 1 mM EDTA) and exposed to light. SOD activities were developed by a photoreaction and were observed as zone of clearing and images were taken using Alpha imager. The different SOD types were identified based on their sensitivity to KCN and H2O2 (Donahue et al., 1997).

112 Results and Discussion

Supercooling point measurements

The objective of part of the present study was to investigate the supercooling capacity of needle palm as one aspect of cold tolerance ( Lee, 1991) and its correlation with certain parameters such as water content, which might have an influential effect on it (Pugh, 1994). There was no significant correlation between the average SCP data of needle palm leaves with increasing duration at 4 0C. According to our data it is in the range of -18.16 0C and -19.3 0C (Table 1 & Figure 1). There as also no significant correlation between relative water content with increasing time at 4 0C (Table 2). According to our observations, needle palm does not increase its supercooling ability upon exposure to low non freezing temperatures. This palm has a relatively high supercooling ability under normal conditions (-19.3 0C +-1.24 (N=6)) when compared with other cold tolerant palms (e.g. Sabal palmetto - -130C Lokuge et al, unpublished data). This constitutive supercooling ability may play a key role in the overall cold tolerance of the palm. However, the biological significance of SCP in much debate (Knight et al., 1986; Turnock and Bracken 1989; Bale, 1993; Larsen et al., 1993; Leather et al., 1993).

The correlation between SCP and water content was investigated by a regression analysis (Figure 2) based on the data obtained for SCP and relative water content for individual plant specimens (Table 2). According the graph of SCP vs individual relative water content, there was no significant correlation between SCP and relative water content of needle palm leaves. Freezing is considered a stochastic event. Therefore at a given temperature the probability of freezing a supercooled liquid should increase along with the volume of the liquid (Franks, 1985). This principle should be valid for plants as well. However, there is little or no data available on supercooling points and their correlation with water content/mass in plants. Rather, much data is available in this regard with animals, especially insects. In certain arthropod species, SCP increases together with the water mass or water content (Cannon et al., 1985; Canon, 1986; Pugh, 1994; Gehrken, 1995). Ring and Danks (1994) also suggested that reduced water content should enhance supercooling capacity which usually occurs if there is a major change in the water content. However, there are also contradictory reports showing a negative relationship, i.e. SCP declined with increasing relative water content with ticks (Dautel and Knulle, 1996).

Proteomic approach to identify putative leaf proteins associated with needle palm cold tolerance

Figure 3 shows representative leaf protein gels obtained after staining with Coomassie brilliant blue stain. (3A= day 0 gel; 3B=day 2 gel; 3C=day 4 gel; 3D=day 8 gel; 3E=day 10 gel and 3F=day 14 gel). Some of the proteins that were shown to be upregulated were identified by MALDI-TOF-MS (Table 4). For the purpose of this study, upregulation was considered as an increase in the quantity of a given protein spot.

113 After 2 DE gel separation and coommassie brilliant blue staining, more than 300 spots were detected in each gel by digital image analysis. Although, nearly 60 protein spots showed an increase in quantity when compare the day 0 gels with other treatment conditions, for nearly 20 spots changes were significant and reproducible changes (Table 3). In terms of numbers, there was little change in the number of spots that were upregulated during the cold treatment on days 2, 4, 8, 10 and 14. Although the majority of these proteins were the same, each day new proteins also appeared to be upregulated (e.g. day 4 gel shows some proteins that are not present in day 2). In our study, we first choose to do MALDI-TOF-MS analysis to identify proteins that were upregulated in day- 2 gels.

Among those proteins that appeared/ were upregulated, did not change, or were downregulated, only a few could be identified (Table 4). The others were either predicted proteins with unknown functions or their identification was not possible due to unreliable results (not enough peptides matched or molecular weight and/ pI out of range). Rubisco activase, small heat shock proteins, oxygen evolving proteins, nudix hydrolase, thioredoxins, peroxidases and glutaraldehyde 3-phosphate dehydrogenase were some of the proteins that appeared to be upregulated in needle palm upon exposure to cold (40 C: 390F ) for 2 days.

Some of the proteins responding to cold stress are linked to antioxidative and/or detoxifying mechanisms. Thioredoxins and peroxidases are two main antioxidants in cells affected by the low temperature stress including mainly antioxidative or detoxifying reaction. Aldehyde dehydrogenases are the enzymes responsible for degradation of aldehydes arising from reactions with reactive oxygen species with lipids and proteins. Nudix forms a protein family whose function is to hydrolyse intracellular nucleotides and so regulate their levels and eliminate potentially toxic derivatives. It hydrolyse predominantly the diphosphate (pyrophosphate) linkage in a variety of nucleoside triphosphates, dinucleoside polyphosphates, nucleotide sugars and nucleotide cofactors having the general structure of a nucleoside diphosphate linked to another moiety. In addition we found some enzymes (oxygen-evolving complex) involved in photosynthesis. The enhancement of expression of these proteins implies that cold stress results in a foundational metabolic alteration.

In summary, the present initial proteomic investigation of needle palm leaf crude extractions reveals a complex cellular network affected by the low temperature stress. The network covers broad metabolic processes, antioxidative/detoxifying reactions and energy production, characteristic to plant cold stress response. However, it is likely that in the case of needle palms defense mechanism against reactive oxygen or toxic species is one of the main mechanisms the plant uses to protect against the stress upon exposure to low non freezing temperatures. So far we were able to identify some of these proteins. However, due to the lack of information in the current databases virtually only a handful of proteins were identified here. To uncover more insight into how this intricate network is operating within the cell, an entire list of stress-responsive protein identification is essential.

114 Immunoblot analysis for dehydrins in needle palm leaves

Dehydrins are family of proteins that are normally induced upon exposure to dehydration stress. In a number of woody plants, dehydrin proteins have been shown to accumulate in barks, xylems, buds, shoot apices and seedlings (Arora and Wisniewski, 1994; Welling et al., 1997) The purpose of this part of study was to identify dehydrins if present in gels from day 0 and day 14 treatments. Western blots obtained for dehydrin detection for leaf protein extracts on day 0 and day 14 are shown in Figures 4A and 4B. According to the Western blots, non-specific binding in the regions where RUBISCO is present can be seen on blots. However, the study proceeded based on the color intensity of reactive spots on the Western blots to detect the proteins on the original gels from the respective regions. Accordingly only one match for dehydrin was detected and others spots did not seem to be good match for dehydrins in existing databases. Various problems may contribute to such results. For example the spots we have identified may involve non-specific binding. Alternately, lack of identity may be due to the non- availability of sequence palm dehydrins in the database. The spot that was putatively identified as dehydrin did not vary in the intensity following cold treatment. This is somewhat in agreement with the Western blot we obtained with 1-D gels for proteins samples from all the treatments which also proved that dehydrin was present in all the samples (data not shown). The band intensity for dehydrins did not seem to vary significantly with the cold treatment according to 1-D Western blots. A new or better antidehydrin antibodies may help to solve non-specific binding problem.

Superoxide dismutase (SOD) activity

Gels obtained for SOD activity determinations for leaf protein extracts from day 0 to 14 are shown in Figures 5A, 5B and 5C. Based on the facts that Cu/Zn-SODs are inhibited by KCN and H2O2; Fe-SODs are inactivated by H2O2 but resistant to KCN and Mn-SODs are resistant to both inhibitors (Fridovich, 1986) we observed at least two different SODs in native PAGE gel (Figure 5A). Activity staining for different isoforms confirmed that one of them could be Mn-SODs (resistant to both KCN and H2O2 activity) and the other one is Cu/Zn isoform (sensitive to KCN and H2O2 activities – Figures 5B and 5C). None of the bands observed seem to be strikingly affected by cold treatment. The quantity of the protein bands do not seem to increase or decrease upon exposure to low non freezing temperatures. This is somewhat different from the results obtained for most of the other plant species studied to date for SOD activity under cold stress. Payton et al. (2001) reported that during moderate chilling at high light intensity cotton photosynthesis is protected by increasing chloroplastic antioxidant activity. However, Prasad et al. (1994) reported that activity of all SODs (4 isoforms) is unaffected by acclimation and /or chilling in mesocotyls of dark grown maize seedlings. Overall, our data suggests that SOD activity in needle palm leaves did not increase upon exposure to low non-freezing temperatures but rather was constitutively expressed. It is possible that the constitutive activity of cold regulatory genes in R. hystrix results in constant upregulation of SODs in this case.

115 Previous research indicate that even when various SOD isoforms are separated by native PAGE, a quantitative determination of each SOD activity may be impeded as a result of co-migration of two or more SODs (Cannon and Scandalios, 1989; Van Camp et al., 1996, Donahue et al., 1997; Kliebenstein et al., 1998). In Figure 5B and Figure 5C we observed two SODs only in the protein sample from day 2 plants. In other samples we did not observe that band in the gels when treated with KCN and H2O2. This behavior may not be due to the fact that it is inhibited by KCN or H2O2 but rather that the two isoforms have co-migrated. In our study, high concentrations of glycerol seem to be essential for the stabilization of minor SOD activity bands since we could not observe the Cu/Zn isoform when glycerol was not used to stabilize the protein sample (unpublished data).

Acknowledgements

This research has been made possible through grants from the Ohio Plant Biotechnology Consortium, Miami University, Department of Botany Academic Challenge program and Sigma Xi Grants-in-Aid of research program. Special thanks are due to Dr. John Hawes from the Department of Chemistry and Biochemistry at the Miami University for his kind help with proteomics work. The ungrudging assistance from Mike Elnitsky from the Ecophysiological and Cryobiology laboratory at the Department of Zoology, Miami University with supercooling measurements is appreciated. We would also like to gratefully acknowledge Dr. T. J. Close (UC-Riverside) for his generous gift of antidehydrin antibodies.

116 References

Anderson MD, Prasad TK, Stewart CR (1995) Changes in isozyme profiles of catalase, peroxidase, and glutathione reductase during acclimation to chilling in mesocotyls of maize seedlings. Plant Physiology 109:1247-1257 Arora R, Wisniewski M (1994) Cold acclimation in generally related (sibling) deciduous and evergreen peach (Prunus persica L. Batsch): a 60-kilodalton bark protein in cold acclimated tissues of peach is heat-stable and related to the dehydrin family of proteins. Plant Physiology 105:95-101 Bale JS (1993) Classes of insect cold hardiness. Func Ecol 7:751-753 Bowler C, Van Montagu M, Inze D (1992) Superoxide dismutase and stress tolerance. Ann Rev Plant Physiol Plant Mol Biol 43:83-116 Bowler C, Van Montagu M, Inze D (1994) Superoxide dismutases in plants. Critical Reviews in Plant Science 13: 199-218 Bradford MM (1976) A rapid and sensitive method for the qualification of microgram quantities of protein utilizing the principle of protein dye binding.Anal. Biochem. 72:248 –254 Cannon RE, Scandalios JG (1989) Two cDNA encode two nearly identical Cu/Zn superoxide dismutase protein in maize. Mol General Genet 219:1-8 Cannon RJC, Block W, Collett GD (1985) Loss of supercooling ability in Cryptopygus antarcticus (Clollembola: Isotomidae) associated with water uptake. Cryo Lett 6: 73-80 Cannon RJC (1986) Effects of contrasting relative humidities on the cold tolerance of an antarctic mite. J Ins Physiol 32:523-534 Dautel H, Knulle W (1996) The supercooling ability of ticks (Acari, Ixodoidea). J Comp Physiol B 166:517-524 Donahue JL, Okpodu CM, Cramer CL, Grabau EA, Alscher RG (1997) Responses of antioxidants to paraquat in pea leaves. Plant Physiology 113:249-257 Guy CL (1980) Cold acclimation and freezing tolerance: role of protein metabolism. Annual review in Plant Physiology and Plant Molecular Biology 41:187-223 Foyer CH, Descourvieres P, Kunert KJ (1994) Protection against oxygen radicals: an important defense mechanism studied in transgenic plants. Plant Cell Environ 17: 507-523 Foyer Ch, Lopez-Delgado H, Dat JF, Scott IM (1997) Hydrogen peroxide-and glutathione-associated mechanisms of acclimatory stress tolerance and signaling. Physiol Plant 100:241-254 Francko DA (2003) Palms Won't Grow Here and Other Myths: Warm-Climate Plants for Cooler Regions. Portland, OR and Cambridge, Timber Press. 307pp Franks F (1985) Biophysics and biochemistry at low temperatures. Cambridge, Cambridge University Press Fridovich I (1986) Superoxide dismutases. Advances in enzymology and related areas of molecular biology. New York, Wiley 5861-5897 Gehrken U (1995) Correlative influence of gut appearance, water content and thermal hysteresis on whole body supercooling point of adult bark beetles, Ips acuminatus. Comp Biochem Physiol 112A: 207-214

117 George MF, Burke MJ, Pellett HM, Johnson AG (1974) Low temperature exotherms and woody plant distribution. HortScience 9:519-522 Gusta L, Tyler NJ, Chen TH-H (1983) Deep undercooling in woody taxa growing north of the -40 0C isotherm. Plant Physiology 72:122-128 Guy CL (1990) Cold acclimation and freezing stress tolerance: role of protein metabolism. Annual review in Plant Physiology and Plant Molecular Biology 41: 187-223 Ishikawa M (1984) Deep supercooling in most tissues of wintering Sasa senanensis and its mechanism in leaf blade tissues. Plant Physiology 75:196-202 Ishikawa M, Sakai A (1981) Freezing avoidance mechanisms by supercooling in some Rhododendron flower buds with reference to water relations. Plant Cell Physiology 22:953-967 Kliebenstein DJ, Monde RA, Last RL (1998) Superoxidedismutase in Arabidopsis: an ecletic enzyme with disparate regulation and protein localization. Plant Physiology 118:637-650 Knight JD, Bale JS, Franks S, Mathias SF, Baust JG (1986) Insect cold hardiness: supercooling points and pre-freeze mortality. Cryo Lett 7:194-203 Kruft V, Eubel H, Jansch L, Werhahn W, Braun HP (2001) Proteomic approach to identify novel mitochondrial proteins in Arabidopsis. Plant Physiology 127:1694- 1710 Larsen KJ, Lee RE Jr, Nault LR (1993) Influence of developmental conditions on cold- hardiness of adult. Entomol Exp Appl 67:99-108 Larson (1988). The antioxidants of higher plants. Phytochemistry 27: 969-978. Leather SR, Walters KFA, Bale JS (1993) The ecology of insect overwintering. Cambridge, Cambridge University Press Lee RE Jr (1991) Principles of insect low temperature tolerance. In: Insects at low temperatures. D. D. Lee RE Jr. (Eds.) New York, Chapman & Hall: 17-46 Levitt J (1980) Response of plants to environmental stresses. Chilling, freezing and high temperature stresses. New York, Academic Press 497 Malan C, Greyling M, Gressel J (1990) Correlation between Cu/Zn superoxide dismutase and glutathione reductase, and environmental and xenobiotic stress tolerance in maize inbreds. Plant Sci 69:157-166 Malone SR, Ashworth EN (1991) Freezing stress response in woody tissues observed using low-temperature scanning electron microscopy and freeze substitution techniques. Plant Physiology 95:871-881 Omran RJ (1980) Peroxide levels and the activities of catalase, peroxidase, and indoleacetic acid oxidase during and after chilling cucumber seedlings. Plant Physiology 65:407-408 Payton P, Webb R, Kornyeyev D, Allen R, Holaday S (2001) Protecting cotton photosynthesis during moderate chilling at high light intensity by increasing chloroplastic antioxidant enzyme activity. Journal of Experimental Botany 52: 2345-2354 Peltier JB, Emanuelsson O, Kalume DE (2002) Central function of the lumenal and peripheral thylakoid proteome of Arabidopsis determined by experimentation and genome-wide prediction. Plant Cell 14: 211-236 Pomeroy MK, Siminovitch D (1971) Seasonal cytological changes in secondary phloem

118 parenchyma cells in Robinia pseudoacacia in relation to cold hardiness. Canadian Journal of Botany 49: 789-795 Prasad TK, Anderson MD, Martin BA, Stewart CR (1994) Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell 6:65-74 Pomeroy MK, Siminovitch D (1971) Seasonal cytological changes in secondary phloem parenchyma cells in Robinia pseudoacacia in relation to cold hardiness. Candian Journal of Botany 49:789-795 Prime TA, Sherrier DJ, Mahon P, Packman LC, Dupree P (2000) A proteomic analysis of organelles from Arabidopsis thaliana. Electrophoresis 21:3488-3499 Pugh PJA (1994) Supercooling points and water content in Acari. Acta Ecol 15:71-77 Rasmussen DH, MacKenzie AP (1972) Effect of solute on ice-solution interfacial free energy; calculation from measured homogeneous nucleation temperature. In: Water structure at the water polymer interface. Jellnek. HHG. (Ed.)New York, Plenum Publishing Corporation: 126-145 Renaut J, Lutts S, Hoffmann L, Hausman JF (2004) Responses of poplar to chilling temperatures: Proteomic and physiological aspects. Plant Biology 6: 81-90 Ring RA, Danks HV (1994) Desiccation and cryoprotection: over lapping adaptations. Cryo Lett 15:181-190 Rossignol M (2001) Analysis of plant proteome. Curr. Opin. Biotechnol. 12:131-141. Sakai A, Larcher W (1987) Frost survival of plants. Responses and adaptation to freezing stress. Ecological Studies. Vol. 62 Berlin, Springer Verlag 321pp Scandalios JC (1990) Response of plant antioxidant defense genes to environmental stress. Adv Genet 28:1-41 Thomashow MF (1998) Role of cold-responsive genes in plant freezing tolerance. Plant Physiology 118:1-8 Thomashow MF (2001) So what's new in the field of plant cold acclimation? Lots! Plant Physiology 125:89-93 Tsugita A, Masaharu K (1999) 2-D electrophoresis of plant proteins. Methods Mol biol. 112:95-97 Turnock WJ, Bracken CK (1989) Effects of low non-freezing temperatures on pupae of two species of diapausing, freeze-intolerant insects. Cryo Lett 10:189-196 Van Camp W, Capiau k, Van Montagu M, Inze D, Slooten L (1996) Enhancement of oxidative stress tolerance in transgenic tobacco plants overproducing Fe- superoxide dismutase in chloroplast. Plant Physiology 112:1703-1714 Welling A, Kaikuranta P, Rinne P (1997) Photoperiodic induction of dormancy and freezing tolerance in Betula pubescens. Involvement of ABA and dehydrins. Physiologia Plantarum 100:496-505 Wise RR, Naylor AW (1987) Chilling-enhanced photooxidation. The peroxidase destruction of lipids during chilling injury to photosynthesis and ultrastructure. Plant Physiology 83:272-277 Wisniewski M, Ashworth EN (1986) A comparison of seasonal ultrastructural changes in stem tissues of peach (Prunus persica) that exhibit contrasting mechanism of cold hardiness. Botanical Gazette 147:407-417

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Yoshida S, Uemura M (1990) Responses of the plasma membrane during cold acclimation in mulberry bark cells (Morus bombycis Koidz. cv Goroji). In: The plant plasma membrane.Larsson C, Moller IM (Eds.). Berlin, Springer-Verlag: 293-319

120 0 02481014

-5

-10

SCP (C) SCP -15

-20

-25 Duration at 4 C in days

Figure 1. Average supercooling point (SCP) in needle palm leaves vs days at 4 0C.

121

0 0 1020304050607080

-5

-10 SCP (C)

-15

y = -0.0074x - 18.481 -20

-25 Relative water content (%)

Figure 2. Supercooling point (SCP) vs relative water content in needle palm leaves under 4 0 C cold treatment for 2 weeks.

122 pH 4 7

Figure 3A. 2DE gel analyses of proteins extracts from needle palm leaves before exposure to cold. Proteins were visualized using biosafe coomassie brilliant blue stain Some of the protein spots that have low quantity when compare with day-2 gel are shown here. They were identified by MALDI-TOF-MS. pH 4 7

Figure 3B. 2DE gel analyses of proteins extracts from needle palm leaves after treatment at 40C for 2 days. Proteins were visualized using biosafe coomassie brilliant blue stain. Some of the protein spots that have increased quantity when compare with day-0 gel are shown here. They were identified by MALDI-TOF-MS.

123 pH 4 7

Figure 3C. 2DE gel analyses of proteins extracts from needle palm leaves after treatment at 40C for 4 days. Proteins were visualized using biosafe coomassie brilliant blue stain. pH 4 7

Figure 3D. 2DE gel analyses of proteins extracts from needle palm leaves after treatment at 40C for 8 days. Proteins were visualized using biosafe coomassie brilliant blue stain.

124 pH 4 7

Figure 3E. 2DE gel analyses of proteins extracts from needle palm leaves after treatment at 40C for 10 days. Proteins were visualized using biosafe coomassie brilliant blue stain. pH 4 7

Figure 3F. 2DE gel analyses of proteins extracts from needle palm leaves after treatment at 40C for 14 days. Proteins were visualized using biosafe coomassie brilliant blue stain.

125

1 1

1 2

Figure 4A; Western blot for dehydrin detection from a day 0 gel. 1. Corresponding spot on the original gel gave a match to coffee dehydrins based on MALDI data. 2. Color development due to non specific binding of antibodies to RUBISCO.

1 2

Figure 4B. Western blot for dehydrin detection from day 14 gel. Color development due to non specific binding of RUBISCO.

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C 0 2 4 8 10 14

Figure 5A. Identification of SODs in needle palm leaf extracts on day 0, 2, 4, 8, 10, 14 under cold treatment. C- Bacterial SOD as a control. Gel shows the activity staining of SODs after native PAGE. At least 2 types of SODs can be detected on this gel as indicated by arrows.

C 0 2 4 8 10 14 C

Figure 5B. Activity staining of SODs in needle palm leaf extracts from day 0, 2,4,8,10,14 under cold treatment in the presence of KCN. C- Bacterial SOD as a control. Lower molecular weight SOD seems to be sensitive to KCN treatment indicating that it can be Cu/Zn isoform. SOD (arrows) that are insensitive to KCN and can be ascribed Mn-SODs.

127 C 0 2 4 8 10 14 C

Figure 5C. Activity staining of SODs in needle palm leaf extracts from day 0, 2, 4, 8, 10, 14 under cold treatment in the presence of H2O2. C- Bacterial SOD as a control. Lower molecular weight SOD seems to be sensitive to treatment indicating that it can be Cu/Zn isoform. SOD (arrows) are insensitive to H2O2 and can be ascribed Mn-SODs.

128 Table 1. Supercooling point (SCP) of the leaf samples collected from needle palms from

day 0 to day 14 under cold treatment at 4 0 C.

Plant Day 0 2 4 8 10 14 NP 1 CD -18.8 CD -19.2 -17.1 -18.9 NP4 CD -20.4 CD -19.4 CD -21.3 NP7 CD CD -20.0 -18.6 CD -19.0 NP8 CD -19.3 -18.8 -18.4 -19.0 CD NP6 -18.9 -20.1 -18.6 CD -17.9 CD NP9 -17.1 -18.1 CD -18.1 CD 1NP 2 -19.6 -16.6 -18.9 -20.0 -17.3 CD 2NP3 -19.6 CD CD -17.9 CD -17.8 3NP5 CD -19.5 -17.1 -19.9 CD -19.1 4NP7 -20.8 -18.0 -19.0 CD CD CD 5NP10 -19.8 CD CD CD -18.9 CD 6NP CD CD CD -20.7 -18.8 -19.1 SCP -19.3+- -18.96+- -18.64+- -19.26+- -18.157+- -19.2+- Average 1.24(n=6) 1.31(n=7) 0.888(n=7) 0.931(n=8) 0.774(n=7) 1.141(n=6) NP – Needle Palm; CD- Could Not Determined

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Table 2. Individual relative water contents (%) and supercooloing points of the leaf samples collected from needle palms from day 0 to day 14 under cold treatment at 4 0 C.

Plant Day 0 2 4 8 10 14 NP 1 CA NA 53.44 NA 74.32 56.25 34.09 -18.8 -19.2 -17.1 -18.9 NP4CA NA 51.85 NA 50.79 NA 55.4 -20.4 -19.4 -21.3 NP7CA NA NA 51.56 53.03 NA 42.85 -20.0 -18.6 -19.0 NP8CA NA 55.38 55.38 57.14 67.08 NA -19.3 -18.8 -18.4 -19.0 NP6CA 51.28 50 33.92 NA 40.98 NA -18.9 -20.1 -18.6 -17.9 NP9CA 50.78 NA 20.93 NA 54.41 NA -17.1 -18.1 -18.1 1NP 2 CA 54.71 55.17 55.17 56.45 53.12 NA -19.6 -16.6 -18.9 -20.0 -17.3 2NP3CA 55.17 NA NA 56.25 NA 55.93 -19.6 -17.9 -17.8 3NP5CA NA 49.12 52.54 48.43 NA 50 -19.5 -17.1 -19.9 -19.1 4NP7CA NA 51.72 NA NA NA NA -18.0 5NP10CA 52.54 NA NA NA 48.38 NA -19.8 -18.9 6NPCA NA NA NA 48.43 55.35 48.52 -20.7 -18.8 -19.1

NP – Needle Palm; NA – Not applicable due to the lack of supercooling point data.

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Table 3. Spot number comparison between gels

Day # of spots upregulated # of spots down regulated ( 2-fold) (2-fold) From To 0 2 21 16 0 4 20 24 0 8 22 22 0 10 20 25 0 14 23 15

131 Table 4. Spot identification by MALDI-TOF-MS analysis and database search

Spot Experimental Protein ID Protein source Calculate Database SC # of pI/mass pI/mass(kDa) & Search peptides (kDa) engine matched

1 5-6/37-50 1- RCA Arabidopsis 5.87/51.95 NCBInr- 21 14 (Rubisco thaliana Mascot activase)

2 5-6/15-20 18.1 kDa class I Pisum 5.83/18.07 NCBInr- 35 7 heat shock sativum Mascot protein (HSP 18.1)

3 5-6/25-37 Oxygen- unreadable 5.5/26 NCBInr- 34 4 evolving MS fit complex protein 1 4 5-6/20-22 Hypothetical Arabidopsis 5.6/25.77 NCBInr- 11 3 protein thaliana Profound 5 5-6/20-25 Nudix Arabidopsis 5.35/20.36 NCBInr- 30 5 hydrolase 16, thaliana Mascot mitochondrial (Mouse-ear precursor cress)

6 5-6/10-15 1-Thioredoxin Ricinus 5.57/13.4 Swissprot 15 2 H-type (TRX- communis - Mascot H) - (Castor bean)

7 5-6 /15-20 1-Probable Mesembryant 6.59/18.90 Swissprot- 21 4 phospholipid hemum Mascot hydroperoxide crystallinum glutathione (Common ice peroxidase plant)

(PHGPx) -

8 4-5/25-37 1-Heat shock Arabidopsis 4.66/24.38 NCBInr- 42 8 protein 81-2 thaliana Mascot

9 6-7 /25-37 1- Zea mays 6.41/36.5 Swissprot- 23 5 Glyceraldehyd (Maize) Mascot e-3-phosphate dehydrogenase , cytosolic 2

10 5-6/50-75 ATP synthase Triticum 5.56/59.2 Swissprot- 26 12 beta subunit aestivum Mascot

Proteins from needle palm leaves were extracted and separated by 2DE within pH range 4-7. The proteins spots of interest were excised from gels, digested and peptides generated were analyzed by MALDI-TOF-MS. Search in databases resulted in the proposed candidate proteins listed in the table. For protein search, a window of mass tolerance 0.5 Da was used. SC; Sequence coverage

133 Chapter 6

Conclusions

Palms are a familiar and characteristic feature of tropical landscapes. Although the great majority of palms are tropical to subtropical in range distribution, some palm species survive temperatures below (6.70C) 200 F and few survive temperatures below -17.70C (00 F.). Needle palm (Rhapidophyllum hystrix), cabbage palm (Sabal palmetto) and Chinese windmill palm (Trachycarpus fortunei) are very resistant to cold under USDA Plant Hardiness Zone 6 conditions.

The most economically important members of the family Arecaceae are propagated by seeds. Seedlings can exhibit great variability in selected characters, which cannot be evaluated until the palm is several years old. Thus the advantages to be gained by eliminating this variability are immense, if cloning of selected, high performance palms could be achieved via tissue culture techniques. Palm tissues in general have shown to be highly recalcitrant to in vitro conditions. Nevertheless some palm species have been regenerated successfully via organogenesis as well as via somatic embryogenesis pathways. Therefore I hypothesized that cold-hardy palms can be regenerated and somatic embryogenesis can be induced from cold hardy palm tissues.

The first part of this study was undertaken to develop a tissue culture system for the clonal propagation of cold-hardy palms with desired characters and with the ultimate goal of producing a system for genetic transformation studies. Under the present investigation (Chapter 2), Trachcycarpus fortunei was regenerated from shoot apical meristem tissues via indirect organogenesis, giving rise to viable plants that fully acclimated to greenhouse conditions. Introduction of cytokinin, benzylaminopurine (BAP) at 5 µM to the media containing 2 µM 2, 4-D with tissues induced organogenic callus formation and subsequent shoot induction. That was the optimal BAP concentration which resulted in plant regeneration in T. fortunei. SEM data suggest that acclimatization to greenhouse conditions is critical for better plant survival rate. I used two different explants i.e. zygotic embryos and shoot apical meristem and but only shoot apical meristem tissue gave rise to plantlets. However, there was an induction of non-embryogenic callus from mature zygotic embryos of T. fortunei. Chapter 3 describes our efforts to induce somatic embryogenesis in another cold- hardy palm Sabal palmetto. From that study it was revealed that 1.5 µM dicamba was optimal for the induction of somatic embryogenesis in S. palmetto zygotic embryos. Somatic embryo formation occurs within a relatively short period of time (6 –7 weeks). This suggests that a brief time period is required for somatic embryo formation with less callus proliferation increase in degree and it may reduce the chances of somoclonal variation..

The second part of this study (Chapter 4) was aimed at developing a genetic transformation system for cold-hardy palms. S. palmetto was selected for this study because it is already in widespread use throughout USDA Zone 8, and previous data (Francko, 2003) suggest that with minor increase in degree of cold tolerance this palm could be grown in even colder areas. From this study I first report the successful genetic transformation of S. palmetto zygotic embryos were with the marker genes gfp and gus. The two most common plant

134 transformation methods, biolistic and Agrobacterium-mediated transformation, were used to introduce foreign genes into these tissues. The results are somewhat different from the results reported for oil palm transformation (Abdullah et al., 2005). According to this study Agrobacterium -mediated transformation gave more promising results when compared with the biolistic method. Out of the three gus vectors used for the Agrobacterium-mediated transformation with pCAMBIA 1305.1 and 1305.2 transient expression was much quicker (GUS expression could be observed within 3hrs when using GUS histochemical assay) and stronger than pCAMBIA 1301. The transformation efficiency could be greatly increased (100%) when the surfactant pluronic acid was used. Transformation efficiency could also be enhanced with tissue sonication as well as with the introduction of acetosyringone into the medium. With biolistic transformation GUS transient expression was increased when tissues were incubated in media containing the osmotica mannitol and sorbitol each at 0.2M concentration 4 hrs before and 19 hrs after bombardment. With the advantage of non- destructive visualization method of GFP, I was able to observe the expression of GFP in S. palmetto zygotic embryos for a period of two weeks without any sign of fading characteristic green florescence.

The final part of this dissertation (Chapter 5) was aimed at studying cold tolerance mechanisms using the most cold-hardy palm, the needle palm, as a model system. Plants exhibit two strategies for surviving extremely cold weather: freeze avoidance and freeze tolerance. Both strategies involved supercooling mechanisms and other adaptations that have not been characterized in palms. Our data suggest that needle palm supercooling capacity is already pronounced even in warm-incubated foliage and the palm leaves supercools to a very high degree - about -200 C under normal conditions. To further investigate the molecular mechanisms underlying cold tolerance in needle palm, a proteomic approach was used to examine initial changes of the leaf proteome upon cold acclimation. Proteomics has become one of the fastest growing areas of research because it involves the probing of differential expression of proteins in response to various external stimuli. It is expected to yield more direct understanding of function and regulation than analysis of genes. Results from Chapter 5 indicate that a number of proteins were up-regulated in the leaf tissue and some were down-regulated upon exposure to low non freezing temperature. However, there was little increase in the number of protein upregulated with time at 40 C. According to MALDI-TOF- MS analysis I propose 10 proteins as candidate proteins. Among them are mainly the enzymes involve in plant defense mechanism such as thioredoxin and peroxidases. However, identification of other proteins proved difficult because of the non- availability of a genome sequence database for needle palm. Nevertheless, 2- dimensional gel electrophoresis suggested that significant changes in protein products occur in needle palm leaves when challenged with non-lethal cold.

Needle palm leaves have supercooling ability to a very high degree -200 C (-40 F) and when compare with the supercooling ability other cold-hardy palms in cabbage palm leaves supercooling point is -130 C ( 90F). Needle palm does not seem to increase its supercooling ability upon exposure to low non freezing temperatures. However, according to the analysis of leaf protein profile from plants under cold treatment there was obviously a change in the some of the proteins in leaf protein. These proteins specially the ones that have shown to be upregulated may contribute to the cold tolerance ability upon exposure to these low non

135 freezing temperatures. However, it less likely that these proteins have a direct effect on the supercooling ability on the plant leaves. According to the MALDI data I obtained so far some of these proteins seem to be the ones involve in defense mechanisms (thioredoxins, peroxidases, nudix hydrolase), while in other plants proteins associated with other activities are also induced. Accordingly needle palm seem to prevent the cell damage due to oxidative stress through a repair mechanism involving ROS species. This ability may prove to be a major aspect of cold tolerance adaptation in needle palm. In addition, our data suggests that superoxide dismuatase (SOD) enzyme activity in needle palm leaves are not strikingly affected upon exposure to low non-freezing temperatures but rather was constitutively expressed. It is possible that the constitutive activity of cold regulatory genes in R. hystrix results in constant upregulation of SODs.

Exposure to low temperatures may result in the increased generation of ROS in plants (Prasad et al., 1994). The ROS may attack plant cellular components or may deliver signals for detecting the change environment or both (Fridovich 1991). Previous data on different plant species suggest that increased cold tolerance can be accompanied by increased expression of specific genes encoding antioxidant enzyme. However the data from this study reveals that SODs maintain relatively constant expression and it may be enough to protect the cells for oxidative damage during cold acclimation along with the higher expression of some of the antioxidant enzymes involve in ROS scavenging pathway.

It is important to note that although the resolution of 2 DE seems impressive, it has limitations. They are mainly due to the large number of proteins present in the sample requiring very high resolution power for separating all the peptides, vast chemical diversity of proteins and different expression of proteins in cells or tissues. There are some technical problems that need to be solved (Corthals et al., 2000; Gygi et al., 2000) with 2DE. It is a labor- and time-consuming process, and costly limiting high throughout analysis of protein expression. In addition, complete protein profiles and quantification are not possible due to the limited loading capacity and incomplete staining methods. The one biggest drawback to 2D gel-based approaches is relatively low detection limits. Coomassie staining is simple and fast but lacks sensitivity; collioidal Coomassie is the most sensitive (100ng of proteins can be detected). Silver staining is much more sensitive (1ng of protein could be detected but the staining procedure is much more elaborate. Besides sensitivity, and the linear dynamic range is important for quantification. Silver staining does not have a large linear dynamic range, and has a large protein to protein variability and it makes quantification more difficult. Fluorescent dyes (SyproRuby and Sypro Orange) have been developed with the aim to provide the sensitivity of the silver stain with a large linear dynamic range and the simplicity of use of the Coomassie stain. These days can be used to stain proteins after separation. A second major drawback to our proteomic approach was that only a part of the needle palm proteome (soluble proteins that have a pI of 4-7) was screened in our studies. However, it included the majority of proteins in the leaf proteome. These conditions can be improved using membrane protein solubilization kits, IPG strips with narrower pH ranges and larger SDS-PAGE gels. However, it is well documented that even under perfect conditions, 2D gel based approaches screens only a part of the leaf proteome.

136 2D-DIGE two-dimensional difference gel electrophoresis which uses multiplexed gels with fluorescent labeling is a recent improvement in the gel based techniques. It has the unique advantage of detecting low abundance proteins without saturating the high abundance ones while giving quantitative results. Another method referred as multidimensional protein identification technology (MudPIT) or liquid chromatography (LC)-MS/MS couples capillary, high performance liquid chromatography (HPLC) to MS/MS. It allows automated analyses of peptide mixtures that are generated from complex protein samples (Appella et al., 1995; Washburn et al., 2001; Wolters et al., 2001). That involves protein analysis directly by MS without gel separation and it overcomes 2 DE-associated limitations. More recently, quantitative proteomics become feasible using isotope-coded affinity tag (ICAT), in the LC- MS/MS system (Han et al., 2001). Affinity tag also allows the quantification and discrimination on the mass spectrometer. CAT-based LC-MS offers the ability of selecting certain peptides by derivatization of specific amino acids (typically cysteine residues). Ficarro et al., (2005) report a similar method for enriching phosphoproteins by affinity columns of affinity tag-derived phosphoproteins.

Several methods have been developed toward large-scale studies of protein functions. Protein array technology has emerged, after the successful applications of DNA chip methods (Mitchell, 2002). Protein microarray consists of antibodies, proteins, protein fragments, peptides, aptamers or carbohydrate elements that are coated or immobilized in a grid-like pattern on small surfaces. The arrayed molecules are then used to screen and assess patterns of interaction with samples containing distinct proteins or classes of proteins. Due to different biochemical properties of proteins, protein microarray requires high-throughput methods for expression and purification. Nevertheless, there are some reports of comprehensive protein microarray screening (Houseman and Mrksich 2002; Schweitzer et al., 2002). Analysis of protein-protein interactions and the identification of multiprotein complexes components are necessary to understand most cellular processes. Protein interaction studies are carried out by using two-hybrid systems, (Fields and Song 1989), protein chips and to the large-scale approach of tandem-affinity purification (TAP)-MS. The yeast two-hybrid system is a powerful technique for identifying multiprotein complexes. Using genetically engineered yeast scientists can identify complexes when specific pairs of interacting proteins activate expression of a reporter gene. The two-hybrid system has been expanded to use microarrays of cloned yeast genes. These large-scale yeast two-hybrid assays can provide information on thousands of protein-protein interactions. TAP-MS has been used for the analysis of the yeast proteome, allowing the purification of more than 589 multiprotein assemblies (Gavin et al., 2002). There is a possibility that it can also be applied to higher eukaryotes, avoiding the problem of the competition from endogenous proteins (Forler, 2003). A more recent general method involves the use of florescent resonance energy transfer (FRET) between the florescent tags on interacting proteins. This uses green, cyan and yellow florescent proteins (Phizicky et al., 2003). The great advantage of this approach over other approaches is to be able to apply to in vivo analysis by microscopy. Finally although these gel-free techniques offer many advantages over gel-based techniques, they are not commonly used in plant proteome analysis work. These gel free techniques require the sequence of the entire genome to be able to do the complete analysis of the proteome of the organism of interest.

137

References

Abdulla R, Zainal A, Heng WY, Li LC, Beng, YC, Phing LM, Sirajuddin SA, Ping WYS, Joseph JL, Jusoh SA, Muad MR, Huey YL (2005) Immature embryo; A useful tool for oil palm ( Elaeis guineensis Jacq.) genetic transformation studies. Electronic Journal of Biotechnology 8: 25-34 Appella A, Padlan EA, Hunt DF (1995) Analysis of the structure of naturally processed peptides bound by class 1 and class 11 major histoaompatibility complex molecules. EXS 73: 105-119 Corthals GL, Gygi SP, Aebersold R, Patterson SD (2000) Identification of proteins by mass spectrometry. In: Proteome research: two dimensional gel electrophoresis and identification methods. Rabilloud T (Ed.) New York, Springer, Berlin Heidelberg: 197-231 Fields S, Song O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340: 235-246 Forler D, Kocher T, Rode M, Gentzel M, Izaurralde E, Wilm M (2003) An efficient protein complex purification method for functional proteomics in higher eukaryotes Nat. biotechnol.1:89-92 Ficarro SB, Salomon AR, Brill LM, Mason DE, Stettler-Gill M, Brock A, Peters EC (2005) Automated immobilized metal affinity chromatography/nano liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites. Rapid Commun Mass Spectrom 19: 57-71 Fridovich I (1991) Molecular oxygen: friend and foe. Active Oxygen/Oxidative Stress and Plant Metabolism. S. K. Pell EJ. Rockville, American Society of Plant Physiologists: 1-5 Gygi SP, Rist B, Aebersold R (2000) Mass spectrometry and proteomics. Curr Opin Biotech 11: 396-401 Han DK, Eng J, Zhou H, Aebersold R (2001) Quantitative profiling of differentiation- induced microsomal proteins using isotope-coded affinity tags ad mass spectrometry. Nat Biotechnol 19: 946-951 Houseman BT, Mrksich M (2002) Towards quantitative assays with peptide chips: a surface engineering approach. Trends Biotechnol. 20: 279-281 Mitchell P (2002) A perspective on protein microarrays. Nat Biotechnol 20: 225-229. Phizicky E, Bastiaens PIH, Zhu H, Synder M, Fields S (2003) Protein analysis on proteomic scale. Nature 422: 208-215 Prasad TK, Anderson MD, Martin BA, Stewart CR (1994) Evidence for chilling-induced oxidative stress in maize seedlings and regulatory role for hydrogen peroxide. Plant Cell 6: 65-74 Schweitzer B, Roberts S, Grimwade B, Shao W, Wang M, Fu Q, Shu Q, Laroche I, Zhou Z, Tchernev VT, Christiansen J, Velleca M, Kingsmore SF (2002) Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat Biotechnol 20: 359-365 Washburn MP, Wolters D, Yates JR (2001) Large scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19: 242-247 Wolters DA, Washburn MP, Yates JR (2001) An automated mutidimensional protein identification technology for shotgun proteomics. Anal. Chem 73: 5683-5690

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