33. Selection of streptomycin-resistant cell lines in rice

Tetsuo Mikami and Toshiro Kinoshita

Plant Breeding Institute, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan

An approach involving somaclonal selection in tissue culture and subsequent genetic analysis has resulted in the recognition and identification of cytoplasmically inherited or nuclear-gene dependent resistance to streptomycin in Nicotiana (Maliga et al. 1973; Maliga 1981). Therefore, we have attempted to obtain streptomycin-resistant cell lines of rice, which might prove of value both in an extensive study of somatic cell genetics and in the development of a selection system for somatic hybrizidation.

Primary calli were induced from surface-sterilized seeds in 71 rice genotypes including japonica and indica varieties and their hybrids. Small pieces of callus (each weighing 50 mg) were then placed on MS medium with the addition of 0,125,150,500,750,1000 or 1500 ppm of streptomycin sulphate, in order to evaluate the inhibitory effects of the antibiotic in culture growth. This evaluation was based upon the resistance index, which represented the relative increase in fresh weight of the treated callus, after two months of incubation, given as a percentage of that of the control tissue grown in the absence of streptomycin (Kinoshita and Mikami 1984).

The results indicated that streptomycin was effective as an agent inhibiting the growth of rice tissue cultures (Table 1). Callus cultures at 500 ppm streptomycin enabled us to detect a wide range of varietal difference in drug resistance. The resistance index varied from 0.752 to 0.058, depending on strains. In contrast, the medium containing 1000 or 1500 ppm streptomycin almost completely inhibited the proliferation of inoculated calli, which turned brown or black in the majority of the genotypes examined.

On the basis of the screening test, we have chosen two sensitive genotypes, namely, Iwakogane (resistance index at 500 ppm streptomycin; 0.130 or 0.136) and H-21 (0.058), as suitable materials for selection of resistant cell lines (Table 1). Calli derived from seed and/or immature embryo were exposed to various levels of streptomycin in the initial and subsequent selection cycles. For each cycle in culture the fastest growing sectors of callus were selected for subculture transfers. In this way a total of 27 comparatively vigorous cell lines have been isolated after 7 to 12 subculture passages. As shown in Table 1, three (Iwakogane No. III, No. IV and No. XIV) out of 27 cell lines exhibited a significant increase in resistance index. It remaines to be seen whether this phenomenon may be due to the genetic changes or epigenetic effects. Plant regeneration from resistant cell lines is currently under investigation.

Table 1. Streptomycin-resistant cell lines in rice.

a) Callus from immature embryo culture

==============================================================================
Strain        Cell line   Concentration of streptomycin(ppm)    Resistance index
                        --------------------------------------   after final
                           T-1         T-2       T-3 to T-7         passage
==============================================================================
Iwakogane        I           0          0           0                 0.338
(0.136)*         II        500          0           0                 0.583
                 III      1000        250         250                 0.765
                 IV        250          0         250                 0.810
-----------------------------------------------------------------------------
H-21             I           0          0           0                 0.246
(0.058)          II        500        500         500                 0.249
                 III      1000          0           0                 0.274
                 IV        500          0         500                 0.632
                 V        1000        250         250                 0.633
                 VI        250        250         250                 0.634
==============================================================================
b) Callus from seed culture

==============================================================================
Strain     Cell Line    Concentration of stretomycin (ppm)    Resistance index
                       -----------------------------------    after final passage
                     T-1  T-2  T-3  T-4  T-5  T-6  T-7toT-12
==============================================================================
Iwakogane   XII      250  250  500  250    0  250    250         0.252
(0.130)    XIII      250  500  250  250  500 1000      0         0.624
            XIV      250 1000 1000  250  250  250    250         0.730
==============================================================================
*Resistance index of primary callus

Resistance index=( increase in mean fresh weight at 500 ppm streptomycin) /(increase in mean fresh weight at 0 ppm streptomycin)

References

Kinoshita, T. and T. Mikami, 1984. Alloplasmic effects on callus proliferation and streptomycin resistance in common wheat. Seiken Ziho 32: 31-38.

Maliga, P., 1981. Streptomycin resistance is inherited as a recessive Mendelian trait in a Nicotiana sylvestris line. Theor. Appl. Genet. 60: 1-3.

Maliga, P., A. Sz.-Brenznovits and L. Martion, 1973. Streptomycin resistant plants from callus culture of haploid tobacco. Nature (New Biol.) 244: 29-30.