ЭлБиб

PLANT GENETIC TRANSFORMATION, CELL SELECTION AND SOMATIC HYBRIDIZATION

GENETIC TRANSFORMATION OF

ARABIDOPSIS

AGROBACTERIUM

INOCULATION IN PLANTAASANALTERNATIVE

OFIN VITRO

TRANSFORMATIONMETHOD

T .A .Ezhova, O.P.Soldatova, L.B.Mamanova, I.P.Polyanskaja, E.A.Klimov

Biologicalfaculty, MoscowState University, 119899Moscow, Russia. E-Mail:

For obtaining of insertion mutants the transformation procedure was

performed by inoculation of

Agrobacterium tumefaciens

suspension (strain

LBA4404) at the incision sites of stems of 3 weeks-old Arabidopsis plants

by method of Chang et al. 1994 (Plant J., 5 ,4 :551 -558 ). The vector pLD4

that is a derivative of the binary plasmid vectorpBI121 (Gapeevae tal., 1995,

Russ. J. of Genetics, 31, 8: 1085 - 1091) was used. The screening of

transformed plants was based on assaying the expression of the T-DNA-

encoded marker genes

npt11

and

uidA,

which provided kanamycin (Km)

resistance and B-glucuronidase (GUS) activity. TheT2 seeds were harvested

from individual newly formed shoots and were placed on media containing

100 mg/l Km and on the soil. Km-resistant seedlings were selected in 19%

of 288 progenies. In different progenies the frequency of Km-resistant

seedlings varied from 4 up to 72% .

The mutants with altered morphology of vegetative and generative features

and one chimeric chlorophyll plant were found in 8 of T2 progenies grown in

the soil. Only in two of these mutants the presence o f GUS activity was

found. The progeny plants (T3 generation) obtained from the GUS -positive

T2 mutants were Km-resistant and GUS-positive. The examination of co

segregation o f mutant phenotype and T-DNA marker genes is needed for

final proof o f insertion nature of the mutations.

The high transformation frequency and the possibility to obviate the

need fo r plant regeneration from tissue explants

in vitro

allow to regard the in

planta method as successful alternative to

in vitro

transformation approach.

Acknowledgements. The investigation was partly supported by the grant

o f Russian Foundation of Basic Research and grant from the Russian State

Programme "Frontiers in Genetics".

255

Научная электронная библиотека ЦНСХБ