Generation of embryoids from primary and secondary anther cultures

Anther culture, via the production of haploids, is used to produce isogenic diploid lines. Because corn has responded poorly to attempts at anther culture, a comparison of two different methods was undertaken: the traditional method of anther culture (Primary Anther Culture, PAC) and a second method which produces anthers from tassels grown in liquid culture (Secondary Anther Culture, SAC).

Tassels for PAC were taken from Se60 (Seneca 60, a commercial sweet corn) plants at the 13th leaf stage. Segments of the tassel containing mainly anthers with uninucleate pollen (aceto-carmine stain) were sterilized with 20% Javex for 20 minutes and transferred to sterile dishes. The larger set of anthers from each spikelet was removed and transferred to 60 mm sterile plastic petri dishes containing test medium. After 24 anthers had been added, the dishes were sealed with two layers of Para-film and incubated in the dark at 8 C for 8 days. After this cold treatment the petri dishes were moved to 25 C in the dark for an additional 5 to 12 weeks.

For SAC, tassels of Se60 were first cultured on a modified liquid medium (0.1 M sucrose) as reported by Pareddy and Greyson (MGCNL 59:72-73, 1985). After 10 days of culture the anthers in the spikelets were carefully removed and placed, 24 to a dish, in sterile plastic petri dishes. Several anthers were removed for staging and care was taken to ensure that the anthers contained uninucleate pollen. The dishes were sealed and cold treated for 8 days at 10 C in the dark, then transferred to 25 C in the dark for 5 to 12 weeks.

The medium used for anther culture was a basic Yu-Pei (Y-P) medium (Genovesi and Collins, Crop Sci. 22:1137-1144, 1982) and the variations of that medium were as follows: Y-P (basic Y-P with TIBA, no agar, no charcoal); Y-P, C (Y-P with charcoal).

After 6 to 8 weeks of culture, embryoids or callus began to break out of the anthers. Usually, only one embryoid was produced per anther although a few anthers have produced as many as 3 embryoids. These embryoids were white, densely packed clusters of cells with an ovoid or spherical shape. They ranged in size from 1 mm to 5 mm. The larger embryoids developed roots, shoots or continued to grow as callus when moved to transfer medium. The success of anther culture from Se60 anthers taken directly from the plant (PAC) and anthers taken from tassel culture (SAC) are compared in Table 1.

The average yield of embryoids is given as a percentage (# of embryoids/# of anthers). The individual yields, however, varied greatly, from .3% to 3.5% for different tassels, and many tassels yielded no embryoids at all. This is likely due to variability of some aspect of the mother plant at the time of culture as nutrition, lighting and stress all are reported to play a role in growth of the plant. Although the SAC anthers yielded approximately the same percentage of embryoids as the PAC anthers, the maximum yield for a given experiment was only 1.3% whereas maximum yield for PAC was 3.5%. Looking at the percentage of embryogenic tassels, it can also be seen that a higher percentage of tassels responded in the PAC system. The use of charcoal in the culture medium did not seem to influence the yield for PAC, however, in the SAC system the sample size for the charcoal medium may have been too small. Our yield of embryoids is comparable to some researchers (Genovesi and Collins, ibid.), but is lower than that of others (Ting, Yu and Wan-Zhen, Plant Science Letters 23:139-145, 1981). We conclude that despite the low yield of embryoids from SAC, it appears to be at least as good in yield as is PAC.

Table 1.

R.I. Greyson and J.D. Dunlop
 
 


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