Development of plant varieties with a high level of tolerance to abiotic stresses is crucial for establishing full yield potential and to stabilize production. Due to the multitude of abiotic stresses and their complex genetic control the progress of breeding for tolerance to abiotic stresses using conventional approaches has not been very rewarding. Recent advances in cellular and molecular biology have made it possible to clone important genes and mobilize them in any organism across barriers of sexual hybridization for stable expression and transmission. All living organisms have evolved mechanisms for avoidance and/or tolerance to one or more of the abiotic stresses. Plants producing crucial enzymes or proteins from various organisms involved in abiotic stress tolerance mechanisms have shown significant advantage over their wild type controls under stressed environment. The enhanced level of compatible osmolytes, radical scavengers and other transgene products correlated with the degree of tolerance. Further understanding of the molecular mechanisms of stress perception, signal transduction and response by plants and other organisms may help to engineer plants with high levels of tolerance to multiple stresses. Perspectives and additional approaches for further improving the tolerance to abiotic stresses through genetic engineering are discussed.
Transformed sweet potato plants were obtained from embryogenic calli following Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA101/pIG121-Hm used in the present study contained a binary vector with genes for β-glucuronidase (gusA) and hygromycin resistance (hpt). Around ten hygromycin resistant cell clusters were produced from 1g fresh weight of the infected embryogenic calli. The hygromycin resistant plantlets were regenerated from 53.1% of the hygromycin-resistant calli. Histochemical GUS assay and Southern hybridization analysis indicated that these plants were stably transformed with a copy number of introduced genes of one to three. Transgenic plants grew normally and formed storage roots after 3 months of cultivation in a green house.
To clarify the molecular biological aspects occurring during the induction of somatic embryogenesis, the stress-induced changes in protein pattern and the proteins associated with somatic embryogenesis induced by stress in carrot (Daucus carota L.) were investigated. In two dimensional polyacrylamide gel electrophoresis (2D-PAGE), 6 spots with molecular weight of 63-65 kilo Dalton (KDa) showing pI ranging from 6.0-6.5, and 2 spots of molecular weight 45KDa with a pI of 5.0 were detected in the extracts of apical tip segments treated with cadmium chloride, sucrose and sodium chloride. Three spots out of the 6 spots of 63-65KDa molecular weight and the two 45KDa spots were also detected in the extracts of 2, 4-dichlorophenoxy acetic acid (2, 4-D)-induced embryogenic cells, but not in those of non-embryogenic cells. Furthermore, these 5 spots were also detected in the extracts of hypocotyl segments cultured with 2, 4-D for more than 4 weeks. Judging from the relationship between accumulation of these proteins and acquisition of embryogenic competence, these proteins may be related to acquisition and/or maintenance of embryogenic competence.
Somaclonal variations in flower and inflorescence axis were investigated among the plants micropropagated through protocorm-like bodies induced by flower stalk bud culture of various cultivars of Phalaenopsis and Doritaenopsis. Eighteen hundred to 14, 750 micropropagated plants were grown for each of 11 genotypes in greenhouses for 1-1.5 years till flowering. Somaclonal variations appeared in flower and inflorescence were classified into 9 categories irrespective of cultivars. The frequencies of these somaclonal variations in each genotype ranged from 0 to 100%, but most of the cultivars showed variations less than 10%. Changes in leaf shapes and ploidy level were not detected in these variants. Possible mechanisms involved in these variations and the problems of the micropropagation method used in this study were discussed.
Embryogenic calli induced from leaf segments of grapevine (Vitis vinifera L. cv. Koshusanjaku) were co-cultivated for 5 days with Agrobacterium tumefaciens strains EHA101 (pIG121Hm) or LBA4404 (pTOK233), both of which contained the plasmid carrying the neomycin phosphotransferase II (NPT II), hygromycin phosphotransferase (HPT) and the β-glucuronidase (GUS) genes. Putative transgenic calli were selected on 2g/l gellan gum-solidified Nitsch's medium (1969) containing 50mg/l kanamycin and 20g/l sucrose after co-cultivation with A. tumefaciens. Transformation frequency of the embryogenic calli evaluated by GUS histochemical assay was increased by the addition of acetosyringone to co-culture medium. Complete transgenic plants were selected among secondary embryos formed on the surface of embryos in the presence of kanamycin. Finally, kanamycin-resistant plants expressing GUS gene were obtained. PCR analysis confirmed their transgenic nature by detecting GUS and NPT II genes.