Since 1975 there have been several reports on the induction of haploid plants from cultured anthers. It appears from the literature that information on the factors which control induction of microspore callus followed by embryoid formation, and their cytological behavior, is still fragmentary. The present communication represents some of the positive factors which induce and control microspore de-differentiation and cytological aspects of pollen callus culture.
Zea mays cultivars (compositae and adecuba) were grown to maturity under field conditions. Standard anther culture techniques were applied for inoculating anthers to the basal medium. The basal medium used for anther culture was modified N6. Flower sizes containing pollen grains in the mid to late uninucleate stages were correlated by extensive cytological studies of the anthers using 1% acetocarmine in a drop of ferric acetate. Anthers containing mid to late uninucleate stages were dissected and aseptically inoculated in the medium. Different factors for induction of haploidy so far studied are as follows:
1. Effect of sucrose: Sucrose concentration was found to play an important role for increasing induction frequency in maize anther culture. In order to find the optimal sucrose concentration for callus and embryoid production, four levels of sucrose concentrations (3%, 5%, 9% and 12%) were tested. Of these the 12% level was found to be optimal for the induction of pollen embryoids in both of the cultivars. Maximum induction frequency at this concentration reached up to 2.4%.
2. Effect of cold pretreatment: Cold pretreatment of the flower buds, at 0 C to 7 C for one to four days before excision and culture to the nutrient medium, was found to increase induction frequency. Best success was obtained when the flower buds were pretreated at 7 C for 4 days; the induction frequency increased up to ten times higher than the control.
3. Effect of growth regulators: Auxin (2,4-D), cytokinin (kinetin) and organic component (casein hydrolysate) were tested in various combinations and concentrations along with the basal medium. Effects of these growth regulators for increasing induction frequency are shown in Table 1.
Cytological status of the anther derived callus tissues was also examined. Cytological observations revealed mixoploid chromosome numbers with cells containing haploid chromosome complements predominant. Callus cells contained an average of 52% haploid, 26% diploid, and 22% mixoploid, containing haploid, diploid and aneuploid chromosome complements.
Nitai Kumar Paul and Parthadeb Ghosh
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