CN110881407A

CN110881407A - Construction method of marigold regeneration system

Info

Publication number: CN110881407A
Application number: CN201911091562.0A
Authority: CN
Inventors: 何燕红; 王亚琴; 韦陆丹; 王文静; 张春玲; 刘宝峻; 包满珠
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-11-10
Filing date: 2019-11-10
Publication date: 2020-03-17

Abstract

The invention belongs to the technical field of isolated culture of plants, and particularly relates to a construction method of a marigold regeneration system. The method comprises the following steps: seed disinfection and explant culture, adventitious bud induction, elongation and rooting culture, seedling hardening and transplanting. The tissue culture and rapid propagation of marigold by the method have the advantages that the regeneration rate reaches over 80 percent, the elongation rate reaches 91 percent, the rooting rate is 100 percent, and the vitrification rate is reduced to 10 percent. The invention establishes a high-efficiency and stable regeneration system of marigold, solves the serious vitrification problem in the regeneration process of marigold, and lays a foundation for genetic improvement and functional gene development of marigold.

Description

Construction method of marigold regeneration system

Technical Field

The invention belongs to the technical field of isolated culture of plants, and particularly relates to a construction method of a marigold regeneration system.

Background

Tagetes erecta L is an annual herb of Tagetes in Compositae, and has bright color, long flowering period and good ornamental performance. Marigold is rich in lutein, and can be used as ornamental flowers and functional flowers. At present, the most main breeding method of marigold is still the traditional breeding method such as cross breeding and the like. The method has long period, great contingency, obvious limitation by seasons, great consumption of manpower and material resources, and great limitation on variety improvement. Modern plant genetic engineering can make up for the defects of the traditional method, and a large number of plants with target characters can be obtained in a short time. The genetic improvement of the plant characters by utilizing the transgenic technology can accelerate the cultivation of new varieties.

The precondition for obtaining transgenic plants is to establish a high-efficiency and stable regeneration system. At present, the establishment of marigold regeneration systems is difficult, the regeneration is influenced by factors such as genotype, explant type, exogenous hormone, vitrification and light quality, and the establishment of efficient regeneration systems is very difficult, one reason is that different genotypes of marigold are suitable for the regeneration systems, and no exact rule can be followed. The explant types suitable for marigold tissue culture comprise leaves, leaf shafts, hypocotyls, stem sections and cotyledons, the leaves are taken as the explants mostly, but the regeneration efficiency is obviously influenced by the positions and the states of the leaves, and no relevant report is found at present. The hormone combination is prepared by using high-concentration 6-BA as a core hormone, and properly adding other hormones such as IAA, NAA, GA3 or 2, 4-D, etc., and the high-concentration 6-BA can inhibit the genetic transformation efficiency of marigold, so that the hormone type capable of replacing 6-BA is required to be searched. In addition, the vitrification phenomenon of the adventitious bud in the regeneration process of the marigold is serious, and other than silver nitrate, no other better method for improving the vitrification problem of the adventitious bud of the marigold exists at present.

In addition, the influence of light quality on marigold regeneration has not been reported.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, provides a construction method suitable for a marigold regeneration system, and provides conditions for genetic transformation and gene function identification of marigold. The invention utilizes marigold small leaves as explants, establishes a marigold plant regeneration system by improving the concentration of hormone combination, firming agent and sucrose and improving the culture conditions, reduces the vitrification rate and improves the marigold plant regeneration system compared with the prior method

The regeneration rate.

The purpose of the invention is realized by the following technical scheme:

the construction method of the marigold regeneration system comprises the following steps:

1) seed disinfection: selecting full mature seeds of marigold, soaking the seeds in sterile water for 10min, washing the seeds for 90s by using 0.1% mercuric chloride solution, and finally washing the seeds for 10min for three times by using sterile water to obtain sterilized seeds;

2) culturing the explants: inoculating the sterilized seeds in the step 1) on an MS solid culture medium for germination, wherein the temperature of the seed culture condition is 25 ℃, the illumination intensity is 800-;

3) induction of adventitious buds: cutting 2-3 pairs of compound leaf small leaves at the top of the sterile seedling obtained in the step 2), inoculating the sterile seedling to an induction culture medium, enabling the cut to be in contact with the culture medium during inoculation, and culturing in a culture chamber to obtain regenerated adventitious buds;

4) and (3) elongation and rooting culture: cutting the adventitious bud regenerated in the step 3), inoculating to an elongation and rooting culture medium for culture, carrying out subculture once after 15 days, and starting rooting after 30 days to obtain a regenerated seedling;

5) hardening and transplanting seedlings: placing the regenerated plantlet into a plant tissue culture chamber for 1d by using a half-opened sealing film when the root system of the regenerated plantlet grows to 3-5cm, then removing the sealing film and placing for 1d, taking out the plantlet, washing a culture medium attached to the root by using tap water, and then transplanting the plantlet onto a substrate (the volume ratio of the substrate is perlite to turf is 1: 2);

wherein:

the formula of the adventitious bud induction culture medium in the step 3) is as follows: MS; adding 0.2mg/L Thidiazuron (TDZ); 0.5mg/L indolebutyric acid (IBA); 8g/L agar; 40g/L sucrose; adjusting the pH of the culture medium to 5.8-6.0;

the culture conditions in the step 3) are as follows: the temperature is 25 ℃, the illumination time is 14h/d, and the light source proportion is 4500K warm white LED lamp: a 6000K white fluorescent lamp with a light ratio of 1: 1;

the elongation and rooting culture medium in the step 4) is MS; adding 8g/L agar; 40g/L sucrose; adjusting the pH of the culture medium to 5.8-6.0. The culture conditions are as follows: the temperature is 25 ℃, the illumination time is 14h/d, and the light source is a 4500K warm white LED lamp.

Compared with the prior art, the invention has the following advantages:

the invention has higher inductivity and elongation rate for marigold, obviously reduces vitrification rate, can realize the establishment of a high-efficiency regeneration system of marigold, and provides technical support for the genetic transformation of marigold.

Detailed Description

The invention is further described below with reference to specific embodiments:

example 1, construction of marigold regeneration System:

1. screening of marigold high-efficiency regeneration variety

Material for test 1: the following varieties were obtained from commercial marigold varieties (lines): self-luxury orange yellow, self-luxury golden, cantaloupe orange color, free goddess yellow, miracle yellow, chrysanthemum marigold orange color, discovery yellow, golden shield yellow, graceful golden yellow, abundance orange color, milestone yellow, vanilla, Hawaii, Xinfeng.yellow, found.orange, cut flower type, heavy petal lemon, Guifu pigment, atlas. gold, Indian. orange, fast thunder.yellow, rich.yellow, old Indian. orange, excellent.yellow or perfect.golden yellow, etc. in total 25 different genotype marigold varieties (strains, the invention is not limited to the marigold varieties or strains exemplified above), the marigold seeds sterilized by the conventional method are inoculated on a germination culture medium (MS; 7g/L agar is added; 30g/L sucrose; pH of the culture medium is adjusted to 5.8-6.0) for germination; culturing for 40-45 days to obtain sterile seedling. Taking 2-3 pairs of compound leaf small leaves at the top of the sterile seedling as explants, inoculating the explants to MS minimal medium with the front side of the explants upward, and adding 5.0 mg/L6-benzylaminopurine (6-BA); 3.0mg/L indoleacetic acid (IAA); 3g/L plant gel; on 30g/L sucrose medium, each group was replicated in 5 dishes, each dish being inoculated with approximately 25 explants. The culture temperature of the culture chamber is 25 ℃, the illumination intensity is 800-.

After 4 weeks, the regeneration rate of marigold is counted, and the analysis of variance shows that (P is less than 0.01), the regeneration efficiency of different gene type materials of marigold is very different, and the regeneration rate of partial gene type materials of marigold is zero. The variety with the highest regeneration efficiency of the marigold leaf is the marigold variety milestone yellow, the regeneration rate of the variety reaches 55.88%, and the repeatability is good, so the milestone yellow is selected as the high-efficiency regeneration variety material of the marigold in subsequent tests.

(II) Disinfection of selected varieties of seeds

Selecting plump mature seeds of marigold (variety milestone. yellow), soaking in sterile water for 10min, washing with 0.1% mercuric chloride solution for 90s, and washing with sterile water for 10min for three times.

(III) explant culture

Inoculating sterilized marigold seed on MS solid culture medium (MS; adding 7g/L agar; 30g/L sucrose; adjusting pH of culture medium to 5.8-6.0) for germinating for 40-45 d. The culture temperature is 25 ℃, the illumination intensity is 800-.

(IV) Induction culture of regenerated adventitious bud

Test 2: 2-3 pairs of compound leaf small leaves at the top of sterile seedlings of marigold (the variety is milestone yellow) are cut, the front surface of the small leaves faces upwards, and the small leaves are inoculated on 6 culture media: wherein the concentration of 6-BA in the additional components of the culture medium is fixed at 3.0mg/L, and the IAA concentration is set at 1.0, 1.5, 1.8, 2.0, 2.5 and 3.0mg/L respectively. MS, adding 3g/L of plant gel and 30g/L of cane sugar, and the pH value is 5.8-6.0. Each group was replicated in 4 dishes, each dish was inoculated with 10 explants. The culture temperature is 25 ℃, the illumination intensity is 800-; the light source is a 6000K white fluorescent lamp.

After 4 weeks, the regeneration rate of marigold in each test was counted. As can be seen from Table 1, the addition of 6-BA and IAA combinations with different concentrations has significant influence on the regeneration of the milestone and yellow leaf blades, wherein 3mg/L of 6-BA +3mg/L of IAA is the optimal hormone combination for the regeneration of marigold, the regeneration rate is obviously improved compared with that of the step (I), and the regeneration rate reaches 65 percent. But the regenerated shoots were smaller and vitrified severely. The specific test results are shown in table 1.

TABLE 1 Effect of the addition of 6-BA and IAA concentration combinations on Tagetes Tagetis leaf regeneration

Example 2

2-3 pairs of multi-leaf small leaves at the top of sterile seedlings of marigold (the variety is milestone yellow) are cut and inoculated on 8 test culture media in a positive upward manner to evaluate the effect of the culture media: the specific formula is as follows: let thidiazuron TDZ be 0.1, 0.2, 0.3, 0.4mg/L respectively, and indolebutyric acid (IBA) be 0.25, 0.5mg/L respectively. Each group was replicated with 4 dishes of 10 explants each. 3g/L of plant gel and 30g/L of cane sugar are added into the test culture medium, and the pH value is 5.8-6.0. The culture temperature is 25 ℃, the illumination intensity is 800-; the light source is a 6000K white fluorescent lamp.

Regeneration rate was counted after 4 weeks of in vitro culture. As can be seen from Table 2, the combination of TDZ and IBA at different concentrations had a significant effect on the milestone yellow leaf regeneration. The regeneration rate of the milestone and yellow color in the treatment of 0.2mg/L TDZ and 0.5mg/L IBA reaches 62.5 percent, and compared with the treatment of 3mg/L6-BA and 3mg/L IAA, the adventitious bud is stronger.

TABLE 2 Effect of TDZ and IBA concentration combinations on Tagetes leaf regeneration

Example 3

2-3 pairs of compound leaf small leaves at the top of sterile seedlings of marigold (variety milestone. yellow) are cut, the front faces of the small leaves face upwards, and the small leaves are inoculated on 7 culture media added with curing agents with different concentrations: wherein the plant gel is 3g/L, 4.5g/L and 6 g/L; 7g/L, 8g/L, 9g/L and 10g/L of agar. TDZ is added into the culture medium at a concentration of 0.2 mg/L; 0.5mg/L IBA and 30g/L sucrose, and adjusting the pH value of the culture medium to 5.8-6.0. Each group was replicated 4 dishes with 10 explants per dish and the experiment was repeated 3 times. The culture temperature is 25 ℃, the illumination intensity is 800-; the light source is a 6000K white fluorescent lamp.

After 4 weeks, the regeneration rate and vitrification rate were counted. As can be seen from Table 3, the curing agents of different types and concentrations had no significant effect on the leaf regeneration rate of marigold, but had a significant effect on the vitrification of adventitious buds. When the concentration of the agar is 8g/L, the regeneration rate of the marigold is the highest and reaches 65%, and the vitrification rate of regenerated adventitious buds is reduced to 41.55%. And the regenerated adventitious bud has the best state, less deformity and stable regeneration.

TABLE 3 Effect of different curing agents on Tagetes erecta leaf regeneration

Example 4

Cutting 2-3 pairs of compound leaf small leaves at the top of marigold (variety milestone yellow) seedlings, inoculating the cut leaves with the front side upward on 4 culture media added with different concentrations of sucrose, wherein the concentration of the sucrose is set as: 20. 30, 40 and 50 g/L; adding 0.2mg/L TDZ into the culture medium; 0.5mg/L IBA; 8g/L agar, and the pH value is 5.8-6.0. Each group was replicated 4 dishes with 10 explants per dish and the experiment was repeated 3 times. The culture temperature is 25 ℃, the illumination intensity is 800-; the light source is a 6000K white fluorescent lamp.

After 4 weeks, regeneration rate and vitrification rate of marigold were counted. Table 4 shows that the regeneration rate reached the highest at a sucrose concentration of 40g/L, which was 70%, while the vitrification rate decreased to 16.07%, and the sprouts were large and robust.

TABLE 4 Effect of different sucrose concentrations on Tagetes erecta leaf regeneration

Example 5

Cutting 2-3 pairs of compound leaf small leaves at the top of sterile seedling of marigold (milestone yellow), inoculating to MS minimal medium with the front side upward, and adding 0.2mg/L TDZ; 0.5mg/L IBA; 8g/L agar; and (3) on a culture medium of 40g/L of sucrose, wherein the pH value is 5.8-6.0. Placed under three light sources: 4500K warm white LED lamps, 6000K white fluorescent lamps, 4500K warm white LED lamps, and 6000K white fluorescent lamps are 1: 1. Each group was replicated in 4 dishes, each dish was inoculated with 10 explants, and the experiment was repeated 3 times. The culture temperature was 25 ℃ and the illumination time was 14 h/d.

After 4 weeks, the regeneration rate and vitrification rate were counted. Table 5 shows that different light sources have significant influence on the regeneration efficiency and vitrification of marigold, and the combination of 1:1 of the 4500K warm white LED lamp and the 6000K white fluorescent lamp can ensure that the regeneration rate of the marigold reaches 82.50 percent, the vitrification rate is reduced to below 10 percent, and the marigold has large buds and is strong.

TABLE 5 Effect of different light sources on marigold leaf regeneration

(5) And (3) elongation and rooting culture:

test 7:

cutting off regenerated adventitious buds of marigold, and culturing in 6 kinds of growth and rooting culture media with different hormones, wherein the culture media are numbered as follows: MS1 MS culture medium without hormone; MS2 MS +0.1mg/LTDZ (0.01 mg/LTDZ after 15 d); MS3, MS +0.3mg/L6-BA +1.0mg/L NAA; MS4 MS +0.6 mg/L6-BA +1.0 mg/LNAA; MS5 MS +0.9mg/L6-BA +1.0mg/L NAA; MS6 MS +0.2 mg/L6-BA +0.6 mg/LNAA. Each culture medium contains 8g/L agar and 40g/L sucrose, and the pH of the culture medium is adjusted to 5.8-6.0. And (5) carrying out subculture once after 15d, counting the elongation of the adventitious bud after 30d, transferring the adventitious bud into an MS1 culture medium for rooting, and counting the rooting condition of the regenerated seedling after 15 d.

As can be seen from Table 6, the addition of different hormones to the medium had a significant inhibitory effect on the elongation effect of the regenerated adventitious bud, and the MS1 medium without any hormone added, resulted in robust seedlings with dark green leaf color that could grow to 4-5cm high. And transferring the strong seedlings elongated to 4-5cm to MS1 culture medium without adding any hormone for rooting, wherein the rooting rate is close to 100%.

TABLE 6 Effect of different media on adventitious bud elongation of Tagetes erecta

(6) Hardening and transplanting seedlings: and when the root system of the plantlet grows to 3-5cm, moving the rooted seedling to a seedling hardening shed for hardening and acclimating the rooted seedling, and transplanting the hardened rooted seedling.

EXAMPLE 6 preparation of the culture Medium

MS minimal medium (MS for short, MS solid medium for short).

(1) Marigold seed germination culture medium: MS; 7g/L agar is added; 30g/L sucrose; adjusting the pH of the culture medium to 5.8-6.0.

(2) Adventitious bud induction medium: MS; adding 0.2mg/L Thidiazuron (TDZ); 0.5mg/L indolebutyric acid (IBA); 8g/L agar; 40g/L sucrose; adjusting the pH of the culture medium to 5.8-6.0.

(3) Elongation and rooting medium, MS; adding 8g/L agar; 40g/L sucrose; adjusting the pH of the culture medium to 5.8-6.0.

Claims

1. A construction method of a marigold regeneration system is characterized by comprising the following steps:

1) seed disinfection: selecting full and mature seeds of marigold, soaking the seeds in sterile water for 10min, washing the seeds in 0.1% mercuric chloride solution for 90s, and washing the seeds with the sterile water for three times, wherein each washing time is 10 min;

2) culturing the explants: inoculating the sterilized marigold seeds obtained in the step 1) on an MS solid culture medium to germinate, wherein the seed culture conditions are as follows: the culture temperature is 25 ℃, the illumination intensity is 800-;

3) induction of adventitious buds: cutting 2-3 pairs of compound leaf small leaves at the top of the sterile seedling in the step 2) and inoculating the small leaves on an adventitious bud induction culture medium, wherein a cut is contacted with the adventitious bud induction culture medium during inoculation, and placing the culture medium in a culture chamber for culture until regenerated adventitious buds are obtained;

4) and (3) elongation and rooting culture: cutting the regenerated adventitious bud obtained in the step 3), inoculating to an elongation and rooting culture medium for culture, and subculturing once every 15d to root the bud to obtain a rooted seedling;

5) hardening and transplanting seedlings: after the root system of the rooted seedling grows to 3-5cm, moving the rooted seedling to a seedling hardening shed for hardening and acclimating the rooted seedling, and transplanting the rooted seedling after hardening and acclimating;

wherein:

the formula of the adventitious bud induction culture medium in the step 3) is MS, and 0.2mg/L thidiazuron is added; 0.5mg/L indolebutyric acid; 8g/L agar; 40g/L sucrose; the pH value of the culture medium is 5.8-6.0;

the culture conditions are as follows: the temperature is 25 ℃, the illumination time is 14h/d, and the light source proportion is 4500K warm white LED lamp: a 6000K white fluorescent lamp with a light ratio of 1: 1;

the elongation and rooting culture medium in the step 4) is MS, and 8g/L agar is added; 40g/L sucrose; pH5.8-6.0; the culture conditions are as follows: the temperature is 25 ℃, the illumination time is 14h/d, and the light source is a 4500K warm white LED lamp.