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Author: Dhillon, Navjot Kaur × Start date: 2000 × Type: Thesis × Thesis Type: M.Sc ×
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    ThesisItemOpen Access
    STUDIES ON SOMATIC EMBRYOGENESIS AND GENETIC TRANSFORMATION IN MAIZE
    (Punjab Agricultural University, Ludhiana, 2012) Dhillon, Navjot Kaur
    The present investigation entitled, “Studies on somatic embryogenesis and genetic transformation in maize” was undertaken using maize inbreds viz., LM 5, LM 6, LM 13, LM 15 and LM 16. Callus induction was studied on ten media compositions based on MS (1962) salts using three explants, viz., immature embryos, mature embryos and split seeds. Among five maize inbreds thus investigated, LM 13 was most responsive to tissue culture. Highest (88.7%) callusing was observed from immature embryos cultured on MS + 2,4-D (3.0 mgL-1 ) + Picloram (10.0 mgL-1). Whereas, the maximum (88.6%) callus induction using mature embryos was observed on MS + Picloram (10.0 mgL-1) + BAP (0.5 mgL-1). Subcultured calli exhibited frequent somatic embryogenesis and the highest (72.6%) somatic embryogenesis was observed in inbred LM 13 on MS + Picloram (10.0 mgL-1) + BAP (0.3 mgL-1). The addition of proline, casein hydrolysate, silver nitrate and sucrose enhanced somatic embryogenesis in all the calli. Whereas, the cefotaxime did not have much effect on somatic embryogenesis. Embrogenic calli, upon their transfer to regeneration medium, exhibited shoot bud/shoot and root regeneration. The maximum (71.6%) shoot regeneration in LM 13 from immature embryo-derived calli was achieved on MS + Proline (3.0 mgL-1) + BAP (3.0 mgL-1) + Kin (0.5 mgL-1) + Gelrite (2.0%). Whereas, the mature embryo-derived calli of same inbred cultured on same regeneration medium exhibited poor (26.6%) shoot regeneration. Genetic transformation was attempted with particle gun (Bio Rad) using two target tissues viz. embroygenic calli and immature embryos. The bombardment was done using plasmids carrying glyI, glyII genes (for abiotic stress tolerance) and Cry1A(c) gene (for insect resistance). The bombarded tissues were selected using hygromycin (35 ppm) for two cycles of selection of two weeks each. In case of glyII gene bombardment, a total 146 plants were regenerated from which 42 plants (43.29%) were GUS positive. Further, the PCR analysis revealed the presence of glyII gene in 6 plants (4.10%). Whereas, in case of Cry1A(c) gene, a total 171 immature embryos were bombarded from which 2 plants (1.16%) were found to be PCR positive. The tissue cultured regenerated plants from all the bombarded tissues were grown in the transgenic glass house for collection of seeds. None of the plants carrying glyII gene set any seed. Whereas, four seeds have been collected from a plant carrying Cry1A(c) gene.