Maize
Genetics Cooperation Newsletter 80. 2006.
North Carolina State University
Genetic engineering of maize is dependent on the development of efficient and reliable callus induction and a plant regeneration system. Mature embryos are a potentially useful alternative to immature embryos because they can be stored in the form of dried seeds and are available at all times. In the present work, we investigated the effect of auxin and cytokinin on maize callus induction and plant regeneration from mature embryo of maize.
Inbred lines A188, B73 and their hybrid (A188 X B73) were used as the source for mature embryo culture. The seeds were surface-sterilized for 1 min in 75% ethanol and rinsed three times with sterilized, deionized water. The seeds were then surface–sterilized for 10 min in 10% (v/v) commercial bleach containing two drops of Tween 20 followed by six rinses with sterilized, deionized water. Mature embryos were excised from the seed and placed onto the surface of callus induction medium (IM). The IM was MS medium supplemented with 2,4-D at different concentrations (1, 2, 4, 6, 8, 10 and 12 mg/L), KT (0 to 0.5 mg/L), 3% sucrose, and 0.8% agar. After 8 days, root and bud were removed from mature embryo. Cultures were transferred at two-week intervals to fresh IM medium. All the cultures were incubated at 26�C in darkness. After calli were induced, all calli were transferred to regeneration medium to induce plantlets and placed in a growth chamber at 26�C. Regeneration medium contained MS inorganic salts at half strength supplemented with the same organic nutrients as the callus induction media and the cytokinin benzylaminopurine (BA) at different concentrations (0, 0.2, 0.4, 0.6, 0.8, 1 and 2 mg/L). Regenerating calli were cultured in a growth chamber at 26�C with light. Plantlets with developed roots were transferred to a sand-soil mixture in plastic pots and placed in the growth chamber for two weeks at 26�C. After two weeks, plants were transplanted and grew in the greenhouse till setting seed.
Callus induction was strongly influenced by the type and dose of auxin. Two main types of calli were observed in the cultures (non-embryogenic and embryogenic callus). Fresh weight of calli increased when the concentration of 2,4-D was raised from 1 to 4 mg/L, while the rate of embryogenic calli decreased when the concentration of 2,4-D was changed from 6 to 12 mg/L. The medium containing KT was effective in promoting a greater frequency of embryogenic callus. The combination of 2,4-D (4 mg/L)+ KT (0.5 mg/L) was the most effective for producing embryogenic callus. Genotype was closely related to callus production from the mature embryo. More calli were induced from the mature embryos of inbred lines A188 and B73 than those of hybrid line A188 X B73. For plant regeneration, BA had a significant effect on the regeneration of embryogenic callus; a high concentration of BA (1mg/L and up) caused a decrease in the rate of regenerated plantlets. Rate of plantlet regeneration was the highest in the medium containing 0.6 mg/L of BA. Under the most optimal conditions, the frequency of plant regeneration could be as high as 25.7%, 33.1% and 21.6% for inbred lines A188 and B73, and their hybrid line (A188 X B73), respectively.
In conclusion, it was shown that auxin and cytokinin concentration had significant effects on callus induction and plant regeneration from mature embryos in maize. The greatest callus growth occurred when the medium included 2,4-D, at a 4mg/L concentration, in combination with KT (0.5 mg/L). The addition of BA (0.6 mg/L) in regeneration medium was the most effective in promoting plant regeneration of embryogenic callus in this study.
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