Factors affecting variability in anther culture and in regeneration of androgenic embryos of Solanum phureja

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Virginia Tech

The variation for embryo production in anther culture of Solanum phureja was examined as a function of maximum greenhouse temperature prior to bud harvest and innate responsiveness among anthers within a bud. S. phureja clones PP5, AD2-4, A3P2-6 and AD3-4 were grown in a greenhouse under a 16 h photoperiod. The temperature was monitored continuously using a thermograph. Buds were collected from PPS and AD2-4 and the anthers were cultured in two groups of five flasks. In the first group, each flask contained the 30 anthers from 6 buds; the second group, each flask contained 1 anther from each of 30 buds -- a total of 30 anthers per flask. Significantly smaller coefficients of variation were observed for the second group, suggesting that the variation for embryogenic capacity among buds was greater than that among anthers within a bud.

Variation in embryo yield as a function of greenhouse temperature for clones A3P2-6 and AD3-4 was examined by stepwise regression analysis. Embryogenic capacity of clone A3P2-6 was adversely affected by high temperatures (31-37°C) that occurred 2 and 7 days before bud harvest. However similarly high temperatures appeared to enhance the androgenic response of clone AD3-4.

Regeneration rate of anther-derived embryos over three subcultures to fresh regeneration medium was examined as a function of anther donor or clone, cold pretreatment of embryos, and morphological classification of embryos. Only clonal origin significantly affected regeneration. Regeneration rate declined on each serial subculture. The frequency of regenerable embryos varied from 12.5% for clone BARD 1-3 to 46.0% for clone A3P2-6.

Flow cytometric analyses were performed on several anther-derived monoploids of S. phureja to examine the frequency of nuclei at the 1x, 2x, and 4x levels within and among clones. Significant variation was found among duplicate cultures of the individual clones, but this variation was small enough to allow the detection of significant differences among the clones. Monoploid cell frequency ranged from 22.3% to 35.7%. Diploid cell frequency ranged from 48.6% to 59.9%. Tetraploid cell frequency ranged from 11.9% to 25.3%. Several families of anther-derived monoploid clones of S. phureja were analyzed for differences among clones within a family and among families. Significant differences were found in both categories. Finally, unstained protoplasts of monoploid S. phureja clone AM3 were sorted based on forward angle light scatter (FALS) and autofluorescence. Fractions selected for low FALS and weak autofluorescence appeared to be selectively enriched for monoploid protoplasts.