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In Vitro Clonal Propagation of Grapevine (Vitis Spp.)

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In Vitro Clonal Propagation of Grapevine (Vitis Spp.) In vitro clonal Propagation of grapevine (Vitis spp.) By Magdoleen Gamar Eldeen Osman B.Sc. Agriculture Science (Honours) Sudan University of Science and Technology Thesis submitted in partial fulfillment of the requirements for the degree of master of science in Agric Supervisor :Prof.Abd El Gaffar Al Hag Said Co supervisor :Dr.Mustafa .M.El Balla Department of Horticulture Faculty of Agriculture University of Khartoum October 2oo4 TABLE OF CONTENTS Dedication………………………………. Acknowledgment………………………….……… i Contents…………………………………………… ii Abstract……………………………………………. iv Arabic Abstract…………………………………… vi 1. Introduction ………………………………………. 1 2. Literature review………………………………… 3 2.1 Origin and distribution of grape (vitis sp)………. 3 2.2 Botany……………………………………………... 3 2.3 Soil and Location………………………………… 4 2.4 Yield and storage…………………………………. 4 2.5 Pests and diseases…………………………………. 2.6 World production………………………………… 2.7 Propagation methods…………………………… 7 2.7.1 Traditional propagation ………………………… 7 2.7.2 Propagation by tissue culture……………………. 8 2.7.2.1 The ex-plant……………………………………….. 9 2.7.2.2 Media composition ……………………………….. 11 2.7.2.2.1 Inorganic nutrients ……………………………… 11 2.7.2.2.2 Organic nutrien…………………………………… 13 2.7.2.2.2.1 Carbohydrates…………………………………… 14 2.7.2.2.2.2 Growth regulators………………………………… 15 3. Materials and methods…………………………… 19 3.1 Plant material……………………………………... 19 3.2 Sterilization……………………………………… 19 3.3 Preparation of ex-plants …………………………. 19 3.4 Basal nutrient medium…………………………… 20 3.5 Culture incubation……………………………….. 20 3.6 Experimentation………………………………….. 20 3.6.1 Effect of MS salts mix……………………………. 20 3.6.2 Effect of supplementary phosphate …………………….. 21 3.6.3 Effect of ex-plant……………………………. 21 3.6.4 Growth regulators………………………………… 21 3.6.4.1 Combined effect of kinetin annaphthaleneacetic acid (NAA) on 21 different growth parameters…… 3.6.4.2 Induction of roots…………………………………. 22 3.6.5 Effect of physical status of media on growth and development of 22 nodal cuttings ………………….. 3.7 Data collection ……………………………………. 22 3.8 Parameters measured ……………………………. 22 4. Results and discussion …………………………… 24 4.1 Effect of MS salt mix strengths on growth parameter measures 24 on two types of ex-plant …. 4.2 Effect of K2H2PO4 on growth and development of two types of 33 ex-plants ………………………………………………….. 4.2.1 Effect of KH2PO4 on growth and development of segment nodal 33 cutting ex-plant…………………... 4.2.2 Effect of KH2PO4 on growth and development of shoot-tip ex- 33 plant………………………………. 4.3 Combined effect of naphthalene acetic acid (NAA) and kinetin on45 different growth parameters ……. 4.4 Effect of indole – butyric acid (IBA) at different concentration on 57 growth and development of nodal cutting 4.5 Effect of physical status of medium on growth and development 63 of nodal cutting of grapes……. 5. References………………………………………… 69 Conculation…………………. 83 DEDICATION This thesis is dedicated with love and best wishes to my parents, husband and all those who encouraged me to achieve this goal ACKNOWLEDGMENTS I would like to express my gratitude and sincere appreciation to my supervisor prof. Abd-El-Gaffar. Alhag Said for the keen interest, sincere guidance and invaluable supervision. Appreciation extended to Dr. Mustafa M.A. El Balla the co supervisor. Special gratitude is extended to Dr. Ilham A.G. Hansen for helping with computer falsities and to Dr. Esa E. for his continuous helps. I am also sincerely indebted to the staff of the tissue culture laboratory, Commission of Biotechnology and Genetic Engineering especially miss Hind E.M. Elmna and prof. Anas A. El Hassan who where great help all the time with their opinion and consultation. Special gratitude’s are extended to Dr. El Sadig for helping with his computer falsities and to my friends especially Mrs Hala E.Abdella who helped me in my work or had the will to help. My last of sincere thanks to my husband Mohammed for his contentious encouragement that help me in preparing this thesis. Abstract Grapes are considered by many scientists as of the promising crops in Sudan . Its cultivation was successful under Sudan climatic conditions . The main single factor that limits grape cultivation in Sudan is the availability of stock mother plant of known varieties . This has led to the scarcity of planting material that is disease free . Introduction of plant from aboard is expensive . The objective of this study was to tailor a nutrient medium suitable for the clonal propagation of grape using tissue culture techniques . Shoot – tips and nodal cutting were cultured on various concetrations of MS salt mit (0.25X, 0.5X, 1.0X, 2.0X). The addition , potassium hypophosphate (KH2PO4) to the medium was also tried as a supplement to test its effact on growth and propagation of grapes . Various combinations of the auxin (NAA) and the cytokinin (kinetin) were tested for the propagation purpose of grapes . The auxin (IBA) was also used for the rooting of in vitro produced plantlets before transfer to soil . Nodel cutting s proved to be the ex – plant of chice for the clonal propagation of grapes when cultured on full MS salt mix . Further enhancements were gained with suppliment of KH2PO4 giving best results in all parameters measured this was adopted as the ex- plant for culture initiation in all experiments conducted in this study . The result also showed that the best combination of auxin and cytokinin for high propagation rate was 0.1mg/1 NAA and 1.0mg/1 kinitin .Best rooting on the other hand was obtianed by the addition of 0.3mg/1 IBA. Growth and development of cultured nodal cutting in liquid medium supported by cotton proved to be the best for grape propagation compared toagar or other solidifying agent . ﻣﻠﺨﺺ اﻷﻃﺮوﺣﺔ ﻳﻌﺘﺒﺮ ﻧﺒﺎت اﻟﻌﻨﺐ ﻣﻦ اﻟﻤﺤﺎﺻﻴﻞ اﻟﻮاﻋﺪة ﻓﻰ اﻟﺴﻮدان ﺣﻴﺚ اﺛﺒﺖ ﻧﺠﺎح زراﻋﺘﻪ ﺗﺤﺖ اﻟﻈﺮوف و أﺻﻮل ﻣﻌﺮوﻓﺔ، اﻟﻤﻨﺎﺧﻴﺔ ﻓﻰ اﻟﺴﻮدان واﻟﻤﺸﻜﻠﺔ اﻟﺘﻰ ﺗﻮاﺟﻪ ﻧﺠﺎح زراﻋﺘﻪ هﻲ ﻋﺪم ﺗﻮﻓﺮ أﻣﻬﺎت إﺿﺎﻓﺔ اﻟﻰ ﻋﺪم ﺗﻮﻓﺮ ﺷﺘﻮل آﺎﻓﻴﺔ ﺧﺎﻟﻴﺔ ﻣﻦ اﻷﻣﺮاض وﺻﻌﻮﺑﺔ اﺳﺘﻴﺮاد هﺬﻩ اﻟﻨﺒﺎﺗﺎت ﻣﻦ اﻟﺨﺎرج. ﺗﻬﺪف هﺬﻩ اﻟﺪراﺳﺔ اﻟﻰ اﻳﺠﺎد وﺳﻂ ﻏﺬاﺋﻰ ﻣﻨﺎﺳﺐ ﻟﻠﺤﺼﻮل ﻋﻠﻰ ﺗﻜﺎﺛﺮ ﺳﻼﻟﻲ ﻟﻠﻌﻨﺐ ﺑﺎﺳﺘﺨﺪام ﻃﺮق زراﻋﺔ اﻷﻧﺴﺠﺔ. ﺗﻢ ﻓﻰ هﺬﻩ اﻟﺪراﺳﺔ اﺳﺘﺨﺪام اﻟﻘﻤﺔ اﻟﻨﺎﻣﻴﺔ واﻟﻌﻘﺪ اﻟﺴﺎﻗﻴﺔ ﻣﻔﺼﻮﻟﺔ ﻟﻨﺒﻴﺘﺎت ﻣﻨﺘﺠﺔ داﺧﻞ اﻷﻧﺎﺑﻴﺐ ﻹﻧﺸﺎء اﻟﺰراﻋﺎت اﻟﻨﺴﻴﺠﻴﺔ ﻓﻰ اﻟﺘﺠﺎرب اﻷوﻟﻴﺔ. زرﻋﺖ اﻟﻘﻤﻢ اﻟﻨﺎﻣﻴﺔ واﻟﻌﻘﺪ اﻟﺴﺎﻗﻴﺔ ﻋﻠﻰ أﻧﻮاع ﻣﺨﺘﻠﻔﺔ ﻣﻦ اﻟﺒﻴﺌﺎت اﻟﺰراﻋﻴﺔ ﺑﺘﺮاآﻴﺰ أﻣﻼح ﻣﺨﺘﻠﻔﺔ ﺗﻢ اﺧﺘﺒﺎر إﺿﺎﻓﺔ أﻣﻼح اﻟﺒﻮﺗﺎﺳﻴﻮم (MS) (0.0, 0.25, 0.5, 1.0, 2.0) ﻣﻦ وﺳﻂ (Kinetin) واﻟﺴﻴﺘﻮآﺎﻳﻨﻴﻦ(NAA )ﻓﻮﺳﻔﻴﺖ ﻋﻠﻰ ﻧﻤﻮ وﺗﻜﺎﺛﺮ اﻟﻌﻨﺐ, وﺗﻮﻟﻴﻔﺎت ﻣﺨﺘﻠﻔﺔ ﻣﻦ اﻻآﺴﻴﻦ ﻟﻠﺘﺠﺬﻳﺮ ﻓﻰ اﻻﻧﺎﺑﻴﺐ ﻟﻴﺘﻢ ﺑﻌﺪ ذﻟﻚ اﻟﻨﻘﻞ (IBA) ﺑﻐﺮض إآﺜﺎر اﻟﻌﻨﺐ وآﺬﻟﻚ اﺧﺘﺒﺮ اﻻآﺴﻴﻦ اﻟﻰ اﻟﺨﺎرج. ﺛﺒﺖ ﻣﻦ اﻟﺘﺠﺎرب اﻷوﻟﻴﺔ ﺗﻔﻮق اﻟﻌﻘﺪ اﻟﺴﺎﻗﻴﺔ ﻋﻠﻰ ﻗﻤﻢ اﻟﺴﻮق ﻓﻰ اﻻﺳﺘﺠﺎﺑﺔ ﻟﻠﻨﻤﻮ واﻟﺘﻜﺸﻒ داﺧﻞ اﻻﻧﺎﺑﻴﺐ وﻟﻬﺬا اﻋﺘﻤﺪ اﺳﺘﺨﺪاﻣﻬﺎ آﺠﺰء ﻟﻼﺳﺘﺰراع ﻹﻧﺸﺎء زراﻋﺎت اﻷﻧﺴﺠﺔ ﻓﻰ آﻞ اﻟﺘﺠﺎرب اﻟﺘﻰ ﺗﻤﺖ ﻓﻰ هﺬﻩ اﻟﺪراﺳﺔ. اﻟﻤﺤﺘﻮى ﻋﻠﻰ أﻣﻼح اﻟﺒﻮﺗﺎﺳﻴﻮم ﻓﻮﺳﻔﻴﺖ اﻓﻀﻞ اﻟﻨﺘﺎﺋﺞ ﻟﻠﺘﻜﺎﺛﺮ (MS) أﻋﻄﻰ اﻟﺘﺮآﻴﺰ اﻟﻜﺎﻣﻞ ﻷﻣﻼح ﺑﻮاﺳﻄﺔ اﻟﻌﻘﺪ اﻟﺴﺎﻗﻴﺔ . آﺬﻟﻚ أوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ ﺁن أﻋﻠﻰ ﻣﻌﺪل ﺗﻜﺎﺛﺮ ﻳﺘﺄﺗﻰ ﺑﺎﺳﺘﺨﺪام اﻟﺘﻮﻟﻴﻔﺔ ﻣﻦ ﻣﻠﺠﻢ/ﻟﺘﺮ، آﻤﺎ ﺗﻢ اﻟﺤﺼﻮل ﻋﻠﻰ Kinetin) 1.0 ) ﻣﻠﺞ/ل اﻟﺴﺎﻳﺘﻮآﺎﻳﻨﻴﻦ NAA) 0.1 ) اﻻآﺴﻴﻦ .(IBA) أﻋﻠﻰ ﻣﻌﺪل ﻟﻠﺘﺠﺰﻳﺮ ﻋﻨﺪ اﺳﺘﺨﺪام 0.1 ﻣﻠﺠﻢ/ﻟﺘﺮ آﺎن ﻧﻤﻮ وﺗﻜﺸﻒ اﻟﻌﻘﺪ اﻟﺴﺎﻗﻴﺔ ﻓﻰ اﻟﻮﺳﻂ اﻟﻐﺬاﺋﻲ اﻟﺴﺎﺋﻞ اﻟﻤﺪﻋﻮم ﺑﺎﻟﻘﻄﻦ اﻓﻀﻞ ﻟﺘﻜﺎﺛﺮ اﻟﻌﻨﺐ ﻣﻘﺎرﻧﺔ ﺑﻼﺟﺎر او اﻟﻌﻮاﻣﻞ اﻟﻤﺼﻠﺒﺔ اﻷﺧﺮى. Introduction Grape (Vitis sp.) belongs to the family Vitaceae and several known types of grapes are named after their place of origin. European grapes have been grown in cultivation throughout recorded history. Wine is the primary use for European grapes, but they can be used for eating out of hand, as raisins, and can be made in to juice. Among the many species of grapes known the most commercially exploited and utilized are: Vitis Vinifera (The old world grape) used for wine, raisins, and for table purposes and V. labrusca grown commercially for table use, grape juice, and to a lesser extent in wine production, (Christopher, 1958). Grapevines are natives to temperate zones. They grow well in Mediterranean climate, and are theis found around the Mediterranean on the coasts of South Africa and South West Australia, California and Chile. They can also be cultivated under irrigation in desert areas and under tropical climates but here they require intensive and constant care, where the production of two harvests per year is normal (Jordens, 1987). The economic importance of grapes is in the exploitation of unsuitable lands for growing other fruits, such as sandy, low fertility soils and low-in- depth lands. Sudan is now heading towards an agricultural revolution and the use and spread of technology so as to maximize the benefit of its resources. In the field of fruit production many promising types of fruits exist, among them are grapes where preliminary trials, showed good potentials for their cultivation especially in the Northern region. Grapes are well known in Sudan and are cultivated in many areas but the yield is of low quantity and quality. This may be attributed to the repeated and continuous use of vegetative propagation that lead to the accumulation of many diseases a reason that necessitates obtaining disease free stocks or importing diseases-free mothers from abroad which is costly and laborious and passes through various complicated quarantine steps. The objective of this research was to study the propagation of a local variety of grape (Vitis sp.) through tissue culture technique and find the most appropriate conditions of medium formulation for mass propagation, using two types of explants: shoot-tip and nodal segments as an initial step aiming at the production of disease-free stock mother plants of commercially known varieties. 2-Literature review 2.1 Origin and distribution: - Grape originated in Middle Asia between the Black and Casbian sea. These places are considered as the origin of European grapes (Vitis Vinifera L.) or the old world grape. From this species all other types of grapes were produced before the discovery of the new world.
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