Induction of Haploid Callus from Isolated Microspores of Peony in vitro

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1 Plant & Cell Physiol. 22(2): (98) Short communication Induction of Haploid Callus from Isolated Microspores of Peony in vitro Kanji Ono and Shuichi Harashima Department of Biology, Faculty of Science, Kumamoto University, Kurokami 2, Kumamoto 86, Japan The in vitro culture of isolated microspores of two cultivars of Paeonia, P. lactiflora cv. "Kumoinotsuru" (2n=) and cv. "Toyonoakari" (2n = ), was attempted. With both cultivars, some cultured microspores (3-6%) began to divide after five to seven days of culture. Those of "Kumoinotsuru" grew into calli, whereas the others degenerated. Cytological examination of the callus developed from a microspore revealed haploids, diploids or a mixture of both. No direct embryo formation from microspores was observed in this experiment. Key words: Haploid callus Microspore culture Peony Pollen culture. Isolated microspore culture is superior to anther culture as a means of obtaining a haploid plant or a haploid cell line, because it offers advantages in terms of the removal of anther-derived inhibitors and the avoidance of competition from somatic callus development. However, this technique has only had limited success using pretreated or precultured anthers in several angiosperm species, e.g., Nicotiana (Sharp et al. 972, Nitsch 974, Reinert 975), Petunia (Binding 972), Datura (Nitsch and Norreel 973), Solatium (Weatherhead and Henshaw 979).. In recent years, Sangwan and Norreel (975) succeeded in inducing haploid calli and haploid plants from isolated Petunia pollen grains without anther pretreatment. In a previous paper (Ono and Tsukida 978), we reported that haploid calli were induced from anther culture of Paeonia lactiflora cv. "Kumoinotsuru" (2n = ) and might have originated from microspores judging from their chromosome complements. The present study details the conditions and processes leading to callus formation from isolated microspore cultures of the same Paeonia cultivar ("Kumoinotsuru") reported previously and another one ("Toyonoakari"). Flower buds of two cultivars containing uninucleate and early binucleate microspores in their anthers were sterilized in a filtered suspension of % sodium hypochlorite solution for min then rinsed three times with sterile distilled water. Isolation of microspores from anthers was carried out according to Nitsch's method (Nitsch 977). Microspore pellets were suspended in liquid culture media and the final concentration of the suspension was adjusted to 4xlO 2 microspores per ml. Downloaded from at Penn State University (Paterno Lib) on September 6, 26 Abbreviation: NAA, naphthaleneacetic acid. 337

2 338 K. Ono and S. Harashima The microspore suspension was cultured as 2 ml per 6 X 5 mm Falcon Petri dish sealed with Parafilm (Fig. 2A). The dishes were maintained at 25 C for seven days in the dark, then illuminated continuously with 5 lux at 27 C. Microspore development was observed with an Olympus inverted microscope every few days. The basal medium for microspore cultures consisted of major and minor salts and vitamins according to Murashige and Skoog (962) and 3% sucrose. The following five types of media, in which the basal medium was supplemented with various combinations of phytohormones and growth regulators, were used: Ml supplemented with NAA (2 mg liter" ), 2,4-D ( mg liter" ) and kinetin (. mg liter" ); M2 supplemented with NAA ( mg liter" ), 2,4-D ( mg liter" ) and kinetin (. mg liter" ); M3 supplemented with 2,4-D ( mg liter" ) and kinetin (. mg liter" ); supplemented with NAA (2 mg liter" ) and coconut milk (%); M5 supplemented with NAA (2 mg liter" ) and casamino acids (.2%). The ph of the media was adjusted in all cases to 5.6 before autoclaving. For cytological experiments, the materials were fixed in ethanol-acetic acid (3 : ) for hr after treatment with.5% colchicine solution for 2 hr. Chromosomes were stained with % aceto-orcein and squashed. At the beginning of microspore cultures of the two cultivars, the uninucleatebinucleate ratio of "Kumoinotsuru" was : 4.7, while that of "Toyonoakari",.3 :. After 5 to 7 days of culture, some microspores of both cultivars began to divide (Fig. 2B and C) and after 2 weeks of culture they developed into cell aggregates consisting of several cells (Fig. 2D and E). After 2 days of culture, the proportion of dividing microspores per microspores observed was examined for each culture in five kinds of media (Fig. ). In each experiment, about to 4 microspores were counted. The microspore cultures of both cultivars represented dividing CO 6 o a. CO 5 O oc O 4 O 3 5 > 2 a n B '.Toyonoakari [Hj!Kumoinot*uru M n n I JI I l M2 M3 MEDIUM i pi P I M5 Fig. The proportion of division in isolated microspore of Paeonia lacliflora cv. "Kumoinotsuru" and cv. "Toyonoakari" in different media after 2 days of culture. Each value represents the percentage of dividing microspores per about to 4 microspores observed. Downloaded from at Penn State University (Paterno Lib) on September 6, 26

3 Haploid callus from isolated microspores of peony 339 -«mm F Fig. 2 In vitro behavior of isolated microspores of P. lactiftora cv. "Kumoinotsuru" and cv. "Toyonoakari". (A): Isolated microspores of "Kumoinotsuru" at the time of inoculation. (B): Two-and three-cell stages of "Toyonoakari" microspores after five days of culture. (C): Four-cell stages of "Kumoinotsuru" microspores after seven days of culture. (D) and (E): Cell aggregates from "Kumoinotsuru" microspores after two weeks of culture. (F): Calli from "Kumoinotsuru" microspores after two months of culture. microspores in the 2.9% to 5.9% range. After two months of culture, some dividing microspores of "Kumoinotsuru" cultured in M2, M3 and media developed into calli of. to 2 mm in diameter (Fig. 2F and 3A). In culture, 7 calli were formed from among 24 dividing microspores. In M3 culture, ten calli were formed from among 6 dividing microspores and in M2 culture two calli from among 2 dividing microspores. On the other hand, dividing microspores of "Kumoinotsuru" cultured in the other media and those of "Toyonoakari" degenerated gradually. Each callus derived from a microspore was subsequently transferred to M3 agar medium to allow it to proliferate. Sunderland (974) reported pollen embryos Downloaded from at Penn State University (Paterno Lib) on September 6, 26

4 34 K. Ono and S. Harashima ft D Fig. 3 Calli and their chromosomes developed from isolated microspores of P. lactiflora cv. "Kumoinotsuru" in vitro. (A): Calli from microspores after two months of culture. (B): Callus cells from microspores after three months of culture. (C) and (D): Somatic metaphase chromosomes in haploid cells (n = 5) after three months of culture. and calli from anther cultures of Paeonia hybrida. However, no direct embryo formation from microspores was observed in the present experiment. After three months of culture, chromosomes of callus cells were examined for seven calli (No. to 7), which were chosen arbitrarily from those produced on M3 and media (Table ; Fig. 3B). In calli No., 2, 3 and 4, all of the mitotic cells had the haploid chromosome number of n=5 (Fig. 3C and D), while in the No. 5 callus it was diploid (2n = ) and in No. 6 and 7 calli, both of mitotic haploid and Table Chromosome numbers of calli obtained from isolated microspore cultures in Paeonia lactiflora cv. "Kumoinotsuru" No. of calli used for chromosome counts 3 s ; 7 No. of cells having chromosome Q fi 6 s 2 numbers of others D & fl Induction medium M3 M3 Downloaded from at Penn State University (Paterno Lib) on September 6, 26

5 Haploid callus from isolated microspores of peony 34 diploid cells were mixed. Mitotic polyploid cells other than diploid and aneuploid ones were not found in the calli examined. The karyotype of a haploid callus cell corresponded to a half chromosome set of the normal diploid observed in root tip cells in a previous study (Ono and Tsukida 978). The diploid callus cells in the present study appear to result from one of the following routes as reported for anther cultures of Nicotiana and Datura by Sunderland (974), that is, polyploidization in a haploid callus, nuclear fusion in a haploid microspore and induction of growth in a diploid microspore. The techniaue of anther pretreatment such as by low temperature (3-5 C) or cholchicine solution (.5%), usually has been used to obtain a plant derived from a microspore or a pollen (Nitsch and Norreel 973, Nitsch 977). The first successful report on induction of haploid calli and plants from isolated pollen grains without anther pretreatment was published by Sangwan and Norreel (975) for Petunia hybrida. In the present study, we also successfully induced the formation of haploid calli from isolated microspore cultures of Paeonia lactiflora cv. "Kumoinotsuru" without any pretreatment. Since this callus is monohaploid, having a low chromosome number (n=5), it appears to be usable for genetic research and plant breeding. The haploid callus cells are now being subcultured in order to establish a genetically stable haploid cell line by suspension culture. In addition, experiments on their morphogenesis are also being carried out to induce the production of haploid plants. References Binding, H. (972) Nuclear and cell divisions in isolated pollen of Petunia hybrida in agar suspension cultures. Nature 237: Murashige, T. and F. Skoog (962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiot. Plant. 5: Nitsch, C. (974) Pollen culture A new technique for mass production of haploid and homozygous plants. In Haploid in Higher Plants Advances and Potential. Edited by K.J. Kasha, p Univ. of Guelph. Nitsch, C. (977) Culture of isolated microspores. In Applied and Fundamental Aspects of Plant Cell, Tissue and Organ Culture. Edited byj. Reinert and Y. P. S. Bajaj. p Springer- Verlag, Berlin. Nitsch, C. and B. Norreel (973) Effet d'un choc thermique sur le pouvoir embryogene du pollen de Datura innoxia cultiv dans l'anthere ou isole de Panthere. C. R. Acad. Sci., Paris 276: Ono, K. andt. Tsukida (978) Haploid callus formation from anther cultures in a cultivar of Paeonis. Jap. J. Genetics 53: Reinert, J., Y. P. S. Bajaj and E. Hererle (975) Induction of haploid tobacco plants from isolated pollen. Protoplasma 84: Sangwan, R. S. and B. Norreel (975) Induction of plants from pollen grains of Petunia cultured in vitro. Nature 257: Sharp, VV. R., R. S. Raskin and H. E. Sommer (972) The use of nurse-culture in the development of haploid clones of tobacco. Planta 4: Sunderland, N. (974) Anther culture as a means of haploid induction. In Haploid in Higher Plants Advances and Potential. Edited by K.J. Kasha, p Univ. of Guelph. Weatherhead, M. A. and G. G. Henshaw (979) The induction of embryoids in free pollen culture of potatoes. Z. Pflanzenphysiol. 94: Downloaded from at Penn State University (Paterno Lib) on September 6, 26 (Received December, 98; Accepted January 6, 98)

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