47. High frequency plantlet regeneration in rice anther callus cultures

Consistent plantlet regeneration from pollen calli in sufficient numbers enhances the efficiency and use of anther culture technique in high volume rice breeding. Factors that influence the differentiation of pollen callus are: 1) age of the pollen callus, 2) sucrose concentration, and 3) ratio of auxin to cytokinin in the regeneration medium (cf. Chu 1982). Stimulation of shoot bud formation and plant regeneration by a two step culture method (Inoue and Maeda 1981) and high frequency long term plant regeneration (Heyser et al. 1983) were, however, reported in somatic callus cultures of rice. This note describes high frequency rengeneration of plants from anther callus cultures on a regeneration medium with proper manipulation of plant growth substances. We obtained a large number of regenerants from anther callus cultures of indica and japonica genotypes of O. sativa as well as from interspecific hybrids between genome A species of genus Oryza (Fig. 1).

Pollen calli were induced using N\6\ induction medium with addition of 2,4- dichlorophenoxy acetic acid (2,4-D, 2mg/1), kinetin (1 mg/1) and coconut milk (CM, 10% v/v). For plant regeneration, Murashige and Skoog's (MS) basal medium supplemented with different concentrations of cytokinins (kinetin and benzyladenine) was used in a series of step-wise regeneration experiments. In the first set of treatments MS medium was supplemented with a-napthalene acetic acid (NAA, 1mg/1) and the levels of kinetin (K) and benzyladenine (BA) varied (0 to 3mg/1). Judged on the results of these trials further regeneration experiments were carried out using MS medium supplemented with a combination of K and BA in different proportions but keeping the NAA level constant (1mg/1). It was observed that presence of both K and BA is not only important in the regeneration medium but also their ratio is critical in obtaining very high frequency regeneration. MS medium with the addition of K (0.5mg/1), BA (2 mg/1) and NAA (1 mg/1), i.e. K : BA : NAA in the ratio of 1:4:2 gave excellent regeneration from anther calli (Fig. 1). The combination of K, BA and NAA which stiumlated high frequency regeneration was further tested with the addition of organic additives - yeast extract (YE), casein hydrolysate (CH) and coconut milk (CM). Of these three organic additives tested addition of CM 10% (v/v) has shown promotive effect and enhanced the regeneration further. This modified MS regeneration medium was tested for its efficiency in terms of plant regeneration over an array of genotypes of O. sativa, subspecies indica and japonica, intervarietal hybrids of O. sativa and two interspecific hybrids. The regeneration frequencies obtained using this protocol are presented in Table. 1. From these data it is clear that through manipulation of kinetin and benzyladenine ratio in the regeneration medium it is possible to obtain consistently high frequency regeneration of plantlets from anther callus of a wide range of genotypes especially indicas which are considered to be recalcitrant.

Table 1. Regeneration frequencies from anther callus culture of different genotypes

Chu, C.C., 1982. Anther culture of rice and its significance in distant hybridization. In Rice Tissue Culture Planning Conference, 47-53, 1982, IRRI.

Inoue, M. and E. Maeda, 1981. Stimulation of shoot bud and plantlet formation in rice callus cultures by two-step culture method using abscissic acid and kinetin. Japan J. Crop Sci. 50: 318-322.

Heyser, J.W., T.A. Dykes, K.J. DeMott and M.W. Nabors, 1983. High frequency long term regeneration of rice from callus culture. Plant Science Letters 29: 175-182.