阅读说明：本技术 一种罗布麻无菌苗茎段高效诱导试管苗的培育方法 (Cultivation method for apocynum aseptic seedling stem section high-efficiency induction test-tube seedling ) 是由 梁美霞 张洪霞 宋志忠 李建召 刘晓华 贺奥 李权龙 岳红丽 孟凡雅 于 2021-08-17 设计创作，主要内容包括：本发明公开了一种罗布麻无菌苗茎段高效诱导试管苗的培育方法,包括罗布麻初始无菌苗的获得、罗布麻初始无菌苗的茎段进行愈伤组织和芽的诱导、愈伤组织上产生的新芽进行茎尖增殖培养获得大量的二代芽、二代芽同时进行下一轮的增殖培养和生根培养获得试管苗等步骤。本发明对于罗布麻的繁殖不受季节时间限制,一年四季均可进行,平均一代繁殖系数≥34,分化出大量的新芽可用于继代循环,实现了试管苗数目呈几何级数增长,有利于罗布麻优质种苗的大量快速繁殖,简化了罗布麻的育苗程序,大大节约了人力和生产成本,为罗布麻种苗工程和基因工程提供了强有利的技术支持和保障。(The invention discloses a cultivation method of apocynum aseptic seedling stem section high-efficiency induction test-tube plantlet, which comprises the steps of obtaining apocynum aseptic seedling, inducing callus and buds of the stem section of the apocynum aseptic seedling, performing stem tip enrichment culture on new buds generated on the callus to obtain a large number of second-generation buds, simultaneously performing the next round of enrichment culture and rooting culture on the second-generation buds to obtain the test-tube plantlet and the like. The method is not limited by season time for the apocynum venetum breeding, can be carried out all the year round, has the average generation breeding coefficient of more than or equal to 34, differentiates a large number of new buds for subculture circulation, realizes the increase of the number of test-tube seedlings in geometric progression, is favorable for the rapid breeding of a large number of apocynum venetum high-quality seedlings, simplifies the seedling raising procedure of the apocynum venetum, greatly saves labor and production cost, and provides strong and favorable technical support and guarantee for apocynum venetum seedling engineering and genetic engineering.)

1. A cultivation method of apocynum venetum aseptic seedling stem section high-efficiency induction test-tube seedlings is characterized by comprising the following steps: comprises the following steps:

(1) obtaining of initial aseptic seedling of apocynum venetum

Taking kendir stem segments with the stem segment size of 1.5-2 cm under field or greenhouse growth conditions, sterilizing with 70-75% ethanol on a superclean bench for 30-40 s, and then sterilizing with 0.1% HgCl₂Disinfecting for 7-10 min, washing with sterile water for 3-5 times, and finally inoculating the bacteria on a No. 1 culture medium under the culture conditions that: the illumination time is 16h/d, the illumination intensity is 1500-2000 lx, the temperature is 25 +/-1 ℃, and axillary buds of the stem segments sprout to form new buds after 20d of culture; shearing the stem tip of the germinated sprout, inoculating the stem tip on a No. 2 culture medium for continuous culture, and obtaining an initial aseptic seedling of the apocynum venetum after 20 days;

(2) inducing stem of initial aseptic seedling of kendir to obtain callus and bud

Cutting stem of the above herba Apocyni Veneti initial aseptic seedling with size of 1cm-1.5cm, inoculating on No. 3 culture medium under the culture conditions: the illumination time is 12h/d, the illumination intensity is 1500-2000 lx, the culture temperature is 25 +/-1 ℃, a small amount of red callus is generated at the incisions at the two ends of the stem segments after one week, a large amount of red callus is generated at the incisions at the two ends of the stem segments after continuous culture for 10d, and new buds are generated by induction from the callus;

(3) carrying out stem tip enrichment culture on the new buds generated on the callus to obtain a large number of second-generation buds;

shearing new buds induced to germinate on the callus, inoculating the new buds on a No. 4 enrichment medium for culturing, after culturing for 20 days, generating a small amount of callus at the base of the new buds, generating a large amount of second-generation buds with different heights from the base, wherein the height of the new buds is different from 1cm to 10cm, and each new bud can be proliferated to more than 34 on average, namely the proliferation coefficient is more than or equal to 34;

(4) the second generation bud is simultaneously subjected to the next round of enrichment culture and rooting culture to obtain the test-tube plantlet

Culturing the obtained large amount of second generation buds according to the height of the buds; for all the second generation buds with the height less than 2cm, cutting off the second generation buds respectively, inoculating the cut second generation buds to a No. 4 multiplication culture medium, and continuously culturing and multiplying the second generation buds for later use; directly cutting the second generation bud with height greater than or equal to 3cm to obtain stem of aseptic seedling with size of 1-1.5 cm, inoculating on No. 3 culture medium, and performing the next round of circular culture; the second generation bud with height of 2-3cm can be directly cut and placed on No. 5 culture medium for rooting culture, and cultured for 15-20 days to obtain lots of rooted test-tube plantlets.

2. The cultivation method of apocynum venetum aseptic seedling stem section high-efficiency induction test-tube plantlet as claimed in claim 1, which is characterized in that: the composition of the No. 1 culture medium is as follows: MS + 6-BA 0.6mg/L + NAA 0.1mg/L + sucrose 30g/L + agar 7.5g/L, pH value is 5.8; the composition of the No. 2 culture medium is as follows: MS + 6-BA 3mg/L + TDZ0.1mg/L + NAA 0.1mg/L + sucrose 30g/L + agar 7.5g/L, pH value is 5.8; the composition of the No. 3 culture medium is as follows: MS + 6-BA 3mg/L + IBA 0.4mg/L + TDZ0.6mg/L + sucrose 20g/L + agar 7.5g/L, and the pH value is 5.8; the No. 4 proliferation medium consists of: MS + 6-BA 2mg/L + IBA 0.2mg/L + TDZ0.4mg/L + sucrose 20g/L + agar 7.5g/L, pH value is 5.8; medium No. 5 consists of: 1/2MS + NAA0.5mg/L + sucrose 30g/L + agar 7.5g/L, pH 5.8.

Technical Field

The invention relates to a cultivation method of apocynum aseptic seedling stem section high-efficiency induction test-tube seedling.

Background

Apocynum venetum L is a precious plant resource in China and is named after being produced in Apocynum venetum L.

The apocynum venetum has high medicinal value and has the effects of improving immunity, soothing nerves, helping sleep, reducing blood pressure and blood fat, dispelling the effects of alcohol, protecting liver, relieving cough and asthma, softening blood vessels and the like. The apocynum venetum is called as the king of fiber, the fiber is flexible and slender, the apocynum venetum has the softness of cotton, the smoothness of the hemp and the luster of silk, and the textile has good prevention and treatment effects on skin diseases and gynecological diseases. Because apocynum venetum has important value in the aspects of medicine, textile and the like, people grab wild apocynum venetum under the drive of economic benefits, so that the germplasm resources of the wild apocynum venetum are increasingly deficient. Therefore, the artificial planting and domestication of apocynum venetum is an effective way for solving the shortage of wild resources. A large amount of high-quality apocynum venetum seedlings need to be obtained in a short time when apocynum venetum is artificially planted, and the traditional apocynum venetum seedling breeding method comprises seed breeding, root cutting and plant division breeding. The traditional propagation method has the defects of poor seedling quality, inconsistent seedlings, irregular seedlings, long period, low propagation coefficient, high cost and the like. In addition, in order to improve the effective components and the fiber yield of the apocynum venetum, the important economic characters of the apocynum venetum can be improved in a short time by the modern biotechnology, namely the gene editing technology.

Disclosure of Invention

The invention aims to provide a cultivation method for apocynum venetum aseptic seedling stem section high-efficiency induction test-tube plantlet, which can accelerate the propagation speed of high-quality apocynum venetum wild resources, improve the propagation coefficient and shorten the propagation period.

The technical solution of the invention is as follows:

a cultivation method of apocynum venetum aseptic seedling stem section high-efficiency induction test-tube seedlings is characterized by comprising the following steps: comprises the following steps:

(1) obtaining of initial aseptic seedling of apocynum venetum

Taking kendir stem segments with the stem segment size of 1.5-2 cm under field or greenhouse growth conditions, sterilizing with 70-75% ethanol on a superclean bench for 30-40 s, and then sterilizing with 0.1% HgCl₂Disinfecting for 7-10 min, washing with sterile water for 3-5 times, and finally inoculating the bacteria on a No. 1 culture medium under the culture conditions that: the illumination time is 16h/d, the illumination intensity is 1500-2000 lx, the temperature is 25 +/-1 ℃, and axillary buds of the stem segments sprout to form new buds after 20d of culture; shearing the stem tip of the germinated sprout, inoculating the stem tip on a No. 2 culture medium for continuous culture, and obtaining an initial aseptic seedling of the apocynum venetum after 20 days;

(2) inducing stem of initial aseptic seedling of kendir to obtain callus and bud

Cutting stem of the above herba Apocyni Veneti initial aseptic seedling with size of 1cm-1.5cm, inoculating on No. 3 culture medium under the culture conditions: the illumination time is 12h/d, the illumination intensity is 1500-2000 lx, the culture temperature is 25 +/-1 ℃, a small amount of red callus is generated at the incisions at the two ends of the stem segments after one week, a large amount of red callus is generated at the incisions at the two ends of the stem segments after continuous culture for 10d, and new buds are generated by induction from the callus;

(3) carrying out stem tip enrichment culture on the new buds generated on the callus to obtain a large number of second-generation buds;

shearing new buds induced to germinate on the callus, inoculating the new buds on a No. 4 enrichment medium for culturing, after culturing for 20 days, generating a small amount of callus at the base of the new buds, generating a large amount of second-generation buds with different heights from the base, wherein the height of the new buds is different from 1cm to 10cm, and each new bud can be proliferated to more than 34 on average, namely the proliferation coefficient is more than or equal to 34;

(4) the second generation bud is simultaneously subjected to the next round of enrichment culture and rooting culture to obtain the test-tube plantlet

Culturing the obtained large amount of second generation buds according to the height of the buds; for all the second generation buds with the height less than 2cm, cutting off the second generation buds respectively, inoculating the cut second generation buds to a No. 4 multiplication culture medium, and continuously culturing and multiplying the second generation buds for later use; directly cutting the second generation bud with height greater than or equal to 3cm to obtain stem of aseptic seedling with size of 1-1.5 cm, inoculating on No. 3 culture medium, and performing the next round of circular culture; the second generation bud with height of 2-3cm can be directly cut and placed on No. 5 culture medium for rooting culture, and cultured for 15-20 days to obtain lots of rooted test-tube plantlets.

The composition of the No. 1 culture medium is as follows: MS + 6-BA 0.6mg/L + NAA 0.1mg/L + sucrose 30g/L + agar 7.5g/L, pH value is 5.8; the composition of the No. 2 culture medium is as follows: MS + 6-BA 3mg/L + TDZ0.1mg/L + NAA 0.1mg/L + sucrose 30g/L + agar 7.5g/L, pH value is 5.8; the composition of the No. 3 culture medium is as follows: MS + 6-BA 3mg/L + IBA 0.4mg/L + TDZ0.6mg/L + sucrose 20g/L + agar 7.5g/L, and the pH value is 5.8; the No. 4 proliferation medium consists of: MS + 6-BA 2mg/L + IBA 0.2mg/L + TDZ0.4mg/L + sucrose 20g/L + agar 7.5g/L, pH value is 5.8; medium No. 5 consists of: 1/2MS + NAA0.5mg/L + sucrose 30g/L + agar 7.5g/L, pH 5.8.

The method comprises the steps of utilizing stem segments of apocynum aseptic seedlings to culture and induce in vitro to generate callus, regenerating new buds through the callus, then carrying out enrichment culture on stem tips of the new buds to generate a large number of new buds, carrying out enrichment, propagation, rooting and other treatments on the new buds with different heights, and finally obtaining the method for producing test-tube seedlings in batches. The method is not limited by season time for the apocynum venetum breeding, can be carried out all the year round, has the average generation breeding coefficient of more than or equal to 34, differentiates a large number of new buds for subculture circulation, realizes the increase of the number of test-tube seedlings in geometric progression, is favorable for the rapid breeding of a large number of apocynum venetum high-quality seedlings, simplifies the seedling raising procedure of the apocynum venetum, greatly saves labor and production cost, and provides strong and favorable technical support and guarantee for apocynum venetum seedling engineering and genetic engineering.

The invention overcomes all the defects of the traditional apocynum venetum propagation through seeds, plant division, root cutting and stem tip, such as inconsistent seedling quality, long propagation period, low propagation coefficient and the like. In addition, good technical support is provided for character improvement through agrobacterium-mediated apocynum venetum stem genetic transformation in the future. The cultivation process can be carried out all the year round, not only can obtain a large number of apocynum venetum excellent seedlings with consistent characters in a short time, but also can lay a foundation for obtaining a transgenic plant line with improved genetic characters by means of genetic engineering, and provides technical support for improving the medicinal components of the apocynum venetum and high-quality fiber plants by means of a gene editing technology.

Drawings

The invention is further illustrated by the following figures and examples.

FIG. 1 is a schematic representation of the germination of new shoots from field stems after 20 days of culture on medium.

FIG. 2 is a schematic representation of the initial sterile plantlets obtained after cutting the germinated shoots for 20 days of culture.

FIG. 3 is a schematic representation of the stem section of an initial sterile shoot after being cut and grown on a medium for 15-18 days.

FIG. 4 is a schematic diagram of the second generation shoots obtained after culturing the shoot tips cut from the callus for 20 days.

FIG. 5 is a schematic of a rooted test tube plantlet.

Detailed Description

A cultivation method of apocynum venetum aseptic seedling stem section high-efficiency induction test-tube seedlings is characterized by comprising the following steps: comprises the following steps:

(1) obtaining of initial aseptic seedling of apocynum venetum

Taking kendir stem segments with the stem segment size of 1.5-2 cm under field or greenhouse growth conditions, sterilizing with 70-75% ethanol on a superclean bench for 30-40 s, and then sterilizing with 0.1% HgCl₂Disinfecting for 7-10 min, washing with sterile water for 3-5 times, and finally inoculating the bacteria on a No. 1 culture medium under the culture conditions that: the illumination time is 16h/d, the illumination intensity is 1500-2000 lx, the temperature is 25 +/-1 ℃, and after 20d of culture, axillary buds of the stem segment sprout to form new buds (figure 1); shearing the stem tip of the germinated sprout, inoculating the stem tip on No. 2 culture medium for continuous culture, and obtaining an initial aseptic seedling of apocynum venetum (figure 2) after 20 days;

(2) inducing stem of initial aseptic seedling of kendir to obtain callus and bud

Cutting stem of the above herba Apocyni Veneti initial aseptic seedling with size of 1cm-1.5cm, inoculating on No. 3 culture medium under the culture conditions: the illumination time is 12h/d, the illumination intensity is 1500-2000 lx, the culture temperature is 25 +/-1 ℃, a small amount of red callus is generated at the incisions at the two ends of the stem segments after one week, a large amount of red callus is generated at the incisions at the two ends of the stem segments after continuous culture for 10d, and new buds are generated by induction from the callus (figure 3);

(3) carrying out stem tip enrichment culture on the new buds generated on the callus to obtain a large number of second-generation buds;

shearing new buds induced to germinate on the callus, inoculating the new buds on a No. 4 enrichment medium for culturing, after culturing for 20 days, generating a small amount of callus at the base of the new buds, generating a large amount of second generation buds with different heights from the base, wherein the height of the new buds is different from 1cm to 10cm, and each new bud can be proliferated to more than 34 on average (figure 4), namely the proliferation coefficient is more than or equal to 34;

(4) the second generation bud is simultaneously subjected to the next round of enrichment culture and rooting culture to obtain the test-tube plantlet

Culturing the obtained large amount of second generation buds according to the height of the buds; for all the second generation buds with the height less than 2cm, cutting off the second generation buds respectively, inoculating the cut second generation buds to a No. 4 multiplication culture medium, and continuously culturing and multiplying the second generation buds for later use; directly cutting the second generation bud with height greater than or equal to 3cm to obtain stem of aseptic seedling with size of 1-1.5 cm, inoculating on No. 3 culture medium, and performing the next round of circular culture; the second generation bud with height of 2-3cm can be directly cut and placed on No. 5 culture medium for rooting culture, and cultured for 15-20 days to obtain a large amount of rooted test-tube plantlets (figure 5).

The composition of the No. 1 culture medium is as follows: MS + 6-BA 0.6mg/L + NAA 0.1mg/L + sucrose 30g/L + agar 7.5g/L, pH value is 5.8; the composition of the No. 2 culture medium is as follows: MS + 6-BA 3mg/L + TDZ0.1mg/L + NAA 0.1mg/L + sucrose 30g/L + agar 7.5g/L, pH value is 5.8; the composition of the No. 3 culture medium is as follows: MS + 6-BA 3mg/L + IBA 0.4mg/L + TDZ0.6mg/L + sucrose 20g/L + agar 7.5g/L, and the pH value is 5.8; the No. 4 proliferation medium consists of: MS + 6-BA 2mg/L + IBA 0.2mg/L + TDZ0.4mg/L + sucrose 20g/L + agar 7.5g/L, pH value is 5.8; medium No. 5 consists of: 1/2MS + NAA0.5mg/L + sucrose 30g/L + agar 7.5g/L, pH 5.8.