Studies On Micropropagation and Plant Regeneration of Sweet Potato (Ipomoea Batatas)

Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas)
Mamatha M Pillai M Unnikrishnan ( Principal scientist ) CTCRI Trivandrum
ACKNOWLEDGEMENT I express my deep sense of gratitude and personal indebtedness to my guide M. Unnikrishnan, Principal Scientist, Division of Crop Improvement, Central Tuber Crops Research Institute, Trivandrum for his valuable guidance, constructive criticism and sincere help in the conduct of project and preparation of thesis. I express my heartful thanks to Dr. Winny Varghese, Principal, Mar Athanasius College, Kothamangalam for his sincere encouragement in conducting the study. I record my heartful gratitude to Dr. Yamuna Anu Joseph, Head of the Department of Biotechnology, Mar Athanasius College, Kothamangalam for her whole hearted support and timely help to carry out the study. I am most grateful to Mr. Paul George, Lecturer, Department of Biotechnology, Mar Athanasius College, Kothamangalam for his sincere guidance and help in completing this project successfully. .I express my thanks to all the Staff members of the Department of Biotechnology, Mar Athanasius College, Kothamangalam. I am extremely indebted to my Parents and Friends for their consistent encouragement and unfailing help rendered to me without whose help this work would not have been possible. I am thankful to Each and Everyone who helped me to complete this work successfully. I thank God Almighty who has given me strength, courage, and blessings to carry out the study successfully.
Mamatha M Pillai
PREFACE
Vegeculture, tropical food production based on vegetatively propagated energy crops, probably emerged before agriculture based on cereals and grains. The tropical root and tuber crops (cassava, sweet potato, yams and aroids) are among the oldest on earth. In many areas, especially in the wet tropics, they were the only staples and fed extensive populations before the introduction of cereals. Today, they represent the second most important set of food crops in developing countries, closely following the cereals. They are produced with low inputs but are an important source of income and employment in marginal areas, especially for women. Consumed mostly by the poorest, they contribute greatly to food security and are held in high esteem culturally. They are also cash crops and are used for animal feed or as raw material for industrial processing.
Sometimes considered as plants of the past, they are, on the contrary, crops of the future since they allow local production of carbohydrates, which can substitute expensively imported cereals. With world population projected to increase from the present 6.6 bn to 8 bn by 2025, it may be argued that the demand for carbohydrates will soon exceed the production potential of areas devoted to the cultivation of cereals. This is especially critical in the wet tropics, where the majority of the world population lives. In circumstances of global climatic change, such a scenario may render increased production of tropical root and tuber crops imperative. This may come about all the sooner if some countries decide to retain their harvests of cereals, to divert it into the production of biofuels, or if the ever-increasing cost of energy causes imported foodstuffs to become too expensive.
As a group, the tropical root and tuber crops are efficient plants and if marginal land is to be exploited to support burgeoning populations, their potential, clearly untapped, will need to be developed. Although these species belong to different botanical families, they are grouped together because they are vegetatively propagated, bulky and perishable. Despite these constraints, they have proven surprisingly transferable and are now cultivated throughout the world. In many places, they are grown together within the same plots, in home gardens or in mixed cropping systems, complementing each other throughout the year to produce a steady supply of energy.
Vegeculture is very much alive and adapting to changing environments. However, compared to other crops of equivalent economic importance, the tropical root and tuber crops are seriously under-researched. Considered in most developing countries as of lesser priority, well below traditional export commodities inherited from the colonial era, these food crops do not receive from governments the attention they deserve. More widely, western ethnocentric prejudices have induced an even more striking neglect of their essential food security role.
Food scarcity and high level of malnutrition remains continued to be a developmental puzzle for the Republic of the Marshall Islands because of a major problem of limited land resources accompanied with poor quality, nutrient deficient soil. Vitamin A deficiency in Marshallese children is highest in the world according to World Health Organization (WHO). The main theme of the proposed research program is to introduce sweetpotato using emerging innovative plant tissue culture to ensure food and nutritional security. The technology generated would also lead to in vitro germplasm conservation of sweetpotato.
CONTENTS
1. 2. 3. 4. 5. 6. 7. 8.
INTRODUCTION .. 4 OBJECTIVES 8
REVIEW OF LITERATURE 9 MATERIALS AND METHODS 11 RESULTS DISCUSSION SUMMARY .19 .. 38 .. 9
BIBLIOGRAPHY 40
1. INTRODUCTION
1.1 ROOT CROPS
The major tropical root crops of the world are Cassava (Manihot esculenta), Sweet Potato (Ipomoea batatas), Yams (Dioscorea spp) and Taro (Colocasia esculenta). In terms of world production, these tropical root crops are fifth behind Wheat (510 million t), Maize (490million t) Rice (466 million t) and White potatoes (299 million t) (FAO Production year book 1998.) Tuber crops are the most important food crop of mankind after cereals and legumes. The importance of tuber crops is mainly because of the high starch content, which make them high caloric value food and also a rich source of starch. Starch characters make them valuable in food and industry.
1.2 IMPORTANCE OF CROP PRODUCTION AND CROP IMPROVEMENT
With the growing population, pressure on agricultural land and available food costs of rice directly affect the low income populations which are already deficient in calories besides escalating costs of rice. In this context, root crops are the only potential supplementary food crops as they can provide more energy per unit area than any other field crop and are cheap source of energy. In order to fill the anticipated energy gap, production has to be increased. Thus, expansion of area of cultivation and crop improvement are two important and concurrent prerequisites to increase crop production.
1.3. IMPORTANCE OF SWEET POTATO
Sweet potato (Ipomoea batatas L.) ranks seventh among all food crops worldwide, with an annual income of 115 million metric tons. Of the root and tuber crops the Sweet potato ranks third in acreage (7.9 million ha) behind the potato and cassava. Sweet potato is grown in more than 100 countries and among the worlds root and tuber crops, it ranks second in importance. It is consumed as a fresh vegetable (roots, petioles, leaves and stems), staple food, snack food and it is also used for industrial starch extraction and fermentation. Sweet potato is industrially dehydrated and used as an important component of bread flour. Sweet potato is consumed as a substitute to rice and wheat flour, especially by the low income classes of the population in Africa and many Asian countries.
1.3.1. MORPHOLOGY OF SWEETPOTATO
Sweet potato (Ipomoea batatas) is a dicotyledonous plant that belongs to the family Convolvulaceae. Its large, starchy, sweet tasting tuberous roots are an important root vegetable (Woolfe, 1992). Root System: The sweet potato root system consists of adventitious roots that absorb nutrients and water, and anchor the plant, and storage roots that are lateral roots which store photosynthetic products Tuber in sweet potato it is specialized root tubers which can be used as propagating material as its sprouts and produces plants. Stem: A sweet potato stem is cylindrical and its length, like that of the internodes, depends on the growth habit of the cultivar and of the availability of water in the soil. Leaves: The leaves are simple and spirally arranged alternately on the stem in a pattern. Depending on the cultivar, the edge of the leaf lamina can be entire, toothed or lobed. Flowers: The inflorescence is generally a cyme. The gynoecium consists of a pistil with a superior ovary, two carpels, and two locules that contain one or two ovules. Fruit and Seeds: The fruit is a capsule, more or less spherical with a terminal tip, and can be pubescent or glabrous. Sweet potato shows self incompatibility. Seed is having hard testa which requires scarification for germination.
1.4. RELEVANCE OF TISSUE CULTURE
Sweet potato and many other vegetatively propagated plants are frequently characterized by their inability to produce seed due to the presence of one more factors, such as incompatibility, dichogamy, abnormal seed and seeding development, seed dormancy and environmental condition which affect flowering and seed settings. Presence of these factors poses some limitations on the use of environmental techniques for improvement of these crops. Therefore as it has been exploited for many other crops, tissue culture technology could offer a very valuable tool for improvement of these crops. Tissue culture system is capable of creating genetic variability and producing plants with novel characters, which could be more favourable than the existing crop varieties. Apart from that, culture techniques which were
Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) first developed by Robbins in 1972 accomplished several important applications such as germplasm conservation, exchange of germplasm and virus elimination. At present, germplasm of root crops is conserved by maintaining them in the field through annual propagation. In the case of sweetpotato propagation is required every three months. Thus, the crop is mostly exposed to the hazards of environmental stresses, pests and diseases. Conservation of germplasm through seed is impossible due to highly heterozygosity nature of the seedling progeny. On the other hand, conservation of plantlets regenerated through meristem culture has several advantages. The germplasm is traditionally exchanged through tubers and cuttings which are susceptible for external as well as internal infestations. Exchange through meristem culture holds great promise for national and international dissemination of germplasm as it assures freedom from infestations. Adoption of this technique poses less quarantine problem too.
1.5 ADVANTAGES OF IN VITRO GENE BANK: Low labour costs. Absence of field infection
Protection against unfavorable climatic conditions. Timely access to material under maintenance. Timely access to material for pathogen clean up. Permanent availability of (when pathogen tested) material for exchange and multiplication of disease free
planting material. omoea batatas)
2. OBJECTIVES OF THIS WORK This work entitled Studies on Micropropagation, Plant regeneration, Development of Media for in vitro flowering and Molecular characterization of Sweet potato (Ipomoea batatas) has the following objectives,
In vitro culturing of plants. Plant regeneration through somatic embryogenesis Germplasm conservation through slow growth cultures. Production of virus free plants through meristem culture.
Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) 3. REVIEW OF LITERATURE
3.1. TISSUE CULTURE
Development of science of tissue culture is historically linked to the discovery of the cell and subsequent propounding of cell theory. Plant tissue culture is the science of growing plant cells, tissues or organs isolated from the mother plant, on the artificial media. It includes techniques and methods used to research into many botanical disciplines and has several practical objectives. The in vitro techniques were developed initially to demonstrate the totipotency of plant cells predicted by Haberlandt in 1902. Totipotency is the ability of a plant cell to perform all the functions of development, which are characteristic of zygote, i.e., ability to develop into a complete plant. In 1902, Haberlandt reported culture of isolated single palisade cells from leaves in Knop's salt solution enriched with sucrose. The cells remained alive for up to 1 month, increased in size, accumulated starch but failed to divide. Efforts to demonstrate totipotency led to the development of techniques for cultivation of plant cells under defined conditions.
The brilliant contributions from RJ. Gautheret in France and P.R. White in U.S.A. during the third and the fourth decades of 20th century may be considered a foreword for the discovery of plant tissue culture. An important breakthrough for continuously growing tip cultures came from White (1934, 1937), who initially used yeast extract in a medium containing inorganic salts and sucrose. Most of the modern tissue culture media derive from the work of Skoog and co-workers during 1950s and 1960s. Since the 1960s, research on the propagation of plants by tissue culture at an ever increasing pace.
3.2. SWEET POTATO MICROPROPAGATION
3.12.1. Meristem culture The meristem tip is meristem together with 1-2 primordial leaves and measuring between 0.1 -0.5 cm in height (Biggs et al. 1985). In vitro cultures could be established in sweet potato from meristem tips 0.1 mm excised from shoots derived from tuber sprouts (IBPGR 1987; Alconero et al. 1975).Meristem tip culture is used successfully to remove viruses, bacteria, and fungi from plants.
3.12.2. Nodal culture Sweet potato cultures could be initiated from nodal explants as well as internode derived callus. Nodal
culture could grow and further multiplied on Murashige Skoog medium without growth regulators where they developed roots and could hardened and transplanted (Unnikrishnan et al. 1990). Growth of in vitro cultures of sweet potato improved under optimal photoautotrophic condition (without sugar in the medium and under 100 M m-2 s-1 PPFD and enriched CO2 concentration) (Kozai et al. 1996). A two stage protocol is reported to be more effective for micropropagation of 27 sweet potato genotypes. Initially, leaf explants are grown on MS medium with 2, 4-D (0.1 mg/l) and zeatin (0.2 mg/l) until the base of the petiole begins to swell (2-4 days). Then, they are transferred to a medium with zeatin (0.8 mg/l) wherein high-frequency shoot regeneration occurs (Raja Sree et al.2001).
Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) Shoot tip meristem along with a leaf primordium cultured on Murashige Skoog medium with GA and kinetin produced plantlets, which, on testing were found to be virus-free. GA and Kinetin in combination, or GA alone, was found to be more effective for the fast development of meristem cultures (Unnikrishnan et al. 1990).The meristem derived plants were subjected to virus indexing through serodiagnostic methods as well as by grafting onto indicator hosts (Alconero et al. 1975). Pathogen free germplasm accessions have been used for safe exchange of germplasm ( IBPGR 1987).
3.12.3. In vitro conservation under Slow- Growth There are several methods by which slow growth can maintained. It is possible to limit growth by modifying the culture medium, mainly by reducing the sugar or mineral elements concentration and reducing of oxygen level available to culture by covering explants with a layer of liquid medium or mineral oil ( Nyman et.al, 1987). Sweet potato cultures could be kept under slow growth at 3% concentration of mannitol. Slow growth could be also be induced by limiting incubation temperature at 16-18C. (IBPGR 1987). The cultures could be isolated in Murashige Skoog media having 3% sucrose and mannitol each supplemented with NAA, BA (0.1 M) and GA (0.3M), for upto 12 months at 25-28C (Unnikrishnan et al.1990). Use of osmotic retardants and low temperature was found to induce slow-growth in sweet potato cultures. Use of low sugar medium (2%) alone, as well as mannitol (2% and 3%), in Murashige Skoog medium was found to be effective in stretching subculture intervals upto 14 months (Chandel et. al 1997).
3.12.4. Anther Culture Response to anther culture was found to depend on genotype as well as on media as observed in Cassava. Callus induction was obtained on Murashige Skoog medium supplemented with NAA and cytokinins (BA/ Kin/2ip). Regeneration was obtained on subsequent cultures on anthers on Murashige Skoog medium with 2, 4 D and Murashige Skoog basal medium without growth regulators. The plants were transferred and established successfully. They showed hetroploidy (chromosome number 70-80) with anomalies like lagging chromosome and disturbed polarity of metaphase plates. Lower ploidy (2n=51) was also noticed (Mukherjee et al. 1991). Embryoid formation and plant development from sweet potato anther-derived callus has been reported by other workers too (Kobayashi 1991).
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4. MATERIALS AND METHODS
4.1. MATERIALS
4.2. EXPERIMENTAL PLANT MATERIAL: Indigenous and exotic collection of sweet potato maintained in the Central Tuber Crop Research Institute (CTCRI) of Indian Council of Agricultural Research (ICAR), Trivandrum, Kerala, India were used as the source of experimental plant material. 4.3. MEDIA: The successful plant tissue culture depends upon the choice of effective nutrient medium. Virtually all tissue culture media were synthetic or chemically defined. The cells of the most plant species can be grown on completely defined media. The nutrient medium for most tissue culture was comprised of five groups of ingredients, inorganic nutrients, carbon source, vitamins, growth regulators and organic supplements. Inorganic Nutrients: Inorganic nutrients consist of macro and micro elements and their salts. Usually nutrient media contain 25mM each of nitrate and potassium. a. Macro Nutrients They include nitrogen, phosphorous, calcium, potassium, magnesium and sulphur. b. Micronutrients: Mineral elements were very important for the growth the plant. They include iron, manganese, zinc, boron, copper, molybdenum, cobalt, and iodine. These elements are needed only in small quantities, so they are called micro elements or miner elements. Vitamins To achieve the best growth of the tissue it was after essential to supplement the medium with one or more vitamins. These include nicotinic acid (Vitamin B 1), pyridoxine (vitamin B6) and myoinositol. Carbon Source: Carbohydrates were used as the carbon source. Sucrose and glucose were commonly used one. The source in the medium was rapidly converted into glucose and fructose. The glucose was absorbed first followed by fructose.
Plant Growth Regulators: A balanced combination of plant growth regulators was required for substantial growth. Auxins (IAA,
NAA, 2, 4-D) were commonly used to support cell division and callus growth. Cytokinins like (TDZ, BAP) were employed to promote cell division, regeneration of shoots, often somatic embryoids induction and to enhance proliferation and growth of axillary buds. Gibberellins (GA3) promote shoot elongation and somatic embryoids germination. Plant growth regulators used were made in to stocks and stored at refrigerated conditions (4+1C). Concentration of plant growth regulators used in various modification of Murashige and Skoogs medium (1962) was shown in the following table;
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SI. No.
Medium (MS)
NAA
BA
GA3
IBA
2,4-D
TDZ
1.
Propagation Medium HM1 HM3 0.5M -
2.
Meristem culture medium 0.1M 0.1M 0.1M -
3.
Callusing Medium 0.2M 0.2M
Agar:
Solidifying agent or gelling agent used were commonly of two types; agar and phytagel in which any one of them was used. Agar was mainly used to prepare solid and semisolid plant tissue culture media. Activated charcoal
Activated charcoal was carbonized wood which has been heated for several hours in steam. It poses strong adsorption properties. It absorbs phenolic compounds secreted by the explants in to the tissue culture media. pH: The pH of the medium was usually adjusted between 5.6 and 5.8 before sterilization and pH of 5.7 was most preferable.
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4.4. PREPARATION AND STERILIZATION OF MEDIUM.
4.4.1. Preparation of Stock Solutions: To prepare Roca et.al. CIAT 1984 medium for micro propagation. (Based on Murashige and Skoog, 1962). Stock code No. solution Constituent chemicals Quantity Volume of stock to be used for preparing 1 litre
medium. (1) A Macronutrients NH4.NO3 KNO3 MgSO4.7H2O KH2PO4 (Dissolve in 1000ml distilled water) (2) B To be Freeze Stored. Micro nutrients H3BO3 MnSO4.H2O ZnSO4. 7H2O Na2. MoO4.242O CuSO4. 5H2O CoCl2. 6H2O (dissolve in 1000ml distilled water) (3) C (4) D (5) E KI (Dissolve in 1000ml distilled water) CaCl2.2H2O (Dissolve in 100ml Distilled water) a) Na2 EDTA( Chelating agent) b) Fe SO4. 7 H2O (Dissolve in 1000ml distilled water) (6) F (7) G Vitamins Thiamine. HCl myo-inositol (Dissolve in 200ml of distilled water) 0.8g 6.25 ml 10mg 5.0 ml 1.492g 1.114g 5.0ml 15.0g 2.9ml 0.075g 1.0 ml 0.62 g 2.176g 0.86g 0.025g 0.0025g 0.0025g 1.0 ml 82.5g 95.0g 18.5g 8.5g 20.0 ml
Stocks (2) and (6) should be kept frozen; all others stored at 8-10oc, kept stock (5) protected from light. Separately dissolve a) and b) in 50ml water each; heat up b) in a water bath; mix both solutions well; let cool and then add water to complete to 200ml.
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Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) Auxins and Gibberellic acid were dissolved initially in minimum volume of absolute alcohol and cytokinines in KOH/NaOH (1N). Then they were made up to required volume by adding double distilled water. pH of the medium was adjusted to 5.7 by IN NaOH or IN HCl prior to autoclaving. Then l g/l of activated charcoal was added. Agar was added and agar in the medium was dissolved by boiling. Then the medium was dispensed into the autoclaved/sterilized tubes and sealed with aluminum foil. The tubes containing medium was finally sterilized by autoclaving at 15 lbs pressure at 121oC for 15 minutes. The sterilized media were kept at 25oC prior to use. (In order to check if there was any visible microbial infection).
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Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) METHODS
4.5. PROCEDURE FOR SWEET POTATO MERISTEM CULTURE:
Meristem culture is the in vitro culture of a generally shiny special dome-like culture measuring less than 0.1mm in length and only in one or two pairs of the youngest leaf primordia, most often excised from shoot apex.
4.5.1 Surface Sterilization and Dissection of Explants.
The apical meristem is usually a dome of tissue located at the tip of shoot and measures approximately 0.1 mm in diameter and 0.2-0.3 mm in length. The explants were quickly rinsed in 70% alcohol for 1-3 minutes in a sterilized Erlenmeyer flask. They were then sterilized with Mercuric chloride (0.1%) for five minutes. Then it was washed 2-3 times using sterilized distilled water.
After rinsing in distilled water, placed the material under the dissection microscope. Using the forceps, hold the stem steady to remove the largest of the young leaves. Removed the underlying leaf primordia by inserting the tip of the scalpel into the base of each primordium and flicking the tip of the scalpel away from the stem axis.
At this point, the apical dome should be visible, flanked by two or three of the youngest leaf primordia. Removal of these primordia can be accomplished by scraping them off with the cutting edge or back edge of the scalpel blade. It was important that all leaf primordia should be removed and only the apical dome (0.1mm in depth) excised in order to increase the probability of obtaining plants, free of viruses.
4.5.2. Media Used and Its Composition: Meristem culture was tried in Murashige and Skoogs media supplemented with NAA, BA and GA 3 denoted as HM2. The composition of HM2 used here was.
Concentration in g/l MS I bottle Sucrose 30 Agar 8
M / l NAA 0.1M BA 0.1M GA3 0.1M
4.5.3 Inoculation and Incubation:
The excised dome was then quickly transferred to the tube containing medium. The dome will just be visible to the naked eye, and care must be taken to ensure that it was placed on the surface of the medium rather than adhering to the tip of the scalpel.
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Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) The culture tubes containing explants were maintained in the culture room at 252 C. They were exposed to artificial illumination of 2000-3000 Lux by placing them at 20-30 cm below fluorescent light for 16 hours every day 50-90% humidity was also maintained.
4.6. METHODS FOR SWEET POTATO NODAL CULTURE Nodal culture is an in vitro culture of node which is a part of stem where leaf arises. 4.6.1. Explant Collection and Surface Sterilization: Explants were taken from sweet potato of different accessions and were collected in different test tubes. The leaves, internodes etc were removed and the remaining nodes were washed in running tap water for removing any adherent particle. Thoroughly washed nodes were then immersed in 5% (v/v) Teepol for 20 minutes and after this, the nodes were again washed well in tap water several times for the complete removal of detergent solution. Then it was dipped in Bavistin (fungicide) for 10 minutes. After 10 minutes the nodes were washed thoroughly with tap water and then it was rinsed three times with sterile distilled water. The explants were then brought inside the laminar airflow cabinet and surface sterilization was done with surface sterilant Mercuric chloride [0.1% (w/v)] for 5 minutes and rinsed 3 times with sterile distilled water to remove all traces of sterilant. 4.6.2. Media Used and Its Composition: Nodal culture was tried in two Murashige and Skoogs (MS) basal media, denoted as HM 1. The composition of MS basal media used here was; Concentration in g/l MS I bottle Sucrose 30 Agar 8 Charcoal 1 CaCl2 2.9ml
4.6.3. Inoculation and Incubation.
Immediately after surface sterilization the plant material was aseptically transferred to solidified MS medium. The explant was inoculated in horizontal position. The inoculation was done with care that the base of the node was touched at the surface of the medium. Then the cultures were incubated at 22C to 27C in light for 16 hours at the intensity of light (1800 lux) and 8 hours dark for 15-20 days. 50-90% relative humidity was also given.
4.7. METHODS FOR PLANT REGENERATION
4.7.1. Explant Source: Young leaves, mature leaves, nodes, internodes, anther, ovary, ovule, stigma, pith of stem, root and shoot tip of sweet potato were used as explants which was grown at Central Tuber Crops Research Institute for callus induction. Callus initiation occurred within two weeks of culture.
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4.7.2. Media Used and Its Composition: Two combinations of media were used for callus culture in sweet potato. These include Murashige and Skoogs medium supplemented with 2, 4 D denoted as S1 i.e. callus induction medium and Murashige and Skoogs medium supplemented with TDZ denoted as (S2) i.e. regeneration medium. The composition of S1 used here was;
Concentration in g/l MS I bottle Sucrose 30 Agar 8 2,4-D 0.2mg/l CaCl2 2.9ml
The composition of S2 used here was; Concentration in g/l MS I bottle Sucrose 30 Agar 8 TDZ 2ml CaCl2 2.9ml
4.7.3. Inoculation and Incubation:
Small pieces of explants were inoculated on to freshly prepared sterile medium1 (S 1) and. After 4 days, the swollen enlarged explants were then transferred to MS medium containing TDZ (Thidiazuron, 0.2mg/l). Hence the procedure involved placing of explants on two step media. The cultures were incubated at 25 oC and 12 hours light.
4.8. PROTOCOL FOR IN VITRO CONSERVATION (SLOW GROWTH CULTURE)
4.8.1. Explant Collection and Surface Sterilization: Explants were taken from sweet potato of different accessions and were collected in different test tubes. The leaves, internodes etc were removed and the remaining nodes were washed in running tap water for removing any adherent particle. Thoroughly washed nodes were then immersed in 5% (v/v) Teepol for 20 minutes and after this the nodes were again washed well in tap water several times for the complete removal of detergent solution. Then it was dipped in Bavistin (fungicide) for 10 minutes. After 10 minutes, the nodes were washed thoroughly with tap water and then it was rinsed three times with sterile distilled water. The explants were then brought inside the laminar airflow cabinet and surface sterilization was done with surface sterilant Mercuric Chloride [0.1 %( w/v)] for 5 minutes and rinsed 3 times with sterile distilled water to remove all traces of sterilant.
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4.8.2. Media Used and Its Composition: Nodal culture was tried in Murashige and Skoogs (MS) basal media, denoted as SG 3. The composition of MS basal media used here was;
Concentration in g/l MS I bottle Sucrose 20 Agar 8 Mannitol 20
Concentration in M/ l NAA 0.5 BAP 0.1 GA3 0.3
4.5.3. Inoculation and Incubation.
Immediately after surface sterilization, the plant material was aseptically transferred to solidified MS medium. The explant was inoculated in horizontal position. The inoculation was done with care that the base of the node was touched at the surface of the medium. Then the cultures were incubated at 22C to 27C in light for 16 hours at the intensity of light (1800 lux) and 8 hours dark for 15-20 days. 50-90% relative humidity was also given.
4.9. METHODS FOR SUB CULTURING:
After a period of time, it becomes necessary to transfer the cultures to fresh media. A portion of tissue was used to inoculate new culture tubes or flasks; this is known as sub culturing. For the initiation of subculture, it was necessary to raise a population of healthy plants. In many experiments, the culture was started from a stock plant raised by micro propagation. The established propagated plants were selected as the source of explant for sub culturing. For the preparation of explant in subculture, the plants were taken from the medium carefully with sterile forceps and the basal regions were cut off with sterile scissors. Then the leaves and other unnecessary regions were removed leaving the explants with a single internode. From the explant, each node was separated and inoculated to fresh medium and incubated under 25C and 1000 lux illumination.
4.9.1 PROCEDURE FOR HARDENING AND FIELD ESTABLISHMENT OF PLANTS.
Sweet potato plants with well developed root and 5-6 nodes were selected. This was transferred to the laminar air flow work station where the plantlet was carefully removed from the medium. It was washed with running tap water in order to remove the agar residues so that we can check up the chance of contaminants on the soil. Then it was transferred into plastic cups containing sterilized vermiculate and then covered with polythene bags in order to maintain humidity and water. After one week, the polythene bags are removed and it was taken to field trials (Fig. 39).
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5. RESULTS 5.1. MERISTEM CULTURE
Sweet potato Meristem cultures were done in HM2 medium.
5.1.1. Studies on HM 2 medium
Isolated meristems of five sweet potato varieties were cultured in HM2 medium for 8-12 hours per day at 3000 lux illumination and 25C temperature. The observations were taken once in every two weeks and repeated upto 30 days. During the first week of incubation, morphological changes were observed, slight increase in tissue volume and they differentiated in the following weeks. After one month plantlets were observed.
5.1.2. Varietal response in Sweet potato
Out of five of varieties of sweet potato, sweet potato 23 showed fast response of growth and S.823, Sree Arun, S.665, S.685 showed medium response of growth after two months of incubation 23 variety of sweet potato were grown upto 2cm with 4 leaves while in Sree Arun and S.685 showed shoot emergence of 1.5 cm with 2 leaves and nodes and S.823, S.685 showed lowest growth comparing to other varieties (table 1, graph 1, figure 1).
TABLE 1: OBSERVATION OF MERISTEM CULTURE IN DIFFERENT VARIETIES OF SWEET POTATO.
Name of variety S.823
Sl no:
No: cultures
of
No:
of
Observation after 15 days
Observation after 30 days
Remarks
cultures obtained 4
inoculated 1 6
No response
Enlargment, green coloration
Medium response
S.23
Green coloration
Development of buds Green coloration
Fast response Medium response Medium response Medium response
Sree arun S.665
No response
No response
Green coloration
S.685
No response
Green coloration
Sweet potato varieties showed a delayed response of growth in S 2 medium. Out of the five varieties S.23 showed a fast response within 15 days morphological changes were observed compared to other varieties which gave response within 30 days.
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GRAPH 1: MERISTEM CULTURE IN DIFFERENT VARIETIES OF SWEET POTATO
S. 685
SreeArun
S.665
S.23
0 10 20
% of30 Growth 40
50
60
FIGURE 1: MERISTEM CULTURE
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5.2. NODAL CULTURE. Sweet potato nodal culture was tried in Murashige and Skoogs basal media (HM 1).
5.2.1.. Studies on HM 1 medium In HMI, the separated nodes of five different accessions were inoculated in such a way that each accession had five replications. Studies involving in vitro culture of nodes of five different accessions of sweet potato genotypes revealed different growth responses which were interpreted in the table 2, graph 2 and figure 2.
5.2.1.1. Varietal response to nodal culture
In all the accessions, the initial response was observed after one week of inoculation. The best response was observed in genotype Gautham (graph2), which attained maximum shoot length of 5.8cm and also had well developed root system; while response of others was recorded such as in accession S16(graph 2). i.e., 3cm , Kishan 3.2cm(graph2), Sree Retna 2.6cm(graph 2) and Sree Bhadra 3.1cm (graph 2) length of shoot after one month of inoculation ( table2, figure 2). In HM1, Gautham showed the best response. The average length of shoot while considering the entire genotype cultured was found as 1.78cm after 15 days while the average length recorded after 30 days is 3.54cm .The mean number of leaves recorded was 1.44 and 3.65 after 15 days and 30 days respectively while mean number of root was 2.82 cm and 4.44 cm. The overall rate of development in sweet potato was found good. The positive growth response can possibly be due to the presence of charcoal. It also promotes root formation due to its ability to exclude light from the medium. The various physical factors and Murashige and Skoog basal medium provided were found to be effective in the multiplication of sweet potato.
Various parameters studied for nodal culture were no. of leaves, no. of nodes and root and shoot length. The three species responded very well after 2 months.
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Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) Table2: OBSERVATION OF NODAL CULTURE IN DIFFERENT VARIETIES OF SWEET POTATO Observation After 15 Days Variety No: of replica No: of Leaves No: of Nodes Mean shoot length(cm) Mean root length (cm) 1 2 S.16 3 4 5 1 2 Gautham 3 4 5 1 2 Kishan 3 4 5 1 2 Sree Rethna 3 4 5 1 2 Sree Bhadra 3 4 5 1 2 1 1 1 1 3 2 3 3 1 1 2 2 1 2 1 1 1 2 1 3 1 2 1 1 1 1 3 2 3 3 1 1 2 2 1 2 1 1 1 2 1 3 0.8 1.7 1.8 0.6 0.9 1.2 1.9 1.2 2.1 1.8 1.6 0.7 1.2 0.9 0.4 1.2 2.2 0.7 0.6 0.4 1.5 0.7 1.6 0.8 1.6 2.5 2 2.7 1.2 2 1.8 2.5 2 3.2 4 3 2.1 2 1.4 2 2.6 2.5 1.3 1 1.8 2.3 1.6 2.8 1.9 2.5 5 7 4 3 2 6 4 4 6 5 4 2 3 4 2 2 3 1 3 2 4 6 3 2 4 5 7 4 3 2 6 4 4 6 5 4 2 3 4 2 2 3 1 3 2 4 6 3 2 4 1.6 2.8 3 1.8 1.9 2.6 2.3 1.6 5.8 2.8 3.2 1.9 1.6 1.8 2.8 2.4 1.3 2.4 2.6 0.9 1.6 2.8 2.2 1.2 3.1 Observation After 30 Days No: of Leaves No: of Nodes Mean shoot length(cm) Mean root length (cm) 4 5 4.2 4.4 3.4 5.2 4.8 3.2 5.5 4.2 4.8 3.2 4.3 2.8 3.8 3.5 3.6 4 3.2 3 2.8 4 3.5 2.5 3.5
22
GRAPH 2: NODAL CULTURE IN DIFFERENT VARIETIES OF SWEET POTATO
5 4 3 2 1 0 No of leaves No of nodes Shoot length Root length
FIGURE 2: DIFFERENT STAGES OF NODAL CULTURE
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5.4. PLANT REGENERATION
Sweet potato plant regeneration was studied in S1 and S2 media.
5.4.1.. Studies on S1 medium The callus response of each variety was observed and studied.
Varietal difference in callusing in S1 media In Sweet potato callus culture, different varieties showed differential response .Retna showed very good response as callus induction happened within 4 days. It was observed that Vardhini and Gautham exhibited callus induction in 6 days which was a moderate response. Nandini took 8 days to respond which was comparatively low response(Table 3. Graph 3). Induction of callusing on explants may be due to the influence of 2, 4 D hormone present in the medium. In this medium, no embryoid development occurred. So it was inoculated into another medium i.e. callus regeneration medium (S2 medium). Response is shown in Table 3, graph 3 and figure 3.
5.4.1. Studies on S2 medium. 5.4.1.2. Varietal difference in callusing in S2 media Variation in response in callus development was observed among the varieties as well as different explants. Only the variety Retna responded with the formation of embryoids and plant regeneration using the explants - young leaf, internode and petiole. Root formation was observed in the variety Nandini from the explant shoot tip fig. 18. Variety Response Good response within 6 days and Vardhini inoculated into S2 medium Good response within 8 days and Nandini inoculated into S2 medium Very Good response within 4 days and Rethna inoculated into S2 medium Good response within 6 days and Gautham inoculated into S2 medium
TABLE 3: RESPONSE OF DIFFERENT VARIETIES IN CALLUSING OF SWEET POTATO IN S 1 MEDIA
24
5.4.1.2. Response of different explants for callus development
Different explants of different varieties showed different responses. Young leaf explant of Vardhini and Nandini initiated callusing within 13 days. Gautham responded within 16 days and Retna within 14 days (Table 4, fig4). Mature leaf explants Vardhini and Gautham initiated callusing within 15 days. Nandini and Retna responded within 13 days (Table 4, fig5). Young petiole of Vardhini initiated callusing within 12 days and Gautham initiated callusing within 15 days. Nandini and Retna responded within 13 days (Table 4, fig 6). Internode of Vardhini initiated callusing within 16 days. Gautham and Nandini responded within 14 days and Retna within 17 days (Table 4, fig 7). Root tip of Vardhini initiated callusing within 15 days. Gautham in 16 days, Nandini in 13 days and Retna in 14 days (Table 4, fig 8). Ovary of all varieties responded within 18-19 days. Anther explant of all varieties responded within 13 days. Ovule from immature seed as explant responded within 22 days of all varieties (Table 5, fig 9). Stigmata of variety Vardhini responded within 24 days and Gautham responded in 23-24 days of incubation. Nandini and Retna responded within 22 and 23 days respectively (Table 4, fig 12). Pith explant in Vardhini, Retna, and Nandini showed response within 14 days and Gautham responded in 17 days. Shoot tip as explant in all varieties responded within 13 days of incubation (Table 6, fig 14).
5.4.1.3. Relation between Callus colour and Plant Regeneration
Cream colored calli was observed in explants such as young leaf, shoot tip, petiole, ovary, anther, ovule and stigma of the sweet potato varieties : Sree Vardhini, Gautham, Sree Nandini and Sree Retna (Table 4, 5, 6, fig 8-14). Pale yellow colored calli was observed in explants : mature leaf, internode, and pith of stem of the sweet potato varieties Sree Vardhini, Gautham, Sree Nandini and Sree Retna. Younger parts of plant showed cream colored callus and continued dividing throughout the experiment. But mature part of plant in callus showed pale yellow color and they became older cells and appeared brown in color. So it may be inferred that a young part of the plant that has rapidly dividing cells responds faster in plant regeneration than mature parts.
5.4.1.4. Plant regeneration from callus Plant regeneration was observed in the callus developed from Sree Retna only. It was observed that within 20 days, plantlets regenerated from young leaf, petiole and mature internode. Plants regenerated from internode developed into mature plants within 30 days (Fig 15, 16). Organ regeneration was observed in Sree Nandini. In Sree Nandini only root development was observed from shoot tip (Fig 18).
25
Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) TDZ (N phenyl 1, 2, 3 thidiazol 5yl) urea has been used extensively in tissue culture studies. It exhibits strong cytokinin like activity and promotes the proliferation of axillary shoots as well as stimulated adventitious organ regeneration and induces somatic embryo genesis. Achievement of crop improvement through plant cell and tissue culture techniques depends upon success in plant regeneration.
GRAPH 3:
Response of Different Varieties in S1 medium

Vardhini Nandini Retna Gautham
20%
25%
17% 33%
FIGURE 3 : EXPLANTS IN S1 (INDUCTION) MEDIUM
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Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) Table 4: RESPONSE OF DIFFERENT EXPLANTS) IN CALLUSING IN S2 MEDIA A. Vegetative parts
Variety Young Leaf Callus colour Vardhini Callus initiatio n After 13 Cream days Pale
Response of Explants Mature leaf Callus colour Callus initiatio n After 15 days Cream After days 12 Pale Yellow colour After 14 days Cream After days 13 Pale Yellow colour After 15 days Cream After days 15 Pale Yellow colour After 15 days Cream After days 13 Pale Yellow colour After 13 days Cream After days 13 Pale Yellow colour After 13 days Cream After days 13 Pale Yellow colour After 13 days Cream After days 13 Pale Yellow colour After 13 days Crea m Responded Shoot length 3.9 cm after 20days of inoculation After 13 days Pale Yellow colour Responded Shoot length After 17 days Crea m After 14 days After 17 days Cream After 14 days After 14 days Cream After 13 days After 14 days Cream After 13 days After 14 days Cream After 16 days After 14 days Cream After 16 days After 16 days Cream After 15 days Young Petiole Callus colour Callus initiation Mature Internode Callus colour Callus initiatio n After 16 days Cream Root tip Callus colour Callus initiatio n After 15 days
Yellow colour
After 13 Cream days
Pale Yellow colour
After 16 Cream Gautham After 16 Cream days days
Pale Yellow colour Pale Yellow colour
After 13 Cream Nandini After 13 Cream days days
After 14 Cream Rethna After Cream 14 days Regener ation of plant in S2 No response days
Responded Shoot length
No response
4.2cm after 20days of inoculation
3.2cm after 30days of inoculation
medium (Retna only respond ed)
27
28
Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) Response of Various explants in S2 Medium
Graph 4:
Response of Vegetative Parts in Callusing In S2 Medium

20 15 10 5 0
VARDHINI Gautham Nandini
Retna
A. Vegetative organs
Figure 4: Young Leaf
Figure 5: Mature Leaf
29
Figure 6 : Young Petiole
Figure 7: Mature Internode
Figure 8: Root Tip
TABLE 5: RESPONSE OF DIFFERENT EXPLANTS IN CALLUSING IN S2 MEDIA
30
Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) A. Floral organs
Response of different explants Variety Ovary Callus colour Vardhini After Cream days After Cream days After Cream Gautham Cream days After days After Cream Nandini Cream days After days Cream After days Retna Cream After days Regene ration of plant in S2medi um No response No response No response No response 18 Cream 18 Cream 19 Cream 19 Cream 18 Cream 19 Cream 19 Cream 19 Cream After days After days After days After days After days After days After days After days 13 Cream 13 Cream 13 Cream 13 Cream 13 Cream 13 Cream 13 Cream 13 Cream After 22 days After 22 days After 22 days After 22 days After 22 days After 22 days After 22 days After 22 days Cream Cream Cream Cream Cream Cream Cream Cream Callus initiation Anther Callus colour Callus initiation Ovule Callus color Initiatio n Stigma Callus colour Callus initiatio n After 24 days After 22 days After 24 days After 23 days After 22 days After 22 days After 23 days After 23 days
31
Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas) Graph 5:
Response of floral organs in Callusing

25 20 15 10 5 0 OVARY ANTHER VARDHINI Gautham Nandini Retna
OVULE
STIGMA
B. Floral organs
Figure 9 : Ovary
Figure 10: Anther
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Figure 11: Ovule
Figure 12: Stigma
TABLE 6: RESPONSE OF DIFFERENT EXPLANTS IN CALLUSING IN S2 MEDIA A. Pith and Shoot tip
Response of explant Varieties Pith Callus colour Pale Yellow colour Vardhini Pale Yellow colour Pale Yellow colour After 14 days After 17 days Cream Cream After 16 days After 16 days Callus initiation After 14 days Shoot tip Callus colour Cream Callus initiation After 16 days
Gautham
Pale Yellow colour Pale Yellow colour Pale Yellow colour Pale Yellow colour
After 17days After 14 days After 14 days After 14 days After 14 days
Cream Cream Cream Cream Cream
After 16 days After 16 days After 16 days After 16 days After 16 days
Nandini
Retha Pale Yellow colour Regeneration of plant in HM1medium No response Root was well developed in Nandini after 20 days.
33
Graph 6:
Response of Pith and Shoot tip in Callusing
20 15 10 5 0 VARDHINIGautham Nandini Retna PITH SHOOT TIP
Figure 13: Shoot tip
igure 14: Pith
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Plant regeneration from Callus Variety Retna
Figure 15 :Plant regeneration from Mature Leaf
Figure 16 : Plant regenerated from Young Petiole
Figure 17: Plant regeneration from Mature Internode
Figure 18: Root development from Variety Nandini
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5.5 IN VITRO CONSERVATION (SLOW GROWTH CULTURE)
Sweet potato slow growth cultures were done in SG 3 medium.
5.5.1. Studies on SG3 medium In SG3, the separated nodes of four different accessions were inoculated in such a way that each accession had five replications. Studies involving slow growth culture of nodes of four different accessions of sweet potato genotypes revealed different growth responses, which were interpreted in the table given below. 5.5.1.1. Varietal response of sweet potato In all the accessions, the initial response was observed after one of the three inoculations. The best response was observed in genotype Thripthi (graph 7), which attained maximum shoot length of 2.1cm and also had well developed root system; while low response was recorded in accession Sourin( graph 7). i.e., 1.2 cm length of shoot after one month of inoculation. The best response was observed in genotype Thripthi which attained a maximum mean root length of 3.2cm after 30 days. The mean root length of genotype Sourin was 2.1cm and that of Kishan (graph 7) was 1.83cm and genotype IC440221 (graph 7) was 1.08 cm (table 7, graph 7, Fig 19). The culture remained dominant for up to three weeks. After 23 days, shoot development was seen. Therefore it was further observed for one more week. Slow-growth of plantlets in vitro provides an attractive alternative to freeze
36
Table 7: Growth response of sweet potato in slow growth (SG3) medium Observation After 15 Days Variety No: of replica No: of Leaves No: of Nodes Mean shoot length(cm) Mean root length (cm) 1 2 Thripthi 3 4 5 1 2 Sourin 3 4 5 1 2 3 4 5 1 2 IC440221 3 4 5 No Response No Response No Response No Response 2 3 3 1 1 2 2 1 1 2 2 1 2 1 2 1 1 1 2 3 3 1 1 2 2 1 1 2 2 1 2 1 2 1 1 1 1.2 2.1 1.8 1.6 0.7 1.2 0.9 1 1.2 2.2 1.2 0.6 1.4 0.8 1.7 1.8 0.6 0.9 2 3.2 4 3 2.1 2 1.4 2 2.6 2.5 1.3 1 1.8 2.5 2 2.7 1.2 2 1 2 1 2 1.2 1.9 Observation After 30 Days No: of Leaves No: of Nodes Mean shoot length(cm) Mean root length (cm) 1.8 2.5
Kishan
preservation of germplasm as it is simpler, cheaper and very effective. Slow growth may be achieved by maintaining the plantlets either at a low temperature or on a medium having high osmotic concentration (Mannitol 20%) or both. In addition, the nutritional status of the medium may be lowered to restrict the growth of plantlets. Under the conditions of slow-growth, cultures may be attended to only once in several months, and subculture may, be necessary only after long periods say, once every 12-36 months.
In the present study, Thripthi gave best response. Due to high osmoticum, the explants of all the four varieties remained dormant for two weeks after that growth was observed
37
Graph 7:
Growth response of Different Varieties of Sweet Potato in Slow Growth medium

3 2 1 0 Thripthi Sourin Kishan
440221 Root length
No of leaves
No of nodes
Shoot length
Figure 19: In Vitro Conservation (Slow Growth Medium)
38
5.DISCUSSION
The result of the present study was significant for rapid propagation of diverse sweet potato genotypes for obtaining genetically stable propagules. Here in five different sweet potato accessions healthy clones were obtained. Though the aseptic manipulation and procedure of sweet potato meristem culture was difficult, results of the experiments were comparatively good rather than nodal cultures of sweet potato. Once a pathogen free culture had been established new batch of cultures for propagation could be started with shoot tip culture because they responded rapidly and readily. The most important application of meristem culture was to produce pathogen free plants, which were genetically identical. The totipotency of the apical meristem cells forms the basis of the meristem culture technique. The major advantages of meristem culture are that it provides:
Clonal propagation in vitro with maximal genetic stability. The potential for removal of viral, bacterial, and fungal pathogens from donor plants. The meristem tip as practical propagules for cryopreservation and other techniques of culture storage. A technique for accurate micropropagation of chimeric material. Cultures those are often acceptable for international transport with respect to quarantine regulations.
The maintenance of aseptic conditions was necessary for obtaining contamination free cultures. Besides these factors, temperature, humidity and light intensity also played an important role in micro propagation and meristem culture. Therefore these techniques could be used as an effective Biotechnological tool for the crop improvement.
39
6. SUMMARY
The results of the present study were highly significant for rapid propagation of diverse sweet potato genotypes for obtaining genetically stable propagules. The nodal culture, meristem culture as well as plant regeneration of sweet potato was successfully developed in Murashige and Skoog basal media and modified MS media with Plant growth regulators such as NAA, GA3,BAP,, 2,4 -D and TDZ. Mercuric chloride (0.1%) was used as surface sterilant which gave a way to reduce contamination.
Inclusion of activated charcoal in the medium helped to absorb phenolic compounds and also to increase the culture viability. The maintenance of aseptic condition was necessary for obtaining contamination free cultures. Besides these factors, temperature, humidity and light intensity was found to be important in micro propagation and meristem culture. Slow cultures provided an ideal method for germplasm conservation.
Plant regeneration is the process of growing an entire plant from a single cell or group of cells due to the influence of plant growth regulators. Plant regeneration through somatic embryogenesis shows several advantages as compared to other in vitro propagation systems, including its high multiplication rates, possibility of cryopreservation of embryogenic callus, the potential for scale-up in liquid suspension cultures, the use of bioreactors and somatic synthetic seed technologies and the fact that embryogenic cultures are suitable target tissues for gene transfer.
Therefore these techniques can be used as an effective Biotechnological tool for the crop improvement for the production of new traits and varieties having desirable characters such as high yield, resistance to diseases etc.
40
7. Conclusion Sweet potato is an edible tuber with high nutritional profile. It has high starch content and dietary fiber content. In most parts of Africa and in some parts of Latin America, Sweet potato has assumed the importance of a staple food. So it is important to have effective propagation and conservation tools to utilize this high profile tuber crop to the maximum extent. Here in this study, micropropagation and plant regeneration of sweet potato have been attempted. Meristem culture carried out here was proved effective as it produced pathogen-free plants. Nodal culture also came up with flying colours as the inclusion of charcoal in the culture medium removed the excess phenolic compounds liberated into the medium by the plantlets. Also it excluded the light from the medium thus inducing rooting in plantlets. Thus nodal culture emerged as a promising vegetative propagation tool for sweet potato. Plant regeneration through somatic embryogenesis also has given promising signs as an effective propagation method As the dietary needs of the people around the world is soaring high, the dependence on tuber crops like Sweet potato is expected to increase. Also serious research has been invested behind exploring the functional values and value added products from Sweet potato. Hence the present study assumes paramount significance and relevance as it sheds light into the conservation and propagation of a crop which can prove beneficial to the growing dietary and health demands of the growing populations across the globe especially in India and African countries.
41
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