| Alfalfa ( Medicago sativa L. ) is the most important fodder and water and soil conservation plant. Anther culture of alfalfa, to generate haploid plants from pollen microspores, by doubling chromosomes in haploid plants, it is possible to obtain completely homozygous lines in a short time, thereby providing a method for speeding up and increasing the selection efficiency.Anther culture of alfalfa was experimented with microspores were in the middle development stage and /or in the side development stage of single nucleus, to improve the anther culture efficiency, The main results were as following:(1) Flower buds were rinsed with water for 30 minutes and dipped in 70% ethanol for 2 seconds and then dipped in 0.1% hypochlorite for 4 minutes and subsequently leached five times with sterilized water, was a good sterilization method for plant materials and callus induction rate is higher.(2) The callus induction rate increased with 4℃cold pretreatment 3 days or 15℃low temperature culture for 3 days.(3) The solid-liquid-double-layer floating culture was more suitable for alfalfa anther culture in comparison with the solid culture and liquid floating culture. The suitable plating density is that a total of 45 anthers were placed onto the solid-liquid-double-layer medium in each 50 mL culture bottle, and the callus induction rate is higher. The optimum basic medium for callus induction of anther culture in alfalfa was modified Nitsch 2. Callus induction rate is higher on the medium supplemented with 2,4-D 2.0 mg/L and 6-BA 0.5 mg/L and maltose 6%~9%.(4) The best medium for green plantlet regeneration was MS containing NAA 0.5 mg/L and KT 2mg/L and sucrose 3% or sucrose 1.5% + maltole 1.5%.(5) Supplemented with casein hydrolysate 700 mg/L or glutamine 700 mg/L or proline 600 mg/L on medium was benefit for the callus induction and green plantlet differentiation.(6) The medium for rooting is 1/2MS containing sucrose 1%. The efficient regeneration system of anther culture of alfalfa was established.
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