PlantCell Reports
Plant Cell Reports (I993) 12:320-323
9 Springer-Verlag 1993
Plant regeneration from protoplasts of durum wheat (Triticum durum Desf. cv. D6962) Y.M. Yang, D.D. He, and K.J. Scott Department of Biochemistry, The University of Queensland, Brisbane, Australia 4072 Received N o v e m b e r 25, 1992/Revised version received January 20, 1993 - C o m m u n i c a t e d by H. L f r z
ABSTRACT. Suspension cultures of durum wheat were established from embryogenic callus mainfained in liquid medium for 30 months. Protoplasts were readily isolated from the suspension cultures with yields as high as 3 X 107 protoplasts per g fresh weight suspension cells. When incubated in a modified MS medium containing half strength of the macr0elements, 5 ~tM 2,4-D (2,4dichlorophenoxyacetic acid) and 0.6 M glucose, protoplast-derived cells divided at frequencies ranging from 1.4 to 10.0 %. After transfer to a solid subculture medium, the protoplast-derived colonies formed embryogenic protuberances, from which green plants have been regenerated.
on a solid medium (Harris et.al. 1988, Wang et a1,1990, Redway et al. 1990). In the second method, primary embryogenic callus is transferred directly to a liquid medium and the suspension cultures are derived from callus which are maintained in liquid medium (Wang and Nguyen et al. 1990, Yang et al. 1991).
Key words: embryogenesis - liquid callus cultures plant regeneration - protoplast - suspension cultures Triticum durum
MATERIALS AND METHODS
INTRODUCTION The tetraploid durum wheat (Triticum durum Desf.) is an important food crop with a harvest area amounting to about 9% of the total wheat area in the world and 32% in the Mediterranean region (Bennici 1986). The first report on tissue culture of this wheat appeared in 1978 (Bennici and D'Amato); the induction of embryogenic callus from scutellum of immature embrTos ~s now a routine procedure (Bennici et al. 1988, He et al. 1988, Borrelli et ai.-1991), and recently, friable callus of durum wheat was established (Borrelli et al. 1991). However, the establishment of embryogenic suspension cultures and plant regeneration from protoptasts have not been reported. During the last four years, success in plant regeneration from protoplasts of the hexaploid bread wheat (T. aestivum L.) has been reported by several groups (Harris et al. 1988; Ren et al. 1989; Wang et al. 1990: Chang et al. 1991, Vasil et al. 1991; Yang et al. 1991; Li et al. 1992) and in most of these studies protoplasts were isolated from embryogenic suspension cultures. Two methods were used for the establishment of suspension cultures of hexaploid wheat; the first approach involves the establishment of a special type of callus (friable callus) through long-term subculture and the selection of callus Correspondence to: K. J. Scott
This paper reports the establishment of embryogenic suspension cultures of a durum wheat (T. durum cv. D6962) through the liquid culture approach and the subsequent plant regeneration from suspension culture protoplasts. To our knowledge, this is the first report on plant regeneration from protoplasts of durum wheat.
Definitions, Callus clumps, 3-10 mm in diameter, were formed when primary embryogenic callus of wheat was cultured in liquid medium. Such cultures were defined as liquid callus cultures as distinct from fine suspension cultures which consisted mainly of small cell clusters, containing a few hundred cells, less than 1 mm in diameter. In this study, however, the fine cell clusters of suspension cultures tended to grow into larger aggregates (1-3 mm in diameter) to form mixed suspension cultures, i.e. cultures containing both fine cell clusters and the larger aggregates.
Establishment of suspension cultures. For the initiation of suspension cultures, primary embryogenic callus (0.5g), which had been induced from the scutellum of immature embryos of D6962 (He et al. 1988), was transferred to 40 ml liquid 1/2 MS medium (Yang et al. 1991) containing 5 ~tM 2,4-D (2,4-dichlorophenoxyacetic acid). The cultures were maintained under diffuse light in 250mi flasks on a rotary shaker (80-100 rpm). These cultures were subcultured at weekly intervals ha fresh liquid medium. Fine suspension cultures were derived from fine cell clumps, which appeared in 30-month-old liquid callus cultures. These fine clusters were separated from the larger clumps by filtration through stainless steel mesh (pore size t.8 mrn, Swiss Screen), transferred to fresh medium and subcultured at weekly intervals.
Isolation and culture of protoplasts.
To collect cells for protoplast isolation, suspension cultures were filtered through a 180 ban stainless steel mesh and the cells retained on the mesh were used. The ceils were suspended in a solution (1 : 7 v/v) containing 2% Cellulase RS (Yakult, Tokyo), 0.2% Pectolyase Y23 (Seishin, Tokyo), 20 mM CaC12 and 0.6M mannitol. The mixture was maintained on a rotary shaker (ca 50 rpm)
321 for 1-3 hours and then left stationary for another 1-3 hours. In experiments determining the protoplast yields, the suspension cells were blotted dry with filter paper and weighed prior to the addition of enzyme solution. The cells were left on the shaker for exactly 3 hours and then left stationary for 3-9 hours. The density of the protoplasts in the digestion mixture was determined using a haemocytometer. The protoplasts were purified by filtration through 53 gm and 38 p,m stainless steel meshes foUowed by washing (3 times) with a solution containing 20 m M CaC1 z and 0.6M mannitol. Purified protoplasts were resuspended in 1/2 MS medium containing 5btM 2,4-D and 0.6 M glucose at a density of 1-10 X 105 protoplasts/mh In some experiments, the medium was gelled with 1.2% agarose. The cultures were incubated in 3.5 cm Petri dishes (Falcon, California) in the dark at 25 ~ To measure the frequencies of cell division and colony formation, the numbers of freshly embedded protoplasts, dividing cells, and colonies were counted from designated areas of the agarose medium.
Differentiation of plants.
Cell clumps from the liquid callus cultures were individually transferred to the solid differentiation medium (contaJning 0.1 btM 2,4-D). Cells frorn the fine suspension cultures were first collected by filtration through stainless steel mesh (pore size 300850 l-tm) and then spread on a subcuhure medium. Similarly, protocoloaies (proloplast-dcdved colonies) which were larger than 1 mm were individually transferred to a subculture medium, while smaller colonies were spread on the medium. The cmbr3,oids forming on these subcultured cells were transferred to a fresh differentiation medium to promote shoot formation.
RESULTS Long-term maintenance of embryogenic liquid callus cultures When primary embryogenic scutellum callus from immature embryos was subcultured weekly in liquid medium containing 5 ~M 2,4-D, the regeneration potential of the callus rapidly decreased and in 5 - 6 months was totally lost. However, one culture line, which was re-initiated from selected callus, did maintain its embryogenic potential. For initiation of the line, primary callus, which had been maintained in liquid medium [or four months, was transferred to solid differentiation medium; approximately 5% of the callus formed embryogenic nodules on solid medium, which were selected and transferred back into liquid medium. Subsequent differentiation tests, 2 and 4 months later, showed high plant regeneration (56% and 38% from the cell clumps), whereas the regeneration potential of the original primary calhls was totally lost. This selection process was repeated 4 times within a period of 30 months. Further selection was perlormed during subculture when those cell clumps forming roots, turning brown or becoming loose in texture were discarded. A reduction of 2,4-D concentration from 5 ~J.M to 1-2 btM facilitated this selection as more non-embryogenic callus clumps formed roots at the lower 2,4-D concentration and were therefore easily identified. In addition, the low concentration of 2,4-D enhanced the R)rmation of white protuberances. The callus clumps with white protuberances were selected since differentiation tests showed that up to 59 % of the white protuberances re;reed plants.
Establishment of suspension cultures Within the first two years, several attempts were made to initiate fine cultures from the liquid callus cultures by dicing large cell clumps into small pieces (1 mm) were individually transferred to a solid subculture medi.m, the majority turned watery with elongated cells, however, 7% formed many solid, smooth surfaced protuberances (Fig. 1H). When colonies (< 1 mm) were spread on a subculture medium, a thick lawn of cells developed and a few solid protuberances appeared. After transfer to differentiation
323 medium, these embryoids formed watery callus and more embryoids. Following 2 to 3 transfers to fresh differentiation medium a few embryoids germinated into green shoots. To date more than 500 embryoids have been obtained from 3 experiments, from which 34 green plants and shoots (Fig. 1K, 1L) have been recovered.
establishment of suspension cultures from this callus. Our success in regeneration of plants from protoplasts of durum wheat is a significant advance towards transformation of this wheat through direct gene transfer.
DISCUSSION Fine suspension cultures of cereals and grasses are established by two approaches: firstly by the establishment of friable callus on solid medium or secondly by direct transfer of the primary callus into liquid medium. The more common approach is to establish a friable callus (Redway et al. 1990); this method requires long-term subculture of the embryogenic callus on solid medium until the desired callus type appears, which is then transferred to a liquid medium (Harris et al. 1988, Wang et al. 1990, Redway et al. 1990, Li et al. 1992). The alternative approach of direct transfer of primary embryogenic callus into liquid medium has been successfully used in barley (Lights and Lorz, 1988) and more recently in hexaploid wheat (Wang and Nguyen, 1990, Yang et al. 1991). The establishment of suspension cultures of cv. D6962 has now been achieved using this method, suggesting that the liquid callus culture approach is reproducible for different species of cereals.
We thank Dr J. Scott for criticism of the manuscript. Financial assistance from the Australian Research Council, the Alumni Association of the University of Queensland, and the Investors in the Queensland Technology Partnership, established under the Commonwealth Syndicated R & D program, is gratefully acknowledged.
The major problem encountered with both approaches is the difficulty in converting the solid primary embryogenic callus of wheat into a particular type of embryogenic callus which is granular and loose. This callus appears friable on solid medium and is readily dispersed in liquid medium. However, with both approaches, the appearance of this callus is unpredictable and requires many months of subculture and selection of primary embryogenic callus on either solid (Wang et al, 1990, Redway et al. 1990), or in liquid medium (Wang and Nguyen 1990, Yang et al. 1991). This results not only in the difficulty in the establishment of suspension cultures from any desired cultivars, but also in the accumulation of somaclonal variation during the long term subculture (Wang and Nguyen 1990). Obviously, a deeper understanding of factors determining the formation of the friable callus is required. This study has shown that protoplasls isolated from the large cell clumps of suspension cultures have the potential for sustained cell division. However, these large cell clumps from the suspension cultures and those from liquid callus cultures differed. Firstly, the former had many tiny protuberances which continuously split off from the large clumps to form fine cell clusters, i_e. fine suspension cultures. In contrast, the large cell clumps from liquid callus cultures were solid and usually would not disperse. Secondly, although several attempts were made to culture protoplasts isolated from the large cell clumps of the liquid callus culture, cell division was never observed. This was similar to hexaploid wheat cv. Hartog, where protoplasts isolated from liquid callus cultures also failed to divide whereas sustained cell division was observed with protoplasts isolated from the large clumps of suspension cultures (He et al. 1992). Embryogenic protoplasts have been considered as one of the most promising target cells for genetic transformation of cereals and grasses (Potrykus 1990)_ Although Borrelli et al_ (1991) have recently reported the establishment of friable callus of durum wheat, they did not report the
ACKNOWLEDGEMENTS
REFERENCES Bennici A (1986) Durum wheat (Triticum durum Desf.). In Biotechnology in Agricuhure and Forestry (ed. Bajaj YPS) Vol 2. Springer-Verlag, Berlin, pp 89-104 Bennici A, D'Amato F (1978) In vitro regeneration of durum wheat plants: Chromosome numbers of regenerated plantlets. Z Pflanzenzuchtg 81 : 305-311 Bennici A, Carfare L, Dameri RM, Profumo P (1988) Callus formation and plantlet regeneration from immature Triticum durum Desf. embryos. Euphylica 39:255-263 BorrellJ GM, Lupotto E, Locatelli E, Wittmer G (t991) Long-term optimized embryogenic cultures in durum wheat (Triticum durum Desf.) Plant Cell Reports 10:296-299 Chang YF, Wang WC, Warfield CY, Nguyen HT, Wong JR (1991) Plant regeneration from protoplasts isolated from long-term cell cultures of wheat (Triticum aestivum L.). Plant Cell Reports 9: 611614 Harris R, Wright M, Byme M, Vamum J, Brightwell B, Schubert K (1988) Callus formation and plantlet regeneration from protoplasts derived from suspension cultures of wheat (Tritiet~n aestivum L.). Plant Cell Reports 7:337-340 He DG, Yang YM, Scott KJ (1988) A comparison of epiblast callus and scutellum callus induction in wheat: the effect of embryo age, genotype and medium. Plant Sci 57:225-233 He DG, Yang YM, Scott g J (1992) Plant regeneration from protoplasts of wheat (Triticum aestivum cv. Ham)g). Plant Cell Reports 11: 16-19 Li ZY, Xia GM, Chen HM (1992) Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.). Plant Cell Tissue Organ Cult 28:7%85 Lchrs R, L~rz H (1988) Initiation of morphogenic cell-suspension and protoplast cultures of barley. Planta 175:71-81 Potrykus I (1990) Gene transfer to cereals: an assessment. Bi&technol 8:535-542 Redway FA, Vasil V, Vasil IK (1990) Characterization and regeneration of wheat (Triticum aestivum L.) embryogenic cell suspension cultures. Plant Cell Reports 8:7t4-717 Ren YG, Jia JF, Li MY, Zhen GC (t989) Ptant regeneration from protoplasts isolated from callus cultures of immature inflorescence wheat (Triticum aestivum L.). Chinese Sci Bull 34:1648-1652 Vasit V, Redway F, Vasil IK (1991) Regeneration of plants from embryogenic suspension culture protoplasts of wheat (Triticum aestivumL.). Bio\Technol 8:429-434 Wang HB, Li XH, Sun Y R, Chen J, Zhu Z, Fang R, Wang P, Wei JQ (1990) Culture of wheat protoplast. Scientia Shfica, Series B 33: 294-302 Wang WC, Nguyen HT (1990) A novel approach for efficient plant regeneration from long-term suspension cultures of wheat. Plant Cell Reports 8:639-642 Yang YM, He DG, Scott KJ (1991) The establishment of embryogenic liquid cultures and suspension cultures of wheat by continuous callus selection. Aust J PlantPhysiol t8:445-452
Plant Cell Reports (I993) 12:320-323
9 Springer-Verlag 1993
Plant regeneration from protoplasts of durum wheat (Triticum durum Desf. cv. D6962) Y.M. Yang, D.D. He, and K.J. Scott Department of Biochemistry, The University of Queensland, Brisbane, Australia 4072 Received N o v e m b e r 25, 1992/Revised version received January 20, 1993 - C o m m u n i c a t e d by H. L f r z
ABSTRACT. Suspension cultures of durum wheat were established from embryogenic callus mainfained in liquid medium for 30 months. Protoplasts were readily isolated from the suspension cultures with yields as high as 3 X 107 protoplasts per g fresh weight suspension cells. When incubated in a modified MS medium containing half strength of the macr0elements, 5 ~tM 2,4-D (2,4dichlorophenoxyacetic acid) and 0.6 M glucose, protoplast-derived cells divided at frequencies ranging from 1.4 to 10.0 %. After transfer to a solid subculture medium, the protoplast-derived colonies formed embryogenic protuberances, from which green plants have been regenerated.
on a solid medium (Harris et.al. 1988, Wang et a1,1990, Redway et al. 1990). In the second method, primary embryogenic callus is transferred directly to a liquid medium and the suspension cultures are derived from callus which are maintained in liquid medium (Wang and Nguyen et al. 1990, Yang et al. 1991).
Key words: embryogenesis - liquid callus cultures plant regeneration - protoplast - suspension cultures Triticum durum
MATERIALS AND METHODS
INTRODUCTION The tetraploid durum wheat (Triticum durum Desf.) is an important food crop with a harvest area amounting to about 9% of the total wheat area in the world and 32% in the Mediterranean region (Bennici 1986). The first report on tissue culture of this wheat appeared in 1978 (Bennici and D'Amato); the induction of embryogenic callus from scutellum of immature embrTos ~s now a routine procedure (Bennici et al. 1988, He et al. 1988, Borrelli et ai.-1991), and recently, friable callus of durum wheat was established (Borrelli et al. 1991). However, the establishment of embryogenic suspension cultures and plant regeneration from protoptasts have not been reported. During the last four years, success in plant regeneration from protoplasts of the hexaploid bread wheat (T. aestivum L.) has been reported by several groups (Harris et al. 1988; Ren et al. 1989; Wang et al. 1990: Chang et al. 1991, Vasil et al. 1991; Yang et al. 1991; Li et al. 1992) and in most of these studies protoplasts were isolated from embryogenic suspension cultures. Two methods were used for the establishment of suspension cultures of hexaploid wheat; the first approach involves the establishment of a special type of callus (friable callus) through long-term subculture and the selection of callus Correspondence to: K. J. Scott
This paper reports the establishment of embryogenic suspension cultures of a durum wheat (T. durum cv. D6962) through the liquid culture approach and the subsequent plant regeneration from suspension culture protoplasts. To our knowledge, this is the first report on plant regeneration from protoplasts of durum wheat.
Definitions, Callus clumps, 3-10 mm in diameter, were formed when primary embryogenic callus of wheat was cultured in liquid medium. Such cultures were defined as liquid callus cultures as distinct from fine suspension cultures which consisted mainly of small cell clusters, containing a few hundred cells, less than 1 mm in diameter. In this study, however, the fine cell clusters of suspension cultures tended to grow into larger aggregates (1-3 mm in diameter) to form mixed suspension cultures, i.e. cultures containing both fine cell clusters and the larger aggregates.
Establishment of suspension cultures. For the initiation of suspension cultures, primary embryogenic callus (0.5g), which had been induced from the scutellum of immature embryos of D6962 (He et al. 1988), was transferred to 40 ml liquid 1/2 MS medium (Yang et al. 1991) containing 5 ~tM 2,4-D (2,4-dichlorophenoxyacetic acid). The cultures were maintained under diffuse light in 250mi flasks on a rotary shaker (80-100 rpm). These cultures were subcultured at weekly intervals ha fresh liquid medium. Fine suspension cultures were derived from fine cell clumps, which appeared in 30-month-old liquid callus cultures. These fine clusters were separated from the larger clumps by filtration through stainless steel mesh (pore size t.8 mrn, Swiss Screen), transferred to fresh medium and subcultured at weekly intervals.
Isolation and culture of protoplasts.
To collect cells for protoplast isolation, suspension cultures were filtered through a 180 ban stainless steel mesh and the cells retained on the mesh were used. The ceils were suspended in a solution (1 : 7 v/v) containing 2% Cellulase RS (Yakult, Tokyo), 0.2% Pectolyase Y23 (Seishin, Tokyo), 20 mM CaC12 and 0.6M mannitol. The mixture was maintained on a rotary shaker (ca 50 rpm)
321 for 1-3 hours and then left stationary for another 1-3 hours. In experiments determining the protoplast yields, the suspension cells were blotted dry with filter paper and weighed prior to the addition of enzyme solution. The cells were left on the shaker for exactly 3 hours and then left stationary for 3-9 hours. The density of the protoplasts in the digestion mixture was determined using a haemocytometer. The protoplasts were purified by filtration through 53 gm and 38 p,m stainless steel meshes foUowed by washing (3 times) with a solution containing 20 m M CaC1 z and 0.6M mannitol. Purified protoplasts were resuspended in 1/2 MS medium containing 5btM 2,4-D and 0.6 M glucose at a density of 1-10 X 105 protoplasts/mh In some experiments, the medium was gelled with 1.2% agarose. The cultures were incubated in 3.5 cm Petri dishes (Falcon, California) in the dark at 25 ~ To measure the frequencies of cell division and colony formation, the numbers of freshly embedded protoplasts, dividing cells, and colonies were counted from designated areas of the agarose medium.
Differentiation of plants.
Cell clumps from the liquid callus cultures were individually transferred to the solid differentiation medium (contaJning 0.1 btM 2,4-D). Cells frorn the fine suspension cultures were first collected by filtration through stainless steel mesh (pore size 300850 l-tm) and then spread on a subcuhure medium. Similarly, protocoloaies (proloplast-dcdved colonies) which were larger than 1 mm were individually transferred to a subculture medium, while smaller colonies were spread on the medium. The cmbr3,oids forming on these subcultured cells were transferred to a fresh differentiation medium to promote shoot formation.
RESULTS Long-term maintenance of embryogenic liquid callus cultures When primary embryogenic scutellum callus from immature embryos was subcultured weekly in liquid medium containing 5 ~M 2,4-D, the regeneration potential of the callus rapidly decreased and in 5 - 6 months was totally lost. However, one culture line, which was re-initiated from selected callus, did maintain its embryogenic potential. For initiation of the line, primary callus, which had been maintained in liquid medium [or four months, was transferred to solid differentiation medium; approximately 5% of the callus formed embryogenic nodules on solid medium, which were selected and transferred back into liquid medium. Subsequent differentiation tests, 2 and 4 months later, showed high plant regeneration (56% and 38% from the cell clumps), whereas the regeneration potential of the original primary calhls was totally lost. This selection process was repeated 4 times within a period of 30 months. Further selection was perlormed during subculture when those cell clumps forming roots, turning brown or becoming loose in texture were discarded. A reduction of 2,4-D concentration from 5 ~J.M to 1-2 btM facilitated this selection as more non-embryogenic callus clumps formed roots at the lower 2,4-D concentration and were therefore easily identified. In addition, the low concentration of 2,4-D enhanced the R)rmation of white protuberances. The callus clumps with white protuberances were selected since differentiation tests showed that up to 59 % of the white protuberances re;reed plants.
Establishment of suspension cultures Within the first two years, several attempts were made to initiate fine cultures from the liquid callus cultures by dicing large cell clumps into small pieces (1 mm) were individually transferred to a solid subculture medi.m, the majority turned watery with elongated cells, however, 7% formed many solid, smooth surfaced protuberances (Fig. 1H). When colonies (< 1 mm) were spread on a subculture medium, a thick lawn of cells developed and a few solid protuberances appeared. After transfer to differentiation
323 medium, these embryoids formed watery callus and more embryoids. Following 2 to 3 transfers to fresh differentiation medium a few embryoids germinated into green shoots. To date more than 500 embryoids have been obtained from 3 experiments, from which 34 green plants and shoots (Fig. 1K, 1L) have been recovered.
establishment of suspension cultures from this callus. Our success in regeneration of plants from protoplasts of durum wheat is a significant advance towards transformation of this wheat through direct gene transfer.
DISCUSSION Fine suspension cultures of cereals and grasses are established by two approaches: firstly by the establishment of friable callus on solid medium or secondly by direct transfer of the primary callus into liquid medium. The more common approach is to establish a friable callus (Redway et al. 1990); this method requires long-term subculture of the embryogenic callus on solid medium until the desired callus type appears, which is then transferred to a liquid medium (Harris et al. 1988, Wang et al. 1990, Redway et al. 1990, Li et al. 1992). The alternative approach of direct transfer of primary embryogenic callus into liquid medium has been successfully used in barley (Lights and Lorz, 1988) and more recently in hexaploid wheat (Wang and Nguyen, 1990, Yang et al. 1991). The establishment of suspension cultures of cv. D6962 has now been achieved using this method, suggesting that the liquid callus culture approach is reproducible for different species of cereals.
We thank Dr J. Scott for criticism of the manuscript. Financial assistance from the Australian Research Council, the Alumni Association of the University of Queensland, and the Investors in the Queensland Technology Partnership, established under the Commonwealth Syndicated R & D program, is gratefully acknowledged.
The major problem encountered with both approaches is the difficulty in converting the solid primary embryogenic callus of wheat into a particular type of embryogenic callus which is granular and loose. This callus appears friable on solid medium and is readily dispersed in liquid medium. However, with both approaches, the appearance of this callus is unpredictable and requires many months of subculture and selection of primary embryogenic callus on either solid (Wang et al, 1990, Redway et al. 1990), or in liquid medium (Wang and Nguyen 1990, Yang et al. 1991). This results not only in the difficulty in the establishment of suspension cultures from any desired cultivars, but also in the accumulation of somaclonal variation during the long term subculture (Wang and Nguyen 1990). Obviously, a deeper understanding of factors determining the formation of the friable callus is required. This study has shown that protoplasls isolated from the large cell clumps of suspension cultures have the potential for sustained cell division. However, these large cell clumps from the suspension cultures and those from liquid callus cultures differed. Firstly, the former had many tiny protuberances which continuously split off from the large clumps to form fine cell clusters, i_e. fine suspension cultures. In contrast, the large cell clumps from liquid callus cultures were solid and usually would not disperse. Secondly, although several attempts were made to culture protoplasts isolated from the large cell clumps of the liquid callus culture, cell division was never observed. This was similar to hexaploid wheat cv. Hartog, where protoplasts isolated from liquid callus cultures also failed to divide whereas sustained cell division was observed with protoplasts isolated from the large clumps of suspension cultures (He et al. 1992). Embryogenic protoplasts have been considered as one of the most promising target cells for genetic transformation of cereals and grasses (Potrykus 1990)_ Although Borrelli et al_ (1991) have recently reported the establishment of friable callus of durum wheat, they did not report the
ACKNOWLEDGEMENTS
REFERENCES Bennici A (1986) Durum wheat (Triticum durum Desf.). In Biotechnology in Agricuhure and Forestry (ed. Bajaj YPS) Vol 2. Springer-Verlag, Berlin, pp 89-104 Bennici A, D'Amato F (1978) In vitro regeneration of durum wheat plants: Chromosome numbers of regenerated plantlets. Z Pflanzenzuchtg 81 : 305-311 Bennici A, Carfare L, Dameri RM, Profumo P (1988) Callus formation and plantlet regeneration from immature Triticum durum Desf. embryos. Euphylica 39:255-263 BorrellJ GM, Lupotto E, Locatelli E, Wittmer G (t991) Long-term optimized embryogenic cultures in durum wheat (Triticum durum Desf.) Plant Cell Reports 10:296-299 Chang YF, Wang WC, Warfield CY, Nguyen HT, Wong JR (1991) Plant regeneration from protoplasts isolated from long-term cell cultures of wheat (Triticum aestivum L.). Plant Cell Reports 9: 611614 Harris R, Wright M, Byme M, Vamum J, Brightwell B, Schubert K (1988) Callus formation and plantlet regeneration from protoplasts derived from suspension cultures of wheat (Tritiet~n aestivum L.). Plant Cell Reports 7:337-340 He DG, Yang YM, Scott KJ (1988) A comparison of epiblast callus and scutellum callus induction in wheat: the effect of embryo age, genotype and medium. Plant Sci 57:225-233 He DG, Yang YM, Scott g J (1992) Plant regeneration from protoplasts of wheat (Triticum aestivum cv. Ham)g). Plant Cell Reports 11: 16-19 Li ZY, Xia GM, Chen HM (1992) Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.). Plant Cell Tissue Organ Cult 28:7%85 Lchrs R, L~rz H (1988) Initiation of morphogenic cell-suspension and protoplast cultures of barley. Planta 175:71-81 Potrykus I (1990) Gene transfer to cereals: an assessment. Bi&technol 8:535-542 Redway FA, Vasil V, Vasil IK (1990) Characterization and regeneration of wheat (Triticum aestivum L.) embryogenic cell suspension cultures. Plant Cell Reports 8:7t4-717 Ren YG, Jia JF, Li MY, Zhen GC (t989) Ptant regeneration from protoplasts isolated from callus cultures of immature inflorescence wheat (Triticum aestivum L.). Chinese Sci Bull 34:1648-1652 Vasit V, Redway F, Vasil IK (1991) Regeneration of plants from embryogenic suspension culture protoplasts of wheat (Triticum aestivumL.). Bio\Technol 8:429-434 Wang HB, Li XH, Sun Y R, Chen J, Zhu Z, Fang R, Wang P, Wei JQ (1990) Culture of wheat protoplast. Scientia Shfica, Series B 33: 294-302 Wang WC, Nguyen HT (1990) A novel approach for efficient plant regeneration from long-term suspension cultures of wheat. Plant Cell Reports 8:639-642 Yang YM, He DG, Scott KJ (1991) The establishment of embryogenic liquid cultures and suspension cultures of wheat by continuous callus selection. Aust J PlantPhysiol t8:445-452
