'Plant Breeding: Sustaining the Future' Abstracts of the XVIth EUCARPIA Congress, Edinburgh, Scotland, 10-14 September 2001 ENGINEERING ABIOTIC STRESS TOLERANCE IN TOBACCO T.K. KONSTANTINOVA, D.P. PARVANOVA, A.I. ATANASSOV, D.L. DJILIANOV AgroBioInstitute, 2232-Kostinbrod, Bulgaria

Drought, low and high temperatures are the major environmental stresses that affect crop productivity.  To cope with dehydration the common consequence of abiotic stresses, micro-organisms and wild plants often accumulate compatible solutes, thus maintaining cellular membranes integrity upon stress and rehydration.  Our aim was to trigger overaccumulation of proline and to induce fructan or glycinebetaine accumulation in a non-producing crop to improve its abiotic stress tolerance.  The tobacco cultivars Nevrokop 1146, Burley 21, Coker 254, (oriental, Burley and flue-cured types, resp.) were used in our study.  We transferred the genes encoding δ-Pyrroline-5-carboxylate-synthetase controlled by VacP5Cs or AtP5Cs, levansucrase – by SacB and choline oxidase – by codA.  The leaf disc transformation method was used.  Antibiotics were added to MS for screening.  PCRs with specific primers were performed.  Regenerated transformants were cloned for further studies, adapted and grown in plastic pots.  We developed a fast screening system for chilling and freezing tolerance.  In vitro cloned plants were potted in greenhouse (24˚C).  After 20 days the potplants were transferred to 2˚C for 120 hours and then to -2˚C for at least 24 hours under normal light/dark conditions.  After freezing, plants were allowed to recover at normal temperature.  Proline, fructan and glycinebetaine resp. was measured before stress, after acclimatisation and after freezing.  More than 650 T0 transformants were selected as Kn or Hyg resistant.  Further on 48 were confirmed as AtP5Cs-transformed, 97 – as VacP5Cs-carrying, 105 – as SacB-containing and 41 – as codA-transformed.  Transformants carrying the respective osmoprotectants survive 50 days in vitro on 40%PEG and recover, while the wild-type plants lack roots and die. Young wild-type tobacco seedlings tolerate chilling temperature (2˚C) easily.  Their proline content increases 5-6 times during acclimatisation.  Sub-zero temperatures, however, are damaging for them even for several hours.  Some of our transformants survive both chilling and freezing showing significantly high levels of respective osmoprotectants under chilling and frost stresses.  The freezing tolerance is steadily inherited in T1 progenies and the levels of osmolytes are much higher.  Further studies are in progress.