In vitro regeneration of two Kenyan bread wheat (triticum aestivum l.) cultivars

Abstract

Wheat (Triticum aestivum L.) is the second most important food crop in Kenya by production and consumption, after maize. However, national wheat productivity has been on the decline due to its vulnerability to a number of biotic and abiotic stresses. Tissue culture can be exploited for improvement of this crop for better yields and other desired attributes including superior grain quality. Due to the fact that tissues culture is highly dependent on genotype and explant type, this study was done to establish a regeneration system for two Kenyan wheat cultivars (Njoro BW II and Eagle 10) using mature seed embryo explants. Three disinfectants Jik® (3.85% w/v NaClO), 0.1% w/v mercuric chloride and chlorine gas were investigated for their effectiveness in establishment of axenic cultures. Murashige and Skoog (MS) medium with 0.5-8mg/l of 2, 4-dichlorophenoxyacetic acid (2, 4-D), 1naphthaleneacetic acid (NAA) and 4-chlorophenoxyacetic acid (4-CPA) were tested for callus induction. After 7-14 days of initial callus induction, calli obtained were sub-cultured to fresh MS medium of the same composition for proliferation. After 30 days, calli were subcultured to hormone free MS medium or MS medium supplemented with 6benzylaminopurine (BAP), kinetin (KN) and thidiazuron (TDZ) at 0.25-2 mg/l alone or in combination with 0.1 mg/l indole acetic acid (IAA) for in vitro shoot or plantlet regeneration. The plantlets obtained were acclimatized using sterile vermiculite for 14 days followed by vermiculite with soil (1:1) in the culture room for 14 days and eventually unsterile soil alone in the green house. Data on percentage callus induction, callus fresh weight and regeneration were subjected to one way analysis of variance (ANOVA) and means separated by Tukey’s HSD test at p ≤ 0.05. All the auxins 4-CPA, NAA and 2, 4-D tested were able to induce callus in 20-90% of mature seed embryos in both cultivars within 1-4 days. However, 2, 4-D induced higher frequencies of callus than 4-CPA or NAA at all concentrations used. Combining 2 mg/l 2, 4-D with either 0.2 mg/l NAA or 4-CPA produced up to 13% higher frequency of callus induction in both cultivars than when 2, 4-D was used alone. Somatic embryogenesis was observed in calli inoculated in medium with 1-8 mg/l 2, 4-D. The highest embryogenic callus induction frequency (i.e. 63%) was observed in MS medium containing 2 mg/l 2, 4-D in combination with 0.2 mg/l 4-CPA. Plant regeneration was achieved after 12-24 days on MS hormone free medium. The highest plant regeneration frequency of 67% was achieved on medium containing 0.5 mg/l BAP plus 0.1 mg/l IAA. A total 9% of plantlets acclimatized survived to maturity. It took 90-100 days from callus initiation to plantlet establishment. This study shows that mature seed embryos can be used to regenerate fertile wheat plantlets. The findings have the potential to inform future effort in the application of tissue culture for expedited wheat cultivar improvement in Kenya. Key words: Triticum aestivum, mature seed embryos, somatic embryogenesis