20. Germination o f Seeds Obtained from a Clone o f Garlic, Allium sativum L.

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1 No. 4] Proc. Japan Acad., 59, Ser. B (1983) Germination o f Seeds Obtained from a Clone o f Garlic, Allium sativum L. By Takeomi ETOH Laboratory of Vegetable Crops, Faculty of Agriculture, Kagoshima University, Kagoshima City 890 (Communicated by Naohide HIRATSUKA, M. J. A., April 12, 1983) Garlic, Allium sativum L., is the most widely used, next to common onion, of the cultivated Alliums in the world. In spite of its long cultivated history from time immemorial, garlic has never been crossbred because it has been regarded as a typical obligate apomict3 ' or a sterile plant. No mature pollen is formed in garlic, and the cause of its pollen-degeneration is still uncertain. The author found out a pollen-fertile clone, so that it was attempted to set seeds in the clone. Materials and methods. At Kagoshima, complete bolting and flower bud formation were observed in 72 of 153 collected clones of garlic. The rest were incomplete- or non-bolting clones, and eight of the former developed flower buds. Accordingly, flower bud formation was observed in 80 clones in total. In seven of the 80 clones, flower buds developed so poorly that meiosis could not be observed. In two other clones, meiosis was not observed, but degeneration of malformed microspores was remarked. Meiosis of pollen mother cells was observed in the rest clones, and multivalent chromosomes or desynapsis was noticed in 63 clones,'? `'~ whose microspores or pollen grains always degenerated by the first pollen mitosis. On the other hand, two clones showed normal meiosis, and one of the two produced viable pollen grains,'' while the other failed to develop microspores. The pollen-fertile clone, as well as other sterile clone, developed bulblets together with flower buds in the inflorescences. Three plants developed flower buds in the pollen-fertile clone, no From one of the three plants, bulblets among the flower buds in the inflorescence were extirpated far before anthesis. One of the other two plants bore only malformed sterile flowers. Therefore, two plants, one with bulblets and the other without, opened normal flowers. Flowers of both plants were self-pollinated by hand during anthesis from July 9 to 23, Meanwhile, the karyotype of no. 130 was compared with those of other sterile clones to ascertain that no. 130 belongs to A, sativum L. The root tips of no. 130 Moscow, no. 40 Kokotsu, and no. 64 Shanhai-

2 84 T. ETOH [Vol. 59(B), wase were pretreated in mol 8-hydroxyquinoline solution, and the squashed materials were observed after stained by means of Feulgen reaction. The average idiograms were obtained from 10 cells in no.130, from 13 cells in no. 40, and from nine cells in no. 64. Results. 1) Germination o f seeds. After pollination, some ovaries developed into fruits, but the others failed to develop (Fig. 1). Only a few ovaries developed into fruits in the plant bearing bulblets without extirpation, whereas 13 of 76 ovaries developed into fruits in the plant bearing no bulblet. The seeds were harvested from August 19 to 30, when the black seeds were found in the dehiscent capsules. The harvested seeds were counted 27 and weighed about 46 mg in total. The enriched mature seeds of common onion weighed about 93 mg at the same number. Fig. 2 shows the biggest of harvested garlic seeds and the enriched mature seeds of common onion. All the seeds were planted in the pots about two months after harvest, and two seeds germinated 11 and 13 days after planting, respectively. The first foliage leaf appeared in one seedling 32 days after planting (Fig. 3). Figs : Developing fruits of no : Seeds of no. 130 (A. sativum L.) and of common onion (A. capa L.). 3 : A seedling of no. 130, showing the first foliage leaf. 4: Somatic chromosomes of no Arrows indicate satellite chromosomes. The rest of the seeds were dug out about one month after planting and were put in the refrigerator at about 5 C, except for dead seeds. After one-month cold storage, eight seeds were put on the wet filter paper in Petri dishes kept at 20 C, but no seeds germinated within ten days. Therefore, the seeds were rubbed on sandpaper, and three seeds germinated two days after scarification, though some

3 No. 4] Germination of Garlic Seeds 85 seeds might be injured seriously. Later two more seeds germinated. Thus, at least seven seeds in total were germinable, though the seedlings from the seeds did not grow so vigorously. 2) Karyotype analysis. The karyotypic characteristics common to the three clones were as follows. Without secondary constrictions, ten longer chromosomes had approximately median centromeres. The shortest two of 16 chromosomes had centromeres on the border of median and submedian region. Secondary constriction was situated on the short arms in two pairs of chromosomes, though no. 64 lacked it in one chromosome. The short arm of each satellite chromosome was divided into a small proximal segment and a big satellite. The longer pair of the satellite chromosomes had the smaller satellites than the shorter pair (Fig. 5). The secondary constrictions, or nonstaining gaps, were often long, as shown in Fig. 4. Fig. 5. sterile Idiograms of the fertile clone (no. 130) and the clones (nos. 40 and 64). Discussion. Kononkov4> reported that germination was induced in the garlic seeds obtained by extirpation of bulblets. His paper seems to be the sole report on viable garlic seeds. However, no one has confirmed this, and it has been still attempted to obtain viable pollen grains or seeds.>' 7 In this trial, no. 130 clone produced 27 seeds in 18 fruits, of which at least seven germinated. Careful growing of the plants may induce good production of seeds in this clone, but it is sure that bulblets among flowers inhibit development of fruits or seeds, because the plant without extirpation of bulblets produced only two small shriveled seeds which could not germinate. Besides

4 86 T. ETON [Vol. 59(B), this, some female gametes may have degenerated early, as Weber stated.' The seeds which did not germinate in the first trial were presumed to be dormant, but they did not germinate also after cold storage. Scarification, not cold storage, is probably effective in germination of garlic seeds. The karyotypes of somatic chromosomes in various clones of garlic have been studied by many investigators. The karyotypic characteristics common to the three clones examined here agreed very well with those of the clones studied by the investigators fundamentally, although a few reported only two satellite chromosomes besides three or four. Especially, the satellite chromosome which has a small proximal segment and a big satellite on the short arm is called sativum-type in Allium, and it is known as a karyotypic component of garlic, A. sativum L.8> Therefore, the karyotypes of the three clones examined here suggested that they belonged to A. sativum L., though their chromosomes behaved at meiosis as 8II in no. 130, 1X+3II in no. 40, and 1VIII+4II in no. 64, respectively.l~'2~ Thus, this fertile clone, no. 130, was ascertained to be one of the clones of A. sativum L., karyologically. In the report mentioned above, Kononkov neither observed meiosis and microsporogenesis of his fertile clone nor identified it as A. sativum L. However, as he described scarification of seeds and the reliable source of the materials, Institute of Genetics (Academy of Science, USSR), Kononkov must have used one of fertile garlic clones and obtained viable seeds.4~ By his report and the author's, it was confirmed that garlic has fertile clones besides sterile ones. As the author obtained the fertile clone from the Central Botanic Garden of Moscow, USSR, it is presumable that some more fertile clones will be found in USSR, though many sterile clones in USSR have been reported.~~ This fertile clone found here will be used for many purposes. It is useful as an important material for crossbreeding, for clarifying the true cause of sterility in garlic, for studying the relationship between A. sativum L. and the allied species, for studying the origin and the differentiation of A. sativum L., and also for investigating the variation of breeding system within a species. Summary. Viable seeds of garlic, Allium sativum L., were obtained after self-pollination in a pollen-fertile clone found by the author. Some of the seeds germinated without any treatment, whereas the others needed scarification for germination. This fertile clone was identified as one of garlic clones karyologically. Acknowledgements. The author wishes to thank Prof. H.

5 No. 4] Germination of Garlic Seeds 87 Ogura helpful of Kagoshima Univ. and Prof. S. Iwasa of Iwate Univ. for suggestions and advices. References 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) Etoh, T. (1979) : Mem. Fac. Agr. Kagoshima Univ., 15, (1983) : ibid., 19, Fryxell, P. A. (1957) : Bot. Review, 23, Kononkov, P. F. (1953) : Sad i Ogorod, 8, Konvicka, 0., Nienhaus, F., and Fischbeck, G. (1978) : Z. Pflanzenzuchtg., 80, Krivenko, A. A. (1938) : Biologicheskij Zhurnal, 7, Novak, F. J., and Havranek, P. (1975) : Biol. Plant. (Praha), 17, Ved Brat, S. (1965) : Chromosoma, 16, Verma, S. C., and Mittal, R. K. (1978) : Cytologia (Tokyo), 43, Weber, E. (1929) : Bot. Arch., 25, 1-44.