阅读说明：本技术 一种姜荷花品种玉如意愈伤诱导及高效再生的方法 (Callus induction and efficient regeneration method for curcuma species of curcuma longa ) 是由 毛俐慧 邹清成 董青 史小华 胡伟 曹雪蕊 于 2021-08-23 设计创作，主要内容包括：本发明公开了一种姜荷花品种玉如意愈伤诱导及高效再生的方法,以姜荷花玉如意种球萌发出的芽点作为外植体,或者以姜荷花玉如意的嫩叶叶鞘作为外植体,消毒,暗培养后产生愈伤组织,两周后愈伤组织块接入新配的愈伤诱导培养基进行增殖,再过两周后转入愈伤再分化成芽培养基,再过两周后进行继代。愈伤诱导培养基为：MS+0.5mg/L TDZ+0.5mg/L BA+0.3mg/L 2,4-D,蔗糖30g/L,琼脂0.6g/L,遮光暗培养,温度为26±2℃；愈伤再分化成芽培养基为：MS+0.5mg/L TDZ+2.0mg/L BA+0.2mg/L NAA,蔗糖30g/L,琼脂0.6g/L,光照时间为16h每天,温度为26±2℃,光照强度1900-5000 LX；继代培养基为：MS,0.5mg/L BA,蔗糖30g/L,琼脂0.6g/L,光照时间为16h每天,温度为26±2℃。本发明通过愈伤诱导和分化的方法进行姜荷花“玉如意”的组培,繁殖效率大大提高。(The invention discloses a callus induction and efficient regeneration method of a curcuma lotus variety jade Ruyi, which takes bud points emitted by the sprouting of the curcuma lotus jade Ruyi as explants or young leaf sheaths of the curcuma lotus jade Ruyi as explants, disinfects and performs dark culture to generate callus, a callus block is inoculated into a newly-prepared callus induction culture medium for proliferation after two weeks, then is transferred into callus to be differentiated into a bud culture medium after two weeks, and then is subjected to subculture after two weeks. The callus induction culture medium is: MS +0.5mg/L TDZ +0.5mg/L BA +0.3 mg/L2, 4-D, sucrose 30g/L and agar 0.6g/L, and culturing in dark in shade at 26 +/-2 ℃; the callus re-differentiation into bud culture medium is as follows: MS +0.5mg/L TDZ +2.0mg/L BA +0.2mg/L NAA, 30g/L sucrose and 0.6g/L agar, wherein the illumination time is 16h per day, the temperature is 26 +/-2 ℃, and the illumination intensity is 1900-; the subculture medium is: MS,0.5mg/L BA, 30g/L sucrose and 0.6g/L agar, the illumination time is 16h per day, and the temperature is 26 +/-2 ℃. The invention carries out the tissue culture of the curcuma alismatifolia 'Jaruyi' by a callus induction and differentiation method, and greatly improves the propagation efficiency.)

1. A callus induction and high-efficiency regeneration method of a curcuma lotus variety Jade Ruyi is characterized in that,

taking bud points emitted by the sprouting of the seedballs of the curcuma, the lotus and the jade as explants, disinfecting with 70% alcohol for 30s, disinfecting with 0.1% mercury bichloride for 10 minutes, washing with distilled water for 4 times, carrying out dark culture for one week to generate callus, inoculating callus blocks into a newly-prepared callus induction culture medium for proliferation after two weeks, transferring the callus into a callus culture medium again after two weeks to differentiate into a bud culture medium, and carrying out subculture after two weeks;

the callus induction culture medium is: MS +0.5mg/L TDZ +0.5mg/L BA +0.3 mg/L2, 4-D, sucrose 30g/L and agar 0.6g/L, and culturing in dark in shade at 26 +/-2 ℃;

the callus re-differentiation into bud culture medium is as follows: MS +0.5mg/L TDZ +2.0mg/L BA +0.2mg/L NAA, 30g/L sucrose and 0.6g/L agar, wherein the illumination time is 16h per day, the temperature is 26 +/-2 ℃, and the illumination intensity is 1900-;

the subculture medium is: MS,0.5mg/L BA, 30g/L sucrose and 0.6g/L agar, the illumination time is 16h per day, and the temperature is 26 +/-2 ℃.

2. A callus induction and high-efficiency regeneration method of a curcuma lotus variety Jade Ruyi is characterized in that,

taking the young leaf sheath of the Jade Ruyi of the Hedychium coronarium as an explant, disinfecting for 30s by 70% alcohol, disinfecting for 6 minutes by 0.1% mercury bichloride, and cleaning for 4 times by sterile water; inoculating the explant to a callus induction culture medium and hormone combination 2.0 mg/L2, 4-D +0.5mg/L BA, sucrose 30g/L and agar 6.5g/L, culturing at the temperature of 27 +/-2 ℃, performing dark culture, and inducing callus after two weeks;

after two weeks, the callus blocks are inoculated into a newly-prepared callus induction culture medium for proliferation, then transferred into callus after two weeks and then differentiated into a bud culture medium, and then subcultured after two weeks;

the callus induction culture medium is: MS +0.5mg/L TDZ +0.5mg/L BA +0.3 mg/L2, 4-D, sucrose 30g/L and agar 0.6g/L, and culturing in dark in shade at 26 +/-2 ℃;

the callus re-differentiation into bud culture medium is as follows: MS +0.5mg/L TDZ +2.0mg/L BA +0.2mg/L NAA, 30g/L sucrose and 0.6g/L agar, wherein the illumination time is 16h per day, the temperature is 26 +/-2 ℃, and the illumination intensity is 1900-;

the subculture medium is: MS,0.5mg/L BA, 30g/L sucrose and 0.6g/L agar, the illumination time is 16h per day, and the temperature is 26 +/-2 ℃.

Technical Field

The invention relates to a callus induction and efficient regeneration method for a curcuma species of curcuma longa.

Background

Jianghuahua 'yuruyi' (ginger and lotus flowerCurcumahybrid 'Laddawan') which is a hybrid of Curcuma aromatica and Hedychium spicatum ((Laddawan)Curcuma alismatifolia x Curcuma cordata). The plant shape and flower type of the method are greatly different from the traditional variety of the curcuma alismatifolia, the jade Ruyi columnar inflorescence has long flower period and high plant height, the leaves are wide and can be used as a leaf-viewing ground cover, and the flower color is unique light pink.

The jade Ruyi has high ornamental value, but has the problems of high price of the seed ball and difficult large-area popularization. The tissue culture efficient propagation technology is necessary for large-area popularization and application, but the tissue culture method of ruyi is not researched at present.

At present, the prior art application on the tissue culture of the curcuma alismatifolia is available, but the Yuruyi is a hybrid of the curcuma alismatifolia and the curcuma longa, has great difference with the traditional curcuma alismatifolia, and the prior art mainly directly carries out bud induction without callus induction.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a callus induction and high-efficiency regeneration method for the variety Jade Ruyi of the curcuma alismatifolia.

A callus induction and high-efficiency regeneration method of a curcuma lotus variety Jade Ruyi,

taking a bud point emitted by the sprouting of a seedball of the curcuma alismatifolia as an explant, disinfecting with 70% alcohol for 30s, disinfecting with 0.1% mercury bichloride for 10 minutes, washing with distilled water for 4 times, carrying out dark culture for one week to generate callus, inoculating a callus block into a newly-prepared callus induction culture medium for proliferation after two weeks, transferring the callus block into a callus re-differentiation culture medium after two weeks (namely 4 weeks), and carrying out subculture after two weeks (namely 6 weeks);

the callus induction culture medium is: MS +0.5mg/L TDZ +0.5mg/L BA +0.3 mg/L2, 4-D, sucrose 30g/L and agar 0.6g/L, and culturing in dark in the dark at the temperature of 26 +/-2 ℃.

The callus re-differentiation into bud culture medium is as follows: MS +0.5mg/L TDZ +2.0mg/L BA +0.2mg/L NAA, 30g/L sucrose and 0.6g/L agar, wherein the illumination time is 16h per day, the temperature is 26 +/-2 ℃, and the illumination intensity is 1900-;

the subculture medium is: MS,0.5mg/L BA, 30g/L sucrose and 0.6g/L agar, the illumination time is 16h per day, and the temperature is 26 +/-2 ℃.

In addition, a callus induction and efficient regeneration method of the curcuma lotus variety jade Ruyi takes the tender leaf sheath of the curcuma lotus jade Ruyi as an explant, is sterilized by 70 percent alcohol for 30s, is sterilized by 0.1 percent mercuric chloride for 6 minutes, and is cleaned by sterile water for 4 times; inoculating the explant to a callus induction MS culture medium and hormone combination 2.0 mg/L2, 4-D +0.5mg/L BA, sucrose 30g/L and agar 6.5g/L, culturing at the temperature of 27 +/-2 ℃, performing dark culture, and inducing the callus after two weeks;

after two weeks, the callus blocks are inoculated into a newly-prepared callus induction culture medium for proliferation, then are transferred into callus after two weeks (namely 4 weeks) and are differentiated into a bud culture medium, and then are subcultured after two weeks (namely 6 weeks);

the callus induction culture medium is: MS +0.5mg/L TDZ +0.5mg/L BA +0.3 mg/L2, 4-D, sucrose 30g/L and agar 0.6g/L, and culturing in dark in the dark at the temperature of 26 +/-2 ℃.

The callus re-differentiation into bud culture medium is as follows: MS +0.5mg/L TDZ +2.0mg/L BA +0.2mg/L NAA, 30g/L sucrose and 0.6g/L agar, wherein the illumination time is 16h per day, the temperature is 26 +/-2 ℃, and the illumination intensity is 1900-;

the subculture medium is: MS,0.5mg/L BA, 30g/L sucrose and 0.6g/L agar, the illumination time is 16h per day, and the temperature is 26 +/-2 ℃.

The invention has the beneficial effects that:

the invention carries out the tissue culture of the curcuma alismatifolia 'Jaruyi' by a callus induction and differentiation method, and greatly improves the propagation efficiency. In addition, the plant callus has multiple functions, namely, the callus is a new material for germplasm preservation, the callus is an ideal material for plant genetic improvement and genetic engineering, and the callus is often used as a material to obtain higher success rate when colchicine double breeding, chemical mutation breeding and transgenic molecule breeding are carried out.

Drawings

Figure 1 is a 'Yuruyi' field photograph of Hemsleya amabilis.

FIG. 2 shows callus of Hedychium coronarium.

FIG. 3 is leaf sheath explant induced callus.

FIG. 4 shows callus differentiation of Hedychium coronarium.

FIG. 5 is a newly differentiated shoot growing healthily after 8 weeks of subculture.

FIG. 6 is hardening off of tissue culture seedlings in a greenhouse.

FIG. 7 is a plant that is growing healthily outdoors and the underground seedball has begun to expand.

Detailed Description

The invention is further illustrated below with reference to the figures and examples.

The invention carries out the tissue culture of the 'Yuruyi' variety of the curcuma alismatifolia through callus induction, thereby greatly improving the propagation efficiency. A 'Yuruyi' field photograph of Hemsleya amabilis is shown in figure 1.

Example 1

The method takes the bud point emitted by the seed ball of the curcuma alismatifolia as an explant, carries out callus induction by dark culture, disinfects for 30s by 70 percent alcohol and then disinfects for 10 minutes by 0.1 percent mercury, cleans for 4 times by distilled water, generates callus after dark culture for one week, and carries out subculture proliferation after two weeks. The result shows that the callus can be efficiently induced by proper hormone proportion. The callus of Hemerocallis zingiberensis Wolff is shown in figure 2.

The callus induction culture medium is: 0.5mg/L TDZ +0.5mg/L BA +0.3 mg/L2, 4-D, 30g/L sucrose and 0.6g/L agar, wherein the illumination condition is light-shading dark culture, the temperature is 26 +/-2 ℃, and the illumination intensity is 1900-. The callus subculture multiplication medium was the same as above.

The callus re-differentiation into bud culture medium is as follows: 0.5mg/L TDZ +2.0mg/L BA +0.2mg/L NAA, 30g/L sucrose and 0.6g/L agar, the illumination time is 16h per day, the temperature is 26 +/-DEG, and the light quality is 1900-5000 LX. The callus differentiation of Hemerocallis zingiberensis is shown in figure 4.

Selecting a differentiation bud subculture medium: 0.5mg/L BA, 30g/L sucrose, 0.6g/L agar, 16h illumination time per day, and 26 +/-2 ℃ temperature. The seedlings after subculture are shown in FIG. 5.

After 6 weeks, the cells proliferated and reached 312-fold differentiation.

Example 2

In addition, the leaf sheath explant can also induce callus, according to the research progress of the tissue culture and rapid propagation technology of the alpinia oxyphylla and the like (alpinia oxyphylla, which is lovely, forest golden water, and the like, Fujian hot work technology, 2019, 44(4): 53-56.), the ginger lotus tissue culture explant uses seedball and bud at present, and has high pollution rate. The explants are inoculated to a callus induction MS culture medium, three controls are pollution-free, 2.0 mg/L2, 4-D +0.5mg/L BA, 30g/L sucrose and 6.5g/L agar are selected as hormone combinations, the culture temperature is 27 +/-2 ℃, dark culture is carried out, and the callus can be induced after two weeks (the arrow position is shown in figure 3). After 2 weeks, callus proliferation was still performed using 2.0 mg/L2, 4-D +0.5mg/L BA medium. The leaf sheath-induced callus is shown in FIG. 3.

The callus re-differentiation into bud culture medium is as follows: 0.5mg/L TDZ +2.0mg/L BA +0.2mg/L NAA, 30g/L sucrose and 0.6g/L agar, the illumination time is 16h per day, the temperature is 26 +/-DEG, and the light quality is 1900-5000 LX.

Selecting a subculture medium: 0.5mg/L BA, 30g/L sucrose and 0.6g/L agar, the illumination time is 16h per day, and the temperature is 26 +/-DEG.C.

After 6 weeks, the cells proliferated and reached 81-fold differentiation.

The survival rate of the tissue culture seedlings after hardening is more than 95 percent, the blade tips have slight coke edge when the plant height is less than 10cm, the coke edge phenomenon does not exist after the plant height reaches 10cm, and the growth vigor is good. The hardening off of the tissue culture seedlings is shown in FIG. 6. After hardening seedlings in the greenhouse for one month, the seedlings are placed outdoors and grow on a shading net, and after about 1 month, the seed balls begin to expand to the diameter of about 0.8cm (figure 7). The enlargement of the seed ball of the tissue culture seedling after the outdoor growth is shown in figure 7.