Use of a Protoplast Regeneration System for African Violet Improvement
Traud Winkelmann, Institute for Breeding of Ornamental Species Ahrensburg, Germany
This article first appeared in African Violet Vol. 46(6), Pp. 50-52 (1993).
IntroductionSince African violet growing began in Germany in 1893, breeders have improved this species in many ways. Vegetative habit, time to flowering, and flower retention have been altered. In addition, a wide spectrum of flower colors, patterns, and shapes is available in the modern African violet. This was done mainly by making crosses and subsequently selecting the desirable seedlings. Traditional breeding methods are limited by the range of species which can be combined, and certain desirable features, particularly the introduction of true red and yellow flowering plants, has not been achieved (the Blansit violets appear to be an exception).
Research demonstrating that African violets could be propagated easily in vitro under sterile conditions has opened new ways for increasing genetic variability through biotechnology (Start and Cumming, 1976; Grunewaldt, 1977). Some of these techniques require that plants be regenerated from protoplasts (naked cells without cell walls) rather than from leaf tissue. Before being able to use methods like direct DNA transfer into protoplasts or fusion of protoplasts of otherwise incompatible species (Saintpaulia and Episcia, for example) it is necessary to develop a reliable method for obtaining whole plants from protoplasts. The aim of our research was to establish such a protoplast regeneration system for African violets.
Isolation of Protoplasts, Protoplast Culture, and Plant RegenerationProtoplasts can be released from plant tissue by one of two methods. The first method involves mechanically isolating the naked cells by dissection or rupture of the cell walls (Bilkey and Cocking, 1982). The more common method involves treating the plant tissue with enzymes that digest the cell wall material. For our work we used a combination of three enzymes: 0.5% macerozyme, a pectinase, to dissolve the tissue; and, 2 % cellulase R10 and 0.1% driselase, two cellulases, to dissolve the cell walls (Winkelmann and Grunewaldt, 1992).
The starting plant material employed for a source of protoplasts proved to be very important for successful regeneration. Only when young shoots from tissue culture were used as the starting material were plants able to be regenerated from protoplasts.
After removing the enzymes by centrifugation, the protoplasts were embedded in alginate. Protoplasts plated in liquid or in a medium solidified with agarose did not develop. The successful medium contained macro- and micronutrients, organic acids, vitamins, and high concentrations of different sugars to stabilize the naked protoplasts until the cell walls reformed. Cell walls were formed and the cells began to divide after 8 to 10 days of growth in the dark (see Figure 1). The medium also contained two plant growth regulators; 1 mg/liter naphthaleneacetic acid (an auxin); and, 1 mg/liter benzyladenine (a cytokinin). The complete details of the protoplast culture procedure can be found in the reference by Winkelmann and Grunewaldt (1992).
After 14 days growth on the initial culture medium, the osmotic strength and the concentration of growth regulators was reduced. The osmotic strength was reduced again 10 days later. Then, after about 4 weeks of culture, small clumps of unorganized cells, or calli, could be removed and plated on a medium solidified with agarose (see Figure 2). These calli were grown in the dark until they reached a size of 3 to 4 mm in diameter, and then, they were transferred to a medium containing 2 mg/liter benzyladenine to induce plant formation.
As soon as the young plants were visible under a stereomicroscope, the cultures were moved to the light and placed on a shoot elongation medium (see Figure 3). The number of plants per callus varied, ranging between 5 and 50. These plants rooted easily and could be grown on in the greenhouse with few losses. The scheme presented in Figure 4 summarizes the process from protoplast isolation to growth of the plants in the greenhouse.
Different cultivars of African violet responded differently in this system. Four of five of the cultivars (from Fischer Company, Hannover-Isernhagen, Germany) we tested produced plants from protoplasts . Plants were regenerated from protoplasts of the cultivars 'Heidrun hell', 'Sarosa', 'Gracia', and 'Rokoko rosa'. Protoplasts of the cultivar 'Blanca' produced callus, but the callus died before shoots were formed.
More than 2,000 plants have been transferred to the greenhouse. These are growing vigorously, and appear to be uniform (see Figure 5). A few plants showed chlorophyll deficiencies (either albino or with variegated leaves), and some appear to be polyploid with thick, succulent leaves and peduncles. Chromosome counts will determine whether these are really polyploid. In total, about 95% of the plants appeared to be true to cultivar, indicating that this regeneration technique is a stable one.
Applications for African Violet ImprovementProtoplasts are useful in genetic manipulations because they do not have cell walls. They are ideal targets for taking up naked DNA and for fusing with protoplasts of other related species. In the related genus, Episcia, some species and selections have true yellow and red flowers. We have applied successfully our procedure for plant regeneration from African violet protoplasts to protoplasts of Episcia cupreata 'Tropical Topaz' (Winkelmann and Grunewaldt, 1993). As was suggested by Bilkey and McCown (1978), protoplast fusion between African violet and Episcia may lead to the production of new flower colors which have not been possible because of genetic barriers. Research toward this goal is now in progress.
AcknowledgmentsThe experiments reported here are part of the Ph. D. thesis of T. Winkelmann, which was supported by the Federal Ministry of Research and Technology (BMFT, Bonn) and Fischer Company (Hannover-Isernhagen). The author would like to thank Prof. R. D. Lineberger for his critical review of the manuscript.
ReferencesBilkey, P. C. and E. C. Cocking. 1982. A non-enzymatic method for isolation of protoplasts from callus of Saintpaulia ionantha (African violet). Z. Pflanzenphysiol. 105:285-288.
Bilkey, P. C. and B. H. McCown. 1982. Towards true red, orange and yellow-flowering African violets - asexual hybridization of Saintpaulia and Episcia. African Violet Magazine 31:64-65.
Grunewaldt, J. 1977. Adventivknospenbildung und pflanzenregeneration bei Gesneriaceae in vitro. Gartenbauwissenschaft 42:171-175.
Start, N. D. and B. G. Cumming. 1976. In vitro propagation of Saintpaulia ionantha Wendl. HortScience 11:204-206.
Winkelmann, T. and J. Grunewaldt. 1992. Plant regeneration from protoplasts of Saintpaulia ionantha H. Wendl. Gartenbauwissenschaft 57:284-287.
Winkelmann, T. and J. Grunewaldt. 1993. Plant regeneration from protoplasts of Saintpaulia ionantha H. Wendl. XVIIth Eucarpia Symposium Ornamental Section, SanRemo Italy, March 1 - 5, 1993. Abstract of presentation.