PlantCell Reports
Plant Cell Reports (1992) 11:16-19
9 Springer-Verlag1992
Plant regeneration from protoplasts of wheat ( Triticum aestivum cv. Hartog) D. G. He, Y. M. Yang, and K. J. Scott Department of Biochemistry, The University of Queensland, Brisbane, Qld. 4072, Australia Received October 31, 1991/Revised version received November 6, 1991 - Communicated by H. L6rz
ABSTRACT Morphologically normal green plants have reproducibly been regenerated from protoplasts of an Australian wheat (Triticum aestivum cv. Hartog). The protoplasts were isolated from fine embryogenic suspension cultures which were initiated from embryogenic callus. Protoplasts were incubated in a modified liquid MS medium containing half strength o f the macroelements, 5 ~ M 2,4-D and 0.6 M glucose. Colonies were formed at frequencies ranging from 0.1% to 5%. The frequency of colonies forming fully developed plants varied between 1% and 25 %. More than eighty green plants with morphologically normal shoots and roots have been obtained and there was no difficulty in establishing these plants in soil. A cytological study of several randomly selected regenerated plants showed the normal chromosome c o m p l e m e n t for wheat (2n = 42). Key words: Embryogenesis - Plant regeneration Protoplast - Suspension cultures - Wheat
INTRODUCTION Protoplasts provide one of the most promising avenues for obtaining transgenic cereals and transgenic plants have been successfully obtained from protoplasts o f rice and maize through direct gene transfer (Potrykus 1990). Protoplasts are readily transformed by exogenous DNA, and plants derived from transformed single protoplasts are homogeneously transformed. However, protoplast culture o f the economically important cereals has been very difficult to achieve (Vasil 1987, Lorz et al. 1988, Potrykus 1990). Due to the world-wide economic importance of wheat, many attempts have been made to achieve plant regeneration from wheat protoplasts. However, wheat is one o f the most recalcitrant cereal species for protoplast culture ( M a d d o c k 1987). Recently there have been a few successful reports o f plant regeneration from wheat protoplasts (Harris et al. 1989, Ren et al. 1990, W a n g et al. 1990, Vasil et al. 1990, Chang et al. 1991). However, in most cases the experiments were not reproducible (Harris et al. 1989, Ren et al. 1990, W a n g et al. 1990, G u o et al. 1991). In only two instances were protoplastderived plants successfully transferred to soil (Vasil et al. 1990, Chang et al. 1991) and in one the plants were p r o b a b l y not fertile due to chromosomal deletion (Chang et aL 1991). Offprint requests to: K. J. Scott
W e now report the highly reproducible regeneration of morphologically normal green plants from protoplasts of an Australian wheat (Triticum aestivum cv. Hartog). These plants are readily established in soil and have the normal c h r o m o s o m e c o m p l e m e n t o f wheat (2n = 42).
MATERIALS AND METHODS Cell cultures. In this paper, liquid cultures refer to cultures maintained
in a hquid medium and consisting of callus clumps larger than 1 mm in diameter;, suspension cultures refer to finely dispersed cultures consisting mainly of small cell clusters, containing tess than a few hundred cells. The establishment and subculture of embryogenic liquid cultures and suspension cultures of wheat cv. Hartog were as previously reported (Yang et aL 1991). The non-embryogeuic suspension cultures used in this study were initiated from embryo-derived primary callus of cv. Oxley and cv. Timmo; and from protoplast-derived callus of cv. Timmo (He et al. 1990). Isolation and culture ofprotoplasts. Cell clusters were collected from either liquid cultures or suspension cultures by filtering through a stainless steel mesh (Swiss Screen), usually 53 lain for fine suspension cultures and 200 gm for liquid cultures. The cells retained on the mesh were transferred to 5 cm Petri dishes (Disposable Products, South Australia) to which a solution containing 2% Cellulase RS (Yakult, Tokyo), 0.2% Pectolyase Y23 (Seishin, Tokyo), 20 mM CaC12 and 0.6M mannitol was added (solution : cells = 5-10 : 1, v/v). The mixture was maintained on a rotary shaker (ca 50 rpm) for 3-5 hours and then left stationary for another 2-5 hours. The protoplasts were purified by filtering through 53 lain stainless steet mesh once and 38 lain mesh twice followed by 3 washes in a solution containing 20 mM CaC12 and 0.6M mannitol. Purified protoplasts were resuspended in 1/2 MS medium (Yang et al. 1991) containing 5~VI 2,4-D (2,4dichlorophenoxyacetic acid) and 0.6 M glucose at a density of 1-10 X l0 s pmtoplasts/ml. The cultures were incubated in 3 cm (Falcon, California) or 5 cm (Disposable, Australia) Petri dishes in the dark at 25 ~ Enzyme solutions, washing solutions and all media for protoplast culture were filter-sterilized by passage through 0.22 ~rn filters (Millipore). Differentiation of plants. Protoplast-derived colonies larger than 0.Smm were transferred to a differentiation medium (1/2MS medium, either hormone free or supplemented with different combinations of phytohormones as specified in the tex0. After 1-4 weeks, the regenerated plants were transferred to a second medium (hormone free 1/2 MS medium containing 1% sucrose). In this paper, fully developed plants refer to those regenerants which had both normal shoots and roots and were at least 3 cm in height, whereas plantlets included both those regenerants which subsequently developed into plants and those which formed only leafy structures. All differentiation media were sterilised by autoclaving.
17 Cytological studies. Root tips were collected from plants grown in Petri dishes or pots and left in ice-water (0 ~ for 24 hours, 0.03% (w/v) 8hydroxyquinoline for 3 hours at room temperature and then fixed in acetic alcohol (70% ethanol : acetic acid = 3:1). The fixed tissue was thoroughly washed and then incubated in an enzyme solution (2% Cellulase RI0, 1% Macerozyme RI0, 0.1% Peetolyase Y23) for 20 min at 37 ~ The cells were immediately fixed in acetic alcohol again. To observe the chromosomes, the cells were squashed in a drop of acetic carmine.
groups by filtering the suspension cultures through stainless steel meshes of decreasing pore size (1200 to 53 gm). The yield of protoplasts from these groups varied from 0.3 to 5 x 107 (Fig. 2); the group (175 to 750 pan) had the highest yields; with the large clusters (> 1200 pm) spontaneous fusion and rupture of protoplasts were frequently observed.
Isolation of protoplasts
The freshly isolated protoplasts often contained large starch granules. The protoplasts containing these granules were quite fragile, resulting in their rupture during centfifugation. However, the number of ceils with these granules decreased as the suspension cultures aged.
Protoplasts were readily isolated from fine embryogenic suspension cultures of cv. Hartog (Fig. 1.A) yielding as many as 5 X 107 protoplasts per gram (fresh weight suspension cells). The embryogenic suspension cultures of cv. Hartog tended to grow into large clumps (Yang et al. 1991), a phenomenon also noted in other wheat cultivars (Wang et al. 1990). After 7-10 hours digestion, these large cell clusters (>lmm) were still quite solid and the yield of protoplasts was low. In one experiment, the suspension cells were separated into five different size
First cell division (Fig. 1.B) was observed following one week incubation. As the dividing cells usually contained dense cytoplasm and many small granules, the newly formed cell walls between two daughter cells were not always visible, and frequently the small colonies showed a irregular contour indicating the outer cell walls (Fig. 1.C). After 30-40 days incubation, colonies became macroscopically visible (Fig. 1.D). The frequency of colony formation varied from 0.1% to 5%.
RESULTS
Fig. 1. Plant regeneration from embryogenic protoplasts of cv. Hartog. A. Freshly isolated protoplasts from the embryogenic suspension cultures of cv. Hartog. B. First cell division of protoplast-derived cells. C. Microscopic colonies (two weeks of incubation). D. Visible colonies after 40 days of incubation. E. Formation of embryogenic callus on 2,4-D containing (5 p.M) medium. F. Typical embtyoid forming on protoplast-derived embryogenic callus (v. ventral scale). G. Formation of a plantlet from a bipolar structure. H. Plants with morphologically normal shoots and roots. I. A root-tip cell from protoplast-derived plant showed the normal wheat chromosome complements (2n = 42). (Bar represents 20 ~un in A,B and C; 1 mm in D, E and G; 0.25 mm in F; and 1 cm in H)
18 Table 1.
Effect of 2,4-D concentration in differentiation medium on plant regeneration and shoot formation. Each treatment used 25 protoplast-derived colonies. O
Dif~renfiafion
Concentration of 2,4-D (la.M) in differentiation media
>,
w
i
A
B
C
D
i
i
I::
Fig 2. Yield of protoplasts (X 107/gm fresh weight) from different size clusters of suspension cultures. Suspension cultures of cv. Hartog were filtered through a series of stainless steel meshes of varying pore size (1200 bun to 53 gm). The cells retained on each mesh were collected, blotted with filter paper, and weighed. The cells were then incubated in enzyme solution (7 volumes per fresh weight of the cells) for three hours on a rotary shaker (50 rpm) and left stationary for another 4 hours. The five size groups were: A, >1200; B, 750-1200; C, 315-750; D, 185315; E, >53.
With the embryogenic protoplasts of cv. Hartog, following one week incubation, there was a marked increase in the viscosity of the medium; even when the Petri dishes were inverted, the 'liquid' medium remained in situ. By contrast, there was no change in the liquid medium containing the non-embryogenic protoplasts of the wheat cultivars Oxley and Timmo. Differentiation o f plants
More than 30%, and in some experiments up to 90%, of the protoplast-derived colonies were solid with a smooth surface and morphologically similar to the embryogenic callus of wheat. When transferred to differentiation medium some colonies formed bipolar slluctures with a shoot and a root (Fig. 1G). On medium containing 2,4-D the colonies proliferated into callus on which the formation of typical embryoids (Fig. 1E) was observed. Typical embryoids were easily identified by the well developed scutellum with a ventral scale (Fig.IF). However, as with the embryogenic wheat callus from immature embryos, or other explants, the formation of typical embryoids was rare (He et al. 1988). The frequency of colonies forming fully developed plants was low when compared to that of immature-embryoderived callus, where up to 100% of callus has the potential for plant regeneration (He et al. 1988). During the first week on a differentiation medium, more than 30% of the colonies developed one or two short, white or green leafy structures; subsequently most of these structures formed swollen cells and shoot formation ceased. This was exacerbated when the newly germinated embryoids were transferred to a fresh medium. Some of these stunted embryoids were white but were not true albinos as in several instances, these eventually formed green leaves. Only a small percentage of embryoids (125%) finally formed plants with normal shoots (Fig. 1H). In contrast to the regenerated wheat plants described by Chang et al. (1991) root formation occurred readily in our plants.
0
0.05
0.I0
0.50
5.00
A
12
16
20
12
0
B
4
12
12
32
20
16
28
32
44
20
C (A+B)
A: % of colonies forming whole plants; B: % of colonies forming leafy structures; C: % of colonies forming either whole plants or leafy structures.
Several differentiation media were tested to increase the differentiation frequency of embryoids and the effect of 2,4-D concenllation on proliferation and differentiation of colonies was examined (Table 1); it was found that colonies grew better on media containing a low concenlration of 2,4-D (0.05 to 0.5 pM). The differentiation frequency of plants was not significantly different between those incubated on medium containing 0.05, 0.1 and 0.5 ~ 2,4-D. On medium containing 5 ~tM 2,4-D, colonies developed into embryogenic callus, which differentiated into whole plants when transferred to hormone free medium. However, compared to the colonies incubated on media containing 0.05-0.5 ~tM 2,4D, more colonies on 5 laM 2,4-D medium became watery. The presence of kinetin (0.2mg/l) inhibited development and most of the colonies became brown; however, some did form green leaves and when transferred to a differentiation medium without kinetin, they formed normal plants. The age of colony (days since protoplast isolation) affected shoot formation; when 40-day-old colonies were transferred to differentiation medium, 25 of 150 (17%) formed whole plants, whereas in 54-day-old colonies only 1 of 184 (0.5%) formed a whole plant. Several hundred green plantlets have been obtained in a series of experiments. Over eighty of these regenerants have grown into plants greater than 5 cm high and no albinos have been observed; these plants were readily established in soil. Root tips were collected from several plants for cytological examination and the normal complement of wheat chromosomes (2n = 42) was observed (Fig. 1I). This is consistent with the norm,'il chromosomal complement observed in fine suspension cultures of cv. Hartog (Yang et al. 1991). DISCUSSION Although plant regeneration from wheat protoplasts has recently been reported by several authors, it is still far from a routine procedure. In these reports, only the formation of small plants was observed and information on the frequency of plant regeneration was scant. In addition, plant regeneration was not reproducible (Harris et al. 1989, Ren et al. 1990, Wang et al. 1990, Guo et al.
19 1991), or the regenerants were aneuploids (Chang et al. 1991), or embryogenic suspension reverted to a nonembryogenic state (Vasil et al. 1990, 1991). In contrast, our system has the advantage of producing normal green plants with a high reproducibility. In previous reports on plant regeneration from wheat protoplasts, the culture medium was usually complex and frequently some special conditions were considered to be essential, such as the use of Ficol, MES buffer (2-[Nmorpholino] ethanesulphonic acid) and special aeration (Harries et al. 1989). In contrast, our system was very simple; the medium was the commonly used MS medium with only minor modification; there were no complex organic additives and only one phytohormone (2,4-D) was present. The protoplasts were maintained in a liquid medium; no agarose or other gelling agents, or nurse cells were used. This procedure is probably not optimal but its simplicity emphasises the importance of the source of the protoplasts rather than the culture conditions: once embryogenic suspension cultures are established, it appears relatively simple to regenerate plants from the protoplasts derived from these cultures. Our success is another example demonstrating that the establishment of embryogenic suspension cultures is indeed a successful approach for obtaining totipotent cereal protoplasts (Vasil 1987). In our study, the embryogenic suspension cultures of cv. Hartog were initiated from embryogenic liquid cultures, which were also used for the isolation of protoplasts. Although there were difficulties, it was possible to isolate protoplasts from these liquid cultures; however, sustained cell division in the cells derived from these protoplasts has not been observed. We have also cultured protoplasts from primary embryogenic callus of cv. Hartog. Despite repeated attempts, however, cell division was rarely observed. Therefore, under present experimental conditions it appears that the finely dispersed state of the embryogenic cells, rather than genotype, is the most important factor for the successful induction of plant regeneration from wheat protoplasts. The major disadvantage of using fine suspension cultures is that their establishment is not only time-consuming but also unpredictable (Potrykus 1990). In contrast, the induction of primary embryogenic callus is a routine procedure and can be obtained using practically any wheat genotype (He et al. 1988). However, although protoplasts can readily be isolated from embryogenic callus, there had previously been only one report (Hayashi and Shimamoto 1988) where callus-derived protoplasts formed albino embryoids, but no plant formation and the result was not reproducible. Ren et al. (1990) and Guo et al. (1991) have reported plant regeneration from wheat protoplasts isolated from embryogenic callus. However, the callus used in these two studies was not primary callus but callus which had been subcultured on solid medium for a prolonged period. Although there is uncertainty about the reproducibility of these reports, it is of interest to note that similarities exist between the cells from these two studies and fine suspension cultures; i.e. the cells were highly friable and in a dispersed state, contrasting to the compact orientation of embryogenic cells in either primary callus or liquid cultures of wheat. More
information regarding the significance of the dispersed state of cells to the subsequent cell division of protoplasts may aid in overcoming the difficulties encountered in culturing protoplasts isolated directly from primary callus; this callus is a far more convenient starting material for the isolation of protoplasts than using embryogenic suspension cultures. ACKNOWLEDGEMENTS 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. Fig 1B and 1D were reproduced by courtesy of the Australian Joumal of Plant Physiology. REFERENCES 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 Guo GQ, Xia GM, Li ZY, Chen HM (1991) Direct somatic embryogenesisand plant regeneration from protoplast-derivedceils of wheat. ScientiaSinica, Series B 34:438-444 Harris R, Wright M, Byme M, Vamum J, Brightwell B, Schubert K (1989) Callus formation and plantlet regeneration from protoplasts derived from suspension cultures of wheat (Triticum aestivum L.). Plant Cell Reports7:337-340 Hayashi Y, Shimamoto K (1988) Wheat protoplast culture: embryogeniccolonyformationfrom protoplasts. Plant Cell Reports7: 414-417 He DG, Yang YM, Scou KJ (1988) A comparison of epiblast callus and scutellum callus induction in wheat: the effect of embryo age, genotypeand medium. Plant Science 57:225-233 He DG, Yang YM, and Scott KJ (1990) Factors affecting colony formation from protoplasts of wheat. The 30th Annual Meeting of Australian Societyof Plant Physiologists. Abstr P17 Lorz H, Gobel E, Brown P (1988) Advancesin tissue culture and progress towards genetic engineering in cereals. Plant Breeding 100: 1-25. Maddock SE (1987) Suspension and protoplast culture of hexaploid wheat (Triticum aestivura L.). Plant Cell Reports6:23-26 Potrykus I (1990) Gene transfer to cereals: an assessment. Bio/technology8:535-542 Ren YG, Jia JF, Li MY, Zhen GC (1990) Plant regeneration from protoplasts isolated from callus cultures of immature inflorescensesof wheat (Triticum aestivum L.). ChineseSci Bull 34:1648-1652 Yang YM, He DG, Scott KJ (1991) The establishmentof embryogenic suspension cultures of wheat by continuous callus selection. Aust J Plant Physiol. 18:445-452 Vasil IK (1987) Developing cell and tissue culture system for improvementof cereal and grass crops. J Plant Physio1128:193-218 Vasil V, Redway FA, Vasil IK (1990) Regenerationof plants from embryogenic suspension culture protoplasts of wheat (Triticum aestivum L). Bio\Technol8:429-434 Vasil V, Brown SM, Re D, Fromm ME, Vasil, IK (1991). Stably transformed callus lines from microprojectile bombardment of cell suspension cultures of wheat. Bio\Technol.9:743-747 Wang HB, Li XH, Sun YR, Chen J, Zhu Z, Fang R, Wang P, Wei JQ (1990) Culture of wheat protoplast. Scientia Sinica, Series B 33: 294-302
Plant Cell Reports (1992) 11:16-19
9 Springer-Verlag1992
Plant regeneration from protoplasts of wheat ( Triticum aestivum cv. Hartog) D. G. He, Y. M. Yang, and K. J. Scott Department of Biochemistry, The University of Queensland, Brisbane, Qld. 4072, Australia Received October 31, 1991/Revised version received November 6, 1991 - Communicated by H. L6rz
ABSTRACT Morphologically normal green plants have reproducibly been regenerated from protoplasts of an Australian wheat (Triticum aestivum cv. Hartog). The protoplasts were isolated from fine embryogenic suspension cultures which were initiated from embryogenic callus. Protoplasts were incubated in a modified liquid MS medium containing half strength o f the macroelements, 5 ~ M 2,4-D and 0.6 M glucose. Colonies were formed at frequencies ranging from 0.1% to 5%. The frequency of colonies forming fully developed plants varied between 1% and 25 %. More than eighty green plants with morphologically normal shoots and roots have been obtained and there was no difficulty in establishing these plants in soil. A cytological study of several randomly selected regenerated plants showed the normal chromosome c o m p l e m e n t for wheat (2n = 42). Key words: Embryogenesis - Plant regeneration Protoplast - Suspension cultures - Wheat
INTRODUCTION Protoplasts provide one of the most promising avenues for obtaining transgenic cereals and transgenic plants have been successfully obtained from protoplasts o f rice and maize through direct gene transfer (Potrykus 1990). Protoplasts are readily transformed by exogenous DNA, and plants derived from transformed single protoplasts are homogeneously transformed. However, protoplast culture o f the economically important cereals has been very difficult to achieve (Vasil 1987, Lorz et al. 1988, Potrykus 1990). Due to the world-wide economic importance of wheat, many attempts have been made to achieve plant regeneration from wheat protoplasts. However, wheat is one o f the most recalcitrant cereal species for protoplast culture ( M a d d o c k 1987). Recently there have been a few successful reports o f plant regeneration from wheat protoplasts (Harris et al. 1989, Ren et al. 1990, W a n g et al. 1990, Vasil et al. 1990, Chang et al. 1991). However, in most cases the experiments were not reproducible (Harris et al. 1989, Ren et al. 1990, W a n g et al. 1990, G u o et al. 1991). In only two instances were protoplastderived plants successfully transferred to soil (Vasil et al. 1990, Chang et al. 1991) and in one the plants were p r o b a b l y not fertile due to chromosomal deletion (Chang et aL 1991). Offprint requests to: K. J. Scott
W e now report the highly reproducible regeneration of morphologically normal green plants from protoplasts of an Australian wheat (Triticum aestivum cv. Hartog). These plants are readily established in soil and have the normal c h r o m o s o m e c o m p l e m e n t o f wheat (2n = 42).
MATERIALS AND METHODS Cell cultures. In this paper, liquid cultures refer to cultures maintained
in a hquid medium and consisting of callus clumps larger than 1 mm in diameter;, suspension cultures refer to finely dispersed cultures consisting mainly of small cell clusters, containing tess than a few hundred cells. The establishment and subculture of embryogenic liquid cultures and suspension cultures of wheat cv. Hartog were as previously reported (Yang et aL 1991). The non-embryogeuic suspension cultures used in this study were initiated from embryo-derived primary callus of cv. Oxley and cv. Timmo; and from protoplast-derived callus of cv. Timmo (He et al. 1990). Isolation and culture ofprotoplasts. Cell clusters were collected from either liquid cultures or suspension cultures by filtering through a stainless steel mesh (Swiss Screen), usually 53 lain for fine suspension cultures and 200 gm for liquid cultures. The cells retained on the mesh were transferred to 5 cm Petri dishes (Disposable Products, South Australia) to which a solution containing 2% Cellulase RS (Yakult, Tokyo), 0.2% Pectolyase Y23 (Seishin, Tokyo), 20 mM CaC12 and 0.6M mannitol was added (solution : cells = 5-10 : 1, v/v). The mixture was maintained on a rotary shaker (ca 50 rpm) for 3-5 hours and then left stationary for another 2-5 hours. The protoplasts were purified by filtering through 53 lain stainless steet mesh once and 38 lain mesh twice followed by 3 washes in a solution containing 20 mM CaC12 and 0.6M mannitol. Purified protoplasts were resuspended in 1/2 MS medium (Yang et al. 1991) containing 5~VI 2,4-D (2,4dichlorophenoxyacetic acid) and 0.6 M glucose at a density of 1-10 X l0 s pmtoplasts/ml. The cultures were incubated in 3 cm (Falcon, California) or 5 cm (Disposable, Australia) Petri dishes in the dark at 25 ~ Enzyme solutions, washing solutions and all media for protoplast culture were filter-sterilized by passage through 0.22 ~rn filters (Millipore). Differentiation of plants. Protoplast-derived colonies larger than 0.Smm were transferred to a differentiation medium (1/2MS medium, either hormone free or supplemented with different combinations of phytohormones as specified in the tex0. After 1-4 weeks, the regenerated plants were transferred to a second medium (hormone free 1/2 MS medium containing 1% sucrose). In this paper, fully developed plants refer to those regenerants which had both normal shoots and roots and were at least 3 cm in height, whereas plantlets included both those regenerants which subsequently developed into plants and those which formed only leafy structures. All differentiation media were sterilised by autoclaving.
17 Cytological studies. Root tips were collected from plants grown in Petri dishes or pots and left in ice-water (0 ~ for 24 hours, 0.03% (w/v) 8hydroxyquinoline for 3 hours at room temperature and then fixed in acetic alcohol (70% ethanol : acetic acid = 3:1). The fixed tissue was thoroughly washed and then incubated in an enzyme solution (2% Cellulase RI0, 1% Macerozyme RI0, 0.1% Peetolyase Y23) for 20 min at 37 ~ The cells were immediately fixed in acetic alcohol again. To observe the chromosomes, the cells were squashed in a drop of acetic carmine.
groups by filtering the suspension cultures through stainless steel meshes of decreasing pore size (1200 to 53 gm). The yield of protoplasts from these groups varied from 0.3 to 5 x 107 (Fig. 2); the group (175 to 750 pan) had the highest yields; with the large clusters (> 1200 pm) spontaneous fusion and rupture of protoplasts were frequently observed.
Isolation of protoplasts
The freshly isolated protoplasts often contained large starch granules. The protoplasts containing these granules were quite fragile, resulting in their rupture during centfifugation. However, the number of ceils with these granules decreased as the suspension cultures aged.
Protoplasts were readily isolated from fine embryogenic suspension cultures of cv. Hartog (Fig. 1.A) yielding as many as 5 X 107 protoplasts per gram (fresh weight suspension cells). The embryogenic suspension cultures of cv. Hartog tended to grow into large clumps (Yang et al. 1991), a phenomenon also noted in other wheat cultivars (Wang et al. 1990). After 7-10 hours digestion, these large cell clusters (>lmm) were still quite solid and the yield of protoplasts was low. In one experiment, the suspension cells were separated into five different size
First cell division (Fig. 1.B) was observed following one week incubation. As the dividing cells usually contained dense cytoplasm and many small granules, the newly formed cell walls between two daughter cells were not always visible, and frequently the small colonies showed a irregular contour indicating the outer cell walls (Fig. 1.C). After 30-40 days incubation, colonies became macroscopically visible (Fig. 1.D). The frequency of colony formation varied from 0.1% to 5%.
RESULTS
Fig. 1. Plant regeneration from embryogenic protoplasts of cv. Hartog. A. Freshly isolated protoplasts from the embryogenic suspension cultures of cv. Hartog. B. First cell division of protoplast-derived cells. C. Microscopic colonies (two weeks of incubation). D. Visible colonies after 40 days of incubation. E. Formation of embryogenic callus on 2,4-D containing (5 p.M) medium. F. Typical embtyoid forming on protoplast-derived embryogenic callus (v. ventral scale). G. Formation of a plantlet from a bipolar structure. H. Plants with morphologically normal shoots and roots. I. A root-tip cell from protoplast-derived plant showed the normal wheat chromosome complements (2n = 42). (Bar represents 20 ~un in A,B and C; 1 mm in D, E and G; 0.25 mm in F; and 1 cm in H)
18 Table 1.
Effect of 2,4-D concentration in differentiation medium on plant regeneration and shoot formation. Each treatment used 25 protoplast-derived colonies. O
Dif~renfiafion
Concentration of 2,4-D (la.M) in differentiation media
>,
w
i
A
B
C
D
i
i
I::
Fig 2. Yield of protoplasts (X 107/gm fresh weight) from different size clusters of suspension cultures. Suspension cultures of cv. Hartog were filtered through a series of stainless steel meshes of varying pore size (1200 bun to 53 gm). The cells retained on each mesh were collected, blotted with filter paper, and weighed. The cells were then incubated in enzyme solution (7 volumes per fresh weight of the cells) for three hours on a rotary shaker (50 rpm) and left stationary for another 4 hours. The five size groups were: A, >1200; B, 750-1200; C, 315-750; D, 185315; E, >53.
With the embryogenic protoplasts of cv. Hartog, following one week incubation, there was a marked increase in the viscosity of the medium; even when the Petri dishes were inverted, the 'liquid' medium remained in situ. By contrast, there was no change in the liquid medium containing the non-embryogenic protoplasts of the wheat cultivars Oxley and Timmo. Differentiation o f plants
More than 30%, and in some experiments up to 90%, of the protoplast-derived colonies were solid with a smooth surface and morphologically similar to the embryogenic callus of wheat. When transferred to differentiation medium some colonies formed bipolar slluctures with a shoot and a root (Fig. 1G). On medium containing 2,4-D the colonies proliferated into callus on which the formation of typical embryoids (Fig. 1E) was observed. Typical embryoids were easily identified by the well developed scutellum with a ventral scale (Fig.IF). However, as with the embryogenic wheat callus from immature embryos, or other explants, the formation of typical embryoids was rare (He et al. 1988). The frequency of colonies forming fully developed plants was low when compared to that of immature-embryoderived callus, where up to 100% of callus has the potential for plant regeneration (He et al. 1988). During the first week on a differentiation medium, more than 30% of the colonies developed one or two short, white or green leafy structures; subsequently most of these structures formed swollen cells and shoot formation ceased. This was exacerbated when the newly germinated embryoids were transferred to a fresh medium. Some of these stunted embryoids were white but were not true albinos as in several instances, these eventually formed green leaves. Only a small percentage of embryoids (125%) finally formed plants with normal shoots (Fig. 1H). In contrast to the regenerated wheat plants described by Chang et al. (1991) root formation occurred readily in our plants.
0
0.05
0.I0
0.50
5.00
A
12
16
20
12
0
B
4
12
12
32
20
16
28
32
44
20
C (A+B)
A: % of colonies forming whole plants; B: % of colonies forming leafy structures; C: % of colonies forming either whole plants or leafy structures.
Several differentiation media were tested to increase the differentiation frequency of embryoids and the effect of 2,4-D concenllation on proliferation and differentiation of colonies was examined (Table 1); it was found that colonies grew better on media containing a low concenlration of 2,4-D (0.05 to 0.5 pM). The differentiation frequency of plants was not significantly different between those incubated on medium containing 0.05, 0.1 and 0.5 ~ 2,4-D. On medium containing 5 ~tM 2,4-D, colonies developed into embryogenic callus, which differentiated into whole plants when transferred to hormone free medium. However, compared to the colonies incubated on media containing 0.05-0.5 ~tM 2,4D, more colonies on 5 laM 2,4-D medium became watery. The presence of kinetin (0.2mg/l) inhibited development and most of the colonies became brown; however, some did form green leaves and when transferred to a differentiation medium without kinetin, they formed normal plants. The age of colony (days since protoplast isolation) affected shoot formation; when 40-day-old colonies were transferred to differentiation medium, 25 of 150 (17%) formed whole plants, whereas in 54-day-old colonies only 1 of 184 (0.5%) formed a whole plant. Several hundred green plantlets have been obtained in a series of experiments. Over eighty of these regenerants have grown into plants greater than 5 cm high and no albinos have been observed; these plants were readily established in soil. Root tips were collected from several plants for cytological examination and the normal complement of wheat chromosomes (2n = 42) was observed (Fig. 1I). This is consistent with the norm,'il chromosomal complement observed in fine suspension cultures of cv. Hartog (Yang et al. 1991). DISCUSSION Although plant regeneration from wheat protoplasts has recently been reported by several authors, it is still far from a routine procedure. In these reports, only the formation of small plants was observed and information on the frequency of plant regeneration was scant. In addition, plant regeneration was not reproducible (Harris et al. 1989, Ren et al. 1990, Wang et al. 1990, Guo et al.
19 1991), or the regenerants were aneuploids (Chang et al. 1991), or embryogenic suspension reverted to a nonembryogenic state (Vasil et al. 1990, 1991). In contrast, our system has the advantage of producing normal green plants with a high reproducibility. In previous reports on plant regeneration from wheat protoplasts, the culture medium was usually complex and frequently some special conditions were considered to be essential, such as the use of Ficol, MES buffer (2-[Nmorpholino] ethanesulphonic acid) and special aeration (Harries et al. 1989). In contrast, our system was very simple; the medium was the commonly used MS medium with only minor modification; there were no complex organic additives and only one phytohormone (2,4-D) was present. The protoplasts were maintained in a liquid medium; no agarose or other gelling agents, or nurse cells were used. This procedure is probably not optimal but its simplicity emphasises the importance of the source of the protoplasts rather than the culture conditions: once embryogenic suspension cultures are established, it appears relatively simple to regenerate plants from the protoplasts derived from these cultures. Our success is another example demonstrating that the establishment of embryogenic suspension cultures is indeed a successful approach for obtaining totipotent cereal protoplasts (Vasil 1987). In our study, the embryogenic suspension cultures of cv. Hartog were initiated from embryogenic liquid cultures, which were also used for the isolation of protoplasts. Although there were difficulties, it was possible to isolate protoplasts from these liquid cultures; however, sustained cell division in the cells derived from these protoplasts has not been observed. We have also cultured protoplasts from primary embryogenic callus of cv. Hartog. Despite repeated attempts, however, cell division was rarely observed. Therefore, under present experimental conditions it appears that the finely dispersed state of the embryogenic cells, rather than genotype, is the most important factor for the successful induction of plant regeneration from wheat protoplasts. The major disadvantage of using fine suspension cultures is that their establishment is not only time-consuming but also unpredictable (Potrykus 1990). In contrast, the induction of primary embryogenic callus is a routine procedure and can be obtained using practically any wheat genotype (He et al. 1988). However, although protoplasts can readily be isolated from embryogenic callus, there had previously been only one report (Hayashi and Shimamoto 1988) where callus-derived protoplasts formed albino embryoids, but no plant formation and the result was not reproducible. Ren et al. (1990) and Guo et al. (1991) have reported plant regeneration from wheat protoplasts isolated from embryogenic callus. However, the callus used in these two studies was not primary callus but callus which had been subcultured on solid medium for a prolonged period. Although there is uncertainty about the reproducibility of these reports, it is of interest to note that similarities exist between the cells from these two studies and fine suspension cultures; i.e. the cells were highly friable and in a dispersed state, contrasting to the compact orientation of embryogenic cells in either primary callus or liquid cultures of wheat. More
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