Development of Cadmium Tolerant Rice Cultivars Through Agrobacterium Mediated Genetic Transformation

Abstract

Rice (Oryza sativa L.) is one of the most common and widely used food cereals, which supports the world population. In vitro micropropagation, breeding and genetic transformation tools are the best techniques for the rice development. Cadmium (Cd) is the most dangerous heavy metal present in the environment and poses a significant risk to human health. Cadmium is accumulated through the excessive use of water for irrigation. The situation gets worst even for the use of mineral fertilizers. Besides being toxic to the plants, cadmium may be absorbed by some crops through the roots, translocation to the seedling and finally accumulated into edible parts such as kernels, creating long term disastrous impacts on human and animal health. In this regard, rice is particularly at risk due to the peculiar agricultural practices. To determine a suitable media composition for callus induction and plant regeneration of rice through somatic embryogenesis, MS media composition and phytohormones (i.e., auxin; 2,4-D, IAA, IBA and cytokinin; BAP, Kin) were tested for high callus induction and plant regeneration. The combinations of 2.0 mg/l 2,4-D + 0.5 mg/l NAA were found suitable inducing high amount of calli and 2.0 mg/l BAP + 1.0 mg/l NAA + 1.5 mg/l Kin were found to be most effective for plant regeneration. Among the experimental varieties BRRIdhan28 shows the highest result for calli induction. Histological analysis is one of the most important parts of Agrobacterium-mediated genetic transformation. In microscopic observations, embryogenic cells and nonembryogenic cells were found. The combination 2.0 mg/l 2,4-D + 0.5 mg/l NAA was found suitable inducing high amount of embryogenic calli. The investigation of targeted YCF1 gene transformation was carried out for GUS gene through Agrobacterium-mediated genetic transformation in calli of rice. Incubation period of 30 minutes was found to be the most effective for infection and integration of foreign genes into callus. GUS expression was also observed in the leaf of putative plants prior to final confirmation of transformation through PCR amplification. After 4 days of co-cultivation in calli and the leaves from putative plants were taken and incubated in X-gluc buffer. Indigo blue coloration was observed in calli and leaves. GUS gene was amplified in transformed plant DNA and plasmid DNA where forward and reverse primer amplified 880 bp GUS gene segment. An efficient Agrobacterium-mediated genetic transformation method was established for Oryza sativa by investing several factors responsible for successful gene transfer. Fifteen to twenty days old calli explants from in vitro grown were cocultivated with Agrobacterium strain GV3101 harboring the binary vector EnPCAMBIA1302-YCF1, which contained the hygromycin phosphotransferase (HPTII) gene as a selectable marker and the yeast cadmium factor 1 (YCF1) gene. Three days co-cultivation period on shoot induction medium (MS medium supplemented with 2.0 mg/l BAP, 1.o mg/l NAA and 1.5 mg/l Kin) containing 20 mg/l acetosyringone and five days delaying exposure of explants to selective agent enhanced transformation efficiency significantly. A two steps selection strategy was developed to select hygromycin (15 mg/l and 20 mg/l) resistant shoots. The hygromycin resistance selected shoots were transferred sub-sequently into the rooted media which contained with 1/2 strength of MS medium and 0.4 mg/l IBA. After 10-12 days, the shoots were shown in rooted. Rooted plantlets were transferred into potsoil, hardened and grown in the well decorated net house until maturity of the plant. Using the optimized transformation procedure, transformation efficiency reached at 16.2% in the study. Segregation analysis of T1 progeny showed that the transgenes were stably integrated and transmitted to the progeny in a Mendelian fashion. Another result also observed in regenerated green shoots turned into white or purple when high concentration of hygromycin (30 mg/l) was used in MS media. Expectedly PCR analysis, transgenic lines viewed 734 bp bands representing the YCF1 fragment and a 501 bp band representing the HPTII fragment. Morphological characteristics also observed through control and newly transform (YCF1) gene into rice genotypes. Into the experiment BRRIdhan29 (control) showed leaf length 48.33 ± 0.90 cm, leaf width 1.24 ± 0.02 cm, plant height 78.39 ± 1.34 cm, number of tiller 24.77 ± 0.84, flag leaf length 29.80 ± 1.00 cm, flag leaf width 1.56 ± 0.02 cm, panicle length 27.00 ± 0.57 cm, spikelet number 12.80 ± 0.26, number of grains 163.00 ± 1.36, 100 grains weight 2.42 ± 0.01 gm, grain length 1.04 ± 0.01 cm and grain width 0.21 ± 0.02 (cm). Whereas it was found 48.33 ± 0.85 cm, 1.24 ± 0.02 cm, 78.06 ± 1.34 cm, 17.10 ± 0.84, 35.40 ± 0.97 cm, 1.94 ± 0.02 cm, 27.80 ± 0.57 cm, 11.60 ± 0.21, 150.60 ± 0.21, 1.85 ± 0.002 gm, 0.86 ± 0.01 cm and 0.19 ± 0.002 cm was found transgenic rice cultivar of BRRIdhan29. The same parameters also observed control and transgenic BRRIdhan28 and Localdhan Lalmota in the experiment. Off the study BRRIdhan28 and BRRIdhan29 were also noted the dwarf characteristics, chlorosis and sterility male and female. The germination rate, coleoptiles growth and root growth of collected seeds from transgenic plants were recorded where used different concentrations of CdCl2.H2O (0.5 mg/l to 9.5 mg/l), BAP (2.0 mg/l) and kinetin (1.5 mg/l) were also applying in MS media for the experiment. Seed germination rate were decreased proportionately with increasing of cadmium chloride concentration. At the end of 72 hours in incubation period of with 9.5 mg/l CdCl2.H2O decreased the germination rate 80%. When the CdCl2.H2O concentration was 9.5 mg/l inhibited the germination rate. On the other hand, the root growth was also inhibited when used 8.5 mg/l to 9.5 mg/l CdCl2.H2O and 7.5 mg/l to 9.5 mg/l CdCl2.H2O concentrations the coleoptiles growth also inhibited in same compositions of media. On the basis of molecular characterization, the present investigations were carried out to analyze genetic diversity among the six rice genotypes. Genetic variation (three controls and three transgenic) were evaluated using RAPD, ISSR and SSR as molecular markers. Among the four RAPD primers tested, one produced polymorphic bands with an average of 5.75 bands per primer, ranging from approximately 434 to 1200 bp and 66.66% was polymorphic. Four ISSR primers tested one produced polymorphic bands with an average of 7 bands per primer, ranging from approximately 308 to 1500 bp and 84.52% were polymorphic. Among the tested three SSR primers, only one amplified polymorphic SSR alleles with an average number of 4.33 bands per primer, ranging from approximately 233 to 950 bp and 100% polymorphism was observed. Based on band polymorphisms generated by RAPD, ISSR and SSR after using primers, the highest similarities were showed between BRRIdhan28 and BRRIdhan29 (control and transgenic), similarity co-efficient value 0.85. Compared to the transgenic plant (YCF1) and control varieties BRRIdhan28, BRRIdhan29 and Localdhan Lalmota play an important role in germination rate, coleoptiles growth and root growth in the MS medium containing with CdCl2.H2O. Tillering, flag leaf length, leaf width and plant height is better in transgenic plants than control varieties.