Plant Cell Reports (1984) 3:149-150

Plant Cell Reports ©

Springer-Verlag 1984

Somatic embryogenesis and plant regeneration in cultured immature inflorescences of Setaria italica Xu Zhi-hong 1, W a n g D a - y u a n 2, Yang Li-jun 1, and Wei Zhi-ming 1 1 Shanghai Institute of Plant Physiology, Academia Sinica, Shanghai, China 2 Chinese National Rice Research Institute, Hangzhou, China Received March 6, 1984 / Revised version received June 20, 1984 - C o m m u n i c a t e d by I. K. Vasil

ABSTRACT Young inflorescence explants of Setaria italica in culture showed high capacity for regenerating plantlets through somatic embryegenesis. Embryogenic callus formation was initiated from the explants cultured on Murashige and Skoog's medium with 2 mg/1 2,4-D and 0.2-0.5 m g / 1 KT or BAP, but it was better for the maintenance of embryegenic growth to subculture the calli on the medium with 2,4-D and KT/BAP and on the medium with 2 mg/1 2iPA and 0.2 mg/1 NAA alternately. A number of plantlets were regenerated when embryogenic calli were transferred onto the same basic medium but with 2 mg/1 BAP and 0.5 mg/1 NAA. Plant regeneration capacity has been maintained in some embryogenic calli during fourteen months of subculture. ABBREVIATIONS 2,4-D, 2,4-dichlorophenexyacetic acid; NAA, naphtha~ene#cetic acid; IAA, 3-indoleacetic acid; 2iPA, N -[~-isepentenyl)adenosine; BAP, 6-benzylaminopurine; KT, kinetin; CH, casein hydrolysate. INTRODUCTION Young inflorescence tissue has proven to be an excellent starting material for initiating tissue cultures of cereals [see Vasil et al., 1982; Wang, 1984; Loo and Xu, 1984). Plant regeneration through somatic embryogenesis in inflorescence culture has been reported in the following species of cereals and grasses: Echinechloa crus-galli (Wang and Kong, 1984); Panicum maximum [Lu and Vasil, 1982), P. miliaceum, P. miliare (Rangan and Vasil, 1983); ffennisetum americanum, P. americanum x P. purpureum (Vasil and Vasil, 1981), ~. purpureum [Wang and Vasil, 1982); Sorghum bicolor, (Brettell et al., 1980); and Triticum aestivum [Ozias-Akins and Vasil, 1982). Setaria italica (millet) is one of the principal cereal crops in North China. Except for anther culture (Ban et al., 1971), to our knowledge, there is no other report on tissue culture of this species. Recently we have briefly reported our observation on multishoot regeneration from the inflorescence cultures of Setaria italica and S. lutescens (Xu et al., 1983). This paper deals with

somatic embryogenesis and plant regeneration from cultured inflorescence segments of Setaria italica. MATERIALS AND METHODS The shoots of Setaria italica (cvs. Yugu No i and Xinneng 761) with immature, unemerged inflorescences, about 1-2 cm in length, were collected from field or green-house grown plants. After stripping away the outermost leaves, the shoots were rubbed with 70~ ethanol for surface sterilization. Then the panicles were dissected out, cut into 2-5 mm segments, and were cultured on MS agar medium [Murashige and Skoog, 1962) with the following supplements: [1) 2 mg/1 2,4-D + 0.2 m g / 1 KT (or BAP) ~ 2 0 0 m g / 1 CH, and (2) 2 mg/1 NAP 0.5 mg/1 NAA or IAA. The media used for callus subculture contained [1) 2 mg/1 2,4-D and 0.2-0.5 mg/1 KT (or BAP), and (2) 2 mg/1 2iPA and 0.2 mg/1 NAA. Cultures were incubated at 28 ° C, under fluorescent light (10 h, lOOn lux). Specimens for scanning electron microscopy were prefixed in 2~ glutaraldehyde in O.i M phosphate b u f f e r (pH 7.2) for 2 h at room temperature, post-fixed in i~ osmium tetroxide, dehydrated in a graded ethanol series, dried at the critical point and coated with gold. Observation and photography were made with a JEOL JSM-T20 Scanning Electron Microscope operating at 20 KV. RESULTS AND DISCUSSION The young inflorescence segments of Setaria italica became swollen and enlarged one week after culture on MS medium with 2,4-D and KT (or BAP). Segments callused at the cut ends and on most spikelets of the explants within two weeks. Addition of CH to the medium had no apparent effect on the initial formation of callus. There were two types of calli initiated in the cultures of inflorescence explants, as reported in the other cereals and grasses [0zias-Akins and Vasil, 1982; Wang and Vasil, 1982; Rangan and Vasil, 1983). One was soft, friable and nen-embryogenic, which usually was formed on the inflorescence axis and on the explants from very young (smaller than 0.5 cm in length) or older (longer than 2 cm) inflorescences. The other was white, compact and nodule-like, from which embryogenic callus lines were established (Fig. 1). The embryogenic calli could be maintained on MS medium supplemented with

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FIGURE LECENDS Fig. I, Embryogenic callus derived from cultured inflorescence segment of Setaria italica after 25 days of culture on MS medium with 2 mg/l 2,4-D and 0.5 mg/l HAP. x 9.4; Fig. 2, Embryoid formed in vitro [cl, coleoptile ;cr, coleorhiza; sc, scutellum), x 185; Fig. 3, Plantlets regenerated from embryogenic callus.

2 mg/l 2,4-D and 0.2-0.5 mg/l KT [or BAP), or with 2 mg/1 2iPA and 0.2 mg/1 NAA, but roots readily formed on the latter. Embryogenic growth was maintained better if the calli were alternately subcultured on both the media described above. Scanning electron microscopy showed that embryoids with coleoptile and scutellum were differentiated from the callusing segments or subcultured embryogenic calli [Fig. 2). The embryoids formed on the above media could not develop further. When the calli with embryoids were transferred onto MS medium with 2 mg/1 BAP and 0.5 mg/1 NAA, embryoids germinated and grew into plantlets [Fig. 3). Some embryogenic calli have maintained the capacity for plant regeneration for over fourteen months. However, the vigour of plant regeneration decreased during the subcultures, and after one year's subculture, only very few shoots formed with poorly developed roots. Our previous work on S. italica and S. lutescens [Xu et al., 1983) has showed that a great number of shoots and plantlets could also be directly regenerated from the inflorescence explants cultured on the medium with 2 mg/1 HAP + 0.5 mg/1 IAA [or NAA). In such a case, green leafy structures usually formed first. In order to know how this development differs from somatic embryogenesis in the present report, histological observations are needed. The results described above provide additional evidence to support the viewpoint that somatic embryogenesis may occur in tissue cultures of most species of cereals and grasses [Vasil et al., 1982). The capacity to regenerate plants from the young inflorescence segments through somatic embryogenesis provides a useful method for the rapid multiplication of cereal crops. - -

ACKNOWLEDGEMENTS We thank Mr. Y. L. Li [Luoyang Institute of Agricultural Sciences, Henan) for providing the seeds, and Mr. N. X. Chen for his photography. REFERENCES Ban, Y., T. Kokuba & Y. Miyaji, 1971, B u l l Fac. Agric. Kagoshima Univ., 21: 77-81. B r e t t e l l , R. I. S., W. W e r n i c k e & E. Thomas, 1980, Protoplasma 104: 141-148. Loo, S. W. & Z. H. Xu, 1984, R e v i e w of Chinese Sciences [in press). Lu, C. & I. K. Vasil, 1982, Amer. J. Hot. 69: 77-81. 0zias-Akins, P. & I. K. Vasil, 1982, Protoplasma ii0: 95-105. Murashige, T. & F. Skoog, 1962, Physiol. Plant. 15: 473-497. Rangan, T. S. & I. K. Vasil, 1983, Z. Pflanzenphysiol. 109: 49-53. Vasil, V. & I. K. Vasil, 1981, Amer. J. Bot. 68: 867-872. Vasil, I. ~., V. V a s i l , C. Y. Lu, P. Ozias-Akins, Z. Haydu & D. Y. Wang, 1982, In: V a r i a b i l i t y in Plants Regenerated from Tissue Culture. [Eds. E. Earle & Y. Demarly) Praeger Press, New York, pp. 3-21. Wang, D. Y., 1984, Chinese J. Cell Biol. 6: 17-20. Wang, D. Y. & Y. Kong, 1984, PI. C e l l Re. kin Press). Wang, D. Y. & I. K.Vasil, 1982, P l a n t Sci. Lett. 25: 147-154. Xu, Z. H., Z. M. Wei & L. J. Yang, 1983, P l a n t Physiol. Comm. [Shanghai) [5): 40.

Somatic embryogenesis and plant regeneration in cultured immature inflorescences of Setaria italica.

Young inflorescence explants of Setaria italica in culture showed high capacity for regenerating plantlets through somatic embryogenesis. Embryogenic ...
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