In vitro propagation of Scilla natalensis planch.
In South Africa, large quantities of Scilla natalensis are harvested from wild populations and sold as traditional medicine, which is reducing the density, distribution and genetic diversity of wild populations. The enforcement of existing legislation, however, has proved ineffective with plants being traded locally and internationally. It has therefore, been suggested that ex situ conservation through cultivation may alleviate pressures on natural resources. Conventional propagation of these plants, however, is usually fairly slow. In vitro propagation provides a rapid means of propagating selected chemotypes or cultivars, serving both conservation and commercial interests. In the first part of the study, continuous culture systems were established for the three forms of Scilla natalensis, S. natalensis sensu stricto (Form A), S. natalensis syn. S. kraussii (Form B) and S. natalensis syn. S. dracomontana (Form C). The efficiency of the systems was strongly influenced by genetic factors, viz the form and epigenetic factors, viz the explant type, carbohydrate source, plant growth regulators and gelling agents. The form, Form A, Form B or Form C respectively, influenced shoot initiation with the larger forms generally producing more shoots than the smaller forms (Form A > Form B > Form C). The data confirmed that the three forms are significantly different in terms of their physiological response to carbohydrates, plant growth regulators and gelling agents in vitro. Since the effect of form could not be compensated for by the addition of either carbohydrates, plant growth regulators or gelling agents, this may provide some support for the reinstatement of these forms as three species, Scilla natalensis Planch., S. kraussii Bak. and S. dracomontana Hilliard & Burtt. The explant type, that is bulb or leaf explants respectively, significantly influenced shoot initiation. Leaf explants generally produced more shoots than bulb explants. The carbohydrate source significantly influenced shoot initiation. The explants generally produced more shoots when cultured on media containing glucose or sucrose than on media containing fructose, lactose, maltose and particularly mannitol. The combination of cytokinins and auxins significantly influenced shoot initiation. Shoot initiation was higher for combinations of kinetin: IAA than for combinations of kinetin: NAA or TDZ: NAA. Optimal shoot initiation for Form A, Form B and Form C occurred on media containing 1 to 2 mg I-1 kinetin and 1 to 2 mg I-1 IAA. The gelling agent also influenced shoot initiation with media solidified with Gelrite® producing more shoots than media solidified with Oxoid or Unilab agar. Shoots were then rooted on media containing IAA, IBA or NAA and the plantlets were successfully acclimatised. These continuous culture systems can be used to produce large quantities of plantlets, which may alleviate pressures on natural resources and provide an alternative source of high quality plants for the burgeoning medicinal plant market. In the second part of the study, the effect of carbohydrate source and concentration on growth and development of shoots of S. natalensis syn. sensu stricto (Form A) were determined. This has applications for the acclimatisation and germplasm storage of bulbous plants. The carbohydrate source and concentration significantly influenced the growth and development of shoots. In the absence of carbohydrates, the shoots were short with spindly leaves and short roots. When media were supplemented with high concentrations of fructose, the shoots were long with broad leaves, small bulbs, and few short to medium length roots at low concentrations. At higher fructose concentrations, however, the shoots were robust and short with narrow, sometimes deformed leaves, large bulbs, and few stunted, brown roots. When sucrose was substituted for fructose, the shoots were robust and long with narrow and often red-pigmented leaves, large bulbs, and many long, thick roots. When AC was used in combination with sucrose, however, the shoots were robust and short with few, and occasionally red-pigmented leaves, small to medium bulbs, and few, severely stunted roots. Optimal shoot growth and development in terms of shoot weight (FW) and quality occurred on media containing glucose or sucrose (40 to 60 g I-1). The carbohydrate source and concentration also significantly influenced the physical properties of media particularly pH, electrical conductivity (EC) and gel-strength. The pH decreased slightly with increasing glucose concentration but decreased significantly with increasing fructose concentration when fructose was used alone or in combination with glucose. The pH also decreased significantly with increasing sucrose concentrations when sucrose was used in combination with Sigma AC. The EC decreased significantly with increasing fructose concentration when fructose was used alone but remained fairly constant irrespective of glucose concentration when glucose was used alone or in combination with fructose. The EC also remained fairly constant irrespective of the sucrose concentration but decreased with increasing sucrose concentration when used in combination with AC. The gel-strength remained fairly constant irrespective of glucose. The gel-strength decreased with increasing fructose concentration when used alone or in combination with glucose. The gel-strength of media increased with increasing sucrose concentration although the addition of Sigma AC significantly decreased the gel-strength of media, which decreased with increasing sucrose concentration. The brand and concentration of AC also influenced gel-strength. The matrix plot suggested that the effect of carbohydrate source and concentration on the growth of shoots may be largely due to the indirect effects of these physical properties such as hydrolysis of carbohydrates, the spectrum and quantity of the breakdown products and the availability of nutrients, plant growth regulators and water rather than the direct effects of pH, EC and gel-strength per se.