Effect of Nutrient Medium Strength, Cytokinins and Auxins on Micropropagation of Gardenia (Gardenia Jasminoides L.) Using Nodal Explants

Effect of Nutrient Medium Strength, Cytokinins and Auxins on Micropropagation of Gardenia (Gardenia jasminoides L.) using Nodal Explants

Amjad Suleiman Khader Elyan B. Sc. in Agriculture (Plants Production) University of Jarash, Jordan (1999)

A Dissertation Submitted to the University of Gezira in Partial Fulfillment of Requirements for the Award of the Degree of Master of Science

in Horticultural Sciences (Plant Tissue Culture)

Department of Horticultural Sciences Faculty of Agricultural Sciences

October, 2018

Effect of Nutrient Medium Strength, Cytokinins and Auxins on Micropropagation of Gardenia (Gardenia jasminoides L.) using Nodal Explants

Amjad Suleiman Khader Elyan

Supervision Committee Name Position Signature Dr. Ibtisam Basheir Abdalla Main supervisor………….. Prof .Mohamed Ahmad Ali Co- Supervisor ………. …..

Effect of Nutrient Medium Strength, Cytokinins and Auxins on Micropropagation of Gardenia (Gardenia jasminoides L.) using Nodal Explants

Amjad Suleiman Khader Elyan

Examination committee Name Position Signature

Dr. Ibtisam Basheir Abdalla Chairperson ………………….

Prof. Adil Omer Salih Abdelrahim External examiner…………….

Dr. Osman Abdalla Ali Internal examiner …………….

Date of Examination: 11 / 10 / 2018

Dedication

All praise to Allah, today, the fatigue of the days and the salvation of the journey between the cover of this humble work.

To those who sought to enjoy the comfort and happiness that did not spare anything to push me in the rising of the ladder of life by virtue and patience, to my dear father.

To the spring that never stops giving, to my mother who weaves my happiness with strings from her merciful heart... to my mother.

To whose love flows in my veins and my heart always remembers them, to my wife

Amjad Suleiman Elyan

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ACKNOWLEDGMENTS

I wish to express my deep sense of gratitude to my supervisor Dr. Ibtisam Basheir, for her outstanding guidance and support, which helped me in completing my thesis work. I would also like to thank Prof. Mohamed Ahmad Ali, for his valuable assistance and help to fulfill my work. Words are inadequate in offering my thanks to my friends in Plant Tissue Culture Laboratory, Agricultural Research Corporation, wad Medani, Gezira State. Last, but not least, I would like to express my heartfelt thanks to my parents, my brother for their unconditional support and encouragement to pursue my interests, for listening to my complaints and frustrations, and for believing in me, my friends and colleagues for their help and wishes for the successful completion of this project. Amjad Suleiman Elyan

Effect of Nutrient Medium Strength, Cytokinins and Auxins on Micropropagation of Gardenia (Gardenia jasminoides L.) using Nodal Explants

Amjad Suleiman Khader Elyan

Abstract

Gardenia is one of the most important ornamental plants worldwide due to its beautiful flowers. Propagation of gardenia by conventional methods is slow and difficult. The main objective of this study was to develop a rapid propagation method using micropropagation techniques. Experiments were conducted to study the effect of different strengths (quarter, half, full) of Murashige and Skoog medium (MS) on gardenia morphogenesis. The effect of different concentrations (0.0, 0.5, 1.0, 2.0 and 3.0 mg/l) of both benzylaminopurine (BAP) and Isopentenyladenine (2ip) on shoot morphogenesis were also tested. The effect of different concentrations (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/l) of indole 3-butyric acid (IBA), on quarter (MS) medium containing 2.0 mg/l BAP, on multiplication rate of gardenia using nodal explants were tested. Rooting of gardenia plantlets was investigated on different concentrations (0.0, 0.5, 1.0, 2.0 and 3.0 mg/l) of naphthalene acetic acid (NAA). The effect of different types of media ( silt, peat moss, silt and Peat moss 1:1, silt & sand 2:1 ) were tested for the acclimatization of the plantlets, All experiments were arranged in a completely randomized design. Results showed that the best medium strength for micropropagation of gardenia was quarter MS medium. Application of 2.0 mg/l BAP gave higher number of shoots (4.0) and shoot length (14.7 mm) while 0.5 mg/1 BAP gave higher shoot length (27.5 mm). Also, 2.0 mg/l 2ip gave 4.0 shoots/explant. Combination of 0.2 mg/l IBA with 2.0 mg / l BAP gave the highest number of shoots (5.8) and shoot length (8.4 mm). The highest number of roots (14) and root length (47.50 mm) were obtained on quarter MS medium containing 1.0 mg/l NAA. It is recommended to propagate gardenia using quarter MS supplemented with 2.0 mg/l BAP and 0.2 mg/l IBA while rooting initiation on quarter MS with 1.0 mg /l NAA. Silt: sand (2:1) was recommended for acclimatization of regenerated plants .

تأثير تركيز الوسط المغذي و السيتوكينيات و االوكسينات على اإلكثار الدقيق للغاردينيا (.Gardenia jasminoides L) باستخدام العقد السالمية أمجد سليمان خضر عليان

ملخص الدراسة

الغاردينيا (Gardenia jasminoides ( هي واحدة من أهم نباتات الزينة في جميع أنحاء العالم بسبب جمال أزهارها. اكثار الغاردينيا بالطرق التقليدية يعد بطيئا وصعبا. الهدف من هذا البحث إيجاد طريقه اكثار للغاردينيا باستخدام تقنيه االكثار الدقيق , تم اجراء تجارب لدراسة تأثير التراكيز المختلفة ) ربع ، نصف ، كامل( للوسط المغذي موراشيجى واسكوج (MS ( على تشكل الغاردينيا وتم اختبار تأثير تراكيز مختلفة (0.0 و 0.5 و 1.0 و2.0 و3.0 ملغم / لتر( لكل من بنزيل امينو بيورين )BAP) و ايزو بنتايل ادينين (2ip) على تخفيز النمو الخضري وتأثير تراكيز مختلفة ) 0.0 و 0.2 و 0.4 و 0.6 و 0.8 و 1.0 ملغم / لتر( من اندول حمض البيوتريك ) IBA) في ربع الوسط )MS( الذي يحتوي على 2 ملغم / لتر من BAP على معدل تضاعف نبيتات الغاردينيا باستخدام العقد السالمية . تم اختبار تجذير نبيتات الغاردينيا في تراكيز مختلفة (0.0 و 0.5 و 2.0 و1.0 و 3.0 ملغم / لتر( من نفثالين حمض الخليك (NAA (, تم اختبار تأثير أنواع مختلفة من البيئات الطمي و البيتموس و طمي مع بيتموس )1:1( و طمي مع الرمل )2 : 1( على االقلمة و تم استخدم التصميم العشوائي الكامل لكل التجارب. أظهرت النتائج أن أفضل تركيز للوسط المغذي كان ربع تركيز 2.0 ملغم / لترBAP أعطى اعلى عدد لألفرع )4.0( مع طول لألفرع )14.7 ملم ( في حين تركيز 0.5 ملغم / ليتر BAP أعطي أعلى طول لألفرع )27.5 ملم( كذلك 2.0 ملغم / لتر 2ip أعطى عدد افرع )4.0( . أظهر تركيز IBA 0.2 ملغم/ لتر مع 2.0 ملغم/ لتر BAP اعلى معدل تضاعف )5.8( مع طول لألفرع )8.4 ملم( . تم الحصول على أكبر عدد من الجذور)14( واطول جذور (mm 47.50) فى ربع الوسط المغذى مضافا اليه 1.0 ملغم / ليتر NAA. يوصى باستخدام ربع وسط مواشيجى واسكوج المحتوي على 2.0 ملغم / لتر BAP و 0.2 ملغم/لتر IBA بينما يحث التجذير في ربع تركيز الوسط المغذى مضافا اليه 1.0 ملغم / لتر NAA الكثار الغاردينيا كذلك يوصى باستخدام الطمي و الرمل ) 1:2( لالقلمة نبيتات الغاردينيا .

Table of Content Title Page

Dedication ...... III

Acknowledgments ...... IV

Abstract ...... V

خطأ! اإلشارة المرجعية غير مع ّرفة...... Arabic Abstract

Table of Content ...... VII

List of Tables...... X

List of Plates ...... XI

List of Abbreviations...... XII

Chapter One ...... 1

Introduction ...... 1

Chapter Two ...... 2

Literature Review ...... 2

2.1 Origin and distribution ...... 2

2.2 Taxonomy ...... 2

2.3 Botany ...... 2

2.4 Morphology ...... 2

2.5 Cultivars ...... 2

2.6 Uses of gardenia ...... 4 2.6.1 Landscape uses ...... 4 2.6.2 Medical uses ...... 4 2.6.3 Agricultural uses ...... 5 2.6.4 Other uses of gardenia ...... 5

2.7 Cultural requirements ...... 5 2.7.1 Soil ...... 5 2.7.2 Sunlight and temperature ...... 5 2.7.3 Irrigation ...... 5

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2.7.4 Fertilization ...... 6 2.7.5 Pruning ...... 6

2.8 Diseases and insects ...... 6

2.9 Propagation of gardenia ...... 7

2.9.1 Conventional methods ...... 7 2.9.1.1 Seeds ...... 7 2.9.1.2 Cuttings ...... 7 2.9.1.3 Grafting ...... 7 2.9.2 Tissue culture methods ...... 7 2.9.2.1 Organogenesis ...... 8 2.9.2.2 Embryogenesis ...... 8

2.9.2.3 Micrografting technology of gardenia ...... 9

2.10 Growth regulators...... 9

2.11 Effect of light quality ...... 10

2.12 Acclimatization ...... 10

Chapter Three ...... 11

Materials and methods ...... 11

3.1 Plant materials of gardenia ...... 11

3.2 Medium preparation ...... 11

3.3 Sterilization ...... 11 3.3.1 Explants ...... 11 3.3.2 Culture medium and glass ware ...... 11 3.3.3 Forceps and dissecting blades ...... 11 3.3.4 Culture room ...... 12

3.4 Incubation conditions ...... 12

3.5 Research work ...... 12

3.5.1 Morphgenic effect of different MS medium strengths ...... 12 3.5.2 Shoot proliferation ...... 12 3.5.2.1 Morphgenic effect of different concentrations of BAP on nodal explant. . 12

3.5.2.2 Morphgenic effect of different concentrations of 2ip on nodal explant. .. 13

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3.5.2.3 Morphgenic effect of different concentrations of IBA combined with 2.0 mg/l BAP on nodal explant ...... 13 3.5.3 Rooting ...... 13 3.5.3.1 Effect of different concentrations of NAA on rooting of gardenia plantlets ...... 13

3.6 Effect of some growing media on acclimatization of in vitro regenerated plantlets of gardenia ...... 13

3.7 Experimental design and analysis ...... 14

Chapter Four ...... 15

Results and Discussion ...... 15

4.1: Morphgenic effect of different MS medium strengths on nodal explants ...... 15

4.2: Morphgenic effect of different concentrations of BAP on nodal explants...... 15

4.3: Morphgenic effect of different concentrations of 2ip on nodal explants...... 19

4.4: Morphgenic effect of different concentrations of IBA combined with 2.0 mg/l BAP on nodal explants...... 23

4.5: Effect of different concentrations of NAA on in vitro rooting of gardenia plantlets...... 26

4.6: Effect of some growing media on acclimatization of in vitro regenerated plantlets of gardenia under cooled plastic house conditions ...... 29

Conclusions and future work ...... 32

Conclusions ...... 32

Future work ...... 32

References ...... 33

List of Tables

Table # Description Page

Table 1: Morphgenic effect of different MS medium strengths on nodal explants . 16

Table 2: Morphgenic effect of different concentrations of BAP on nodal explants. . 18

Table 3: Morphgenic effect of different concentrations of 2ip on nodal explants. .... 21

Table 4: Morphgenic effect of different concentrations of IBA combined with 2.0 mg/l BAP on nodal explants after 8 weeks...... 24

Table 5 : Effect of different concentrations of NAA on in vitro rooting of gardenia plantlets...... 28

Table 6: Effect of some growing of media on acclimatization of in vitro regenerated plantlet of gardenia under cooled plastic house...... 30

List of Plate

Plate # Description Page

Plate 1: Morphgenic effect of different MS medium strengths on nodal explants of gardenia after 8 weeks...... 17

Plate 2: Morphgenic effect of different concentrations of BAP on nodal explants after 8 weeks...... 20

Plate 3: Morphgenic effect of different concentrations of 2ip on nodal explants after 8 weeks...... 22

Plate 4: Morphgenic effect of different concentrations of IBA combined with 2.0 mg/l BAP on nodal explants after 8 weeks...... 25

Plate 5: Effect of different concentrations of NAA on in vitro rooting of gardenia plantlets after 8 weeks...... 27 plate 6: Acclimatized of gardenia plantlets after 4 weeks...... 31

LIST OF ABBREVIATIONS

2iP 6-(γ,γ-Dimethylallylamino) purine

BAP 6-Benzylaminopurine

°C Degree of Celsius

DMRT Duncan's Multiple Range Test

IBA Indole-3-Butric Acid l Litter mg Milligram

MS Murashige and Skoog Medium mm Millimeter ml Milliliter

NAA Naphthalene acetic acid pH Hydrogen ion concentration

PGR’s Plant Growth Regulator et al. And others

GenStat Statistical Computer Program

TCM Traditional Chinese Medicine

GA3 Gibberellic acid

¼ MS Quarter strength Murashige and Skoog medium

½ MS Half strength Murashige and Skoog medium

MS Full strength Murashige and Skoog medium

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CHAPTER ONE INTRODUCTION

Gardenia (Gardenia jasminoides Ellis) is one of the most important ornamental plants scattered in abundance in the world (Rauch, 1996). Due to its flowers beauty it became one of the most important cut flowers in the United States and many European countries. Gardenia Ellis is an evergreen tropical plant, in subtropical regions and member of family Rubiaceae and belongs to the genus gardenia (Wilkins,.1986). It is a shrub cultivated in many temperate regions and favorite to gardeners throughout the world. Gardenia has very fragrant creamy-white flowers and glossy, dark-green leaves. White gardenia blooms are borne from mid spring to early summer, and a number of flowers opening over a fairly long season. The tree reaches a height of up to 3 meters and width 1.2 meters in the suitable areas of tropical and subtropical, adapted to address medium-fertility sandy soil and clay fertile land as favorite acidic and high lighting. This kind of the shady trees bloom in the fall, produce flowers white in color with a long tube (Bradshaw, 2003). It is used as a cut flower and a garden shrub. It is a popular pot plant in the US and many European countries. There are over 200 species of gardenias. In Florida, two species are of primary importance: G. jasminoides which is native to China containing many cultivars, and G. thunbergia which is native to South Africa, grown primarily as a rootstock. The latter species is valuable due to its nematode resistance and the vigor it imparts to species grafted on its root (Wilkins, 1986; Joan, 2003; He et al., 2006; Lee et al., 2009; Duhoky and Rasheed, 2010 and Dirr, 1990). This plant was chosen for this research due to its medical uses as well as magical uses for treating jaundice, hemorrhage, hepatitis, toothaches, wounds, sprains, and skin conditions (Choi et al., 2007, Lelono et al., 2009). The traditional methods of propagation of gardenia (seeds and soft cuttings), but not recommended by seeds being heterozygous, and gives genetically variable plants. The conventional propagation methods of gardenia are slow with low proliferation rates. Therefore, the Objective of the study was to develop technique for micro propagation of Gardenia jasminoides using nodal explants.

CHAPTER TWO LITERATURE REVIEW 2.1 Origin and distribution There are about 250 species of gardenia in tropical and subtropical regions of Africa, Asia, Madagascar, and Pacific islands; five species (one endemic) in China. Several species from Asia and the Pacific are occasionally cultivated, but gardenia, which is native to Middle East, is very popular worldwide for its flowers (Rauch, 1996). They are native to the tropical and subtropical regions of Africa, Southern Asia, Australia and Oceania (Neal, 1965).

2.2 Taxonomy The genus gardenia belongs to Rubiaceae family and there are about 200 species of this genus. The name gardenia was given to commemorate Dr. Alexander Garden 1780-1791 (Green, 1965).

2.3 Botany

Gardenia is native to the south of Japan and China. The leaves are opposite, thick, dark green with lanceolate shape to ovate and can reach 10 cm in length. The sweet fragrant, terminal flower, 8-10 cm across, consists of a calyx (with five green fascinated teeth) and a corolla (with six whorls and five to nine white waxy petals). Frequently, stamens and pistil(s) are transformed, resulting in sterile flowers. Flower induction and development are influenced by different factors (Hutchinson, 1980).

2.4 Morphology This species is an evergreen shrub with dark green leaves. The blooms are waxy and the color ranges from pale yellow to creamy white. The gardenia has very fragrant creamy-white flowers and glossy, dark-green leaves (Kent et al., 2006).

2.5 Cultivars The most common species is Gardenia jasminoides, Gardenia thunbergia, Gardenia rathmannia and Gardenia august (Kent et al., 2006). In the United States and China, Gardenia Jasminoides is one of the most popular species, where the shrubs grows up to 1.5 m high and are evergreen shrub and flowers with a smell. Blooms from mid-May through June may continue until September, giving flowers

to double white color and a strong aromatic smell. Gardenia originated in South Africa, are expensive, because of their resistance to nematodes. Varieties that grow in Florida, mostly cross-breeding, are produced only by mutations, and there is a significant difference in the shape and size of flowers, as well as in the time of flowering, continuity and growth volume between varieties. The most important are the following according to (Kent et al., 2006): 1- Aimee Yashioka: with dark green leaves glimmer, a large flower diameter ranges from 10- 15 cm and produces flowers profusely in late spring. 2- Auguust Beauty: with parchment or a dense white flower with a large double blossom from spring to fall, plant height of 120-160 cm. 3- Belmont: With a parchment or dark green with flowers large diameter 10-12.5 cm blooms throughout the growing season. 4- Coral Gables: with parchment or a dark green with big flowers, flowering throughout the summer months. 5-Fortuneiana: bright with a pink double the diameter of up to 10 cm 6-Glazerii: with parchment or a medium evergreen and flowering summit in April in South Florida 7-Golden Magic with pure white flowers and with double the age may turn into a golden yellow, plant height of 90 cm and width 60 cm in 3 years. 8-Miami Supreme securities graded color than the average green to dark green and large diameter of flowers 15-10 cm diameter. 9-Mystery Plant height 120-150 cm bright with a white double the diameter of 10 -12.5 cm these need to be trimmed to maintain an appropriate appearance of the plant. 10-Radicans: small leaves, flowers and a large double diameter 2.5 cm, height 30-60 cm and 120 cm wide. 11-Radicans variegata: verse color copy with creamy white margins of leaves. 12-Veitchii: plant height 60-120 cm, white flowers with a diameter of 2.5-4 cm and be abundant from spring to autumn 13-Veitchii improved growth longer up to 15 cm produces bright largest with a diameter of 5.6 to 5.7.

2.6 Uses of Gardenia Gardenia an evergreen shrub and is one of the most important plants used in landscaping, as it is characterized by dark green leaves and are important sources of perfumes in gardens (Edward.,1990 ). Gardenia veitchii used as a hard wood for the manufacture of some agricultural tools in South America (Dumanois et al., 1984). aside from being used a cut flower, in landscape designs its oil & scent (flower) is use for traditional Chinese Medicine (TCM). Gardenia growing up to 1-2 meter tall, with sweetly fragrant flower and can be used as a cut flower and landscape shrub. Also is one of the most popular plants in the USA and many of the European countries (Green, 1965). The dried fruit of gardenia, commonly used in heat signs such as Irritability, restlessness. According to Traditional Chinese Medicine (TCM), there are some other uses for gardenia:

2.6.1 Landscape uses

Gardenia can be used as screens, hedges, borders, or ground covers. They also may be used as free-standing specimens or in mass plantings. These shrubs are excellent choices for fragrant flowers and handsome foliage. To enjoy the flowers' fragrance, plant gardenia in areas with good air circulation near patios or windows, where the fragrance will be noticed. Many cultivars bloom in the spring, while others bloom throughout most of the growing season. Planting of gardenia in full sun, partial shade, or shifting shade for best flower production. Prolonged shade may reduce flowering (Dirr,. 1990).

2.6.2 Medical uses

The effect of ethanol extract of cape jasmine could be useful in preventing vascular disease (Hwang et al., 2010). The importance pigments of gardenia fruit as natural colorants in food science was studied by Mortensen in 2006. G. jasminoides extract could be used as chemopreventive agent in Alzheimer‟s disease (Choi et al., 2007). The fleshy fruit of this plant is a diuretic, stimulant, an emetic and used in lung, jaundice and kidney disorders (George et al., 1993). Gardenia seed clears internal heat and eliminates heart vexation, remove the pathogenic fire, and induce dieresis (Xinrong, 2003).

2.6.3 Agricultural uses

G. jasminoides has antifungal activity against agricultural pathogens with no environmental side effects (Lelono et al., 2009).

2.6.4 Other uses of Gardenia

The importance of pigments of gardenia fruit as natural colorants in food was reported by Mortensen (2006).

2.7 Cultural requirements

2.7.1 Soil

The best soil for gardenias growth is a mixture of peat moss and compost with the soil to maintain moisture. The soil needs to be moist at all times, Soil should be covered with a material like sawdust to make sure the soil maintains its moisture throughout the day. (Kidder et al., 1991). Soil pH is an important factor for gardenia. Soil pH determines the availability of mineral elements and should be between 5.0 and 6.5 for gardenia. Where soil pH increase 7.0 increases the possibility of micronutrient deficiencies, particularly iron. Gardenia has poor to low salt tolerance. Continuous effort is needed to avoid micronutrient deficiencies, most notably iron (Shober, and Denny., 2008).

2.7.2 Sunlight and temperature

Gardenia are semi-tropical plants that perform well in a mild, humid climate. Gardenia will grow best in full sunlight while it is preferred to be shaded on hot days during the summer months. High temperatures will stop gardenia flowers from blooming. The ideal temperatures for gardenia are 18 C° to 21 C° during the daytime and at night around 16 C°. The maximum flowering of gardenia occur under full sun or light shade in an open flowerbed (Dirr, 1990).

2.7.3 Irrigation

Watering during dry periods is necessary for healthy gardenias and is important because water determines the number of floral buds that remain on a plant to maturity. If water stress occurs in a heavily budded plant, many buds will fall before opening. Therefore, while the plant is in bud, large variations in soil moisture should

be avoided. Establishing and maintaining a layer of mulch 2 - 3 inches deep around the plant will help maintain consistent soil moisture (Shober, and Denny, 2008)

2.7.4 Fertilization

Gardenia plants are fertilized two to three times a year, the first fertilization in February, the second in October, and the third in summer. Added nutrients should be in the following ratios 2:1:3 or 3:1:3 NPK of the basic elements (Yeager and Gilman, 1991).

2.7.5 Pruning

Pruning keeps plants shapely and in scale with the landscape. Pruning should be done just after the plant finished blooming. Researcher in Florida suggests that a combination of long nights, low temperatures, and age of wood aid in bud initiation and development. Pruning should be early enough to allow new growth to be at least 4 - 6 inches long by October. Pruning after October decreases next year's blooms. Young plants, growing vigorously during their first year, may be pinched once in June and again in August to encourage heavy branching. (Dirr, 1990).

2.8 Diseases and insects

1- Nematodes: or worms caecilians, are one of the serious pests that attack the roots of gardenia causing big problems since they live in and feed on gardenia roots;

damages roots and prevents normal uptake of water and nutrients.( Kent and Kaufman, 2006 ) . 2- Root Rots: Fungal diseases of gardenia roots, cause malfunction, and decay (Kent and Kaufman, 2006). 3- Powdery Mildew: A fungal disease of leaves, favored by relatively cool nights and warm days (Kent and Kaufman, 2006). 4- Scale Insects: insects that are usually green or brown attached to the stems or under sides and upper sides of leaves (Kent and Kaufman, 2006 ) 5- Chlorosis: A lack of normal green pigmentation in foliage, generally due to deficiency of one or more micronutrients usually iron (Kent and Kaufman, 2006)

2.9 Propagation of gardenia

2.9.1 Conventional methods

The traditional methods of propagation of gardenia are seeds, soft cuttings, and grafting.

2.9.1.1 Seeds

It is not recommended to propagate gardenia by seeds, being Heterozygous, and gives plants genetically variable. The main objective of the multiplication by seed is to produce new varieties. Seeds are not used for propagation purposes (Husseini, 1993). Seeds can be grown on pots containing a 50:50 combination of peat moss and perlite or a 50:50 combination of peat moss and sand. (Dirr,.1990).

2.9.1.2 Cuttings In conventional propagation, terminal cutting of Gardenia jasminoides results in a low proliferation rate (Dirr,. 1990). Cuttings can be taken any time during the year, but are most successful in June, July, and August. Gardenia thunbergia can be propagated from seeds or cuttings (Economou, and Spanoudaki, 1985). Tip or midsection cuttings with wood 6 - 8 weeks old should be cut 4 - 5 inches long with at least two or three sets of leaves. Cuttings can be taken at or between nodes as they root from the cut end. Leaf removal is unnecessary and undesirable because it results in a longer rooting period. Rooting of cuttings is best under intermittent mist or in a closed-case propagating device, such as an old aquarium or clear plastic bag. Rooting media should be a 50:50 combination of clean, sharp builders' sand and peat moss; or a 50:50 combination of peat moss and perlite (Dirr,. 1990). Stem cutting after flowering is the conventional method for propagation (Hutchinson, 1980).

2.9.1.3 Grafting

In Central Florida and South Florida, propagation of gardenia is mostly by grafting rootstock of Gardenia thunbergia (Dirr,. 1990).

2.9.2 Tissue culture methods

Tissue culture technique is a useful tool, which allows rapid production of many genetically identical plants using a relatively small space, supplied at a time

(Amin and Jaiswal, 1987). Tissue culture techniques play a major role in the propagation of trees and shrubs difficult to be propagated by traditional ways. Various experiments on in vitro propagation of ornamental plant by root culture have been reported (Kubota et al., 1995). Shoot tip culture was successfully used in many of ornamental and medicinal plants.There are numerous reports and experiments about micropropagation of different species of ornamental. (Liu & Li, 2001).

2.9.2.1 Organogenesis

Micropropagation of G. jasminoides by shoot tips (Economou & Spanoudaki, 1986a; Serret et al., 1996; Sayd et al., 2010) and microshooting (Hatzilazarou et al., 2006) were reported. (Chuenboonngarma et al., 2001) used the young shoots of G. jasminoides as explant. In vitro culture of single nodes and shoot tip from G. jasminoides have been reported by Duhoky and Rasheed (2010). Abdullah et al (2003) stated that many plantlets were obtained by culturing shoot cuttings of Gardenia in MS nutrient media with 30 g/l sucrose, 7.0 g/l agar, and different concentrations of BA and IAA. The best combination was 1mg/l BA with 0.5mg/l IAA. This treatment gave the best shoot growth suitable for rooting in primary and secondary culture by re-culturing the rootstock cutting every 6 weeks and for many times. Successful utilization of in vitro techniques for propagation, maintenance, and manipulation of plant germplasmas has been possible for a great number of plant species. Several studies have documented that in vitro micropropagation can be used for clonal propagation of G. jasminoides, the efficiency remained low. Anyway, micropropagation of G. jasminoides via in vitro organogenesis using modified Murashige and Skoog (1962) medium (MS) offers higher proliferation rate of G. jasminoide (Suprasanna and Bapat, 2005 and Wu et al., 2012).

2.9.2.2 Embryogenesis

Young leaves of G. jasminoides have been studied for callus induction (Al- Juboory et al., 1998; Mizukami et al., 1987). There were some successful reports on development of plantlets from callus raised through leaf cultures of Gardenia jasminoides Ellis another member of Rubiaceae (Al-Juboory et al., 1998). (Reddy and Saritha, 2012) reported that in callus induction from leaves of in vitro grown plantlets of Gardenia latifolia high frequency was observed on MS medium supplemented with 3.0 mg/l 2,4-D . (George et al., 1993) reported that ovary culture

can be used for callus initiation via immature ovary portion of flower of G. jasminoides. There were some successful reports on development of plantlets of gardenia from callus raised through embryogenic callus formation observed on MS medium with various combinations of BA (1.0-2.0 mg/l), Kin (2.0mg/l) and IAA (0.1 mg/l) (Reddy and Saritha, 2012).

2.9.2.3 Micrografting technology of gardenia

There are many reports about production of virus-free- gardenia via meristem culture method (Tyagi et al., 2010). The developed Micrografting technology using for ridding planting materials of diseases. The problem of low metal absorption in G. jasminoides can be solved at low temperatures by grafting on G. thunbergia; Since G. thunbergia was able to absorb iron at low temperatures (Mastalerz.1977). In vitro an excellent number and percentage of successful grafted on G. thunbergia in greenhouse were obtained scions derived from Gardenia jasminoides using MS medium supplemented with 0.50 mg/l kin (Nower et al., 2013).

2.10 Growth regulators

Plant hormones are a naturally occurring organic material in very small quantities and affect the growth and development of plants. It has been possible to find a group of synthetic organizations, which resemble exactly the impact of those naturally produced. The multiple physiological effects of the known growth regulators such as auxins Indole butyric acid (IBA) and Indole acetic acid (IAA) and Naphthalene acetic acid (NAA), as well as cytokinins such as Kinetin (KIN) and Benzyladenine (BA) which is organizing the evolution of plant organs and revealed in many parts of the plant or plant parts. BAP showed high proliferation of G. jasminoides compared to 2ip and kinetin Sayd et al (2010). Using IBA in micro cuttings of G. jasminoides, high percentages of root in vitro and ex vitro were obtained (Pontikis, 1983; Hatzilazarou et al., 2006). Dumitrescu (2002) carried out a successful combination of 0.1 mg/l IAA and 1.0 mg /l BAP for chlorophyll extract on G.jasminoides, and a higher range of shoot proliferation of this plant via BAP was reported by Chuenboonngarma, et al (2001). Pontikis (1983) revealed long and high quality shoots in jasmine via 2iP, but Chuenboonngarm, et al (2001) reported 2iP produced 100% chimeric plants of G. jasminoides. Economou & Spanoudaki (1986a) reported that BA induced axillary buds in G. jasminoides. However, BAP

and NAA showed high proliferation compared to 2ip and kinetin on G. jasminoides (Sayd et al., 2010). In addition, the use of IBA in micro cuttings of G. jasminoides produced high percentages of roots in vitro and ex vitro (Pontikis, 1983; Hatzilazarou et al., 2006).

2.11 Effect of light quality

Light plays an important role in plant growth and development processes. The morphogenesis processes are affected by both light quality and intensity. Growth materials have also an enormous influence (Gabarkiewicz and Rudnicki, 1995). Danuta Kozak, (2011) reported that yellow light increased the number of axillary shoots of Gardenia jasminoides from explants cultivated on medium containing 25 μM BA and it promoted shoot elongation most strongly on medium with the addition of 5 μM BA. Red and yellow light increased the number of roots on control medium, whereas yellow light significantly promoted root elongation growth. However, yellow and white light had the most positive effect on the average fresh weight of roots of gardenia.

2.12 Acclimatization

The process of acclimatization of the plants reproduce inside the tubes is the last stage of exact propagation, in which the Plants are prepared to gradually adapt to relative humidity out tube. Plants in this period are leaves soft, thin, and inactive in photosynthesis. The leaf stomata are open at this stage and this causes a great water stress for the plant especially in the early periods after the plants exit the tube. Therefore, the use of polyethylene covers around the plant is usually used to increase the relative humidity. (Muriithi et al., 1982) used transparent polyethylene covers to provide high moisture around the plant when acclimatize for plants grown inside the tubes. High humidity can also be provided by spraying with spray and mist , The formation of a good root system should help the buds produced in the tubes for some moisture stress, and the more successful the transfer of plants without damage the roots, the greater the number of plant roots. Using a mixture of peat moss and perlite was effective in the success of the process. As it has an active role in the preservation of moisture, and does not prefer the use of pure sand alone to adjust the plants because it requires repeated irrigation and may cause the death of many plants that are transferred to the pots.

CHAPTER THREE MATERIALS AND METHODS 3.1 Plant materials of gardenia One-year-old Gardenia jasminoides, plants were collected from private nursery in Wad Medani, Gezira State. These plants were kept at the Horticultural Research Section, Agricultural Research Corporation, under plastic house condition and fertilized regularly. Shoots were collected from the plants; selected shoots were washed under tap water to remove the dust, followed by tap water + liquid soap for 20 minutes, and then rinsed with distilled water. The shoots were cut into shorter sections (1.5 cm long) including single node with axillary bud. 3.2 Medium preparation MS medium (Murashige and Skoog 1962) was prepared supplemented with 30g/l sucrose and 8.0 g/l agar. The pH was adjusted to 5.8±0.1 with Na OH or HCL drops prior to addition of the gelling agent. 3.3 Sterilization

3.3.1 Explants

The single nodes with axillary buds were transferred to Laminar air-flow to be sterilized by soaking in two concentration (50 % and 75% ) of Clorox (sodium hypochlorite 5% active ingredient ) with one drop of Tween 20 per 100 ml for 10 and 15 minutes. The explants were washed three times with sterile distilled water to remove the sterilant. The results showed that the best concentration for sterilization of the nodal explants was 50 % Clorox for 10 minutes. The results did not record any contamination.

3.3.2 Culture medium and glass ware

Glassware, nutrient medium and distilled water were sterilized by autoclaving at 121°C and 1.21 kg /cm for 20 minutes and then was transferred to the culture room.

3.3.3 Forceps and dissecting blades

Forceps and dissecting blades were dipped in ethanol followed by flaming; they were dipped and reframed after every use.

3.3.4 Culture room

The laminar airflow cabinet was switched on 15 minutes before use. The surface of the working area of the laminar airflow cabinet was wiped with ethanol from time to time during work. Sometime the UV lamp was switched on during night to disinfect the culture room.

3.4 Incubation conditions

All cultures were incubated at 25 °C, sixteen hours light and eight hours dark. Light was supplied from cool white fluorescent lamps at 1000-lux density.

3.5 Research work

Experiments were conducted using nodal cutting explants to study the effect of different strengths of MS medium and different concentrations of cytokinins on morphogenesis of Gardenia jasminoides and rooting of plantlets using different concentrations of auxins.

3.5.1 Morphgenic effect of different MS medium strengths

Nodal explants were cultured on different strengths (1/4, 1/2, and full) of MS salts. The cultures were incubated under the above-mentioned conditions. Shooting percentage, number of leaves, number of shoot, and length of shoot were recorded after 4, 6, and 8 weeks.

3.5.2 Shoot proliferation

3.5.2.1 Morphgenic effect of different concentrations of BAP on nodal explants.

Nodal segments of about 1.0 cm length were cultured on MS medium supplemented with different BAP concentrations (0.0, 0.5, 1.0, 2.0 and 3.0 mg/l). The cultures were incubated under the above-mentioned conditions. Shooting percentage, number of leaves, number of shoots, and length of shoot were recorded after 4, 6, and 8 weeks.

3.5.2.2 Morphgenic effect of different concentrations of 2ip on nodal explants.

Nodal segments of about 1.0 cm length were cultured on MS medium supplemented with 2ip at different concentrations (0.0, 0.5, 1.0, 2.0, and 3.0 mg/l). The cultures were incubated under the above-mentioned conditions. Shooting percentage, number of leaves, number of shoots, and length of shoot were recorded after 4, 6, and 8 weeks.

3.5.2.3 Morphgenic effect of different concentrations of IBA combined with 2.0 mg/l BAP on nodal explants

Nodal segments of about 1.0 cm length were cultured on quarter MS medium supplemented with IBA at different concentrations (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/l) in combined with 2.0 mg/l BAP. The cultures were incubated under the above- mentioned conditions. Shooting percentage, number of leaves, numbers of shoots, length of shoot, were recorded after 4, 6, and 8 weeks.

3.5.3 Rooting

3.5.3.1 Effect of different concentrations of NAA on rooting of gardenia plantlets

Plantlets with 3.0 cm length were cultured on quarter MS medium containing different concentrations (0.0, 0.5, 1.0, and 3.0 mg/l) of NAA. The cultures were incubated under the above-mentioned conditions. Rooting percentage, number of roots per explant, root length were recoded after 4, 6, and 8 weeks.

3.6 Effect of some growing Media on acclimatization of in vitro regenerated plantlets of gardenia

The experiment conducted in the greenhouse to evaluate the effect of some growing media on the survival percentage of gardenia plantlets during the acclimatization stage. The in vitro regenerated plantlets were carefully removed from the culture tube and rinsed with water to remove the remaining of nutrient medium and then transplanted in small pots containing different type of media, silt, peat moss , silt + peat moss (1:1) and silt+ sand (2:1), The survival percentage was recorded after 4 weeks.

3.7 Experimental design and statistical analysis Completely Randomized Design (CRD) was used in all experiments with 6 replicates, the data obtained were analyzed using the GenStat computer program and means were separated using Duncan's Multiple Range Test (DMRT).

CHAPTER FOUR RESULTS AND DISCUSSION 4.1: Morphgenic effect of different MS medium strengths on nodal explants The effect of different MS medium strengths (¼ MS, ½MS, full MS) on shoot initiation of nodal explants were shown in (Table 1). Shoot proliferation occurred by axillary branch from buds present in the original nodal explant. The results indicated that explants proved to form shoots on the different MS medium strengths (Plate 1), but the highest number of leaves (8.0) and shoot length (47.9), which were significantly higher than all other strength, were obtained with ¼ MS. Full MS strength gave the lowest number of leaves and shoot length. In addition to coloring, the leaves had brown color on half and full MS strength and then the death of the plantlets (Plate 1). ¼ MS medium proved to give the best results with respect to number of leaves and shoot length while the worst results was produced by full MS medium. Pierik (1997) reported high salt content of medium is not necessarily always optimal for growth and development of explants and plantlets in vitro cultures of higher plants. Study by Fadel et al (2010) proved that half-strength MS could result in maximum number of shoots and roots of Mentha spicata. Wan et al (2012) showed that MS media strength could affect the number of shoots obtained in Pogostemon cablin. Tetsumura et al (2008) also observed that a reduction in the strength of MS medium resulted in the increase of in vitro shoot and root formation from blueberry (Vaccinium corymbosum and V. virgatum). In the cases of other orchids such as Dendrobium tosaense and Paphiopedilum armeniacum, the optimum basal medium for seed germination and development was, respectively, half-strength and eight-strength MS medium (Udomdee et al., 2014; Zhang et al., 2015).

4.2: Morphgenic effect of different concentrations of BAP on nodal explants. Different BAP concentrations (0.0, 0.5, 1.0, 2.0 and 3.0 mg/1) had significant effect on shoot initiation from nodal explants cultured on quarter MS medium. Shoot proliferated from axillary buds present in original nodal explants (Table 2).

Table 1: Morphgenic effect of different MS medium strengths on nodal explants

weeks

MS medium Shooting 4 6 8 strengths % Number Shoot Number Shoot Number Shoot of length of length of length leaves (mm) leaves (mm) leaves (mm)

¼ MS 100 2.5 a 10.0 a 3.4 a 21.4 a 8.0 a 47.9 a

½ MS 100 1.9 b 6.1 b 2.9 ab 10.4 b 6.8 ab 25.7 b

Full 100 1.2 c 4.0 c 2.6 b 7.9 b 6.0 b 17.0 c

Grand mean 1.9 6.7 3.0 13.2 7.0 30.2

CV ( % ) 13.4 19.0 18.3 17.0 16.7 12.9

SE ± 0.3 1.3 0.5 2.2 1.0 3.9

Means in columns with the same letters are not significantly different at 5 % probability level according to DMRT.

Plate 1: Morphgenic effect of different MS medium strengths on nodal explants of gardenia after 8 weeks.

Table 2: Morphgenic effect of different concentrations of BAP on nodal explants.

weeks Shooting 4 6 8 BAP Conc. % mg/l Number Shoot Number Shoot Number Shoot of length of length of length shoots (mm) shoots (mm) shoots (mm) 0.0 100 1.0 c 2.0 d 1.5 c 2.4 d 1.9 c 4.7 d

0.5 100 1.6 b 9.0 a 2.5 b 13.8 a 2.9 b 27.5 a

1.0 100 1.4 bc 7.0 b 2.0 bc 10.7 b 2.4 bc 21.0 b

2.0 100 2.5 a 2.8 c 3.7 a 7.3 c 4.0 a 14.7 c

3.0 100 1.6 b 2.8 c 2.4 b 4.2 d 2.7 b 8.4 d Grand 1.6 4.7 2.5 7.7 2.7 15.4 mean CV ( % ) 19.6 19 15.3 18.1 16.1 19

SE ± 0.4 1.0 0.4 1.4 0.4 2.9

Means in columns with the same letters are not significantly different at 5 % probability level according to DMRT.

The results indicated that shoots regeneration was induced on quarter MS with and without addition of BAP (Plate 2). The highest number of shoots, which was significantly higher than all other treatments, was obtained on 2.0 mg/l BAP. BAP concentrations from 0.0 – 1.0 mg/l were comparable in the percentage of explants with shoot, but when BAP concentrations increased more than 2.0 mg/l the number of shoots decreased significantly. The lowest number of shoots was obtained on quarter MS medium devoted from BAP. The number of shoot regenerated per explants increased significantly with the increase in BAP concentrations from 0.5 to 2.0 mg/1, whereas 2.0 mg/l gave significantly the highest number of shoots per explant compared with all other BAP concentrations. Shoot height was also significantly variable on different BAP concentrations, where 0.5 mg/l gave the highest length of shoots and BAP at 3.0 mg/l and 0.0 mg/l gave the lowest shoot length (Table 2). BAP at 2.0 mg/l proved to give the best results with respect to number of shoots whereas 0.5 mg/l gave the highest length of shoot; on the other hand, quarter MS medium without BAP gave the lowest results in all measured parameters. These results are in agreement with those of Duhoky and Rasheed (2009) who indicated that 2.0 mg/l BAP gave the highest number of shoots, leaves and length of new shoots. However, Nower (2007) reported that, 3.0 mg/l BAP was more effective on G. jasminoides than other treatment for shoot number/explant. Jhansi Lakshmi and Jaganmohanreddy (2013) reported that the multiple shoots were induced on MS medium in combination of 2.0 mg/l BAP with 0.1mg/l NAA, which produced 2.0 shoots per nodal segments. Duhoky and Rasheed (2010) showed that the highest growth length was achieved on MS medium supplemented with 2.0 mg/l kin and 0.1 mg/l NAA.

4.3: Morphgenic effect of different concentrations of 2ip on nodal explants.

Different 2ip concentrations (0.0, 0.5, 1.0, 2.0 and 3.0 mg/1) had significant effect on shoot initiation from nodal explants cultured on quarter MS medium (table 3). The highest number of shoots was induced on 2.0 mg/l 2ip, and all the treatments showed significant differences compared with control (Plate 3). The lowest number of shoots was obtained on quarter MS medium without 2ip, medium

Plate 2: Morphgenic effect of different concentrations of BAP on nodal explants after 8 weeks.

Table 3: Morphgenic effect of different concentrations of 2ip on nodal explants.

weeks

2ip Shooting 4 6 8 Conc. mg/l % Number Shoot Number Shoot Number Shoot of length of length of length shoots (mm) shoots (mm) shoots (mm) 0.0 100 1.0 a NA 1.0 c 5.6 d 1.0 d 11.7 d 0.5 100 1.0 a NA 1.3 c 8.0 c 1.7 c 13.5 c 1.0 100 0.8 a NA 1.9 b 10.0 b 3.0 b 15.7 b 2.0 100 1.0 a NA 2.8 a 12.5 a 3.8 a 17.8 a 3.0 100 0.8 a NA 1.3 c 6.3 d 1.8 c 11.8 d Grand mean 0.9 1.7 8.5 2.3 13.9 CV ( % ) 17 17 9.4 15 5.5 SE ± 0.2 0.3 0.8 0.3 0.76

Means in columns with the same letters are not significantly different at 5 % probability level according to DMRT.

Plate 3: Morphgenic effect of different concentrations of 2ip on nodal explants after 8 weeks.

With 0.5 and 3.0 mg/l, 2ip gave similar number of shoots. The number of shoots regenerated per explant increased significantly with the increase in 2ip concentrations from 1.0 to 2.0 mg/1 , where. 2.0 mg/l induced significantly the highest number of shoots per explant compared with all 2ip concentrations. Shoot height was also significantly variable on different 2ip concentrations, where 2.0 mg/l gave the highest length of shoots and 2ip at 3.0 mg/l and 0.0 gave the lowest shoot length (Table 3). Ngarmnij et al (2011) reported that number of shoots in medium with 7.5 mg/l 2iP was 4 times greater than in 2iP free medium and all explants receiving 2iP gave somaclonal variation in G. jasminoides leaf. Al-Juboory (1998) showed that 2iP and kinetin can be used for promotion of axillary bud development from shoot tip explants of gardenia in vitro. Chuenboongarma, et al (2001) reported that the highest propagation rate of gardenia was when using 10 mg /l BA or 7.5 mg /l 2ip compared with the control.

4.4: Morphgenic effect of different concentrations of IBA combined with 2.0 mg/l BAP on nodal explants. The results in table (4) showed the effect of the different concentrations of IBA combined with 2.0 mg /l BAP on morphogenesis induced significant results of number of shoot, length of shoots and number of leaves. Increasing the concentration of auxin decreased the rate of reproduction significantly compared to the low concentration of auxin (IBA). The best combination of BAP and IBA for shoot morphogenesis was 2.0 mg/l BAP with 0.2 mg/l IBA (Plate 4). This combination gave significantly higher number of shoots and leaves per explant than all other combinations. Shoot height was significantly higher on 0.4 mg/l IBA combined with 2.0 mg/l BAP. Pierik (1987) pointed out that the use of a high concentration of cytokinins with a low concentration of auxin stimulates growth, formation of new growths and increased propagation rate on higher plants. Conti et al (1991) indicated that according to the species, the physiological state of the mother plant, the type of explant used, source, and the type and concentration of the hormone, but generally the low concentration of auxins is important at propagation stage because of its direct

Table 4: Morphgenic effect of different concentrations of IBA combined with 2.0 mg/l BAP on nodal explants after 8 weeks.

IBA Conc. Shooting mg/l % Number of Number of Shoot length Shoots leaves (mm)

0.0 100 1.0 e 3.2 d 3.4 c

0.2 100 5.8 a 15.8 a 8.4 b

0.4 100 3.4 b 8.4 b 12.4 a

0.6 100 2.5 c 7.0 bc 6.8 b

0.8 100 1.7 d 6.2 cd 6.6 b

1.0 100 1.7 d 5.2 cd 5.8 bc

Grand mean 2.7 8 7.2

CV ( % ) 11 17.9 18

SE ± 2.9 1.3 1.3

Means in columns with the same letters are not significantly different at 5 % probability level according to DMRT.

Plate 4: Morphgenic effect of different concentrations of IBA combined with 2.0 mg/l BAP on nodal explants after 8 weeks.

physiological effect in cellular permeability and osmosis increase, due to altering the osmotic system, increasing the rate of transcription of the genetic material, and the formation of new proteins. This corresponded to what was indicated by Rossignol et al. (1990). Jhansi Lakshmi and Jaganmohanreddy (2013) reported that combination of 2.0 mg/l BAP+ 0.25 mg/l NAA on Gardenia latifolia gave two shoots with average shoot length 3.75 cm and 2.0 BAP +0.1 mg/l IAA also gave two shoots with average shoot length of 3.53cm. 4.5: Effect of different concentrations of NAA on in vitro rooting of gardenia plantlets. All NAA concentrations produced roots on gardenia plantlets after 6 weeks (Plate 5). The results in table (5) showed the effect of the different NAA concentrations (0.0, 0.5, 1.0, 2.0 and 3.0 mg/1( on rooting of gardenia plantlets. The number of root / explant and root length were significantly higher on 1.0 mg/l NAA compared with all other concentrations of NAA. The number of roots and root length decreased significantly, when NAA concentrations decreased below or increased above 1.0 mg/l (Table 5). NAA at 0.5 and 2.0 mg/l gave similar results on root parameters. The results of this research were in consistent with the results of many previous research works (Gupta, 1986; Fitch, 1987 and Vuylsteke, 1989) who reported the NAA was best at the stage of rooting. The effect of the auxin in the rooting process varies according to its concentration and type and the presence of one or more auxin in the medium. Rossignol et al. (1990) reported that an Auxin works to increase the rate of cellular permeability, enzymatic activity and ion transport rate, which increases the rate of root formation on cultivated branches due to its important and direct role in the rooting phase (The Root exit phase), and this interpretation is in consistence with the findings of Haissing (1986). Study on Gardenia latifolia by Reddy et al. (2013) reported that 70 % of root induction occurred in half-strength MS medium supplemented with IBA (4.0 mg/l) at 6 weeks. Patel and Shah (2009) reported that root number and root length of Stevia rebaudiana plant cultures were significantly influenced by the strength of MS medium (¼ MS, ½ MS and full strength MS) and treatment combinations of IBA, NAA and BAP. Such combinations may have affected cell differentiation and elongation.

Plate 5: Effect of different concentrations of NAA on in vitro rooting of gardenia plantlets after 8 weeks.

Table 5 : Effect of different concentrations of NAA on in vitro rooting of gardenia plantlets.

Weeks NAA 6 8 Conc. mg/l Number of Root length Number of Root length Roots (mm) Roots (mm) 0.0 1.0 d 0.8 d 1.0 d 6.0 d 0.5 5.8 b 11.7 c 9.0 b 27.5 b 1.0 9.0 a 26.7 a 14.5 a 47.5 a 2.0 6.8 b 16.3 b 10.5 b 29.0 b 3.0 4.0 c 8.7 c 6.0 c 15.0 c Grand mean 5.3 12.8 8.3 25.1 CV ( % ) 16.4 19.3 19.4 16.2 SE ± 0.9 2.5 1.6 4.0

Means in columns with the same letters are not significantly different at 5 % probability level according to DMRT.

4.6: Effect of some growing media on acclimatization of in vitro regenerated plantlet of gardenia under cooled plastic house conditions

One of the most important steps is successful moving of plantlets from culture tubes to the soil. The results of the present study revealed the ability of plants to depend on themselves the results in table (6) showed the effect of different types of media on acclimatization of gardenia plantlets. The results showed that the best medium for the acclimatization was silt + sand (2: 1) resulted in 90 % successful hardening and then silt, depending on the percentage success in growth, gave in the end plants full, and mature plate (6). Muriithi et al (1982) used transparent polyethylene covers to provide high moisture around the plant when acclimatized fig plants growing inside the tubes. In the pepper plant, the plants were transferred to a mixture of sand and vermiculite )1 :1( size volume / volume and covered with plastic polyethylene caps and saved in the culture room for two weeks before transferring them to their permanent place and 84% success in acclimatized of producing plants ( Venkataiah et al., 2006).

Table 6: Effect of some growing of media on acclimatization of in vitro regenerated plantlet of gardenia under cooled plastic house.

Medium Successfully acclimatized plantlets (%)

Peat moss 40

Silt 70

1 : 1 Silt : peat moss 50

2 : 1 Silt : sand 90

Plate 6: Acclimatized of gardenia plantlets after 4 weeks.

CONCLUSIONS AND FUTURE WORK

CONCLUSIONS

1. This study confirmed the possibility of in vitro propagation of gardenia and a large number of plants have been obtained from mature single nodes. 2. The best medium for the micropropagation for the studied species is quarter concentration of Murashige and Skoog (1962) medium. 3. BAP and 2ip induced shoot morphogenesis at 2.0 mg/ l after 8 week; however, BAP was faster (6week). 4. Combination of BAP 2.0 mg/l plus 0.2 mg/l IBA improved the shoot regeneration from 4.0 to 5.8. 5. The best medium for rooting is 1/4 MS with NAA at 1.0 mg/l. 6. The plantlets were successfully hardened in cooled plastic house on mixture of silt + sand (2:1).

FUTURE WORK

1. GA3 can be tested to enhance plant elongation when using BAP. 2. Other factors can be tested to increase the rate of multiplication.

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