INTRODUCTION AND REVIEW OF LITERATURE

1.1. INTRODUCTION

Sweet potato (Ipomoea batatas (L). Lam), is a member ofthe morning

glory family, Convolvulaceae, and the only member of the genus
Ipomoea whose roots are edible. It is speculated to be a native of
South America but presently grown throughout the tropical and
subtropical regions ofthe world.

Sweet potatoes form a large part ofthe food ofthe people in many
countries and is the sixth most important food crop of the world with
an annual production of about 126.19 million tonnes from 9.26 million
hectares (FAO1992).It can grow under a wide range of agroecological
conditions from 40°north to 32° south latitudes and from sea level up
to 9000 ft elevations. The major growing areas occur where the
average temperature is about 23.8° C or more with a well distributed
annual rainfall of 500 to 700 mm and anabundance of sunshine. The
crop growth cycle is 3 to 4 months under congenial conditions.
Because ofits wide adaptability and high yield potential and high dry
matter production per unit area per unit time, sweet potato
cultivation is gaining worldwide importance day by day as base
material for1food, feed and industrial products. To meet the required
demand, foodproduction need to be doubled by 2020 just to maintain
present per capita food consumption (2.6 billion tonnes for 8.5 billion
people). To achieve this target of food production, sweet potato can
play a significant role to secure foods for future.
Theproduction gap of other food crops in a country like ours can be
filled up by proper exploitation of yield potential of sweet potato.
However, low productivity ofsweet potato is a major hurdle towards
achieving the goal.

The major causes of low productivity in many countries identified are:

(i) non-availability of high yielding as well as disease and pest tolerant

varieties, (ii) shortage of healthy planting materials of high yielding
varieties resulting from perpetuation and transmission of diseased
propagules through continuous cycles of vegetative propagation, and
(iii) lack of varieties tolerant to abiotic stresses. All these account for
reduction in yield about 70-80%. The major objectives ofsweet potato
research are thus the area of improvements interms of yield, storage
ability, nutritional composition, culinary properties,

disease and insect resistance. Despite the fact that considerable work
have

been carried out in sweet potato research, very little progress has
been made in

those areas as compared to other crops. Much of this stagnation can

be

attributed to the limited genetic base that most breeders have used.
Genetic improvement of sweet potato by conventional methods has

been associated with the genetics of polyploidy. Sweet potato is a

hexaploid

crop. Though sexual breeding in sweet potato was initiated in early of

this

century (Stout 1926; Tioutine 1935), the cause of poor seed set i.e.
selfincompatibility was understood much latter (Martin 1965; 1967).
Further

studies indicated that incompatibility in sweet potato is a complex

phenomenon of multiallelic nature and can be explained cytologically

with

bivalent pairing and segregation on the basis of multifactor models

(Jones
1965; 1969). The other studies show existence of continuous and
overlapping4variation towards the quantitative inheritance for all the
characters in sweet

potato. Tuber yield is the most complex and variable character.

Heritabilty

estimate for tuber yield was low indicating non-additive variance

(Jones

1977). The studies of Zheng et al. (1985) indicated that red skin colour
was

controlled by additive genes. Hernandez et al. (1967) and Zhang and

Xie

(1988) reported a negative association between flesh colour and dry

matter.

Starch and dry matter ofthe tuber were governed by additive gene
effects (Dai
et al. 1988). Genetic studies have concluded that resistance to most of
the

diseases is quantitative and the variability is due to the outcrossing

behaviour.

Most ofthe morphological characters do not have any stable

correlation with

the tuber yield. Sometimes the genotypic and environmental

variations may

affect the characters through physiological mechanisms (Vimala and

Nair

1988).

Despite the heavy breeding emphasis on yield increase, disease and

insect resistance, progress through classical breeding is impeded due

to the
major constraints like incompatibility and sterility associated with
hexaploidy

as mentioned before. In addition to that, a narrow genetic base

resulting from

continuous vegetative mode of propagation is also affecting the

productivity.

Though some high yielding varieties have been developed

conventionally,

high disease severity and pest attacks during field propagation and
unhealthy

propagules due to poor maintenance are the major constraints. Thus

it

becomes imperative to opt for novel techniques for propagation and

improvement ofthis polyploid and clonally propagated crop sweet

potato. In
this context, biotechnological tools - tissue culture techniques in
particular can

play a major role in overcoming the problems associated with those

classical

mode of propagation and improvement. Meristem and shoot tip

culture

techniques are very useful in developing virus free materials. In vitro

micropropagation and storage techniques can be used effectively to

maintain a

large collection of genetically pure material in a relatively small area

as

7breeders’ active collection. Approximately 10 % ofthe consumable

product is

often used to provide healthy propagules to maintain the continuous

cycle of
planting and replanting of this vegetatively propagated crop. Tissue
culture

techniques could save a large portion of this expense both in storage

and

bedding so also ensure safer storage against diseases and pests in the
field

(Henderson et al. 1984). Micropropagation can be achieved either

through

shoot organogenesis from pre-formed meristems or through de novo

organogenesis and somatic embryogenesis. In vitro methods have

been

developed for propagation ofsome popular varieties and also for

conservation
and exchange of genetic resources in sweet potato (Love et al. 1987;
Dodds et

al. 1992). However, wide application of in vitro propagation protocols

is still

being hindered due to low regeneration frequencies resulting from

influence of

genotypes and long duration of cultures (Kuo 1991; Prakash et al.

1993).

Information on genetic stability ofregenerants during maintenance is

lacking.

Such knowledge is essential to exploit in vitro techniques for storage

and

propagation of sweet potato. A reliable and efficient in vitro

regeneration
system is an absolute prerequisite for production of genetically
transformed

plants. This has always been a problem in sweet potato (Hill et al.
1992).

Moreover, regeneration from tissues has been reported mostly

through a callus

phase, which is likely to affect genetic stability of the regenerated

plants.

Informations on cytogenetic situations ofthe regenerants are lacking

which is

deemed desirable for genetic manipulation. More recently,

development of

transgenics and global issues on patenting genes are causing more

concern for
the development of efficient propagation and storage protocols not
only to

cater present food demand but also to secure food for future. Work on

regeneration protocols may alleviate the problems or at least raise

regeneration

rates to acceptable levels. Knowledge on cyto-histo and morphological

changes associated with regeneration would also help to develop and

maintain

a highly stable regenerating system monitoring the process of

regeneration at

cell level.11

1.2. Review ofliterature

In vitro propagation of sweet potato (Ipomea batatas) has not been as

successful as other food and vegetable crops. However, several
reports on

tissue culture studies in sweet potato have appeared in different

journals in

recent years which mainly covers a) regeneration of plants by

embryo/ovule,

shoot tip, lateral bud and meristem cultures; b) callus induction in

tissue

explants followed by regeneration through organogenesis and somatic

embryogenesis; c) protoplast culture and genetic transformation

experiments;

as well as d) in vitro conservation and cryoconservation aspects. Some

authors

like Henderson et al. (1984) and Torres (1989) have tried to review the
progress in tissue culture studies of sweet potato. However, a review
of the

literature available on the recent work on sweet potato tissue culture

has been

attempted in the following sections.

1.2.1. In vitro culture of meristems, axillary buds and shoot tips for
virus

elimination and micropropagation.

Viral diseases often affect the genetic resources ofsweet potato, and
the

spread of such diseases, through vegetative propagation, affects

productivity

and storage. Normally the apical meristem (0.3-0.5 mm) ofthe plant
infected
with virus is almost disease free. Meristem and shoot tip culture
techniques

coupled with thermotherapy and chemotherapy can eliminate viruses.

Hence

most of the tissue culture experiments conducted so far with sweet

potato

meristems, shoot tips and lateral buds were aimed primarily at

elimination of

virus from different cultivars; and once this was achieved the
experiments

were, however, directed towards in vitro production of sweet potato

propagules. The best-known work in this direction is probably that of

Mori

(1971) who was able to establish virus-free meristem cultures of

several
species, including the sweet and white potato. He was also able to
eliminate

15three ofthe most prevalent and damaging viruses from the plants
infected with

internal cork, rugouse mosaic and feathery mottle viruses. Prior to

Mori’s

work, however, Elliot (1969) used excised meristem tips from the cv.
Kumara

and observed that the length of the explant influenced the initiation
of roots

and subsequent development ofthe plantlets. MS medium

supplemented with

various growth regulators and auxins accelerated the growth and

development
ofshoot, root and eventually plantlet production (Henderson et al.
1984).

Alconero et al. (1975) regenerated complete plants from shoot tips

(0.4-

0.8mm long) of axillary shoots of 10 cultivars of sweet potato. Use of

NAA

(5.4 pM) could shorten the time necessary to produce complete plants
from

meristem tips. Litz and Conover (1978) propagated two varieties of

whitefleshed sweet potato (White Star and PI 315343) by using
explants from shoot

tips and axillary buds grown on MS medium containing BA, IAA, Kn,
and

activated charcoal. Optimum shoot regeneration from White Star

explants was
induced by BA (4.4pM) and from PI 315343 using Kn (4.6 pM) and IAA
(5.7

pM).

Production of virus-free plants by a combination of heat treatment

and

shoot tip culture (Liao and Chung 1979) or by meristem culture (Frison
1981)

have been reported in sweet potato. For heat therapy regenerated

plants were

exposed to 38-42 0 C for 28 days prior to excision of the explants.

Later,

meristems were grown in modified MS medium containing 17.7-35.5

pM BA

and 5.7-11.0 pM IAA under a light intensity of 150 lux or below. The
developed shoots did not show any virus symptom on indexing on
Ipomoea nil

or Ipomoea setosa. Symptom free plants were multiplied by single

node

cuttings on MS medium (Frison 1981). Production of single shoot from

leaf

axil cultures on MS medium has also been reported (Sihachakr 1982)

Excised apical meristems devoid of leaf primordia (to avoid virus

carry-over) when cultured on MS medium supplemented with BA and

NAA

transferred subsequently to liquid medium on a filter paper bridge

provided

virus free plant. Plants were indexed for virus by grafting onto
Ipomoea setosa

seedlings (Henderson et al. 1984).

Alkhalifa and Chambliss (1985) evaluated different conventional and
in

vitro propagation procedures using 3 cultivars ofsweet potato.

Morphological

abnormalities were higher in all cultivars when propagated by tissue

culture

than those propagated by conventional methods. Plants propagated

from vine

cuttings produced the lowest percentage ofoff-type roots. Ng and

Hahn (1985)

suggested that in vitro propagation technique is the most advanced

application

of plant tissue culture, which provides a fast multiplication rate

generally
using node cuttings or shoot tips as explants. Shoot tip culture may be

effectively adapted to free improved varieties of sweet potato from

mosaic

virus.

Love and Rhodes (1985) improved the methods for meristem culture
of

sweet potato. The basic medium contained salt base ofthe MS with
0.7% agar.

An increase in sucrose concentration from the standard 30 g/1 to 50

g/1 resulted

in a marked increase in meristem survival and regeneration

percentage, and

reduced the average regeneration time. Optimum growth regulator

concentrations, over a wide array of genotypes, were determined to
be 0.3

mg/1 of BA and 0.03 mg/1 of NAA. Transfer to a growth regulator free

medium, following the formation of shoot primordia, sped up

regeneration.

Optimum pH of the nutrient medium was found nearer 5.2 than the
standard

5.7. Changes in MS salt concentration had no effect.

Ng (1986) reported the use of meristem culture and thermo-therapy

to

free sweet potato from a number of viral diseases such as mosaic virus
and21feathery-mottle virus. Excised apical and lateral buds were
cultured in MS

medium containing 0.2 ppm IAA and 0.5 ppm BA. Henderson (1987)
reported
the advantages of gelrite over agar for meristem culture and
propagation.

Gelrite is completely transparent and solidifies at concentrations as

low as

0.05%. However, one disadvantage of gelrite is that it requires a

certain level

of salt concentration, especially the cations of calcium and magnesium

for

proper gelling. As most culture media contain one or both ofthese

salts, this is

normally not a problem.

Green et al. (1989) reported meristem culture protocol of sweet

potato
to clean witches broom disease. A high percentage of stem tips
originating

from cuttings previously treated with 25, 50 and 100 mg/1 tetracycline
for 3

days developed into healthy plants. More than 90% ofthe plants
derived from

stem tips, which were excised from cuttings and had received 1-month
heat

treatment at 37° C remained symptomless for 9 months. Griffith and

Slack

(1990) cultured axillary buds from sweet potato (Ipomoea batatas cv.
Georgia

Red) infected with sweet potato-feathery-mottle-virus (SPFMV) on a

medium
supplemented with 20 g/1 ribavirin. Cultures were heat treated under
a4h

alternating 35° C light/31°C dark regime for 28 days, and were tested
for the

presence of virus using dot-blot ELISA. Their results indicated that

SPFMV

free plants could be obtained either by in vitro culture methods using

ribavirin

and heat, or by excising apical buds, which were rooted in vitro prior
to

transferring to the green house.

Ohkoshi (1991) has developed plant propagation system from

meristem

to eliminate viruses. The rate ofsuccess ofmeristem culture varied

among the
cultivars. Some effective culture medium components common to all
cultivars

were identified. Virus free planting stock gave an increased yield and
better

quality plants.

825

1.2.2. Ovule and zygotic embryo culture

Embryo endosperm incompatibility and seedling lethality are the

bottlenecks in interspecific crosses. Sterility at post fertilization stages

due to

seed non-viability and seedling lethality was reported in compatible

crosses
(Martin 1982) and in interspecific crosses ofsweet potato
(Wedderbum 1967;

Vijayabai and Hrishi 1977). Ovule and zygotic embryo culture

techniques can

help to overcome the problems associated with intervarietal and

interspecific

sterility at post fertilization stages.

Mukherjee et al. (1991a) reported recovery of plantlets from

immature

embryos produced 7 days after pollination in sweet potato. They

observed that

V,

GA3 was the most essential for initial morphogenesis of globular and
heart
shaped embryos, and recovered seedlings from late globular stage
onwards.

Multiple shoots were obtained from differentiated embryos on MS

medium

supplemented with 1 pM NAA, 2 pM BA and 0.5 pM GA3. Rojas and

Thompson (1991) reported in vitro fertilization and embryo rescue

techniques

for sweet potato to overcome the incompatibility between the

cultivars Regal

and MD-708. They reported the highest percentage of embryos

rescued on MS

medium. Establishment and propagation of exotic Ipomoea species

viz. I.

trifida , I. cairica, through in vitro seed germination techniques have

been
reported by Mukherjee and Vimala (1994). Wang and Lu (1996)
cultured

ovule to overcome incompatibility between A genome and B genome

in

section batatas of the genus Ipomoea. They could recover hybrid

plantlets

from/, triloba xI. batatas andI. triloba xl. littorilis.

928

1.2.3. Callus and cell suspension culture

Most of the culture work on the sweet potato starts with the

establishment of callus tissue either from small segments ofleaf, stem,

root or
from anthers (Henderson et al. 1984). For callus formation several
media

formulations like those ofWhite (1943), Gamborg et al.(1968),

Murashige and

Skoog (1962) and Linsmaier and Skoog (1965) are used but most
frequently

used medium is the MS medium (Murashige and Skoog 1962) or its

modifications.

Yamaguchi and Nakajima (1972) used a modified White’s medium to

produce callus and adventitious roots and buds. Medium was

supplemented

with 5 g/1 Difco yeast extract, and 146 mM sucrose. Callus cultured on
medium containing 5.4 pM NAA produced adventitious roots after 6
weeks.

Probably the earliest and most thorough research on callus culture

was carried

out by Gunckel et al. (1972) on the influence of polarity in the sweet

potato

tuber. They also studied the influence of a number of organic nutrient

variants

on root and shoot initiation in relation to callus production from the

explant.

Three cultivars (Yellow Jersey, Jersey Orange, and Centennial) were

cultured

on White’s basal salt medium in various combinations ofthe growth

regulator
supplements like IAA, NAA, 2,4-D, adenine sulfate, Kn, GA3, and
Coconut

water (CW). The amount of callus or number of roots formed

depended upon

tissue polarity, explant orientation, cultivar response, and culture

medium.

Hozyo (1973) also reported callus formation from 4 cultivars of sweet

potato

and the influence of medium supplements, especially 2,4-D and Kn.

Tsai and

Lin (1973 a, b) experimented with callus produced from the anthers

ofseveral

sweet potato cultivars. They used Blaydes (1966) medium. They

showed the
influence of IAA, 2,4-D, Kn, and several plant extracts, including CW.
The

most vigorous callus was obtained in Blaydes medium supplemented

with 2,4-

D, CW, and pea extract. The culture medium composition and cultural

3210

1.2.3. Callus and cell suspension culture

Most of the culture work on the sweet potato starts with the

establishment of callus tissue either from small segments ofleaf, stem,

root or

from anthers (Henderson et al. 1984). For callus formation several

media

formulations like those ofWhite (1943), Gamborg et al.(1968),

Murashige and
Skoog (1962) and Linsmaier and Skoog (1965) are used but most
frequently

used medium is the MS medium (Murashige and Skoog 1962) or its

modifications.

Yamaguchi and Nakajima (1972) used a modified White’s medium to

produce callus and adventitious roots and buds. Medium was

supplemented

with 5 g/1 Difco yeast extract, and 146 mM sucrose. Callus cultured on

medium containing 5.4 pM NAA produced adventitious roots after 6

weeks.

Probably the earliest and most thorough research on callus culture

was carried
out by Gunckel et al. (1972) on the influence of polarity in the sweet
potato

tuber. They also studied the influence of a number of organic nutrient

variants

on root and shoot initiation in relation to callus production from the

explant.

Three cultivars (Yellow Jersey, Jersey Orange, and Centennial) were

cultured

on White’s basal salt medium in various combinations ofthe growth

regulator

supplements like IAA, NAA, 2,4-D, adenine sulfate, Kn, GA3, and
Coconut

water (CW). The amount of callus or number of roots formed

depended upon
tissue polarity, explant orientation, cultivar response, and culture
medium.

Hozyo (1973) also reported callus formation from 4 cultivars of sweet

potato

and the influence of medium supplements, especially 2,4-D and Kn.

Tsai and

Lin (1973 a, b) experimented with callus produced from the anthers

ofseveral

sweet potato cultivars. They used Blaydes (1966) medium. They

showed the

influence of IAA, 2,4-D, Kn, and several plant extracts, including CW.
The

most vigorous callus was obtained in Blaydes medium supplemented

with 2,4-

D, CW, and pea extract. The culture medium composition and cultural
3611

growth. Tisch (1981) used cell suspension to study sulphur

metabolism in cv.

Red Jewel. Callus and cell suspension cultures developed by different

researchers for organogenic and embryogenic regeneration studies in

sweet

potato are dealt in respective sections ofreview.

1.2.4. Organogenesis/ organogenic regeneration

Organogenesis is the formation of individual organs such as shoots or

roots and is either adventitious or de novo in origin. Gunckel et al.

(1972) were

able to produce roots and shoots from sweet potato explants by a

combination
of auxins, Kn, adenine, and GA3 from which plantlets were recovered.

Yamaguchi and Nakajima (1972, 1973) were able to regenerate

plantlets by

varying eytokinins and ABA. In order to obtain adventitious roots from

freshly

harvested tubers, eytokinins (Kn and Zea) were needed in the medium
at

varying concentrations (0.5-5.0 pM). While eytokinins inhibited

adventitious

bud formation, abscisic acid (ABA) in combination with low

concentrations of

2,4-D stimulated adventitious bud formation in sweet potato tubers in

some

varieties. It was concluded that eytokinins play an important role in

organ
formation and that ABA have an antagonistic effect on endogenous
eytokinins

in sweet potato tissue as far as adventitious bud and root formation

are

concerned.

Sehgal (1975) was able to produce roots and shoots from leaf callus

tissue. Later he used anthers of sweet potato at various stages to

produce

profuse callus on MS supplemented with adenine and 2,4-D. However,

only

diploid tissue was developed into plantlets when the callus was
transferred to

regular MS medium.
The origin of organs (roots and shoots) from specific (anatomical)

sections ofthe root was investigated by Hwang et al. (1983). Segments

taken

3912

from lateral sections formed significantly more shoots than segments

taken

from central cylinders. The authors attributed the origin of organs to

anomalous cambia (secondary and tertiary) within the inner section

ofthe root.

Henderson (1987) reported multiple plant production from root and

stem explants of cv. Jewel when cultured on MS medium with or

without

gelrite. A factorial design experiment was conducted to understand

the culture
medium effect. The medium containing cytokinins, NAA and abscisic
acid

was used to initiate cultures. The budding medium had no growth

factor

supplementation; Kn, NAA and gibberellin were used for subculturing.

Houndonougbo (1989) studied the influence of different

concentrations

of IAA, NAA, 2,4-D and Kn on callusing and organogenesis in vitro from

intemode segments oftwo sweet potato varieties. Optimal callus

formation in

culture medium containing 0.45pM 2,4-D and 4.6 pM Kn for pink

variety, and

0.045 pM 2,4-D and 0.0046 pM Kn for the white variety were

reported. While
Kn improved root formation in media containing IAA and 2,4-D, bud

formation required Kn and 2,4-D. In vitro tuberization of the roots was

achieved in presence of Kn and auxin. Ozias and Perera (1990) could

regenerate shoot from adventitious roots in MS medium

supplemented with 1-

3% sucrose or 1-6 % fructose. A low concentration of eytokinin (0.02

mg/1)

promoted shoot formation, while higher concentration (0.1-0.5 mg/1)

encouraged callus growth. The average number of shoots produced

per root

explant was 0.5.

Rivas et al. (1991) obtained regeneration from intemode and leaf

explants of sweet potato cv. Beauregard. Plantlet regeneration
occurred when

complete leaves and intemode explants (10 mm) were cultured in

liquid and

solid media for a 8 week period. The effect of NAA and various

concentrations ofthidiazuron (TDZ) on intemode culture was examined

using42

13

MS medium at 16 h photoperiod. Mostly plantlets were regenerated

using 0.05

mg/1 NAA and 0.01 mg/1 TDZ. Kobayashi et al.(1992) reported

enhanced

shoot regeneration using p-chlorophenoxy indole butyric acid in leaf

cultures

ofIpomoea species in the section batatas.

Shen and Henderson (1993) discussed the critical factors for plant

regeneration from petiole and lamina cultures in sweet potato cv.

Jewel. The

critical factors were growth factors viz. 2,4-D, BA and explant

orientation on

the culture medium. The base of the explants should touch MS

medium

containing 1 mg/1 2,4-D for 12 h, transfer of a petiole explant to MS

medium

with 0.1 mg/1 2,4-D and transfer of a lamina explant to MS medium

with 0-

0.05 mg/1 BA, top face up. Within 5 weeks of incubation at 26 ± 2° C

with

illumination by 40 pmol/sec.sq.m fluorescent light, whole plants were

regenerated. Prakash et al. (1993) developed a protocol to produce

adventitious plants ofsweet potato. They used 27 genotypes ofwhich 5

were

identified as highly regenerative. They got regeneration from 60-80%

explants

when leaf explants with intact petioles from the apical portions ofthe
in vitro

shoots were cultured on MS medium supplemented with 0.2 mg/1

2,4-D for 3

days and transferred to a medium containing 0.2 mg/1 zeatin riboside.

Regeneration was also possible from petiole by substituting zeatin

with 0.2

mg/1 thidiazuron (Ramana Murthy et al. 1993). Increasing

concentrations of ,
TDZ (more than 0.4 mg/1) lowered the shoot and root regeneration.
Shoot

regeneration was also recorded in most explants (78.2%) of genotype

P1318846-3 within 28 days when grown on thidiazuron at 0.2 mg/1

(Gosukonda et al. 1995 a). Explants from apical portion showed higher

regeneration than basal portion and response was better with the use
of

thidiazuron and was effective in diverse genotypes of sweet potato

(Gosukonda et al. 1995 b). Petiole explants when cultured following a

twostage protocol on medium with only 2iP but without TDZ failed to
regenerate

4514

shoots. The genotype PI 318846-3 was identified to be most

regenerative
producing 1-3 shoots per explant.

Weerakoon et al. (1994) regenerated sweet potato plants in vitro from

leaf explant and cultured on MS medium containing 1.0 mg/1 glycine,

1.5 mg/1

nicotinamide, 0.5 mg/1 pyridoxin-HCl, 0.1 mg/1 thiamine-HCl, 2%

sucrose and

0.2 % phytagel; with 0.2, 0.5, 2.0 mg/1 zeatin in combination with 1.0
and 2.0

mg/1 NAA in a factorial experiment. They reported best root growth

in

medium with 0.2 mg/1 zeatin and 1.0 mg/1 NAA. The medium
containing 0.2

mg/1 zeatin and 0.1 mg/1 NAA was considered optimal for shoot
formation.
Alloufa (1994) reported callus formation and plant development in
vitro from

root tip culture on MS medium supplemented with BA (2 mg/1), NAA

(0.1

mg/1), sucrose (30 gm/1) and agar (10 gm/1) in sweet potato cultivars

‘Leucorhiza’ and Torfhirohiza’.

1.2.5. In vitro tuberization

Houndonougbo (1989) had studied the influence of different

concentrations ofIAA, NAA, 2,4-D and Kn on callusing and

organogenesis in

vitro from intemode segments of two varieties of sweet potato. Roots,

buds
and complete plants were obtained. Kn improved root formation in
media

containing indole acetic acid and 2,4-D. Bud formation required Kn

and 2,4-

D. In vitro tuberization of the regenerated roots occurred exclusively

in the

presence ofKn and auxin.

Aboul and Bouwkamp (1990) used fibrous root explants for in vitro

tuberization. MS-salts, vitamins and sucrose (2,4,8, and 12%) were

used with

three concentrations (0, 1 and 5 ppm) of BA. They observed storage

root

formation in presence of 8% sucrose in combination with 1 or 5 ppm

BA and
4 concentrations ofsucrose (2,4,8 and 12%) in combinations with BA
i.e., 0, 1

4915

and 5 ppm. Microtuber formation and shoot organogenesis from the

roots

induced from leaf explants in MS and MS with lpM NAA at 12 h

photoperiod

have been reported in sweet potato (Mukherjee et al. 1993, 1996).

1.2.6. Somatic embryogenesis

Somatic embryogenesis results in the most rapid mode of plant

production (Evans et al. 1981) and is defined as nonsexual

developmental

process which produces a bipolar embryo from the somatic tissues.

Somatic
embryogenesis was first reported in carrot (Steward et al. 1958) and
thereafter

regeneration protocols through somatic embryogenesis have been

developed in

many crops. Considerable progress has also been made in sweet

potato

regeneration through somatic embryogenesis. Tsai and Tseng (1979)

investigated embryo formation and plant regeneration from anther

callus of

five cultivars of sweet potato. They experimented with two basal

media,

Blaydes (1966) and MS (1962), supplemented with various

concentrations of

LAA, 2,4-D, Kn, ABA, 2iP, BA, and yeast extract. Cultures were grown at
27°C. Callus induction (in darkness) occurred in both media containing
auxins

and Kn, and was especially successful in the presence of 2,4-D and Kn.
For

embryo formation the medium required ABA. They suggested that in

the

presence of ABA, as in the work of Yamagauchi and Nakajima (1972)

endogenous cytokinins were antagonized and regulated and thus

permitted the

formation of embryos.

Liu and Cantliffe (1984) reported formation of somatic embryos from

leaf, shoot tip, stem and root explants of sweet potato (cv. White star
and
GaTG-3) through callusing on MS medium containing 0.5-2.0 mg/12,4-
D. The

embryos could develop into plantlets on transfer to hormone free

ntedium.

Conversion of embryogenic callus to heart or torpedo-shaped

embryoids was

achieved in MS medium containing 2 mg/1 2,4-D, Kn (2 mg/1) and

20%

5216

and 5 ppm. Microtuber formation and shoot organogenesis from the

roots

induced from leaf explants in MS and MS with lpM NAA at 12 h

photoperiod

have been reported in sweet potato (Mukherjee et al. 1993, 1996).

1.2.6. Somatic embryogenesis

Somatic embryogenesis results in the most rapid mode of plant

production (Evans et al. 1981) and is defined as nonsexual

developmental

process which produces a bipolar embryo from the somatic tissues.

Somatic

embryogenesis was first reported in carrot (Steward et al. 1958) and

thereafter

regeneration protocols through somatic embryogenesis have been

developed in

many crops. Considerable progress has also been made in sweet

potato

regeneration through somatic embryogenesis. Tsai and Tseng (1979)

investigated embryo formation and plant regeneration from anther

callus of
five cultivars of sweet potato. They experimented with two basal
media,

Blaydes (1966) and MS (1962), supplemented with various

concentrations of

LAA, 2,4-D, Kn, ABA, 2iP, BA, and yeast extract. Cultures were grown at

27°C. Callus induction (in darkness) occurred in both media containing

auxins

and Kn, and was especially successful in the presence of 2,4-D and Kn.
For

embryo formation the medium required ABA. They suggested that in

the

presence of ABA, as in the work of Yamagauchi and Nakajima (1972)

endogenous cytokinins were antagonized and regulated and thus

permitted the
formation of embryos.

Liu and Cantliffe (1984) reported formation of somatic embryos from

leaf, shoot tip, stem and root explants of sweet potato (cv. White star
and

GaTG-3) through callusing on MS medium containing 0.5-2.0 mg/12,4-

D. The

embryos could develop into plantlets on transfer to hormone free

ntedium.

Conversion of embryogenic callus to heart or torpedo-shaped

embryoids was

achieved in MS medium containing 2 mg/1 2,4-D, Kn (2 mg/1) and

20%

5517

stage in suspension culture required immobilization with sodium

alginate. On
average 9.7 embryos were produced from 1.0 mg of callus. Further
studies on

callusing and morphogenesis were carried out (Chee and Cantliffe

1989 a,

1989 b and 1989 c) with different growth regulators like 2,3,5-

triiodobenzoic

acid (TIBA), 2,4-D, p-chlorophenoxy isobutyric acid (PCIB), 7-aza-indole

(AZI). While 1-5 pM AZI and PCIB did not affect morphogenesis, 5 pM

TIBA and 2,4-D inhibited morphogenesis and promoted embryogenic

callus

growth. Such response may be a consequence ofthe disruption by

exogenous

auxins or endogenous IAA efflux from embryogenic loci. Chee et al.

(1990)
have also studied the effect of BA, NAA and sucrose on plant recovery
from

somatic embryos in the cv. White Star and recorded positive effect
with

addition of NAA and negative effect by addition of BA and sucrose.

The

frequency of plant development was increased to 38% by addition of

lpM

NAA to the culture medium. Reducing the sucrose concentration to

1.6%

increased the frequency of plant development to 32%. Medium

containing 60

mM potassium was found effective to enhance embryogenic response,

additions of upto 40 mM NaCl to basal medium did not affect callus

proliferation (Chee et al. 1992). Chee and Cantliffe (1992) improved

production procedure of sweet potato by fragmenting embryogenic

callus.

About 14% ofsuch aggregrates were embryogenic.

DeWald and Cantliffe (1988) undertook histological studies of callus

initiation and somatic embryogenesis in sweet potato. The

embryogenic cells

were isodiametric with densely staining cytoplasm. A fast-growing

white

friable callus commonly associated with embryogenesis was formed

from the

older leaf primordia of the shoot apices. It consisted of large-

elongated
vacuolated cells.

5918

Mukherjee et al. (1991 b) reported regeneration from sweet potato

anthers through eallusing and embryogenesis in MS medium

supplemented

with 1 pM of 2,4-D. It was suggested that 2,4-D is a better growth

component

compared to ABA or NAA to interact with endogenous cytokinins for

stimulating embryogenesis.

Bieniek et al. (1991) developed suspension cultures of Ipomoea

batatas. The effect of endogenous plant growth factor (PGF) content

on the

embryogenic callus in 2,4-D free media was also demonstrated at the

time of
subculture. The addition of 0.5 pM BA to cell suspension cultures for
one

generation prior to culture in PGF-free medium increased subsequent

embryo

formation 100% over suspension grown" with 2,4-D alone. Bieniek et

al.

(1995) had produced somatic embryos in an airlift culture vessel and

tried to

optimise the conditions from semisolid to liquid system for application

in a

bio-reactor. Somatic embryogenesis was recorded in liquid cultures

containing

20 mM NH4NO3 and 30 mM KC1. Embryo formation and development

was
limited in culture vessel but the regulation of culture vessel gases
could allow

embryo formation comparable to shaker flasks.

Sonnino and Mini (1993) had tested different cultivars of sweet potato

for their capacity to produce somatic embryos. The mean number of

embryos

per embryogenic callus was low for cv. 288-6B (56.7) and W 190 F
(12.4) and

very low for cv.Q 23836 (3.3) and CN 1489-89.(1.3); and the number of

completely developed plants was 66 for cv. 288-6B, 48 for Q 23836, 7

for W

190 F and 0 for CN. No obvious morphological variations were evident

between these plants and control plants.

CavalcanteAlves et al. (1994) had identified the isozyme related to

embryogenic and non-embryogenic callus. They maintained

embryogenic

callus on MS medium supplemented with 30g/l sucrose and 10 pM

2,4-D for

6219

6-8 weeks. The frequency of embryogenic response was low and

varied with

genotypes ranging from 0 to 17%. After several subcultures, 0.5-12 %

of

embryogenic callus could be reverted irreversibly into friable fast-

growing

non-embryogenic callus. They could separate the isozymes of esterase

(EC-
3.2.1.1) , peroxidase (EC-1.11.1.7) glutamate-oxaloacetate-
transaminase (EC-

2.6.1.2) and acid phosphatase (EC-3.1.3.2) to distinguish compact

embryogenic callus from friable non-embryogenic callus in sweet

potato.

Desamero et al. (1994) had shown direct somatic embryogenesis in

sweet potato induced by picolinic-acid (PA). Apical meristem ofsweet

potato

cv. Jewel and Regal when cultured on medium containing picolinic

acid (PA)

0.2 mg/1, Kn 1 mg/1 and BA 2 mg/1, shoot growth was suppressed

and callus

proliferation was induced. Increased amounts of PA (2 and 3 mg/1)

and Kn
(0.25 and lmg/1) induced direct somatic embryogenesis in cv. Regal.
The

primary embryos matured and germinated bipolarly yielding whole

plantlet

and unipolarly producing embryogenic hyperhydrated-fasciated

shoots. These

shoots when grown in PA and kinetin enriched medium produced

secondary

embryos.

Harrell et al. (1992, 1994) tried to develop a mathematical model to

maintain cell suspension culture of sweet potato shoot tips in airlift

culture

vessel. Growth rates did not vary with time during culture. Growth
rates and
viable to non-viable ratios decreased as the fraction size increased.
While

attempting to make an automated system to harvest embryos they

suggested

that improvements to somatic embryogenic section processing time,

and

increased culture population density and homogenity should be

explored prior

to fabricate the system.

Norton and LaBonte (1996) studied the cultural conditions affecting

the

frequency of embryoid formation in sweet potato using 1 responsive

and 3

6520

recalcitrant cultivars. The effects of auxin, nitrogen, carbon source,

explant
source and desiccation during somatic embryogenesis were reported.

Ammonium and nitrate promoted the pro-embryo formation in all

cultivars.

Ammonium was essential for somatic embryogenesis; replacing it with

proline, glutamine, asparagine, and glycine resulted in poor or no pro-

embryo

formation. More pro-embryos were formed on medium supplemented

with

sucrose than with glucose, fructose or maltose. Gowda and Prakash

(1996)

could enhance somatic embryogenesis in sweet potato using herbicide

‘glyphosate’ in leaf explants of genotype PI 318846-3. Somatic

embryos
developed faster on explants grown with glyphosate (200 pM) than
those

grown with ABA (2.5 mg/1) alone. A rapid and repetitive

embryogenesis

system in sweet potato cv PI 318846-3 was developed by Zheng et al.

(1996).

MS medium containing a factorial combination of auxin (2,4-D) and

cytokinins (BA) at various levels was used. Addition of 2.5 mg/1

abscisic acid

enhanced the development of somatic embryos. Secondary somatic

embryo

production was recorded on culture ofprimary embryos on medium

containing
various levels of 2,4-D, while, somatic embryos germinated into full
plantlet

on basal MS medium. They suggested that addition of ABA was critical

for

enhancing somatic embryogenesis.

Al Mazrooei et al. (1997) screened five auxins to study their effects on

sweet potato somatic embryogenesis. Of the five auxins tested, 2,4-D

and

2,4,5- trichlorophenoxy acetic acid were the most effective. Use of

2,4,5-T

could induce somatic embryogenesis in the cultivars, which responded

poorly

or not at all to 2,4 - D.

Padmanabhan et al. (1994) examined the serial sections of somatic

embryos and revealed that lack ofshoot development could be

attributed to the

lack of an organized apical meristem. Attempts were also made to

select the

competent embryos using computer vision and canonical discriminant

function

6921

CDA) analysis. Diagnostic separations of those competent embryos

were

based on the analysis of external morphology (captured via machine

vision)

and internal anatomy (Padmanabhan et al, 1998 a; Padmanabhan et

al.1998 b).

1.2.7. Artificial seeds

Artificial seeds, also known as synthetic seeds, are good quality

somatic embryos or propagules enclosed in protective nutrient

coating.

Artificial seed technology provides a rapid, high volume and cost

competitive

method. Schultheis and Cantliffe (1988) studied the effect of gelling

agents for

artificial seed production in sweet potato. Torpedo-stage embryos

were placed

in 2% (w/v) N-gel 250 HHR, Liqua-gel, American Colloid (AC) and

Viterra

gels supplemented with MS medium and 2 % sucrose. Over 80% of the

embryos died in AC and Viterra gels after 6 days while plant formed in
the Ngel. Glucose, fructose, galactose, sucrose and maltose (47-93
mM) were added
to N-gel containing MS. Plant formation was recorded maximum (20-
25 %)

with maltose, sucrose, glucose while no plants were obtained with

galactose.

No plants were produced with sucrose alone. Plants were obtained

from the

full complement ofMS macronutrients and sucrose.

Schultheis et al. (1990) selected the embryos based on shape and size.

To optimize plantlet formation the gels used for fluidized sowing were

potassium starch polyacrylamide (PSA), potassium acrylate (PC), a

copolymer

of potassium acrylate and acrylamide (PA) and hydroxyethylcellulose

(HEC).
Embryos placed in HEC were healthy after 6 days. Nearly all embryos
were

dead in PC and PA gels, while the PSA had 40 % healthy embryos. The

percentage of embryos converted to plantlets at 31 days was 38 % for

elongated torpedo stage and 21% and 17 % for torpedo and

cotyledonary

stages respectively. Schultheis and Cantliffe (1992) studied the growth

of

somatic embryos of sweet potato in hydroxyethyl cellulose gel

amended with

22

salts and carbohydrates. MS salt concentrations at 1/2 . strength and

Ml

strength resulted in 40% plantlet production, while at 1/4 strength 80

%
produced roots but no shoots. Both macro and micro-nutrients were
necessary

for maximum plant conversion. Plantlet formation was improved with

fructose, maltose or sucrose as C-source. Concentrations ranging from

23-93

mM were best for plantlet formation.

1.2.8. Protoplast culture

Studies on protoplast culture have opened up avenues for genetic

transformation as well as to understand the physiology and genetics

at cell

level. Progress ofwork on isolation, fusion and transformation ofsweet

potato
protoplasts is given below.

Isolation of viable protoplast from sweet potato and some of its wild

progenitors have been reported by many researchers (Wu and Ma

1979;

Shepard 1980; Bidney and Shepard 1980; Eilers et al. 1986; Sihachakr
and

Ducreux 1987; Otani et al. 1987; Eilers et al. 1988; Ozias and Perera
1990;

Kobayashi et al. 1990; Buchheim and Evans 1991; Perera and Ozias
1991;

Kobayashi et al. 1992; Belarmino et al., 1992, 1994 and 1996). Stem,
petiole

and leaf explants or callus tissue produced from those explants were
digested
either with eellulase (2%) alone or cellulase (2%) with macerozyme
(0.05%),

pectolyase (0.3%), hemicellulase (0.5%) and driselase (1%) at pH 5.6 to

release protoplast. Belarmino et al. (1994) regenerated plants from

stem and

petiole protoplasts ofsweet potato and Ipomoea lacunosa. They also

reported

asymmetric protoplast fusion between sweet potato and Ipomoea

trifida by

electrofusion and polyethelene glycol (PEG) treatment. They could

regenerate

only from electrofused protoplast and suggested that PEG might be

toxic to

the system. They characterized the interspecific hybrids through

isozyme
analysis.

2376

Methods for isolation of viable protoplasts, formation of morphogenic

callus, regeneration through somatic embryogenesis and transient

expression

of the gusA gene in the regenerants introduced by electroporation

have been

described by Dhir et al (1998). A combination of cellulase (1%)

macerozyme

(2%) and pectolyase (0.3%) was used for enzymatic digestion for
protoplast

isolation. Regeneration from protoplasts was achieved on subsequent

culture
on KP8 (Kao and Michayluk, 1975) media containing the combinations
like

2,4-D (0.9 pM) and zeatin (2.3 pM); 2,4-D (11.3 pM) and BAP (2.2 pM);

GA3 (3.5 pM) alone.

1.2.9. In vitro conservation and cryo-conservation

In vitro methods are currently being practiced for the conservation of

root and tuber-crops germplasm in the gene banks of IITA (Nigeria),

CATIE

(Costa Rica), CIAT (Colombia), INRA (Guadalope) and CIP (Lima Peru).

Different methods like use ofminimal media, controlled environments,

growth

retardants have been found effective for short to medium term

storage of
cultures. Minimal media free of growth regulators and vitamins
supplemented

with sucrose (3%) and mannitol (3%) could extend culture life more
than 6

months in sweet potato (Unnikrishnan et al. 1991). Jarret and Gawel

(1991)

induced growth inhibition in sweet potato in vitro using abscisic acid

(ABA).

ABA at concentrations of 0.01, 0.1, 1.0 or 10 mg/1 inhibited axillary

bud and

root development and subsequent plantlet growth. ABA at 10 mg/1

completely

inhibited shoot development but did not affect the viability. The
growth
retarding effect and the toxicity to explants were genotype dependent
and can

be used for conservation.

Supplementation of MS medium with some growth regulators as well

sucrose and mannitol could extend culture life for more than 12
months

(Mukheijee et al. 1994) in 200 genetic resources ofsweet potato. MS

medium

7924

with addition of 10, 15, 20 g/1 mannitol or reduction ofsucrose from

30 to 20

or 25 g/1 could store cultures for 12 months (Mandal and Chandel

1996).

Cryostorage of shoot tips as well as embryogenic tissues have also

been
attempted (Towill and Jarret 1992; Blakesley et al. 1996; Blakesley et
al.

1997).

1.2.10. Improvement through transgenics

Development oftransgenics has become the ultimate choice to

improve

certain crop species. The methods used for genetic transformation are
either by

electroporation or by physical bombardment of cells with

microprojectiles

coated with DNA or cocultivation with the bacterial vector

Agrobacterium

tumefaciens or A. rhizogenes. Attempts have been made to develop

useful
transgenics in sweet potato.

Hattori et al. (1985) and Hattori et al. (1990) had reported molecular

cloning and nucleotide sequence of cDNA for sporamin, the major

soluble

protein of sweet potato tuber. About 80 % of total soluble protein was

accounted for sporamin. A full-length cDNA clone for sporamin has

been

isolated and identified by E. coli vectorprimed cDNA synthesis and

screening

of the library by immunological assay. Nishiguchi et al. (1992) reported

genetic transformation of sweet potato by electroporation. They

studied
electroporation optimization for sweet potato protoplast culture
transformation

for hygromycin-resistance selectable marker. They confirmed that

some calli

cells containing DNA encoding hygromycin-resistance through

southern

hybridization analysis. Otani et al. (1993) had shown Agrobacterium

rhizogenes-mediated transformation in sweet potato. Transgenic

plants

expressed the genes obtained by infection with A. rhizogenes

containing a

binary vector plasmid. Garcia et al. (1995 a) had reported transgenic

sweet

potato plants resistant to pest (Cylasformicarius). The expression ofBt

genes
8325

was detected by Western Blot using polyclonal antibodies.

Methodology for

regeneration and genetic transformation of sweet potato for storage

protein

(sporamin) have been developed in two cultivars (Garcia et al. 1995 b).
They

tested the transgenic plants for sporamin gene by polymerase chain

reaction.

Prakash et al. (1995) reported high efficiency transformation and

regeneration

of transgenic sweet potato plants. Cipriani and Golmirzaie (1996)

reported

transformation ofsweet potato cv. Regal and Huachano using

Agrobacterium
tumefaciens and A. rhizogenes, for insect resistance. Walls et al.
(1996)

observed fungal resistance in sweet potato by genetic transformation

with

chitinase and glucanase genes. Integration and expression of genes

were

assessed through Southern, Northern and Western analysis. Genetic

transformation of Ipomoea trichocarpa was reported by Otani et al

(1996).

Maingi et al. (1996) could confer resistance to feathery-mottle virus in

sweet

potato through genetic transformation. They reported an efficient

transformation system using A. tumefaciens strain containing marker

for
SPFMV coat protein genes. Egnin and Prakash (1997) reported
transgenic

sweet potato expressing a synthetic storage protein gene for high

levels oftotal

protein and essential aminoacids. Storage roots of transgenic lines had

9%

protein on a dry weight basis over control (2.25%) protein. The

transgenic

plants had smaller storage roots compared to control.

1.2.11. Induction ofstress tolerance

In vitro methods provide the most systematic and efficient routes to

develop stress tolerant plants by way of adapting the cells to stress

conditions
and regenerating the whole plants from those cells. Salgado et al.
(1985) could

isolate NaCl resistant variant cells from sweet potato cell suspension
which

could tolerate 1% NaCl. They established callus cultures on MS

medium

supplemented with 10 jiM 2,4-D and 0.01 pM BA and sucrose. Cell

suspension were prepared in liquid MS. Harvested cells were

suspended with86

26

or without 1% NaCl and incubated at 25° C with illumination for 15

days.

Cells were sub-cultured 11 times and tested for salt resistance.

Resistance to

1% NaCl was stable for 3 passages in NaCl-free medium. Ekanayake

and
Dodds (1993) had tested the effects of salt stress on growth and
survival of

sweet potato in vitro. Results showed significant variations among the

cultivars and breeding lines for salt tolerance.

1.2.12. Evaluation of performance ofin vitro raised plants

Schultheis et al. (1993) had evaluated yield potential of plants grown

from somatic embryos. They suggested high yield potential ofroot

bulking of

plants grown from somatic embryos. Schultheis et al (1993) also

studied

effects ofmicro-propagation and soma-clonal propagation on plant

growth and
yield ofsweet potato. A trend towards early bulking was consistently
observed

in micro-propagated Jewel, ‘White delight’ and ‘Beauregard’. Early

micropropagation and vine cuttings were compared. Plants derived

from

somatic embryos grew slower and yielded less root weight than cut
plants.

More storage roots were obtained from plants derived from somatic
embryos

indicating high yield potential of root bulking. Schultheis et al. (1994)

compared the yield of plants derived from somatic embryogenesis,

zygotic

embryos and from vegetative cuttings. Plants from somatic embryos

required
more time for roots to bulk. Root yield of somatic embryos derived
plant

showed a 14-fold increase when harvest was delayed at least 53 more

days.

Such plants yielded 1.8 kg tuber /plant. The morphology of these

plants was

identical to stock plants.

1.2.13. Genetic stability or variability ofin vitro raised plants

In vitro technique offers a new method for propagation and storage of

vegetatively propagated crops such as sweet potato. However,

somaclonal

8927

variations arising in some ofthe culture systems offer serious problems

as they
are known to have genetic basis (Heinz and Mee 1971; Larkin and
Seowcroft

1981). Therefore, monitoring the genetic stability ofin vitro raised

propagules

is most important. Genetic stability could be studied either by

cytobiochemical methods or employing the advanced techniques like
isozyme or

DNA polymorphisms. Isozymes are the forms of enzymes whose

polypeptides

are coded by different gene loci and have been used to identify the
somaclonal

variants in sugar cane (Heinz and Mee 1971), potato (Denton 1977)
and celery

(Orton 1983). Isozyme studies were also carried out in sweet potato
by
Lakhanpaul et al. (1991). They have recorded qualitative and
quantitative

differences in banding patterns of acid phosphatases, peroxidases and

malate

dehydrogenases in callus regenerated plants in contrast to direct

regenerants.

Belarmino et al. (1994) characterized the interspecific hybrid of sweet

potato

and I. trifida through isozyme analysis. Cavalcante Alves et al. (1994)

characterize the embryogenic and nonembryogenic calli with specific

isozyme

activity.

DNA marker provides a direct insight into the genomic constitution, it

is very useful for genetic analysis. Random Amplified Polymorphic
DNA

(RAPD) marker have the advantages of being a very simple, less time

consuming and relatively inexpensive compared to other DNA marker

techniques. Moreover, RAPD analysis are independent of

environmental

influences and tissue type. Villordon et al. (1996) studied genetic

uniformity

in the regenerants propagated through adventitious and nodal

propagation.

They used 15 decamer primers and got 64 scorable amplified

fragments in a

PCR based assay. Their results confirmed that clonal plants derived
from preexisting meristematic regions are more genetically uniform
than the plants
propagated from adventitious origins.

2893

1.3. Objectives and scope of present study

With the background ofthe inherent problems ofsweet potato

breeding

and with the realisation of the potential usefulness of modem plant

biotechnological tools to overcome such problems in sweet potato,

the present

programme of research study has been undertaken with the following

major

objectives:

i) To work out an in vitro protocol for mass propagation of different

sweet potato genotypes through all possible routes ofpropagation.

Such studies would allow utilisation ofthe in vitro propagation

methods

widely in diverse genotypes.

ii) To establish a plant regeneration system from different explants

through

organogenesis and somatic embryogenesis.

This will enhance the plasticity of propagation to use different parts as

propagules and to utilize those judiciously for propagation, storage

and

improvement.

iii) To enhance regeneration frequencies in all possible routes of

propagation.

As each mode ofpropagation has its merits and demerits, all

propagation

protocols can be combined to optimise the regeneration efficiency and

use those either for propagation, storage or for improvement

purposes.

iv) To test the applicability of synthetic seed technology in sweet

potato

genotypes.

Work in this direction would definitely be beneficial for production of

artificial seeds, which could be the novel substitute of true seeds in a

clonally propagated crop like sweet potato.

9529
v) To investigate the possible morphological, histological, cytological
and

biochemical changes associated with embryogenic regeneration.

Such studies would help to assess the morpho-histo-cyto and

biochemical situation during regeneration and to develop and

maintain a

stable regenerating system.

vi) To evaluate the cytogenetic situations of regenerants with special

emphasis on genetic stability and variability.

Evaluation of cytogenetic situation is a must to assure stability or

variability of regenerants for their further utilization in propagation,

storage and genetic manipulation.

vii) To evaluate the field performance of the regenerants with respect

to

yield potential and tuber quality.

In order to provide a stable propagation protocol with stable

qualitative

and quantitative characteristics, field evaluation is essential for any in

vitro mediated regeneration protocol.

3097