National Research Council, Prairie Regional Laboratory, Saskatoon, Saskatchewan, S7N OW9, Canada

Isolation, Fusion and Culture of Sorghum and Corn Protoplasts

D. S. BRAR, S. RAMBOLD, F. CONSTABEL and o. L. GAMBORG

With 8 figures

Received July 31,1979 . Accepted August 20,1979

Summary

Procedures have been developed for the isolation, culture and growth of protoplasts of cell cultures derived from sorghum (Sorghum bicolor L. MOENCH cv. GPR-168). Fusion be­tween protoplasts from cell cultures of sorghum and leaf protoplasts of corn (Zea mays L. cv. Punjab Local) has been achieved using polyethylene glycol. The fusion products under­went divisions and formed colonies consisting of 8-10 cells in 15-18 days.

Key words: Sorghum, corn, protoplast, culture, fusion.

Introduction

Sorghum and corn are important cereal crops of the world. Both feature specific attributes and limitations. Somatic hybridization involving protoplast isolation, fusion, culture and plant regeneration from fusion products is being considered as a new approach for combining characteristics between otherwise incompatible species (BAJAJ, 1974; GAMBORG et a1., 1974; SCOWCROFT, 1977; BROCK, 1977). Viable intergeneric fusion products have been obtained with protoplasts from a number of plant species (CONSTABEL, 1978). However, limited information is available on culture and fusion of cereal protoplasts with no apparent success on subsequent plant regeneration (POTRYKUS et a1., 1977; KOBLITZ, 1976; BHOJWANI et a1., 1977; CAl QI-GUI et a1., 1978).

This communication reports on procedures for i) the culture and sustained division of protoplasts from sorghum resulting in callus formation; and ii) the formation of viable fusion products of sorghum and corn protoplasts.

Materials and Methods

1. Protoplast Isolation

1) Sorghum cell culture protoplasts: A cell suspension culture of sorghum (Sorghum bico­lor L. MOENCH cv. GPR-168) was established from explants of immature embryos (BRAR et al., 1979). Protoplasts were isolated by incubating 5 ml of a 3-4 day-old subculture in

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100 X 15 mm petri dishes with 5 ml enzyme solution consisting of 0.5 Ofo pectinase (Sigma), 0.5 Ofo Driselase (Kyowa Hakko Kogyo, Japan), 1.0 Ofo Onozuka-RI0 (Yakult Biochemical Co., Japan), 5 mM CaCl2 • 2H20, 0.7 mM NaH2P04 • HP, 3 mM 2-(N-morpholino)-etha­ne sulfonic acid, 0.45 M glucose, pH adjusted to 5.8 (cf. GAMBORG, O. L. and WETTER, L. R., 1975). The petri dishes were sealed with parafilm® and incubated at room temperature (20-22 0c) for 8-1 0 h. Protoplasts were passed through a stainless steel sieve, 67 p,m pore size, to remove debris and the filtrate was centrifuged at 100 X g for 3 minutes. The supernatant was discarded, and the protoplasts were resuspended and washed three times in 10 ml cul­ture medium (Table 1). Protoplasts were cultured in droplets in 60 X 15 mm petri dishes and incubated in a growth cabinet at 25°C and in diffuse light at photoperiods of 16 hi day.

II) Corn leaf protoplasts: Protoplasts were isolated from leaf tissue of 10-15 day-old seedlings of corn (Zea mays L. cv. Punjab Local). The seedlings were grown under green­house conditions, i. e. daylight and, if required, fluorescent and incandescent light of 20 W'm-2 over a light and dark cycle of 18/6 h, at about 22/16 °c. Before isolation of pro­toplasts, the seedlings were kept in the dark for 36-48 h. Leaves were thoroughly cleaned in running tap water, then surface-sterilized by immersion in 70 Ofo ethanol for 2-3 min. The leaves were washed three times in sterile water. They were cut into fine longitudinal strips and approximately 0.2 g tissue was incubated in 5 ml enzyme solution consisting of 0.5 Ofo pectinase, 1.0 Ofo Driselase, 5 mM CaCl2 • 2H20, 1 mM CaH4(P04)2 • H 20, and 0.5 M sorbitol, pH adjusted to 6.2. After 5-6 h of incubation the protoplasts were collected, washed and cultured as described for sorghum protoplasts.

2. Protoplast Aggrega'tion and Fusion

After incubation with enzyme solutions cell culture protoplasts of sorghum and leaf pro­toplasts of corn were washed twice with a solution containing 0,45 M glucose, 5 mM CaCl2 • 2H20, pH adjusted to 5.8. Protoplasts of the two species, approximately 5 X 104/ml, were then mixed at a ratio of 1 : 1, centrifuged and resuspended in 1 ml of the above solution. Droplets of 200 p,l of the protoplast suspension (104 to 5 X 104 protoplasts per ml) were transferred to 100 X 15 mm petri dishes. After about 5 minutes when the pro­toplasts had settled, two 100,al drops of PEG solution were gently added. The solution consisted of 5 g of PEG-1540 dissolved in 10 ml of water containing 10 mM CaCl2 • 2H20, 0.7 mM KH2P04, 0.1 M glucose, pH adjusted to 5.8. After 15 minutes, PEG was slowly eluted by dropwise addition of rinse solution containing 50 mM CaCl2 • 2H20, 50 mM gly­cine, 0.3 M glucose, pH adjusted with 0.5 N KOH to 10.5. Ten minutes after the start of the elution each petri dish was flooded with 2-3 ml of rinse solution. After another 5 mi­nutes the supernatant was discarded, the protoplasts gently washed 5 times with the proto­plast culture medium (Table 1) and finally covered with a thin layer of medium. The pro­toplasts were incubated under the same conditions as described for sorghum protoplasts.

The frequency of protoplast fusion was examined after a culture period of 16-24 h. The fusion products were identified by the simultaneous occurrence of chloroplasts from corn leaf protoplasts and leucoplasts and dense cytoplasm from sorghum protoplasts.

3. Chromosome Count

Cells regenerated from protoplasts were fixed in glacial acetic acid: alcohol, 1 : 3, and stained with carbol-fuchsin (KAO, 1975).

Results and Discussion

Protoplast culture: Protoplasts of sorghum (Fig. 1) cultured in liquid medium (Table 1), at a density of 5 X 104_105 protoplasts per ml, regenerated cell walls

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Fusion of Sorghum and corn protoplasts 271

Table 1: Culture medium found suitable for sorghum and corn protoplasts and fusion products.

mg/l

Mineral salts and vitamins after GAMBORG et al. 1968.

glucose xylose ribose L-arginine L-asparagine CaCl2 '2HP D-calcium pantothenate folic acid p-aminobenzoic acid biotin choline chloride riboflavine ascorbic acid vitamin A vitamin D3 vitamin B12 2,4,5-Trichlorophenoxyacetic acid 2,4-Dichlorophenoxyacetic acid*) casamino acids (vitamin free)") pH 5.8

81,000 500 500 210 300 735

1 0.4 0.02 0.Q1 1 0.2 2 0.01 0.Q1 0.02 0.5 1

250

*) The cell culture medium contained 2 mg/l 2,4-D and 500 mg/l N-Z amine (Humko­Sheffield, Norwich, N.Y. 13815).

within 48 h and started dividing after 5-6 days of culture. Generally 5-12 Ofo of the protoplasts underwent mitosis. Gradual lowering of the osmoticum after 8-10 days of culture by adding 0.2 ml of protoplast culture medium containing 2.5 Ofo sucrose at 3 day intervals enhanced further divisions (Fig. 2), and cell clusters and callus were obtained in 6-7 weeks (Fig. 3). The importance of the cell source or genotype for protoplast culture is supported by the observation that protoplasts from cell cultures of corn as well as protoplasts from leaves and roots of sorghum and corn failed to show consistent cell divisions even under a wide range of cultural conditions (d. BRAR et al., 1979). Callus from sorghum protoplasts were maintained on standard cell culture agar medium with 2.5 % sucrose.

Cells regenerated from proroplasts of sorghum had 64-66 chromosomes (Fig. 4) corresponding to that of the original cell culture initiated from immature embryos. The chromosome number has been stable in liquid culture during 80 subcultures. Apparently the cells which were selectively favoured and became rapidly growing callus or cell suspension cultures tended to be polyploid.

Protoplast Fusion: Protoplasts from cell cultures of sorghum and leaf protoplasts of corn (Fig. 5) have been induced to fuse using polyethylene glycol. The fusion

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272 D. S. BRAR, S. RAMBOLD, F. CONSTABEL and O. L. GAMBORG

Plate 1:

Figs. 1-4: Culture of sorghum (GPR-168) protoplasts.

Fig. 1: Protoplasts from cell culture. X230. - Fig. 2: Cells after 18 days of culture. X480. - Fig. 3: Callus after 52 days of culture. Fig. 4: Mitosis in protoplast-derived cell suspen­sion. X 3000.

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Fusion of Sorghum and corn protoplasts 273

Plate 2:

Figs. 5-8: Protoplast fusion (sorghum + corn).

Fig. 5: Protoplasts of sorghum (clear) and corn (dark with chloroplasts) before PEG-treat­ment. X 480. - Fig. 6: Fusion product after 48 hr of culture. X 800. - Fig. 7: Daughter cells of fusion product. X 800. - Fig. 8: Callus formation of a fusion product. X 800.

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274 D. S. BRAR, S. RAMBoLD, F. CONSTABEL and O. L. GAMBORG

frequency ranged from 3.6-12.1 Ofo in different experiments. In one experiment 101 out of 835 protoplasts were identified as fusion products after 24 h of culture. Judged by the acquisition of an ellipsoidic shape and fluorescence after treatment with 0.1 Ofo Calcofluor White (American Cyanamid Co.) the fusion products regenerated cell walls within 48-60 h (Fig. 6). Most of the non-fused corn protoplasts did not survive PEG treatment. No mitosis was observed in any of the corn pro top lasts.

The fusion products underwent the first division (Fig. 7) after 7-8 days of culture, 'sorghum protoplasts usually started dividing after 5-6 days. CONSTABEL et al. (1976) have observed intermediate timing of the first mitosis of fusion products and the two kinds of parental protoplasts. Within 10-12 days of culture, fusion products reached the 4-6 cell-stage (Fig. 8). Small colonies consisting of 8-10 cells were obtained with 15-18 days of culturing. After 3 weeks of culturing it became difficult to identify the fusion products, because the chloroplasts gradually lost their pigmentation. The number and size of sorghum and corn chromosomes in meta phases of fusion products did not allow to clearly claim the presence of sorghum + corn hybrid cells.

Acknowledgements

Research funds granted to one of the authors (D.S.B.) by the International Development Research Center, Ottawa, Canada is gratefully acknowledged. The views expressed in this publication are those of the authors and do not necessarily reflect the views of the Interna­tional Development Research Center. We are grateful to Dr. HUGH DOGGETT, Associate Director, IDRC, Nairobi, Kenya, and Dr. AMARJEET S. KHERRA, MAIZE BREEDER, Punjab Agricultural University, Ludhiana, India for the generous supply of sorghum and corn seeds respectively.

References

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