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African Crop Science Journal
African Crop Science Society
ISSN: 1021-9730 EISSN: 2072-6589
Vol. 6, Num. 2, 1998, pp. 149-158
African Crop Science Journal, Vol

African Crop Science Journal, Vol. 6. No. 2, pp. 149-158, 1998

THE ROLE OF AMINO ACIDS ON MATURATION AND CONVERSION OF GERANIUM SOMATIC EMBRYOS

R. M. Madakadze, T. Senaratna1 and P. K. Saxena1

Department of Crop Science, University of Zimbabwe, Private Bag MP 167, Mount Pleasant, Harare, Zimbabwe
1 Department of Horticultural Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada

(Received 21 November, 1997; accepted 24 March, 1998)

Code Number:CS98017
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ABSTRACT

The effect of various concentrations of 4 amino acids (cysteine, glutamine, methionine and proline) on maturation and conversion of thidiazuron - induced somatic embryos were investigated. All the amino acids used improved maturation frequency, protein content and plantlet dry weight of converted embryos indicating a general improvement of the nutritive status of the somatic embryos. Addition of methionine, proline, cysteine(3mM) and glutamine (10 mM) in the maturation media also improved conversion frequency of somatic embryos. Inclusion of amino acids in a maturation media improves both morphological maturity and physiological maturity of geranium somatic embryos. This study shows the importance of inclusion of amino acids in a maturation media for improved somatic embryo quality.

Key Words: Electrophoresis, embrogenesis, maturation frequency, proteins

RÉSUMÉ

La présente étude porte sur l'effet des différentes concentrations de 4 acides aminés (cystéine, glutamine, méthionine et proline) sur la maturation et la transformation de thidia zuron - embryons somatiques induits. Tous les acides aminés utilisés ont amélioré la fréquence de maturation, la teneur en protéine et le poids sec des plantules des embryons transformés indiquant une amélioration générale de l'état nutritif des embryons somatiques. L'addition des méthionine, proline, cystéime (3mM) et glutamine (10mM) dans les milieux de maturation a aussi amélioré la fréquence de transformation des embryons somatiques. L'introduction des acides aminés dans les milieux de maturation améliore à la fois la maturité morphologique et physiologique des embryons somatiques du geranium. Cette étude montre ainsi l'importance de l'inclusion des acides aminés dans les milieux de maturation afin d'améliorer la qualité des embryons somatiques.

Mots Clés: Electrophorèse, embryogénèse, frequence de maturation, protéines

INTRODUCTION

Somatic embryos are bipolar structures similar to zygotic embryos. They originate from somatic cells, and develop through the same ontogeny as zygotic embryos. In view of their bipolar nature somatic embryos can be developed into artificial seeds by either desiccation or gel encapsulation (Senaratna, 1992). In geranium, attempts at developing artificial seeds by desiccation (Marsolais et al.,1991) and gel encapsulation (Gill et al.,1994) have been made, but the vigour of plantlets produced was considerably less than that of true seedlings indicating lower quality of the somatic embryos. The low quality of somatic embryos has been established to be due to very low levels of triacylglycerols, proteins, starch and sugars in the somatic embryos compared to seeds (Madakadze, 1996).

Amino acids have been used in several embryogenesis systems to improve both numbers and quality of somatic embryos (Stuart and Strickland, 1984; Stuart et al., 1985 ). In alfalfa somatic embryogenesis optimisation of regeneration occurred when 100 mM proline and 25 mM ammonia were added to the embryogenesis medium. In the maturation medium, glutamine stimulated storage protein synthesis in alfalfa (Lai et al., 1992). The objective of this study was to study the effects of amino acids on geranium somatic embryo maturation and conversion into plantlets.

MATERIALS AND METHODS

Plant material. Geranium (Pelargonium x hortorum Bailey) seeds (cv. Scarlet Orbit Improved obtained from Stokes Seed Company, St. Catharines, Ontario, Canada) were surface sterilised by immersing first in 70% (v/v) ethanol for 30 seconds followed by agitation for 20 min in a 30% (v/v) solution of commercial sodium hypochlorite containing one drop (per 200 mL) of Tween-20. Seeds were then rinsed five times with sterile distilled water. Ten sterilised seeds were germinated in a Petri dish (100 x 15 mm) containing 25 mL water-agar medium (0.8% Sigma purified agar in distilled water). Petri dishes were sealed with Parafilm, and incubated in the dark at 24° C for six days in an incubator (Percival, Boone, Iowa, USA).

Somatic embryo culture. Hypocotyls from six-day-old etiolated seedlings were excised into 0.8-1 cm long explants. Nine explants per Petri dish were cultured on an induction medium containing MS salts (Murashige and Skoog, 1962), B5 vitamins (Gamborg et al.,1962), 30 gL-1 sucrose, 20 µM N-phenyl-N'-1,2,3-thidiazol-5-ylurea (thidiazuron, TDZ) (Hutchinson and Saxena, 1995), and 3 gL-1 gelrite (Scott Laboratories, Carson, CA, USA). The pH of the medium was adjusted to 5.5 before autoclaving at 120°C and 1.2 kg.m-1 for 20 min. The Petri dishes were sealed with Parafilm and incubated at 24° C under 16 hr photoperiod (20-25 µmol m-2 s-1) provided by cool white fluorescent tubes (Phillips Canada, Scarborough, Ontario, Canada). Three days after culture on the induction medium the hypocotyls were transferred onto basal medium for the development phase of somatic embryogenesis. All hypocotyls were subcultured on maturation phase medium (basal medium with different amino acid treatments) after 21 days of culture. Amino acid treatments used were cysteine, glutamine, methionine and proline each at 3, 10 and 50 mM.

All somatic embryos were counted 35 days after initial culture using a stereo-microscope. Cotyledon stage somatic embryos were also counted. The maturation frequency, an indicator of morphological maturity and developmental synchrony, was calculated as the percentage of somatic embryos that were at the cotyledonary stage (number of somatic embryos at the cotyledonary stage/ total number of somatic embryos x 100). Fifty somatic embryos from each treatment were harvested into 1.5 mL microcentrifuge tubes and freeze dried to determine the somatic embryo dry weight.

Conversion of somatic embryos into plantlets. Hypocotyls with somatic embryos from all treatments were cut into 2.5 mm sections 35 days after explant culture. Five sections were cultured on basal medium in each Petri dish and three replications were used for each treatment. After 21 days, total numbers of plantlets and ungerminated somatic embryos on each section were counted. The conversion frequency was calculated as the percentage of somatic embryos that converted into plantlets.

Protein analysis. Somatic embryos at the cotyledonary stage, harvested after 35 days from culture initiation, were used for all protein analysis. Each sample weighed approximately 60 mg dry weight. Samples were stored at -70°C until analysis. Storage proteins of somatic embryos were extracted according to Krochko (1988) with minor modifications. Each sample was ground in 1 mL of buffer 1 (0.05 M NaCl in 25 mM Potassium phosphate buffer, pH 7.0 with protease inhibitors, 1mM phenylmethylsulphonyl fluoride (PMSF) and 10 µM leupeptin) using a Duall ground glass homogeniser. The supernatant (S1) proteins were collected after centrifugation at 14,000g for 5 min. The remaining pelleted material was resuspended vigorously in 1 mL of buffer 2 (1.0 M NaCl in 25 mM potassium phosphate buffer, pH 7.0, 1 mM PMSF and 10 µM leupeptin) and the supernatant (S2 protein) was collected after centrifugation. Protein quantification was carried out using the bicinchoninic acid assay (BCA) (Pierce, Rockford, Il) (Smith et al.,1985) using bovine serum albumin (BSA) as a standard. Protein content was calculated for each sample by getting the sum of S1 and S2 proteins. One dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS - PAGE) was carried out on buffer 2 extracted protein according to Krochko and Bewley (1988). This fraction was used because it contains most of the seed specific protein in geranium zygotic embryos (Madakadze, 1996). All samples were treated with 5 % mercaptoethanol (ME) to break any disulphide bonds before loading them on the gel. Fifteen microliters of sample were loaded per lane.

Experimental design and statistical analysis. This experiment was designed as a factorial experiment with 4 amino acid treatments each at three concentrations (3, 10 and 50 mM) and a control with no amino acids. Each treatment was replicated 3 to 5 times and the experiment was repeated three times. Analysis of variance of pooled results from the three repeats was performed using PC SAS version 6 (SAS Institute, 1995) and the means were compared by the protected least significance differences (LSD0.05) test.

RESULTS

The number of somatic embryos formed varied with different concentrations of amino acids tested (Fig. 1A). Glutamine and proline at all concentrations had no effect on somatic embryo numbers compared to the control. However, for cysteine and methionine the numbers of somatic embryos decreased with increase in concentration. Proline at all concentrations caused a lot of precocious germination of somatic embryos (data not shown). All concentrations of amino acids used significantly improved maturation frequency compared to the control except 50 mM cysteine and methionine (Fig. 1B). Fifty mM cysteine significantly reduced maturation frequency compared to the control. The optimum amino acids for maturation frequency were 3 mM cysteine and 50 mM glutamine. These concentrations of amino acids also caused very good synchronisation of somatic embryo compared to the control as shown in Figure 2.

    Figure 1. The effects of different concentrations of amino acids applied to the basal medium at the maturation phase of geranium somatic embryos on (A) the mean number of somatic embryos produced per hypocotyl section, and (B) the maturation frequency.

    Figure 2. The effects of 3mM cysteine (A) and 50mM glutamine (B) on somatic embryo appearance and synchronisation compared to the control (C).

Glutamine at all concentrations had no significant effect on somatic embryo dry weight compared to the control (Fig. 3A). Cysteine at 50mM, 3mM proline and 3 and 10 mM methionine significantly reduced somatic embryo dry weight. Cysteine, glutamine and methionine at all concentrations and proline (3 mM) significantly improved protein content in somatic embryos with the greatest improvement at 10 and 50 mM Cysteine (Fig. 3B). SDS-polyacrylamide gel electrophoresis of proteins from several amino acids at different concentrations indicates improved protein content with the greatest improvement observed with cysteine(10 mM), glutamine (3 and 50 mM) and methionine (3 and 50 mM) (Fig. 4).

Methionine and proline at all concentrations significantly improved conversion frequency compared to the control (Fig. 5A). Three mM cysteine and 10 mM glutamine also significantly improved conversion frequency compared to the control. All concentrations of amino acids used significantly improved the vigour of plantlets (plantlet dry weight) compared to the control with 3 mM cysteine and glutamine more than doubling the plantlet dry weight of the control (Fig. 5B).

    Figure 3. The effects of different concentrations of amino acids applied to the basal medium at the maturation phase of geranium somatic embryos on (A) the somatic embryo dry weight (µg), and (B) the protein content (µg mg-1).

    Figure 4. SDS-PAGE of proteins from somatic embryos matured on media containing different concentrations of amino acids.

    Figure 5. The effects of different concentrations of amino acids applied to the basal medium at the maturation phase of geranium somatic embryos on (A) the conversion frequency and (B) the plantlet dry weight.

DISCUSSION

Storage proteins in seeds function to provide amino acids required for seed germination and early seedling growth (Muntz, 1987). For this reason amino acids have been used in different phases of somatic embryogenesis media to provide raw materials for proteins needed in regeneration and for storage protein synthesis. In this study, amino acids improved maturation frequency, protein content and plantlet dry weight of converted embryos indicating improved morphological and physiological maturity of somatic embryos resulting in improved vigour of plantlets from these somatic embryos. The inclusion of glutamine, alanine, proline or arginine at a concentration of 30 mM in the regeneration medium of alfalfa increased the yield, dry weight, and rate of conversion of somatic embryos into plantlets (Stuart et al.,1985). In maize somatic embryogenesis system, addition of proline improved the somatic embryo maturation (Armstrong and Green, 1985). Reduction of somatic embryo numbers and weights by high concentration of cysteine and methionine suggests possible toxicity of these amino acids at these levels. Cysteine and methionine were also found to be inhibitory to somatic embryogenesis in alfalfa (Stuart et al., 1985).

In alfalfa a glutamine supplement of 50 mM increased embryo size, raised protein accumulation and somatic embryo vigour of seedlings (Lai and McKersie, 1993; Zhang, 1992). These authors suggested that glutamine either meets a nutritional requirement of the somatic embryo by providing reduced amino groups for the synthesis of other amino acids which limit storage protein synthesis or it has a regulatory role involving transcription or translational regulation of the storage protein genes. In this study, although glutamine improved protein content, this protein was not similar to the 11S and LMW proteins found in the zygotic embryos.

Inclusion of S - containing salts in alfalfa maturation media also stimulated the synthesis of 11S and 2S storage proteins in somatic embryos (Lai, 1994) also suggesting a regulatory role of S in these protein synthesis. Cysteine and methionine, S-containing amino acids used in this study also significantly improved protein content but this protein was also not similar to the 11S and LMW proteins of geranium zygotic embryos characterised in an earlier study (Madakadze, 1996) . Selectively limiting the inorganic S supply decreased the synthesis of the S-rich seed storage proteins in favour of the S-poor proteins in peas , wheat (Moss et al., 1981) and barley (Rahman et al., 1983), (Spencer et al., 1990). In addition to this nutritional role, S also has a regulatory role because its deficiency decreases transcription of legumin genes and the stability of the legumin mRNA, indicating that gene expression is affected both at transcriptional and posttranscriptional levels (Higgins et al., 1986). It is therefore possible that in geranium somatic embryogenesis supply of both S and N does not influence regulation of storage protein synthesis but just meets an important nutritional role.

Improvement of conversion frequency by some concentrations of amino acids used in this study is probably due to the significantly improved morphological maturity and protein content of somatic embryos at these concentrations. However, improvement of conversion frequency at 10 and 50 mM methionine is possibly due to lack of competition of somatic embryos, since the numbers of somatic embryos were considerably reduced compared to the control. Zhang (1992) also reported improvement of somatic embryo conversion and vigour after maturation in medium supplemented with amino acids.

Three mM cysteine and 50 mM glutamine significantly improved maturation frequency, protein content, conversion frequency and plantlet dry weight and could therefore be included in possible maturation media of geranium.

ACKNOWLEDGMENTS

Financial support from the Natural Sciences and Engineering Research Council of Canada to PKS and from International Development Research Centre to RMM are gratefully acknowledged.

REFERENCES

Armstrong, C.L. and Green, C. E. 1985. Establishment and maintenance of friable, embryogenic maize callus and the involvement of L- proline. Planta 164:207-214.

Gamborg, O.L., Miller, R. A. and Ojima, K. 1968. Plant cell cultures: 1. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 50:151-158.

Gill, R., Senaratna, T. and Saxena, P. K. 1994. Thidiazuron-induced somatic embryogenesis enhances viability of hydrogel-encapsulated somatic embryos of geranium. Journal of Plant Physiology 143:726-729.

Higgins, J.T.V. 1984. Synthesis and regulation of major proteins in seeds. Annual Review of Plant Physiology 35:191-221.

Hutchinson, M. J. and Saxena, P. K. 1995. Acetylsalicylic acid enhances and synchronizes thidiazuron - induced somatic embryogenesis in geranium (Pelargonium x hortorum Bailey) tissue cultures. Plant Cell Reports.

Lai, F-M. 1994. Maturation and Conversion of Alfalfa Somatic Embryos. Ph. D. Thesis, University of Guelph, Department of Crop Science. 270pp.

Lai, F-M. and McKersie, B. D. 1993. Effect of nutrition on maturation of alfalfa (Medicago sativa L.) somatic embryos. Plant Science 91: 87-95.

Lai, F-M., Senaratna, T. and McKersie, B. D. 1992. Glutamine enhances storage protein accumulation in Medicago sativa L. somatic embryos. Plant Science 87:69-77.

Madakadze, R. M. 1996. Maturation of Geranium Somatic Embryos. Ph. D. Thesis, University of Guelph, Department of Horticulture. 233pp.

Marsolais, A. A., Wilson, D. P. M., Tsujita, M. J. and Senaratna, T. 1991. Somatic embryogenesis and artificial seed production in Zonal (Pelargonium x hortorum Bailey) and Regal (Pelargonium x domesticum Bailey) geraniums. Canadian Journal of Botany 69: 1188-1193.

Moss, H. J., Wrigley, C. W., MacRitchie, F. and Randall, P. J. 1981. Sulphur and nitrogen fertilizer effects on wheat. 11. Influence on grain quality. Australian Journal of Agricultural Research 32:213-226.

Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15:473-497.

Rahman, S., Shewry, P. R., Forde, B. J., Kreis, M. and Miflin, A. B. J. 1983. Nutritional control of storage protein synthesis in developing grain of barley (Hordeum vulgare L.). Planta 159:366-372.

Senaratna, T. 1992. Artificial seeds. Biotechnological Advances 10:377-392.

Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. and Klenk, D. C. 1985. Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150:76-85.

Spencer, D., Rerie, W. G., Randall, P. J. and Higgins, T. J. V. 1990. The regulation of pea seed storage genes by sulphur stress. Australian Journal of Plant Physiology 17:355-363.

Stuart, D. A. and Strickland, S. G. 1984. Somatic embryogenesis from cell cultures of Medicago sativa L. The role of amino acid additions to the regeneration medium. Plant Science Letters 34:165-174.

Stuart, D. A., Nielsen, J., Strickland, S. G. and Nichol, J. W. 1985. Factors affecting developmental processes in alfalfa cell cultures. In: Tissue Culture in Forestry and Agriculture. Proceedings of the 3rd Tennessee Symposium on Plant cell and tissue culture held Sept. 9-13, 1984 at University of Tennesse, Knoxville, Tennessee. Henke, R.R., Hughes, K. W. Constantin, M.P. and Hollaender, A. (Eds.), pp. 59-73. New York: Plenum Press, c1985.

Zhang, Y. L. 1992. Induction and Maturation of Alfalfa Somatic Embryos. MSc Thesis, Universty of Guelph, ON, Canada. 179pp.

Copyright 1998, African Crop Science Society


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