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Genetic engineering approach to develop male sterility in Jute (Corchorus spp)

plantTirthartha Chattopadhyay, ALPGE and Mrinal K. Maiti, Department of Biotechnology, IIT, Kharagpur

Figure 1: Jute plants,

fibre and some of the

jute-products

ale sterility (inability to produce

nctional male gamet) phenomenon in

nt system is crucial for hybrid seed

oduction. Limitations of naturally

urring male-sterility demand

plication of genetic engineering

hnique to develop transgenic male

rility in important crop plants.

veral genetic

gineering

ategies have

en devised in

ferent crops but

thing is available

developing a

le sterility

tem in Jute

orchorus spp.) ,

important fibre

p. Here, a

ategy of

veloping

netically engineered male-sterile Jute

nt is elucidated and progress made

this regard is documented.

stract

male sterile plant lacks functional

len grains (male gamets) but has

nctional female part. Therefore, all of

genetic engineering strategies to

velop male-sterile plants intend to

erfere precisely with the anther orlen development (Mariani et al.,1990,

ena et al., 1992, Poovaiah et al., 2002).

wever, transgenic male-sterile plants

oduce 50% male fertile plants due to

regation in the filial progeny. In a

brid seed production programme,

se fertile plants are considered highly

desirable. So, any strategy to develop

nsgenic male-sterile plants should

lude a plan for selective elimination of

se fertile plants.

the basis of this background

ormation, the following objectives are

ed

solation and charaterization of a novel

her specific promoter

solation of a few candidate genesfrom

e plant or other sources

reparation of a suitable gene construct

pable of causing male sterility

tandardization ofjute plant

nsformation protocol

reparation of a linked herbicide

istance gene construct to facilitateemical rouging of fertile plants

roduction

Isolation of genomic DNA, RNA and cDNA

synthesis:

The plant genomic DNA isolation was carried out

as described by Doyle and Doyle, 1990. Total

RNA from jute seedlings was isolated by Trizol

method (Simms et al., 1993). cDNA was prepared

by using anchored oligo-dT primer with the help

of Transcriptor First Strand cDNA Synthesis Kit

(Roche).

Standard molecular biological techniques:

Carried out as described by Sambrook et al.,

1989.

Agrobacterium mediated Plant transformation:

Suitable explants (leaf disc for tobacco and

embryonic meristem with attached petiole for

jute) were surface sterilized and taken for

transformation by Agrobacterium LBA4404

strain bearing the concerned genetically

engineered plasmid. After transformation and 3

days of co-cultivation, transformed explants were

placed on agar solidified MS basal media(containing 8mg/l IAA, 2.5mg/l kinetin, 50mg/l

hygromycin and 250mg/l cefatoxin for tobacco

whereas 0.5mg/l IBA, 0.5mg/l BAP, 15mg/l

hygromycin and 250mg/l cefatoxin for jute). After

8 weeks of subculture, individual plantlets were

transferred to glass bottles containing sand for

hardening and root development. After 2 weeks

of hardening, individual plants were transferred

to field.

Histochemical GUS assay:

As described by Jefferson, 1987.

Materials and methods

Results- 1. Anther specific novel promoter isolation

On the basis of the bioinformatics analysis with

the Pea END1 anther specific protein (Gomez et

al., 2004), a putative anther specific promoter

(OsASP) was isolated from rice genome by PCR

mediated cloning (Figure 2). OsASP-GUS-NOS

transformed transgenic tobacco plants showed

anther-predominant GUS expression in T1 and

T2 generations. Strong GUS expression was

observed in the anthers of 13.5mm to 45mm long

flower buds of transgenic tobacco plants (Figure

3).UncutPCR

produ

ct

pUC18/H

inf1

marker

AccI

ClaI

BamH

I

HindIII

1419bp

517bp396bp

214bp

UncutPCR

produ

ct

pUC18/H

inf1

marker

AccI

ClaI

BamH

I

HindIII

UncutPCR

produ

ct

pUC18/H

inf1

marker

AccI

ClaI

BamH

I

HindIII

pUC18/H

inf1

marker

AccI

ClaI

BamH

I

HindIII

1419bp

517bp396bp

214bp

HindIII BamHI

BglII

BglII ClaI

PstI

GUS

HygromycinR

LB

CaMV35S polyA

CaMV35S promoter

NospolyA

RBE ND p ro mo te r

HindIII BamHI

BglII

BglII ClaI

PstI

GUS

HygromycinR

LB

CaMV35S polyA

CaMV35S promoter

NospolyA

RBE ND p ro mo te r

HindIII BamHI

BglII

BglII ClaI

PstI

GUS

HygromycinR

LB

CaMV35S polyA

CaMV35S promoter

NospolyA

RBE ND p ro mo te r

AB

C D

Figure 2: A. Restriction

analysis of OsASP PCR

product B. Schematic

diagram of pCAMBIA-

OsASP-GUS construct,

C. Restriction analysis of

this construct and D.

OsASP-GUS

transformmed putative

transgenic tobacco plants

Results-2. Isolation, cloning, partial

characterization and preparation of

siRNA mediated gene silencing construc

of 3 potential candidate genes from Jut

seedling cDNA

Potential candidate genes are selected

the basis of the following strategies:

A. Deregulating the energy metabolism

the cells in the anther tissue (for t

purpose partial CDS of the gene encodi

beta subunit of F1 ATPase is cloned)

Deregulation of lipid metabolism in anth

tissue (for this purpose partial CDS of t

gene encoding Stearoyl ACP Desaturase

cloned) C. Cell ablation mediated by cy

skeletal damage (for this purpose part

CDS of the Actin gene is cloned)

A B C DFigure 4: Amino acid sequence alignment of the 3 cloned p

genes (A), partial characterization of them by South

hybridization (B), respective schematic diagram of the siR

mediated gene silencing construct s (C), and restrict

analysis of the concerned constructs.

Results-3. Standardization of a proto

for plantlet regeneration and gene

transformation in jute plantBest organogenic media for multi

shooting was found to be MS basal me

supplemented with 0.5mg/l IBA a

0.5mg/l BAP (taking embryonic merist

with attached petioles as the explant).

Transient GUS expression was found

the explants infected with Agrobacteri

LBA4404 strain harboring pCAMBIA13

2X35S-GUS-NOS plasmid. However,

plantlets could be recovered throu

hygromycin selection.

pUC

18/Hi

nf1m

arke

r

-ve

contr

ol

1 2 3 4 5 6 7 8

517bp

pUC

18/Hi

nf1m

arke

r

-ve

contr

ol

1 2 3 4 5 6 7 8

517bp

A

B

C

D

Figure 3: A. PCR

screening of transgenic

lines B. Southern

hybridization of PCR

positive plants C.

Comparative

histochemical GUS assay

of transgenic lines D. GUS

assay of transgenic tobacco

anther at different

developmental stages

Figure 5: Composition of different multiple shooting media

jute, their relative performance and transient histochemica

GUS assay of transformed jute explants

References:

1. Mariani C., De Beuckeleer M., Truettner J., Leemans J. and Goldberg R.B. (

Induction of male sterility in plants by a chimaeric ribonuclease gene. Nature. 3

737-741.

2. Spena A., Estruch J.J., Prensen E., Nacken W., Van Onckeler H. and Somme

(1992): Anther specific expression of the rolB gene ofAgrobacterium rhizogenes

increases IAA content in anthers and alters anther development and whole flow

growth. Theor. Appl. Genet. 84: 520-527.

3. Poovaiah B.W., Liu Z., Patil S. and Takezawa D. (2002): Compositions and

methods for production of male sterile plants.Patent No. US6.362.395.

4. Gomez, M.D., Beltran J.P. and Canas L.A. (2004): The pea END1 promoter d

anther specific gene expression in different plant species. Planta. 219: 967-981.

5. Doyle J. and Doyle J. (1990): Isolation of plant DNA from fresh tissue. BRL

FOCUS. 12: 13-15.

6. Sambrook, J., Fritisch, E.F. and Maniatis, T. (1989): Molecular cloning: a

Laboratory Manual, 2nd edition. Cold Spring Harbor, NY: Cold Spring Harbo

Laboratory.

7. Jefferson R A (1987): Assaying Chimeric Genes in Plants: The GUS Gene Fu

System.Plant Molecular Biology Reporter. 5: 387-405.

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