Somatic embryogenesis and genetic transformation of sugarcane variety CoJ 64 using Cry1A(c) gene
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Date
2011
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Punjab Agricultural University, Ludhiana
Abstract
The present investigation entitled, “Somatic embryogenesis and genetic transformation of sugarcane variety CoJ 64 using Cry1A(c) gene” was undertaken on a high sugared variety of sugarcane viz., CoJ 64. Maximum per cent somatic embryogenesis and regeneration was observed on MS medium supplemented with NAA (5 mgL-1) + Kin (0.5 mgL-1) + sucrose (3%) + agar (8 gL-1), i.e. 79.63 and 75.17, respectively. Changing the agar (8 gL-1) concentration to 16 gL-1 in above medium improved somatic embryogenesis (84.87%) and regeneration (80.68%). Sucrose replaced with maltose significantly increased the frequency of somatic embryogenesis (97.61%) and plant regeneration (93.74%). Use of glutamine also enhanced per cent somatic embryogenesis (89.97) and percent plant regeneration (85.82). Activated charcoal (1.0 gL-1) along with citric acid (100 mgL-1) controlled the problem of browning of medium and also promoted somatic embryogenesis (90.33%) and plant regeneration (86.19%). For genetic transformation, particle gun method was employed. The already standardized parameters were used for carrying out bombardments. The embryogenic regions of cultured leaf rolls were used as the target tissue and co-bombardment was done using two plasmids: one carrying gusA and hpt genes, and the other carrying Cry1A(c) gene. The selection of bombarded embryogenic leaf segments was carried out on MS + NAA (5 mgL-1) + Kin (0.5 mgL-1) + Sucrose (3%) + hygromycin B (40 mgL-1). Out of 860 embryogenic leaf segments bombarded, 96 putative transgenic plants were obtained. Histochemical GUS assay revealed two GUS-positive plants (2.08% expression). PCR analysis of all the putative transgenics with a gene-specific primer confirmed the integration of the gene in 24 plants (25.00% expression). The plants were hardened and successfully transferred to the glasshouse.
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