Plant Cell Reports
Plant Cell Reports (1991) 10: 200- 203
9 Springer-Verlag1991
Plant regeneration from protoplasts of wild rice (Oryza rufipogon Griff.) Abdul B a s e t 1, R o b e r t P. Finch 2, and E d w a r d C. Cocking Plant Genetic Manipulation Group. Department of Botany, University of Nottingham, Nottingham NG7 2RD, UK 1 On leave from the Plant Breeding Division, Bangladesh Rice Research Institute, Bangladesh, India 2 Present address: Department of Plant Science, Scottish Agricultural College, Auchincruive, Ayr KA6 5HW, Scotland Received February 19, 1991/Revised version received March 23, 1991 - Communicated by I. Protrykus
Abstract Embryogenic callus initiated from basal segments o f micropropagated shoots of Oryza rufipogon were used to initiate cell suspension cultures. After approximately 3 months these cultures were capable o f yielding large numbers o f protoplasts w h i c h u n d e r w e n t sustained division in agarose-solidified medium at a frequency comparable to that observed with Japonica rice protoplasts in previous studies. O. rufipogon plants were reproducibly regenerated from the protoplast--derived callus and are currently being ~ o w n to maturity. This is the first report o f plant regeneration from protoplasts of a wild species of Or3,za~
Abbreviations BAP = 6-benzyalamino purme; 2,4-D = 2,4-dichlorophenoxyacetic acid; MES = 2[N-morpholino] ethanesulphonic acid; NAA = a-naphthalene acetic acid; PAR = photosynthetically active radiation; SCV = settled-cell volume. Introduction
Or3,za rufipogon is a perennial wild rice species distributed from tropical Asia to Australia (Vaughan 1989). It is closely related to Asian cultivated rice (Oryza sativa), having the same A A genome, but with a number of distinct genetic characters important to rice breeders, including resistance to s t a ~ a n t flooding and acid sulphate soils (Jena and Khush 1987). Some accessions also offer novel sources of cytoplasmic male sterility (Sitch et al. 1990). In the past 5 years a number o f protocols have been published for the regeneration of plants from protoplasts of cultivated rice. The development of these procedmes has led to the production o f somatic hybrids/cybrids (Terada et al. 1987; Toriyama and Hinata 1988; Hayashi et al. 1988; Finch et al. 1990; Yang et al. 1989; Kyozuka et al. 1989; A k a ~ et al. 1989) and transgenic plants (Zhang et al. 1988; Toriyama et al. 1988; Zhang and Wu 1988; Shirnamoto et al. 1989; Peng et al. 1990; Offprint requests to." E. C. Cocking
Datta et al. 1990) througJa protoplast filsion and direct uptake of foreign DNA respectively. Although protoplast regeneration systems are available for a wide range o f Japonica rice varieties, and a "true Indica' variety (Lee et al. 1989), there have been no reports, as yet, of plant regeneration from protoplasts o f wild rice species which are an important source of genetic diversity. Here we present the first report o f plant regeneration from protoplasts o f a wild rice species, Oryza t~tfipogon. Our protocol is b a ~ t on previous work with Japonica rice (Abdullah et al. 1986; Finch et al. 1991). However, the use of friable embryogenic callus derived from micropropagated shoots o f O.rufipogon (Finch unpublished) was critical for the production o f suitable cell suspension cultures, and a strict subculture regime was essential for their maintenance.
Materials and Methods Initiation of micropropagules Seeds of O. rufipogon (Ace.no. [03522) supplied by T.T. Chang, IRRI, were after-ripened for 5d (50 vC), dehusked, surfaee-sterilized (20% (vPr Domestos for 30 rain, 5 washes in sterile distilled water) and germinated on autoclaved MS-based medium (Murashige and Skoog 1962) containing 4% (w/v) sucrose and solidified with 0.8% (w/v) agar atpH 5.8 without hormones (='MS0') in 170 ml glass jars in the light (125 lzmol/m2/s PAR, 16h daylength) at 25 ~C. The endosperm and radicle were excised from 7-day-old seedlings which were then inoculated to a depth of 5 mm into 30 ml MS medium (4 explaaats per 170 ml jar) containing 6%(wN) sucrose and 2 mg/1 BAP, solidified with 0.4% (w/v) Sigma type I agarose (pH 5.8, autoelavedl and maintained as before. Mulliple shoots or 'micropropagules" developed at the base of the explants.
Callus inductionfrom micropropagules Segments (2 mm thick) cut from firm white tissue at the base of micropropagules were inoculated onto 25 ml autoclaved Linsmaier and Skoog (1965) medium containing 2.5 mg/l 2,4-D and solidified with 0.4% (w/v) agarose (='LS2.5') in 9 cm plastic Petri dishes and maintained in the dark at 28 ~C. Pale yellow callus with a dry appearanee was selectively subcultured onto fresh LS2.5 medium every four weeks. Such callus resembled embryogenie callus of Japonica rice.
201 Initiation of cell suspension culturesfrom micropropagule callus Soft embryoid structures on the surface of the callus (lg, 3months-old) were inoculated into a 100ml conical flask containing 10 ml AA2 liquid medium (Abdullah et at. 1986) and shaken (120 rpm) in the dark at 28 ~ Seventy percent of the medium was replaced at 5day intervals. After 6 weeks, a 7 ml aliquot (1 ml SCV, 6 ml old medium) was subeultured into 21 ml fresh AA2 medium, using a 10 ml plastic pipette (Sterilin), and the resulting culture maintained as before. This subculture procedure was subsequently carried out at weekly intervals. Suspension cultures were suitable for protoplast isolation after 2 months.
Transfer ofplants to the glasshouse Shoots of approximately 10 cm in height were washed to remove the agarose from the roots and transl~rred to 10 cm plastic pots containing pearlite in plastic propagator trays with transparent lids, watered twice daily with a 0.v-2% (v/v) solution of Maxicrop organic feed (Maxicrop Ltd., Tonbridge, UK) and maintained in a t 2h day:night cycle at 28 'C:24~C respectively with a relative humidity of 70%. The propagator lids were removed after 1 week, and the plants transferred to a 3:1 mixture of compost [t:1 mixture of John Innes No. 3 (Fisons pie., Suffolk, UK.) and Levington soil-less compost (J. Bentley Ltd., South Humberside, UK)] and perlite in 20 cm pots after a further 2 weeks for growth to maturity.
Isolation and cultureof protoplasts Protoplasts were isolated from freely-divided celt suspensions, 5 or 6d after subculture, using an enzyme mixture containing CPW salts (Frearson et al. 1973), 0.25% (w/v) CeUulase RS, 0.ff25% (w/v) Pectolyase YZ3 and 5 mM MES at pH 5.8 (18h at 28 ~ with shaking at 30 rpm), pipetted through a 30//m nylon sieve, collected at 80 g (5 min) and washed twice in liquid KPR cul0are medium (Abdullah et at. 1986). The purified protoplasts were heat-shocked at 45 ~ for 5 rain (Thompson et al. 1987) and cultured at a density of 3.5 x 10 5/ml in KPR medium, containing 0.6% (w/v) Sea-plaque agarose (FMC Corp., Rockland, ME, USA) in the dark at 28 C. Plating efficiencies were determined after 7d (percentage of protoplasts dividing) and t4d (final percentage of protoplasm undergoing sustained division to give microcolonies).
Plant regenerationfrom protoplast-derived microcolonies Microcolonies of between 1 and 2 mm diameter were transferred into differentiation medium (~MSKN': MS salts, 4% (w/v) sucrose, 2 mgJlkinctin, 05 mg/I NAA, 0.4% (w/v) Sigma type t agarose, pH 5.8); 4 colonies were placed onto 2 ml aliquots in separate chambers of 25-well plastic Sterifin dishes and maintained in the dark at 28:C. Somatic embryos which were visible on the surface of the eaUus within 5d began to germinate after a further 7d. The differentiating colonies were transferred to 'MSB2A' medium (MS salts, 6% (w/v) sucrose, 2 mgJl BAP, 0.4% (w/v) Sigma type I agarose, pH 5.8) for 1 week and fmatty transferred to 'MSN1.5A' medium (as MSB2A without BAP but with 1.5 mg~ NAA), both at 28 ~C with a t6h daylength (125/.tmol/m-'/s PAR), to promote shoot and root development respectively.
Results Initiation of callus and cell suspension cultures Embryogenic callus initiated from micropropagated shoots o f O.rufipogon grew rapidly on LS2.5 medium to form numerous globular sWuctures (Fig.la) and was capable of plant regeneration when transferred to regeneration medium (Finch unpublished). After 2 subcultures onto fresh LS2.5 medium the callus was suitably friable for the initiation of cell suspension cultttres. Established cell suspensions exhibited a doubling in settled cell volume every 3 or 4d and were composed o f clusters o f small, cytoplasmic cells (Fig.lb).
Isolation amt culture of protoptasts Protoplast yields from cell suspensions o f at least 3months-old averaged 2.5 x 10 6/g ft. wt. (Fig. lc). Initial divisions o f plated protoplasts were observed after 3d o f culture. Plating efficiencies o f between 45 and 50% were observed after 7d (Table 1). The frequency o f sustained protoplast division after t4d, resulting in colony formation (Fig. ld/e), averaged 0_5 %. The compact microcolonies ~m'ewto a diameter of l m m within 3 weeks enabling transfer o f individual protoclones to differentiation medium.
Plant regeneration from protoptast-derived microcalh~ White globular structures resembling the somatic embryos previously observed on the surface o f Japonica rice
Table 1. Plant regeneration from protoplast-derived cell colonies of
O.rufipogon
Cell line
Age of cell suspension
Plating efficiency at 5d (%)
Plating efficiency No. colonies transferred No. calluses at 14d (%) transferred to regener- that prod=sustained division ation medium uced plants
1(a) 1(b) l(b) 1(b) l(b) 1(c)
6 2 3 3 4 4
43 not tested (nt) 42 51 53 42
0.46 0.38 0.54 0.60 0.38 0.36
months months months months months months
100 60 80 100 80 60
t5 4 nt 5 3 3
202
Figure 1 a) Fanbryogenie mieropropagule-derived callus. Bar = 2.5cm. b) Embryogenic ceil suspension composed of densely-cytoplasmic cells, 2 months after initiation. Bar = 120~m. c) Freshly isolated cell suspension-derived pmtoplasts. Bar = 60~tm. d) Protoplast-derived cell r alter 2 weeks of culture. Bar = 120gm. e) Protoplast-derived microcalluses after 3 weeks of culture. Bar = 7mm, t) Development of root and shoot primordia in the surface of pmtoplast-derived callus on MSKN differentiation medium. Bar = 2ram. g) Plant development in MSN1.5A medium. Bar = 1.3cm. h) Protoolast-derived plant in soit. Bar = 4.5cm.
203 protoptast-derived callus (Abdullah et al. 1986) developed on the surface o f approximately 6% o f O.rufipogon protoplastderived calluses within 5d of transfer to MSKN medium, and germinated to form well defined coleoptiles within 12d (Fig.1 f). Transfer of differentiating callus to MSB2A medium was essential for optimal shoot development, but few roots were formed. Subsequent transfer to MSN1.5A promoted root development and shoot elongation (Fig.lg) and plants were ready for transfer to pots after 3 weeks (Fig.lh). An average of 4 plants developed from each regenerating callus. No albino regenerants were produced.
Discussion Green plants were successfully produced from protoplasts o f the wild rice species O.rufipogon. The techniques used were similar to those adopted for Japonica rice by Abdullah et al. (1986) but micropropagule callus was used to initiate the cell suspensions utilised as a source of protoplasts. We suggest that the micropropagation procedure conditioned the tissues for growth in vitro which resulted in the production of highly embryogenic friable callus from mieropropagule explants. Nurse cultures were not necessary to sustain the division o f O.rufipogon protoplasts although specialised nurse cultures were an absolute requirement for sustained division of protoplasts of Japonica and Indica rice in some protocols (Kyozuka et al. 1987; Lee et al. 1989). Plant regeneration frex!uencies from O.rufipogon protoplasts were in the range 4 to 6.6% (expressed as the percentage of protoplast-derived calluses ~ving plants). However, we were able to promote the development o f several plants from each regenerating callus through the use o f a 3-step regeneration procedure. Although this involved the use o f a cytokinin-containing regeneration medium, normally required for organogenesis, the protoplastderived callus possessed many globular embryoid structures which appeared to concomitantly produce roots and shoots in response to their removal from 2,4-D-containing medium. We found that the separate use of BAP and N A A promoted shoot and root production respectively. The plants obtained have been transferred to the glasshouse for growth to maturity and appear phenotypically normal. O.rufipogon usually produces few seeds and is daylength sensitive. Plants regenerated from protoptasts are currently being grown in short-day conditions to promote flowering. The production of plants from protoptasts isolated from 3 different cell suspension lines of this species (Table 1) demonstrates the reproducibility of our technique. The ability, to produce friable, embryogenic, micropropagule-callus from other important Oryzae, such as O.granulata which is shade and drouglit tolerant, may allow the development o f similar protoplast regeneration techniques for other such wild rice ~ecies. The availability o f a regenerating protoplast system for O.ntfipogon will facilitate the transt~ of nuclear and cytoplas-
mic genes into cultivated rice through protoplast fusion. Since protoplasts o f the wild rice species are capable of regeneration there would be no need to develop protoplast regeneration systems for the cultivated rice varieties used in fusion studies. Thus the possibility exists to transfer useful genes from wild rice into those important Indica rice varieties which cannot, as yet, be regenerated from protoplasts. The production o f transgenic wild rice following direct DNA uptake into protoplasts also offers an opportunity for the development o f selection systems for somatic hybridisation studies.
Acknowledgements The authors are grateful to BV Case for photographic assistance. AB is on leave from the Plant Breeding Division, Bangladesh Rice Research Institute and is in receipt of a British Council studentship. The f'maneiat assistance of the UK Overseas Development Administration and the Rockefeller Foundation is gratefully acknowledged.
References Abdullah R, ThompsonJA, Cocking El: (1986) Bio/Technolngy 4:1087- 1090. AkagiH, SakamotoM, NegishiT, Fujimura T (1989) Mol. Gen. Genet. 215:501 - 506. Datta SK. Peterhans A, Datta K. Potrykus I (1990) Bio/Technology 8:736 - 740. Finch RP, Lynch trr, Jotham JP, CockingEC (1991b) In: Bajaj, YPS (ed.): Biotechnolog3~in A~oricultureand Forestry.,Vol.14. Spdnger-Verlag, Heidelberg (in press). Finch PP. Slamet IH, Cocking EC (1990) J.Plant Physiok t36:592_- 598. Frearson EM. PowerJB, Cocking EC (1973) 33:130 - t37. Hayashi Y. KyozukaJ, ShimamotoK (1988) Mol. Gen. Genet. 214:6 - 10. Jena KK. Khush GS (1987) Proc. Int. Rice Res. Con.IRRLPhilippines KyozukaJ, HayashiY, ShimamotoK (1987)Mol. Gen. Genet. 206:408 - 4t3. KyozukaJ, KanedaT, ShomamotoK (1989) Bio/Technology 7:1171- 1174. Lee L SchrollRE, Grimest-ID.HodgesTK (1989) Ptanta t70:3~_5- 333. Linsmaier EM, SkoogF (1965) Physiol. Plant. 8:100 - t27. Murashige T, Skoog F (f962) Physiol.Plant. 15:473 - 497. Pang J, Lyznik,HodgesTK (1990) Proc.2nd Inl, Rice Genet Syrup. IRRI, Philippines (in press). ShimamotoIr Terada R, IzawaT. FujimotoH (1989) Nature338:274 - 276. Sitch LA. DalmacioRD, Elloran R, RomeroGO. Amante AD, Leung H. Nelson R, Klaush GS (1990) Proc. 4th Annual Meeting Rockefeller Foundation's Internationallh'ogrammeon Rice Biotechnology,IRRL Philippines (abstract). Terada R, KyozukaJ, NishibayashiS, ShirnamotoK (1987)Mol. Gen, Genet. 210:39 - 43. ThompsonJA, AbduUahR. Chen W-H, GartlandKMA (t987) J. Plant Physiol. 127".367- 370. ToriyamaK, Hinata K (1988) Theor. Appl. Genet. 76:665 - 668. ToriyamaK. ArimotoY, UchimiyaH, Hinata K (1988) Biofl'eclmolo~o3r 6:1072- 1074. Vaughan DA (1989) IRRI research paper series no. t 38. The IntentionalRice Research Institute. P.O.Box933, Manila, Philippines. Yang Z-Q, Shikanai T, Mori K, Yamada Y (1989)Theor. AppLGenet. 77:305 - 310. Zhang HM, Yang H, Rech EL, Colds TJ, Davis AS, Mulligan BJ. Cocking EC, DaveyMR (1988) Plant Cell Rep. 7".379- 384. Zhang W, Wu R (1988) Theor. Appl. Genet. 76:835 - 840.
Plant Cell Reports (1991) 10: 200- 203
9 Springer-Verlag1991
Plant regeneration from protoplasts of wild rice (Oryza rufipogon Griff.) Abdul B a s e t 1, R o b e r t P. Finch 2, and E d w a r d C. Cocking Plant Genetic Manipulation Group. Department of Botany, University of Nottingham, Nottingham NG7 2RD, UK 1 On leave from the Plant Breeding Division, Bangladesh Rice Research Institute, Bangladesh, India 2 Present address: Department of Plant Science, Scottish Agricultural College, Auchincruive, Ayr KA6 5HW, Scotland Received February 19, 1991/Revised version received March 23, 1991 - Communicated by I. Protrykus
Abstract Embryogenic callus initiated from basal segments o f micropropagated shoots of Oryza rufipogon were used to initiate cell suspension cultures. After approximately 3 months these cultures were capable o f yielding large numbers o f protoplasts w h i c h u n d e r w e n t sustained division in agarose-solidified medium at a frequency comparable to that observed with Japonica rice protoplasts in previous studies. O. rufipogon plants were reproducibly regenerated from the protoplast--derived callus and are currently being ~ o w n to maturity. This is the first report o f plant regeneration from protoplasts of a wild species of Or3,za~
Abbreviations BAP = 6-benzyalamino purme; 2,4-D = 2,4-dichlorophenoxyacetic acid; MES = 2[N-morpholino] ethanesulphonic acid; NAA = a-naphthalene acetic acid; PAR = photosynthetically active radiation; SCV = settled-cell volume. Introduction
Or3,za rufipogon is a perennial wild rice species distributed from tropical Asia to Australia (Vaughan 1989). It is closely related to Asian cultivated rice (Oryza sativa), having the same A A genome, but with a number of distinct genetic characters important to rice breeders, including resistance to s t a ~ a n t flooding and acid sulphate soils (Jena and Khush 1987). Some accessions also offer novel sources of cytoplasmic male sterility (Sitch et al. 1990). In the past 5 years a number o f protocols have been published for the regeneration of plants from protoplasts of cultivated rice. The development of these procedmes has led to the production o f somatic hybrids/cybrids (Terada et al. 1987; Toriyama and Hinata 1988; Hayashi et al. 1988; Finch et al. 1990; Yang et al. 1989; Kyozuka et al. 1989; A k a ~ et al. 1989) and transgenic plants (Zhang et al. 1988; Toriyama et al. 1988; Zhang and Wu 1988; Shirnamoto et al. 1989; Peng et al. 1990; Offprint requests to." E. C. Cocking
Datta et al. 1990) througJa protoplast filsion and direct uptake of foreign DNA respectively. Although protoplast regeneration systems are available for a wide range o f Japonica rice varieties, and a "true Indica' variety (Lee et al. 1989), there have been no reports, as yet, of plant regeneration from protoplasts o f wild rice species which are an important source of genetic diversity. Here we present the first report o f plant regeneration from protoplasts o f a wild rice species, Oryza t~tfipogon. Our protocol is b a ~ t on previous work with Japonica rice (Abdullah et al. 1986; Finch et al. 1991). However, the use of friable embryogenic callus derived from micropropagated shoots o f O.rufipogon (Finch unpublished) was critical for the production o f suitable cell suspension cultures, and a strict subculture regime was essential for their maintenance.
Materials and Methods Initiation of micropropagules Seeds of O. rufipogon (Ace.no. [03522) supplied by T.T. Chang, IRRI, were after-ripened for 5d (50 vC), dehusked, surfaee-sterilized (20% (vPr Domestos for 30 rain, 5 washes in sterile distilled water) and germinated on autoclaved MS-based medium (Murashige and Skoog 1962) containing 4% (w/v) sucrose and solidified with 0.8% (w/v) agar atpH 5.8 without hormones (='MS0') in 170 ml glass jars in the light (125 lzmol/m2/s PAR, 16h daylength) at 25 ~C. The endosperm and radicle were excised from 7-day-old seedlings which were then inoculated to a depth of 5 mm into 30 ml MS medium (4 explaaats per 170 ml jar) containing 6%(wN) sucrose and 2 mg/1 BAP, solidified with 0.4% (w/v) Sigma type I agarose (pH 5.8, autoelavedl and maintained as before. Mulliple shoots or 'micropropagules" developed at the base of the explants.
Callus inductionfrom micropropagules Segments (2 mm thick) cut from firm white tissue at the base of micropropagules were inoculated onto 25 ml autoclaved Linsmaier and Skoog (1965) medium containing 2.5 mg/l 2,4-D and solidified with 0.4% (w/v) agarose (='LS2.5') in 9 cm plastic Petri dishes and maintained in the dark at 28 ~C. Pale yellow callus with a dry appearanee was selectively subcultured onto fresh LS2.5 medium every four weeks. Such callus resembled embryogenie callus of Japonica rice.
201 Initiation of cell suspension culturesfrom micropropagule callus Soft embryoid structures on the surface of the callus (lg, 3months-old) were inoculated into a 100ml conical flask containing 10 ml AA2 liquid medium (Abdullah et at. 1986) and shaken (120 rpm) in the dark at 28 ~ Seventy percent of the medium was replaced at 5day intervals. After 6 weeks, a 7 ml aliquot (1 ml SCV, 6 ml old medium) was subeultured into 21 ml fresh AA2 medium, using a 10 ml plastic pipette (Sterilin), and the resulting culture maintained as before. This subculture procedure was subsequently carried out at weekly intervals. Suspension cultures were suitable for protoplast isolation after 2 months.
Transfer ofplants to the glasshouse Shoots of approximately 10 cm in height were washed to remove the agarose from the roots and transl~rred to 10 cm plastic pots containing pearlite in plastic propagator trays with transparent lids, watered twice daily with a 0.v-2% (v/v) solution of Maxicrop organic feed (Maxicrop Ltd., Tonbridge, UK) and maintained in a t 2h day:night cycle at 28 'C:24~C respectively with a relative humidity of 70%. The propagator lids were removed after 1 week, and the plants transferred to a 3:1 mixture of compost [t:1 mixture of John Innes No. 3 (Fisons pie., Suffolk, UK.) and Levington soil-less compost (J. Bentley Ltd., South Humberside, UK)] and perlite in 20 cm pots after a further 2 weeks for growth to maturity.
Isolation and cultureof protoplasts Protoplasts were isolated from freely-divided celt suspensions, 5 or 6d after subculture, using an enzyme mixture containing CPW salts (Frearson et al. 1973), 0.25% (w/v) CeUulase RS, 0.ff25% (w/v) Pectolyase YZ3 and 5 mM MES at pH 5.8 (18h at 28 ~ with shaking at 30 rpm), pipetted through a 30//m nylon sieve, collected at 80 g (5 min) and washed twice in liquid KPR cul0are medium (Abdullah et at. 1986). The purified protoplasts were heat-shocked at 45 ~ for 5 rain (Thompson et al. 1987) and cultured at a density of 3.5 x 10 5/ml in KPR medium, containing 0.6% (w/v) Sea-plaque agarose (FMC Corp., Rockland, ME, USA) in the dark at 28 C. Plating efficiencies were determined after 7d (percentage of protoplasts dividing) and t4d (final percentage of protoplasm undergoing sustained division to give microcolonies).
Plant regenerationfrom protoplast-derived microcolonies Microcolonies of between 1 and 2 mm diameter were transferred into differentiation medium (~MSKN': MS salts, 4% (w/v) sucrose, 2 mgJlkinctin, 05 mg/I NAA, 0.4% (w/v) Sigma type t agarose, pH 5.8); 4 colonies were placed onto 2 ml aliquots in separate chambers of 25-well plastic Sterifin dishes and maintained in the dark at 28:C. Somatic embryos which were visible on the surface of the eaUus within 5d began to germinate after a further 7d. The differentiating colonies were transferred to 'MSB2A' medium (MS salts, 6% (w/v) sucrose, 2 mgJl BAP, 0.4% (w/v) Sigma type I agarose, pH 5.8) for 1 week and fmatty transferred to 'MSN1.5A' medium (as MSB2A without BAP but with 1.5 mg~ NAA), both at 28 ~C with a t6h daylength (125/.tmol/m-'/s PAR), to promote shoot and root development respectively.
Results Initiation of callus and cell suspension cultures Embryogenic callus initiated from micropropagated shoots o f O.rufipogon grew rapidly on LS2.5 medium to form numerous globular sWuctures (Fig.la) and was capable of plant regeneration when transferred to regeneration medium (Finch unpublished). After 2 subcultures onto fresh LS2.5 medium the callus was suitably friable for the initiation of cell suspension cultttres. Established cell suspensions exhibited a doubling in settled cell volume every 3 or 4d and were composed o f clusters o f small, cytoplasmic cells (Fig.lb).
Isolation amt culture of protoptasts Protoplast yields from cell suspensions o f at least 3months-old averaged 2.5 x 10 6/g ft. wt. (Fig. lc). Initial divisions o f plated protoplasts were observed after 3d o f culture. Plating efficiencies o f between 45 and 50% were observed after 7d (Table 1). The frequency o f sustained protoplast division after t4d, resulting in colony formation (Fig. ld/e), averaged 0_5 %. The compact microcolonies ~m'ewto a diameter of l m m within 3 weeks enabling transfer o f individual protoclones to differentiation medium.
Plant regeneration from protoptast-derived microcalh~ White globular structures resembling the somatic embryos previously observed on the surface o f Japonica rice
Table 1. Plant regeneration from protoplast-derived cell colonies of
O.rufipogon
Cell line
Age of cell suspension
Plating efficiency at 5d (%)
Plating efficiency No. colonies transferred No. calluses at 14d (%) transferred to regener- that prod=sustained division ation medium uced plants
1(a) 1(b) l(b) 1(b) l(b) 1(c)
6 2 3 3 4 4
43 not tested (nt) 42 51 53 42
0.46 0.38 0.54 0.60 0.38 0.36
months months months months months months
100 60 80 100 80 60
t5 4 nt 5 3 3
202
Figure 1 a) Fanbryogenie mieropropagule-derived callus. Bar = 2.5cm. b) Embryogenic ceil suspension composed of densely-cytoplasmic cells, 2 months after initiation. Bar = 120~m. c) Freshly isolated cell suspension-derived pmtoplasts. Bar = 60~tm. d) Protoplast-derived cell r alter 2 weeks of culture. Bar = 120gm. e) Protoplast-derived microcalluses after 3 weeks of culture. Bar = 7mm, t) Development of root and shoot primordia in the surface of pmtoplast-derived callus on MSKN differentiation medium. Bar = 2ram. g) Plant development in MSN1.5A medium. Bar = 1.3cm. h) Protoolast-derived plant in soit. Bar = 4.5cm.
203 protoptast-derived callus (Abdullah et al. 1986) developed on the surface o f approximately 6% o f O.rufipogon protoplastderived calluses within 5d of transfer to MSKN medium, and germinated to form well defined coleoptiles within 12d (Fig.1 f). Transfer of differentiating callus to MSB2A medium was essential for optimal shoot development, but few roots were formed. Subsequent transfer to MSN1.5A promoted root development and shoot elongation (Fig.lg) and plants were ready for transfer to pots after 3 weeks (Fig.lh). An average of 4 plants developed from each regenerating callus. No albino regenerants were produced.
Discussion Green plants were successfully produced from protoplasts o f the wild rice species O.rufipogon. The techniques used were similar to those adopted for Japonica rice by Abdullah et al. (1986) but micropropagule callus was used to initiate the cell suspensions utilised as a source of protoplasts. We suggest that the micropropagation procedure conditioned the tissues for growth in vitro which resulted in the production of highly embryogenic friable callus from mieropropagule explants. Nurse cultures were not necessary to sustain the division o f O.rufipogon protoplasts although specialised nurse cultures were an absolute requirement for sustained division of protoplasts of Japonica and Indica rice in some protocols (Kyozuka et al. 1987; Lee et al. 1989). Plant regeneration frex!uencies from O.rufipogon protoplasts were in the range 4 to 6.6% (expressed as the percentage of protoplast-derived calluses ~ving plants). However, we were able to promote the development o f several plants from each regenerating callus through the use o f a 3-step regeneration procedure. Although this involved the use o f a cytokinin-containing regeneration medium, normally required for organogenesis, the protoplastderived callus possessed many globular embryoid structures which appeared to concomitantly produce roots and shoots in response to their removal from 2,4-D-containing medium. We found that the separate use of BAP and N A A promoted shoot and root production respectively. The plants obtained have been transferred to the glasshouse for growth to maturity and appear phenotypically normal. O.rufipogon usually produces few seeds and is daylength sensitive. Plants regenerated from protoptasts are currently being grown in short-day conditions to promote flowering. The production of plants from protoptasts isolated from 3 different cell suspension lines of this species (Table 1) demonstrates the reproducibility of our technique. The ability, to produce friable, embryogenic, micropropagule-callus from other important Oryzae, such as O.granulata which is shade and drouglit tolerant, may allow the development o f similar protoplast regeneration techniques for other such wild rice ~ecies. The availability o f a regenerating protoplast system for O.ntfipogon will facilitate the transt~ of nuclear and cytoplas-
mic genes into cultivated rice through protoplast fusion. Since protoplasts o f the wild rice species are capable of regeneration there would be no need to develop protoplast regeneration systems for the cultivated rice varieties used in fusion studies. Thus the possibility exists to transfer useful genes from wild rice into those important Indica rice varieties which cannot, as yet, be regenerated from protoplasts. The production o f transgenic wild rice following direct DNA uptake into protoplasts also offers an opportunity for the development o f selection systems for somatic hybridisation studies.
Acknowledgements The authors are grateful to BV Case for photographic assistance. AB is on leave from the Plant Breeding Division, Bangladesh Rice Research Institute and is in receipt of a British Council studentship. The f'maneiat assistance of the UK Overseas Development Administration and the Rockefeller Foundation is gratefully acknowledged.
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