Method for increasing efficiency of tissue culture of Medicago sativa L.
A technique of tissue culture and alfalfa, applied in the field of plant biology, can solve problems such as insufficient callus formation and differentiation, long induction time of alfalfa tissue culture, explant contamination, etc., to retain differentiation ability and increase callus induction rate , The effect of low seed pollution rate
Inactive Publication Date: 2013-08-28
GRASSLAND RES INST OF CHINESE ACAD OF AGRI SCI
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Problems solved by technology
[0013] At present, in the tissue culture of alfalfa, there are mainly two technical problems: serious contamination of explants, insufficient callus formation and differentiation, which lead to long induction time, low efficiency, poor quality and serious browning of alfalfa tissue culture.
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[0036] 1. Seed sterilization and acquisition of sterile seedlings
[0037] Select seeds with full grains, soak them in 75% alcohol for 1 min, soak in 1% mercury for 5 minutes, then soak in 30% hydrogen peroxide for 30 minutes, and then rinse with distilled water for 3-4 times. Put the washed seeds on the filter paper to absorb the water. , Inoculate in solid medium (pH=6.0). Medium formula: MS + agar (7.5g / L) + sucrose (30g / L). Cultivation conditions, 25°C, light intensity 2500lk, divided into light and dark cycles of 16 / 8h, culture for 5-7 days.
[0038] By adopting the combined disinfection method of mercury and hydrogen peroxide, the goal of thorough disinfection effect, low pollution rate and minimal damage to seeds is achieved, and the current serious problem of explant pollution is finally solved.
[0039] 2. Induction of callus
[0040] Select robust seedlings and cut out hypocotyls (the part above the root and below the stem). Cut it into 1cm pieces and place them on callus...
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The invention belongs to the technical field of plant biology, in particular relates to a method of tissue culture of Medicago sativa L. By way of developing a novel tissue culture technical method, the invention focuses on improvement and innovation of two technical nodes (seed disinfection and callus formation and differentiation) in culture steps. With adoption of a mercury bichloride and hydrogen peroxide compound disinfection method, the invention realizes the purposes of thorough disinfection effect and low pollution rate and minimum damage to seeds. Finally, the invention solves the problem of severe pollution to current explants. Meanwhile, a method of TDZ and 6-BA is adopted, thereby, the callus induction rate and the formation rate are remarkably increased, the callus quality is improved, the differentiation capacity of the callus is reserved to a maximum extent, the formation of embryoid sprouts is promoted so that the novel technical method of the tissue culture of the Medicago sativa L. which is efficient, saves time, and has low toxicity and low browning, is obtained.
Description
Technical field [0001] The invention belongs to the field of plant biotechnology, and specifically relates to a method for tissue culture of alfalfa (Medicago sativa L.). The present invention develops a new tissue culture technology method, focusing on the improvement and innovation of the two major technical nodes of seed disinfection and callus formation and differentiation in the culture step, thereby obtaining high-efficiency, low-toxicity, and low-browning alfalfa tissue culture new Technical method. Background technique [0002] Alfalfa (Medicago sativa L.) is an important legume forage. It is known as the "king of forage" because of its high nutritional value and good palatability. Tissue culture of alfalfa mainly refers to a technical method of plant regeneration using alfalfa isolated organs (cotyledons, radicles, etc.) through artificial cultivation. This method is of great significance to the propagation and preservation of alfalfa precious genetic material and the ...
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IPC IPC(8): A01H4/00
Inventor 任卫波郭慧琴李晶王茅雁刘雅学张文静赵海霞解继红
Owner GRASSLAND RES INST OF CHINESE ACAD OF AGRI SCI