Downing College, University of Cambridge, Cambridge CB2 1 DQ, UK; Plant Biotechnology Centre, Punjab. Agricultural University, Ludhiana 141004, India.
139
Euphytica 81 : 1 3 9 -142,1995 . © 1995 Kluwer Academic Publishers . Printed in the Netherlands .
Micropropagation of Indica rice through proliferation of axillary shoots
J.S . Sandhu l , S .S. Gosal, M .S . Gill & H.S. Dhaliwal ' Downing College, University of Cambridge, Cambridge CB2 1 DQ, UK ; Plant Biotechnology Centre, Punjab Agricultural University, Ludhiana 141004, India Received 16 March 1993 ; accepted 26 October 1994
Key words : Oryza sativa, indica rice, shoot proliferation, micropropagation, cytokinins
Summary Week old seedlings of indica rice variety Jaya obtained on basal MS medium and further sub-cultured on agar solidified MS medium supplemented with cytokinins, sucrose (3% w/v) and mannitol (1 % w/v) lead to development of multiple shoot buds . Shoot cultures were maintained and multiplied in liquid medium containing BAP 5 mg 1-1, sucrose (3% w/v) and mannitol (1% w/v) . Profuse rooting was obtained on transfer to MS liquid medium containing IBA 1 mg 1-1 and sucrose (3% w/v) . Complete plants were successfully transferred to soil and grown to maturity.
Introduction
Materials and methods
Protocols ensuring reproducible plant regeneration from somatic cells (Abe & Futsuhara, 1985 ; Brar et al ., 1985), gametic cells (Chen et al ., 1981 ; Datta et al ., 1990) and protoplasts (Abdullah et al ., 1986 ; Yamada et al ., 1986) and transgenic plants have been developed both in japonica and indica rice (Peng et al ., 1992) . The technique of micropropagation is independent of the seasonal constraints and ensures rapid, true to type and year around propagation of plants under disease-free environment . Moreover, plants derived through micropropagation using meristem culture usually become free even from systemic pathogens . Micropropagation techniques in cereals, particularly in japonica rice through seed (Hisajima et al ., 1987), shoot base segments (Greco et al ., 1990) and wild rice species (Finch et al ., 1992) have been established whereas, the technique needs to be standardized in indica rice . The present communication deals with the development of a protocol ensuring efficient propagation of indica rice through rapid proliferation of axillary shoots using mature seeds as explant.
Dehusked seeds of a commercial rice variety Jaya after surface sterilization with HgC12 (0 .1%) for 8 minutes were rinsed thrice in sterile water and were cultured on basal MS (Murashige & Skoog, 1962) medium for germination . The seeds were placed in the medium with embryo facing upward and endosperm anchored inside. One week-old seedlings were subcultured on agar solidified MS medium supplemented with high concentration of cytokinins to induce multiple shoots . Twenty five ml of liquid medium was dispensed in flasks (Borosil, 250 ml) for multiplication of shoot buds . Solid and liquid MS basal media supplemented with sucrose (3% w/v), mannitol (1% w/v) various concentrations of BAP (1, 2, 3, 4, 5 mg 1 -1 ) and kin (1, 2, 3, 4, 5 mg 1 -1 ) were used to induce multiple shoot buds . Liquid cultures were continuously shaken by placing on Gyrotory shaker maintained at 90 RPM under fluorescent light (5000 Lux) with 16 hours light and 8 hours dark regimes at 25 ± 1° C .
140 Results and discussion Stage I: Establishment of shoot cultures Week-old seedlings obtained on basal MS medium and subcultured on agar solidified MS medium supplemented with high concentration of BAP (5 mg 1 -1 ) lead to induction of multiple shoot buds . Less than 10% of the shoots cultured on high BAP medium showed shoot proliferation (Fig . 2A) . Likewise, Hisajima et al . (1987) have also reported that frequency of seedling exhibiting multiple shoot buds was low (40%) . This shows that variation exists among the seedlings for their ability to form multiple shoot buds in primary culture . Stage II: Shoot multiplication The established shoot cultures from Stage I were subcultured in liquid MS medium, and these multiple shoots were highly proliferative in successive subcultures, however, the proliferation rate depended on the composition of culture medium . MS medium supplemented with various concentrations of BAP showed significantly better shoot proliferation than with Kinetin (Fig . 1) . Similar observations have been made by Hisajima et al . (1987) and Greco et al . (1990). Among the various media compositions, MS medium supplemented with BAP 5 mg 1 -1 , sucrose (3% w/v) and mannitol (1% w/v) lead to intense proliferation and formation of shoot clumps in liquid medium (Fig . 2C) . Each clump (55-65 shoot buds/shoots) was separated into about 20 smaller clumps consisting of 3-4 shoots . Subsequent serial subculturing of clumps after every 4 weeks lead to about 20 fold increase in shoot buds during each subculture . It is estimated that starting with one clump (55-65 shoots), 192 x 10 6 plants can be obtained within six months using this protocol . Rapid shoot multiplication has also been reported in japonica rice by Hisajima et al . (1987) and Greco et al . (1990) but with further scope of improvement . The high frequency of multiple shoot buds obtained at a much rapid rate in the present study may be attributed to continuous gentle shaking as compared to stationary cultures grown in liquid or on agar solidified medium .
1
2
3
4
Media concentration (rng I
® BAP
1\\\\\\\\
5
-' )
KIN
Fig. 1. Effect of various concentrations of cytokinins on shoot bud proliferation .
Stage III: Pretransplant This stage involves the induction of roots in vitro by transferring the shoot clumps (3-4 shoots) into liquid MS medium supplemented with IBA (1 mg 1 -1 ) and sucrose (3% w/v) . The root primordia were initiated within a week and profuse rooting was obtained in about 4 weeks on all the shoots (Fig . 2D) . Stage IV- Transplant The plantlets were removed from the culture vessels, washed thoroughly in running tap water and were transferred to plastic pots containing autoclaved sandy loam soil (Fig. 2E) under good light intensity and high humidity. Established plants were transferred to field (Fig . 2 F-G) in normal crop season and the survival rate was 73% . Fertile micropropagated plants have been successfully grown to full maturity. The scheme for the micropropagation through proliferation of axillary shoots is shown in Figure 3 . In conclusion, the developed protocol can be hopefully exploited for the mass propagation of important plant materials such as male sterile lines which are
141
Micropropagation of rice through proliferation of axillary shoots . A . Establishment of shoot cultures . B . Shoot proliferation after 2 weeks of incubation . C . Shoot proliferation and formation of clumps after 4 weeks of incubation . D. Induction of rooting in vitro . E. Transfer of plantlets to soil . F. Field performance of tissue culture propagated rice plants . G . Closer view of tissue culture propagated field grown plants . Fig . 2 .
142 Germination of seeds on MS basal medium
I
Culture initiation
1 Week-old seedlings subcultured on agar solidified MS medium supplemented with cytokinin
1 Inoculation on liquid MS medium
Shoot maintenance and
supplemented with high concentration of cytokinin/BAP (5 ml l - t )
multiplication cytokinin
1 Separation of shoot buds into smaller clumps of 3-4 shoots
1 Induction of rooting in vitro
Pretransplant
1 Transfer of complete plantlets to soil
Transplant
1 Fertile micropropagated plants grown to full maturity
Fig . 3.
Seed production
Flow scheme for the micropropagation of indica rice variety Jaya through proliferation of axillary shoots .
required in large numbers for production of hybrid seeds as reported by Kumari et al . (1988) . Besides the technique can also be used for the mass multiplication of Fl hybrids, somatic hybrids/cybrids, transgenic plants, haploids, doubled haploids produced through anther culture and endangered wild species for their maintenance, early assessment and release .
References Abdullah, R ., E .C . Cocking & J.A . Thompson, 1986 . Efficient plant regeneration from rice protoplasts through somatic embryogenesis . Bio/Technology 4: 1087-1090 . Abe, T. & Y. Futsuhara, 1985 . Efficient plant regeneration by somatic embryogenesis from root callus tissues of rice (Oryza sativa L .). J . Plant Physiol . 121 : 111-118 . Brar, D .S ., D .H. Ling & S . Yoshida, 1985 . Plant regeneration from somatic cell cultures of some IR varieties of rice . In : Biotechnology in International Agricultural Research, IRRI Pub., pp . 169177 . Chen, Y., Q . Zoo, L .S.D. Lu & K.L. Zheng, 1981 . Green plant regeneration from isolated rice pollen grains in vitro and the induction factors . Acta Genet . Sci . 8 : 158-163 .
Datta, S .K ., K. Datta & T. Potrykus, 1990. Embryogenesis and plant regeneration from microspores of both indica and japonica rice (Oryza sativa). Plant Sci . 67 (1) : 83-88 . Finch, R .P, A . Baset, I .H. Slamet & E.C . Cocking, 1992, In vitro shoot culture of wild Orygae and other grass species . Plant Cell, Tissue and Organ Culture 30 : 31-39 . Greco, B ., A.A . Lomonaco, B . Boggini, C. Tomassini & O .A . Tanzanella, 1990 . Clonal propagation of rice through proliferation of axillary shoots . Euphytica 48 (2) : 123-127. Hisajima, S ., P. Chongpraditnum & Y. Arai, 1987 . Microplant propagation of rice plant in vitro . Japan J. Trop . Agri . 31 (1) : 12-15 . Kumari, D.S ., N .P. Sharma & G .J .N. Rao, 1988 . Micropropagation of cytosterile rice stocks . Intl . Rice Res. New. L . 13 : 5-6. Murashige, T. & F Skoog, 1962 . A revised medium for rapid growth and bioassays with tobacco tissue culture . Physiol . Plant 13 : 473479 . Peng, H., H . Kononowicz & T.K . Hodges, 1992. Transgenic indica rice plants . Theor. Appl . Genet . 83 : 855-863 . Yamada, Y., Z .Q . Yang & D .T. Tang, 1986. Plant regeneration from protoplast-derived callus of rice (Oryza sativa L.) . Plant Cell Rep . 5 (NZ) : 85-88 .
