II.52 In vitro germination of barley pollen.

T. Chakrabarti, N.C. Subrahmanyam and C.H. Doy. Genetics Department, Research School of Biological Sciences, The Australian National University, Canberra, A.C.T. 2601, Australia. "R"

In vitro germination of pollen has been used as a powerful tool for genetical, physiological, biochemical and cytochemical studies for a wide range of plant species belonging to different families (Heslop-Harrison, 1971). However, for graminaceous plants, pollen of only four species have been successfully germinated (Pennisetum, Vasil, 1960; Zea mays, Pfahler, 1967; Setaria sphacelata, DeBruyn, 1966; Lolium, Ahloowalia, 1973). No information is available for barley.

Haploids can be produced for any line of Hordeum vulgare (2n = 14) following hybridization with H. bulbosum (2n = 14) where bulbosum chromosomes are selectively eliminated (Subrahmanyam and Kasha, 1973). Recently, germinating Nicotiana and Petunia pollen were shown to take up DNA (Hess, 1974). This suggests a new approach for genetic munipulation of higher plants. With a view to the use of H. bulbosum pollen as a vector, attempts have been made to germinate pollen of diploid and tetraploid H. bulbosum. A brief account of a technique for in vitro germination of bulbosum pollen is presented.

Monohaploid and dihaploid pollen (from diploid and tetraploid cytotypes of H. bulbosum respectively) at anthesis were used to study germination on media varying from sucrose solutions to a solution of CaCl₂, H₃B0₃, EDTA and sucrose (Ahloowalia, 1973). No germination was observed. However, dihaploid pollen collected by squeezing anthers prior to anthesis gave a maximum germination of 55% in Ahloowalia's (1973) medium. Since it was difficult to monitor the exact developmental stage of the pollen for consistent success, we developed a medium suitable for pollen. This medium consists of 20-25% sucrose, 0.01% boric acid, 10 ppm di-sodium salt of EDTA and 0.03-0.04% calcium nitrate adjusted to pH 7.0 with NaOH. For dihaploid and monohaploid pollen respectively, a maximum of 72.3% and 51.5% germination and 1.456 mm and 1.39 mm pollen tube length were obtained after 2 hours at room temperature (approximately 22°C).

Effects of Ca²⁺ and EDTA on the germination of bulbosum pollen are summarised in Table 1 and 2. Ca²⁺ is necessary for germination of both monohaploid and dihaploid pollen of bulbosum. Without Ca²⁺ there is poor germination and short pollen tubes. A Ca²⁺ concentration above the optimum (0.04%) results in short pollen tubes and for monohaploid pollen decreased germination. Low levels of EDTA (10 ppm) (Table 2) are probably beneficial for the germination of both monohaploid and dihaploid pollen, but levels of 15 ppm and above are inhibitory to monohaploid pollen. Lack of an inhibitory effect of EDTA on dihaploid pollen may be due to a genetic buffering capacity of heterozygous pollen from an outbreeding autotetraploid.

Table 1. Effect of Ca²⁺ on germination of barley pollen. Basal medium contains 22% sucrose, 0.01% H₃B0₃, 10 ppm EDTA, pH 7.0 + .1, incubated at 22° for 2 hours.

Table 2. Effect of EDTA on germination of barley pollen. Basal medium 22% sucrose, 0.01% H₃B0₃, 0.04% Ca(N0₃)₂, 4H₂0, pH 7.0 + .1; incubation as above.

The success of this technique depends not only on the medium but also on the vigour, health and growing conditions of the plant material; the time of collecting the pollen and the quality of the pollen. Considering these points, studies with various combinations of Ca²⁺, EDTA and boric acid are continuing.

Acknowledgement:

The authors with to thank Dr. R. Oram, Division of Plant Industry, CSIRO, Canberra for supplying the seeds of H. bulbosum lines used in this study.

References:

Ahloowalia, B.S. 1973. Germination in vitro of ryegrass pollen grains. Euphytiea. 22: 573-581.

deBruyn, J.A. 1966. The in vitro germination of pollen of Setaria sphacelata, I. Effects of carbohydrates, hormones, vitamins and micronutrients Physiol. Plant. 19: 365-376.

Heslop-Harrison, J. (Ed.) 1971. Pollen development and physiology, Butterworths, London.

Hess, D. 1974. Uptake of DNA and bacteriophage into pollen and genetic manipulation. In: Genetic Manipulation with Plant Material (ed. Ledoux, L.) Plenum Press, 519-538.

Pfahler, P.L. 1967. In vitro germination and pollen tube growth of maize (Zea mays) pollen. I. Calcium and boron effects Can. J. Bot : 45 : 839-845.

Subrahmanyam, N.C. and K.J. Kasha 1973 Selective chromosomal elimination during haploid formation in barley following interspecific hybridization. Chromosoma (Berl.) 42 :111-125.

Vasil, I.K. 1960. Pollen germination in some Gramineae: Pennisetum typhoideum. Nature, 187: 1134-1135.

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