26. Plant regeneration from protoplasts in wild Oryza species

Koh-ichi MORI, Yasushi OKINAKA and Toshiro KINOSHITA

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

Plant regeneration from rice protoplasts has been reported by a number of workers (Fujimura et al. 1985 ; Yamada et al. 1986; Toriyama et al. 1986; Abdullah et al. 1986; Coulibaly et al. 1986). However, regeneration from protoplasts of wild Oryza species has been reported only by Hayashi et al. (1988).

For isolating protoplasts, we induced callii from husked seeds of 65 strains of 11 wild species, which were made available through the courtesy of Dr. Y. Sano of National Institute of Genetics. Callii were induced from mature seeds on MS agar medium with 2 mg/l 2, 4-D, 20 g/l sucrose and 8 g/l agar, and were cultured in R2 liquid medium with 2 mg/l 2, 4-D and 30 g/l sucrose. Protoplasts were isolated from suspension cells by treatment with 4% Cellulas Onozuka RS, 1%

Table 1. Comparison of callus type, initiation of suspension cells and isolation of protoplasts among Oryza species

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                              callus No. of  Suspension    Isolation of
Species        Genome Origin  type   strains cells induced protoplasts
=======================================================================
rufipogon         AA  Asia     F       10        9             6
                               C        9        2             0
                      America  M        6        0             0
                               C        2        0
                      Oceania  FM       5        2             0
                               C        1        0
longistaminata    AA  Africa   FM       1        1             0
glaberrima        AA  Africa   F        1        1             1
breviligulata     AA  Africa   F        2        1             1
punctata          BB  Africa   F        1        1             1
                  BBCC do.     F        2        2             2
Officinalis       CC  Asia     F        4        2             1
                               C        2        nt
eichingeri        CC  Africa   F        1        1             nt
minuta            BBCC Asia    F        1        1
alta              CCDD America C        2        0
latifolia         CCDD America C        1        0
australiensis     EE Australia F        1        1             nt
                               C        4        nt
brachyantha       FF  Africa   F        5        3             nt
                               C        1        nt
=======================================================================
Note: F-friable, C-compact, M-morphogenic, FM-friable and morphogenic nt-not tested

Table 2. Plating efficiency and plant regeneration from protoplast-derived calli in wild Oryza species

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                    Plating    No. of   No. of plants  Frequency(%)
Species      Strain efficiency plating  regenerated    Green  Albino
                        (%)    calli    green  albino  plant  plant
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rufipogon    W0120      1.20     60     20       3     33.3   5.0
             W0122      4.05    220      2       3      1.0   1.4
             W0124      1.53    140      0       0       0      0
             W0133      1.31     50      0       0       0      0
             W0157      4.65    120      0       0       0      0
punctata     W1515      4.73    110      0      10       0    9.0
             W0015      5.80    230      0      37       0   16.1
             W1564      2.05    170      0       0       0      0
officinalis  W0002      4.23    170      0       4       0    2.4
minuta       W1331      0.41    190      0       0       0      0
sativa       Kitaake    4.52    310     67       0    21.6      0
====================================================================
Macerozyme R-10, 0.5% MES and 0.4 M Mannitol. Isolated protoplasts were solidified by R2 medium with 2 mg/l 2, 4-D, 136 g/l sucrose and 1.25% Sea plaque agarose, and were initiated by nurse culture method. Removing nurse cells after 10 days, growing cell colonies were placed on N6 hormone-free medium with 10 g/l agarose.

Callii were obtained from 62 out of 65 strains tested (Table 1). They were classified into four kinds of callus types, i.e., friable (F), compact (C), morphogenic (M), and friable and morphogenic (FM). In the common wild rice, O. rufipogon, callus types differed according to strains (Table 1). Induction of suspension cells was successful in 16 strains, but were more difficult and more culture time was needed in the wild strains than in Japonica strains. Suspension cells were not induced from any M-type callus. A large number of protoplasts were obtained from suspension cells of 10 strains, but the isolation was quite difficult ftom C- and F-type callii. In the succeeding culture, all protoplasts divided to form colonies (Table 2). After being transferred to the regeneration medium, green plants were regenerated from two strains of O. rufipogon, while only albino plants were obtained from O. punctata and O. officinalis. We will attempt to produce somatic hybrids between Oryza species on the basis of these results.

References

Abdullah, R. and E. Cocking 1986. Efficient plant regeneration from rice protoplasts through somatic embryogenesis. Biotechnology 4: 1087-1090.

Coulibaly, M. Y. and Y. Demaly 1986. Regeneration of plant from protoplasts of rice, Oryza sative L. Z. Pflanzenzuchtung. 96: 79-81.

Fujimura, T., M. Sakurai, H. Akagi, T. Negishi and A. Hirose 1986. Regeneration of rice plants from protoplasts. Plant Tissue Culture Lett. 2: 74-75.

Hayashi, Y., J. Kyozuka and K. Shimamoto 1988. Hybrids of rice (Oryza sativa L.) and wild Oryza species obtained by cell fusion. Mol. Gen. Genet. 214: 6-10.

Yamada, Y., Z-Q. Yang and D. Tang 1986. Plant regeneration from protoplast-derived callus of rice (Oryza sativa L.) protoplasts. Theor. Appl- Genet. 76: 801-808.