CN103168684B - Tissue culture intermediate propagation method of chickpeas - Google Patents

Abstract

The invention discloses a rapid propagation method of chickpea tissue culture. Firstly, a basic culture medium and a culture medium of each stage are prepared; seeds of wrinkled chickpea with uniform size are selected for disinfection and then germinate; the chickpea seedlings grow to about 10 cm , cutting the stem section with axillary buds and culturing them in the induction medium; inoculating the first-generation young shoots on the proliferation medium to cultivate them as secondary young shoots; inoculating the secondary young shoots into the rooting medium for rooting culture. The present invention provides a new method for rapidly propagating chickpeas by using mature chickpea seeds to germinate and form small sections with axillary buds as explants. This research provides technical support and theoretical basis for future research on chickpea transgenics, genetic breeding, etc.; It plays a certain role in promoting the improvement of the economic benefits of the chickpeas and the improvement of the yield and quality of the chickpeas.

Description

A kind of chickpea tissue culture rapid propagation method

technical field

The invention belongs to the technical field of plant tissue culture, and in particular relates to a rapid propagation method of chickpea tissue culture.

Background technique

Chickpea (Cicer arietinum.L) is one of the edible bean crops with the largest cultivation area in the world, mainly distributed in more than 40 countries in the world. In my country, it is mainly planted in Xinjiang, Gansu and other provinces. Chickpea is a leguminous plant belonging to the genus Chickpea, which originated in western Asia and the Near East. The global cultivation area exceeds 10 million hectares, and most of them are distributed in arid or semi-arid areas such as India, Pakistan, and Turkey. It is the third soybean family in the world. crop. Chickpea is also a kind of non-staple food favored by the Uighur people in my country. It has a history of 2,500 years in Xinjiang, and the planting area is about 300,000 mu. Because chickpea has the characteristics of high nutritional value, high yield and drought tolerance, upright resistance to lodging, relatively high temperature resistance and biological nitrogen fixation, the above-mentioned excellent traits have attracted the attention of commercial producers and researchers of chickpea.

At present, domestic research on chickpea is still in its infancy, and domestic research on the genetic transformation system of chickpea has not yet been reported. At the same time, we have geographical and resource advantages, and there is a strong market demand for chickpea, so we have the conditions to develop Research work on the genetic transformation system of chickpea, and lay the foundation for future related research. This research will provide technical support and theoretical basis for the research of chickpea transgenic, genetic breeding, in vitro tissue culture, etc.; it will promote the improvement of the economic benefits of chickpea and the improvement of the yield and quality of chickpea It has a positive effect on the promotion and development of chickpea genetic breeding.

Contents of the invention

The present invention provides a new method for rapidly propagating chickpeas by using the mature chickpea seeds to germinate and form small sections with axillary buds as explants. This research provides technical support and theoretical basis for future research on chickpea transgenics, genetic breeding, etc. ; It plays a certain role in promoting the improvement of the economic benefits of the chickpea and the improvement of the yield and quality of the chickpea.

The embodiment of the present invention is achieved in this way, a chickpea tissue culture rapid propagation method, the method is carried out according to the following steps:

Preparation of basic culture medium and culture medium of each stage;

Selecting uniform size wrinkled chickpea seeds to germinate after disinfection;

When the chickpea seedlings grow to about 10 cm, the stem segments with axillary buds are cut and cultured in the induction medium;

Inoculate the first-generation young shoots on the proliferation medium and cultivate them as secondary young shoots;

The subcultured shoots were inoculated into rooting medium for rooting culture.

Further, the tissue culture medium includes basic medium and various stages of medium, and the components and the weight of each component in each liter are:

1) Basic medium: MS or 1/2MS medium, in which Gelzan 2g/L, MgCl ₂ 1.97g/LpH5.7-6.0;

2) Seed germination medium: 1/2MS;

3) Induction medium: MS+2,4-D0.5mg/L+6-BA3mg/L;

4) Proliferation medium: MS+6-BA2mg/L+IBA1mg/L;

5) Rooting medium: 1/2MS (10g sucrose)+6-BA2mg/L+IBA1mg/L.

Further, the disinfection treatment and germination method of chickpea seeds are as follows:

Select chickpea seeds with uniform size and size, sterilize with 1% sodium hypochlorite solution for 10 minutes, wash with sterile water for 5-6 times, soak in sterile water for a day and night, and soak in 0.1% mercury liter for 1 minute in the ultra-clean workbench the next day. After rinsing with sterile water and absorbing the surface moisture with sterile filter paper, inoculate in 1/2MS medium, culture at 26±2°C, 16/8 light and dark alternately.

Further, the induction culture method is as follows: when the chickpea seedlings grow to about 10 cm, cut the stem segment with axillary buds and culture them in the induction medium; at 26±2°C, 16/8 light and dark alternate culture, after about 20 days of cultivation The explants were induced to germinate the first generation of young shoots.

Further, the proliferation culture method is as follows: inoculate the first-generation young shoots on the proliferation medium, and cultivate them for 15-20 days under the same conditions until they are differentiated to form secondary young shoots with 2-5 times of bud length and 3-5 cm in length.

Further, rooting culture: Inoculate the subcultured young shoots into the rooting medium, and cultivate them for 30-40 days under the same conditions until the seedling base grows 8-15 capillary root systems, and then cultivate them for 20-30 days until the root length is 7 -10 cm.

The present invention provides a new method for rapidly propagating chickpeas by using mature chickpea seeds to germinate and form small sections with axillary buds as explants. This research provides technical support and theoretical basis for future research on chickpea transgenics, genetic breeding, etc.; It plays a certain role in promoting the improvement of the economic benefits of the chickpeas and the improvement of the yield and quality of the chickpeas.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The invention provides a method for rapid propagation of chickpea tissue culture, which is carried out according to the following steps:

(1) Preparation of tissue culture medium: including the basic medium and the components of each stage medium and the weight of each component in each liter:

1) Basic medium: MS or 1/2MS medium, in which Gelzan 2g/L, MgCl ₂ 1.97g/LpH5.7-6.0;

2) Seed germination medium: 1/2MS;

3) Induction medium: MS+2,4-D0.5mg/L+6-BA3mg/L;

4) Proliferation medium: MS+6-BA2mg/L+IBA1mg/L;

5) Rooting medium: 1/2MS (10g sucrose)+6-BA2mg/L+IBA1mg/L;

(2) Sterilization and germination of chickpea seeds: select the uniform size of wrinkled chickpea seeds, sterilize with 1% sodium hypochlorite solution for 10 minutes, wash with sterile water for 5-6 times, soak in sterile water for a day and night, and supercharge the seeds the next day. In a clean bench, soak in 0.1% mercury chloride for 1 minute, rinse with sterile water and absorb the surface moisture with sterile filter paper, inoculate in 1/2MS medium, and culture at 26±2°C, 16/8 light and dark alternately;

(3) Induction culture: when the chickpea seedling grows to about 10 cm, the stem section with axillary buds is cut out and cultured in the induction medium. Under the temperature of 26±2℃, 16/8 light and dark alternate culture, cultivated for about 20 days until the explants induced to germinate the first generation of young shoots;

(4) Proliferation culture: the first-generation young shoots are inoculated on the proliferation medium, and cultivated under the same conditions for 15-20 days to differentiate and form 2-5 times, 3-5 cm long shoots of the second generation;

(5) Rooting culture: Inoculate the subcultured young shoots into the rooting medium, cultivate 8-15 capillary roots at the base of the seedlings under the same conditions for 30-40 days, and then cultivate them for 20-30 days to root length 7-10 cm;

The present invention will be further described below in conjunction with specific examples.

[Example 1] Chickpea seed germination and explant preparation (1)

In order to compare the effects of different media on the germination effect of chickpea seeds, MS, 1/2MS, B ₅ and 1/2B ₅ media were prepared. Select chickpea seeds with uniform size and size, sterilize with 1% sodium hypochlorite solution for 10 minutes, wash with sterile water for 5-6 times, soak in sterile water for a day and night, and soak in 0.1% mercury liter for 1 minute in the ultra-clean workbench the next day. After rinsing with sterile water and absorbing the surface moisture with sterile filter paper, inoculate in each of the above mediums, culture at 26±2°C, 16/8 light and dark alternately, and select the most suitable medium.

[Example 2] Chickpea seed germination and explant preparation (2)

Treat with concentrated sulfuric acid for 15 minutes, rinse with running water for 30 minutes, wash with 75% alcohol for 1 minute, wash with sterile water for 5-6 times, soak in sterile water for a day and night, and soak in 0.1% mercury liter for 1 minute in an ultra-clean workbench the next day, inoculate in MS, 1/2MS, B ₅ and 1/2B ₅ medium.

[Example 3] Screening of induction medium

The stem segments with axillary buds were respectively inoculated on the following medium, and the most suitable medium for inducing chickpea germination was screened.

[Example 4] Screening of Proliferation Medium

Inoculate the first-generation shoots on the following propagation medium, and select the most suitable medium for producing secondary shoots.

[Example five] the screening of rooting medium

The subcultured shoots were inoculated into the following rooting medium, and the most suitable medium for rooting was screened.

I1, 1/2MS+1mg/lIBA (10gSucrose+1gPVP)

I2, 1/4MS+0.4mg/lIBA (30gSucrose+1gPVP)

I3, 1/4MS+1mg/l IBA (30gSucrose+1gPVP)

I4, MS+1mg/l6-BA+0.1mg/l IBA (10gSucrose+1gPVP)

MNI, 1/2MS+B5+1.2mg/lNAA+0.4mg/l IBA(10gSucrose+1gPVP)

RB1, MS+B5+1mg/l6-BA (10gSucrose+1gPVP)

RB1N, MS+B5+1mg/l6-BA+0.05mg/l NAA(10gSucrose+1gPVP)

RB2, MS+B5+2mg/l6-BA (10gSucrose+1gPVP)

RB2N, MS+B5+2mg/l6-BA+0.05mg/l NAA(10gSucrose+1gPVP)

RB3, MS+B5+3mg/l6-BA (10gSucrose+1gPVP)

RB3N, MS+B5+3mg/l6-BA+0.05mg/l NAA(10gSucrose+1gPVP)

Control, MS+B5+1mg/l NAA (without Sucrose+1gPVP)

The screening results showed that medium I1 was the most suitable for the rooting of chickpea sprouts, and the rooting rate was 32.1%.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (1)

1. a chickpea tissue culture quick propagation culturing method, is characterized in that, the method is carried out according to the following steps:

Preparation minimal medium and each stage medium;

After choosing the disinfection of big or small homogeneous wrinkle grain chickpea seed, sprout, utilize 1% liquor natrii hypochloritis's sterilizing 10 minutes, sterile water wash 5-6 time, sterile water soaks diel, and in next day superclean bench, 0.1% mercuric chloride soaks 1 minute, with aseptic water washing and with after aseptic filter paper absorption surface moisture, be inoculated in 1/2MS medium, 26 ± 2 DEG C, 16/8 alternation of light and darkness is cultivated;

Treat that chickpea seedling grows to 10cm left and right, cut with the stem section of axillalry bud and cultivate in inducing culture, at 26 ± 2 DEG C of temperature, 16/8 alternation of light and darkness is cultivated, and is cultured to explant induction and sprouts just for young shoot through about 20 days;

To just be inoculated on proliferated culture medium for young shoot, under the same condition, be cultured to through 15-20 days be differentiated to form 2-5 doubly, the subculture young shoot of the long 3-5 of bud centimetre;

Subculture young shoot is inoculated in root media and carries out culture of rootage, be cultured under the same conditions seedling base portion through 30-40 days and grow 8-15 bar capillary root system, then be cultured to the long 7-10 centimetre of root through 20-30 days;

Group training medium comprises minimal medium and each stage medium, and each component contained weight in every liter is:

1) minimal medium: MS or 1/2MS medium, wherein Gelzan2g, MgCl ₂1.97g, pH5.7-6.0;

2) seed germination medium: 1/2MS;

3) inducing culture: MS+2,4-D0.5mg+6-BA3mg;

4) proliferated culture medium: MS+6-BA2mg+IBA1mg;

5) root media: 1/2MS+10g sucrose+6-BA2mg+IBA1mg.