African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 5, Num. 2, 1997, pp. 197-200

SHORT COMMUNICATION

A comparative assessment of the purification techniques for etiolated seedling protoplast of Sorghum bicolor

E. O. UMOH

University of Calabar, P. O. Box 3608, Calabar, Cross River State, Nigeria

(Received 19 February, 1996; accepted 7 April, 1997)

Code Number: CS97025
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ABSTRACT

Protoplasts of Sorghum bicolor Moench are usually obtained from suspension cells and purified using either the sedimentation or the sucrose floatation methods. Effects of these two methods were assessed using etiolated shoot protoplats of two cultivars of sorghum. Recovery rates were higher in the sedimentation method than in the sucrose floatation method. The ratios of vacuolated to etioplastic in the initial and purified samples of protoplasts were not markedly changed with the sedimentation method. On the other hand, these ratios were largely titled in favour of vacuolated protoplast when the sucrose floatation method was used. This finding could be exploited and an appriopriate purification method selected based on the specific experimental requirements.

Key Words: Etiolated shoots, protoplast purification, sorghum

RESUME

D'une facon generale, on obtient les protoplastes de Sorghum bicolor Moench a partir des cellules en suspension et purifiees a l'aide des methodes de sedimentation ou de flottaison de sucrose. Les effets de ces deux methodes ont ete evalues en utilisant les protoplastes de la tige etiolee de deux cultivars de sorgho. Les taux de recuperation se sont averes plus eleves dans la methode de sedimentation que dans celle de flottaison du sucrose. La methode de sedimentation n'a pas montre un changement significatif des rapports protoplastes vacuoles sur protoplastes etioles dans les echantillons initiaux et purifies. Par ailleurs, ces proportions ont ete en grande partie titrees en faveur du protoplaste vacuole par la methode de sedimentation du sucrose. On pourrait ainsi exploiter a resultat et selectionner la methode de purification appropriee en se basant sur les besoins experimentaux specifiques.

Mots Cles: Tiges etiolees, purification des protoplastes, sorgho

INTRODUCTION

The successful isolation of protoplasts from plant tissues is a prerequisite for their use as model systems for physiological, biochemical and virological studies. Sterile shoot cultures are becoming increasingly popular as protoplast source. One advantage is that the material is already sterile and does not have to be surface-sterilised prior to incubation in the digestive enzyme mixture. Also, within the highly protected and stress-free environment of the culture vessel, the young shoots are in optimal physiological condition, and they develop a very thin cuticle, thus giving little resistance to the penetration of the enzyme mixture (Bengochea and Dodds, 1986).

A key step in the use of these protoplasts is the initial provision of clean, pure samples of protoplasts. Common purification protocols for cereals are mainly based on two methods which commence with an initial filtration step. The protoplasts are then either sedimented by low speed (500 to 800rpm) centrifugation, often in a solution containing mannitol or sorbitol for three to five minutes (Wei and Xu 1990; Quayle et al., 1991), or purified by floatation on sucrose solutions of 0.5 to 0.6 molarity (Chang et al., 1991).

The aims of this paper are to provide detailed comparative data for protoplast numbers and subpopulation profiles in two cultivars of Sorghum bicolor Moench and to assess losses and effect on final culture attributable to the different methods of protoplast purification.

MATERIALS AND METHODS

Plant materials.

Two cultivars of Sorghum bicolor (CH 9 and MH 51) supplied by Dr. Naidu of Spic Science Foundation, Madras, (India) were used in this study. Mature seeds were surface-sterilised in 2% sodium hypochlorite for 10 minutes. They were rinsed four times with sterile distilled water and soaked in 0.1% HgCl2 solution for seven minutes. They were again rinsed four times with sterile distilled water and germinated in sterile magenta boxes at 25 C in the dark.

Protoplast isolation.

Etiolated seedlings, between 6 - 10 days old were used as the protoplast source. Hypocotyl sections, about 3cm in length were cut once or twice longitudinally and presoaked in 0.6M mannitol (or sorbitol) for 3.5 hrs. This had previously been found to be optimal for protoplast yield and viability in these cultivars (E. O. Umoh, unpubl.). The isolation medium contained 3% cellulase (Onozuka R. S.), 0.5% macerozyme (R-10) and 0.1% pectolyase (Y-23) dissolved in protoplast wash solution based on CPW medium (Frearson et al., 1973). Hypocotyl sections were incubated at 27 C and at a pH of 5.7 for 2.5hr in the dark, interrupted by gentle manual agitation at 20 min intervals. One ml of IN Na2 CO3 solution was added as buffer to the reaction mixture. Protoplasts were separated from undigested tissue by filtration through 30um steel mesh.

Protoplast purification.

Two procedures were used for protoplast purification, Sedimentation and floatation methods. In the Sedimentation method, the protoplast suspension (pH 5.7) was centifuged at 720g for four minutes in 10 ml falcon tubes. The resultant pellet was resuspended in 5ml of 0.6M mannitol and the sequence repeated thrice (Millam et al., 1991).

For the second procedure (floatation method), the initial protoplast suspension (pH 5.7) was centrifuged at 720g for 4 min. in 10 ml falcon tubes. The resultant pellet was resuspended in 1-2ml of 0.6M mannitol which was then layered on 5mls of 0.6M sucrose in a 10ml falcon tube. This was centrifuged at 225g for 10 min. Floating protoplasts were collected with a pasteur pipette and transfered to a 5ml of 0.6M mannitol in a 10 ml falcon tube. This was centrifuged at 720g for 4 min. (pH 5.7). The pellet was suspended in 1ml of 0.6M mannitol for analysis.

Initial and final protoplast numbers were obtained by counting using a 0.2 Fuchs-Rosenthal haemocytometer. Sub-populations were characterised as vacuolated (containing few or no etioplasts but a large vacuole) or etioplastic (containing many etioplasts and no large vacuole).

In both cases, ten counts per replicate were made and the means and standard errors computed therefrom. A t-test was used to determine significance levels where necessary.

RESULTS

Total protoplast counts both before and after purification by (i) sedimentation and (ii) floatation methods are given in Table 1 for the two cultivars of Sorghum bicolor studied. Recovery percentages of protoplasts were found to be significantly higher (P<0.05) in the sedimentation method for both cultivars. Plates 1 and 2 show the protoplasts in their unpurified and purified forms, respectively. The comparative subpopulation contents of initial and purified samples are presented in Table 2. The ratios of vacuolated to etioplastic in the initial and purified samples of protoplasts were not significantly changed (P>0.05) in the two cultivars when the sedimentation method of purification was used. On the other hand, these ratios were significantly tilted in favour of vacuolated protoplasts in the two cultivars when the sucrose floatation method was used.

Plates 1 and 2 - Freshly isolated protoplasts of Sorghum bicolor. 1. Unpurified protoplasts; 2. Purified protoplasts.

--------------------------------------------------------------------- 
Purification   Cultivar   Initial count    Purified count    Recovery 
  method                (10^6/g fresh wt) (10^6/g fresh wt)    (%)
----------------------------------------------------------------------      
                 CH 9     1.98+/-8.71      0.85+/-6.87       42.92
Sedimentation    MH 51    2.01+/-8.53      0.98+/-7.84       44.28
  
                 CH 9     1.94+/-7.86      0.74+/-8.72       38.14
Floatation       MH 51    1.95+/-6.84      0.68+/-5.76       34.87
----------------------------------------------------------------------  

--------------------------------------------------------------------------- 
Cult-  Purifi-   Initial count      Ratio      Purified count      Ratio
ivar   cation  ------------------- ------- --------------------- ---------- 
       method    A           B      (A:B)        A           B       A:B
--------------------------------------------------------------------------- 
CH 9     I  0.59+/-8.2  0.89+/-8.4  0.66:1  0.30+/-8.5  0.42+/-7.2  0.71:1 

        II  0.65+/-7.5  0.94+/-6.8  0.69:1  0.45+/-5.6  0.25+/-5.4  1.80:1  
 
MH 51    I  0.67+/-8.2  1.04+/-6.2  0.64:1  0.42+/-8.2  0.60+/-7.8  0.70:1 

        II  0.45+/-8.5  0.72+/-4.7  0.63:1  0.46+/-7.4  0.29+/-6.7  1.59:1 

DISCUSSION

While Millam et al.'s report of 1991 is in total agreement with the lower percentage recovery of protoplasts recorded in the sucrose floatation method here, it disagrees with the present report in that the ratios of vacuolated to etioplastic protoplasts between initial and purified (by sedimentation) samples are similar.

The tilt in favour of vacuolated protoplasts when the sucrose floatation method was used might have been caused by the difference in the specific densities of the two protoplast types. Mehrle et al. (1989) have previously noted that the two protoplast types are not usually suspended in the same medium due to their different specific densities.

The sucrose floatation method has earlier been reported to cause less loss due to damage than the sedimentation method (Bengochea and Dodds, 1986). Accordingly, since the Sorghum bicolor cultivars used in the present study had a higher proportion of etioplastic protoplasts (Table 2), and since the sucrose floatation method gave a higher proportion of vacuolated protoplast, a greater percentage of the etioplastic protoplasts might have sedimented with the debris. This is further supported by the report that the highly meristematic protoplasts are difficult to purify as they sink into the sucrose solution and sediment with the debris (Davey, 1983).

The proportion of the protoplast subpopulations present in the purified samples is particularly relevant to protoplast fusion and electroporation studies. A marked increase in the fusion ability of vacuolated chloroplasts has been reported (Millam et al., 1991). Other workers observed a decrease in sample viability when vacuolated chloroplast was used (Zimmermann and Scheurich, 1981). Protoplast regeneration which commences with cell wall formation is reported to be slower with the chloroplastic protoplasts (Mehrle et al., 1989). Such inherent properties of the protoplast subpopulations may thus be exploited by future workers and the protoplast sample purified according to specific experimental requirements.

ACKNOWLEDGEMENT

The author thanks SPIC Science Foundation Madras (India) for providing the facilities used, TWAS (Italy), COSTED (Madras, India) and RMDC (Nigeria) for financial assistance.

REFERENCES

Bengochea, T. and Dodds, J. H. 1986. Plant Protoplasts : A biotechnological tool for plant improvement. Chapman and Hall, London, NY 89pp.

Chang, Y., Wang, W. C., Warfield, C. Y., Nguyen, H. T. and Wong, J. R. 1991. Plant regeneration from protoplasts isolated from long term cell cultures of wheat (Triticum aestivum L.). Plant Cell Reports 9:611- 614.

Davey, M. R. 1983. Recent developments in the culture and regeneration of plant protoplasts In: Proceedings of the 6th International Protoplast Symposium Exp. Supp 46, pp. 2 - 30. Birkhauser Verlag, Basel.

Freason, E. M., Power, J. P. and Cocking, E. C. 1973. The isolation, culture and regeneration of Petunia leaf protoplasts. Developmental Biology 33:130-137.

Mehrle, W., Hampp, R., Naton, B. and Grothe, D. 1989.Effects of microgravitation on electrofusion of plant cell protoplasts. Plant Physiology 89:1172-1177.

Millam, S., Burns, A. T. H. and Hocking, T. J. 1991. A comparative assessment of purification techniques for mesophyll protoplasts of Brassica napus L. Plant Cell, Tissue and Organ Culture 24:43 - 48.

Quayle, T. J. A., Hetz, W. and Felix, G. 1991. Characterization of a maize endosperm culture expressing zein genes and its use in transient transformation assays. Plant Cell Reports 9: 544-548.

Wei, Z. and Xu, Z. 1990. Regeneration of fertile plants from embryogenic suspension culture of protoplasts of Sorghum vulgare. Plant Cell Reports 9:51-53.

Zimmerman, U. and Scheurich, P. 1981. High frequency fusion of protoplasts by electric fields.Planta 151:26-32.

Copyright 1997 The African Crop Science Society

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