Culturing of endosperm tissue

The culturing of endosperm tissue has been abetted by the use of inbred R168 (Illinois) as the background genotype (J. C. Shannon and J. W. Batey, 1973). These tissues have been cultured on modified MS medium in our continuing study of anthocyanin genes and controlling elements in tissue culture (M. B. Gorman and P. A. Peterson, 1978). Several conditions were varied in testing the efficacy of growth conditions for field-grown materials. These include culturing of different genotypes at different post-pollination stages and different times of collection (7:00 a.m., Noon, and 8:00 p.m.). Assessing the hour of culturing was done in order to evaluate the effect of age difference on tissue growth and callus initiation in terms of hours rather than in days after pollination. Time effects (morning, noon, or evening) on the tissue performance in vitro could not be compared due to the confounding effect of age differences in hours from pollination, which is done normally at approximately 10:00 a.m.

The two phases of endosperm tissue growth: Two phases of tissue growth were recognized in vitro. The initial growth (first 7 to 10 days) resulted in organized and compact tissue which is comparable to the in vivo endosperm. In contrast, the second phase of growth (after 10 to 15 days) is disorganized and localized proliferations of growth appear.

The first phase can simply be identified as the growth phase, whereas the second phase can be considered the callus phase. The timing of appearance of the callus phase differed from genotype to genotype. Callus initiation seems to depend on the initial tissue growth.

Effect of age and genotype on tissue growth: Using an index method to assess tissue growth performance, it could be determined that the first and second phases of growth of different genotypes were better when cultured between 202 hours (8-days old at 8:00 p.m.) and 226 hours (9-days old at 8:00 p.m.) than when cultured at later stages after pollination. Genotypic differences were more noticeable for these two phases of growth with endosperms of earlier stages than with older endosperms. Also, the differences among genotypes were more significant in callus phase (15 days) than in growth phase (until a week).

In addition to the inbred R168 background, some genotypes in the present study contained the controlling element allele wx-m-8 which was backcrossed two to three times to R168 [wx-m-8(R168)]. The genotypes with R168 background showed better tissue growth and callus initiation than that of wx-m-8(R168). Inbred R168 by itself had been previously reported to be good for endosperm callus initiation (A. R. Reddy and P. A. Peterson, 1977) and was found to perform better than wx-m-8(R168) in the present study.

Considerable variation occurred within the progeny of a selfed ear in growth and callus phases. This may be attributed to the heterozygosity of the segregating alleles.

In conclusion, the conditions for the most efficient growth of callus include genotype and age of tissue (hours after pollination).

L. V. Reddy and P. A. Peterson


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