Regeneration of two maize genotypes (Zea mays L) from mature embryos through callus initiation using split seed technique
Plant regeneration from single or few cells is a prerequisite for effective selection of transformed cells and to minimize the event of chimeras during transformation. This can only be achieved if plants are regenerated through callus initiation. To date immature embryos have been widely used as explants for maize (Zea mays L.) plant regeneration through callus initiation and transformation work. However, the utilization of immature embryos has been hampered by their strictly limited suitable stage for culture, 12-17 days after pollination. In contrast mature seeds are ubiquitous. Therefore, use of mature embryos as an explant can significantly reduce the time required to generate immature embryos and hence the overall time required to regenerate maize plant. However, tropical maize genotypes and mature embryos have been considered as the most recalcitrant for tissue culture work. Consequently tropical maize line regeneration using mature embryos has not been reported so far. The purpose of this study was to regenerate two tropical maize lines, CML 216 and Katumani, from mature embryo. Splitting maize seeds longitudinally exposes three different tissues of the embryo simultaneously: scutellum, coleoptile-ring and shoot apical meristem. In the present study up to 92.6% germination and 0% contamination rate was attained for mature embryos harvested directly from open field or screen house by soaking sterilized seeds in 1% NaOCI solution for 2-3 hours. Seeds were germinated on MS media supplemented with 2 mg r' 2,4-D. Both the amount and frequency of callus produced by splitting mature seeds early (one day after germination) was found to be low, 43.3 % and 57.4 % for Katumani and CML 216 genotypes respectively, as compared to 66.3 % and 75.7% for Katumani and CML 216 respectively when splitting was done late (3-5 days after germination). The maximum average callus induction recorded was 90% for CML 216, 80% for Katumani and 34.3% for A188. When 2,4-D was combined with lower levels of Kinetin ( cytokinin) both the amount and frequency of callus induction was reduced to 52.5% for CML 216 and Katumani and to 34.3% for A188. The media used was LS salts and B5 vitamins supplemented with 900 mg t', 250 mg r' and 3-4 mg r' of 2,4-D. The average production of Type II and Type I callus was 75.6% and 62.3% respectively. The media used was LS salts and B5 vitamins supplemented with 900 mg r', 250 mg r' and 2 mg r' of 2,4-D. The frequency of regenerable calli produced was 21.14% for CML 216 and 16.51% for Katumani. The number of shoots regenerated per callus induced from single split seed ranged from 1-5. The media used was LS salts and B5 vitamins supplemented with 900 mg r', 250 mg r' and 4 mg r' of BAP and 2 mg rl of Kinetin. Plants were acclimatized in pots contained pit moss. This regeneration protocol gives an alternative explant source for maize researchers of the tropics in transgenic maize production to tackle different production constraints.