31. Plant regeneration from protoplasts of cold tolerant rice varieties


Division of Plant Breeding, ICAR Research Compex for North-Eastern Hill Region, Barapani 793103, India

Plant regeneration from protoplasts is still a prerequisite for genetic manipulations like somatic hybridization and cybridization and for direct gene transfer. There are a number of reports on plant regeneration from Japonica as well as indica varieties (Vasil, 1992). We report here plant regeneration from protoplasts of cold tolerant Japonica and Indica varieties from higher hills 1000 msl of North-Eastern India.

Embryogenic calli were induced from scutella of immature and mature embryos on MS medium supplemented with 2 mgl-1 2,4-D and 0.2 mgl-1 BAP and from anthers on G5 medium (Gupta and Borthakur 1987). Cell suspension cultures were established from embryogenic calli of group I (Indica) rice variety, Mandri and Group VI (Japonica) rice varieties viz. RCPL 1-1C, RCPL 1-2C, Nami and Dullo-10, after repeated subcultures in AA medium with 4 mgl-1 2,4-D at 2-3 day interval for 3-4 months. Protoplasts were isolated in an enzyme mixture containing 1% cellulase RS, 0.1% Pectolyase Y-23 and 5 mM MES in CPW 13 M (Abdullah et al., 1987). They were purified on 1M sucrose and cultured in the wells (surrounded by feeders) in (i) liquid N6 medium supplemented with 1.5 ml-1 2,4-D, 0.2 mgl-1, zeatin and 500 mgl-1 casein hydrolysate (N6PCMZ) (ii) N6PCMZ solidified with 0.15% agarose (N6PCMZ Ag) and (iii) N6PCMZ Ag on 0.8 μ- pore membrane filter kept on the top of feeder. Protocalli (3-4 mm dia.) were transferred to regeneration medium (MS + 2 mgl-1 BAP + 0.5 mgl-1 NAA) at 25 ± 2°C in 16/8 hr light/dark period, where plantlets differentiated. They were allowed to grow for 20-30 days, hardened and then transferred to soil following the method described by Gupta and Pattanayak (1993).

Microscopic examination of the protoplasts plated in liquid as well as agarose showed cell wall formation within 48 hours. First cell division was generally seen on 3rd or 4th day. Subsequent divisions gave rise to microcolonies which became macroscopic after 25-30 days. Early divisions in the protoplasts plated on membrane filter could not be seen due to opaqueness of the membrane. However, calli were seen after 30-40 days (Fig. 1) which were allowed to grow further. Protoplasts plated at different densities ranging from 3x105 to 1X106/ ml, formed colonies in all the plating densities. The plating densities of 4X105/ ml were found to be optimum for obtaining highest plating efficiency (0.13 to 0.23% at day 30) in all varieties (Table 1).

Table 1. Frequency of colony formation on 30th day in protoplasts plated in liquid N6PCMZ

Protoplast  ================================================================
  density   RCPL          RCPL           Dullo-10       Nami        Mandri
            1-1C          1-2C 
3x105       160 (0.05)    134 (0.05)    128 (0.04)   130 (0.04)  180  (0.06)
4X105       812 (0.20)    633 (0.16)    520 (0.13)   592 (0.15)  900  (0.23)
6X105       890 (0.15)    683 (0.12)    592 (0.1O)   630 (0.10)  985  (0.16)
8X105       920 (0.12)    738 (0.10)    623 (0.09)   689 (0.09)  1022 (0.13)
1X106       972 (0.10)    803 (0.08)    690 (0.07)   780 (0.08)  1270 (0.13)
Figures given in parenthesis are plating efficiency on 30th day of cultures.

Table 2. Plating efficiency of suspension-derived protoplasts and frequency of plant regeneration in the protocolonies

                                       Protoplast      Plating     Plant re-
                  Age of cell          culture         efficiency  genera-
Variety  Varietal suspension  Explant  method in       on the      tion
         group     (months)            presence of     30th day   ==========
                                       feeders*                    No.   %
RCPL 1-1C  VI       8     Microspore     Liquid           0.2%     63    22
                          derived        Agarose          NC       72    46
                          callus         Membrane filter  NC       46    14

Nami       VI       5     Mature seed    Liquid           0.15%    68    28
RCPL 1-2C  VI       4     Mature seed    Liquid           0.16%    70    29

Dullo-10   VI       5     Mature seed    Liquid           0.13%    65    18
Mandri     I        6     Immature seed  Liquid           0.22%    75    20
*Feeder: 1.5 ml packed cell volume+20ml N6PCMZ+0.8% agarose.
NC: not counted.

Protocalli transferred to regeneration medium differentiated and formed plantlets after 25-30 days through somatic embryogenesis. So far more than 150 plants have been grown to maturity. Data on plating efficiency and plant regeneration are presented in Table 2.

The work was supported by the grant from the Department of Biotechnology, Government of India. We thank Dr. G.S. Khush and Dr. D.S. Brar of IRRI, Philippines for isoenzymatic grouping of rice varieties used in this study.


Abdulla, R., E.C. Cooking and J. A. Thompson, 1987. Efficient plant regeneration from rice protoplasts through somatic embryogenesis. Bio/Technology 4: 1087-1090.

Gupta, H.S. and D.N. Borthakur, 1987. Improved rate of callus induction from rice anther culture following microscopic staging of microspores in iron alum-haematoxylin. Theor. Appl. Genet. 74: 95-99.

Gupta, H.S. and A. Pattanayak, 1993. Plant regeneration from mesophyll protoplasts of rice (Oryza sativa L.) Bio/Technology 11 (in press).

Vasil, I.K. 1992. Advances in cereal protoplast research. Physiol. Plant. 85: 279-283.