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Studies on Micropropagation and Plant regeneration of Sweet Potato (Ipomoea batatas)

1

Studies on Micropropagation and Plant regeneration of

Sweet Potato

(Ipomoea batatas)

Mamatha M Pillai

M Unnikrishnan ( Principal scientist )

CTCRI Trivandrum


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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 toDr. Winny Varghese, Principal, Mar Athanasius College, Kothamangalam

for his sincere encouragement in conducting the study.

I record my heartful gratitude toDr. Yamuna Anu Joseph, Head of the Department of Biotechnology, MarAthanasius 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 andFriends for their consistent encouragement and unfailing help

rendered to me without whose help this work would not have been possible.

I am thankful toEach andEveryone 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


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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 exportcommodities 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.


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CONTENTS

1. INTRODUCTION .. 42. OBJECTIVES 83. REVIEW OF LITERATURE 94. MATERIALS AND METHODS 115. RESULTS .196. DISCUSSION .. 387. SUMMARY .. 98. BIBLIOGRAPHY 40


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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.


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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 ofa 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
http://en.wikipedia.org/wiki/Dicotyledonoushttp://en.wikipedia.org/wiki/Convolvulaceaehttp://en.wikipedia.org/wiki/Starchhttp://en.wikipedia.org/wiki/Tuberous_roothttp://en.wikipedia.org/wiki/Root_vegetablehttp://en.wikipedia.org/wiki/Root_vegetablehttp://en.wikipedia.org/wiki/Tuberous_roothttp://en.wikipedia.org/wiki/Starchhttp://en.wikipedia.org/wiki/Convolvulaceaehttp://en.wikipedia.org/wiki/Dicotyledonous


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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 OFIN 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 cleanup. Permanent availability of (when pathogen tested) material for exchange and multiplication of disease free

planting material.

omoea batatas)


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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.


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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 ofin 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).


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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 Dand 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.

VitaminsTo 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 B1), 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 0.1 M 0.1 M - - -

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 charcoalActivated 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 solution

code No.

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)

82.5g

95.0g

18.5g

8.5g

20.0 ml

(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)

0.62 g

2.176g

0.86g

0.025g

0.0025g

0.0025g

1.0 ml

(3)

C

KI


0.075g 1.0 ml

(4)

D

CaCl2.2H2O

(Dissolve in 100ml Distilled water)

15.0g 2.9ml

(5)

E

a) Na2 EDTA( Chelating agent)

b) Fe SO4. 7 H2O


1.492g

1.114g

5.0ml

(6)

F

Vitamins

Thiamine. HCl

10mg 5.0 ml

(7)

G

myo-inositol

(Dissolve in 200ml of distilled water)

0.8g 6.25 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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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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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.

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.

Concentration in g/l M /l

MS Sucrose Agar NAA BA GA3

I bottle 30 8 0.1M 0.1M 0.1M


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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.


Nodal culture was tried in two Murashige and Skoogs (MS) basal media, denoted as HM1.

The composition of MS basal media used here was;

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.

Concentration in g/l

MS Sucrose Agar Charcoal CaCl2

I bottle 30 8 1 2.9ml


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Two combinations of media were used for callus culture in sweet potato. These include Murashige and

Skoogs medium supplemented with 2, 4D denoted as S1i.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;


MS Sucrose Agar 2,4-D CaCl2

I bottle 30 8 0.2mg/l 2.9ml

The composition of S2 used here was;


MS Sucrose Agar TDZ CaCl2

I bottle 30 8 2ml 2.9ml

4.7.3. Inoculation and Incubation:

Small pieces of explants were inoculated on to freshly prepared sterile medium1 (S1) 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 25oC 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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Nodal culture was tried in Murashige and Skoogs (MS) basal media, denoted as SG3. The composition of

MS basal media used here was;

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).

Concentration in g/l Concentration in M/l

MS Sucrose Agar Mannitol NAA BAP GA3

I bottle 20 8 20 0.5 0.1 0.3


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5. RESULTS

5.1. MERISTEM CULTURE

Sweet potato Meristem cultures were done in HM2 medium.

5.1.1. Studies on HM2 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.

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.

Name

of

variety

Sl no: No: of

cultures

inoculated

No: of

cultures

obtained

Observation

after 15 days

Observation

after 30 days

Remarks

S.823 1 6 4 No response Enlargment,

green

coloration

Medium

response

S.23 2 8 6 Green

coloration

Development

of buds

Fast

response

Sree

arun

3 6 5 No response Green

coloration

Medium

response

S.665 4 7 4 No response Green

coloration

Medium

response

S.685 5 6 4 No response Green

coloration

Medium

response


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GRAPH 1: MERISTEM CULTURE IN DIFFERENT VARIETIES OF SWEET POTATO

FIGURE 1: MERISTEM CULTURE

0 10 20 30 40 50 60

SreeArun

S.665

S.23

S. 685

% of Growth


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5.2. NODAL CULTURE.

Sweet potato nodal culture was tried in Murashige and Skoogs basal media (HM1).

5.2.1.. Studies on HM1 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 numberof 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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Table2: OBSERVATION OF NODAL CULTURE IN DIFFERENT VARIETIES OF SWEET POTATO

Variety No: of

replica

Observation After 15 Days Observation After 30 Days

No: of

Leaves

No:

of

Nodes

Mean

shoot

length(cm)

Mean

root

length

(cm)

No: of

Leaves

No:

of

Nodes

Mean

shoot

length(cm)

Mean

root

length

(cm)

S.16

1 1 1 0.8 2.5 5 5 1.6 4

2 2 2 1.7 2 7 7 2.8 5

3 1 1 1.8 2.7 4 4 3 4.2

4 1 1 0.6 1.2 3 3 1.8 4.4

5 1 1 0.9 2 2 2 1.9 3.4

Gautham

1 1 1 1.2 1.8 6 6 2.6 5.2

2 3 3 1.9 2.5 4 4 2.3 4.8

3 2 2 1.2 2 4 4 1.6 3.2

4 3 3 2.1 3.2 6 6 5.8 5.5

5 3 3 1.8 4 5 5 2.8 4.2

Kishan

1 1 1 1.6 3 4 4 3.2 4.8

2 1 1 0.7 2.1 2 2 1.9 3.2

3 2 2 1.2 2 3 3 1.6 4.3

4 2 2 0.9 1.4 4 4 1.8 2.8

5 - - 0.4 2 2 2 2.8 3.8

Sree

Rethna

1 1 1 1.2 2.6 2 2 2.4 3.5

2 2 2 2.2 2.5 3 3 1.3 3.6

3 - - 0.7 1.3 1 1 2.4 4

4 1 1 0.6 1 3 3 2.6 3.2

5 - - 0.4 1.8 2 2 0.9 3

Sree

Bhadra

1 1 1 1.5 2.3 4 4 1.6 2.8

2 1 1 0.7 1.6 6 6 2.8 4

3 2 2 1.6 2.8 3 3 2.2 3.5

4 1 1 0.8 1.9 2 2 1.2 2.5

5 3 3 1.6 2.5 4 4 3.1 3.5


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GRAPH 2: NODAL CULTURE IN DIFFERENT VARIETIES OF SWEET POTATO

FIGURE 2: DIFFERENT STAGES OF NODAL CULTURE

0

1

2

3

4

5

No of leaves

No of nodes

Shoot length

Root length


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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.

TABLE 3: RESPONSE OF DIFFERENT VARIETIES IN CALLUSING OF SWEET POTATO IN S1 MEDIA

Variety Response

Vardhini

Good response within 6 days and

inoculated into S2 medium

Nandini



Rethna

Very Good response within 4 days and


Gautham




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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).

Pithexplant 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).


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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:

FIGURE 3 : EXPLANTS IN S1 (INDUCTION) MEDIUM

25%

17%

33%

20%

Response of Different Varieties in S1

medium

Vardhini Nandini Retna Gautham


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27

Table 4: RESPONSE OF DIFFERENT EXPLANTS) IN CALLUSING IN S2 MEDIA

A. Vegetative parts

Variety Response of Explants

Young Leaf Mature leaf Young Petiole Mature Internode Root tip

Vardhini

Callus

colour

Callus

initiatio

n

Callus

colour

Callus

initiatio

n

Callus

colour

Callus

initiation

Callus

colour

Callus

initiatio

n

Callus

colour

Callus

initiatio

n

Cream

After 13

days

Pale

Yellow

colour

After 15

days

Cream After 12

days

Pale

Yellow

colour

After 16

days Cream

After 15

days

Cream

After 13

days

Pale

Yellow

colour

After 14

days

Cream After 13

days

Pale

Yellow

colour

After 16

days Cream

After 15

days

Gautham

Cream

After 16

days

Pale

Yellow

colour

After 15

days Cream

After 15

days

Pale

Yellow

colour

After 14

days Cream

After 16

days

Cream

After 16

days

Pale

Yellow

colour

After 15

days Cream

After 13

days

Pale

Yellow

colour

After 14

days Cream

After 16

days

Nandini

Cream

After 13

days

Pale

Yellow

colour

After 13

days Cream

After 13

days

Pale

Yellow

colour

After 14

days Cream

After 13

days

Cream

After 13

days

Pale

Yellow

colour

After 13

days Cream

After 13

days

Pale

Yellow

colour

After 14

days Cream

After 13

days

Rethna

Cream

After 14

days

Pale

Yellow

colour

After 13

days Cream

After 13

days

Pale

Yellow

colour

After 17

days Cream

After 14

days

Cream

After

14

days

Pale

Yellow

colour

After

13

days

Crea

m

After 13

days

Pale

Yellow

colour

After 17

days Crea

m

After

14

days

Regener

ation

of plant

in S2

medium(Retna

only

respond

ed)

No response

Responded

Shoot length

4.2cm after 20days

of inoculation

Responded

Shoot length 3.9

cm after 20days of

inoculation

Responded

Shoot length

3.2cm after 30days

of inoculation

No response


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29

Response of Various explants in S2 Medium

Graph 4:

A. Vegetative organs

Figure 4: Young Leaf Figure 5: Mature Leaf

0

5

10

15

20

Response of Vegetative Parts in Callusing In

S2 Medium

VARDHINI

Gautham

Nandini

Retna


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30

Figure 6 : Young Petiole Figure 7: Mature Internode

Figure 8: Root Tip

TABLE 5: RESPONSE OF DIFFERENT EXPLANTS IN CALLUSING IN S2 MEDIA


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31

A. Floral organs

Variety

Response of different explants

Ovary Anther Ovule Stigma

Vardhini

Callus

colour

Callus

initiation

Callus

colour

Callus

initiation

Callus

color

Initiatio

n

Callus

colour

Callus

initiatio

n

Cream

After 19

days

Cream After 13

days

Cream After 22

days

Cream After 24

days

Cream

After 19

days

Cream After 13

days

Cream After 22

days

Cream After 22

days

Gautham

Cream

After 19

days

Cream After 13

days

Cream After 22

days

Cream After 24

days

Cream

After 18

days

Cream After 13

days

Cream After 22

days

Cream After 23

days

Nandini

Cream

After 19

days

Cream After 13

days

Cream After 22

days

Cream After 22

days

Cream After 19

days

Cream After 13

days

Cream After 22

days

Cream After 22

days

Retna

Cream After 18

days

Cream After 13

days

Cream After 22

days

Cream After 23

days

Cream After 18

days

Cream After 13

days

Cream After 22

days

Cream After 23

days

Regene

ration

of plant

in

S2medi

um

No response No response No response No response


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Graph 5:

B. Floral organs

Figure 9 : Ovary Figure 10: Anther

0

5

10

15

20

25

OVARYANTHER

OVULESTIGMA

Response of floral organs in Callusing

VARDHINI

Gautham

Nandini

Retna


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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

Varieties

Response of explant

Pith Shoot tip

Callus colour Callus initiation Callus colour Callus initiation

Vardhini

Pale Yellow colour After 14 days Cream After 16 days


Gautham


Pale Yellow colour After 17days Cream After 16 days

Nandini



Retha



Regeneration

of plant in HM1medium

No response

Root was well developed in Nandini

after 20 days.


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Graph 6:

Figure 13: Shoot tip igure 14: Pith

0

5

10

15

20

VARDHINIGautham

Nandini Retna

Response of Pith and Shoot tip in Callusing

PITH

SHOOT TIP


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Plant regeneration from CallusVariety Retna

Figure 15 :Plant regeneration from Mature Leaf Figure 16 : Plant regenerated from

Young Petiole

Figure 17:Plant regeneration from Figure 18: Root development from Variety

Mature Internode Nandini


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5.5IN VITRO CONSERVATION (SLOW GROWTH CULTURE)

Sweet potato slow growth cultures were done in SG3 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 bestresponse 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


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37

Table 7: Growth response of sweet potato in slow growth (SG3) medium

Variety No: of

replica

Observation After 15 Days Observation After 30 Days

No: of

Leaves

No: of

Nodes

Mean shoot

length(cm)

Mean

root

length

(cm)

No: of

Leaves

No: of

Nodes

Mean shoot

length(cm)

Mean

root

length

(cm)

Thripthi

1

No Response

1 1 1.2 1.8

2 2 2 1.9 2.5

3 2 2 1.2 2

4 3 3 2.1 3.2

5 3 3 1.8 4

Sourin

1

No Response

1 1 1.6 3

2 1 1 0.7 2.1

3 2 2 1.2 2

4 2 2 0.9 1.4

5 1 1 1 2

Kishan

1

No Response

1 1 1.2 2.6

2 2 2 2.2 2.5

3 2 2 1.2 1.3

4 1 1 0.6 1

5 2 2 1.4 1.8

IC440221

1

No Response

1 1 0.8 2.5

2 2 2 1.7 2

3 1 1 1.8 2.7

4 1 1 0.6 1.2

5 1 1 0.9 2

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 fourvarieties remained dormant for two weeks after that growth was observed


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38

Graph 7:

Figure 19: In Vitro Conservation (Slow Growth Medium)

0

1

2

3

ThripthiSourin

Kishan440221

Growth response of Different Varieties of Sweet

Potato in Slow Growth medium

No of leaves No of nodes Shoot length Root length


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39

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.


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40

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, GA 3,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.


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41

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.


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