Regeneration of sugarcane plants from protoplasts

BUREAU OF SUGAR EXPERIMENT STATIONS QUEENSLAND,AUSTRALIA

FINAL REPORT

SRDC PROJECTS BS3S AND BS43S

REGENERATION OF SUGARCANE PLANTS FROM PROTOPLASTS

by P W J Taylor

This Project was funded by the Sugar Research Council/Sugar Research and Development Corporation during the 1988/89, 1989/90 and 1990/91 financial years.

December 1991

CONTENTS

SUMMARY

OBJECTIVES

BACKGROUND

MATERIALS AND METHODS

RESULTS AND DISCUSSION

ACHIEVEMENTS

DIFFICULTIES

RECOMMENDATIONS

PUBLICATIONS ARISING FROM PROJECT

ACKNOWLEDGMENTS

REFERENCES

Page No.

1

1

1

2

5

8

9

9

9

10

10

SUMMARY

For 18 sugarcane cultivars, four distinct callus types developed on leaf explant tissue.

Cell suspension cultures were initiated from embryogenic callus and developed in three stages. In stage 1 the callus adapted to the liquid medium; then a heterogeneous cell suspension culture formed in 14 cultivars after five to eight weeks of culture; and a homogeneous cell suspension culture developed in six cultivars after 10-14 weeks as the third stage. Plants were regenerated from cell aggregates in heterogeneous cell suspension cultures but not from homogeneous cell suspension cultures.

. High yields of totipotent protoplasts were obtained from homogeneous cell suspension cultures incubated in an enzyme mixture containing cellulase and pectinases, compared to heterogeneous cell suspension cultures. Protoplasts were isolated from heterogeneous cell suspension cultures treated with pectolyase enzyme or from cultures incubated with silver nitrate (inhibitor of physiological action of ethylene), but these protoplasts failed to develop. Higher yields of pro top lasts were obtained from homogeneous cell suspension cultures for cultivars Q63 and Q96 after regenerating callus from the cell suspension cultures, then recycling this callus to liquid medium (S-cell suspension cultures) or recycling callus regenerated from protoplasts to liquid medium (SP-cell suspension cultures). Callus developed from protoplasts for Q63, Q96 and Q138 S- and SP-cell lines. From Q63-SP protoplast callus, small, photosynthetically active, shoot-like structures regenerated from embryogenic type callus.

OBJECTIVES

• Develop a protoplast and callus regeneration system which will yield high plating efficiencies with several Australian commercial sugarcane cultivars.

• Optimise the conditions for rapid and efficient regeneration of whole plants of the selected cultivars and so minimise somaclonal variation among these plants.

BACKGROUND

In the BSES plant breeding program, agronomically superior sugarcane cultivars are obtained after a 10 year selection process at a cost exceeding $O.5m each. However, some new cultivars are abandoned near the end of the selection process because of a single fault such as disease susceptibility. Novel genes from micro-organisms or other plants may become available for use in sugarcane. The capacity to introduce specific genes such as disease resistance genes or herbicide tolerance genes into parental lines or commercial cultivars will be of long-term value to the sugarcane industry.

Most direct gene transfer techniques for crop improvement depend on the regeneration of transformed plants from protoplasts. For the cereals there are few examples of plant regeneration from protoplasts. Plants have been regenerated from protoplasts isolated from cell suspension cultures of pearl millet (Vasil and Vasil, 1980), barley (Luhrs and Lorz, 1988), rice (Abdullah et aI, 1986; Yamada et aI, 1986), maize (Rhodes et aI, 1988)

2

and wheat (Vasil et ai, 1990; Wang et ai, 1990). In all these cases embryogenic cell suspensions were an important source of protoplasts which were capable of cell division and plant regeneration.

In sugarcane, Srinivasan and Vasil (1986) regenerated five green plants from protoplasts of the cultivar B4362 derived from two- to six-month-old embryogenic cell suspension cultures. Chen et ai, (1988) also regenerated plants from protoplasts derived from embryogenic cell suspension cultures of the cultivar FI64. However, both groups have been unable to repeat their earlier successes (l Vasil, pers. comm., W Chen, pers.

comm.).

Large numbers of protoplasts are required to regenerate genetically transformed plants from protoplasts because of the low plating efficiencies associated with the culture of protoplasts (Thompson et ai, 1986) and reduced viability of protoplasts during gene transfer procedures (Fromm et ai, 1985).

This study aimed to develop a protoplast regeneration system for sugarcane so that gene transfer techniques could be applied for cultivar improvement. Published techniques for embryogenic callus culture, cell suspension culture, protoplast culture and plant regeneration in sugarcane were developed and tested using only a few cultivars. These techniques were further developed, evaluated and adapted for application to a range of commercial sugarcane cultivars.

MATERIALS AND METHODS

Callus cultures

Callus cultures for 18 sugarcane cultivars were established from leaf explant tissue and subcultured at three-week intervals by selectively transferring embryogenic callus onto the same medium (Taylor et ai, 1991).

Cell suspensiou cultures

Cell suspension cultures were initiated for cultivars by incubating embryogenic callus in liquid medium on an orbital shaker at 120 rpm in darkness at 27°C. Cell suspension cultures were developed in three stages (Taylor et ai, 1991). First, the callus adapted to the medium; then five to eight weeks after transfer of callus to liquid medium, a heterogeneous cell suspension culture developed. These were maintained by dividing the culture equally into two flasks and adding fresh medium to 35 mL volume every seven days. Finally, homogeneous cell suspension cultures were developed from the second stage cell suspension cultures by enrichment for actively dividing cells. These were maintained by transferring 10 mL of culture into 25 mL of fresh medium each three to four days.

3

A secondary homogeneous cell suspension culture, designated an S-cell suspension culture, developed after callus regenerated from homogeneous cell suspension cultures was transferred to liquid medium.

A third type of homogeneous cell suspension culture, designated an SP-cell suspension culture, was developed after callus, regenerated from protoplasts isolated from homogeneous S-cell suspension cultures of Q63, Q96 and Q138, was transferred to liquid medium.

Protoplast isolation

Protoplast isolation was attempted within one week of the establishment of heterogeneous and homogeneous cell suspension cultures, and repeated at two-week intervals until protoplasts were isolated (faylor et aI, 1991). Enzyme mixtures containing various concentrations of cellulases and pectinases, and the optimum time for incubation in enzymes, were assessed.

Protoplast culture

Isolated protoplasts were cultured at a density of approximately 5 x

105

protoplasts mL'\

in agarose droplets, in petri dishes bathed in K and M medium (Srinivasan and Vasi!, 1986). The culture medium was replaced weekly with fresh medium. Regenerated microcallus was transferred to solid MS medium containing 1 mg 2,4-D L-1.

Protoplasts from some cell lines failed to develop into cells, divide and form microcolonies. Therefore, various protoplast culturing techniques were assessed to improve plating efficiency. These included:

(a) culturing protoplasts in agarose as a thin layer on the base of the petri dish;

(b) CUlturing protoplasts in liquid K and M without agarose;

(c) culturing protoplasts in agarose droplets, bathed in liquid K and M medium supplemented with 1.5 mL of cells from a three to four-day-old homogeneous cell suspension culture. The addition of actively dividing cells to protoplast culture may result in the cells producing chemicals conducive to promoting protoplast division (Somers et aI, 1987). Cells from suspension culture were replaced when the liquid medium was replaced;

(d) culturing of protoplasts in agarose droplets dispensed onto a sterilised filter placed on top of 0.5 g of actively dividing homogeneous suspension cells suspended in 2 mL of cell suspension medium (Rhodes et aI, 1988; Shillito et aI, 1989). The suspension cells were pipetted over a thin layer of solid K and M medium in petri dishes. The filters supporting the protoplasts were transferred at seven-day intervals to agar with progressively reduced levels of osmolarity and lower 2,4-D concentrations;

4

(e) increasing the osmolarity of the enzyme mixture and protoplast culture medium;

decreasing osmolarity of the protoplast culture medium (Chen et aI, 1988);

(f) increasing agarose concentration and or auxin concentration in protoplast culture medium (Chen et aI, 1988).

Protoplast callus culture

To induce embryo, shoot and plantlet development, protoplast callus was sequentially transferred, at three to four-week intervals, to MS medium supplemented with 1 % charcoal and various combinations of plant growth regulators:

(a) (b) (c)

1 mg 2 4-D L·1.

, ,

0.5 mg BAP L-\

0.5 mg BAP L-¹ and 0.5 mg fluridone L-¹ (Srinivasan and Vasil, 1986).

Callus which failed to regenerate organised structures was incubated on medium containing various concentrations of these plant growth regulators.

The morphology and ultrastructure of roots and shoots differentiated from protoplast callus were examined using scanning electron microscopy and histology. In an attempt to induce shoots from roots regenerated from protoplast callus, 1 cm long roots were cut and incubated on MS medium containing charcoal, various concentrations of plant growth regulators, adenine (enhancer of endogenous cytokinin synthesis) and cefotaxime (stimulator of embryogenesis) (Xiang-Can et al, 1989).

Factors limiting the isolation and culture of protoplasts

As viable protoplasts were not readily isolated from heterogeneous cell suspension cultures, factors limiting the isolation of protoplasts were investigated. These included an assessment of endogenous ethylene production by cell suspension cultures and the effect of regulating ethylene production on cell suspension growth and protoplast isolation.

Ethylene measurements were made at 0, 1, 2, 3, 5, 7, 14, 21, 28 and 35 days after the transfer of embryogenic callus of cultivar Q63 to liquid MS medium. Treatments involved the addition to the medium of chemicals known to inhibit ethylene biosynthesis (aminoethoxyvinylglycine, A VG), to promote ethylene biosynthesis (aminocyclopropane-l- carboxylic acid, ACC), or to inhibit the physiological action of ethylene (silver nitrate, AG). Treatment effects were assessed by growth measurements made at weekly intervals and protoplasts were isolated at weeks five to eight.

5

RESULTS AND DISCUSSION

Establishment of embryogenic callus cultures

Four callus types developed on sugarcane leaf explant tissue from a range of genetically diverse sugarcane cultivars: Type 1 - semi-translucent, Type 2 - mucilaginous, Type 3 - embryogenic with globular structures, and Type 4 - friable with clusters of semi-organised structures. Types 1 and 2 callus were non-morphogenic whereas plants regenerated from Type 3 and to a lesser extent Type 4 callus. Embryogenic callus appears to represent an ideal target for gene transfer by microprojectile bombardment.

Establishment of heterogeneous and homogeneous cell suspeusion cultures

Heterogeneous cell suspension cultures formed within five to eight weeks after transfer of embryogenic callus to liquid medium, and were capable of plant regeneration. For some cultivars, homogeneous cell suspension cultures could be selected after 10 to 14 weeks, but these were not capable of plant regeneration (Taylor et al, 1991).

Homogeneous cell suspension cultures consisted of small, compact, aggregates of densely cytoplasmic and actively dividing cells with prominent nuclei. The cells resembled the embryogenic cells described by Ho and Vasi! (1983) and Srinivasan and Vasi! (1986) for cultivars 68-1067 and B4362 respectively. S- and SP-homogeneous cell suspension cultures were established within two weeks of transfer to liquid medium of callus regenerated from homogeneous cell suspension cultures, or callus regenerated from protoplasts isolated from homogeneous cell suspension cultures respectively. This approach allowed the establishment of stable homogeneous cell suspension cultures from cultivars Qll0 and Q138 that otherwise died in liquid culture. Plants could not be regenerated from S- and SP-cell suspension cultures.

Protoplast isolation

Protoplast yield from heterogeneous cell suspension cultures, incubated in the enzyme mixture containing cellulase in combination with various pectinases, was low compared to homogeneous cell suspension cultures (Table 4, Taylor et al, 1991). Replacement of the pectinases with the more active pectolyase resulted in higher yields of protoplasts from both heterogeneous and homogeneous cell suspension cultures. These results suggest that there are differences in the composition of the walls of cells from homogeneous and heterogeneous cell suspension cultures. The highest yield of protoplasts was obtained after incubation of cells of either culture type in enzyme mixture for three to five hours.

Protoplasts isolated from heterogeneous cell suspension cultures rarely developed beyond the cell division stage. Protoplasts isolated from homogeneous cell suspension cultures generally reformed cell walls, divided and formed microcolonies. However, pectolyase decreased the totipotency of protoplasts and reduced the plating efficiency. Hence, the enzyme mixture containing cellulase and pectinases was used in all further isolations.

6

Higher yields of protoplasts were obtained from homogeneous cell suspension cultures of the cultivars Q63 and Q96 after recycling regenerated callus to liquid medium. The high yield of viable protoplasts from S- and SP-cell suspension cultures makes these cultures suitable for direct gene transfer studies that require large numbers of protoplasts.

Protoplast culture

Protoplasts isolated from homogeneous cell suspension cultures of Q63, Q96, Q63-S, Q96-S, Q138-S, Q63-SP, Q96-SP and Q138-SP developed microcallus of 0.5-1 mm diameter in the agarose droplets after four weeks of culture. Callus developed after three to four weeks incubation of microcallus on solid medium. Frequently, the development of microcallus from protoplasts or the regeneration of callus from microcallus required several sequential protoplast isolations from the one cell suspension culture. This may have been due to a lack of developmental maturity of the cells in suspension culture.

Cells of polyploid species such as wheat undergo continual genetic changes in suspension culture (A Karp, pers. comm.), and these changes may affect the capability of cells to regenerate plants as well as the totipotency of protoplasts isolated from these cultures.

In one experiment, protoplasts isolated from a six-month-old homogeneous cell suspension culture of Q63 formed microcolonies only when cultured in agarose droplets and incubated with actively dividing cells (technique c). The cells may have produced chemicals that promoted cell division. Conditioning factors have been found in medium from four- to five-day-old maize cell suspension cultures which has increased colony formation from maize protoplasts (Somers et aI, 1987). Nevertheless, protoplasts isolated from the same cell suspensions one month later divided and formed microcolonies both with and without actively dividing cells in protoplast culture. Other cultures and cell lines failed to respond to the presence of actively dividing cells in protoplast culture.

Generally, plating efficiency was not improved when protoplast culture techniques other than the standard one were used.

Protoplast callus culture

Protoplast callus was morphologically similar to Type IV callus which regenerated from cell suspension cultures (Taylor et aI, 1991). A compact nodular yellow callus developed after three to four-weeks incubation of Q96-S, Q63-SP and Q138-SP protoplast callus on medium containing charcoal and 2,4-D. Prolonged incubation of this callus resulted in the regeneration of white roots 10-30 weeks after protoplast isolation. Replacement of 2,4-D with BAP resulted in the proliferation of roots.

From one protoplast isolation of Q63-SP, a small amount of compact, nodular, white callus with smooth globular surfaces similar to embryogenic callus also developed on medium containing BAP. Incubation of this callus on medium containing charcoal, BAP and fluridone (an inhibitor of carotene and abscissic acid synthesis), under diffuse light and a 12 h photoperiod, resulted in the development of green and white shoot primordia, first noted after 66 weeks.

Shoot-like structures containing chlorophyll and albino shoots grew to 5 mm in length.

Transfer of shoots to medium without plant growth regulators, or to medium with various concentrations of BAP and/or fluridone, resulted in the necrosis of the shoots and death

7

within four weeks. However, subculture of the callus containing shoots to fresh medium resulted in the regeneration of more green shoots. Shoot-like structures were regenerated from this callus for over six months of subculture.

Histological studies of regenerated roots showed well-defined vascular system containing xylem, phloem and endodermis cells. Shoot-like structures contained an outer layer of epidermal cells but no vascular tissue. Shoots could not be regenerated from roots incubated on a selective medium.

Factors limiting the isolation and culture of protop1asts

Plants have been regenerated from heterogeneous but not from homogeneous cell suspension cultures. Therefore, protop1asts isolated from heterogeneous cell suspension cultures may be competent for plant regeneration. However, viable protop1asts have not been readily isolated from these cultures and this may have been due to the effects of accumulated gases, such as ethylene, in the culture containers.

Ethylene is generally produced by cells in response to environmentally induced stress, such as in vitro culture conditions (Benson, 1990). Accumulation of ethylene in the headspace of culture containers can affect cell growth and morphogenesis (Adkins et aI, 1990). Ethy1ene,production by heterogeneous cell suspension cultures peaked 1 day after the transfer of callus to liquid medium and then rapidly decreased by day 7 (Figure 1).

The peak in ethylene production correlated with the period of callus adaptation to liquid medium. As the callus adapted to the culture medium, the level of ethylene biosynthesis was reduced. Protop1asts could not be isolated from these cultures.

Compared to the untreated cells, the production of ethylene by heterogeneous cell suspension cultures was significantly increased by the addition of ACC, and significantly decreased by the addition of AVG (Figure 1). Protop1asts were not isolated from these cultures. These results indicated that there was no correlation between ethylene synthesis and protoplast isolation in sugarcane cell suspension cultures.

However, the addition of AG to the culture medium resulted in increased ethylene production by the cells (Figure 1) and the isolation of protop1asts. Further studies showed that two weeks after removal of AG from the culture medium, protop1asts could no longer be isolated from the culture. However, protoplast isolation capability was restored within one week after the addition of AG to the culture. The reason for the isolation of protop1asts only from cultures incubated with AG may have been due to AG's direct effect on the composition of the cell walls rather than an effect on ethylene biosynthesis.

Earlier studies with enzymes indicated that difficulties in isolating protop1asts from heterogeneous cell suspension cultures were due to components of the cell walls in these cultures. According to Per! et al (1988), silver ions protect cells from the stress caused by the enzymatic maceration of tissue. AG has been reported to inhibit the physiological action of ethylene and promote somatic embryogenesis in cell suspensions of carrot (Roustan et aI, 1990). It also induced shoot regeneration in callus of wheat (Pumhauser et aI, 1987) and maize (Songstad et al, 1988).

Protop1asts isolated from heterogeneous cell suspension cultures incubated with AG did not develop beyond the microco10ny stage.

Figure 1

8

90

o control

80 _v AG

c ACe

70 ^~ AVG

~ 60

$ _'" "

~

⁵⁰

0 S ₄₀

~ .:

Q) .:

"

³⁰

>, .a ...,

~

20

10

0

0 7 14 21 28 35

Time (day.)

Effect of ethylene regulators on ethylene production of Q63 heterogeneous cell suspension cultures

ACHIEVEMENTS

Significant achievements have been made in the development of sugarcane tissue culture techniques suitable for gene transfer for cultivar improvement. These included:

Development of an embryo culture technique which has been used for direct gene transfer studies using microprojectile bombardment (Franks and Birch, 1991).

Development of cell culture techniques to isolate protoplasts from young, highly morphogenic, cell suspension cultures.

Development of cell suspension cultures with high protoplast yields, which have been used to study gene expression during optimisation of the microprojectile bombardment technique (Franks and Birch, 1991).

9

Isolation of high numbers of totipotent protoplasts. These have been used in direct gene transfer studies using electroporation to produce stably transformed sugarcane callus (Rathus and Birch, 1992), in assessing gene control signals which require an effective transient expression system (Rathus and Birch, unpubl.), and may be used in somatic hybridisation studies.

Regeneration from protoplasts of callus which differentiated roots and shoots, the latter containing chlorophyll.

DIFFICULTIES

The major difficulty of this project was the lack of success in developing plants from the shoot-like structures that regenerated from protoplast callus. This may have been due to major genetic changes occurring in the plant tissue during the formation of homogeneous cell suspension cultures.

RECOMMENDATIONS

Protoplast culture technique is regarded as the best system for obtaining stable incorporation of'novel genes into plant cells. However, this system is limited by difficulties in regenerating plants from protoplasts in some plant species. Plants were not regenerated from protoplasts in this project, but tissue culture techniques were developed which provided the basis for further research into development of genetic transformation systems for sugarcane. The recommendations are:

• Conduct further research into the isolation of protoplasts from young morphogenic, heterogeneous cell suspension cultures so that competent cells may be regenerated to plants.

• Initiate a project to obtain embryogenic protoplast callus and plant regeneration, involving a study of the effect of gases in the culture environment on cell growth and embryogenesis, and additional modification to culture conditions.

PUBLICATIONS ARISING FROM PROJECT Accepted or published

Taylor, P W J, Ko, H-L, Adkins, SW, Rathus, C and Birch, R G (1991). Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum SPP hybrids). Plant Cell Tiss. Organ Cult. (In press).

Taylor, P W J (1991). Tissue culture of sugarcane for crop improvement (abstract). 4th Aust. Branch Inter. Assoc. Plant Tiss. CuI. Meet., Launceston, p 41.

10

Taylor, P W J, Ko, H-L, Adkins, S and Birch, R G (1990). Factors affecting regeneration of sugarcane plants from protoplasts. 7th Inter. Congo on Plant Tiss. and Cell Cult., Amsterdam, (abstract), p 39.

Taylor, P W J and Ko, H-L (1989). Development of a method for regeneration of sugarcane plants from protoplasts (abstract). Proc. Aust. Soc. Sugar Cane Technol. 1989 Conf., p 263.

Taylor, P W J, Gordon, G H and Birch, R G (1988). Development of a method for regeneration of sugarcane plants from protoplasts. 9th Aust. Plant Breed. Conf., Wagga Wagga, p 141-142.

Being written currently

Taylor, P W J (1992). Application of tissue culture techniques for sugarcane crop improvement. Aust. J. Bot.

Taylor, P W J, Ko, H-L and Adkins, S W (1992). The effect of silver nitrate on sugarcane cell suspension growth, plant regeneration, protoplast isolation and ethylene production. J. Plant Physiol.

ACKNOWLEDGMENTS

I wish to thank Tracy Fraser, Lien Ko and Niall Masel for their assistance, and Robert Birch and Steve Adkins for helpful discussions during the project.

REFERENCES

Abdullah, R, Cocking, E C and Thompson, J A (1986). Efficient plant regeneration from rice protoplasts through somatic embryogenesis. Bio/Technol., 4: 1087-

1090

Adkins, S W, Shiraishi, T and McComb, J A (1990). Rice callus physiology - Identification of volatile emissions and their effects on culture growth. Physiol.

Plant, 78:526-531

Benson, E E (1990). Free radical damage in stored plant germplasm. International Board for Plant Genetic Resources, Rome, pp 128.

Chen, W H, Davey, M R, Power, J B and Cocking, E C (1988). Sugarcane protoplasts:

factors affecting division and plant regeneration. Plant Cell Rep., 7: 344-347.

Franks, T and Birch, R G (1991). Gene transfer into intact sugarcane cells using microprojectile bombardment. Aust. J. Plant Physiol., 18:471-480.

11

Fromm, N, Taylor, L P and Walbot, V (1985). Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA, 82:5824-5828

Ho, W J and Vasil, I K (1983). Somatic embryogenesis in sugarcane (Saccharum officinarum L.). Growth and plant regeneration from embryogenic cell suspension cultures. Ann. Bot., 51:719-726.

Luhrs, R and Lorz, H (1988). Initiation of morphogenic cell suspension and protoplast cultures of barley (Hordeum vulgare L.). Planta, 175:71-81.

Ped, A, Aviv, D and Galun, E (1988). Ethylene and in vitro culture of potato:

Suppression of ethylene generation vastly improves protoplast yield, plating efficiency and transient expression of an alien gene. Plant Cell Rep., 7:403-406.

Purnhauser, L, Medgyesy, P, Czako, M, Dix, P J and Marton, L (1987). Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Vivo tissue cultures using the ethylene inhibitor AgN0₃• Plant Cell Rep., 6:1-4.

Rathus, C and Birch, R G (1992). Optimisation of conditions for electroporation and transient expression of foreign genes in sugarcane protoplasts. Plant Sci., (In press). ,.

Rhodes, C A, Lowe, K S and Ruby, K L (1988). Plant regeneration from protoplasts isolated from embryogenic maize cell cultures. Bio/TechnoI., 6:56-60.

Roustan, J-P, Latche, A and Fallot, J (1990). Control of carrot somatic embryogenesis by AgN0₃, an inhibitor of ethylene action: effect on arginine decarboxylase activity. Plant Sci., 67:89-95.

Shillito, R D, Carswell, G K, Johnson, C M, DiMaio, J J and Harms, C T (1989).

Regeneration of fertile plants from protoplasts of elite inbred maize.

Bio/TechnoI., 7:581-587.

Somers, D A, Birnberg, PR, Petersen, W L and Brenner, M L (1987). The effect of conditioned medium on colony formation from 'Black Mexican' sweet corn protoplasts. Plant Sci., 53:249-256.

Songstad, D D, Duncan, D R and Widholm, J M (1988). Effect of I-amino cyclopropane-I-carboxylic acid, silver nitrate and norbornadiene on plant regeneration from maize callus cultures. Plant Cell Rep., 7:262-265.

Srinivasan, C and Vasil, I K (1986). Plant regeneration from protoplasts of sugarcane (Saccharum officinarum L.). J. Plant PhysioI., 126:41-48.

Taylor, P W J, Ko, H-L, Adkins, S W, Rathus, C and Birch, R G (1991). Establishment of embryogenic callus and high protoplast yielding suspension cultures of sugarcane (Saccharum SPP hybrids). Plant Cell, Tiss. Organ CuI., (In press).

12

Thompson, J A, Abdullah, R and Cocking (1986). Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose. Plant Sci., 47:123-133.

Vasil, V, Redway, F and Vasil, I K (1990). Regeneration of plants from embryogenic suspension culture protoplasts of wheat (Triticum aestivum L.). Bio/Technol., 8:429-434.

Vasil, V and Vasil, I K (1980). Isolation and culture of cereal protoplasts. Part 2:

Embryogenesis and plantlet formation from protoplasts of Pennisetum americanum. Theor. Appl. Gen., 56:97-99.

Wang, H B, Li, X H, Sun, Y R, Chen, J, Fang, R, Wang, P and Wei, J K (1990).

. Culture of wheat protoplast - high frequency microcolony formation and plant regeneration. Sci. China (Series B), 33:294-302.

Xiang-Can, Z, Jones, D A and Kerr, A (1989). Regeneration of shoots on root explants of flax. Ann. Bot., 63:297-299.

Yamada, Y, Yang, Z Q and Tang, D T (1986). Plant regeneration from protoplasts - derived callus of rice (Oryza sativa L.). Plant Cell Rep., 5:85-88.