Propagation of Uapaca Kirklana Using Tissue· Culture Techniques

Abstract

A study on propagation of Uapaca kirkiana using tissue culture-techniques was done G ' at Bunda College of Agriculture. Experiments were conducted on In Vitro and In Vivo seed germination; axillary shoot proliferation using seedling stem nodal sections; direct organogenesis and callus development from stem shoot sections, adult tree leaf and cotyledonous explants; and root development. Explants were fruits obtained from a local market, some fruits direct from trees and adult tree explants were obtained from Uapaca kirkiana provenance near the College. Treatments in the seed germination experiments included surface sterilization of seeds with four different concentrations of sodium hypochlorite (NaOel) and four exposure times; primary and secondary seed-coat removal; split-plot treatments of two fruit sources (market and direct from trees) as main factors, and two seed germination media - Murashige & Skoog (MS) and Woody Plant Medium (\VPIYf) as subplot factors. Another seed germination experiment was set up in a green house with the two fruit sources as main factors and primary seed-coat removal and non-removal as sub- factors. The axillary shoot proliferation experiment used seedling stem nodal sections as explants. Treatments included 0, 5, 10, 20, 40, 80, and 160 mg/l of kinetin growth regulator supplement on WPM media in a preliminary test. A detailed experiment followed with a split-plot design which included five indole butyric acid (IBA) levels (0.0,0.25,0.50, 1.00,2.00 mgll) as main factors and the same five levels of kinetin as sub-factors. The direct organogenesis and callus experiments tested three types of tree organs which included adult stem shoot sections, leaf sections and cotyledonous VI leaf sections. MS basal salts media were supplemented with 2mgll lAA +2mgll kinetin+ 1Omgll inositol and White vitamins and glycine + 80mg(l adenine sulphate + IOmg/1tyrosine for direct organogenesis; and 2mglllAA + O.5mgJIkinetin for callus development. Explants for rooting experiment were obtained from axillary shoot proliferation plantIets and tested on rooting media in a split-plot design. The main factors were two basal salts (MS and \VPM) and subfactors were five IBA growth regulator levels (0.0 IBA, 0.5 IBA, 0.5 IBA + 0.5 NAA + 0.5 BAP, 1.0 IBA and 2.0 IBA in mgll. The containers used for media were 25mm x 150mm belli co tubes with 20 ml quantities. Each treatment had N=20 cultures. Incubation of cultures was done under 16 hours light illumination at 4511EM~-1 except for callus cultures which were incubated under 24 hour darkness. Temperatures were maintained within the range of 25 to 28°C. The data collected was subjected to Analysis of Variance and regression analysis where necessary. "... ,. Optimum concentration of NaOCl "for surface sterilization for the U kirkiana seeds was 2% for 20 minutes. Removal of both primary and secondary seed-coats promoted the number of aseptic seedlings. The !viS medium gave significantly higher frequency of normal seedlings (90%) and multiple seedlings/seed (90%) than \VPM medium in the first experiment. When the experiment was repeated the effect of the two media did not significantly differ. Primary seed-coat removal improved in vivo germination from 55% to 780/0and from 35% to 95% for market fruits and fruits direct from trees respectively. The seeds exhibited apomixis with a maximum of 9 seedlings/seed for VIl in vitro, and 2 seedlings/seed for in vivo germination. The high number of shoots that developed per axillary nodal bud section was achieved with supplementation of 0.50 mg/l kinetin alone (1.3 shoots) or combined with 0.25 mg/l IBA (I.Ishoots), 2.0 mgll kinetin alone (1.1shoOLS)or combined with 0.5 mgll IBA (1.3 shoots). The direct organogenesis and callus cultures did not regenerate into adventitious plantlets but just developed callus and embryoids. The stem sections had a high callus development frequency (67.5% partial and 15% full) followed by cotyledonous sections (27.5% partial and 200/0 full). Adult tree leaf explant callus development failed because all cultures had fungal contamination. The subcultured calli proliferated fastest on \VPM supplemented with 3.0 mg/l IAA. Higher ratios of kinetin to auxin (IAA) slowed calli proliferation. In the rooting experiments, MS media containing 1.0 and 2.0 mg/l IBA promoted rooting at 65% (1.7 roots/plantlet) and 60% (1.3 roots/plantlet) respectively, than lower levels of IBA. The MS media promoted better rooting (0.7roots/plantlet) than the WPM media (0.4 roots/plantlet). Further investigation on media protocols to improve adventitious axillary shoot proliferation and rooting performance to ensure high field survival of the U. kirkiana plantlets is suggested. Chemical names used: 6-furfurylaminopurine (kinetin); benzyladenine (BA); N6-benzylaminopurine (BAP); 1 H-indole-3-butyric acid (IBA); 2-naphthaleneacetic acid (NAA); indole-3-acetic acid (IAA); and gibberellic acid (GA3)·