The Study Of Genetic Transformation, Plant Regeneration And Acdclimation Of Cucumber (Cucumis Sativus L.)

Cucumber (Cucumis sativus L.) is grown worldwide as an important vegetable. On the one hand, establishing an efficient cucumber transformation system is important for functional genomics research through techniques such as gene over-expression, reporter gene expression and RNA interference. On the other hand, Improving the biotic or abiotic resistance of cucumber through conventional breeding is limited by its narrow genetic basis and incompatibility barriers to wild Cucumis species. Therefore, it would be desirable to apply genetic engineering to expand the germplasm base of this crop. In this study, a European genotype EP-6and a Northern China genotype Jinchun4were selected to study the callus fomation, transformation, heterogenous gene transient expression and plant regeneration technology of cucumber.1Two kinds of cucumber a-galactosidase-EGFP fusion expression vector controlled by the CaMV35S promoter were constructed. CaMV35S promoter sequence was amplified by polymerase chain reactions (PCR) from vector pCambia1303; The entire coding region of EGFP was amplified by PCR from vector pEGFP-Nl; The entire coding region of cucumber acid a-galactosidase I and alkaline a-galactosidase I were amplified by Reverse transcript PCR using cucumber leaf total RNAs as template. The three above-mentioned fragments were Inserted into the mμltiple cloning sites of expression vector pCambia1381*c. Then two a-galactosidase-EGFP fusion expression vectors were constructed respectively and the EGFP gene was located at the C-terminal of the target genes.2To obtain the suitalbe material for transient expression of heterogenous genes, friable calli can be produced on the medium with6-BA1.0mg/L,NAA0.1mg/L,NH4NO32.0g/L and Yeast extrat5g/L. Two recombinant plasmids were transformed into onion epidermal cells and cucumber callus cells by particle bombardment. Green fluorescence signals were monitored by a confocal microscope. The results showed that cucumber acid a-galactosidase I is located in the whole cells includeing the vacuole, while cucumber alkaline a-galactosidase I is located at the nucleus and the peripheral region of the cell.3Regeneration of cucumber plants:Cotyledon is a suitable explant for in vitro shoot induction of EP-6, while for Jinchun4, highest shoot induction rate was obtained if stem node was used as explant. The optimal culture medium of shoot induction of EP-6cotyledons was MS+0.5mg/L ABA+2.0mg/L6-BA+2.0mg/L AgNO3, while the best culture medium for Jinchun4shoot induction was MS+0.5mg/L6-BA. The highest root induction rate can be observed when the regenerated shoots were cultured on the medium1/2MS+15g/L sucrose+0.6mg/L IBA.4The transfomation system of EP-6cotyledons:After2days of pre-culture on the optimized shoot induction medium, the cotyledons were incubated with the suspension of pCAMBIA2201/EHA105(Agrobacterium tumefacieus stains/binary vector) for20min, then co-cultured for2days on the same medium containing50μmol/L AS in the dark. Explants were further transferred to the plant selection medium(optimal shoot induction medium+400mg/L Cef) for about25d, the antibiotics-resistant shoots were transferred to shoot elongation medium until they were2cm long. Shoots were excised from the explants and placed on the optimal root induction medium. Both shoot elongation medium and rooting-culture medium contained60mg/L Kan and400mg/L Cef.5The transfomation system of Jinchun4stem nodes:After2days of pre-culture on the optimized shoot induction medium, the stem nodes were incubated with the suspension of pCAMBIA2201/EHA105(Agrobacterium tumefacieus stains/binary vector) for20min, then co-cultured for2days on the same medium containing50μmol/L AS in the dark. Explants were further transferred to the plant selection medium(optimal shoot induction medium+400mg/L Cef) for about12d, the antibiotics-resistant shoots were transferred to shoot elongation medium until they were2cm long. Shoots were excised from the explants and placed on the optimal root induction medium. Both shoot elongation medium and rooting-culture medium contained80mg/L Kan and400mg/L Cef.6From explant co-culture to well rooted putative transgenic plantlets, the transfomation systems of Jinchun4stem nodes and EP-6cotyledons needed42d and59d respectively. The transformation efficiency can be evaluated as0.83%for the Jinchun4stem node system and0.36%for the EP-6cotyledon system by RT-PCR positive plantlets.7The highest survival rate of98.7%of transgenic plants can be obtained when the plantlets were rooted in the1/2MS with6%sucrose for4weeks, then transferred to the1/2MS with3%sucrose for1week, and finally transplanted to the substrate peat:perlite:sand=1:1:1.