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S O M A T IC H Y B R ID IZ A T IO N
STRATEGY FOR CROP IMPROVEMENTBY TRANSFER O FTUBULIN
GENES CONFERRING RESISTANCE TO HERBICIDES-MITOTIC
DISRUPTERS
A.Yemets, O.Kundelchuk, V .Solodushko, A.Smertenko, Ya.B.Blume
Inst. Cell Biol. & Genet. Eng., Natl. Acad. Sci. o fUkraine, acad. Zabolotny str., 148, Kiev
GSP-22,252650, Ukraine ;
H erb icid e resistant plants rep re sen t one o f the first com m e rcia lized
products on the agricultural m arket created by plant biotechnology. Previously
m u ta n ts o f N ic o tia n a p lu m b a g in ifo lia r e s is ta n t to h e r b ic id e s w ith
a n tim ic r o tu b u la r a c tiv ity - a m ip ro p h o s m e th y l, А Р М , (fro m
phosphoroam idates) and trifluralin, TFL, (from dinitroanilines) - w ere obtained
by in vitro selection in our lab. These lines are nuclear mutants w ith altered
β-tubulin, w hich provide resistance o f m icrotubular arrays to АРМ or TFL,
respectively.
On next step we tried to tra n s fe r th is h e rb icid e -re sista n ce to o the r
species o f S o la n a c e a e by m eans of asymm etric hybridization. M esophyll
protoplasts of N. s y lvestris and A . belladonna were utilized as recipients for
fusion o f protoplasts in interspecific and intertribal com binations w ith mutant
material, which was irradiated before fusion. The different АРМ - and TFL-
resistant clones of N. plumbaginifolia + N. sylve stris and N. plumbaginifolia
+ A. belladonna were obtained after appropriate selection. The regenerated
clones in these com b ina tion s had recipient phenotypes. The cytogene tic
analysis of chromosome sets of hybrids confirm ed their highly asymmetric
n a tu re . Im m u n o flu o re s c e n t m icro scop y a n a lysis show n th a t in te rph a se
m icrotubular networks and m itotic figers of hybrid protoplasts don’t destroyed
a fte r АРМ or TFL treatm ents as m utant ones. 2-D electrophoresis results
th a t АРМ -re sistan t as w ell as TF L-re sistant hybrids expressed m utant β-
tubu lin isoform s from respective parents. It w as shown that purified tubulins
o f hybrids binds herbicides more slightly than tubulin of N . plumbaginifolia
w ild type. АРМ and TFL resistance inh e rite d in F1 o f se le cte d hybrids.
These results allow us to assume that produced hybrids inherited altered β-
tubu lin genes from mutant N.plumbaginifolia.
S ince these m utant genes o f tubulin conferring resistance to respective
h e rb ic id e s d o n 't isola ted yet, a sym m e tric h yb rid iza tio n can be used fo r
tra n s fe r o f such genes to d iffe re n t crops. A t p resent w e are ca rrying out
e xpe rim en ts on tra n sfe r of d in itroaniline resistance from natural resistant
b io typ e E leu sin e indica, w h ich e xpresses m u ta n t isoform o f tu b u lin , to
m onocotyledon species. As alternative w e plan to identify and clone mutant
genes o f tubulin from mutant N. plumbaginifolia fo r genetic transform ation of
plant material.
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