AVOCADO SUNBLOTCH VIROID (ASBVd) IN CELL AND
TISSUE CULTURES OF AVOCADO ( Persea americana Mill.)
By
ISIDRO ELIAS SUAREZ PADRON
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2003 Copyright 2003
by
Isidro E. Suarez Padron To my wife Sahara,
for her love and comprehensive patience during these difficult years of sacrifice,
and to my sons, Sebastian and Elias David for my absence in the most
precious moments.
A mi esposa Sahara
por su amor y comprensiva paciencia durante estos dificiles anos de sacrificio,
y a mis hijos Sebastian y Elias David por mi ausencia en los
momentos mas hermosos de sus vidas. AKNOWLEDGMENTS
I wish to express my deepest gratitude to Dr. Richard Litz, my major professor, for his unlimited support, advice, help, patience and time, and particularly for his direction in the completion of this dissertation. His encouragement, guidance and permanent stimulation for better work will always be remembered.
I want to extend my gratitude for their help, guidance, and stimulating discussion
to the members of my graduate committee, Drs. Raymond J. Schnell, Dennis Gray, Paul
Lyrene, and Nigel Harrison.
Sincere thanks go to Mrs. Pamela Moon for her help with statistical analyses, graphic designs and lab support. The invaluable cooperation of Drs. David N. Kuhn,
Martha Heath and Alan Meerow, and Cecile Olano, Wilber Quintanilla, Cheryl Krol,
Mike Winterstein, Jason Clayton, Emilio Power, Tyrone McArthy, Wilhelmina Wasik and Nannette Langiven at the Plant Sciences lab of the ARS-USDA in Miami, FL, will always be appreciated. Special appreciation goes to Dr. Simon Raharjo, Darda Efendi and his lovely wife, Neneng, and Sadanand Dekhney for making this long time more enjoyable and unforgettable. The help and friendship of other staff and faculty members at TREC will be remembered.
The development of this doctoral program was granted by the support of my
home institution, La Universidad de Cordoba, and I am honored to acknowledge a scholarship provided by the Instituto Colombiano de la Ciencia y la Tecnologia
IV (COLCIENCIAS) and the Fulbright Commission, and administered by the Latin
American Program for American Universities (LASPAU). Last but not least, a scholarship provided by the Dade County Agri-Council and a research assistantship provided by the California Avocado Society were critical for the completion of this program and therefore are deeply appreciated.
IV TABLE OF CONTENTS
ACKNOWLEDGMENTS iv
LIST OF TABLES viii
LIST OF FIGURES «
ABSTRACT xi
CHAPTERS
INTRODUCTION 1
LITERATURE REVIEW 5
Avocado 5 Somatic Embryogenesis 13 Micrografting 19 Avocado Cell and Tissue Culture 22 Viroids 32
MICROGRAFTING OF ASBVd INFECTED PLANTS 52
Introduction 52 Materials and Methods 53 Results 61 Discussion 67
REGENERATION OF AVOCADO PLANTS FROM THE NUCELLUS AS A STRATEGY FOR ELIMINATING ASBVd 73
Introduction 23 Materials and Methods 24 Results 84 Discussion 102
SUMMARY AND CONCLUSION 116
Summary H6 Conclusion 118
vi APPENDIX 120
LIST OF REFERENCES . 134
BIOGRAPHICAL SKETCH 155
vii LIST OF TABLES
Table2- page 3- 2-1. Avocado production and area planted (in parenthesis) by region during 3- 1996-2002 12 4-
2-2. Largest country avocado producers during 1996 - 2002 13
3. Viroid Classification 34
1 Percentages of success of micrografting of avocado 62
2. ASBVd in micrografted plants and scion-donor plants 65
86 1 . Number and type of embryogenic cultures
4-2. Average number and standard errors (in parenthesis) of proembryonic masses (PEMs), globular somatic embryos (GSE), hyperhydric and opaque somatic embryos (HSE) and total number of somatic embryos (SE) proliferated on MSP medium in 2002 and 2003 87
4-3. Settled cell volumes (SCVs) of embryogenic cultures (in ml) in liquid medium 91
4-4. ANOVA of the total somatic embryos that developed on SED medium 93
4-5. t-test of hyperhydric somatic embryos that developed on SED medium 94
4-6. Sequence variants of ASBVd isolated from ‘Vero Beach’ SE2 99
4-7. Distribution of the number of variants isolated from in vitro tissues with respect to nucleotide exchanges in the ASBVd molecule 104
A-l. ANOVAS of number and type of embryogenic cultures that developed on MSP medium 121
A-2. ANOVAs of settled cell volumes (SCVs) in liquid medium 122
viii 2- LIST OF FIGURES 3- Figure page
1. ASBVd nucleotide sequence and secondary structure showing the areas of the hammerhead formation in the plus (+) strand 38
1. Micrografting showing localization of the scion on the rootstock (left), initial stages of elongation (center) and growth and development (right) 62
3-2. Electropherograms of RT-PCR positively indexed leaves (left) of a and b:
micrograft No. 103 and its respective scion-donor seedling; c and d: 3- micrograft No. 133, and its respective scion-donor seedling and e: flower tissue of positive control ‘Vero Beach’ SE2, and negatively indexed 4- leaves (right) of f and g: micrograft No. 10 and its scion-donor seedling; h
and i: micrograft No. 1 1 1 and its scion-donor seedling; and j: leaf tissue of negative control ‘Simmonds’ 64
3-3. Gel agarose electrophoresis of PCR amplified fragments of micrografts
12, 15 and 52 (Lines 1, 3 and 5) and their respective scion-donor plants (lines 2, 4, 6). Line 7 = positive control ‘Vero Beach’ SE2 floral tissue. M = 100 bp ladder 66
4. ASBVd variant J02020 secondary structure molecule (Daros and Flores, 2000) isolated from micrograft No. 133 67
1. Induction and proliferation of embryogenic cultures on solid medium. Embryogenic culture induction from nucellus (left) and PEM proliferation (right) 85
4-2. Mean and standard errors from the ANOVA of a = somatic embryo development; b = globular somatic embryos; c = hyperhydric and opaque somatic embryos <0.5 cm diam.; d = hyperhydric and opaque somatic
embryos >0.5 cm dia., and e = PEMs of cultivars ‘Irwin’, ‘Vero Beach’ 1, ‘Vero Beach’ SE2 and ‘Yama’ 381 87
4-3. Suspension cultures of ‘Vero Beach’ SE2 90
4-4. Means and standard errors of settled cell volumes (SCVs) of suspension cultures 92
4-5. Average of somatic embryos that developed on SED medium in each dish 93
IX 4-6. Average number of hyperhydric somatic embryos per Petri dish that developed on SED medium from approx. 100 mg embryogenic culture inoculum 94
4-7. Shoot development (left) and plantlet recovery from somatic embryos of ‘Vero Beach’ SE2 95
4-8. Electropherograms of RT-PCR amplified fragments of: a: PEMs of ‘Hass’
11.4.3, b: somatic embryo of ‘Vero Beach’ SE2, c: ‘Vero Beach’ SE2 PEMs one year after induction, d: PEMs of ‘Yama’ 381 one year after
induction, e: ‘Vero Beach’ SE2 nucellar plant number 1 and f: floral tissue of ‘Vero Beach’ SE2 97
= 4-9. Agarose gel electrophoresis of RT-PCR amplified products. 1 and 10 PEMs of ‘Hass’ 11.4.3; 2 to 4 = PEMs of ‘Irwin’, ‘Yama’ 381 and ‘Vero = Beach’ 1 respectively; 5 = Somatic embryo of ‘Vero Beach’ SE2; 6 to 8
Nucellar plants number 1, 2 and 3 respectively; 9 = Floral tissue of ‘Vero Beach’ SE2; M= 100 bp ladder 98
4-10. Nucleotide variation, frequency of changes and number of variants with
these changes in a: embryogenic cultures, b: floral tissue, c: nucellar plant
1, d: nucellar plant 2 and e: nucellar plant 3 of ‘Vero Beach; SE2 105
A-l. ASBVd variants isolated from micrograft No 133 123
A-2. ASBVd variants isolated from PEMs, somatic embryos, floral tissue and nucellar plants 124
x Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
AVOCADO SUNBLOTCH VIROID (ASBVd) IN CELL AND
TISSUE CULTURES OF AVOCADO (Persea americana Mill.)
By
Isidro Elias Suarez Padron
December, 2003
Chairman: Richard E. Litz Major Department: Horticultural Sciences
Avocado sunblotch is an important disease of avocado (Persea americana Mill.).
The symptoms of the disease include marked decrease in the health of the plant, streaks on stems, bleaching and variegation of leaves and sunken yellow or reddish areas on the fruit surface. The disease is caused by the avocado sunblotch viroid (ASBVd), a single- stranded RNA molecule with a rod-like circular secondary structure. ASBVd-affected avocado trees have >25% reduction in fruit yield and >50% undergraded fruits. ASBVd can be transmitted via seeds, vegetative tissue and pollen, and can withstand high temperatures when thermotherapy treatments have been applied. Nucellus culture and micrografting have been used for the recovery of healthy plants of several citrus species
infected with citrus exocortis viroid (CEVd). Likewise, healthy potato ( Solanum
tuberosum ) and grapevine ( Vitis vinifera ) plants have been recovered from potato spindle
xi tuber viroid (PSTVd)-infected and grapevine viroid-infected materials, respectively. This study has examined the use of nucellar culture and micrografting in order to recover healthy avocado plants from ASBVd-infected material.
Embryogenic cultures were induced from the nucellus of ASBVd-infected
cultivars ‘Irwin’, ‘Vero Beach’ 1, ‘Vero Beach’ SE2 and ‘Yama’ 381. The cultures proliferated and nucellar plants were recovered from white-opaque somatic embryos of cultivar ‘Vero Beach’ SE2. Embryogenic nucellar cultures and regenerated plants were indexed for the presence of ASBVd using RT-PCR. ASBVd was persistent in embryogenic cultures and nucellar plants.
Shoot tips from ASBVd-infected seedlings were micrografted onto ASBVd-free seedlings and plants were recovered under in vitro conditions. RT-PCR indexing established that ASBVd was persistent in scions that developed from shoot tips <0.5 mm.
Pospiviroidae viroids such as PSTVd and CEVd move in the phloem and therefore cannot infect the nucellus and the shoot apical meristem, both of which lack vascular tissue. These viroids can therefore be eliminated either by micrografting or nucellar culture. However, the inability of these two methods to eliminate ASBVd indicates that a different procedure must be applied for elimination of Avsunviroidae viroids.
ASBVd replication in embryogenic avocado cultures has significant implications for the study of this pathogen in vitro.
xii CHAPTER 1 INTRODUCTION
Avocado sunblotch is a disease that affects the avocado (Persea americana Mill.) and was first reported in 1928 in California (Coit, 1928). The symptoms of avocado sunblotch include reduction in plant health, bleaching and variegation of leaves, streaks
on stems and new shoots, and sunken yellow and reddish areas on the fruits (Coit et al.,
1928; Home and Parker, 1931; Parker and Home, 1932; Dale et ah, 1982). Symptoms can be permanent or appear periodically, although asymptomatic trees occur and trees that once presented symptoms can “recover” and become asymptomatic (Semancik and
Szychowsky, 1994; Schnell et ah, 2001). Avocado trees affected with avocado sunblotch can have approx. 27% reduction in fruit yield and >50% undergraded fruits in comparison with healthy plants (Da Graca et ah, 1983).
Avocado sunblotch was initially believed to be the result of sunburn of neglected trees and was later attributed to be a physiological or genetic disorder (Coit, 1928; Home and Parker, 1931). The possibility that a vims might be the causal agent of avocado sunblotch was considered for long time (Parker and Home, 1932; Wallace and Drake,
1962); however, a viroid was observed to be associated with infected avocado tissue by
Palukaitis et ah (1979) and it was designated the avocado sunblotch viroid (ASBVd).
Infectivity studies using purified ASBVd induced typical avocado sunblotch symptoms among inoculated plants, establishing that this viroid was the causal agent of the disease
(Allen et ah, 1981). Sequence analysis demonstrated that ASBVd is a 247 bp length
1 2
RNA molecule with a rod-like circular secondary structure, and shared little similarity with other viroids sequenced at that time (Symons, 1981). Analyses of ASBVd-infected plants have revealed that multiple variant sequences of ASBVd can affect a single tree in
a phenomenon that resembles the quasispecies evolutionary concept (Duarte et al., 1984;
Pallas et ah, 1988; Rakowsky and Symons, 1989; Ambros et ah, 1999). Specific ASBVd
sequences have been found to cause differential symptom expressions; however, there is no consensus about the specific changes in the nucleotide sequence that can produce a specific type of symptom (Semancik and Scyzowsky, 1994; Schnell et ah, 2001).
Currently, ASBVd is classified as a member of the Avsunviroidae family, a group of viroids that can self-cleave in vivo and in vitro through hammerhead ribozymes, localize preferentially in the chloroplasts and do not possess a central conserved region characteristic of the Pospiviroidae family of viroids (Flores et ah, 1998; Navarro et ah,
1999).
ASBVd threatens not only commercial avocado orchards but also germplasm repositories that secure genetic resources. A recent survey using RT-PCR indexing
(Schnell et ah, 1997) at the National Germplasm Repository (NGR) found that approx.
19% of the trees were ASBVd-infected (Olano et ah, 2002). ASBVd affects all avocado cultivars, and transmission has been experimentally confirmed via seed, vegetative
material, grafting tools, pollen, and is believed to occur via root grafting as well
(Whitsell, 1952; Wallace and Drake, 1962; Desjardins et ah, 1980; Desjardins et ah,
1987). No insect is known to act as an ASBVd vector and eradication is the only method available to control the transmission of the disease.
Several in vitro techniques have been reported for eliminating Pospiviroidae viroids from infected material; however, the low sensitivity of indexing techniques 3
generates doubt about the effectiveness of such methods. Bitters et al. (1972) reported the elimination of citrus exocortis viroid (CEVd) in plants regenerated from the nucellus of a CEVd-infected tree. CEVd elimination in this case could be explained by the fact
that Pospiviroidae viroids have been observed to move through the phloem (Zhu et al.,
2001). Since the nucellus has no vascular connections with the rest of the plant (Esau,
1977), the absence of viroid in this tissue explains the recovery of viroid-free nucellar
plants. However, it is known that CEVd mild strain can cause late symptom development or a non-symptomatic infection in indicator plants (Visvander and Symons, 1985).
Therefore, a more sensitive indexing technique should be used to confirm these earlier results. Duran-Vila et al. (1988), using polyacrylamide gel electrophoresis (PAGE), reported elimination of viroid-like RNAs from infected citrus material using meristem tip culture; however, Wan et al. (1997), using highly sensitive RT-PCR indexing, showed that grape plants recovered by the same technique were still infected with low levels of
grapevine yellow speckle viroid 1 (GYSVdl), grapevine yellow speckle viroid 2
(GYSVd2) and hop stunt viroid (HSVd) that could not be detected by PAGE indexing.
RT-PCR-confirmed viroid elimination has been reported for pear plants affected with apple skin scar viroid (ASSVd) (Postman and Hadidi, 1995). In this case, plants were recovered from meristem tip culture after the infected material was maintained in vitro
either at 38 °C for 55 days or at 4 °C for 40 days. These results can be explained by the
fact that ASSVd accumulates at temperatures between 18°C and 28°C and is inactivated below or above that range (Skrzeczkowski et al., 1993).
Desjardins et al. (1980) attempted to eliminate ASBVd from infected material
using thermotherapy; however, like CEVd (Semancik and Weathers, 1970), potato
spindle tuber viroid (PSTVd) (Singh and Bagnall, 1968) and hop latent viroid (HLVd) 4
(Barba et al., 2003), ASBVd was able to withstand higher temperatures than the tissue before showing any sign of inactivation. Nel and Lange (1985) attempted to use micrografting to eliminate ASBVd; however, the study primarily focused on refinement of a technique that would allow recovery of micrografted avocado plants; indexing of plants to demonstrate ASBVd elimination was not reported. Avocado cell culture applied to ASBVd research was attempted by Desjardins (1958) and Blickel et al. (1988); however, these experiments focused on the development of a protocol to study ASBVd replication rather than viroid elimination
In the current study, two in vitro techniques, i.e., micrografting and nucellar culture, have been applied in attempting to eliminate ASBVd in infected avocado. CHAPTER 2 LITERATURE REVIEW
Avocado (Persea americana Mill.)
Use and Importance
Avocado (.Persea americana Mill.) is considered to be one of the most nutritious of
all fruits and has long been a traditional staple in Guatemala and other Central American countries (Popenoe, 1920; Purseglove, 1968). The avocado pulp is yellow, with smooth and soft texture, a nutty flavor and surrounds a solitary seed. In contrast to other fruits,
avocado is consumed as an uncooked savory dish and as an ingredient for salads, desserts, milkshakes and guacamole, a traditional companion of Mexican cuisine
(Popenoe, 1920; Verheij and Coronel, 1991; Bergh and Lahav, 1996).
Depending on the cultivar, the avocado pulp can have an oil content of 3-30% fresh weight with 95% of the content corresponding to four fatty acids: 42% to 81% of monounsaturated oleic acid, 0% to 8% of monounsaturated palmitoleic acid, 6.0% to
18.5% of polyunsaturated linoleic acid and 7.2% to 25% of saturated palmitic acid
(Bergh, 1996; Mazliak, 1965). Consumption of avocado on a regular basis has beneficial effects on reduction of blood cholesterol levels and improvement of heart conditions
(Grant, 1960; Colquhoun, 1990). Avocado oils are used as ingredients for the production of cosmetics, soaps, shampoos and skin moisturizer (Smith et ah, 1992).
5 6
Origin, History and Distribution
Avocado may have originated in a broad geographical area that today extends from
Chiapas (Southern Mexico) through Guatemala and to the Pacific coast of Central
America (Popenoe, 1920; Storey et al., 1986; Kopp, 1996). The cultivation of avocado
may be 10,000 years old according to archeological evidence found in the Tehuacan
Valley in Puebla (Mexico); the findings support selection for larger fruits over time since
fossils found in upper soil layers have larger seeds than those found in deeper excavations
(Smith, 1966; 1969).
During the course of evolution, three different botanical varieties of avocado,
known as horticultural races, developed in geographical isolation from each other and
with differential climatic adaptation (Popenoe, 1920). The Mexican race is the most
tolerant of low temperatures, and most probably originated in the highlands of South-
Central Mexico where primitive forms of avocado have been found (Bergh and Ellstrand,
1986; Storey et al., 1986). The Guatemalan race has an intermediate tolerance of low
temperatures and may have developed in the highlands north of Guatemala City and
Antigua (Storey et al., 1986; Bergh and Ellstrand, 1986). The West Indian race, now
known as the Lowland race, is the best adapted to tropical conditions and was initially
believed to have originated in the Antilles (Popenoe, 1920); however, it is now known to
have evolved in an area of the Pacific coast of Central America between Guatemala and
Panama (Scora and Bergh, 1992).
th The current name, avocado, was officially adopted and approved in the early 20
century by the American Pomological Society and the U. S. Department of Agriculture,
and is the final result of a sequence of variations of the original Aztec name aoactl, 7
wrongly spelled as ahuactl by initial writers and corrupted by the Spaniards to ahuacate
and aguacate (Popenoe, 1934).
At the time of the Spanish conquest of the New World in 1492, avocado was
already grown from Mexico to the Pacific coast of Peru; it was soon brought by the
Spaniards to the Caribbean islands and was first planted in Spain in 1601 (Popenoe,
1920; Purseglove, 1974). The avocado was introduced into Indonesia and probably the
Philippines by 1750 (Morton, 1987), but was not introduced into Brazil, Israel, Hawaii,
th South Africa and Australia before the 19 century (Oppenheimer, 1947; Ludman, 1965;
Whiley, 1982; Donadio, 1984). The first introductions into the U.S.A. are recorded to
have occurred from Mexico in 1833 into Florida and in 1850 into California (Koch, 1983;
Crane et al., 1992).
Taxonomy
= = The avocado (Persea americana Mill.) (2n 2x 24) (Garcia, 1975) is a species of the genus Persea, a member of the Lauraceae family, a group of plants that together with the Annonaceae, Magnoliaceae and Proteaceae families are among the oldest flowering
plants (Scora et al., 2002). Persea is divided into two vegetatively and sexually
incompatible subgenera: Persea and Eriodaphne. Some species of subgenus Eriodaphne
are resistant to the oomycete Phytophthora cinnammomi Rands., the main limiting factor
in most avocado producing areas (Bergh and Ellstrand, 1986). The subgenus Persea is
composed of three species: P. schideana Nees, P. parvifolia Williams and P. americana
Mill. (Scora et al., 2002). The botanical varieties or subspecies are commonly referred to
as horticultural races. According to Kopp (1966), the Mexican race should be considered 8
as a different species (P . drymifolia), while the Guatemalan and West Indian races are close enough to be subspecies of P. americana. Williams (1977) considered that the
is Guatemalan race different enough to be a distinct species (P. nubigena) and that West
Indian and Mexican races are subspecies of P. americana. Bergh and Ellstrand (1986) and
Scora and Bergh (1990), using morphological, physiological and geographical data and isozyme analysis, supported the division of P. americana into three horticultural races as proposed by Popenoe (1941). Morphological and biochemical characteristics, and climatic adaptations have long been used to associate cultivars with their racial background. A well
known example of this practice is the identification of Mexican cultivars based on their
anise-scented leaves, which is the result of the presence of a chemical compound known as stragol (King and Knight, 1987).
The avocado flowering habit promotes outcrossing and the three horticultural races
can hybridize freely in the wild or under cultivation since no sexual incompatibilities
have been reported (Popenoe and Williams, 1947; Bergh, 1969). Most of the
commercially grown cultivars are interracial hybrids that have been randomly selected
from seedling populations and therefore are clonally propagated.
The racial background of avocado cultivars is of special interest for breeding
purposes and the characteristics they confer to the selected progeny; however,
classification based on morphological characters can be influenced by environmental
conditions or can be difficult to determine in closely related genotypes and complex
interracial hybrids (Mhameed et al., 1997; Schnell et al., 2003). Molecular markers have
allowed better analysis and more reliability in determining racial background of certain
cultivars. Sharon et al. (1997), using simple sequences repeats (SSRs), random amplified
polymorphic DNA (RAPD) and minisatellite DNA fingerprinting (DFP) to screen two 9
highly heterozygous parents and 50 progenies, developed a linkage map containing 12
linkage groups.
Mhameed et al. (1997) used DNA fingerprinting (DFP) to analyze 24 avocado
cultivars with respect to their racial origin, and confirmed the previous racial
classification of 19 accessions while five were relocated into a different racial group.
Fiedler et al. (1998), using random amplified polymorphic DNA (RAPD) analysis, found
specific polymerase chain reaction (PCR)-amplified products for every race of avocado, which could eventually be used as race-specific markers for cultivar classification and
germplasm evaluation; the same study also found that the three avocado races are equidistant from each other, supporting the division of this species into three different races. Davis et al. (1998) showed that random fragment length polymorphism (RFLP) markers were useful for determining the parental origin of ‘Gwen’. Schnell et al. (2003), using simple sequence repeats (SSRs) markers to evaluate 223 accessions, changed the racial designation or interracial status of 50 accessions at the National Germplasm
Repository (NGR) in Miami, FL. This study also showed that the races are equally
separated, although no race-specific markers were detected.
The most recent classification of the subgenus Persea according to Scora et al.
(2002) is as follows:
Persea americana Mill.
var. americana Mill. (West Indian)
var. drymifolia (Schlet. + Cham.) Blake (Mexican)
var. guatemalensis Williams (Guatemalan)
var. nubigena (Williams) Kopp
var. steyermarkii Allen 10
var. zentmyerii Schieber and Bergh
var. tolimanensis Zentmyer and Schieber
Persea parviflora L. (Williams)
Persea schiedeana Nees
Botany
Botanically, the avocado plant is a polyaxial tree with a synchronous growth
pattern characterized by alternating shoot and root flushes (Verheij, 1986; Whiley et al.,
1988; Thorp and Sedgley, 1993). The tree has an evergreen dome-shaped canopy that can reach 30 m height; the roots can penetrate 3-4 m in deep soils, although the feeder root system is restricted to the top 60 cm (Schaffer and Andersen, 1994). The leaves are
simple, variable in shape and dark green, and there is a characteristic anise scent in
Mexican cultivars (Morton, 1987). The flowers are borne in panicles of terminate or indeterminate cymes that grow on terminal or subterminal buds. The individual flowers are hypogynous with two alternating whorls of three sepals and petals, two whorls of three stamens and staminods, and a unicarpelar gynoecium with a superior ovary (Reece,
1939; Scora et al., 2002). There are two types of flowers, A and B, which are specific for each cultivar and result in a flowering habit, known as protogynous diurnal dichogamy that promotes outcrossing (Bergh, 1969), although self-pollination has been reported to
occur in some cultivars (Davenport et al., 1994).
The avocado fruit is a pyriform or globose berry with an exocarp of variable texture
and a thick and fleshy mesocarp that surrounds a solitary seed. Fruit size can range
between 50 g and 200 g, and time to maturity depends on the horticultural race; 11
Guatemalan cultivars are the last and Mexican the earliest to reach maturity. Fruits of
West Indians cultivars are the most sensitive to cold storage while Mexican cultivars have the thinnest skin (Schaffer and Andersen, 1994; Bergh and Lahav, 1996; Valmayor,
1997).
Production
World avocado production averaged 2,392,793 million tons per annum during the
lh period 1996-2002 (Table 2-1), and ranks ll among fruits after Musa (banana and plantain), citrus, grape, apple, mango, pear, pineapple, peach, plum and strawberry
(FAOSTAT Database, 2002).
Central America with the Caribbean Region is the largest avocado producing region in the world with >1 million tons per annum, followed by Asia with a production of approx. 340,000 tons per annum, and North America (USA) with approx. 200,000 tons per annum (FAOSTAT Database, 2002). The largest increase in planted area has taken place in South America, where >10,000 hectares have been established in the last eight
years (Table 2-1).
Mexico remains the largest avocado producer with nearly 1 million tons per annum
(Table 2-2), followed by U.S.A. and Colombia with 205,000 and 143,538 ton a year
respectively (FAOSTAT, 2002). Chile has had the fastest growing avocado industry in
the last 6 years, and has doubled its production from 55,000 tons in 1996 to 1 10,000 tons
in 2002 (Table 2-2). The avocado industry is expected to continue expansion in countries
such as China, where an increase in planted area seems very likely (Knight, 2002). 12
Table 2-1. Avocado production and area planted (in parenthesis) by regions during 1996- 2002
Region 1996 1997 1998 1999 2000 2001 2002
World 2,268,557 2,192,966 2,261,218 2,360,699 2,485,910 2,596,976 2,583,226
(332,689) (311,418) (317,592) (331,295) (334,669) (345,567) (348,769)
Africa 178,581 172,990 222,813 187,372 212,242 213,296 214,296
(29,325) (28,863) (34,0420 (34,970) (36,125) (36,665) (36,765)
Asia 312,545 302,598 287,009 293,009 319,089 329,375 330,875
(68,341) (51,297) (51,191) (52,745) (53,129) (53,479) (53,979)
Europe 66,806 67,181 88,468 80,479 61,350 74,295 74,300
(17,935) (18,821) (18,300) (18,387) (19,084) (19,012) (19,012)
USA 173,000 161,270 144,500 164,500 217,100 205,000 205,000
(26,630) (26,730) (26,560) (26,360) (26,385) (26,385) (26,385)
C A&C 1,093,796 1,008,909 1,118,537 1,108,336 1,145,762 1,205,903 1,218,193
(122,483) (112,064) (122,370) (122,487) (122,800) (125,333) (130,442)
Oceania 20,986 25,100 26,491 34,731 37,802 36,816 38,263
(5,740) (6,362) (6,162) (7,267) (8,713) (8,713) (8,713)
South America 422,843 454,918 373,400 492,272 492,565 532,291 502,299
(62,235) (67,281) (58,967) (69,079) (68,433) (75,980) (73,473)
Source: FAOSTAT (2002); C A & C = Central America and the Caribbean 13
Table 2-2. Largest avocado producers during 1996-2002.
Country 1996 1997 1998 1999 2000 2001 2002
Mexico 837,787 762,336 876,623 879,083 907,439 940,229 950,174
USA 173,000 161,270 144,500 164,500 217,100 205,000 205,000
Colombia 114,000 126,000 74,000 158,505 131,664 137,065 143,538
Indonesia 143,151 129,952 130,950 126,480 128,000 130,000 130,000
Dominican Republic 114,000 90,000 85,000 71,158 81,736 111,058 111,058
Chile 55,000 68,000 60,000 82,000 98,000 120,000 110,000
Brazil 80,731 84,043 84,231 86,418 86,146 88,000 89,000
Israel 76,000 79,347 65,684 66,099 81,303 85,868 85,868
South Africa 53,801 44,586 92,161 55,900 80,546 81,000 81,000
China 45,000 48,000 51,000 70,000 70,000 74,500 76,000
Source: FAOSTAT (2002).
Somatic Embryogenesis
An embryo is the simplest multicellular level of an organism previous to the development of the morphogenic process characteristic of a given species (Gray, 1996).
Somatic embryos are unique to the plant kingdom. In contrast to zygotic embryos that result from the fusion of the gametes within the embryo sac, they can originate from cells of any tissue (Litz and Gray, 1995). Similar to zygotic embryos, somatic embryos are bipolar, have a single cell origin and lack any vascular connection with the mother plant
(Haccius, 1978). The first reports of somatic embryo production were attained using citrus ovule cultures (Stevenson, 1956; Maheshwari and Ranga Swamy, 1958) and carrot cell cultures (Reinert, 1958; Steward, 1958). Advances in the field of cell and tissue 14
culture have increased significantly the number of species that can form somatic
embryos. It is believed that somatic embryogenesis can be accomplished in any species with the appropriate explant, culture medium and environmental conditions (von Arnold et al., 2002). Somatic embryo production can occur in vitro and in vivo; apomixis, a
characteristic of polyembryonic citrus and Mangifera species, is a spontaneous somatic embryogenic event that results in the development of an embryo from a somatic cell
within the embryo sac (Marshall and Brown, 1981; Koltunow et al., 1995) while
Bryophyllum and Malaxis species produce somatic embryos from the leaf margins
(Yarbrough, 1932; Taylor, 1967).
The somatic embryogenesis pathway follows a sequence of hierarchical stages that are initiated with the explanting of the plant tissue and terminates with the development of cotyledonary somatic embryos and plantlet recovery:
1 . Induction
2. Maintenance
3. Development
4. Plantlet recovery
Induction
A major difference between zygotic and somatic embryogenesis is the
characteristic of the initial cell. The zygote is intrinsically preprogrammed to follow the embryogenic pathway while the somatic cell needs to be induced to acquire embryogenic
competence (Ammirato, 1987; Dodeman et al., 1997). Two pathways have been observed to occur when inducing embryogenic cultures: direct or permissive, in which 15
embryogenic cultures develop directly from cells within the explant without intermediate callus formation, and the indirect pathway, in which cells within the explant undergo a directive induction that drives the cell to acquire embryogenic competence (Haccius,
1973; Ammirato, 1987; Christianson, 1985, 1987). In the directive induction pathway, rapid cell division and changes in gene expression (including asymmetric cell division) drives the cell to acquire embryogenic competence.
Directive induction pathway
In the directive induction pathway, differentiated tissues are explanted onto induction medium containing auxins (usually 2,4-D) (Ammirato, 1985). Differentiated cells are determined to carry out specific functions controlled by the synthesis of specific gene products. In order to acquire embryogenic competence, gene expression in differentiated cells must be reprogrammed and embryogenesis-related products must be synthesized. Auxins are believed to induce down-regulation of current gene expression by stimulating DNA methylation and promoting chromatin rearrangement that allows expression of new gene products (LoSchiavo, 1989; Feher et al., 2003) resulting in rapid
cell proliferation, unequal cell division and cell dedifferentiation.
Auxins not only induce the growth of new cells but also influence the occurrence
of asymmetric cell division (Kohlenbach, 1978; Dudits et al., 1991). Asymmetric cell
division results in a basal highly vacuolated cell and a small richly cytoplasmic cell with
a re-determined cell function and acquired embryogenic competence (Christianson, 1985;
Litz and Gray, 1995). This stage resembles the unequal cell division of the zygote that
results in the small cytoplasmically rich upper cell that will form the embryo and the 16
highly vacuolated basal cell that will become the suspensor during zygotic embryogenesis (Scheres and Benfey, 1999). A study using antibody-labeled carrot cells showed that only small isodiametric cells resulting from asymmetric cell division are able to develop into somatic embryos while large vacuolated cells die soon after division
(McCabe et al., 1997). The embryogenic cell will continue dividing and develop onto a proembryonic cell cluster. Continuous subculture of proembryonic clusters in the presence of 2,4-D results in the loss of integrative development as proembryos and the clusters become proembryonic masses (PEMs). If transferred to an auxin-free medium, somatic embryos can develop from PEMs (Halperin, 1966). Directive somatic embryogenesis induction has been observed to occur from explants of monoembryonic mango species (Litz et al., 1982), longan (Litz, 1988) and Carica pentagona (Vega de
Rojas and Kitto, 1991).
Permissive induction pathway
In the direct pathway, somatic embryos or proembryonic masses develop directly from competent cells present in the explant without cell division and redetermination
(Christianson, 1985). These cells already have embryogenic competence, but their
expression is inhibited by the surrounding tissue (Litz and Gray, 1995), i.e., immature
zygotic embryos, nucellus, hypocotyls, etc. Permissive induction usually requires culture of the explants on a medium without auxins for induced cells to express their embryogenic potential; these cells have also been referred to as pre-embryogenic
determined cells (PEDCs) (Sharp et al., 1980; Ammirato, 1986; Williams and
Maheswaran, 1986). Induction of the embryogenic pathway has been reported from 17
explants consisting of the nucellus of polyembryonic mangos (Litz, 1984) and the
nucellus of monoembryonic citrus species (Rangan et al., 1969).
Maintenance of Embryo genic Cultures
Embryogenic cultures are usually maintained as proembryonic masses (PEMs) by continued subculture on semisolid or in liquid medium containing 2,4-D or another auxin
(Litz and Gray, 1995). Auxins disrupt the polarized cell division required for the development of singulated somatic embryos from PEMs and proembryos causing
proliferation of embryogenic cells (Ammirato, 1987; De Vries et al., 1988; Litz and Gray,
1992; Kawahara and Komamaine, 1995; Williams and Maheshwaran, 1986). Certain
embryogenic cultures can lose the sensitivity to auxin after they start to synthesize their
own auxin, i.e., habituation (Gautheret, 1942), and this has been observed in citrus
embryogenic cultures (Button et al., 1974; Grosser and Gmitter, 1990). PEM
proliferation during maintenance of embryogenic cultures is desirable over secondary
somatic embryo proliferation in terms of rate of multiplication and for use in cell studies
such as protoplast isolation and genetic transformation (Gray, 1996; Merkle, 1995).
However, depletion of auxin or insensitivity of certain species can affect the uniformity
of embryogenic cultures, resulting in the occurrence of PEMs of different sizes and
morphologies and somatic embryo development (Merkle, 1995; von Arnold, 2003).
Since variability in the degree of development from embryogenic cultures can occur,
sieving of suspension cultures is used to improve uniformity (Litz and Gray, 1995). pH
has been shown to affect the degree of development in maintenance medium; Smith and
Krikorian (1990) found that a decrease of the medium pH favors PEM maintenance while 18
a pH of 5.8 caused the PEMs to develop into globular, heart, torpedo and cotyledonary stages.
Somatic Embryo Development and Plant Recovery
The somatic embryo development pathway resembles that of zygotic embryos, and includes the globular, heart, torpedo and cotyledonary stages (Zimmerman, 1993).
Because auxins generally inhibit somatic embryo development (Zimmerman, 1993), they usually are omitted from somatic embryo development media. Abnormal development of somatic embryos, consisting of monocotyly, polycotyly, fused cotyledons, fasciation and
multiple somatic embryo can occur (Ammirato, 1987; Litz et al., 1995; Gray, 1996); however, a high frequency of singulated somatic embryos can be recovered by sieving and plating the smaller fraction of PEMs (Ammirato, 1987; Litz and Gray, 1995). In contrast with zygotic embryos, somatic embryos always behave as recalcitrant embryos and cannot withstand desiccation (Bewley and Black, 1985; Gray, 1996). Abscisic acid
(ABA) has been shown to delay precocious germination in caraway somatic embryos
(Ammirato, 1974, 1985) and improve desiccation tolerance of celery somatic embryos
(Kim and Janick, 1989). Treatment with ABA reduced secondary somatic embryo
development (Monsalud et al., 1995) in mango but had no beneficial effects on
improving desiccation tolerance (Pliego-Alfaro et al., 1996). Late embryogenic abundant proteins (LEA) are believed to enable zygotic embryos to resist desiccation and are
synthesized by a group of ABA-induced genes (Dure et al., 1989). Treatment of carrot somatic embryos with ABA has been shown to increase the synthesis of LEAs
(Hatzopoulus, 1990; Zimmerman, 1993; Dodeman et al, 1997). Increasing medium 19
osmolarity also has beneficial effects for somatic embryo maturation (Lee and Thomas,
1985).
Plant recovery from somatic embryos varies among species. Low plant recovery from somatic embryos has been one of the major constraints of the somatic embryogenesis pathway. Plant recovery from somatic embryos is typically low (<50%) when compared to zygotic embryogenesis (>90%) of commercial seeds (Gray, 1996).
Plant recovery from citrus is reported to be between 30 to 50% (Kochba et al., 1972). In some cases the number of plants recovered has not been reported; however, recommendations for improving the protocols and increasing the number of recovered
plants suggest that a low number of plants have been recovered (Rangan et al., 1968;
Bitters et al., 1972). Sondahl and Sharp (1977) reported a rate of 30-40% of plants recovered from Coffea arabica. Vega de Rojas and Kitto (1991) reported the recovery of
a single rooted babaco ( Carica pentagona) plant from nucellus culture. De Wald et al.
(1989) reported 35% of normal germination of mango somatic embryos. Recovery of avocado plants from somatic embryos has been reported to be low (0-5%) (Pliego Alfaro and Murashige, 1988; Mooney and Van Staden, 1988; Witjaksono and Litz, 1999b); however, significant improvements of plant recovery have been made by
micropropagating somatic embryo-developed shoots (Witjaksono et al., 1999a) and micrografting (Raharjo and Litz, unpublished reports).
Micrografting
Micrografting consists of the in vitro grafting of very small shoot apices onto decapitated rootstock seedlings, and has been used to recover healthy plants from 20
pathogen-infected material (Starrantino and Carusso, 1988). The first report of pathogen elimination using in vitro culture of meristematic tissues occurred when White (1934) obtained free-virus root cultures in an attempt to multiply a plant virus in tomato roots.
White noticed that in order to maintain infected tissue, larger segments of roots than the
tip alone were needed. Elimination of systemic pathogens using meristem tip cultures is likely to be due to the lack of vascular connections in the apical meristems; however; the fact that not all meristem-derived plants are healthy may be due to the erratic distribution of the pathogen in the plant (Hollings, 1965). The first application of virus elimination with a commercial crop involved dahlia and potato plants, which were freed of viruses following meristem tip culture (Morel and Martin, 1952; 1955). Meristem tip culture was routinely used for viral infection elimination mostly with herbaceous ornamental species
(Hollings, 1965). The first report of micrografting to recover healthy clones of a woody species from pathogen-infected material was Murashige et al. (1972), who attempted to bypass the prolonged juvenile phase of the virus-free plants recovered from the nucellus of citrus plants. The scions (meristem tip plus 4-6 leaf-primordia) were excised from plants infected with citrus tristeza virus and citrus exocortis viroid (CEVd), and the micrograftings tested negative for both infections when indexed using indicator plants.
Plants did not develop any thorns and flowered after 1 8 months, indicating bypass of the juvenile phase. A similar study involved <0.2 mm long meristem tips which were inserted into an inverted-T made at the top of decapitated rootstocks and allowed the recovery of citrus plants free of citrus exocortis viroid, tristeza and psorosis viruses and
stubborn spiroplasma without reversion to the juvenile phase (Navarro et al., 1975). The study also showed that a higher number of plants could be recovered when rootstock seedlings were germinated in darkness. Healthy apple plants were recovered when 0.1- 21
0.2 mm long shoot tips isolated from stem grooving virus (SGV)-infected trees were micrografted onto in vitro germinated seedlings of ‘Golden Delicious’ (Huang and
Millikan, 1980); in this study the micrograft was cultured in liquid MS (Murashige and
Skoog, 1962) medium, and larger scions favored a higher rate of plant recovery. Jonard et al. (1983) observed that a higher number of plants were recovered from citrus micrograftings when the shoot tips and the scions were soaked in different cytokinin solutions. Starrantino and Carusso (1988) recovered healthy citrus plants from viroid and virus-infected material, and observed that the rate of plant recovery increased when the scion and the rootstock were dipped into a solution containing 0.5 ppm of 6- benzylaminopurine (BAP).
The development of a micrografting technique that would permit the recovery of avocado plants free of avocado sunblotch viroid (ASBVd) was proposed by Nel and
Lange (1985). The report showed that approx 50% of plants could be recovered when all the cuts in the rootstock and the scions were made under water and both parts were maintained submerged for 24 h before performing the micrografting; however, the application of the technique to remove ASBVd from infected material was never reported. Pliego-Alfaro and Murashige (1987) restored rooting in adult avocado material by micrografting lateral buds isolated from flowering plants onto in vitro germinated seedlings. Approx. 50% of the micrografted scions regained rooting capacity, indicating reversion to the juvenile phase. In a similar study, Tranvan et al. (1991) reported development of juvenile characteristics in micrograftings of Sequoia sempervirens. In this study, the scion obtained from shoots of a 500-year-old tree showed juvenile morphological and physiological characters. A study intended to induce phase reversal
using micrografting in cashew (Anacardium occidentale) attained only partial 22
rejuvenation in recovered plants (Mneney and Mantell, 2001). Micrografting has been used to speed up the detection of systemic infections in indexing. Symptom development occurred 8-12 weeks after healthy LN33 grapevine shoots were micrografted with shoot tips infected with corky bark-like virus while the traditional indexing procedure consisting of grafting of normal size scion onto indicator rootstocks can take as long as 2
year to provide results (Tanne et al., 1993).
Avocado Cell and Tissue Culture
Avocado Cell Culture
Callus initiation
Cell culture of avocado tissue was first reported by Schroeder (1956) using fruit pericarp to study fruit cell growth. Proliferation of non morphogenic callus was described from fruit tissue explants of ‘Fuerte’ cultured on artificial medium
(composition not reported) supplemented with IAA, several amino acids and coconut milk. Desjardins (1958), attempting to develop an in vitro system to study ASBVd,
reported callus development at the cut-end of seedling stem segments used as explants and cultured on media similar to that of White (1934) and Gautheret (1942) and supplemented with nicotinic acid, pyridoxine, calcium panthothenate, thiamine, 2% sucrose, naphthaleneacetic acid (NAA) and coconut milk. Schroeder (1961) studied the anatomy of cell cultures derived from pericarp tissue and observed differentiation of cells into tracheids and parenchyma cells. Schroeder (1968), using fleshy cotyledon pieces 23
explanted onto Nitsch medium with a substitution of FeEDTA for iron citrate and IAA
(10 ppm), initiated a callus that could be maintained for 2 years. Schroeder (1971) observed that callus that originated from pericarp tissue explanted on a culture medium with a similar composition to the previous report, grew better in darkness at 25°C.
Blumenfeld and Gazit (1971), using several tissues of ‘Fuerte’, found that different explants have differential plant growth requirements for callus proliferation; cytokinins were required for cell proliferation from mesocarp explants; while no cytokinin were necessary when cotyledons were used as explants. Schroeder (1975), in an attempt to study the floral behavior of mature avocado flower buds under controlled conditions, observed profuse callus proliferation followed by root morphogenesis from the cut end of different flower parts explanted onto MS and Nitsch (1969) media supplemented with various concentrations of indoleacetic acid (IAA), NAA and coconut milk, and cultured at 27°C. Similarly, callus was reported to proliferate from the cut ends of leaf buds explanted on an unreported medium composition in an attempt to clonally propagate avocado cultivars (Schroeder, 1976). Schroeder (1977) reported long term maintenance
(17 years) of non morphogenic avocado callus on Nitsch medium. The same study reported maintenance of callus that originated from peduncles for >6 years. Schroeder
(1978) reported that short term exposure of avocado pericarp tissue, explanted on Nitsch medium, to ultraviolet light at 25-27°C stimulates callus proliferation. Schroeder (1979), using stem sections with apical or bud tips isolated from plants grown in the glasshouse or in darkness explanted on MS or Nitsch media, observed that callus proliferation occurred at the cut end in contact with the medium on MS medium but not on Nitsch medium. Young (1983), using leaf and stem sections isolated from seedlings of ‘Waldin’ and ‘Lula’ explanted on Anderson’s (1978) medium supplemented with casein 24
1 1 hydrolysate (1 g l' ), sucrose (30 g l' ) and several concentrations of benzyladenine (BA),
indolebutyric acid (IBA) and 2,4-diclorophenoxyacetic acid (2,4-D), observed that 0.2
1 mg l" of 2,4-D was optimum for initiating friable callus growth from the cut ends of stem
segment explants. Blickel et al. (1988), in an attempt to study ASBVd replication in
vitro, initiated callus from stem seedlings explanted on semi solid MS medium
1 1 supplemented with IAA (1 mg l' ) and BA (0.3 mg l' ).
Somatic embryogenesis
Induction. Induction of avocado embryogenic cultures was first achieved by
Pliego-Alfaro (1981), who reported the development of spherical to oblong adventitious
embryos from zygotic embryos (0.6-0. 8 mm length) excised from 9 mm length ‘Hass’
fruits. The zygotic embryos were explanted on MS plant growth medium supplemented
1 1 1 with 0.41 pM picloram, 30 g f sucrose, 0.4 mg l" thiamine HCL and 100 mg l' myo-
inositol and cultured at 27°C with a 16 h daily exposure to 100 lux. Later reports based
on the work of Pliego-Alfaro (1981) reported that the size of the zygotic embryo explant
affected embryogenic culture induction. Using the protocol reported by Pliego-Alfaro
(1981), Mooney and Van Staden (1987) induced embryogenic cultures from 0.6-0. 8 mm
long zygotic embryos isolated from 3-4 mm length fruits of ‘Fuerte’ and ‘Duke’ and from
0.6-0. 8 mm long ‘Hass’ zygotic embryos, respectively. Raviv et al. (1998) induced
embryogenic cultures from 7-10 mm long cotyledonary zygotic embryos. Witjaksono
and Litz (1999a), used 0.1 to 2.7 mm length zygotic embryos isolated from 7 avocado
cultivars and explanted on B5 (Gamborg et al., 1968) major salts supplemented with MS
1 1 1 minor salts, 0.41 pM picloram, 30 g l' sucrose, 0.4 mg l' thiamine HCL, 100 mg f 25
1 myo-inositol and solidified with 8 g l' TC agar; they reported a 5-25% induction frequency, demonstrating that induction is genotype-dependent. Similar induction frequencies were observed when avocado nucelli from 4 avocado genotypes were
explanted on a similar medium (Witjaksono, 1997; Witjaksono et al., 1999).
Embryogenic cultures develop from the explant 8-25 days after explanting.
Although initially designated as embryogenic callus, later histological analysis has confirmed that these cultures consist of polyembryonic masses (PEMs) and early stages of hyperhydric somatic embryos (Mooney and Van Staden, 1987; Witjaksono and Litz,
1999a; Witjaksono et al., 1999b; Witjaksono and Litz, 2003).
Maintenance . Long term maintenance of avocado embryogenic cultures occurs either on semi solid or in liquid medium (Witjaksono and Litz, 1999a). The optimum growth on semisolid medium occurs when embryogenic cultures are inoculated on MS
1 1 supplemented with 0.41 pM picloram, 30 g l' sucrose, 4 mg l' thiamine HCL, and
1 cultures solidified with 8 g l' TC agar and cultured in darkness at 25°C. Embryogenic are subcultured at monthly intervals and only the smallest PEMs are inoculated onto fresh medium.
Embryogenic suspension cultures are maintained in liquid medium consisting of
1 filter-sterilized MS and supplemented with 30-50 g T sucrose, 0.41 pM picloram and 4
1 mg T thiamine HCL. Suspension cultures are subcultured biweekly by inoculating 0.5 g
flasks. PEMs into 40 ml medium dispensed in 125 ml flasks or 1 g into 80 ml in 250 ml
Suspension cultures are maintained on a rotary shaker at 120 rpm in semi darkness at
25°C. Maintenance in liquid medium has been optimized by using autoclaved modified
1 MS containing 15 mM NH4NO3 and 30 mM KNO3 and supplemented with 45 g f 26
sucrose, 1 1 0.41 pM picloram, 4 mg 1 thiamine HC1 and 100 mg l" myo-inositol under the
same culture conditions described above (Witjaksono and Litz, 1999b; 2003).
Although the presence of auxins in the maintenance medium would be expected
to prevent development of singulated somatic embryos (Litz and Gray, 1995), formation
of heart and later stages somatic embryos can occur. Witjaksono and Litz (1999a)
observed the proliferation of two types of embryogenic cultures in maintenance medium,
PEM and somatic embryo (SE) types, and demonstrated that the responses were
genotype-dependent. Maintenance of SE-type cultures in liquid medium involves the
sieving of the suspension culture to increase the homogeneity. Loss of embryogenic
potential of cultures in maintenance medium has been shown to be genotype-dependent
and is associated with increased disorganization of proembryonic masses. Witjaksono
and Litz (1999a) observed that cultures of ‘Yon’ are embryogenic for 3 months after
induction while those of ‘Esther’ retained embryogenic competence for >2 years.
Cryopreservation of embryogenic cultures followed by recovery on maintenance
medium, development of somatic embryos and plantlet recovery has been attained
(Efendi et al., 2001; Efendi, 2003) using slow cooling and vitrification protocols.
Cryopreserved cultures were plated on MSP and those that proliferated were inoculated
into liquid medium. No differences were detected in terms of proliferation of suspension
cultures and the number of somatic embryos that developed with respect to the two
cryopreservation methods regarded before. Somatic embryos recovered from storage in
liquid nitrogen germinated and converted into plants.
Somatic embryo development and plant recovery . Although somatic embryo development of the SE-type can occur in the presence of auxins, the standard protocol for 27
avocado somatic embryo development involves the transfer of cultures onto medium
without auxin (Pliego-Alfaro, 1981; Mooney and Van Staden, 1987; Pliego-Alfaro and
Murashige, 1988; Witjaksono and Litz, 1999b). Pliego-Alfaro (1981) and Pliego-Alfaro
and Murashige (1988) reported the recovery of avocado somatic embryos after transfer of
embryogenic cultures onto semi solid MS basal medium. Raviv et al. (1998) reported
development of cotyledonary stage somatic embryos on semi solid maintenance media
supplemented with 9.04 pM 2,4-D and 2,2 pM BA. Witjaksono and Litz (1998b)
observed that SE-type cultures can form somatic embryos after explanting and in the
presence of picloram. Development of somatic embryos to maturity occurs with high
frequency are when PEMs explanted onto a modified MS medium with a high N03 :NH4
ratio and without 1 1 (1:0 3:1) auxins, 30 g l' sucrose, 6-7 g l' gellan gum and cultured in
darkness at 25°C (Witjaksono and Litz, 199b). The same study showed that high gellan
gum concentrations and high sucrose content reduce the number of hyperhydric somatic
embryos.
Pliego Alfaro (1981) reported that 5% of somatic embryos formed shoots.
1 1 Medium containing 1 to 3 g L casein hydrolysate, 0.1% activated charcoal or 10 mg l'
abscisic acid (ABA) failed to increase the number of normal embryos. Mooney and Van
Staden (1987) and Witjaksono and Litz (1999b) reported similar rates of shoot
development from somatic embryos. However, Raviv et al. (1998) reported that 11% of
somatic embryos develop shoots and 1% developed both a shoot and a root. According to Pliego-Alfaro (1981), the low rates of shoot development from avocado somatic
embryos are due to the failure of a clearly delineated plumule-radicle axis to develop.
Similar observations have been made by Mooney and Van Staden (1987) and Witjaksno and Litz (1999b). 28
Micropropagation of recovered shoots has been used to improve avocado plant
recovery from somatic embryogenesis. Witjaksono et al. (1999) using stem cuts and
shoot tips from micropropagated shoots reported a 100% survival when the explants were
cultured on MS semisolid medium containing 20 mM KN0 3 without NH4N0 3 although
40 mM total N concentration with a 2:1 ratio of KN0 3 :NH4N0 3 was found to be
optimum for shoot proliferation. The same study showed that CO 2 content in the
environment affects shoot development with better growth and accumulated higher
biomass in a -enriched C0 2 environment than under ambient C0 2 conditions, although
higher C0 2 assimilation (31 was observed in ±7) ambient C0 2 than in the enhanced-C0 2 conditions (17±2). Improvement of the number of somatic embryo-recovered plants has been achieved using micrografting (Raharjo and Litz, unpublished results). Shoot tips are micrografted onto decapitated seedlings germinated in vitro on MS medium
1 1 supplemented with 30 g l' sucrose, 100 mg l' myo-inositol and 4 mg f‘ thiamine HC1.
The micrografts are 2 1 cultured in translucent plastic boxes at 25°C with 50 pmol m" s' illumination provided by white cool fluorescent light. This technique has allowed the recovery of a large number of genetically transformed avocado plants that are now being evaluated under glasshouse conditions.
Protoplast isolation and culture
Protoplast isolation from non morphogenic avocado callus was attempted by
Blickel et al. (1986) in an attempt to study ASBVd replication and from mesocarp tissue by Percival et al. (1986) to study fruit ripening. Isolation, culture and plant regeneration from protoplasts isolated from embryogenic avocado cultures was achieved by 29
Witjaksono al. et (1998). Following their enzymatic isolation and purification, avocado
protoplasts were plated on MS medium supplemented with glutamine (MS 8P), 0.4 M
sucrose and liquid 5 2% coconut endosperm. A plating density of 1.6 x 10 was shown to
be optimum for development of PEMs after one month of culture. PEMs were used as
inocula for somatic embryo development by inoculating 1-2 mm diameter PEMs onto
somatic embryo development medium (SED) (Witjaksono and Litz, 1999b). White-
opaque somatic embryos ( 0.8 cm) that developed on SED were transferred onto
maturation medium where they enlarged to a 1.0- 1.5 cm in diameter before forming a
root and a shoot.
Genetic transformation
Agrobacterium tumefaciens-mediated genetic transformation of embryogenic avocado cultures was first reported by Cruz-Hemandez et al. (1998). Embryogenic cultures were induced from zygotic embryos of ‘Thomas’ and were maintained on semi solid and in liquid maintenance medium. PEMs were co-cultivated for three days with log-phase acetosyringone-activated A. tumefaciens harboring a co-integrative vector pMON9749 containing a kanamycin-resistance marker (nptll) gene and a reportable marker, i.e., /?-glucoronidase (GUS). The PEMs were cultured in liquid medium
1 1 supplemented with 50 mg l' kanamycin and 200 mg f cefotaxime for 2 months.
Kanamycin-resistant PEMs were transferred to liquid medium supplemented with 100 mg
1 1 1 kanamycin and 200 mg l' cefatoxime for 1-2 months. Although white-opaque somatic embryos were recovered, plants were not regenerated. 30
A gene for anti fungal protein (AFP) has been transferred into embryogenic
avocado cultures with the aim to produced plants with increased resistance to
phytophthora root rot (PRR) (Raharjo and Litz, unpublished data). In this case, PEMs
were co-cultivated with A. tumefaciens harboring the pGPTV-BAR-AFP binary plasmid
carrying a phosphinothricin acetyl transferase (bar) gene and a /Tglucoronidase (uidA)
reporter gene (Becker et ah, 1992). Transformed embryogenic cultures were selected for
resistance to phosphinotricin (ppt) and somatic embryos were recovered. The number of
transformed plants recovered was increased by micrografting somatic embryo-recovered
shoots onto in vitro germinated avocado seedlings. Transformed plants are currently
under evaluation for disease resistance. Embryogenic cultures have also been
transformed with A. tumefaciens harboring the pGPTV-BAR-Chi-Glu. This plasmid
harbors two genes, f -glucanase and chitinase, for pathogen resistance (Raharjo and Litz,
unpublished data).
Efendi (2003) transformed embryogenic avocado cultures with the A. tumefaciens
strain EFIA101 carrying the pAG4092 binary plasmid, which contains a SAMase gene
under the control of an avocado fruit ripening-specific cellulase promoter and the nptll
gene. Shoots from somatic embryos cultured on SED medium supplemented with
1 kanamycin levels as high as 400 mg F were recovered after 3-4 months.
Micropropagation
Micropropagation of avocado from nodal sections was attempted by Schroeder
(1980) using different cytokinins, who reported bud elongation and callus production at the cut end of the explants was reported. Pliego-Alfaro (1981) attempted shoot culture 31
with stem tips on MS medium supplemented with BA. Although shoot elongation was accomplished, die back and leaf abscision occurred at a high frequency. Skene and
Barlass (1983) stimulated multiple shoot proliferation from mature zygotic embryos of
1 ‘Fuerte’ on MS medium supplemented with 0.5 mg l" BA. Gonzales-Rosas and Salazar-
Garcia (1984) cultured nodal segments from seedlings of a West Indian cultivar on MS medium supplemented with IBA and kinetin, and attained multiple shoot proliferation in
>90% of the explants. Pliego-Alfaro et al. (1987) addressed micropropagation of adult avocado material of genotypes ‘GA-13’ and ‘IV-8’ using nodal explants on MS medium
1 supplemented with 1 mg l' BA; however, apical necrosis was a significant limiting factor in survival. In vitro rooting of shoots was demonstrated by pulsing shoots on medium
1 with 100 mg l' IBA for three days followed by subculture in MS medium without growth regulator. One hundred (100) % rooting was achieved after 1-2 months (Pliego-Alfaro,
1988). Pliego-Alfaro and Murashige (1987) tested the rooting capacity of young and adult shoots of ‘Topa-Topa’. Whereas 100% of the young shoots formed roots, no root formation occurred in adult material. Barringer et al. (1996) micropropagated in vitro- grown shoots from several types of avocado seedlings on MS medium supplemented with
4.4 pM BA and 0.05 pM NAA. Barcelo-Munoz et al. (1999) stimulated shoot proliferation from nodal explants from pruned ‘IV-8’ by alternating MS and B5 media differentially supplemented with BA. Rooting (90%) of shoots occurred with a double- phase (solid/liquid) MS medium supplemented with 4.9 pM IBA for three days followed
by subculture in medium without plant growth regulators. Witjaksono et al. (1999) reported that juvenile shoots of ‘Gvaran 13’ grew better and had a higher biomass accumulation when grown in a C02-enriched environment when compared to those
maintained under ambient CO2 conditions. 32
Viroids
Although the first report of a disease that later was associated with a viroid
infection occurred in 1922 (Martin, 1922), the description of viroids as a new plant
pathogen occurred only in 1971 (Diener, 1971). Most of the diseases known today to be
caused by viroids were earlier believed to be the result of virus infections; however,
Diener and Raymer (1967) reported that the infectious principle of potato spindle tuber
disease had a significantly lower sedimentation rate than viruses and was sensitive to
ribonuclease. A similar study with citrus exocortis-affected tissue reported that the
causal agent of this disease was probably not a virus because of the low sedimentation rate, sensitivity to ribonuclease and inability to detect virus particles using electron microscopy with infected tissue (Semancik and Weathers, 1968). Zaitlin and
Hariharasubramanian (1970) reported that no protein resembling a virus coat protein was observed in potato spindle tuber-affected tissues. Finally, Diener (1971) using density gradient tests combined with polyacrylamide gel electrophoresis, reported that the causal agent of potato spindle tuber disease is not a virus but is a low molecular weight RNA.
He designated it as the potato spindle tuber viroid, and proposed the term viroid for all pathogenic nucleic acids with similar characteristics.
Viroids are currently defined as unencapsidated, single-stranded, circular RNA molecules with a size between 246 and 375 nucleotide residues (Keese and Symons,
1987). Viroids form rod-like structures that resemble a double strand molecule as a result of the high molecular base-pairing, and with small loops and branches (Riesner, 1987).
With the exception of chrysanthemum chlorotic viroid (CCHVd) and peach latent mosaic viroid (PLMVd) that adopt branched structures (Hernandez and Flores, 1992; Navarro 33
and Flores, 1997), most viroids have two terminal (left and right) regions and a central
(stem) region (Keese and Symons, 1985). Viroids are not known to code for any protein
sequence; therefore, they rely completely on the host for carrying out their basic activities
(Davies et al., 1994).
Since potato spindle tuber viroid (PSTVd) was associated with potato spindle tuber
disease, many other viroids (28) have been reported to infect different plant species.
Viroids have been classified based on their molecular, structural and biological characteristic in two families: Pospiviroidae and Avsunviroidae (Flores et ah, 1998)
(Table 2-4).
The Pospiviroidae are characterized as having a central conserved region (CCR)
(Keese and Symons, 1985), replication using an asymmetric pathway (Hutchins et ah,
1986) and replication in the nucleus (Muhlbach and Sanger, 1979). In contrast,
Avsunviroidae viroids do not have a central conserved region (Symons, 1981), replicate using a symmetric pathway (Daros et ah, 1994) most probably in the chloroplasts
(Bonfiglioli et ah, 1996; Navarro et ah, 1999), and have the ability to self-cleave using hammerhead ribozymes (Hutchins, et ah, 1986; Hernandez and Flores, 1992; Navarro and Flores, 1997).
Viroids can cause different symptoms ranging from severe to asymptomatic. The nature of the symptoms depends on the host, environmental conditions and viroid sequence (Singh and Slack, 1984; Herold et ah, 1992; Hammond, 1992). Viroid diseases can affect agricultural production by causing losses in whole plants or in the tissues that are the main product of the species. Viroid can cause death of plants and become epidemics; coconut cadang-cadang viroid killed millions of palm trees in Philippines, causing enormous losses (Price, 1971). 34
Table 2-4. Viroid Classification (Flores et al., 1998)
FAMILY GENUS SPECIES
PSTVd (potato spindle tuber) TCDVd (tomato chlorotic dwarf) MPVd (Mexican papita) POSPIVIROID TPMVd (tomato planta macho) CSVd (chrysanthemum stunt) TASVd (tomato apical stunt)
IrVd (iresine I)
HOSTUVIROID HSVd (hop stunt) |
CCCVd (coconut cadang-cadang) COCADVIROID CTiVd (coconut tinangaja) POSPIVIROIDAE HLVd (hop latent)
ASSVd (apple scar skin)
CVd-III (citrus III)
ADFVd (apple dimple fruit)
GYSVd 1 (grapevine yellow speckle 1) APSCAVIROID GYSVd 2 (grapevine yellow speckle 2) CBLVd (citrus bent leaf) PBCVd (pear blister canker)
AGVd ( Australian grapevine)
CbVd 1 (coleus blumei 1) COLEVIROID CbVd 2 (coleus blumei 2) CbVd 3 (coleus blumei 3)
AVSUNVIROID ASBVd (avocado sunblotch) AVSUNVIROIDAE |
PLMVd (peach latent mosaic) PELAMOVIROID CChMVd (Chrysanthemum chlorotic mottle
Sometimes the plants are not killed but become unproductive; chrysanthemum
plants affected with chrysanthemum chlorotic mottle viroid (CCHMVd) become
bleached, dwarfed and the flowers are considerably reduced in size (Dimock, 1 947). The
symptoms can appear on the fruits or on other commercial part of a plant e.g., potato
tubers (Diener, 1971) and avocado (Coit, 1928). 35
Viroid transmission can occur in several ways, i.e., seeds (Hunter et al., 1969)
contaminated tools (van Dorst and Peters, 1974), pollen (Desjardins et al., 1979) and
aphids (Galindo et al., 1986). Control mechanisms for infected plants in the field are not
available and eradication is the only procedure to avoid spread of viroid infections.
However, in vitro techniques have been reported to allow the recovery of healthy plants
from infected material; these results will be discussed in Chapters 3 and 4.
Avocado Sunblotch Disease Syndrome
Origin and symptoms
The disease known as avocado sunblotch disease syndrome was first reported in
California in 1928 and was believed to be a consequence of sunburn on weak trees, or
trees that suffer seriously from neglect (Coit, 1928). At that time, it was considered that
excessive exposure of certain tissues to sun rays could cause chimeric shoots and fruits
and the affected condition could be transferred to the progeny and other parts of the plant.
Sunblotch was observed to equally affect all avocado cultivars and no resistance to
this disease has been observed (Coit, 1928; Home and Parker, 1931). The initial
symptoms of the disease include pendulous or weeping branches with scaly bark,
yellowish depressed streaks or stripes on twigs or young branches and sunken areas of
yellow, white, reddish-yellow or red color on fruits sometimes associated with fruit
dwarfing and shape distortion (Coit, 1928). An affected tree can have a stunted or
irregular growth with reduced yields and progressive decline in health (Dale et al., 1982).
Diseased branches appear dwarfed and drooping, with short, slender shoots bearing 36
unequal distribution of leaves. In addition, leaf distortion can occur at the base or one
edge, and bleaching and variegation affect midribs areas between the veins (Home and
Parker, 1931; Stevens and Piper, 1941). Diseased asymptomatic trees and trees that
appear to have “recovered”, i.e., becoming asymptomatic, have been reported, increasing
the opportunity for spread of the disease. Differential and erratic symptom expression
has been related to host:pathogen interactions and effect of environmental conditions
(Home and Parker, 1931, Da Graca and VanVuuren 1981; Semancik and Szychowsky,
1994; Schnell et al., 2001).
Causal agent
The first report of this disease attributed the symptoms to a genetic disturbance
caused by the direct effect of sunburn comparable to the effect of X-rays applied to seeds
(Coit, 1928). Home and Parker (1931) observed that the affected condition could be
transferred from infected scions to healthy rootstocks. Based on this observation,
avocado sunblotch was included in the group of plant diseases called “infectious
chloroses”. Further inoculation experiments eliminated the possibility that either of fungi
or bacteria was the causal agent, but instead suggested that a vims was the most probable
infectious agent (Parker and Home, 1932). The belief that a vims was the causal agent of
avocado sunblotch was supported after an infectivity study showed the graft
transmissibility of the disease in avocado embryos (Home et al., 1941).
The causal agent was identified as a viroid using nucleic acid hybridization with a
32 P-labeled complementary DNA probe, and it was designated as the avocado sunblotch
viroid (ASBVd) (Palukaitis et al., 1979). Further infectivity studies involving the use of 37
electron and light microscopy, thermotherapy to inactivate the infectious agent and
differential centrifugation supported the likely viroid nature of the causal agent of the
avocado sunblotch syndrome (Desjardins et al., 1980). Finally, a study where highly purified ASBVd from infected tissue was inoculated into healthy plants and was able to
induce avocado sunblotch symptoms established that ASBVd was the actual causative
agent of this disease (Allen et al., 1981).
Sequencing of the ASBVd molecule demonstrated that the viroid was a 247-residue single stranded circular RNA molecule with a rod-like secondary structure, and only an
18% sequence similarity to potato spindle tuber viroid (PSTVd) and citrus exocortis viroid (CEVd) (Symons, 1981). Figure 2.1 shows the rod-like secondary structure of
ASBVd with a branched left terminal region and the areas of formation of the hammerhead ribozymes (underlined) (Navarro and Flores, 2000)
Since the first nucleotide sequence (J02020) was published, >50 ASBVd sequence variants have been reported. Pallas et al. (1988) sequenced 4 variants from a ‘Fuerte’ tree
displaying symptoms; the variations were observed at nucleotides 3, 5 and 7 in the left terminal region and between nucleotides 122-128 in the right terminal loop. Sixteen new
ASBVd variants were reported from three different avocado trees with changes in both terminal regions and the stem area (Rakowsky and Symons, 1989). Fifty-eight (58) new
ASBVd sequence variants were isolated from six avocado trees planted at the NGR in
Miami, FL (Schnell et al., 2001). The study showed that most of the new ASBVd variants were exclusive to the tree from which they were isolated.
Multiple sequence variants affecting a single host is a phenomenon that has been
associated with the quasispecies evolutionary concept (Duarte et al., 1994, Ambros et al.,
1999). This concept posits that multiple sequence variants result from the host:pathogen 38
interaction in a specific environment, where a natural selection process is exerted over the
variant population allowing the best fitted sequence to overgrow and predominate over the others. Trees with a long history of ASBVd infection provide an ideal scenario for
this process to take place (Schnell et al., 2001).
U G U A 1-U A 247- G C U A U A C G 120 U- A 80 A 40 AC UU U A u 20 / 100 G(J U i U lliU — G Sg G U U C AG UGAGGAUA GA UAAACUUUGU GAC CAG GU CU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA CG GGUGAACU ACUUCUAU CU AUUUGGAACG CUG GUC CA GA AGGGU GAAAGG GACUU CUG CACU CUCU CUUCU GC UCACUUGA - 240 i -A U CC CU A a va a a/ iA/ r/ncunA u aa i u ( r A- U 220 y 200 A A C G 180 a A U U U U U U U
Figure 2-1. ASBVd sequence and secondary structure showing the areas of hammerhead formation in the plus (+) strand (Navarro and Flores, 2000)
Nucleotide exchanges in the ASBVd molecule have been associated with symptom expression. Foliar symptoms have been classified as bleached (B) variegated (V) or symptomless carrier (Sc) (Semancik and Szychowski, 1994). Exchanges and additions of
A or AA in the 120-128 poly-A stretch, a U addition between nucleotides 115-118 and
UU to AA exchanges at positions 120-121, all result in a pronounced enlargement in the right terminal loop region and are associated with bleached symptoms. Variegated
symptoms have been associated with a single A nucleotide addition at the same 122-128 poly-A right terminal region which produces a less pronounced enlargement. No
differences have been observed between sequences isolated from asymptomatic infected tissue and the wild type sequence J02020 (Semancik and Szychowski, 1994). 39
A study examining symptomatic and asymptomatic tissues found that bleached symptoms appear to be exclusively associated with additions of U or UU between nucleotides 115-118 but not to an increasing number of A residues between nucleotides
122-128 (Schnell et al., 2001). Moreover, no A to U exchanges at position 1 19 or UU to
AA at nucleotides 120-121 were observed in this study, and sequences showing additions of A residues at the 120-128 poly-A region were isolated from asymptomatic tissues.
Viroids are classified according to functional properties, sequence similarities and structures in two broad families: Pospiviroidae and Avsunviroidae (Koltunow and
Rezaian, 1989; Flores et al., 1998) (Table 2-4). Members of the Pospiviroidae family have a central conserved region, do not self-cleave and localize and replicate in the
nucleus, (Flores et al., 2003). In contrast, ASBVd and the other two member of the
Avsunviroidae family do not have a central conserved region, have the ability to self- cleave in vivo and in vitro by forming hammerhead ribozyme structures that are immersed in their molecules and, at least ASBVd and PLMVd replicate and accumulate in the chloroplasts (Keese and Symons, 1985; Hernandez and Flores, 1992; Daros et al.
1994; Navarro and Flores, 1997).
Viroid replication occurs through a rolling circle mechanism and using a symmetric or asymmetric pathway that involves RNA transcription, processing of multimers and
ligation to form the circular molecule (Sanger et al., 1977; Branch and Robertson, 1984).
In contrast to Pospiviroidae viroids that follow the asymmetric pathway, Avsunviroidae viroids replicate using the symmetric pathway, in which a circular plus (+) strand is transcribed into linear multimeric minus (-)-strands that in a second step are processed and ligated to monomeric circular (-)-strand RNA, which therefore serves as the template 40
for the synthesis of the linear multimeric (+)-strand RNAs (Daros et al., 1994; Hutchins
et ah, 1986).
Studies using a cell-free system to localize viroid in different cellular compartments
have found that members of the Pospiviroidae family localize and replicate in the nucleus
(Flores and Semancik, 1982). In situ hybridization using a biotin-labelled RNA probe in
avocado infected leaves found that ASBVd localizes in the chloroplasts, where it is
associated with the thylakoid membranes (Bonfiglioli et ah, 1994).
Navarro et ah (1999), using polyacrylamide gel electrophoresis (PAGE) and
Northern-blot hybridization with nuclear and chloroplast fractions isolated from ASBVd-
infected mesophyll cells and separated by differential centrifugation, also found that most
of the ASBVd transcripts are associated with the chloroplastic fraction. Monomeric,
dimeric, subgenomic and double-stranded ASBVd complexes of both strands were
isolated from the same organelle, indicating that chloroplasts are the most likely
replication sites of ASBVd (Navarro et ah, 1999).
Viroids do not have coding capacity for any protein; therefore, they rely on the host biological machinery to perform the replication process (Davies et ah, 1974). Studies to
determine the kind of enzymes involved in viroid replication have been conducted using
inhibitors that block enzyme activity at specific concentrations. Alpha (oc)-amanitin is a
fungal toxin that at 1 and 200 pg/ml, respectively, has the ability to inhibit the activity of
the DNA-dependent RNA polymerases II and III in animal and plant systems (Marzuff and Huang, 1984; Cox and Goldberg, 1988). Low concentrations of a-amanitin have been shown to inhibit replication of potato spindle tuber viroid (PSTVd), hop stunt viroid
(HSVd) and citrus exocortis viroid (CEVd), indicating the possible involvement of RNA polymerase II in the replication of these viroids (Mulbach and Sanger, 1979; Yoshikawa 41
and Takahashi, 1986; Flores and Semancik, 1982). Marcos and Flores (1992), using cells
isolated from ASBVd-infected avocado leaves, reported that ASBVd replication is
unaffected by the presence of 1 and 200 pg/ml of a-amanitin, eliminating the possibility
that RNA polymerase II and III could be involved in ASBVd replication.
Tagetitoxin is a bacterial phytotoxin that inhibits the activity of the plastid-encoded
RNA polymerase enzyme that transcribes chloroplastic genes (Mathews and Durbin,
1990). In a study where isolated avocado chloroplasts from ASBVd-infected leaves were
treated with 3 pM of tagetitoxin, the transcription of 4 choroplastic genes was completely
inhibited; however, concentrations of tagetitoxin as high as 100 pM did not affect
ASBVd replication (Navarro et al., 2000). Since tagetitoxin inhibits plastid-encoded
polymerase (PEP), but has no effect on nuclear-encoded polymerase (NEP) (Liere and
Maliga, 1999), a NEP or a different tagetitoxin-resistant PEP could be the enzyme
involved in polymerization of ASBVd. That a nuclear-encoded RNA polymerase may be
involved in ASBVd replication comes from the study of Navarro and Flores (2000); the
linear (+) and (-) ASBVd-transcripts are initiated with a UAAAA sequence in both
strands. This sequence is similar to the promoter region of the chloroplastic genes rpoB
and accD. The gene rpoB codes for the plastid encoded polymerase /Tsubunit of a
nuclear-encoded RNA polymerase, while accD codes for the alpha (oc) subunit of the
Acetyl-CoA carboxylase enzyme, but both genes are synthesized by a nuclear encoded
RNA polymerase.
Using a different experimental approach, ASBVd- infected avocado leaves were
UV-irradiated to cross-link proteins that are associated with ASBVd in vivo (Daros and
Flores, 2002). The results showed that at least two proteins were isolated forming covalent adducts with ASBVd (+) strands. The two products were designated as 42
PARBP33 and PARBP35, and belong to a family of nuclear-encoded chloroplastic RNA-
binding proteins that catalyze different processes in the post-transcriptional metabolism
of chloroplast RNAs (Ohta et al., 1995; Schuster and Gruisem, 1991; Nakamura et al.,
2001; Hirose and Sugiura, 2001). The report suggested that the two isolated adduct
proteins can act as facilitators of ASBVd replication by enhancing self-cleavage (Daros
and Flores, 2002).
Transmission
ASBVd can be transmitted via seeds, and transmission in seedlings has been
observed to increase when seeds come from asymptomatic or “recovered” infected trees
(Wallace and Drake, 1962). Pollen transmission was experimentally demonstrated by
caging a healthy tree with an ASBVd-infected cultivar that served as pollinator; several
seedlings that originated from seeds of the healthy tree showed symptoms of infection
(Desjardins et al., 1979). Mechanical infection using tools or infected plant material is
and probably the most common method of ASBVd transmission. ASBVd-infected buds
grafted onto healthy rootstocks were able to induce symptom display in new shoots that
originated from the rootstock (Home and Parker, 1931). Similar results were observed
when infected, macerated tissue was placed under the bark of healthy trees or bark
patches from infected trees were grafted on healthy plants (Parker and Home, 1932;
Bums et al., 1968; Da Graca, 1978). ASBVd transmission via infected tools has also
been reported (Desjardins et al., 1980); however, immersion in formaldehyde (2%),
sodium hypoclorite and hydrogen peroxide (6%) is effective for disinfesting the tools and
preventing contamination of healthy plants (Desjardins et al., 1987). 43
Although avocado sunblotch transmission via root grafting in the field is very
likely (Whitsell, 1952), it has not been experimentally demonstrated; however, presence
of the viroid in avocado roots has been reported, and mechanical transmission from
diseased to healthy root material has been observed (Home et ah, 1941).
Indexing
Visual diagnosis of ASBVd in the field is difficult due to the irregular appearance
of symptoms, and occurrence of asymptomatic trees (Coit, 1928; Home and Parker,
1932; Semancik and Szychowski, 1994; Schnell et ah, 2001). The first indexing methods
to detect ASBVd were based on bioassays consisting of grafting of buds or bark patches
onto indicator seedlings; however, the method was impractical due to the long time
needed for symptom development and low reliability due to the occurrence of
asymptomatic infections (Bums et al., 1968; Da Graca and Van Vuuren, 1978). Grafting
of avocado embryos with adult shoots was attempted to accelerate the indexing
procedure; however, this technique has not been utilized routinely (Drake and Wallace,
1974). Increased temperature ranges (day/night:38/28°C) shortened the time for bioassay
symptom development in symptomatic infections to 8 months after inoculation, while
those maintained a lower temperatures (day/night:20/18°C) required 2 years for symptom
development (Da Graca, 1981). Even so, asymptomatic infection remained as a limiting
factor.
Polyacrylamide gel electrophoresis (PAGE) not only allows isolation of highly
purified viroid RNA but also improves reliability and efficiency of viroid indexing
(Hanold et al., 2003). Polyacrylamide tube gels have been used to detect ASBVd- 44
infected trees that have been previously reported as being free of the viroid (Palukaitis et
al., 1979). Although far better than bioassay indexing methods, PAGE still presents some sensitivity challenges to detect ASBVd in some tissues. Uthermohlen (1981), using
PAGE, readily detected ASBVd in symptomatic trees; however, he failed to detect the infection in a tree that had “recovered” from the symptoms. In addition, Da Graca (1983) reported that PAGE results varied with respect to the tissue used for indexing, i.e., flower tissue was found to be a better source of material for ASBVd indexing than leaf tissue.
Nucleic acid hybridization using labeled complementary probes has been used with good results for ASBVd indexing (Muhlbach et ah, 2003). Palukaitis et al. (1979, 1981) efficiently detected ASBVd using hybridization analysis with P-complementary DNA probes prepared against purified ASBVd RNAs. Similar results were reported by Bar-
Joseph et al. (1981). Mclnnes et al. (1989) detected ASBVd in infected plants using
photobiotin-labelled DNA. Lima et al. (1994) and Bonfiglioli et al. (1994) used a digoxigenin-labelled RNA probe complementary to ASBVd and observed that most of the hybridization signal was detected in the chloroplasts. Radioactive probes were the most sensitive and reliable for detecting ASBVd. Because of the problems associated with their use, radioactively-labeled probes have never been considered routine for
indexing large numbers of avocado plants (Schnell et al., 1995).
Molecular biology has permitted the development of improved and more time- efficient methods for viroid indexing. Reverse transcription-polymerase chain reaction
(RT-PCR) is a technique that synthesizes and amplifies a cDNA sequence from an RNA
template (Freeman et al., 1999). RT-PCR has been shown to be more sensitive and time efficient than PAGE for detecting different viroids. CEVd, citrus viroid II (CVdII) and citrus cachexia viroid (CCaVd) sequences were detected and amplified from nucleic acid 45
extracts of infected citrus plants using primers complementary to the CCR and were
visualized with PAGE (Yang et al., 1992). A similar procedure was applied to isolate
sequence variants of citrus viroids (Visvander and Symons, 1985).
The use of RT-PCR as an indexing method for ASBVd detection was first reported
by Schnell et al. (1995), and was used to estimate the number of avocado accessions
infected at the NGR (Ronning et al., 1996). The study showed that 81 of the 429
(approx. 19%) accessions in the collection were ASBVd-infected. RT-PCR was later
improved to function with partially purified nucleic acids and to detect ASBVd in tissues
where other indexing methods showed less sensitivity, e.g., asymptomatic old and young
leaves, using only 1 ng of total nucleic acids in the RT-PCR reaction (Schnell et al.,
1997). Recent RT-PCR applications have involved the use of labeled primers and
capillary electrophoresis, which have increased the sensitivity of the technique and
permitted detection of ASBVd variants through single-strand conformation
polymorphism (Schnell et al., 2001).
Control
Since the first reported cases, avocado sunblotch was observed to equally affect
cultivars from the three botanical varieties (Coit, 1928; Home and Parker, 1931). This
observation remains valid since no genetic resistance among avocado cultivars has been
reported, and control of the disease has relied on preventing the propagation of affected
material (Coit, 1928; Home et al., 1941). Chemical control of the disease in affected
plants is not available; however, preventive measures, i.e., disinfection of grafting tools
with chemicals can help to avoid spread of the problem (Desjardins et al., 1987). Legal 46
measures to prevent introduction of viroid-infected material are enforced by government
agencies in Europe, U.S.A. and Canada; however, ASBVd is not listed for quarantine
treatment or certification importance (Barba et al., 2003). Self-motivated control
measures by nurseries and grower associations can have a significant effect on
maintaining spread of ASBVd at a low level. In Australia, a voluntary sanitary
certification system that certifies mother trees as being free of several pathogens,
including ASBVd, has been adopted by the avocado nursery industry. The system
focuses on periodical indexing of mother trees only; however ASBVd indexing of young
trees for sale can also be performed at buyer’s expense (Whiley, 2003; Newett et al.,
2003). In California, growers are recommended to purchase the trees from nurseries with
certified ASBVd-free mother trees; however, neither California nor Florida require a
legal ASBVd-free certification for nursery operation.
Cell Culture Studies Involving Viroids
Cell culture techniques have been used to study viroid disease and as a means for their elimination from infected material. In an attempt to study avocado sunblotch viroid
(ASBVd) under in vitro conditions, Desjardins (1958) reported growth of callus from stem segments of avocado; however, callus production from root explants was unsuccessful. The elimination of viral pathogens from nucellar plants recovered from
infected trees was initially reported by Weathers and Calavan (1959). Bitters et al.
(1972) cultured nucellar tissue from citrus infected with several viruses and citrus exocortis viroid. The recovered plants were free of CEVd and the viruses based on tests conducted with indicator plants. Muhlbach and Sanders (1981) demonstrated potato 47
spindle tuber viroid (PSTVd) infection in callus culture of wild potato (Solarium
denissum ) and tomato (Lycopersicum sculentum), and cell suspensions of Solarium
tuberosum. Infection of the cultures was detected >1 year after inoculation using polyacrylamide gel electrophoresis (PAGE) and, PSTVd replication was demonstrated by
32 incorporation of P labeled nucleotide into newly synthesized viroid RNAs.
Blickel et al. (1988) attempted to study ASBVd replication in vitro using stems of
1 1 avocado seedlings on MS medium supplemented with 1 mg f IAA and 0.3 mg f BA;
the study reported the growth of callus tissue on the end-cut of the explants; however no
data on ASBVd replication was reported. In order to develop an efficient protocol for
indexing potato germplasm, protoplasts were isolated from mechanically inoculated
potato plants with cDNAS of PSTVd, potato virus X (PVX), potato virus Y (PVY) and
potato leaf roll virus (Hopp et al., 1991). The study showed that simultaneous indexing
of the 4 pathogens was possible using nucleic acid hybridization. Semancik et al. (1993)
isolated CEVd from callus of a tomato hybrid (Lycopersicum sculentum x .L peruvianum )
that had been mechanically inoculated. The study was conducted in order to compare
CEVd sequence variability from the same sequence inoculated into citron ( Gynura
aurantiaca) leaves, the tomato hybrid plants and the callus tissue. In order to compare
growth patterns, tomato cell cultures of infected and non PSTVd-infected cells were
established. There were no growth differences between the two type of cells; however,
PSTVd-infected cells were more efficient in biomass accumulation and showed greater
longevity (Duran-Vila et al., 1995).
Avocado protoplasts were isolated from ASBVd-infected plants to study the site
of viroid replication (Navarro et al., 1999). In this study, isolated protoplasts were used
for nucleus and chloroplast isolation rather than for cell culture. Protoplasts were isolated 48
from cells of Nicotiana benthamiana in order to study PSTVd structural characteristics and replication (Qi and Ding, 2002). Isolated protoplasts were further electroporated with PSTVd RNA transcripts and infection occurred in 60 to 70% of the cells. The study showed that nucleotide variations occur soon after inoculation without altering replication levels.
Micrografting and Meristem Culture Studies Involving Viroids
Micrografting and meristem tip culture have been employed to eliminate viroids
from infected material. The first report of pathogen elimination using in vitro culture of meristematic tissues occurred when White (1934) obtained free-virus root cultures while attempting to multiply a plant virus in tomato roots. White noticed at that time that in order to maintain infected tissue larger segments of roots than the tip alone were required.
The first application of White’s observation to a commercial crop was accomplished when dahlia and potato plants were freed of viruses using meristem tip culture (Morel and Martin, 1952; 1955).
Viroid elimination using meristem tip culture was reported by Hollings and Stone
(1970) with chrysanthemum based on the absence of symptoms in recovered plants. The meristem tips were isolated from plants maintained at 35°C for at least 14 weeks. Two out of 337 initial plants were reported to be freed of the viroid. Nucellar culture was used to recover citrus exocortis viroid (CEVd)-free plants from plants infected with this viroid
and other citrus viruses (Rangan et al., 1968; Bitters et ah, 1972). However, nucellar plants reversed to the juvenile phase and required a long time to flower and fruit. 49
Murashige et al. (1972) showed that micrografting could be used as an alternative
to recover clonal viroid and virus disease free plants without causing reversal to the
juvenile phase. The study showed that plants regenerated from the apical meristem plus
4-6 leaf-primordia micrografted onto decapitated in vitro germinated seedlings were freed
of CEVd based on absence of symptoms on indicator plants; moreover, plants flowered
within one and half year after being micrografted. This study was confirmed when citrus
micrografts that originated from plants infected with CEVd and tristeza and psorosis
viruses all tested negative for these pathogens using indicator plant indexing (Navarro et
al, 1975). Micrograft-recovered plants flowered and fruited within 1 year after
micrografting. Citrus exocortis viroid elimination using micrografting was attained by
Monteverde et al. (1987), who used the meristem dome plus 2-3 leaf-primordia from
adult plants infected with CEVd and micrografted onto healthy rootstocks. The results
based on indicator plant assays showed that all recovered plants were freed of CEVd.
Reports of viroid elimination using meristem tip culture are associated with cold
and thermotherapy treatments. Lizarraga et al. (1980) reported PSTVd elimination in
potato plants recovered after cold treatment and meristem culture. The shoot-tips,
consisting of the meristem plus one primordial leaf, were isolated from PSTVd-potato
plants maintained at 5°C for at least 3 months and cultured on MS medium. PSTVd
elimination was based on absence of symptom on indicator tomato plants and gel
electrophoresis. Paludan (1984), using cold treatment and meristem-tip culture, reported elimination of chrysanthemum stunt viroid (CSVd) and chrysanthemum chlorotic mottle viroid (CCMVd) in recovered chrysanthemum plants. The shoot tips with one or few
leaf-primordia were isolated from infected plants or in vitro - grown plantlets maintained at 5°C for 5 months and the results were based on absence of symptoms on the recovered 50
plants. Adams et al. (1996) reported meristem culture for elimination of hop latent viroid
(HLVd) in plants recovered from meristem tips (<0.5 mm). Viroid elimination occurred only with those plants recovered from setts maintained in darkness at 2°C for at least 6 months.
Most of the above results were based on the absence of symptoms in the recovered plant or indicator plants, an indexing procedure that is not reliable for viroid diseases due to the possibility of asymptomatic infections (Hollings et al., 1965; Whitsell,
1952; Visvander and Symons, 1985; Semancik and Szychowsky, 1994). Even very sensitive biochemical indexing procedures can fail to detect low levels of viroid
infections. The recovery of healthy grapevine plants was reported from meristem tips
(0.1 to 0.2 mm) that were isolated from plants infected with several viroids (Duran-Vila
et al., 1987) based on polyacrylamide gel electrophoresis (PAGE) indexing; however, a
later study using more sensitive RT-PCR indexing showed that grapevine plants that were
supposed to be “clean” were still infected but with low viroid levels that were undetected
by PAGE (Wan and Symons, 1997).
Thermotherapy has been used to eliminate viroid infections and results have been
confirmed by highly sensitive RT-PCR indexing. Apple scar skin viroid (ASSVd)
elimination was reported using a combination of thermotherapy and meristem tip culture
(Postman and Hadidi, 1995). Meristem tips (0.2-0. 8 mm long) from shoots maintained
for 49 days at 4°C or 55 days at 38°C were utilized. ASSVd elimination was also
reported by Howell et al. (1998), but infected stock plants were maintained at 38°C for 70
days. Thermotherapy success varies according to the characteristics of the viroid. Apple
skin scar viroid accumulates between 18°C and 28°C but not at 38°C (Skrzeczkowsky et
al., 1993). In contrast, potato spindle tuber viroid (PSTVd) requires temperatures >100°C 51
to completely lose infectivity (Singh and Bagnall, 1968) and citrus exocortis viroid is active at 1 10°C (Semancik and Weathers, 1968). The exact point of thermal inactivation of ASBVd is unknown; however, attempts to inactivate it using thermotherapy revealed that heat treatments that were able to kill seeds, budwoods and budlings infected with
ASBVd had no effect on ASBVd inactivation (Desjardins et al., 1980).
General conclusion of the literature review
Avocado sunblotch is an important disease of avocado, and is caused by the
avocado sunblotch viroid (ASBVd). ASBVd affects all avocado genotypes and affected
plants are eradicated since no cure is known. Nucellar tissue culture and micrografting
have been used to eliminate citrus exocortis viroid (CEVd) and healthy citrus plants have
been recovered from infected material. Protocols for the recovery of avocado plants from
the nucellus by somatic embryogenesis and micrografting have been described
previously. The objective of this research has been to recover healthy avocado plants
from ASBVd infected material. CHAPTER 3 MICROGRAFTING OF ASBVd-INFECTED PLANTS
Introduction
Avocado sunblotch viroid (ASBVd) is a member of the Avsunviroidae family of
viroids and causes sunblotch disease of avocado (. Persea americana Mill.) (Palukaitis et
al., 1979). ASBVd affects fruits, leaves, and possibly roots, and the levels of infection in
the plant have been observed to vary according to the tissue of origin and environmental
conditions (Coit, 1928; Whitsell, 1952; da Graca and Moon, 1983; Schnell et al., 1997).
Infected plants are sometimes difficult to recognize visually since asymptomatic trees
occur (Semancik and Szychowsky, 1994) and in some cases infected plants have
appeared to become healthy after indexing. This has been interpreted as being due to
unequal distribution of the viroid, lack of efficiency of the indexing technique and
possible silencing mechanism of the host on the viroid (Olano et al., 2003)
Transmission of ASBVd can occur via seeds, vegetative planting material, pollen and contaminated tools (Parker and Home, 1932; Wallace and Drake, 1962; Desjardins et al., 1980; 1987). Insect transmission has not been reported and the only effective control measure is eradication of affected plants. Based on indexing procedures of low sensitivity, micrografting and meristem tip culture-recovered plants from viroid-infected material have been reported to eliminate viroids of the Pospiviroidae family in citrus,
potato, grape and hop (Murashige et al., 1972; Lizarraga et al., 1980; Duran-Vila et al.,
52 53
1988; Adams et al., 1996). Therefore, micrografting was studied as a means of
eliminating ASBVd from infected material.
Materials and Methods
Plant Material
All avocado (Persea americana Mill.) plant material was obtained from the
National Germplasm Repository (NGR) at the USDA-SHRS in Miami, FL. Seedlings to
be used as rootstocks were obtained from seeds of ASBVd-free ‘Simmonds’ (Miami#
7831) and ‘Wilson Popenoe’ (Miami # 20032). Scion-donor seedlings were obtained
from seeds of ASBVd-infected ‘Donaldson’ (Miami# 20024) and ‘Vero Beach’ GRD
(Miami# 20536).
Seedling Germination
The seeds were removed from mature fruits, the seed coats were removed and
surface-disinfested for 20 min in a 1.5% (v/v) sodium hypochlorite solution containing 5
1 drops of Tween-20® L' and rinsed with three changes of sterile deionized water. Under
axenic conditions in a laminar flow hood, each surface-disinfested seed was bisected
longitudinally and the zygotic embryo was removed 3 with approx. 1 cm portion of a
cotyledon (Pliego-Alfaro and Murashige, 1987). The excised embryos were cultured in
Petri dishes (1 10 x 20 mm) containing 20 ml of semi solid avocado germination medium
(AGM) (Pliego-Alfaro and Murashige, 1987). AGM consisted of Murashige and Skoog 54
(1962) (MS) medium 1 supplemented with (in mg l' ): sucrose (30,000), myo-inositol
(100), thiamine HC1 (0.4) and TC agar (8,000) (Carolina Biological). The medium was
autoclaved 2 at 121°C at 1.1 kg cm' for 15 min and dispensed as indicated above. One
zygotic embryo was inoculated in each Petri dish, the dishes were sealed with Parafilm®
and stored in translucent plastic boxes at 25 °C with 16 h light provided by cool white
fluorescent tubes at 40-50 1 pmol m'Y . A total of 63 embryos of ‘Vero Beach’ GRD, 90
of Simmonds 135 of Donaldson , and 120 of ‘Wilson Popenoe’ were explanted on
AGM. The percentage of germination was calculated for each cultivar 1-2 weeks after
inoculation.
Micrografting
Prior to micrografting, all leaves except the 2-3 smallest leaf-primordia were removed from the scion. For the rootstocks, the leaves were removed from 2-3 cm long seedlings and all lateral shoots were removed, leaving a single shoot. Using a sterile razor blade as a scalpel, the rootstock was decapitated approx. 1 cm above the medium surface. Immediately, and with a different scalpel, a shoot-tip (meristem plus 2-3 leaf primordia) was excised and placed on the decapitated seedling. Since the diameter of the rootstocks was greater than that of the scions, the latter were placed off-center and in contact with the vascular ring (Navarro et al., 1975). In order to prevent cross- contamination, the scalpel used in each cut was dipped in a 1.25% (v/v) sodium hypochlorite solution, air-dried, dipped in 95% ethanol and flamed. At any point, the same scalpel was used to make two consecutive cuts. To avoid dehydration of the micrografted shoot-tip, a piece (approx. 2 mm thick) of semisolid AGM was placed next 55
to the union area but not between the rootstock and the scion. Thereafter, the Petri dish
was covered, sealed with Parafilm® and stored as indicated before. All manipulations
were performed within a laminar flow hood and using a dissecting microscope. Each
micrograft was marked with the same number of the scion-donor seedling for further
indexing correlation. A total of 136 micrograftings were performed: 40 of ‘Vero Beach’
GRD onto ‘Simmonds’ and 90 of ‘Donaldson’ onto ‘Wilson Popenoe’. Two different
sizes of scion were evaluated in each micrografted pair: half of the experimental units
were performed with scions < 0.5 mm long and the other half with scions 0.5 mm but
never larger than 1 mm long. The number of successful micrografts was recorded 5-6
weeks after the micrograftings were performed and the percentages of success with
respect to genotype and scion size were recorded.
Micrograft growth and development
Micrografted plants were transferred into 150 x 25 mm borosilicate test tubes
containing 25 ml of sterile AGM. The test tubes were capped with Kaputs®, sealed with
one layer of Nescofilm® and cultured at 25-26°C with a 16 h photoperiod provided by
cool white fluorescent tubes at 40-50 pmol mV. The surviving m.crografts were
transferred monthly to fresh medium of the same formulation. After three consecutive
subcultures, the micrografts were transferred into 266 ml Qorpak® clear glass bottles
containing 40 ml of AGM supplemented 1 with 100 mg P activated charcoal (Sigma). The
bottles were capped with transparent sterile Petri dish covers, sealed with Nescofilm and
cultured as indicated for the test tubes. Shoots that developed from the rootstock were removed to avoid competition with the scion. Approximately 15-20 ml of warm AGM 56
was added to the top of older medium at 20-day intervals. In order to stimulate a degree
of hardening of the plants, the bottles were opened twice a week in a laminar flow hood
for approx, lh and then placed back in the incubator. The scion-donor seedlings were
cultured as indicated for micrografted plants.
RT-PCR
Plants were indexed for ASBVd infection using RT-PCR (Schnell et al., 1997) 6-
7 months after micrografting. Floral tissue from ASBVd-infected ‘Vero Beach’ SE2
(Miami # 20535) and leaf tissue from ASBVd-negatively indexed ‘Simmonds’ were used
as positive and negative controls, respectively.
RNA extraction
Approximately 100 mg of tissue was ground to a fine powder using liquid
nitrogen in a sterile ceramic mortar; during grinding and while the tissue was still frozen,
1 ml of RNA extraction buffer (Ainsworth, 1995) was added. The extraction buffer
consisted of 500 mM NaCl, 100 sodium mM acetate, 50 mM ethylenediaminacetic acid
(EDTA), 2% (w/v) polyvinylpyrrolidone (PVP) and 5 mM dithiothreitol (DTT). The pH
was adjusted to 5.5 before adding 1.4% (w/v) sodium dodecyl sulfate (SDS). The slurry
was transferred to a sterile 2 ml centrifuge tube using a wide bore pipette and incubated
for 15 min in a water bath at 65°C with occasional shaking before centrifugation at
14,000 rpm for 10 min at room temperature in a Marathon 16 Km centrifuge (Fisher
Scientific). The pellet was discarded and the supernatant was transferred to a 2 ml 57
centrifuge tube to which 1/5 (v/v) 3 5 M M potassium acetate (pH 4.8) was added, and
the mixture was incubated on ice for 30 min. The mixture was centrifuged at 14,000 rpm
for 15 mm at 4°C, the supernatant was transferred to a 2 ml centrifuge tube to which 1/3
(v/v) 8 M LiCl was added and left overnight at 4°C in order to precipitate RNA. The
mixture was centrifuged at 10,000 rpm for 10 mm at 4°C, the supernatant was discarded,
and the pellet was re-suspended in 100 pi of 2 M LiCl by vortexing, and centrifuged at
10,000 rpm for 10 mm at 4°C. After repeating the last step 2x, the pellet was re-
suspended in 50 pi diethyl pyrocarbamate (DEPC)-treated water to which 1/10 (v/v) 3 M
sodium acetate and 2.5 (v/v) cold absolute ethanol were added, and allowed to precipitate
overnight at -20°C. The mixture was thereafter centrifuged at 10,000 rpm for 10 min at
4 C, the supernatant was discarded and the pellet was - washed with 0.5 1 ml cold 70%
ethanol before drying in a vacuum desiccator. The dried pellet was finally dissolved in
20 pi DEPC-treated water and RNA was quantified using a GeneQuant (Amersham
Pharmacia)
cDNA synthesis
Annealing of the primer to the RNA template was performed using 8-10 pi of a
sample containing various concentrations of isolated RNA, 3 pi 5X first strand buffer [50
mM Tris HC1 (pH 8.3), 75 mM KC1, 3 mM MgCl 2 (Gibco BRL), 0.1 M DTT (Gibco
BRL)] and 0.5 pg forward primer (5 ’-AAGTCGAAACTCAGAGTCGG-3)
complementary to nucleotides 68 to 87 of ASBVd variant J02020 (Symons, 1981) in the upper conserved region (Bar-Joseph et al., 1985). The mixture was denatured at 100°C 58
for 5 min in a DNA Thermal Cycler PTC 100 (MJ Research), followed by 2 min
incubation on ice and 1 h at room temperature. The first strand cDNA was synthesized
using 10 pi of a cDNA reaction mixture containing 2 pi of 5X first strand buffer, 25 mM
of each dNTP (ATP, CTP, GTP, TTP), 500 pM p-mercaptoethanol, 20 units of RNAsin
(Promega), and 200 units M-MLV Reverse Transcriptase (Gibco BRL). The cDNA
mixture was added to each annealing reaction and the volume was adjusted to 25 pi with
sterile distilled water. The reactions were incubated for 150 min in a stationary water
bath at 42°C.
PCR amplification
First strand cDNA amplifications were performed using 5 ’-end labeled (6-FAM)
forward primer 5 ’-AAGTCGAAACTCAGAGTCGG-3 ’ complementary to nucleotides
68-87 of the J02020 ASBVd molecule and 5’-end labeled (HEX) reverse primer 5’-
GTGAGAGAAGGAGGAGT-3 homologous , to ASBVd J02020 nucleotides 88-104
(IDT Co.). A PCR mixture containing 10% lOx PCR buffer [lOmM Tris-HCl, (pH 8.3),
50 mM KC1, 15 mM MgCl (w/v) 2 , 0.001% gelatin], 25 mM each of dNTP (ATP, GTP,
CTP and TTP), 1.25 units of Amplitaq DNA Polymerase (Perkin Elmer) and 10 pM of each primer was mixed with 2.5 pi of the first strand cDNA reaction and the volume was adjusted to 25 pi with sterile distilled water. The amplifications were performed in a
DNA Thermal Cycler PTC 100 for 40 cycles under the following conditions: 30 sec at
94°C, 30 sec at 55°C and 30 sec at 72°C, with a final extension of 7 min at 72°C. 59
Capil lary and agarose gel electrophoresis
Amplified PCR fragments were analyzed using capillary electrophoresis (CE) in
an ABI Prism 3100 Genetic Analyzer (Applied Biosystems) using 4% Performance
Optimized Polymer (POP 4) ABI buffer with EDTA as the electrophoresis buffer and a 500 ROX as the internal standard (Applied Biosystems). The capillaries were 36 cm in
length with 50 pm internal diameter. Data were collected and analyzed using Gene Scan
Software (Applied Biosystems). Amplified cDNA products were also fractionated by gel
electrophoresis in 1% molecular grade agarose (Sigma) in 0.5x TBE and visualized with
ethidium bromide.
Cloning and Sequencing
cDNA amplified fragments from RT-PCR-positive indexed micrograft No. 133
were cloned into E. coli competent cells by heat shock treatment using TOPO® Cloning
(Invitrogen). Four (4) pi of PCR-amplified products were mixed with 1 pi of salt
solution (1.2 M NaCl and 0.06 M MgCl and 2 ) 1 pi pUC18 plasmid vector (10 ng/pl), and
incubated at room temperature for 30 mm. Two (2) pi of the previous mixture was added
to a vial containing 50 pi of ice-thawed E. coli, the mixture was incubated on ice for 30
min, heat-shocked at 42 °C for 30 sec in a stationary water bath and incubated on ice for 2 min. Two hundred and 50 pi of (250) SOC broth were added to the vial before incubation on a shaker at 200 rpm. After 1 h, 30 pi aliquots of the bacterial culture were plated in 60 x 15 mm Petri dishes containing 15 ml of LB semisolid medium supplemented with 1 100 mg 1 ampicillin. The plates were incubated overnight at 37°C in 60
darkness. The ampicillin-resistant colonies were individually removed with sterile
toothpicks and incubated overnight 1 in 100 pi of SOC broth containing 100 mg f
ampicillin. Twenty-five (25) pi bacterial aliquots from each cultured colony were
transferred to a new 0.5 ml tube and centrifuged at l,700g for 20 min. The supernatant
was discarded and the bacterial pellets were re-suspended in 50 pi 10 mM Tris-HCl (pH
8.0); these were used as the template for PCR insert amplification. Cloned inserts were
amplified using M13 forward and M13 reverse primers (Promega) in a PCR reaction
mixture containing lOx buffer with 15 mM MgCl 25 2 , mM each of dNTPs ATP, CTP,
GTP and TTP, 10 pM of each primer, 0.3 units of Amplitaq DNA Polymerase (Perkin
Elmer) and the volume was adjusted with 9.24 pi of sterile-filtered DI water. The
amplifications were performed in a DNA Thermal Cycler PTC 100 for 30 cycles under
the following conditions: 30 sec at 94°C, 30 sec at 60°C and 2 min at 72°C, with a final
extension of 7 min at 72°C. The amplified fragments were sequenced using the ABI
Prism Big Dye Terminator Cycle Version 3 (V3) and products were analyzed in an ABI
Prism 3100 Genetic Analyzer (Applied Biosystems) using an ABI Prism DNA sequencing analysis software. The sequence data were manually aligned using Sequencer
4.1 (Gene Codes) and compared with previously reported ASBVd variants. 61
Results
Germination
Germination occurred within two weeks after explanting and the percentages of
gemination were 87.3%, 91.1%, 91.1% and 92.6% for 'Vero Beach' GRD, ‘Sitnmonds'
Donaldson and ‘Wilson Popenoe’, respectively.
Micrografting
Successfully micrografted scions remained green and elongation was observed 7-
10 days after micrografting (Figure 3-1). Production of new leaves was observed 2-3
weeks after micrografting. Table 3-1 shows the percentages of success for micrografting.
Forty-seven (34.56%) plants were recovered from 136 performed micrografts: 10 were
micrografts of ‘Vero Beach’ GRD on ‘Simmonds’ and 37 were ‘Donaldson’ on ‘Wilson
Popenoe’. When the micrografting was performed using 0.5 mm length scions, 40
plants were recovered (58.8%), while only 7 (10.3%) plants survived when the scion was
< 0.5 mm long (Table 3-1).
Among plants recovered from larger scions, 32 were ‘Donaldson’ on ‘Wilson
Popenoe’, and 5 were ‘Vero Beach’ GRD on ‘Simmonds’. The 7 plants recovered from scions <0.5 mm long were distributed accordingly: 5 from ‘Donaldson’ on ‘Wilson
Popenoe’ and 2 from ‘Vero Beach’ GRD on ‘Simmonds’ (Table 3-1). 62
Figiire 3-1 Micrografting showing localization of the scion on the rootstock (left) initiation of elongation (center) and growth and development (right).
- Table . 3 1 Percentages of success of micrograftings of avocado
Genotype Number of Scion Size Plants Success Micrografts recovered (%)
<5mm Success >5mm Success
VB GRD/ 40 20 2 20 8 10 25 Simmonds
Donaldson/ 96 48 5 48 32 37 W.P 38.54
Total 36 68 7 68 40 47 34.55
Micrograft growth and development
Micrografted plants were transferred to fresh medium at monthly intervals during the first three months. Thereafter, they were transferred into 266 ml Qorpak® clear glass bottles, and medium overlays were added at 20-day intervals. Losses of plant material 63
occurred during transfers in the first three months; the losses were reduced when the
micrografted plants were not transferred to fresh medium.
RT-PCR
A total of 24 micrografted plants were indexed for ASBVd infection using RT- PCR. An initial indexing done 4 months after micrografting did not detect ASBVd in the
micrografts or in the scion-donor plants. Plants were indexed 7 months after
micrografting and infection was detected in 19 of the 24 indexed individuals.
Capillary and agarose gel electrophoresis
When electropherograms were analyzed, a total of 19 micrografted plants showed
peaks that migrated at the same size (approx. 250 bp) as the positive control ‘Vero
Beach’ SE2 floral tissue. In contrast, no peak formation was observed in the negative
control ‘Simmonds’ (Figure 3-3). Five of the 24 recovered plants did not generate any
amplification products after RT-PCR indexing (Table 3-2). When RT-PCR was
performed on the scion-donor seedlings of the negative tested micrograftings, the results
showed that the scion donor seedlings were negative for ASBVd infection (Figure 3-3).
In contrast, the indexing of the scion-donor seedlings of the 19 ASBVd positive-tested seedlings showed amplifications consistent with ASBVd infection (Table 3-2). 64
Figure 3-2. Electropherograms of RT-PCR positively indexed leaves (left) of a and b: micrograft No. 103 and its respective scion-donor seedling; c and d: micrograft No. 133, and its respective scion-donor seedling and e: flower tissue of positive control ‘Vero Beach’ SE2, and negatively indexed leaves (right) of f and g: micrograft No. 10 and its scion-donor seedling; h and i: micrograft 1 No. 1 1 and its scion-donor seedling; and j: leaf tissue of negative control ‘Simmonds’. 65
Table 3-2. ASBVd in micrografted plants and scion donor plants.
Micrograft Scion/Rootstock Scion Size ASBVd Infected (mm)
Micrograft Scion- donors
10 VBGRD/Simmonds >0.5 No No 12 VBGRD/Simmonds >0.5 Yes Yes 15 VBGRD/Simmonds >0.5 Yes Yes 25 VBGRD/Simmonds <0.5 Yes Yes 35 VBGRD/Simmonds <0.5 Yes Yes 52 Donaldson/WP >0.5 Yes Yes 57 Donaldson/WP >0.5 Yes Yes 66 Donaldson/WP >0.5 No No 75 Donaldson/WP <0.5 Yes Yes 81 Donaldson/WP <0.5 Yes Yes 84 Donaldson/WP >0.5 Yes Yes 91 Donaldson/WP >0.5 Yes Yes 93 Donaldson/WP >0.5 Yes Yes 95 Donaldson/WP <0.5 No No 99 Donaldson/WP >0.5 Yes Yes 100 Donaldson/WP >0.5 Yes Yes 103 Donaldson/WP >0.5 Yes Yes 104 Donaldson/WP <0.5 Yes Yes 111 Donaldson/WP >0.5 No No 118 Donaldson/WP >0.5 Yes Yes 125 Donaldson/WP >0.5 No No 132 Donaldson/WP >0.5 Yes Yes 133 Donaldson/WP >0.5 Yes Yes 136 Donaldson/WP >0.5 Yes Yes
VBGRD Vero Beach’ GRD; WP — ‘Wilson Popenoe’.
Agarose gel electrophoresis (Figure 3-4) shows the banding patterns that were generated for three ASBVd positively indexed micrografted plants (Figure 3-4 lines 1, 3 and 5) and their respective scion-donor seedlings (Figure 3-4 lines 2, 4 and 6) which are similar in size (approx. 250 bp) to the positive control ‘Vero Beach’ SE2 floral tissue
(Figure 3-4 line 7). The data clearly show that micrografting did not eliminate ASBVd 66
from the infected tissue and that the absence of infection in several micrografted plants
was due to the absence of infection in their scion-donor plants rather than elimination of
ASBVd-free tissue from infected plants.
• • » *
m £ < 600 bn Z
A*** 1 «*#•* < 300 bn
<4 100 bn
Figure 3-4. Gel agarose electrophoresis of PCR amplified fragments of micrografts 12, 15 and (Lines 3 52 1, and 5) and their respective scion-donor plants (lines 2, 4, 6). Line 7 = positive control ‘Vero Beach’ SE2 floral tissue. M = 100 bp ladder.
Cloning and sequencing
The sequencing cycle of one randomly chosen sample that showed fragment amplification demonstrated that the CE-generated peaks were the result of ASBVd infection. A total of 9 clones containing sequences with >97% similarity to ASBVd variant J02020 (Symons, 1981) (Figure 3-5) were isolated from micrograft No. 133. 67
Three clones were similar to variant J02020, 4 were similar to variant CF24 and 2 were
similar to variant CF2 (Schnell et al., 2001).
U 0 U A
247-0 C U A
C- O U A SO A A AC UU u C G A G U U C AG 100 GU U , UU UGAGGAUA GA ,, UAAACUUUGU (SAC ?A ? PU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA CG GGUGAACU ACUUCUAU CU AUUUGGAACG CUG GUC CA GA AGGGU GAAAGG GACUU CUG CACU CUCU CUUCU GC UCACUUGA 240-A l 1 ' cc c CU, A A A A CU A UC A U AA l A U 140 U S U c- o 180 A A A A- U U U u u u u
Figure 3-5. ASBVd variant J02020 secondary structure molecule (Daros and Flores, 2000) isolated from micrograft No. 133.
Discussion
The first example of pathogen elimination from meristematic tissue was reported
by White (1934), who obtained virus-free tomato roots isolated from infected tissue.
Application of this technique in commercial species was first reported with dahlia and
potato (Morel and Martin, 1952, 1955) and subsequently in several herbaceous species
(Hollings, 1965). Micrografting is a meristem culture technique that consists of in vitro
grafting of an excised meristem tips onto a decapitated seedling that serves as the
rootstock. Micrografting has been used to recover healthy plants from virus and viroid
infected citrus (Murashige et al., 1972; Navarro et al., 1975; Monteverde et al., 1987). In the current research, micrografting has been used in an attempt to eliminate avocado 68
sunblotch viroid (ASBVd) from infected avocado plants; however, ASBVd was present
in all scions that originated from infected material.
Plant recovery from micrografting is relatively low. Murashige et al. (1972)
reported that the incidence of success ranges from 5% to 40% depending on the
genotype. Size of the scion and placement on the decapitated rootstock can affect the rate
of micrografting success; Navarro et al. (1975), using micrograftings of ‘Robertson’
navel orange on ‘Troyer’ citrange, observed the lowest success (1.8%) using the
meristem dome alone and the highest recovery rate (47.3%) using the apical meristem
plus 6 leaf-primordia. Success rates of 14.6% and 34.6% were obtained when the scion
consisted of the meristem plus two leaf-primordia and the meristem plus four leaf-
primordia, respectively. With ‘Cadenera’ and ‘Pera’ sweet oranges and ‘Temple’ tangor
scions, Navarro et al. (1975) observed that up to a 50% recovery could be achieved when
the scion is placed in contact with the vascular ring. Although larger scion sizes are
likely to provide better recovery of plants, the possibilities of recovering healthy material are reduced. Even so, citrus exocortis viroid can be eliminated using scions consisting of
the meristem dome plus 6 leaf-primordia (Monteverde et al., 1987).
Nel and Lange (1985) attempted to develop a technique for the recovery of micrografts used for ASBVd elimination. Although the study did not involve the use of
ASBVd-infected material, the results showed that nearly 50% of recovery could be achieved when all cuts were made under water and the scion and the rootstock were maintained submerged for 24 h prior to the micrografting. Preliminary experiments with this technique resulted in very low rates of recovery. Therefore, a technique where the roots of the rootstock were preserved during the early stages of development was preferred. In the current research, all scions were placed in contact with the vascular ring 69
of the rootstock. Therefore, no comparison among placement treatments could be
analyzed. However, the data show similarity with previous reports with respect to scion
size since a total recovery rate of 34.35 % was achieved and a higher recovery rate
(58.8%) was observed when larger scions were used compared to 10% when smaller
shoot tips were utilized.
Murashige et al. (1972), using scions from diseased citrus plants reported that 4
out of 6 recovered ‘Temple’ tangor and 1 out of 2 micrograftings of ‘Eureka’ lemon
tested negative for citrus exocortis viroid (CEVd) infection using indicator plants.
Navarro et al. (1975), using the same bioassay, reported CEVd elimination in all 31
micrografts of ‘Cadenera Fina’ sweet orange, 5 out of 6 micrografts of ‘Temple’ tangor
and 77 out of 99 micrograftings of ‘Robertson’ navel oranges. Monteverde et al. (1987),
using the same indexing method, observed that all six micrografts recovered from CEVd-
infected material were negative for the presence of the viroid. In contrast, the results
with avocado demonstrate that micrografting is ineffective for eliminating ASBVd from
infected material. Since all measures were taken to minimize any risk of cross-
contamination and ASBVd was detected in the scion-donor seedlings of the positive
micrografts while not in the negative ones, the presence ASBVd infection in the
recovered plants most likely indicates that it was the result of ASBVd presence in the
scion.
Elimination of systemic pathogen infections using meristem tip culture is believed
to result from the inability of the pathogens to invade the apical meristem or to erratic
distribution of the pathogen within the plant (Hollings, 1965). Data obtained from studies of Pospiviroidae viroid movements inside the plant, have shown that the apical meristem is not invaded by members of this family (Momma and Takahashi, 1983), and 70
Bonfiglioli et al. (1996) observed that coconut cadang-cadang viroid (CCCVd) and citrus
exocortis viroid (CEVd) localize and move long distances within the plant through the
phloem, where they also replicate. Therefore, the isolation and culture of the shoot apical
meristem plus 1-4 leaf-primordia can be used to regenerate viroid-free plants as a result
of the absence of vascular tissue in this area (Barba et al., 2003). Analysis of
micrografting-recovered plants from viroid-infected material showed that increased levels
of a nuclease in the apical meristem may be related to the absence or low levels of viroid
infection in this meristematic tissue (Matousek et al., 1995). Although no histological
analyses were conducted in the current research, most of the excised shoot tips had either
2 or 3 leaf-primordia, which, based on previous reports and the hypothesis of Barba et al.
(2003), would have been small enough to isolate healthy tissue and recover ASBVd-free
plants.
Pospiviroidae and Avsunviroidae viroids have significant differences, i.e.,
Avsunviroidae lack a central conserved region characteristic in Pospiviroidae , self-cleave using hammerhead structures embedded in their sequences, and in contrast to
Pospiviroidae that replicate in the nucleus, Avsunviroidae replicate in the chloroplast
(Flores et al., 1998). How Avsunviroidae viroids move inside the plant is unknown; however, the inability of rescue ASBVd-free material using scion sizes comparable to those used for CEVd elimination may be an indication that isolation of non vascularized tissue from the avocado shoot apical meristem is more difficult or that most probably,
ASBVd does invade the meristem.
Although not attempted in the current research, cold treatments can possibly assist viroid elimination in conjunction with meristem tip culture. Lizarraga et al. (1980) using polyacrylamyde gel electrophoresis (PAGE), reported potato spindle tuber viroid 71
(PSTVd) elimination in recovered plants when the shoot tips were isolated from in vitro-
grown plants maintained at 5-6°C for 6 months. Adams (1996), based on absence of
symptoms in recovered plants, reported elimination of hop latent viroid (HLVd) using
shoot tip culture (<0.5 mm long) from hop setts stored at 2°C for at least 6 months. No
report on the use of low temperature treatments to eliminate ASBVd is known. Cold
treatments that have been used for viroid elimination require several months to provide
good results (Singh et al., 2003) and this could be a limiting factor in cold-sensitive
avocado.
Thermotherapy followed by meristem tip culture has been successfully applied to
eliminate some viroids. Holling and Stone (1970), basing their results on absence of
symptoms, reported elimination of chrysanthemum chlorotic mosaic viroid (CChMVd) in
2 out of 337 chrysanthemum plants regenerated from meristem tips isolated from plants
maintained for at least 14 weeks at 35°C. Postman and Hadidi (1995), using highly
sensitive RT-PCR, found that approx. 86% of pear plants recovered from meristem tips
isolated from apple skin scar viroid (ASSVd)-infected plants maintained at 38°C for 55
days, were free of viroid. ASSVd elimination was also achieved when thermotherapy
was 77 days at 38°C (Howell et al., 1998).
Reports of meristem tip culture of avocado are unknown, and although no data is provided, preliminary tests to promote growth from small shoot tips explanted directly on semi solid MS (Murashige and Skoog, 1962) were unsuccessful. Thermotheraphy does not work similarly for all viroids. Attempts to eliminate citrus exocortis viroid (CEVd) showed that temperatures as high as 100°C for 20 min do not inactivate the viroid
(Semancik and Weathers, 1970), while exposure of potato spindle tuber viroid (PSTVd) nucleic acids to temperatures >75°C causes some decrease in viroid infectivity (Singh and 72
Bagnall, 1968). Data on the temperature necessary to inactivate ASBVd is unknown;
however, attempts to inactivate the viroid in seed, budwood and budlings by
thermotherapy demonstrated that ASBVd has “a pronounced thermal stability that it
could withstand any heat treatment regimen that the avocado tissue could” (Desjardins et
al., 1980). In addition, ASBVd symptoms develop faster at 30°C than at 20°C (da Graca
and Van Vuuren, 1980), which indicates high temperature tolerance.
With the exception of apple skin scar viroid (ASSVd) elimination from pear
plants (Postman and Hadidi, 1995), the other reports of viroid elimination have been
based on low sensitivity indexing methods. Absence of symptom development cannot be
considered a reliable test (Hollings, 1965). Citrus exocortis viroid (CEVd) mild variants
have been shown to cause asymptomatic infections in tomato indicator plants (Visvander
and Symons, 1985) and meristem tip culture-recovered grape plants were found to be
infected with low levels of yellow speckle viroid 1 (YSVdl), yellow speckle viroid 2
(YSVd2) and Australian grapevine viroid (AGVd) that were undetected using polyacrylamide gel electrophoresis (PAGE) (Wan and Symons, 1997). RT-PCR confirmed the inability of micrografting to eliminate ASBVd using micrografting under the conditions described in the current research.
In a separate chapter this of dissertation, it has been demonstrated that ASBVd is not eliminated using nucellus culture, another technique used to eliminate citrus exocortis viroid (CEVd) from infected material. Lack of infection of the nucellus and the apical meristem are believed to be the result of the same mechanism, i.e., absence of vascular tissue (Esau, 1977; Rangaswami, 1981). Therefore, persistence of ASBVd in both nucellar embryogenic cultures and micrografts could be an indication that Avsunviroidae can localize and replicate in tissues where Pospiviroidae cannot. CHAPTER 4 RECOVERY AND INDEXING OF AVOCADO PLANTS FROM THE NUCELLUS OF ASBVd INFECTED PLANTS
Introduction
Avocado sunblotch is an economically important disease of avocado (Persea
americana Mill.) (Coit, 1928). The causal agent of the disease is the avocado sunblotch
viroid (ASBVd), a circular single-stranded RNA molecule of approx. 247 bp (Palukaitis
et al., 1979; Symons, 1981) and a member of the Avsunviroidae family, a group of
viroids that has the ability to self-cleave in vivo and in vitro through formation of hammerhead ribozymes (Daros et al., 1994). ASBVd localizes and replicates in the chloroplasts (Daros et al., 2000), where it is associated with the thylakoid membranes
(Bonfiglioli et al., 1994).
Symptoms of avocado sunblotch include stunting, leaf variegation and bleaching, fruit malformation and dropping, scaly branches, and generalized decrease in health of the plant (Coit, 1928; Dale et al., 1982). Avocado plants infected with ASBVd have a
27.3% lower yield and produce 52.7% more undergraded fruit than healthy trees (da
Graca et al., 1893). Infection and dissemination of ASBVd is a major threat not only to commercial avocado crops but also to germplasm banks that secure the genetic resources of the species and serve as a source of propagation material. It has been estimated that
1 9% of the trees at the national germplasm repository (NGR) in Miami FL are infected
73 74
with ASBVd (Olano et al., 2002). In this study, we have attempted to eliminate ASBVd from infected stock plants by generating plants from the nucellus, a tissue of maternal origin.
Material and Methods
Plant Material
Avocado embryogenic cultures were induced from the nucellus of ASBVd-
infected tissue according to the protocols of Witjaksono et al. (1999). Immature avocado fruits <0.5 cm diam. were collected from ASBVd-infected trees in the National Avocado
Germplasm Repository at the USDA-SHRS in Miami, FL. Fruits were collected from
cultivars ‘Vero Beach’ SE-2 (M# 20535), ‘Vero Beach’ 1 (M# 18435) and ‘Yama’ 381
(M# 20726) in February 2002; these cultures were used in all studies related to embryogenic culture characterization, suspension culture evaluation, ASBVd persistence over time, plantlet recovery and ASBVd cloning and variant analysis. A second group of embryogenic cultures were induced from ‘Vero Beach’SE2, ‘Yama’381 and ‘Irwin’ (M#
20721) in February 2003, which were used for embryogenic culture characterization and indexing for the presence of ASBVd.
Embryogenic Culture Induction
The petals and sepals were removed and the fruits were wrapped in cheese cloth.
After washing with running tap water for lh, fruits were surface-disinfested for 15 min in a 1.25% (v/v) sodium hypochlorite solution containing five drops of Tween-20®, and 1
75
finally rinsed with three changes of sterile deionized water. Surface-disinfested fruits
were bisected under axenic conditions, and the zygotic embryo and endosperm in each
fruit were removed and discarded. The nucellus together with the integuments were
isolated and inoculated onto avocado induction medium (AIM) (Witjaksono and Litz,
1999a; Witjaksono and Litz, 1999b) with the nucellus in contact with the medium.
Avocado induction medium consisted of B5 (Gamborg et al., 1968) medium
(without NH4NO 3 ) supplemented with MS (Murashige and Skoog, 1962) minor salts,
1 0.41 pM picloram and (in mg l' ) thiamine HC1 (0.4), myo-inositol (100), sucrose
and (30,000) TC agar (8,000) (Carolina Biological Supply). Sterile medium was
dispensed in 10 ml aliquots into sterile disposable Petri dishes (100 x 15 mm).
Four explants were inoculated in each Petri dish. The plates were sealed with
Parafilm® and stored in darkness at 25 °C in a completely randomized design. There
were 628 explants (164 in 2002 and 464 in 2003) of ‘Vero Beach’ SE2, 80 explants from
‘Vero Beach ’ (2002), 328 (32 in 2002 and 296 in 2003) explants from ‘Yama’381, and
308 explants of ‘Irwin’. The number of induced embryogenic cultures was recorded and
the percentage of induction calculated for each cultivar.
Embryogenic Culture Maintenance
Embryogenic cultures induced on AIM were transferred onto MSP semi solid medium (Witjaksono and Litz, 1999a) for maintenance. MSP medium consisted of MS
1 major and minor salts, 0.41 picloram (in pM and mg l' ) thiamine HC1, (0.4), myo- inositol (100), sucrose (30,000) and TC agar (8,000). Autoclaved sterile medium was dispensed in 10 ml aliquots into sterile disposable Petri dishes (100 x 15 mm). 76
Each embryogenic culture was transferred into a single Petri dish containing MSP
medium, sealed with Parafilm® and incubated in darkness at 25°C in a complete
randomized design. The number of PEMs, globular somatic embryos, hyperhydric and
opaque heart-stage somatic embryos (<5 mm diam.), hyperhydric and opaque early
torpedo-stage somatic embryos (>5 mm diam.) and the total number of somatic embryos
in each Petri dish were recorded after four weeks of culture. Data were analyzed with
using ANOVA Yt= n + i + e, statistical model; where, Y was any of the independent
variables noted above, i was the cultivar and e was the experimental error. Means were
separated using Duncan’s Multiple Range Test.
Embryogenic Suspension Cultures
Suspension cultures were established for ‘Vero Beach’ SE2, ‘Vero Beach’ 1 and
for ‘Yama’ 381 from a single embryogenic culture line of each genotype induced in 2002
and maintained on MSP medium. Approx. 200 mg of 14-day-old embryogenic cultures
were inoculated into 125 ml flasks containing 40 ml of MS3:1N liquid medium
(Witjaksono and Litz, 1999a). MS3:1N consisted of MS major salts with 15 mM
1 NH4NO3 and 30 mM KNO3, MS minor salts, 0.41 picloram (in pM and mg l' ) thiamine
HC1 (0.4), myo-inositol (100) and sucrose (45,000). Flasks were sealed with heavy-duty aluminum foil, secured with Parafilm® and maintained in semi darkness at 25°C on a rotary shaker at 120 rpm. Cultures were transferred biweekly to fresh medium of the same formulation.
After three subcultures, the growth of the embryogenic suspension cultures was analyzed and compared to that of ASBVd-free embryogenic line ‘Hass’ 11.4.3. 77
‘Hass’ 11.4.3 was induced from ‘Hass’ zygotic embryos from the avocado germplasm
collection of the University of California, Riverside, CA. ‘Hass’ 11.4.3 had been
maintained in liquid MS3:1N medium for 18 months at the time of evaluation.
Fourteen-day-old embryogenic suspension cultures of each genotype were sieved
through sterile 1.8 mm mesh nylon filtration fabric and the smallest PEM fraction was
collected. Inoculum consisting of 1-ml settled cell volume (SCV) of suspension cultures
was obtained by decanting the cultures into a 50 ml graduated centrifuge tube, and was
inoculated into 125 ml flasks, to which liquid medium was added for a final volume of 40
ml. Cultures were incubated as described above. The increased SCV of suspension
cultures was recorded at 3, 6, 9, 12, 15, 18, 21, 24 and 27 days after inoculation. The
samples were distributed in a complete randomized design with four genotypes and five
replicates for each genotype for a total of 20 experimental units. The data were analyzed
with ANOVA using the T, = i + e, statistical n + model; where, Y was the volume, i was the cultivar and e was the experimental error. Means were separated using the Duncan
Multiple Range Test.
Media Sterilization
The pH of all media was adjusted to 5.7-5. 8 with KOH or HC1 prior to the addition of gelling agent, 2 autoclaved at 121 °C at 1.1 kg cm' for 15 min and dispensed as indicated. 78
Somatic Embryo Development
Embryogenic supension cultures of ‘Vero Beach’ SE2, ‘Vero Beach’ 1 and
‘Yama’ 381 each derived from a single explant after three subcultures and ‘Hass’ 1 1.4.3
suspension cultures were used for somatic embryo development. Samples from each
cultivar were sieved through a sterile 1.8 mm mesh nylon filtration fabric and the
smallest fraction was dried for 1 h on several layers of sterile Kinwipes®. Approx 100
mg of dried embryogenic cultures were inoculated onto 20 ml semi solid somatic embryo
development (SED) medium contained in sterile Petri dishes (100 x 20 mm) (Witjaksono
and Litz, 1 1999b). SED consisted of MS major and minor salts with (in mg l" ) thiamine
HC1 myo-inositol (0.4), (100), sucrose (30,000) and Gel-Gro gellan gum® (6,000) (ICN
Biochemical). Embryogenic culture inocula were evenly spread over the medium, the plates were sealed with Parafilm® and incubated for 8 weeks in darkness at 25°C. A total of 75 plates containing embryogenic cultures of ‘Vero Beach’ SE2, 54 with embryogenic cultures of ‘Vero Beach’ 1 and 51 with embryogenic cultures of ‘Yama’
381 were cultured using a complete randomized design. The number of white-opaque and hyperhydric somatic embryos, and the total number of somatic embryos were recorded.
Data were analyzed with ANOVA using the pi Yt = + i + e,- statistical model, where, Y was the number of somatic embryos developed, i was the cultivar and e was the experimental error. Means were separated using either Duncan’s Multiple Range Test or
/-test. 79
Plant Recovery
Eight-week-old white-opaque somatic embryos of ‘Vero Beach’ SE2 and ‘Vero
Beach’ 1 that developed on SED medium were inoculated onto 50 ml of somatic embryo
germination and maturation medium (SEGM) in 100 mm x 25 mm sterile Petri dishes
(Witjaksono and Litz, 1999b). SEGM consisted of MS3:1N supplemented with 1.0 pM
benzyladenine (BA) and 10 pM gibberellic acid and 1 (GA 3 ), solidified with 3 g l' Gel
Gro® gellan gum. Seven opaque somatic embryos (>0.5 cm diameter) were inoculated in
each Petri dish the and dishes were sealed with Nescofilm® and stored in translucent
plastic boxes at 25°C with a 16 h photoperiod (approx. 50 pmol m'V) provided by cool
white fluorescent tubes. A total of 520 white-opaque somatic embryos of ‘Vero Beach’
SE2 distributed in 75 plates and 85 white-opaque somatic embryos of ‘Vero Beach’ 1
distributed in 1 2 plates were cultured. The number of somatic embryos forming shoots
and/or roots was recorded after 12-16 weeks.
Regenerating somatic embryos were transferred to 118 ml Qorpak® clear glass
bottles containing 25 ml of MS medium solidified with 8,000 mg TC agar. A single
germinated embryo was inoculated in each container, which was capped with 100 x 15
Petri dish mm lids, sealed with Nescofilm® and cultured at 25°C with a 16 h photoperiod
(approx. 2 50 pmol m s ') provided by cool white fluorescent tubes. Approximately three months after germination, plants were transferred to 473 ml Qorpak® clear glass bottles
containing 1 100 ml of the same medium but supplemented with 200 mg f of activated charcoal (Sigma) for root development. Containers were covered with 100 x 15 mm Petri dish lids, sealed with Nescofilm, and cultured as above. 80
RT-PCR Indexing for ASBVd
All plant material was indexed for ASBVd infection using RT-PCR (Schnell et
al., 1997). For embryogenic cultures induced in 2002, suspension cultures of a single
explant origin of ‘Vero Beach’ 1 were indexed at 6 and 9 months after induction.
Suspension cultures of ‘Yama’ 381 originated from a single explant and embryogenic
cultures maintained in MSP medium were indexed at 6, 9 and 12 months after induction.
Single explant-derived embryogenic cultures of ‘Vero Beach’ SE2 cultured on MSP and
in MS3: IN were indexed at 6, 9 and 12 months after induction. Single explant-derived 8-
week old white-opaque somatic embryos of ‘Vero Beach’ SE2 were indexed at 6 and 9
months after induction and leaf tissue from single explant-derived nucellar plants of
‘Vero Beach’ SE2 were indexed approx. 1 year after embryogenic culture induction.
For embryogenic cultures induced in 2003, samples from different embryogenic
lines were indexed 3-4 months after induction as follows: 5 different embryogenic lines of ‘Vero Beach’ SE2, 6 embryogenic lines of ‘Yama’ 381 and 5 embryogenic lines of
Irwin . Embryogenic cultures of ‘Hass’ 1 1.4.3 and flower samples of ‘Vero Beach’ SE2 were indexed as the negative and positive controls, respectively.
RNA extraction
Approximately 100 mg of tissue was processed to a fine powder using liquid
nitrogen to which 1 ml extraction buffer (Ainsworth, 1995) was added. The extraction buffer consisted of 500 mM NaCl, 100 mM sodium acetate, 50 mM ethylenediaminetetraacetic acid (EDTA), 2% (w/v) polyvinylpyrrolidone (PVP) and 5 81
mM ditithreitol (DTT). The pH was adjusted to 5.5 before adding 1.4% (w/v) sodium dodecyl sulfate (SDS). The slurry was transferred to a 2 ml centrifuge tube using a wide bore pipette and incubated in a water bath at 65 °C with occasional shaking for 15 min before centrifugation at 14,000 rpm for 10 min at room temperature in a Marathon 16 Km centrifuge (Fisher Scientific). The supernatant was transferred to a 2 m! centrifuge tube to which 1/5 (v/v) 3 M 5 M potassium acetate (pH 4.8) was added and incubated on ice
for 30 min. The mixture was thereafter centrifuged at 14,000 rpm for 1 5 min at 4°C. The supernatant was transferred to a 2 ml centrifuge tube to which 1/3 (v/v) 8M LiCl was added and left overnight at 4°C to precipitate RNA. The mixture was centrifuged at
10,000 rpm for 10 min at 4°C, the supernatant was discarded, the pellet was re-suspended in 100 pi of 2 M LiCl and centrifuged at 10,000 rpm for 10 min at 4°C. After repeating the last step 2x, the pellet was re-suspended in 50 pi diethyl pyrocarbamate (DEPC)- treated water to which 1/10 (v/v) 3 M sodium acetate and 2.5x (v/v) of cold absolute ethanol were added and allowed to precipitate for 2 h at -70°C or overnight at -20°C.
The mixture was thereafter centrifuged at 10,000 rpm for 10 min at 4°C, the supernatant
was discarded and the pellet was washed with 0.5 to 1 ml cold 70% ethanol before drying in a vacuum desiccator. The dried pellet was finally dissolved in 20 pi DEPC-treated water and RNA quantified using a Gene Quant (Amersham Pharmacia).
cDNA synthesis
First strand cDNA was synthesized using 10 pi of a sample containing various
concentration of isolated RNA, 3 pi 5X first strand buffer [50 mM Tris HC1 (pH 8.3), 75 82 mM KC1, 3 mM MgCfo], 0.1 M DTT (Gibco BRL) and 0.5 pg forward primer (5’-
AAGTCGAAACTCAGAGTCGG-3,) complementary to nucleotides 68 to 87 of ASBVd variant J02020 in the upper conserved region (Bar-Joseph et al., 1985). The mixture was
incubated at 100°C for 5 min, then placed on ice for 2 min and allowed to stand at room
temperature for 1 h. Ten (10) pi of a cDNA reaction mixture containing 2 pi of 5X first strand buffer, 25 mM of each dNTP (ATP, CTP, GTP, TTP), 500 pM of p- mercaptoethanol, 20 units of RNAsin (Promega), and 200 units M-MLV Reverse
Transcriptase (Gibco BRL) was added to each annealing reaction and adjusted to a total
reaction volume of 25 pi with sterile water. The reactions were incubated for 1 50 min in
a stationary water bath at 42°C.
PCR amplification
First strand cDNA fragments were amplified using 5’-end labeled (6-FAM) forward primer (5’-AAGTCGAAACTCAGAGTCGG-3’ complementary to ASBVd
J02020 nucleotides 68-87) and 5’-end labeled (HEX) reverse primer (5’-
GTGAGAGAAGGAGGAGT-3 homologous to ASBVd variant J02020 nucleotides 88-
104) (Integrated DNA Technology) in a 25 pi reaction containing 2.5 pi of first strand cDNA reaction and 22.5 pi of a mixture of 10X PCR buffer, 25 mM of each dNTP, 1.25 units of Amplitaq DNA Polymerase (Perkin Elmer) and 10 pM of each primer. The amplifications were performed in a DNA Thermal Cycler PTC 100 (MJ Research) for 30
cycles under the following conditions: 30 sec at 94°C, 30 sec at 55°C and 30 sec at 72°C,
with a final extension of 7 min at 72°C. 83
Capillary electrophoresis (CE) and agarose gel electrophoresis
Amplified PCR fragments were analyzed by capillary electrophoresis (CE) in an
ABI Prism 3100 Genetic Analyzer (Applied Biosystems) using 4% Performance
Optimized Polymer (POP 4), ABI buffer with EDTA as the electrophoresis buffer and a
500 ROX internal standard (Applied Biosystems). The capillaries were 36 cm in length with 50 pm internal diameter. Data were collected and analyzed using Gene Scan
Software (Applied Biosystems). Amplified cDNA products were also fractionated by gel electrophoresis in 1% molecular grade agarose (Sigma) in 0.5x TBE and visualized with ethidium bromide.
Cloning. Sequencing and Variant Analysis
RT-PCR amplified fragments from different tissues were cloned using TOPO
Cloning (Invitrogen). Four (4) pi of PCR-amplified fragments were mixed with 1 pi of
salt solution (1.2 M NaCl and 0.06 M MgCL) and 1 pi of pUC18 vector (10 ng/pl) and
incubated at room temperature for 30 min. Two (2) pi of the previous mix was added to a vial containing 50 pi of ice-thawed E. coli, maintained on ice for 30 min and heat- shocked at 42 °C for 30 sec in a stationary water bath and incubated on ice for 2 min.
Two hundred and fifty (250) ml of SOC broth were added to the vial before incubating it
at 37°C for 1 h at 200 rpm on a rotary shaker. Aliquots of 30 pi of the bacterial mixture were inoculated onto 60 x 15 mm Petri dishes containing 15 ml of LB medium
1 supplemented with 100 mg l' of ampicillin. Plates were incubated at 37°C in darkness for selection of transformed colonies. The recovered E. coli colonies were cultured 84
1 overnight in 100 pi of SOC broth with 100 mg l' ampicillin. The bacterial cultures were
centrifuged and the pellets were re-suspended in 50 pi 10 mM Tris-HCL (pH 8.0); this was used as the template for PCR insert amplification. Inserts were amplified using M13 forward and M13 reverse primers (Promega) in a PCR reaction mix containing 10X buffer with 15 mM MgCB, 25 mM of each dNTPs, 10 pM of each primer, 0.3 units of
Amplitaq DNA Polymerase (Perkin Elmer); the volume was adjusted with 9.24 pi of sterile filtered deionized water. The amplifications were performed in a DNA Thermal
Cycler PTC 100 for 30 cycles under the following conditions: 30 sec at 94°C, 30 sec at
60°C and 2 min at 72°C, with a final extension of 7 min at 72°C. The amplified
fragments were sequenced using the ABI Prism Dye Terminator Cycle Version 3 (V3) and products were analyzed in an ABI Prism 3100 Genetic Analyzer (Applied
Biosystems) using an ABI Prism DNA sequencing analysis software. The sequence data were manually aligned using Sequencer 4.1 (Gene Codes) and compared with previously reported ASBVd variants.
Results
Induction of Embryogenic Cultures From the Nucellus of ASBVd-lnfected Plants
Embryogenic cultures developed directly from the nucellus of fruits explanted
onto AIM without intervening callus formation (Figure 4-1). Generally, all embryogenic tissues were induced between the second and eight weeks after explanting. The frequency of embryogenic culture induction was 2.5% and 1.5% for ‘Vero Beach’ SE2 in
2002 and 2003, respectively; 6.25% and 4.05% for ‘Yama’ 281 in 2002 and 2003, 85
respectively; 6.25% for ‘Vero Beach’ 1 and 1.98% for irwin’. Due to the low level of induction, no cultivar effect could be statistically inferred from the collected data.
Figure 4-1. Induction and proliferation of embryogenic cultures on solid medium. Embryogenic culture induction from nucellus (left) and PEM proliferation (right).
Embryogenic culture maintenance on semisolid medium
Embryogenic cultures were transferred onto MSP medium for tissue maintenance
1-2 weeks after induction. Table 4-1 shows the number and types of embryogenic cultures that developed from explants explanted in 2002 and 2003.
Table 4-2 shows the average number of embryogenic cultures that were induced
for every genotype. The ANOVAs (Appendix 1) show that there was no variation with respect to PEMs, globular somatic embryos, hyperhydric and opaque somatic embryos and total number of somatic embryos that proliferated on MSP medium (Figure 4-2). In most embryogenic cultures induced in 2002 that consisted predominantly of hyperhydric
somatic embryos, individual somatic embryos expanded but did not proliferate, becoming
brown and died. Proembryonic mass (PEM)-type embryogenic cultures proliferated as 86
PEMs after several subcultures on MSP, with eventual development of very few globular and cotyledonary somatic embryos.
Table 4-1. Number and type of embryogenic cultures
Cultivar Year of Induced PEMs GSE HSE HSE Total SE induction explants <0.5 mm >0.5 mm
VBSE2 2002 4 2 7 7 6 20
VB1 2002 5 5 9 10 8 27
Yama 2002 2 3 3 6 1 10
VBSE2 2003 7 5 6 22 6 34
Yama 2003 12 10 27 37 19 83
Invin 2003 7 12 27 23 14 54
= = = = VBSE2 ‘Vero Beach’ SE2, VB1 ‘Vero Beach’ 1, Yama ‘Yama’ 381, PEMs Proembryonic masses, GSE = Globular somatic embryos, HSE = Hyperhydric somatic embryos, SE = Somatic embryos.
The embryogenic competence of most embryogenic lines was lost after 3-4 subcultures; however, one year after induction, a single line of ‘Vero Beach’ SE2 and
‘Yama’ 381 remained embryogenic on MSP while ‘Vero Beach’ stopped growing. A similar situation was observed with the embryogenic cultures induced in 2003. Many of the embryogenic lines lost their embryogenic capacity after 3-4 subcultures on MSP medium. 87
Table 4-2. Average number and standard errors (in parenthesis) of proembryonic masses (PEMs), globular somatic embryos (GSE), hyperhydric and opaque somatic embryos (HSE) and total number of somatic embryos (SE) proliferated on MSP medium in 2002 and 2003.
Embrvoeenic Cultures
Genotype PEMs GSE HSE HSE Total SE < 0.5 cm >0.5 cm
‘Irwin’ 1.714 3.857 3.285 2.000 9.142 (0.865) (1.454) (1.248) (0.534) (2.729)
‘Vero Beach’ 1 1.000 1.800 2.000 1.600 5.40 (0.632) (0.800) (0.948) (0.400) (1.720)
‘Vero Beach’ SE2 0.818 1.181 2.636 1.363 5.181 (0.352) (0.463) (0.741) (0.387) (1.110)
‘Yama’ 381 1.00 2.142 3.071 1.428 6.642 (0.314) (0.543) (0.821) (0.343) (1.462)
Cultivars
Figure 4-2. Mean and standard errors from the ANOVA of a = somatic embryo development; b = globular somatic embryos; c = hyperhydric and opaque somatic embryos <0.5 cm diam.; d = hyperhydric and opaque somatic embryos >0.5 cm dia., and e =-PEMs of cultivars ‘Irwin’, ‘Vero Beach’ 1, ‘Vero Beach’ SE2 and ‘Yama’ 381. 6
SEs
Globular
’Vero Beach’ 1 'Vero Beach’ SE2 'Yama'
Cultivars
cm
SEs<0.5
HH
Cultivars 3.0
2.5 -
2.0 - cm
>0.5
1.5 -
SEs
HH 1.0 -
0.5 -
0.0
'Irwin' 'Vero Beach' 1'Vero Beach' SE2 'Yama'
Cultivars
PEMs
Cultivars 90
Suspension culture
Established embryogenic suspension cultures were mostly of the PEM-type and
they retained this characteristic in liquid medium (Figure 4-3).
Figure 4-3. Suspension cultures of ‘Vero Beach’ SE2
The analyses of variances (Appendix 2) and DMRT mean separations (Table 4-3)
showed that suspension culture growth varied among the different genotypes. All cultures attained the greatest SCV 24 days after inoculation with 27 ml, 1 1 ml, 7.85 ml and 5.35 ml for ‘Vero Beach’ SE2, ‘Hass’ 11.4.3, ‘Vero Beach’ 1 and ‘Yama’ 381, respectively (Table 4-3). Figure 4-4 shows that cultures of ‘Vero Beach’ SE2 proliferated more rapidly than the other cultivars as early as 3 days after inoculation and maintained the same pattern throughout the evaluation period, while no difference was observed among ‘Vero Beach’ 1, ‘Yama’ 381 and ‘Hass’ 11.4.3 during the first 12 days in culture. After the second week and until the end of the evaluation, ‘Hass’ 11.4.3 showed a higher rate of proliferation than ‘Yama’ 381 and ‘Vero Beach’ 1. After 24 days in culture, ‘Vero Beach’ 1 proliferated more rapidly than ‘Yama’ 381, which showed the lowest suspension cell volume (SCV) (Figure 4-4). The highest daily rates for 91
suspension culture proliferation occurred between days 15 to 18 after inoculation, with
0.933, 0.377 and 0.28 ml for ‘Vero Beach’ SE2, ‘Hass’ 11.4.3 and ‘Vero Beach’ 1,
respectively. ‘Yama’ 381 reached the highest daily rate of proliferation between days 12
to 15 with a 0.197 ml increase. The daily proliferation rates at the end of the culture
period were 0.806, 0.306, 0.221 and 0.144 ml for ‘Vero Beach’ SE2, ‘Hass’ 1 1.4.3, ‘Vero
Beach 1 and Yama 381, respectively. After several subcultures in liquid medium,
PEMs of ‘Vero Beach’ SE2 became smaller, while ‘Vero Beach’ 1 and ‘Yama’ 381 were
disorganized with a lower proliferation rate. After approx, three months, ‘Vero Beach’ 1
and Yama 381 ceased proliferation, while ‘Vero Beach’ SE2 maintained a steady
proliferation rate after one year in culture.
Table 4-3. Settled cell volumes (SCVs) of embryogenic cultures cultures (in ml) in liquid medium.
Davs in Culture Cultivar 3 6 9 12 15 18 21 24 27 30
‘Vero 1.75 3.42 5.57 8.40 12.80 20.40 25.40 27.00 25.80 25.20 Beach’ SE2 a a a a a a a a a a ‘Hass’ 1.06 1.76 2.36 4.16 6.70 9.50 10.60 11.00 10.60 10.20 11.4.3 b b b b b b b b b b
‘Vero 1.14 1.86 2.52 3.88 4.80 6.44 6.81 7.85 7.52 7.64 Beach’ 1 b b b b c c c c c c
‘Yama’ 381 1.14 1.82 2.12 3.20 4.22 5.140 5.290 5.35 5.34 5.34 b b b b c c c d d d
Different letter indicates significant differences at 0.05 level according to DMRT. 92
Figure 4-4. Means and standard errors of settle cell volume (SCV) of suspension cultures.
Somatic Embryo Development
Development of mature white-opaque somatic embryos was attempted from embryogenic suspension cultures of ‘Vero Beach’ SE2, Vero Beach’ 1 and ‘Yama’ 381, each of which was derived from a single nucellar explant, and the embryogenic line
‘Hass’ 1 1.4.3. The ANOVA (Table 4-4) indicates that after 8 weeks in culture there were more ‘Vero Beach’ SE2 somatic embryos (approx. 1148) than ‘Vero Beach’ 1 (approx.
150). No somatic embryos of ‘Yama’ 381 and ‘Hass’ 11.4.3 developed from embryogenic cultures. An average of 15.3 somatic embryos developed in each ‘Vero
Beach’ SE2 Petri dish while there were approx. 2.73 somatic embryos in each Petri dish of ‘Vero Beach’ 1 (Figure 4-5). The t test indicated that ‘Vero Beach’ SE2 developed more hyperhydric somatic embryos that ‘Vero Beach’ 1 (Table 4-5). Nearly half 93
(approx. per 6.8 plate) of the ‘Vero Beach’ SE2 somatic embryos were hyperhydric while
36 (approx. 0.7 per plate) ^ of ‘Vero Beach’ 1 somatic embryos were hyperhydric
(Figure 4-6)
Table 4-4. ANOVA of the total number of somatic embryos that developed on SED medium.
Source DF Sum of Squares Mean Square F Value Pr > F
Total SEs
Cultivar 3 10118.64 3372.88 493.08 <0.0001
Error 217 1484.36 6.84
Total 220 11603.00
Model: Yt = ju + i + e\. = = Y- Somatic embryos; fx Mean; i Cultivar; e = error SEs = somatic embryo
Cultivars
Figure 4-5. Average number of somatic embryos that developed on SED in each dish. 94
Table 4-5. /-test of hyperhydric somatic embryos that developed on SED medium.
Cultivar N Mean Standard. Error
‘Vero Beach’ SE2 77 6.80 0.27
‘Vero Beach’ 1 46 0.69 0.13
t Value = 19.92; Pr> Itl <.0001
Cultivars
Figure 4-6. Average number of hyperhydric somatic embryos per plate that developed on SED medium from approx. 100 mg embryogenic culture inoculum. 95
Plantlet Recovery
Only somatic embryos of ‘Vero Beach’ SE 2 converted into plants. Shoot
development from somatic embryos was observed 3-4 months after inoculation on SEGM
medium (Figure 4-7). A total of 25 (4.8%) somatic embryos developed a shoot, a root or
both a shoot and a root from the 520 somatic embryos distributed in 75 Petri dishes: 15
somatic embryos formed a root, 6 formed a shoot and 4 formed both a shoot and a root.
Four plants were recovered but only 3 of them provided enough tissue for ASBVd
indexing using RT-PCR.
Figure 4-7. Shoot development (left) and plantlet recovery from somatic embryos of ‘Vero Beach’ SE2.
RT-PCR
RT-PCR showed that embryogenic cultures induced from ‘Vero Beach’ SE2,
‘Vero Beach’ 1, ‘Yama’ 381 and ‘Irwin’ were infected with ASBVd. ASBVd was
detected in the embryogenic cultures of ‘Vero Beach’ 1 indexed 6 and 9 months after induction, ‘Yama’ 381 indexed at 6, 9 and 12 months after induction, ‘Vero Beach’ SE2 96
embryogenic cultures from MSP, white-opaque somatic embryos at 6, 9 and 12 months
after induction, and ‘Vero Beach’ SE2 nucellar plants that originated from the same
explant.
ASBVd was also detected in embryogenic cultures that originated from multiple
nucellar explants induced in the year 2003 and maintained on MSP for 3-4 months. RT-
PCR detected ASBVd in 5 different embryogenic lines of ‘Vero Beach’ SE2, 6
embryogenic lines of ‘Yama’ 381 and 5 of ‘Irwin’.
Capillary and agarose gel electrophoresis
Figure 4-8 shows the results of the RT-PCR with embryogenic cultures of ‘Yama’
381 and ‘Vero Beach’ SE2 one year after explanting, somatic embryos of ‘Vero Beach’
SE2 and leaf samples from a nucellar plant of ‘Vero Beach’ SE2. The electropherograms show that the samples generated peaks that migrated at a similar size (approx. 250 bp) as those from floral tissues of ‘Vero Beach’ SE2 (Figure 4-8). In contrast, no signal of product amplification was observed with embryogenic cultures of ‘Hass’ 1 1.4.3.
Agarose gel electrophoresis (Figure 4-9) illustrates the results of RT-PCR on extracted RNAs from different tissues. PEMs of ‘Irwin’, ‘Yama’ 381 and ‘Vero Beach’ 1
4-9 (Figure lines 2, 3 and 4, respectively), somatic embryos of ‘Vero Beach’ SE2 (Figure
4-9 line and leaf tissue 5) from the three nucellar plants (Figure 4-9 lines 6 to 8) migrated in the gel at a similar size (approx. 250 bp) as those of the positive control consisting of floral tissue of ‘Vero Beach’ SE2 (Figure 4-9 line 9). RT-PCR performed on RNAs isolated from the negative control consisting of PEMs of ‘Hass’ 1 1.4.3. (Figure 4-9 lines
1 and 10) show the complete absence of bands. 97
Figure 4-8. Electropherograms of RT-PCR amplified fragments of: a: PEMs of ‘Hass’ 11.4.3, b: somatic embryo of ‘Vero Beach’ SE2, c: ‘Vero Beach’ SE2 PEMs one year after induction, d: PEMs of ‘Yama’ 381 one year after induction, e: ‘Vero Beach’ SE2 nucellar plant number 1 and f: floral tissue of ‘Vero Beach’ SE2. 98 M12 3456 789 10 M
= Figure 4-9. Agarose gel electrophoresis of RT-PCR amplified products. 1 and 10
PEMs of ‘Hass’ 11.4.3; 2 to 4 = PEMs of ‘Irwin’, ‘Yama’ 381 and ‘Vero Beach’ 1 respectively; 5 = Somatic embryo of ‘Vero Beach’ SE2; 6 to 8 = Nucellar plants number
1, 2 and 3 respectively; 9 = Floral tissue of ‘Vero Beach’ SE2; M- 100 bp ladder.
Cloning Sequencing and Variant Analysis
Clones were recovered from RT-PCR amplified products of PEMs, somatic
embryos and leaf tissue of nucellar plants all of which originated from a single nucellar
explant of ‘Vero Beach’ SE2 explanted in 2002 and floral tissue of ‘Vero Beach’ SE2.
Samples of PEMs were obtained from those growing on MSP and MS3:1N approx. 1
year after induction; somatic embryo samples were obtained from 8-week old somatic
embryos while leaf samples were taken from nucellar plants after 3-4 months on MS
medium.
A total of 102 clones harboring sequences with >90% similarity with ASBVd
were isolated and sequenced from several tissues of ‘Vero Beach’ SE2: 7 floral tissue, 10
from PEMs, 6 from somatic embryos, 16 from nucellar plant number 1 (NPT1), 19 from
nucellar plant number 2 (NPT2) and 44 from nucellar plant number 3 (NPT3). 99
The sequenced clone populations yielded 48 variants as a result of several indels
and nucleotide exchanges with respect to the variant J02020 (Symons, 1981) (Table 4-6).
Several variants were present in more than one tissue; however, several others were
unique to a specific sample. The variant J02020 was found in PEMs, somatic embryos
and nucellar plant 1 but not in floral tissue or leaf tissue from the other two nucellar
recovered plants. Figure 4-10 shows the nucleotide variation present in each sample
tissue with respect to the ASBVd J02020 secondary structure molecule. Variation
occurred at a higher frequency in the terminal regions of the secondary structure
molecule.
Table 4-6. Sequence variants of ASBVd isolated from ‘Vero Beach’ SE2.
Embryogenic tissue Sequence Clones Residue in Base Change Number of Reported Variant J20202 Bases J02020 4 247 Symons, 1981 A-2 1 +A 122-128 248 Rakowsky and Symons, 1989 B1 3 U->A 3 248 Rakowsky U->A 5 and Symons, +U 230-236 1989 CF3 2 U->A 3 246 Schnell et al., -A 7 2001 CF24 2 U>A 3 248 Schnell et al., -A 7 2001 +A 122-128
CF56 1 +AA 122-128 249 Schnell et al., 2001 CF62 1 U->A 3 247 No -A 7 +U 230-236 CF63 1 A->G 16 248 No +A 122-128 CF64 1 +U 72-73 248 No c->u 213 100
Floral tissue of ‘Vero Beach’ SE2
Sequence Clones Residue in Base Change Number of Reported Variant J20202 bases B4 3 U ->AA 3 247 Rakowsky U-> A 5 and Symons, 1989
B1 1 - U >AA 3 249 Rakowsky + U 230 - 236 and Symons,
CF65 1 U -> A 3 248 No U->A 5 A -> U 7
+ U 230 - 236 CF66 1 U-> AA 3 248 No U -> A 5 +AA 122-128 CF67 1 U->A 3 249 No U-> AA 5 + A 122-128 U -> A 148
Nucellar plant number 1
Sequence Clones Residue in Base Change Number of Reported Variant J20202 bases
J02020 - - 4 247 Symons, 1981 CF3 2 U->A 3 246 Schnell et al., -A 7 2001
CF24 1 U->A 3 247 Schnell et al., -A 7 2001 +A 122-128
A-2 1 +A 122-128 248 Rakowsky and Symons, 1989 CF68 2 C->U 215 247 No CF69 1 C->U 213 247 No CF70 1 AU->UA 3-4 247 No CF71 1 u->c 150 247 No G->A 186 CF72 1 C->G 165 247 No CF73 1 G->A 174 247 No CF74 1 U->A 3 246 No -A 7 U->C 210 101
Nucellar plant number 2 Sequence Clones Residue in Base Change Number of Reported Variant J20202 bases
A-2 1 +A 122-128 248 Rakowsky and Symons, 1989
CF3 3 U->A 3 246 Schnell et a!.. -A 7 2001
CF13 4 U->A 3 247 Schnell et al., U->A 5 2001
CF24 2 U->A 3 247 Schnell et al.. -A 7 2001 +A 122-128
CF2 2 U->A 3 247 Schnell et al., 2001
CF5 1 U-A 3 246 Schnell et al., -A 7 2001 G->A 207
CF75 1 -A 7 246 No G->A 207
CF76 1 U->AA 3 248 No U->A 5 G-A 207 CF77 2 -A 7 246 No U->A 5 A->G 131 G->A 207
CF78 1 U->A 3 248 No u->c 181 +A 122-128
CF79 1 U->A 3 248 No U->A 5 +U 129-130 102
Nucellar plant number 3
Sequence Clones Base Change Residue in Number of Reported Variant J20202 bases
CF3 16 U-A 3 246 Schnel! et al., -A 7 2001
CF12 4 U->A 5 247 Schnel! et ai., 2001
CF2 4 U->A 3 247 Schnell et al., 2001
CF13 1 U->A 3 247 Schnell et al., U->A 5 2001
CF8 2 U->A 3 248 Schnell et a!., U->A 5 2001 +A 122-128
CF24 3 U->A 3 247 Schnell et al., -A 7 2001 +A 122-128
CF34 1 U->A 3 248 Schnell et al., -A 7 2001 +AA 122-128 CF80 2 UA->AU 3-4 248 No +A 122-128 CF81 2 U->A 5 247 No u->c 150 CF82 2 U->A 3 245 No -A 7 -U 115-118
CF83 1 U->A 3 246 No -A 7 C->U 135
CF84 1 U->A 3 246 No -A 7 U->C 26
CF85 1 U>-A 3 245 No -A 7 -A 122-128
CF86 1 U->A 3 246 No -A 7 A-G 179
CF87 1 U->A 3 246 No -A 7 A->G 240
CF88 1 -U 3 248 No A->U 7 +AA 122-128
CF89 1 U->A 3 247 No U->A 5 u->c 217 103
The most common nucleotide changes detected in the sequence variants were a U
to A exchange at position 3 present in 77 clones, a U to A transition at nucleotide 5
present in 23 clones, an A deletion at position 7 detected in 45 clones and an A or AA
addition between nucleotides 122-128 sequenced in 23 clones. Nucleotide changes in the
stem area were scattered in the region and some occurred within the hammerhead
formation region (Figures 4-10). A reduction in the right terminal loop was observed in
two clones sequenced from nucellar plant 3; one variant had a U deleted between
positions 155 and 118 and one showed an A deleted between nucleotides 122-128.
Nucleotide changes commonly associated with the stage of development of in vitro
maintained material were detected within the cloned population (Table 4-7). Nine of the
10 variants isolated from PEMs had changes at the left terminal loops and only one had a
variation (+A between 122-128 poly-A stretch) at the right terminal loop. In contrast, out
of the 6 variants isolated from the somatic embryos 3 had changes at the right terminal
loop, but all lacked changes at the left terminal loops. In addition, 3 variations in the
stem area were detected in variants isolated from somatic embryos while no variation in
this area was observed in variants isolated from PEMs. Changes in the stem area were
more common among variants isolated from leaf tissue of nucellar plants where 19
variants possessed changes in this region. Only two changes detected in the stem region
were common to more than one tissue, i.e., a U to C exchange at nucleotide 150 was
detected in nucellar plants 1 and 3 and a C to U exchange at 213 occurred in somatic
embryos and nucellar plant 1 (Table 4-7).
Twenty-eight new sequences of ASBVd not previously reported were isolated from the clone population and were designated respectively as Chapman Field 62 (CF62) to Chapman Field 89 (CF89). The new ASBVd variants were unique to the tissue from 104
which they were isolated: CF62 was isolated from PEMs, CF63 and CF64 were isolated from somatic embryos, CF65 to CF67 were isolated from floral tissue, CF68 to CF74
were isolated from nucellar plant 1, CF75 to CF79 were from nucellar plant 2 and CF80 to CF89 were isolated from nucellar plant 3 (Table 4-6). Although, changes at the left and right terminal loops accounted for most the nucleotide exchanges, 20 of the new 28
ASBVd variants were the results of changes detected in the stem region (Table 4-7)
Table 4-7. Distribution of the number of variants isolated from in vitro tissues with respect to nucleotide exchanges in the ASBVd molecule.
Area in ASBVd Position Exchange PEMs SE NPT1 NPT2 NPT3 molecule 3 U->A 4 4 8 15
LTLs 5 U->A 1 4 5 7 -A 3 3 5 10
230-236 +U 1
122-128 +A 1 2 2 2 3
122-128 +AA 1 2
RTL 122-128 -A 1
115-118 -U 1
129 +U 1
16 A->G 1
72 +u 1
213 c->u 1 1
215 c->u 1
150 u->c 1 1
186 G->A 1
165 C->G 1
174 G->A I
SR 210 U->C 1 207 G->A 4
131 A->G 1
181 U->C 1
135 C->U 1
26 U->C 1
179 A->G 1
240 A->G 1
217 U->C 1 148 U->A (LTLs = Left Terminal Loops; RTL = Right Terminal Loop; SR = Stem Region). 105
a Embryogenic tissue
—i u- o U A t-U A x?-ac V A U' A g C <3 U A AC ^ 4 >» UU tllUGC G A GU UCUU AG 1 p u/ Figure 4-10. Nucleotide variation, frequency of changes and number of variants with these changes in a: embryogenic cultures, b: floral tissue, c: nucellar plant 1, d: nucellar plant 2 and e: nucellar plant 3 of ‘Vero Beach; SE2. b Flower tissue 5 1 msjm =qua U A S i-U A ~'2A1 - O' c V A Cl A 2 106 Nucellar plant 1 1-U A 247- G - C U A U A C G 1 HD 20 AC y) uu U u - ~ w u„ V. AG 100 GU U , uW c U GAGGA UA GA UAAACUUUGU GAC CAG GU ,. CU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA A [if] ACUUCUAU CG GGUGAACU CU AUUUGGAACG CUG GUC CA GA AGGGU GAAAGG GACUU CUG u CCT c T CACU CUCU CUUCU GC j c U T uc a 220 u uT aa l T I U GU I | * A A (6>*i u u 1 u u 1 u u Nucellar plant 2 S3 =lU G U A | 1-U A 247- G C U A U‘ A C G U A 80 120 A 40 20 AC UU A G u u lllU 4 G U _ . UU AG 100 GU U .. U U GAGGA UA GA UAAACUUUGU GAC CAG GU CU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA CG GGUGAACU a m ACUUCUAU CU AUUUGGAACG CUG GUC CA GA AGGGU GAAAGG 240-A GACUU^.CUG CACU CUCU CUUCU GC UCACUUGA U . cc C T CU, A A U 220 u A U Ju 200 ' G U C G 1 180 A a A U U u u Nucellar plant 3 H U A I 1-U A 27 1 *7 -° C H U A U A C- © : U A : U i I U U A?®AHA A UAAACUUUGU OAC ? 9 CAG GU CU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA CG GGUGAACU J ACUUCUAU CU AUUUGGAACG CUG GUC <3A CA AGGGU GAAAGG GACUU CUG CACU CUCU CUUCU GC UCACUUGA 240>A C C CU A A A A A CU UC . A UT AA ,^- A L 220 u Gu CO j 200 ij UGU IS*] | GS 2 107 Discussion Induction of embryogenic cultures in vitro was first attained from the nucellus of citrus (Stevenson, 1956). Nucellar seedlings of polyembryonic citrus cultivars are generally free of viroid and viruses (Weathers and Calavan, 1959) and provide a means to recover healthy clones from infected trees. The development of in vitro procedures for recovery of somatic embryos and recovery of plants from the nucellus of monoembryonic citrus species was reported by Rangan et al. (1968) and was the basis for the recovery of healthy nucellar plants from monoembryonic citrus and other non-polyembryonic plant species. Bitters et al. (1972), using somatic embryogenesis, reported the recovery of nucellar plants from several citrus species, including monoembryonic types, and based on the absence of symptoms in indicator plants, demonstrated that the recovered individuals were free of citrus exocortis viroid (CEVd) and several viruses. In the current research, we have attempted to eliminate avocado sunblotch viroid (ASBVd) using an approach similar to that described by Bitters et al. (1972). Embryogenic avocado cultures were induced from the nucellus, the cultures were maintained as embryogenic suspension cultures, somatic embryo development was initiated and nucellar plants were recovered. However, RT-PCR indexing established that ASBVd was persistent in the embryogenic cultures and in plants recovered from the nucellus of ASBVd- infected avocado plants. Avocado nucellar embryogenic cultures were first induced by Witjaksono et al. (1999b). In the current study, nucellar embryogenic cultures were induced from the nucellus by the permissive inductive pathway (Sharp et al., 1980; Ammirato, 1987; Litz and Gray, 1995). The frequency of induction was between 1.5 and 6.5% which is low 108 compared with citrus (10%— (Rangan 30%) et al., 1968; Bitters et al., 1872) and Carica pentagona (>40%) (de la Vega and Kitto, 1991) but comparable to previous avocado reports (Witjaksono, 1997). The collected data do not support any negative effect of ASBVd infection on avocado embryogenic culture induction, and the presence of ASBVd did not inhibit proliferation of embryogenic tissue. Maintenance of the embryogenic cultures occurred by continuous subculture on semi solid and in liquid media. The results of the suspension culture evaluation do not allow the establishment of any correlation between ASBVd infection and growth of embryogenic cultures in liquid medium. The data clearly demonstrated that ASBVd-infected embryogenic cultures have a typical suspension culture growth response with an initial lag phase during the first week, an exponential growth during the second and third weeks that ends between the third and fourth week. Witjaksono and Litz (1999a) observed that maintenance of embryogenic capacity in liquid medium was a genotype-related characteristic. In the current research, suspension cultures of ‘Vero Beach’ 1 and ‘Yama’ 381 ceased proliferation approx. 3 months after inoculation in liquid medium; in contrast, ‘Vero Beach SE2 and the ASBVd-free ‘Hass’ 1 1.4.3 have proliferated for >1 year. White-opaque somatic embryo development has been previously reported by Pliego-Alfaro (1981), Mooney and Van Staden (1987), Pliego-Alfaro and Murashige (1988), Raviv et al. and (1998) Wijaksono and Litz (1999b). In the current research, the presence of ASBVd did not affect the development of somatic embryos from ‘Vero Beach SE2 and Vero Beach’ 1 cultures; whereas, no white-opaque somatic embryos developed from ASBVd-free ‘Hass’ 11.4.3 and ASBVd-infected ‘Yama’ 381. Somatic 109 embryo development from citrus nucellar explants also varied with the genotype and was observed to range between 0 and 7.3 embryos for each explant (Bitters et al., 1972), Plant recovery occurred from a single cultivar at a very low rate (approx. 0.7%) in the current research. Low rates of avocado somatic embryos ( 5%) conversion into plants have been reported by Mooney and Van Staden (1987), Pliego-Alfaro and Murashige (1988) and Witjaksono and Litz (1999b), although >1 1% of somatic embryos forming shoots were reported by Raviv et al. (1998). Vega de Rojas and Kitto (1991) reported that only a single plant recovered from multiple somatic embryos that developed from nucellar explants, while no data on the number of plants recovered is reported by Rangan et al. (1968) and Bitters et al. (1972). Low conversion rates of somatic embryos into plants have been attributed to failure of shoot meristem development in somatic embryos (Mooney and van Staden, 1987; Pliego-Alfaro and Murashige, 1988; Nickel and Yeung, 1993). RT-PCR confirmed that ASBVd was present in embryogenic nucellar cultures that originated from nucellar explants of ‘Vero Beach’ SE2, ‘Irwin’, Yama’ 381 and ‘Vero Beach’ 1 and in somatic embryos and nucellar plants. In contrast to members of the Pospiviroidae family that localize and replicate in the cell nucleus, ASBVd localizes in the chloroplasts, where replication most probably occurs, (Lima et al., 1994; Navarro et al., 1999). Within the chloroplast, ASBVd is associated with the thylakoid membranes (Bonfiglioli et al., 1994). Thylakoid membranes develop only in fully mature chloroplasts during synthesis of chlorophyll (Vothknecht and Westhoff, 2001); however the embryogenic cultures and the somatic embryos in the current research were white or yellow but never green-colored. This suggests that ASBVd can replicate in the proplastid 110 or in an alternative site when chloroplasts are not fully developed and completely functional. ASBVd was detected in cultures that originated from the single explants of ‘Vero Beach’ 1 at 6 and 9 months after inoculation, and in cultures of ‘Vero Beach’ SE2 and ‘Yama’ 381 at 9 and 6, 12 months after inoculation and the presence of the viroid was supported by the cloning and sequencing of multiple sequence variants from different stages of development of in vitro cultures. These results indicate that the continuous subculture of ASBVd-infected embryogenic cultures does not eliminate the infection and that ASBVd multiplies during the different developmental stages of the somatic embryogenic pathway. Elimination of citrus exocortis viroid (CEVd) as determined by evaluation with indicator plants demonstrated that all of the nucellar seedlings of a ‘Robertson’ navel individual affected with CEVd were freed of the viroid. Our data based on RT-PCR indexing of embryogenic cultures that originated from 19 different nucellar explants revealed that ASBVd is not eliminated by nucellus culture. Avocado sunblotch viroid (ASBVd) and citrus exocortis viroid (CEVd) belong to different viroid families with marked differences. CEVd and other members of the Pospiviroidae family move long distances inside the plant through the phloem (Bonfiglioli et al., 1996). Since the nucellus is a non vascularized tissue (Esau, 1977; Rangaswami, it 1981), is possible that CEVd is unable to infect this tissue and that would result in the elimination of the viroid in the nucellar plants. The mechanism of movement of ASBVd and other members of the Avsunviroidae family is still unknown; however, if CEVd elimination occurs by the mechanism described above, the results of the current Ill research may indicate that ASBVd uses a different mechanism rather than vascular movement to infect nucellar tissue. The highest nucleotide variation of the cloned ASBVd population was detected in the terminal loops, which indicates specificity rather than randomization of variability areas as seen in previously reported variant populations (Pallas et al., 1988; Rakowsky and Symons, 1989; Semancik and Szychowsky, 1994; Schnell et al., 2001). A correlation was observed between patterns of nucleotide variation and tissue samples. Variations in the left terminal region, frequently observed in most of the indexed tissues, were absent in variants isolated from somatic embryos. Differential gene expression occurs during the somatic embryogenesis developmental pathway (Zimmerman, 1993); however, the effects of these changes, and the effect of environmental conditions in vitro on ASBVd sequence variability are unknown. Fourteen of the 17 changes in the stem area were located in the lower part between nucleotides 130-226 of the plus (+) strand; several of these changes occurred within the hammerhead formation area; whether there is any difference in stability between the two strands that conform the stem region and the detected changes in this region have any effect on the replication ability of the ASBVd molecule is yet to be elucidated. Viroid sequence variability has been associated with differential symptom expression in affected plants (Singh et al., 2003), and nucleotide changes in the right terminal region of the ASBVd molecule have been linked to bleaching symptoms in avocado leaves. Semancik and Szychowsky (1994) reported that insertions of one or several As in the 122-128 polyA stretch, UU to AA exchange between nucleotides 120- 121, exchange of A to U at position 1 19 or a U addition between nucleotides 115-118, all resulting in an enlargement of the right terminal loop are responsible for the bleaching 112 symptoms in ASBVd-infected ‘Hass’ trees. Studies involving ASBVd-infected trees in the NGR showed that 13 out of 15 samples isolated from leaf bleached tissue of ‘Aycock Red’ had a U or UU addition between nucleotides 115-118, while no UU to AA exchanges at position 120-121 or transition from A to U at nucleotide 119 were detected (Schnell et al., 2001). In the variant population sequenced in the current study, no U or UU additions between nucleotides 115-118 were found in the sequenced clones, but enlargement of the right terminal loop by one or several A residue additions between nucleotides 122-128 or a U addition between nucleotides 129-130 was detected. No bleaching symptoms were observed in the nucellar plants or in the ‘Vero Beach’ SE2 tree. Several nucleotide changes occurred in the stem region of the secondary structure molecule, and some of them were localized within the area of the hammerhead structure formation, whether these variations affect the hammerhead mediated replication process is unknown. Multiple ASBVd sequence variants have been previously isolated from avocado trees in the NGR. In the current research, 28 new ASBVd sequences were found among the 48 isolated variants, and 18 of the 20 previously reported sequences were first isolated from cultivars ‘Lima Late’, ‘Aycock Red’, ‘Wilson Popenoe’ and ‘Young Special (Schnell et al., 2001a; b). ASBVd has been found to localize in the thylakoid membranes of the chloroplasts and evidence that ASBVd replication occurs within this subcellular organelle has been provided (Lima et al., 1994; Bonfiglioli et al., 1994; Navarro et al., 1999). The results of the cloning protocol in this research showed that ASBVd was able to persist in PEMs devoid of chloroplasts >12 months after induction, which indicates that ASBVd may replicate in the proplastid or a different subcellular organelle when chloroplasts are not fully developed. Besides being smaller and lacking 113 thylakoid membranes, proplastids are in much lower copy numbers than chloroplasts, which accounts for lower levels of transcriptional gene activity (Baumgamer et ah, 1989). Plastids contain both nuclear encoded (NE) and plastid encoded (PE) gene products (Martin et ah, 1990; Abdallah et ah, 2000). Although expression of nuclear encoded genes in the proplastids is possible (Harrak et ah, 1995), high levels of transcription occur only during the proplastid to chloroplast conversion and further in fully developed chloroplasts (Bisanz-Seyer et ah, 1989; Baumgartner et ah, 1993). Indication that specific genes control this transition comes from the isolation of mutant genes that block plastid development (Keddie et ah, 1996; Chatteijee et ah, 1996; Mandel et ah, 1996). Studies involving the use of fungal and bacterial toxins that inhibit activity of nuclear and plastid polymerases have suggested that a nuclear encoded polymerase (NEP) is the most likely type of enzyme to be associated with the replication of ASBVd. In addition, Navarro and Flores (2000) reported that the initiation of 5’ sites of both polarity strands of ASBVd reveal similarity to the promoter region of 2 chloroplastic genes transcribed by a nuclear encoded polymerase (NEP), and more recently Daros and Flores (2002) isolated two proteins associated with ASBVd RNAs in vivo that contain transit peptide sequences capable of transporting products from the cytoplasm into the chloroplast and facilitated the ASBVd hammerhead-mediated self cleavage. In the current research, a relative low number of ASBVd variants was isolated from non chloroplastic tissues i.e., 5 from PEMs and 5 from white opaque somatic embryo. In contrast, using a similar protocol, more than twice the number of variants was isolated from green leaf tissue of each nucellar plants (NPTs) i.e., 1 1 from NPT1, 1 1 from NPT2 and 16 from NPT3. Whether any specific gene product involved in the proplastid to chloroplast conversion triggers a high number of ASBVd sequences is beyond the scope 114 of this research; however, the data strongly suggest that one mechanism inherent to this transition could be responsible for not only an increased ASBVd replication level but also a higher rate of mutation. Multiple variants of viroid in a single host has been characterized as quasispecies (Ambros et ah, 1999; Schnell et al., 2001). Quasispecies are believed to be the result of the host:pathogen interaction in a surrounding environment where the best fitted sequence overgrows the others (Duarte et al., 1994). Studies with potato spindle viroid (PSTVd) showed that when several mutant variants infect a single individual, many revert to the wild type sequence (Owens et al., 1986). Therefore, although several viroid variants are present during the early stages of infection, the wild type will predominate over time (Diener, 1996). Deviations from this rule have been demonstrated with ASBVd (Schnell et al., 2001). In the sequence population isolated in this study, several variants occurred more frequently than the J02020 variant. Among nucellar plants, J02020 was observed in one plant (NPT1) and was absent from clones isolated from floral tissue. In contrast, other sequences were overpopulated with respect to the rest of the variant population, i.e., CF3, a variant initially isolated from ‘Lima Late’ (Schnell et al., 2001a), was detected in 16 of the 44 clones isolated from NPT3. These data clearly show that the cloned ASBVd sequence populations resemble a quasispecies, in which the wild type variant is not necessarily the predominant sequence. Schnell et al. (2001) suggested that the ASBVd quasispecies isolated from trees at the National Germplasm Repository have resulted from many years of infections that allow many replication cycles and believed that some trees may have been infected since the mid 1930’s. In the present research, three new ASBVd variants (CF59 - CF61) were isolated from non green embryogenic cultures 9 months after induction and 22 new 115 different variants (CF68 - CF89) were isolated from leaves of nucellar plants 3 months later (12 months after induction). These results indicate that the in vitro somatic embryogenesis pathway can generate a similar degree of sequence variation in the ASBVd molecule observed in field grown plants but in a much shorter period of time (See discussion above regarding effects of the proplastid to plastid transition interaction with ASBVd). Viroid replication in vitro has been reported for PSTVd in potato and tomato cell culture (Muhlbach and Sanders, 1981; Stocker et al., 1993) and CEVd in micropropagated shoots of citron (Greno et al., 1988); however there are no reports of viroid replication in embryogenic cultures. ASBVd can replicate in avocado embryogenic cultures, and in somatic embryo-recovered plants. Levels of ASBVd vary with the environmental conditions and the developmental stage of the plant (da Graca and Moon, 1983; Schnell et al., 1997). ASBVd sequence variation appears to be associated with tissue developmental stage. Infection of embryogenic cultures with a known sequence variant could elucidate the changes that take place in the viroid during the somatic embryogenesis pathway at a specific developmental stage and the effect of those changes on symptom expression of the regenerated plants. CHAPTER 8 SUMMARY AND CONCLUSIONS Summary In this study two approaches were utilized in an attempt to recover avocado sunblotch viroid (ASBVd)-free avocado plants from ASBVd-infected material. These strategies involved the induction of embryogenic cultures and regeneration of avocado plants from the nucellus of 4 avocado cultivars infected with ASBVd, and the micrografting of shoot tips of ASBVd-infected seedlings on in vitro germinated healthy seedlings. Embryogenic cultures were induced on semi solid avocado induction medium (AIM) and were maintained on semi solid MSP medium. Embryogenic suspension cultures were maintained in MS3:1N liquid medium and their growth was characterized and compared with an embryogenic line derived from a healthy avocado cultivar. Somatic embryos developed from embryogenic cultures on semi solid somatic embryo development medium (SED) and conversion into plantlets occurred when somatic embryos were inoculated on somatic embryo germination and maturation medium (SEGM). For micrografting experiments, zygotic embryos were excised from fruits of two ASBVd-infected avocado cultivars and germinated in vitro on MS medium. Scions, consisting of apical shoot tips between 0.2 mm to 0.8 mm length, were micrografted onto decapitated in v/Yro-germinated healthy seedlings on MS medium. 116 117 Micrografted plants were transferred into 266 ml Qorpak clear glass jars and cultured for at least six months. Embryogenic cultures consisting of PEMs from the 4 cultivars, and white-opaque somatic embryos and leaf tissue from nucellar plants were indexed for ASBVd infection using RT-PCR. The electropherograms showed that amplified fragments similar to those of the positive control were generated by all the embryogenic cultures of the 4 cultivars. Amplified fragments from PEMs, somatic embryos and leaves from the nucellar plants were cloned and sequenced. The sequencing cycle allowed the isolation of 102 clones harboring sequences with >97% similarity to the ASBVd wild type molecule. Forty-eight ASBVd variants were isolated and 28 of them were new ASBVd sequences. All of the new variants were unique to the tissue from which they were isolated. The analysis of the variant sequences indicated that somatic embryogenesis may have induced a high level of sequence variability in the ASBVd molecule and that this variability can be correlated with the stage of development of the embryogenic tissue and the conversion of proplastids into chloroplasts. Micrograft recovery was higher when larger scions (0.5-0.9 mm length) were used irrespective of the avocado genotype employed. Leaf samples from micrografted plants demonstrated that ASBVd was transmitted from infected scion-donor seedlings using scions <0.5 mm length. RT-PCR indexing for ASBVd infection showed that for those micrografts that tested positive, ASBVd was also present in their respective scion- donor seedlings regardless of the avocado genotype, while those that tested negative originated from healthy scion-donor plants. Cloning and sequencing showed that the RT- PCR amplified fragments corresponded to ASBVd, and establishes that ASBVd is persistent in micrografts recovered from shoot tips as small as <0.5 mm long 118 Conclusion Nucellar somatic embryogenesis and micrografting failed to eliminate avocado sunblotch viroid (ASBVd) from infected tissue in contrast to citrus exocortis viroid (CEVd) infection in citrus plants which can be eliminated by these two approaches. The information generated in the current study can have significant implications for further studies of host:pathogen interaction. 1. ASBVd is persistent in avocado micrografts originated from <0.5 mm long shoot tips, which indicates that Avsunviroidae viroids infect the shoot apical meristem and may move inside the plant using non-vascularized tissue. 2. ASBVd does not inhibit induction and maintenance of embryogenic tissue, and ASBVd-infected embryogenic cultures showed typical cell suspension growth in liquid medium. 3. ASBVd is persistent in embryogenic cultures and somatic embryo-recovered plants. The former will permit the use of the embryogenic pathway to study host:pathogen interactions. 4. Somatic embryogenesis may cause nucleotide changes in the ASBVd molecule as a result of rapid cell division and development. This information can be used to study the changes in the ASBVd sequence in avocado embryogenic cultures and the evolution of those changes with respect to the stage of development of the embryogenic tissue. 5. ASBVd proliferated in embryogenic cultures devoid of chloroplasts which indicates that ASBVd replication occurs in the proplastid or in another intracellular organelle; however a much larger number of ASBVd sequences 119 and variants occurred in the green leaves of nucellar plants indicating that the transformation of proplastids into chloroplasts may induce a greater rate of ASBVd replication and mutations. APPENDIX ANOVAs OF NUMBER AND TYPES OF EMBRYOGENIC CULTURES THAT DEVELOPED ON MSP MEDIUM, ANOVAs OF SETTLED CELL VOLUMES LIQUID MEDIUM, (SCVs) IN ASBVd VARIANT SEQUENCES ISOLATED FROM MICROGRAFTS ASBVD VARIANT SEQUENCES r'J^ ISOLATED FROM PEMs, SOMATIC EMBRYOS FLORAL TISSUE AND NUCELLAR PLANTS Table A-l. ANOVAS of number and type of embryogenic cultures that developed on Source DF Sum of Squares Mean Sauare F Value Pr > F Total SEs Cultivar 3 74.33 24.77 0.91 0.4459 Error 33 896.90 27.17 Total 36 971.24 SEs < 0.5 cm diam. Cultivar 3 6.12 2.04 0.25 0.85 Error 33 266.90 8.08 Total 36 273.02 SEs > 0.5 cm diam. Cultivar 1 3 2.01 0.67 0.42 0.74 Error 33 53.17 1.61 Total 36 55.18 PEMs Cultivar 3 3.69 1.23 0.57 0.63 Error 33 71.06 2.15 Total 36 74.75 Globular embrvos Cultivar 3 31.31 10.43 1.92 0.14 Error 33 179.00 5.42 Total 36 210.32 Model: Y + i : t - + et Y= n Independent variable i.e., Total SEs, SEs <0.5 cm diam. SEs >0.5 cm diam., PEMs and = Globular embryos; fi Mean; i = Cultivar; = - e error SE somatic embryo; PEM = proembryonic mass 121 122 Table A-2. ANOVAs of settled cell volumes (SCVs) in liquid medium DF Sum of Mean Sauare F Value Pr > F Squares Dav 3 Cultivar 3 1.54 0.51 21.30 <.0001 Error 16 0.38 0.02 Total 19 1.92 Dav 6 Cultivar 3 9.70 3.23 24.53 <.0001 Error 16 2.11 0.13 Total 19 11.81 Dav 9 Cultivar 3 39.69 13.23 44.14 <.0001 Error 16 4.79 0.29 Total 19 44.48 Dav 12 Cultivar 3 83.76 27.87 45.70 <.0001 Error 16 9.76 0.61 Total 19 93.39 Dav 15 Cultivar 3 231.15 77.05 70.25 <.0001 Error 16 17.54 1.09 Total 19 248.70 Dav 18 Cultivar 3 720.77 240.25 72.72 <.0001 Error 16 52.86 3.30 Total 19 773.64 Dav 21 Cultivar 3 1267.38 422.46 199.66 <.0001 Error 16 33.85 2.11 Total 19 1301.24 Dav 24 Cultivar 3 1424.42 474.80 394.13 <.0001 Error 16 19.27 1.20 Total 19 1443.70 Dav 27 Cultivar 3 1282.14 424.38 464.55 Error 16 14.72 0.92 <.0001 Total 19 1296.86 Dav 30 Cultivar 3 1204.04 401.34 264.22 '<.0001 Error 16 24.30 1.51 Total 19 1228.34 Model: Y - + / + Cell t // ef. Y= Settled Volume (CSV): n = Mean; i = Cultivar; e = error G 123 Figure A-l. ASBVd variants isolated from micrograft No 133 J02020 U G U A 1-U A 247- G C U A U A C G U- A 80 40 k 20 u u * AC UU UU G„ so A U AGGA Z.. I G U U C UU AG 100 GU U ? yA ?A UAAA CUUUGU GAC n OUM CU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA ACUUCUAU CU AUUUGGAACG CUG CG GGUGAACU *®SSU aA * A8a OACUUcyCW CACU CC C CU A A CUCU CUUCU GC UCACUUGA 220 A A®* A U AA U 200 U GU 140 180 A A A 180 CF2 U‘ A 1-U A 247- - C C G U- A 20 T. AC U U 60 U A UU G ® *7* , u U GAGGA A G U t C UU AG 100 GU U UA GA UAAA CUUUGU GAC CAG GU CU UUCCG CUUUCC CUGAG GAC ®A °A ®AGGA ACUUCUAU CU AUUUGGAACG CUG CG GGUGAACU GUC CA GA AGGGU GAAAGG GACUU CUQ CACU -CC C CU A 'CU CUCU CUUCU GC UCACUUGA 220 AAA a UC A U AA . J I( 200 , 180 A A A CF24 a u u A AK- m 1 U G U A 1-U A 247-G C U A U A C G U- A A A G UU ° UU<5A? u AG 100 GU U ° 0 = AS 00 c ‘oac^oug. GAGA SfeACUUCUAU CU AUUUGGAACG WW&tWm W? GAGGA CG GGUGAACU CUG GUC CA GA AGGGU GAAAGG GACUU 240-A I CC c CUG CACU CUCU CUUCU GC ' ,^ <=U A UCACUUGA UC i A U G U A U AA Uu C G GU 140 A U A A A U U U U U U i . 124 Figure A-2. ASBVd variants isolated from PEMs, somatic embryos, floral tissue and nucellar plants Polyembrvonic masses (PEMs) CF62 ri UU “a A>— —I U- G U A 1-U A 247- O'C U A U- A C G U A 40 20 * Ac UU U Cfl u UU G G A UGAGGAUA GA ^ i G U U C UU AG 100 GU U UAAACUUUGU GAC CAS GU CU UUCCG CUUUCC CUGAG ACUUCUAU GAC GUGA GAGA GAGGA CG GGUGAACU CU AUUUGGAACG CUG GUC CA GA 240-A U AGGGU GAAAGG GACUU CUG CACU CUCU CC C CU, A t,u CUUCU GC UCACUUGA 220 UC A U U A U AA i U.. 200 140 C G GU , A U 180 U U 160 U U U U T Somatic embryos CF63 U A 1-U A 247- GC U A U* A C G U A J AC 3 / U A ^ U U (!) u , - -U w C UU AG 100 GU U U GAGGA UA GA UAAACUUUGU GAC ?AG GU CU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA ACUUCUAU CU AUUUGGAACG CUG CG GGUGAACU *151 -*' GUC CA GA AGGGU GAAAGG GACUU CUG M0 U ’ CC C CU CACU CUCU CUUCU GC UCACUUGA l * * “ M0 UC I A U u . A U AA CG *» u A A • U a A J u i u U U A 125 CF64 U G U A 1-U A 247-G C U A U A C- G U A A 02 80 40 a a ” AC UU U U l!>U G r ^60 U*u ya*0«u* GA U C GU U U UAAACUUUGU GAC CAG GU CU UUCCG*CUUUC? r„„„ „. ““ T „ ACUUCUAU— CU AUUUGGAACG CUG ““ 9? °^<“ACU 240-A GUC CA GA AGGGU GAA.GG GACUU U CC CU, A CUG «CU CUCU CUUCU GC UCACUUGA A- U J20 ? A n u T «u U AA I Uu C- G ^ G U 140 u i 160 » A' U A a A U U U U Floral tissue CF65 —| U G U A 1-U A 247-G C U- A U A C G U- A A 20 A AC UU U A . . U U.GA®<^UA GA G U U C UU AG 100 GU U UAAACUUUGU^ GAC CUUU?C U ACUUCUAU - 90 CUGAG CG GGUGAACU CU AUUUGGAACG CUG GUC CA tl AGGGU^GAAAGG u ' CC C CU A "CUU^CUG CACU CUCU CUUCU GC 220 ? A A UCACUUGA uc A U AA l . G U 1 I U G U 140 180 , 160 CF66 1 —. U- G U A §J 1-U' 247-G' C U A U A C G U- A 120 „ a UU * U U uA I UU G AG 100 u i, UGAGGA UA GA UAAACUUUGU GAC U C GU u ACUUCUAU CU AUUUGGAACG CUG GUC.CA.GA AGGGU GAAAGG ScUG g* “cU CUUCU CU A A GC S2 ou A U 200 G U AA A a A H1 A C 1 126 CF67 ESI u u 155*1 > . u 6 U A 1-U A 247- G C U A U A 80 „ 40 * 5 * U U UU UU G c G A UGAGGAUA GA UAAACUUUGU G U «. UU AG 100 GU GAC CAG GU CU UUCCG CUUUCC CUGAG ACUUCUAU (SAC GUGA GAGA GAGGA CG GGUGAACU A® CU AUUUGGAACG CUG GUC CA GA AGGGU cc c GAAAGG GACUU CUG CA CU CUCU CUUCU - CU. A CUJ GC UCACUUGA A U 220 u UC A U TaA C G 200 U GU' 140 urso A A A 160 1 Nucellar plant 1 CF68 1-U A 247-0- U A U- A C G U- A A 40 120 20 u u U A^ UU ~ , — " v» A UGAGGAUA GA I w u b UU G 100 GU U UAAACUUUGU^ GAC CAG GU CU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA CG GGUGAACU ACUUCUAU CU AUUUGGAACG CUG CA 240-A U AS CF69 U G U A 1-U - 247- GC U A 80 40 20 U \ .. 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CO 200 G U MO 1 A • U A A A u u 160 u u u u CF71 u u A A U G U A 1-U A 247- G-C U A U A C G U A A 40 A G 20 U U UU c U GAGGA G*®A GU UC AG 100 GU UA GA UAAACUUUGU GAC ?AG GU CU UUCCG - CUUUCC CUGAG GAC ACUUCUAU CU —T —GUGA GAGA GAGGA CG GGUGAACU AUUUGGAACG CUG GUC CA GA AGGGU GAAAGG 240-A U CC C GACUU CUG CACU CUCU CUUCU GC UCACUUGA 220 CU A A A A- U “° “ OU UC u AA C- G I u u 140 A U l U U f CF72 u u A A U- G U A 1-U A 247- G C U A U A C G U- A 80 A 40 20 AC UU A G U U UU G OA UAAACUUUGU G U C UU AG 100 GU U GAC CAG GU CU UUCCG CUUUCC CUGAG y ACUUCUAU GAC GUGA GAGA GAGGA CG GGUGAACU CU AUUUGGAACG CUG GUC CA GA AGGGU GAAAGG GACUU ,,CUGCUG CACUC CUCU CUUCU GC CC C i CU, * AAA A C UCACUUGA 220 U UC A U AA 200 C U °j u GU 140 . A a A 160 U U 128 CF73 U G U A 1-U A 247- G- C U A U' A C G U- A 40 20 UU G UGAGGAUA g iA GU UCUU AG 100 GU GA UAAACUUUQU GAC CAG GU CU UUCCO CUUUCC CUGAG GAC GUGA GAGA GAGGA CG GGUGAACU ACUUCUAU CU AUUUGGAACG CUG GUC CA ' GA AGGGU GAAAGG • GACUU CUG CACU cc c CU. 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AC UU U u G A G AG 100 I UGAGGAUA GA UAAACUUUGU i U w C UU GU 9^9 CAG GU CU UUCCG CUUUCC CUGAG GAC ACUUCUAU GUGA GAGA GAGGA CG GGUGAACU CU AUUUGGAACG CUG GUC CA GA AGGGU GAAAGG 240 -A U CC GACUU CUG CACU CUCU CUUCU GC UCACUUGA C T CU, A A C U A U 220 AAA UC A U AA C G 200 U 140 A A A U U A 129 CF76 u u A U G U- A It;®1-U- 7-G C U A U A C- G 80 40 120 20 AC JU A u u lliu U U ~ ” " G« Uu AG 100 U VpAGGAJJA | UC uu OU UAAACUUUGU GAC CAP GU CU UUCCG ACUUCUAU CUUUCC CUGAG SAC GUGA GAGAGA GA GAGGA CG CU AUUUGGAACG CUG AGGGU GGUGAACu" 240 - A • U cc GAAAGG aACUU CUO cacu C T CU, A A A cu bucu CUUCU GC UCACUUGA A U 220 u u C- G 200 G U A U a A A U U U U U U CF77 1^*1 U G U A 1-U A 247- GC u A U A c- G U A 40 120 20 A U t!iu U y C G I PpAGOAUA GA UAAACUUUGU A G U L C UU AG 100 GU U GAC ?A G GU CU UUCCG CUUUCC CUGAG U ACUUCUAU GAC GUGA_ GAGA GAGGA CG GGUGAACU CU AUUUGGAACG CUG GUC CA GA 240 -A AGGGU GAAAGG GACUU CUG CACU U CC C T CU, A AAA CU CUCU CUUCU GC UCACUUGA A- U 220 U a A U AA C G 2C0 G U 140 A A A 160 CF78 130 CF79 —iU- o U A 5 1-U A 347-0- U A U A C G U- A 120 A AC UU G *7 A G U U GAGGA UA GA UA A A CUUUGU X. „ UCUU AG 100 GU U GAC cag ou cu yucca cuuucc cugag U ACUUCUAU CU gac guga GAGA GAGGA CG GGUGAACu' AUUUGQAACG CUG GUC CA GA AGGGU GAAAGG i GACUU J Ct, CU| CUG CACU CUCU CUUCU GC UCACUUGA 220 y u AA 200 G U 140 ' A A A Nucellar Plant 3 CF80 —'U G U A r 1-U - 247- G C U- A U A C- G U- A 120 40 20 AC UU A U uu 60 “ U , u 240- A l CC ssssyss szj&Ysss- A- U 220 200 Gu u C G l u G u 140 A U 180 A a A U U 160 U U U U CF81 ESI 1 u u A A 1-U- A 247-0 C U A U A C- G U* A 120 4 _ 20 P A AC 3 u U \ „ UU U A i UU G c , i G « A GU UC UU AG 100 GU YPAGGAUA GA UAAA CUUUGU GAC CAG GU CU UUCCG CUUUCC CUGAG ®AC GUGA GAGA ACUUCUAU CU AUUUGGAACG GAGGA CG GGUGAACU CUG GUC CA GA AGGGU GAAAGG GACUU CUG CC C CU. A CACU CUCU CUUCU GC UCACUUGA A 220 A UC i G U UT AA Uu A 200 U GU 140 y ' j A A A 160 U U A 131 CF82 -u 0 H U A “ 1-U A 247-0 C U A U A C 0 U- A 80 k 120 „ 40 a 3 “ AC UU u U (!l(j \ U A o G A (j U .°AGGAUA GA | GU UCUU AG 100 GU UAAACUUUGU QAC CAG GU CU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA ACUUCUAU CU AUUUGGAACG CUO GUC CG GGUGAACU " CA GA AGGGU GAAAGG GACUU CUG CACU '.CC C CU. A AAA GU CUCU CUUCU GC UCACUUGA 220 u A jj£ A4 U AA 200 U G U 140 180 160 U U u u CF83 I U- G U A GC | 1-U- 247-° c m u a U A C- G U- A W 120 A A A g *> u u AC UU u * uu g C I U U GA ®GAUA GA U a UAAACUUUGU GAC CAG GU CU UUCCG CUUUCC CUGAG GAC GUGA GAGA GAGGA CG ACUUCUAU CU AUUUGGAACG CUG GUC CA GGUGAACU 240-A GA AGGGU GAAAGG GACUU CUG U '.CC C CU A CACU CUCU CUUCU GC UCACUUGA A- 220 u uc U AA i CG 200 U 140 A U 180 * A A §S] CF84 'U- G | U- A rr L 1-U- A 247- GC U A U A C G U- A A 1 *? A A A G 20 AC i U u UU G C G “ A 0 AG 100 U U GAGGA UA GA UAAACUUUGU GAC 0 u c uu GU U CAG GU CU UUCCG CUUUCC CUGAG ® u ACUUCUAU CU A ? GUGA ®AGA GAGGA CG GGUGAACU AUUUGGAACG CUG GUC CA GA AGGGU GAAAGG CC C GACUU CUG CACU CUCU CUUCU GC CU A AAA u UCACUUGA 220 i a UC A U AA i J , 200 U GU 140 y 180 A A i A 160 A 1 132 CF85 U U A A -»U 6 U A 1 -U A 247-0 C U A U A C 0 U A 80 40 120 20 AC UU u u UUOU G 60 U A u — I " G« w U' L AG 100 y?.A®9*UA GA UAAACUUUGU GAC C uu GU U CAG GU CU UUCCO CUUUCC CUGAG GAC ACUUCUAU CU AUUUOGAACG GUGA GAGA GAGGA CG GGUGAACU A' CUG GUC CA OA AGGGU OAAAGG 240- U CC C GACUU CUG CACU CUCU CUUCU GC »fl CU A A UCACUUGA A- U ”” U «U UC * u aa CO M U G U 140 A U A A A U U U U U U I ; CF86 u u 11- Q U A S] 1-U A 247-0 C c o u- A 80 120 40 A 20 ac UU A U U l!»U u C O A , U V?AGGAUA OA UAAACUUUOU GU UC AG 100 GU U GAC CAG GU CU UUCCG CUUUCC CUGAG ACUUCUAU GAC GUGA GAGA GAGGA CG GGUGAACU A CU AUUUGGAACG CUG GUC CA GA 240-A U AGGGU GAAAGG GACUU CUG CACU A CC C CU A CU CUCU CUUCU GC UCACUUGA A 220 UC A U U AA i A C 0 200 u G U 1^0 ‘ A U ieoj A A A U U U U SI CF87 U U A A, —n- O U A IS 1-U - 247-0- C U- A U A C G U- A 80 40 120 — 20 u UU uA i u UU G i AG 100 GU U UOAGGAUA GA UAAACUUUGU GAC U A °'J CU fyuucc CUGAG GAC GUGA ACUUCUAU CU AUUUGGAACG GAGA GAGGA CG GGUGAACU CUG OUC CA GA AGGGU GAAAGG GACUU ' CUG CACU CC C CU, A * CUCU CUUCU GC UCACUUGA A ’ U 220 1 A U AA / u Gr u1 C O 200 G U 140 ' A U A A A U U U U U U 133 CF88 ,U,J lU- G mr U A 03 i-u a 247-G- C U A U A C 0 U A A 40 20 A G f cn AC uu I G G A L G r. - i o u U C AG 100 * GGAUA «A UAAA UU GU u . 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Plant Cell 5:1411-1423 Zaitlin M and Hariharasubramanian V (1970) Proteins of tobacco mosaic virus and potato spindle tuber virus-infected plants. Phytopathology 60:1537-1538 BIOGRAPHICAL SKETCH Isidro th E. Suarez Padron was bom on the 20 of February 1971 in Monteria, capital of Cordoba department, in Northern Colombia, the oldest son among four siblings, Carmen, Ana and Sahastid, of Adolfo Suarez Gutierrez and Rosiris Padron Jattin. He grew up in Monteria and attended school at Simon Bolivar Elementary School and Luis Lopez de Mesa High School in the same city. After graduating from high school in 1987, he pursued studies in the Agronomy Department of the Universidad de Cordoba, where he graduated in 1994 as Ingemero Agronomo. After his graduation, he worked as a consultant in several private companies for two years. In November 1996, after a selective competition, he was appointed as Profesor Auxiliar in the areas of Plant Propagation and Tropical Fruit production in the Agronomy Department of the Universidad de Cordoba. After a national draft in 1999, he received a FULBRIGHT-COLCIENCIAS-LASPAU scholarship to study a PhD program in the United States of America. In summer 1999, he attended an intensive English course at the University of Pittsburgh and in August of the same year he was admitted into the Horticultural Science Department of the University of Florida in Gainesville to study for the PhD with specialization in biotechnology of tropical fruit crops. He spent two years taking courses in Gainesville before moving to the Tropical Research and Education Center to begin his research. He is married to Sahara Vasquez and blessed with two lovely sons, 4-year-old Sebastian and 3-year-old Elias David. 155 aVe r, ad thiS StUdy and that in ray °Pinion « conforms to acceptableacceptaW^standarrlV f i , standards of scholarlyH presentation and is fully adequate in ’ scone andd m.alifv as a dissertation for P q the degree of Doctor of Philosophy. ^ Richard E. Litz,' Chair Professor of Horticultural Science 1 ‘ ' S h S,“dy a"d ,ha‘ in my °Pinio" * conforms to accentMe^Ztlacceptable standards of scholarly presentation and is fully adequate in scone and m.alitv as a dissertation P * for the degree of Doctor of Philosophy. • V SclW Raymond J. Sthneli Adjunct Associate Professor of Horticultural Science ‘ ‘ hiS StUdy and that in my inion “ acceptableacceotaLe'^H °P <° standards of scholarly presentation and is fully adequate in scone and nualitv as a dissertation P qUallty> for the degree of Doctor of Philosophy. Dennis J. Gray Professor of Horticultural ScieW" I certify that I have read this study and that in my opinion it conforms acceptable to standards of scholarly presentation and is fully adequate in scope and as a dissertation P qquality for the degree of Doctor of Philosophy. Paul M. Lyrene Professor of Horticultural Science I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a dissertation P Q Y ’ for the degree of Doctor of Philosophy. -hb — Nigel A. Harrison Associate Professor of Plant Pathology This dissertation was submitted to the Graduate Faculty of the College of Apiculture and Life Sciences and to the Graduate School and was accepted as partial lulfillment of the requirement for the degree of Doctor of Philosophy. December 2003 Dean, College of Agriculture and^Life fences Dean, Graduate School