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IN VITRO culture of red clover (TRIFOLIUM PRATENSE L.) and evaluation of regenerated plants Wang, Hong


Red clover (Trifolium pratense L.) cvs 'Altaswede' (2n=2X=14) and 'Norseman' (2n=4X=28) were used in the present study to investigate tissue culture initiation, plant regeneration and the occurrence of somaclonal variation. Hypocotyl explants of aseptic seedlings were inoculated into L2 medium containing 0.06 mg/1 Picloram and 0.1 mg/1 benzyladenine for callus induction. Calli were usually induced after two weeks of culture. Callus induction frequency was 60% to 85% of the explants cultured with 'Altaswede' showing a slightly higher frequency than 'Norseman'. Satisfactory results were obtained under dark or light conditions using either test tubes or petri plates, as culture vessels. After callus induction, an experiment was conducted to regulate shoot induction by subculturing the calli on L2 medium containing 0.01 mg/1 2,4-dichlorophenoxy acetic acid and 2 mg/1 adenine (LSE) and on B₅ medium containing 2 mg/1 naphthalene acetic acid and 2 mg/1 adenine, media which have been reported to be shoot-supportive. However, both media failed to initiate shoots under the present experimental conditions. Further tests confirmed that LSE medium did not induce shoots from these calli and that callus growth on LSE medium steadily deteriorated over several subcultures. Subsequently, various media were tested with an emphasis on different combinations of growth regulators. Root differentiation from these calli was frequently observed. Shoots were initiated from some calli when they were transferred from SCP medium to media containing naphthalene acetic acid and kinetin. Embryogenic callus of one genotype was selected and maintained on LSP medium, leading to the regeneration of numerous plants. Supplementation with arginine, glutamic acid and casein hydrolysate did not show a significant effect on callus growth and differentiation. The source of callus influenced rates of growth and the occurrence of differentiation. Usually 'Norseman' calli grew faster and produced more roots than 'Altaswede' calli, while shoots were induced only from 'Altaswede' calli. Although 'Norseman' had more shoot tips induced to produce multiple shoots, the multiple shoot number per culture of 'Altaswede' was higher than that of 'Norseman'. Shoot tip cultures were also established to induce multiple shoots and to regenerate plants via root organogenesis. Regenerants from initial multiple shoots (RG1), multiple shoots after two subcultures (RG2), three-month calli (RG3) and one-year calli (RG4) were evaluated for chromosome number stability, morphology and several biochemical traits. When 'Altaswede' plants were analysed for chromosome number, RG1 and RG3 plants were normal, while one RG2 plant and 23% of 119 RG4 plants had tetraploid chromosome numbers. Regenerated plants were quite stable regarding their isozyme patterns of malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, phosphoglucomutase and shikimate dehydrogenase and their nodule leghaemoglobin profiles. Morphologically, the leaflet length to width ratio of RG1, RG2 and RG3 plants of 'Altaswede' showed significantly more variation than control plants (P≤0.01), while RG4 plants of 'Altaswede' and RG1 and RG2 plants of 'Norseman' were not different from control plants. It is suggested that the absence of detectable differences in the RG4 'Altaswede' plants was a consequence of their origin from one original genotype. Variability and stability of regenerated plants are discussed.

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