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TitleStudies on tbt in vltro culture and genetic transformation of pigtonpea
LanguageEnglish
File Size29.5 MB
Total Pages241
Table of Contents
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Document Text Contents
Page 1

Studies on tbt in vltro culture and genetic transformation of

pigtonpea (C#mw Nun (L) Millsp.) for induced resistance
to hngal pathogens

Thesis Submitted to the
Osmania University for the award of the degree of

Doctor of Philosophy

G. SUNEETHA

Department OIBotrny Genetic Transformation hboratoy

Oammia University ICRlSAT

Hydenbrd 500 007 A,P., India Patancheru 502324, A.P., India

Page 120

stem segments with Agrobacterrum tumefacrens placed in a horizontal position proved

to be critical for efficient transformation in Brassrca (Kuvshinov et al., 1999)

Genotype of the explant donors has been shown to play a major role in the

regeneration of shoots in several species including legumes (Pederson, 1986, Chen et

al., 1987, Meijer and Brown, 1987, McKently et al., 1989, 1991, Sellars et al., 1990)

Since the development of an ideal regeneration system should be genotype independent,

the choice of a suitable genotype is critical in such studies (Jain et al., 1988, Shoemaker

and Counche, 1986) Shoot product~on In a particular explant donor explant genotype

depends on the culture conditions and principally to the interaction between the

genotype and the culture medtum (Flores et al., 1999) The plant regeneration protocol

reported in the present study has been found to be applicable across a wide range of

pigeonpea genotypes tested, which belonged to different maturity groups Among all

the genotypes tested the genotype ICPL 88039 was found to respond better and hence

was used in the present work Uniform observations were reported in all the three

cultivars of gerbera in terms of efficient shoot bud regeneration with slight variation and

therefore, the regeneration protocol used was genotype independent (Orlikowska et al.,

1999) Genotype independent protocols were reported in guineagrass (Chen et al.,

2002), in melon (Ficcadenti and Rotino, 1995, Yadav, 1996), in gypsophila (Ahroni et

al., 1997), and in Ktrs (Torregrosa and Bouquet, 1996) Genotype dependent response

was reported in all the varieties of onion (Luthar and Bohance, 1999)

Therefore, a highly efficient regeneration protocol has been optimized in the

present study, which is genotype independent and the time involved in raising the

Page 121

plantlets was less that can be further used for the production of transgenic plants with

genes of agronomic importance

5.2 Genetic transformation of pigeonpea

The most important aspect of successful genetic transformation is the induction

of a larger number of regenerable cells that are accessible to the gene transfer methods,

and which continue to proliferate transformed cells under stringent selection conditions

Further, the gene transfer into potentially regenerable cells may not allow recovery of

transgenic plants if the capacity for efficient regeneration is short lived (Birch, 1997)

Transformation efficiency is proportional to the efficiency of the tissue culture and gene

transfer systems (Hiei et aL, 1994, Ishida el al., 1996, Bower et ol., 1996, Li et a[.,

1996) In the present study, an efficient regeneration and transformation protocol was

optimized by using the petiole region of the leaf explant for high frequency production

of multiple shoots at a rapid rate, from the bombarded (microprojectile bombardment)

target cells, without any intervening callus phase

It is reported that the length of the callus phase is negatively correlated with

regeneration ability, where the somaclonal var~ations can influence the phenotype of the

regenerated shoots (Fontanna et al., 1993) The rate of plant regeneration using the

present protocol (>90%) is highly efficient and reproducible and is applicable to a wide

range of pigeonpea genotypes tested

Page 240

Genetic Transformation and Hybridization

An efficient protocol for shoot regeneration and genetic
transformation of pigeonpea [Cajanus cajan (L.) Millsp.] using leaf
explants

(1) Oenetlc TranSfomlUM Laboratory Inkrnatwsi Crop Research Inrtltute for the %rnMr!4 TrWla Pabncheru 502 324 Andhm Pmda
indll

(2) DspsmnenI of Bobny Otmsnla Un1wr61i-y 390 006 Hyddrabsd lndla

Abatnd A protocol for efficrent plant regeneration from leaf explants ofplgmnpca [Cqarmr capn (L ) Mlllsp ] was developed for the
prcducuon of hMsgeruc plants Led explants from 4. to 5-day-old m vlfm rued seedhngs wen most eficteat m productng mulllple

adventltlous shoots In 90% of the explants on shwt ~nduct~on med~um [Murashlgc and Skwg (MS) med~wn t 5 0 M benzyladen~ne t 5

I1,U klnetln] Shoot buds orlglnated from the petlolar cut end of the explants and elongated rap~dly on medlum contalnlng 0 58 1l.U

glbberelllc ac~d Over 8Lph o f the elongued shoots mokd wcll on MS medlum contalnlng I 1 42 / I M mdole-3.acehc acid and were

transplanted WIB 100% success The procedure reported here u very sunple, eEtic~ent and repduclble, and u applicable across dlvene
3enor)pcr o f plgeonpca The uyhlness of BIS r)s!tm ior fbnh~ltr a ~ d l e r on Be geneLC rraruformsl~on o f plgmnpca has been
oernonirrated In b~ol~mco-mcd~aud gmc transfer b) us~ng nprll and u!dA m asmarker genes, where 5OO/o o f l e ulcclcd plan0 showed gene
lntegranon and expresslon

N6-Benzyladenine
GA, Gibberellic acid
IAA Indole-3-acetic acid
l u Indole-3-butyric acid
2 - 8 N6-[2-Isoptenyl]adenine
MS Murashige and Skoog medium
RIM R00t M m medium
RT.~RReverse &ptase polymerase chain reaction

Sh00S elongdm medium
JIM S& i n M m medim

Page 241

To
The Dean
Faculty of Sc~ence
Osman~a Un~vers~ty
Hyderabad-500 007

From
Ms G Suneetha
Research Scholar
(Regd for Ph D )
Department of Botany
Osman~a Unlvers~ty

Through Prooer Channel
Sir,

Thls 1s to ~nform you that I G Suneetha, a reg~stered Research Scholar (Ph D ) v~de
OU letter no 505/DFSC/98-99 Dated 5' July 1999, have completed my research and
presently subm~tt~ng my thew for evaluat~on for the award of Ph D degree I have
worked as Project Fellow In the AP-Netherlands B~otechnology Project
"Development of disease resistant transgenic pigeonpea", under the
supervision of the ~ r i n c i ~ & ~ n v e s t i ~ a t o r Dr B Prathibha Devi and the Co-
investigator Dr Kiran K Sharma The Project work involved the use of several
methods for genetic transformation of pigeonpea and was carried out mostly in
the Genetic Transfomiation Laboratory, International Crops Research Inst~tute
for the Semi-Arid Tropics (ICRISAT), Patancheru, India However, the research
concerning the use of microprojectile method of gene transfer has been comp~led
for my Ph D thesis entitled "Studies on the in vitro culture and genetic
tmnsfornlrt ion of pigeonpea (Calanus cajnn (L.) Millsp.) for induced fungal
patliugcns". T h ~ s is for your ~nformation

Thank~ng you,
Yours s~nqerely

Signature of ~ r inc i~& inves t i~a tor
DP.IC i

L ' ldbw>. K. K, S;(ARMA
Signature o f the Co-investigator , R I A

PATbNCilERU P 0.
ANOHRA PRADESH-SO2 324

S~gnature of

Ikpnnrrent of PMmT
m a n i n UBLW~SIY

.motn.

G
G Suneetha

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