ЭлБиб

PLANT GENETIC TRANSFORMATION, CELL SELECTION AND SOMATIC HYBRIDIZATION

PRODUCTIONOFTHETRANSGENICPHOSPHINOTHRICIN-RESISTANT

PLANTSANDANALYSISOFTHEBARGENEINHERITANCEFOR

COMMERCIALPEALINES

Y.V.Simonenko,N.V.Kuchuk,Y.Y.Gleba

Institute ofcell biology andgenetic engineeringNationalAcademy ofSciences of Ukraine,

Zabolotnogo str., 148, Kiev, DSP-22, 252650

UkraineE-Mail:

iicb@iicb.kiev,

Double transform ation system w as used fo r production o f transgenic

phosphinothricin-resistant plants o f comm ercial pea lines

(Pisum sativum

L .).

This system consists o f two steps: 1) production o f regenerable pea

lines after transform ation w ith "shooty" mutant о

f A.tumefaciens

pGV2206;

2) transform ation of regenerable lines with the plasm id pK3 containing two

genes: bar and nptll. After transformation o f 8 primary pea lines with pGV2206

plasm id three regenerable lines were selected, which were used in the further

tra n s fo rm a tio n expe rim en ts. H e rb icid e -re sista n t pea lines have been

produced after transform ation o f regenerable lines w ith "binary” vectors

A.tumefacience

pMP90 pK3 and pGV2206 pK3. T ransform ed callus w as

selected on the medium containing 10 mg/L o f ammonium glufosinate. Shoot

regeneration via organogenesis began after several passages on regeneration

m edium . T ransform ed plants were selected on the m edium containing 10

m g/L o f amm onium glufosinate. The obtained herbicide resistant shoots

w ere grafted on to normal pea plants in greenhouse conditions fo r seed

production, because root form ation are suppressed in these lines. Besides

this w

have developed the method o f seed production

in vitro.

The bar and

nptll genes expressed in both the prim ary transgenic pea plants and in the

next generation progeny, w here they showed a typ ical 3:1 M endelian

inheritance. The transm ission o f the introduced gene into the progeny o f the

tra n s g e n ic p la n ts w a s stu d ie d o ve r tw o g e n e ra tio n s, and the sta b le

transm ission was shown to take place. Transformation o f regenerated plants

w as confirm ed by P C R -a n a lysis, Southern blot hybridisation and assays

fo r neomycin phosphotransferase.

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