STUDIES ON SOMATIC EMBRYOGENESIS AND GENETIC TRANSFORMATION IN MAIZE
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Date
2012
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Punjab Agricultural University, Ludhiana
Abstract
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.
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