CN-122004133-A - Method for shortening tissue culture time of fructus amomi

Abstract

The invention discloses a method for shortening the tissue culture time of amomum villosum, belonging to the technical field of plant tissue culture. The natural leaching solution is prepared by collecting fructus amomi leaves, pretreating, leaching, filtering and sterilizing, and is added into MS culture medium for explant induction. Experiments show that the leaching solution with the concentration of 75g/L can shorten the induction time of the explant from 4-6 weeks to 19 days, the induction efficiency reaches 54.76 percent, the adventitious bud robustness rate is high, the risk of exogenous hormone residue is avoided, and the mass production efficiency of the fructus amomi tissue culture seedlings is obviously improved.

Inventors

• LI DAN
• QI LINYAN
• SUN SHENGJIE
• WU XIAODONG
• CHEN PEIZHEN
• SHI RUI
• HE XIAHONG

Assignees

• 西南林业大学

Dates

Publication Date

20260512

Application Date

20260325

Claims (4)

1. 1. A method for shortening the tissue culture time of fructus amomi, which is characterized by comprising the following steps: (1) Preparing fructus Amomi leaf extract, namely collecting fructus Amomi leaf, washing with sterile water, drying in the shade, shearing, immersing in sterile water according to a feed liquid ratio of 25-100g/L, leaching at 25-30deg.C, filtering with 4 layers of sterile gauze and 0.22 μm microporous filter membrane, and sterilizing to obtain extract; (2) The explant treatment, namely selecting creeping stems of fructus amomi as the explant, cleaning and sterilizing, inoculating the explant to an MS culture medium containing leaching solution, and culturing under the culture conditions of 25+/-2 ℃ and 2500lx illumination and 12h/d photoperiod until the explant forms callus or adventitious buds.
2. 2. The method according to claim 1, wherein in the preparation of the leaching solution, the blades are sheared into small segments of 0.5-1cm, the leaching time is 24 hours, and the stirring is carried out every 8 hours.
3. 3. The method of claim 1, wherein the step of sterilizing the explant comprises soaking the explant in 75% ethanol for 10 seconds, washing the explant with sterile water 3 times, sterilizing the explant with 0.1% mercuric chloride for 8 minutes, and washing the explant with sterile water 5-6 times.
4. 4. The method of claim 1, wherein in step (2), the MS medium is added to the leach liquor in an amount of 0.5mg/L.

Description

Method for shortening tissue culture time of fructus amomi Technical Field The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for shortening the tissue culture time of fructus amomi. Background Fructus Amomi (Amomum villosum Lour.) is a perennial herb of Amomum genus of Zingiberaceae family, and its dried fruit is a rare Chinese medicinal material, and has edible, medicinal and economic values, and market demand is vigorous. The tissue culture technology is a key means for large-scale seedling raising and fine variety breeding of fructus amomi, but the existing tissue culture process of fructus amomi has the prominent problem that the induction period of an explant is long, the traditional method depends on exogenous hormone (such as 6-BA and NAA) for regulation and control, the induction time from the inoculation of the explant to the formation of callus or adventitious buds usually needs 4-6 weeks, the induction efficiency is low, and the large-scale production process of the tissue culture seedling of fructus amomi is severely restricted. Meanwhile, researches show that the fructus amomi leaves contain rich natural active ingredients (such as volatile oil, flavonoids, phenolic compounds and the like), and the ingredients possibly participate in the growth and development regulation of the fructus amomi and have potential tissue differentiation promoting activity. However, the prior art focuses on optimizing the ratio of exogenous hormone, and there is no report that natural active liquid is obtained by simple leaching by using self-leaf of fructus amomi as raw material and is applied to self-tissue culture to shorten the induction time. The traditional method has the risk of exogenous hormone residue, and has complex hormone proportion adjustment, high cost and difficulty in meeting the requirement of high-efficiency environment-friendly large-scale seedling culture. Disclosure of Invention The invention aims to provide a natural, efficient and low-cost process, which utilizes fructus amomi leaves to prepare leaching solution, is applied to tissue culture of fructus amomi, and realizes shortening of induction time by regulating and controlling differentiation of explants through natural active ingredients. A method for shortening the tissue culture time of fructus amomi, comprising the following steps: (1) Preparing fructus Amomi leaf extract, namely collecting fructus Amomi leaf, washing with sterile water, drying in the shade, shearing, immersing in sterile water according to a feed liquid ratio of 25-100g/L, leaching at 25-30deg.C, filtering with 4 layers of sterile gauze and 0.22 μm microporous filter membrane, and sterilizing to obtain extract; (2) The explant treatment, namely selecting creeping stems of fructus amomi as the explant, cleaning and sterilizing, inoculating the explant to an MS culture medium containing leaching solution, and culturing under the culture conditions of 25+/-2 ℃ and 2500lx illumination and 12h/d photoperiod until the explant forms callus or adventitious buds. Preferably, in the preparation of the leaching solution, blades are sheared into small sections of 0.5-1cm, the leaching time is 24 hours, and the leaching solution is stirred every 8 hours. Preferably, the step of sterilizing the explant comprises the steps of soaking the explant in 75% ethanol for 10 seconds, washing the explant with sterile water for 3 times, sterilizing the explant with 0.1% mercuric chloride for 8 minutes, and washing the explant with sterile water for 5-6 times. Preferably, in step (2), the MS medium is added to the leaching solution in an amount of 0.5mg/L. The invention achieves the technical effects that: 1. The invention utilizes natural growth active substances (such as flavonoid, plant sterol, amino acid and the like) contained in the villous amomum leaves, directly acts on the explant in a leaching solution form, shortens the induction time of the villous amomum explant from 4-6 weeks to 2-3 weeks, improves the induction efficiency to about 54.76%, and greatly accelerates the propagation process. 2. The induced survival rate is improved, the risk of deformity is reduced, the leaf leaching solution is a natural source component, the physiological compatibility with the amomum villosum explant is extremely strong, the problems of browning, deformity and the like caused by improper chemical hormone ratio are avoided, and the adventitious bud growth vigor is strong. 3. The preparation process of the leaching solution is simple, the raw materials are the self-leaves of fructus amomi, the source is convenient and fast, the cost is low, the precise proportioning of complex chemical reagents is not needed, the preparation cost and the operation difficulty of the culture medium are obviously reduced, and the leaching solution is suitable for large-scale popularization and application. 4. The method maintains the excellent shape of the germp