CN109169278B

CN109169278B - Tissue culture method for improving in-vitro regeneration efficiency of broccoli

Info

Publication number: CN109169278B
Application number: CN201811088523.0A
Authority: CN
Inventors: 曾爱松; 李家庆; 严继勇; 宋立晓; 许园园
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2018-09-18
Filing date: 2018-09-18
Publication date: 2021-12-10
Anticipated expiration: 2038-09-18
Also published as: CN109169278A

Abstract

The invention discloses a tissue culture method for improving the in vitro regeneration efficiency of broccoli, which comprises the steps of selecting outer leaves of a mature period of broccoli curd as explants, cutting the explants after disinfection and sterilization to small blocks, inoculating the small blocks onto an induction culture medium, and carrying out illumination treatment by using red and white combined light (white light: red light: 3: 1); transferring the induced adventitious bud to a proliferation culture medium for subculture proliferation; and (4) taking the seedling to transfer to a rooting culture medium for inducing rooting to obtain a rooted seedling. According to the characteristics of the explant, a broccoli efficient regeneration system is established through different hormone ratios of a culture medium and illumination treatment, and the method has the advantages of cheap material, quick adventitious bud induction, high propagation efficiency and the like.

Description

Tissue culture method for improving in-vitro regeneration efficiency of broccoli

Technical Field

The invention relates to a method for establishing an efficient in-vitro regeneration technical system by taking outer leaves of a cauliflower ball in a mature period as explants, belonging to the technical field of biology.

Background

Broccoli, also known as broccoli, cabbage, and cabbage mustard italy, belongs to the brassica genus vegetables of the family brassicaceae, and is served by the curd formed at the top of the main stem and the side branches. Broccoli is rich in various vitamins and minerals, and particularly contains sulforaphane (SF or SFN), which is a cancer preventing and anticancer substance. In recent years, the broccoli planting area is rapidly enlarged in China, and the broccoli planting area becomes an important export vegetable in China. However, about 90 percent of main cultivars used in the current production are imported varieties, and the lack of germplasm resources is one of the key problems limiting the breeding of broccoli varieties in China.

The existing commercial species are basically hybrid species prepared from male sterile lines, and the in vitro preservation of the hybrid species is a necessary link for the preservation of germplasm resources before a better maintainer line or restorer line is found. In vitro preservation of broccoli at home and abroad, in which tissue culture research is carried out by using flower buds, flower organs, leaves, cotyledons with stalks and axillary buds as explants is reported, but the regeneration frequency difference between different genotypes and different explants is large. The leaf used in the studies using leaves as explants was taken from the leaves where the axillary buds of the plants occur, but it is difficult for most varieties to have no or fewer axillary buds (leixuan and henvadao, 1983). The research establishes a simpler, efficient and universal in-vitro regeneration technical system by using the outer leaves of the mature period of broccoli curd as explants, and provides a more applicable method for asexual propagation and propagation of male sterile lines, self-incompatible lines and broccoli materials with difficult seed inoculation.

Disclosure of Invention

According to the invention, the outer leaves of the mature period of the broccoli curd are used as explants, single-factor and multi-factor tests are respectively carried out on a plurality of technical links through experimental research on the basis of the conventional technology in the conventional tissue culture according to the characteristics of the explants, the regeneration technical system is adjusted and improved, a systematic innovation system is provided, and a good effect is achieved.

The purpose of the invention can be realized by the following technical scheme:

1) explant selection

Taking outer leaves of the mature period of broccoli curd, sterilizing the outer leaves with 75% by volume of ethanol for 1-2 min, then sterilizing the outer leaves with 8-9% by volume of sodium hypochlorite for 15-20 min, and finally washing the outer leaves with sterile water for 3-4 times. After sterilization, cutting the leaves into squares with the side length of 1.0-1.5 cm;

2) differentiation Induction

Inoculating the explant to an MS culture medium, and culturing in a culture room with the temperature of 25 +/-0.5 ℃ and the daily illumination time of 14h, wherein the adventitious bud can be directly induced within 15-20 days;

the induction culture medium is MS +0.2 mg/L NAA +2.0 mg/L6-BA + 6-7 g/L agar + 25-30 g cane sugar, and the pH value is 5.8;

the illumination condition is a white light: red light =3: 1;

3) subculture multiplication

Transferring the induced single buds or cluster buds (cutting the cluster buds into single buds) to a subculture multiplication medium, transferring for one time in 25-30 days, and multiplying the buds by about 200 times through 3-4 subcultures; the light culture environment during the period is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;

the subculture multiplication medium is MS +0.1mg/L NAA +1.0 mg/L6-BA + 6-7 g/L agar + 25-30 g sucrose, and the pH value is 5.8;

4) root induction

When the sprouts grow to 3-4 cm high, transferring the sprouts to a rooting culture medium, and inducing the sprouts to root for 15-20 days to obtain rooted seedlings; the light culture environment during the period is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;

the rooting medium is MS +0.1mg/L NAA +0.1mg/L IBA + 7-8 g/L agar + 20-25 g sucrose, and the pH value is 5.8;

5) transplanting and field planting

After hardening off the rooted seedlings at normal room temperature for 24 hours, transplanting the rooted seedlings into a plug tray with 50 holes for growth, wherein the matrix is a cruciferous vegetable seedling culture matrix; and (5) planting 5-7 true leaves of the seedlings in the field.

Has the advantages that:

(1) the explant is convenient to obtain and rich in quantity. The number of outer leaves in the mature period of the broccoli curd is about 20, and the maximum leaf length is 30-60 cm and the maximum leaf width is 20-35 cm according to different varieties. Therefore, the explant is cheap to obtain;

(2) adventitious buds are formed early. According to the invention, adventitious buds can be directly induced within the fastest 15 days by the treatment of culture conditions such as culture medium formula, illumination treatment and the like. In the in vitro regeneration system of vegetables and vegetables of cabbage type, the induction of the general adventitious bud needs 40-50 d (Zhengzi pine, a tissue culture method for improving the in vitro regeneration efficiency of common head cabbage, CN 103125398A; Zhanhui, etc., a propagation method for maintaining the RGMS male sterile line of cabbage, CN 101564009A), and the faster needs 20-25 d (Zhang Zheng super, etc., a method for rapidly propagating the cytoplasmic male sterile line of broccoli, CN 103262795B);

(3) the regeneration frequency is about 95 percent, the number of single buds contained in the directly induced cluster buds is about 8, the regeneration frequency and the differentiation frequency are both higher, and the method is an efficient and convenient in-vitro regeneration technical method.

Drawings

FIG. 1 shows the formation of adventitious buds at an early stage.

FIG. 2 the adventitious bud later stage is formed.

FIG. 3 regenerated plantlets.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The embodiment of the invention is realized by a tissue culture method for improving the in-vitro regeneration efficiency of broccoli, which comprises the following steps:

1) explant selection

Taking healthy outer leaves of the mature period of broccoli curd, sterilizing the healthy outer leaves with 75% by volume of ethanol for 1-2 min, then sterilizing the healthy outer leaves with 8-9% by volume of sodium hypochlorite for 15-20 min, and finally washing the healthy outer leaves with sterile water for 3-4 times. After sterilization, cutting the leaves into squares with the side length of 1.0-1.5 cm;

2) differentiation Induction

Inoculating the explant to a culture medium of MS +0.2 mg/L NAA +2.0 mg/L6-BA + 6-7 g/L agar + 25-30 g sucrose and pH5.8, wherein the temperature of the culture chamber is 25 +/-0.5 ℃, and the illumination is white light: red light =3:1, the illumination time is 14h every day, adventitious buds begin to grow after 7-10 days, and the adventitious buds can be directly induced after 15-20 days;

in an embodiment of the present invention, wherein:

A. differentiation induction medium was prepared by adding different concentrations of 6-BA and NAA on the basis of MS solid medium as follows: the concentrations of 6-BA are 1.0 mg/L, 2.0 mg/L and 3.0 mg/L respectively; concentration of NAA is 0.1mg/L, 0.2mg/L and 0.3mg/L respectively;

TABLE 1 NAA and 6-BA test treatment

Processing number	NAA/ mg/L	6-BA/ mg/L
			A1	0.1	1
A2	0.1	2
			A3	0.1	3
A4	0.2	1
			A5	0.2	2
A6	0.2	3
			A7	0.3	1
A8	0.3	2
			A9	0.3	3

B. In the present embodiment, the light treatment was performed by 2 treatments of B1 (white light) and B2 (white light: red light =3: 1);

single-factor and multi-factor tests are respectively carried out on the A (hormone treatment) and the B (light treatment), and the test results show that the regeneration rate is higher when the hormone combination concentration is lower, but the adventitious bud induction rate is low; the regeneration rate is reduced when the concentration of the hormone combination is higher, and the adventitious bud induction rate is high; under B2 (white light: red light =3: 1), adventitious buds formed early; taken together with the results of the experimental data, the optimal culture conditions for differentiation induction were the combination of a5+ B2, i.e.: MS +0.2 mg/L NAA +2.0 mg/L6-BA, white light: red light =3:1, regeneration frequency is 95% on average, and single buds contained in the directly induced cluster buds are 8 on average.

3) Subculture multiplication

Cutting the induced cluster buds into single buds, transferring the single buds to a culture medium of MS +0.1mg/L NAA +1.0 mg/L6-BA + 6-7 g/L agar + 25-30 g of cane sugar and pH5.8, transferring for 25-30 days once, and carrying out 3-4 times of subculture to proliferate the buds by about 200 times; the light culture environment during the period is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;

in the present example, the subculture multiplication medium was prepared by adding 6-BA and NAA at different concentrations on the basis of MS solid medium as follows: the concentrations of 6-BA are 0.5 mg/L, 1.0 mg/L and 1.5 mg/L respectively; concentration of NAA is 0.1mg/L and 0.2mg/L respectively;

TABLE 2 NAA and 6-BA test treatments

Processing number	NAA/ mg/L	6-BA/ mg/L
			C1	0.1	0.5
C2	0.1	1.0
			C3	0.1	1.5
C4	0.2	0.5
			C5	0.2	1.0
C6	0.2	1.5

Test results show that under the treatment of C2, the regeneration and proliferation coefficient of the adventitious bud is averagely 3.5, the bud grows robustly and no root is generated; the other proliferation coefficients are low, or the growth of adventitious buds is slow; the optimal medium was selected as the C2 combination, i.e.: MS +0.1mg/L NAA +1.0 mg/L6-BA

4) Root induction

When the sprouts grow to 3-4 cm high, transferring the sprouts to a culture medium of MS +0.1mg/L NA +0.1mg/L IBA + 7-8 g/L agar + 20-25 g sucrose and pH5.8, and inducing the sprouts to root for 15-20 days to obtain rooted seedlings (as shown in figure 3); the light culture environment during the period is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;

5) transplanting and field planting

The rooted seedlings are hardened for 24 hours at normal room temperature and then transplanted into a plug tray with 50 holes for growth, and the matrix is a cruciferous vegetable seedling culture matrix; and (5) planting 5-7 true leaves of the seedlings in the field.

In conclusion, healthy outer leaves of broccoli in the mature period are selected, and are best cut into small pieces with the square of 1.0-1.5 cm and inoculated to an adventitious bud induction culture medium with the culture medium of MS +0.2 mg/L NAA +2.0 mg/L6-BA + 6-7 g/L agar + 25-30 g sucrose and the pH value of 5.8, and the light treatment is red combined white light (white light: red =3: 1); after adventitious buds are formed at the edges of the leaves, the adventitious buds are cut and transferred to a subculture multiplication medium of MS +0.1mg/L NAA +1.0 mg/L6-BA + 6-7 g/L agar + 25-30 g sucrose and pH5.8 for culture; when the sprouts grow to 3-4 cm high, transferring the sprouts to a culture medium of MS +0.1mg/L NAA +0.1mg/L IBA + 7-8 g/L agar + 20-25 g sucrose and pH5.8 to induce rooting, and obtaining regenerated seedlings. By the technical system, the adventitious buds are early formed (15-20 days), the regeneration frequency is about 95%, and the number of single buds contained in the cluster buds directly induced is about 8.

Claims

1. A tissue culture method for improving the in vitro regeneration efficiency of broccoli is characterized by comprising the following steps:

(1) explant selection

Taking outer leaves of the cauliflower in the mature period, sterilizing, and cutting the leaves into explant squares with the side length of 1.0-1.5 cm after sterilization;

(2) differentiation Induction

Inoculating the explant blocks onto an induction culture medium, and placing the explant blocks in a culture room at the temperature of 25 +/-0.5 ℃ for 14 hours per day;

(3) subculture multiplication

Transferring the single bud or the cluster bud induced and generated in the step (2) to a subculture multiplication medium for 25-30 days, wherein the illumination culture environment is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;

(4) root induction

When the sprouts grow to 3-4 cm high, transferring the sprouts to a rooting culture medium; the illumination culture environment is as follows: white light, the illumination time is 14h/d, and the culture temperature is 25 +/-0.5 ℃;

(5) transplanting and field planting

Hardening the rooted seedlings at room temperature for 24 hours, and transplanting the rooted seedlings into a plug for growth, wherein the substrate is a cruciferous vegetable seedling culture substrate; after the seedlings grow to 5-7 true leaves, fixedly planting the seedlings in a field;

in the step (2), the induction culture medium is MS +0.2 mg/L NAA +2.0 mg/L6-BA + 6-7 g/L agar + 25-30 g cane sugar, and the pH value is 5.8;

in the step (2), the illumination condition in the culture room is a white light lamp: red light =3: 1;

in the step (3), the subculture multiplication medium is MS +0.1mg/L NAA +1.0 mg/L6-BA + 6-7 g/L agar + 25-30 g sucrose, and the pH value is 5.8;

in the step (4), the rooting medium is MS +0.1mg/L NAA +0.1mg/L IBA + 7-8 g/L agar + 20-25 g sucrose, and the pH value is 5.8.

2. The tissue culture method for improving the in vitro regeneration efficiency of broccoli according to claim 1, wherein the sterilization treatment in step (1) is performed by the following method:

sterilizing the leaves for 1-2 min by adopting ethanol with the volume fraction of 75%, then sterilizing for 15-20 min by using sodium hypochlorite with the volume fraction of 8-9%, and finally washing with sterile water.