Citation
Nguyen, Thi Mai Anh
(2005)
In Vitro Culture Of Maize (Zea Mays L.) Inbred Line Sm5-4.
Masters thesis, Universiti Putra Malaysia.
Abstract
Tissue culture of inbred line SM5-4 maize (Zea mays L.), maternal parent of Putra J-
58 (F1 hybrids) was established using maize zygotic embryo as explant. To obtain
embryogenic callus from mature and immature zygotic embryos of inbred line SM5-
4, manipulations of media components such as carbon, nitrogen, proline, and casein
hydrolysate, and culture conditions such as incubation temperature, light, were
carried out. Immature embryos have the ability to form callus and embryogenic calli,
which can result in plant regeneration.
At tissue culture level, the study aims at establishing the best tissue culture system
via somatic embryogenesis and to overcome plant regeneration problems by
manipulating the sucrose concentration, hormone combination and concentration,
culture age, the type of medium formulation used to grow callus, incubation
temperature, light and media formulation.
Sterilization technique of maize from mature and immature maize seeds of the inbred
line SM5-4 was investigated. Mature seeds (50 days old) and immature seeds (14
days after pollination) were disinfected by washing in different concentrations of
sodium hypochlorite (Clorox) for different duration. Disinfection in 50%(v/v) Clorox
solution (2.27% sodium hypochlorite) for 20 minutes gave 90% of contaminationfree
culture of mature seeds whereas 50%(v/v) Clorox solution (2.27% sodium
hypochlorite) for 15 minutes gave 75% of contamination-free culture of immature
seeds. Reduction in Clorox concentration of 20% (v/v) Clorox (1.05% sodium
hypochlorite) for 20 minutes gave high percentage (67%) of contamination- free
culture of immature seeds that remain viable.
N6 basal medium was found to be the best medium in enhancing both callus
induction and embryogenic calli formation. The highest callus induction frequency
on N6 basal medium supplemented with 9 pM 2,4-D from immature zygotic
embryos was 79.5%. Both plants growth regulators, 2,4-D, IAA and BAP, kinetin,
were capable of switching on the induction of callus necessary for embryogenic
totipotency. The combination of 2,4-D and kinetin were however more effective in
producing callus induction in embryos culture of maize.
The most effective method for producing friable, embryogenic callus was found for
immature zygotic embryos. Maturation of somatic embryos was enhanced by
transferring the embryogenic callus after 4 weeks to medium containing 6% sucrose
and Img/L NAA. During the following 3-4 weeks, as the somatic embryos
developed, the cultures were transferred to the regeneration medium (MSO).
Approximately 80% of immature zygotic embryos produced embryogenic callus and
then plantlets. Immature embryos of inbred line SM5-4 produced the highest
percentage of callus and showed the highest number of plant regeneration compared
to mature zygotic embryos.
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