II.21 Barley pollen culture.
0. W. Pearson and R. A. Nilan. Department of Agronomy and Soils, Washington State University, Pullman, Washington 99163, USA.
Clapham (1971, 1973) reported the first successful culturing of barley pollen. Most of his success was with the varieties Akka and Sabarlis but the proportion of successful cultures was very low. In order to use barley pollen culture in plant breeding or in mutation and other genetic studies, the techniques must be somewhat improved. Attempts were thus made to increase the efficiency of plantlets from barley pollen.
Knowing that the pollen of various varieties have a rather wide range of response to the culturing process (Guha et al., 1970), 16 spring barley varieties and 1 winter barley variety were tested using a modified Linsmaier and Skoog's medium (Clapham, 1973) with 12% sucrose and 1 p.p.m. each of IAA and BAP. Between 300 and 1,200 anthers were plated for each spring variety. All the spring varieties produced callus and several, including Akka, Zephyr, Unitan, Traill, and Trebi produced at least one plantlet with shoot and or roots. The winter variety, Hudson, on the other hand did not respond--not even producing callus, though more than 2,000 anthers were plated. Since none of the varieties appeared superior to Akka, this variety was used in most of the subsequent work.
A principle objective was to induce a large percentage of the pollen grains to divide and thus hopefully start a large number along the developmental course of forming plantlets. Rowell (1972) showed that spraying wheat tillers with ethephon (Ethrel) at the early through late boot stages caused a fairly high percentage of the grains to continue dividing. Thus, though other workers (Bennett and Hughes, 1972; Picard, 1973) have tried unsuccessfully to use ethephon in producing plantlets in wheat, four procedures were used in treating the barley pollen with ethephon prior to plating:
Procedure 1. Plants were sprayed with 1,000, 2,000, and 4,000 p.p.m. ethephon at the early boot stage (approximately the first pollen mitosis) and then examined and plated at anthesis. This procedure resulted in a number of grains which had undergone extra divisions as in Fig. 1. In contrast to normal pollen, the cytoplasm is lightly stained and free of starch. Most of the grains however were dead, and the production of multinucleate grains was inconsistent. Culturing the multinucleate grains obtained by this procedure gave no plantlets or even calluses, though more than 200 anthers were plated.
Procedure 2. Anthers were plated at various stages (just prior to the first mitosis through the three nucleate stage) on medium to which 10-1,000 p.p.m. ethephon had been added. Pollen was relatively unaffected by concentrations up to 100 p.p.m. Concentrations greater than 100 p.p.m. caused a deterioration in the health of the grains, and at 1,000 p.p.m. all the grains died soon after plating.
Procedure 3. Spikes were removed from the boot and dipped in 1,000, 2,000, and 4,000 p.p.m. ethephon for 3 hours, and then plated near the first pollen mitosis stage. At 5 days after treatment and plating, there were very few multinucleate grains. Most were dead or were large with a considerable starch build up.
Procedure 4. Tillers at various stages were clipped off at ground level and the stems placed in solutions of ethephon at concentrations of 500, 1,000, 2,000, and 4,000 p.p.m. for various periods of time. Those which were treated at such early stages as the tetrad stage were placed in aerated Hoaglands Solution to which ethephon had been added. The anthers were plated at stages from the first mitosis through the 3 nucleate stage.
Of the four procedures, the fourth gave the most encouraging results. Those which were left in contact with the ethephon from the tetrad stage for a total of 6 days appeared dead although two dense compact degenerated looking sperm nuclei could usually be seen.
Apparently degeneration did not set in until shortly after the second mitosis. Spikes which were placed in 4,000 p.p.m. ethephon for 24 hours and planted at the two nucleate stage gave a rather high percentage of multinucleate grains (Fig. 2). The stages in the development of ethephon-treated grains plated at the two nucleate stage are shown in Fig. 4. It appears that only the vegetative nucleus takes part in embryo formation. When induced to divide at the two nucleate stage, almost always the vegetative nucleus becomes diploid immediately, presumably due to a failure to divide (Fig. 3, a-d). The generative nucleus does not always divide at the same time in relation to the vegetative nucleus. It generally divides quite early as in Fig. 3b, but can divide rather late or perhaps not at all as in Fig. 3c.
Figures: 1 and 2. Multinucleate pollen grain. 1. Multinucleate pollen from plants sprayed 15 days previously with 4,000 p.p.m. ethephon. The pollen was at a stage between the tetrad and first pollen mitosis when plants were sprayed. 2. High frequency of multinucleate grains after the following treatment: Spikes were clipped off at ground level, placed in a 4,000 p.p.m. ethephon solution for 24 hours and the anthers plated when pollen was at the two nucleate stage.
Figure 3. Stages in the development of ethephon-treated pollen grains plated at the two nucleate stage: (a) Pollen grain 5 days after plating in which the vegetative nucleus is in metaphase. In this case the vegetative nucleus has divided before the generative nucleus. (b) Presumably, a failure of division, results in the vegetative nucleus which was 2 C in (a) becoming 4 C. Pollen grain to the right contains a 4 C vegetative nucleus in prophase, whereas pollen grain to the left contains a 4 C nucleus in metaphase, with 14 chromosomes. In both instances, the generative nucleus has undergone one division. (c) Pollen grain similar to (b) except that the generative nucleus has not yet divided. (d) Pollen grain with two nuclei which are most likely diploid (daughters of the vegetative nucleus) and two sperm nuclei which seldom if ever undergo further division. NOTE: Crack in Fig. 3a occurred during mounting.
Figs. 4, 5, and 6 show stages in the development of plantlets from callus. Most of the plantlets obtained were partially or completely albino. None of the plants lived to maturity but died at various stages from the callus stage up to a height of 15 cm. No chromosome counts were made on these plantlets. Various attempts were made to increase the survival of the plantlets. They were transferred soon after they had formed to Norstog's Barley Medium II, and to the original medium which had various concentrations of growth hormones and sugar, but without success.
Figure 4. Two calluses 15 days after plating. Shown is a small callus along the border of a much larger one. Anthers were treated with 4,000 p.p.m. ethephon for 48 hours by procedure 4 and plated at the two nucleate stage.
Figure 5. Small plantlet derived from pollen 5 weeks
after beginning of culture. Notice shoot which is half green and half albino.
Figure 6. Plantlet derived from pollen, 5 weeks after the beginning of culture.
Most of the plantlets were root deficient, but when present did not survive long. Varying the sugar concentration from 3 to 12% and the auxin and cytokinen concentrations from 0 to 1 p.p.m. had little effect on the health of the roots. Shoot growth on the other hand remained unchanged despite these changes in the medium.
Bennett, M. D. and W. G. Hughes, 1972. Additional mitosis in wheat pollen induced by Ethrel. Nature 240: 566-568.
Clapham, D., 1971. In vitro development of callus from the pollen of Lolium and Hordeum. Z. Pflanzenzucht. 65: 285-292.
Clapham, D., 1973. Haploid Hordeum plants from anthers in vitro. Z. Pflanzenzucht. 69: 142-155.
Guha, S., D. Iyer, N. Gupta, and M. S. Swaminathan, 1970. Totipotency of gametic cells and the production of haploids in rice. Current Sci. 39: 174-176.
Picard, E., 1973. Influence de modifications dans les correlations internes sur le devenir du gametophyte male de Triticum aestivum L. in situ et en culture in vitro. C. R. Acad. Sci. Paris, Ser. D 277: 777-780.
Rowell, P., 1972. The effects of 2-chloroethylphosphonic acid (ethrel) on male sterility, female fertility and anther and pollen development in wheat (Triticum aestivum L.). Thesis. Washington State University.
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