Plant regeneration from somatic culture of apomictic maize-Tripsacum hybrids
--Li, YG, Kindiger, B

In an attempt to transfer apomixis from Tripsacum to maize using classical backcross- hybridization methods, a series of apomictic maize-Tripsacum hybrids have been generated. The Tripsacum chromosome number in these hybrids has been reduced to 9 from 36 in the original crosses between tetraploid maize (2n = 4x = 40) and tetraploid Tripsacum (2n=4x=72) (Kindiger et al., Crop Sci. 36:1108-1113, 1996). Recently, the long arm of Tripsacum chromosome 16 (Tr16L), which carries the apomixis gene(s), has been transferred to the long arm of maize chromosome 6 (Mz6L) via a Mz6L-Tr16L translocation (Kindiger et al., Genome 39:1133-1141, 1997). The stock carries 29 intact maize chromosomes, the Mz6L-Tr16L translocation, and 8 additional Tripsacum chromosomes. Since normal meiotic events are omitted during apomictic development, the genetic constitution of the apomictic hybrids can be changed only by sexual polyploidization or via rarely occurring polyhapoids (Kindiger et al., Genetica 92:197-201, 1994). Consequently, further reduction of the Tripsacum chromosome complement in this stock will be extremely difficult. In an attempt to circumvent this situation, tissue culture techniques were developed for this stock to facilitate chromosome manipulation.

Young unemerged inflorescences from field grown plants were taken in the early boot stage when the inflorescences were 2-3 cm long. The outer leaves were removed and the inner leaves were surface sterilized by misting the leaves several times with 70% ethanol. The remaining inner leaves were removed aseptically to expose the young intact infloresence. The clean inflorescence was then cut into 2-3 mm pieces and placed on the callus induction medium with 1 inflorescence per plate. Immature embryos from greenhouse grown plants were also used as explants to initiate culture. Immature embryos were collected 2 weeks after pollination when the embryos were about 2 mm in length. The caryopses were removed from the spikelets, surface sterilized for 5 minutes in 10% commercial bleach plus Tween 80 and rinsed three times in sterile distilled water. The embryos were then aseptically removed from the caryopses and placed onto the callus induction medium. The callus induction and maintenance medium for both inflorescences and immature embryo cultures was N6 basal salt containing 1.0 mg/l thiamine HCl, 0.5 mg/l each of pyridoxine HCl and nicotinic acid, 2.88 g/l L-proline, 10 µM silver nitrate and 1.0 mg/l 2,4-D. Plates were incubated at 25 C in the dark for callus induction. Calli emerged easily from both inflorescences and immature embryos after three weeks culture and these calli were subcultured on the same medium for two more weeks. They then were transferred to hormone free N6 regeneration medium and incubated at 28 C under 16/8 hours (dark/light) photoperiod. One plant was regenerated from one of the 40 plates initiated from inflorescences after two months culture on regeneration medium. No plant was obtained from calli derived from immature embryos. These results indicate that genetic components controlling regeneration in this Mz6L-Tr16L translocation stock do exist and regeneration frequency in the stock can be improved by further manipulation of culture medium and protocol. Since somatic chromosome numbers do change with duration of culture, cell culture may play a role in the reduction and/or elimination of the extra Tripsacum chromosomes in these maize-Tripsacum hybrids.

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