CN112400693A

CN112400693A - Pretreatment method for improving induction of calluses of mature embryos of indica rice

Info

Publication number: CN112400693A
Application number: CN202011336591.1A
Authority: CN
Inventors: 张维林; 杨玲; 金海芳; 李双香
Original assignee: Zhejiang Normal University CJNU
Current assignee: Zhejiang Normal University CJNU
Priority date: 2020-11-25
Filing date: 2020-11-25
Publication date: 2021-02-26
Anticipated expiration: 2040-11-25
Also published as: CN112400693B

Abstract

The invention belongs to the technical field of transgenosis, and particularly provides a pretreatment method for improving the induction rate of calluses of mature embryos of indica rice. The hulled indica rice seeds are soaked in 30% trichloroisocyanuric acid solution with the mass concentration of 0.5% -1% for 8-12h, and then are disinfected and inoculated in an induction culture medium according to the traditional method, so that the callus growth rate of the indica rice seeds can be obviously improved and the quality of callus can be obviously improved under the conditions of not adjusting the formula, the hormone type, the concentration and the like of the traditional induction culture medium, and a new way is provided for solving the problem of low induction rate of the indica rice. The method is simple and easy to implement, not only saves a large amount of time and energy, but also promotes the genetic transformation process.

Description

Pretreatment method for improving induction of calluses of mature embryos of indica rice

Technical Field

The invention belongs to the technical field of transgenosis, and particularly provides a pretreatment method for improving the induction of calluses of mature embryos of indica rice.

Background

Rice is one of the most important food crops in the world, and the survival of more than half of the world population is maintained. In recent years, with the increase of the population of the world and the reduction of the culturable area, the improvement of the yield and quality of rice has an important meaning for solving the worldwide food crisis. The further development of molecular biology and molecular genetics makes the transgenic technology an important tool for improving the yield and quality of rice. The tissue culture technology is an important component of the transgenic technology and is widely applied to rice genetic transformation and variety improvement. The most used system for rice genetic transformation is a callus regeneration system. The callus induction rate of japonica rice varieties is high, a high-efficiency genetic transformation system is established, and the transgenic breeding has obtained favorable results in japonica rice; however, indica rice which accounts for more than 80% of rice varieties is difficult to genetically transform due to low callus induction rate, and the application of a transgenic technology in indica rice is severely restricted.

The rice somatic tissue culture is a basic technology of rice genetic transformation and plays an important role in rice genetic improvement, so that the establishment of a high-efficiency regeneration system is a prerequisite for realizing genetic transformation of genes. Because the mature embryo of the rice has the advantages of wide source, no limitation of the material selection by seasons and geographical environments, convenient operation, difficult pollution and the like, the mature embryo of the rice is an ideal material for genetic operation, but the callus induction rates of different varieties of rice are very different; even under the theoretically optimal conditions, the callus induction rate of mature embryos of many indica rice varieties with excellent traits is still low. In the traditional method, the callus induction of the mature embryo of the rice is improved by adopting measures on the improvement of a culture medium and other aspects of tissue culture to induce the callus, for example, different culture media, different hormone types and concentrations are tried out, and a small amount of kinetin is added in the induction culture medium to improve the differentiation quality of the callus; organic additives (e.g. hydrolysed casein) are added to improve the callus quality of certain varieties. However, the results of previous studies show that the genetic background of rice determines the induction process in tissue culture, and the main factor influencing the induction of rice callus is the genotype of rice varieties. The rice variety has abundant resources and high-level genetic diversity, and the induction of the calluses of different mature embryos of the indica rice is improved by trying different culture media, different hormone types and concentrations, culture medium improvement and other aspects of tissue culture by using a traditional method, so that the method is time-consuming and labor-consuming, and ideal results are not necessarily obtained.

Based on this, a method widely applicable to pretreatment for callus induction of indica rice materials is highly demanded in the art.

Disclosure of Invention

The invention aims to solve the technical problem that the invention provides a callus induction pretreatment method which is widely suitable for indica rice materials. After the pretreatment, the callus induction rate of the indica rice can be improved without adjusting the formula, the hormone type and the concentration of an induction culture medium used in the traditional method. The pretreatment method of the invention not only saves time and labor, but also accelerates the genetic transformation progress of indica rice.

The invention is realized by the following means:

a pretreatment method for improving callus induction of mature embryos of indica rice comprises the following steps:

(1) hulling mature embryo of long-shaped rice

Selecting full, non-mildew and mature indica rice seeds. Hulling with manual or miniature huller, taking care to keep embryo intact.

(2) Preparation of pretreatment reagent

30 percent of trichloroisocyanuric acid compound and sterilized water, and preparing trichloroisocyanuric acid solution with the mass concentration of 0.5 to 1 percent according to the mass ratio.

(3) Pretreatment of mature embryo

The rice seeds after shelling are washed clean with clear water and rinsed 3-5 times with sterile water. Soaking the hulled rice seeds for 8-12h at 25-37 ℃ by using trichloroisocyanuric acid solution with the mass concentration of 0.5-1%; rinsing with sterile water for 3-5 times, and sucking dry the seeds with sterile filter paper.

(4) Sterilization of mature embryos

Treating the rice seeds soaked in the trichloroisocyanuric acid solution with 75% alcohol for 2min, and rinsing with sterile water for 2-3 times; sterilizing with 5-10% sodium hypochlorite for 15-20min, or sterilizing with 0.15% mercuric chloride for 12min, and rinsing with sterile water for 2-3 times; sucking dry the seeds with sterile filter paper; then inoculated into callus induction medium.

The callus induction culture medium comprises the following components in percentage by weight: NB minimal medium +2,4-D (4mg/L) + sucrose (30g/L) + plant gel phytagel (2.7g/L), pH 5.8.

The invention has the beneficial effects that:

according to the method, husked indica rice seeds are soaked in 30% trichloroisocyanuric acid solution with the mass concentration of 0.5% -1% for 8-12h, then the seeds are disinfected and inoculated in a traditional induction culture medium according to a traditional method, the callus growth rate of the indica rice seeds can be obviously improved, the quality of callus can be obviously improved, the tedious work of trying to adjust the formula of the induction culture medium, the types of hormones, the concentration conditions and the like in order to improve the callus induction rate in the traditional method is avoided, and a new way is provided for solving the problem of low indica rice induction rate; the method is simple and easy to implement, not only saves a large amount of time and energy, but also promotes the genetic transformation process.

Drawings

FIG. 1 shows callus induction conditions of 93-11 indica rice seeds soaked in 1% 30% trichloroisocyanuric acid aqueous solution for 4-72 h; a. the treatment times of b, c, d, e, f and g were 4h, 8h, 12h, 24h, 36h, 48h and 72h, respectively.

FIG. 2 shows callus induction of indica rice seeds treated with 30% trichloroisocyanuric acid aqueous solution with a mass concentration of 1% and water for 12 hours; a. b, treating indica rice seeds by water and 1 percent of 30 percent trichloroisocyanuric acid aqueous solution for 12 hours respectively.

Detailed Description

Example 1

(1) hulling mature embryo of long-shaped rice

(2) Preparation of pretreatment reagent

(3) Pretreatment of mature embryo

The rice seeds after shelling are washed clean with clear water and rinsed 3-5 times with sterile water. Soaking the hulled rice seeds for 8-12h at 25-37 ℃ by using trichloroisocyanuric acid solution with the mass concentration of 0.5-1%; rinsing with sterile water for 3-5 times. The seeds were blotted dry on sterile filter paper.

(4) Sterilization of mature embryos

Treating the rice seeds soaked in the trichloroisocyanuric acid solution with 75% alcohol for 2min, and rinsing with sterile water for 2-3 times; sterilizing with 5-10% sodium hypochlorite for 15-20min, or sterilizing with 0.15% mercuric chloride for 12min, and rinsing with sterile water for 2-3 times; the seeds were blotted dry on sterile filter paper. Inoculating to callus induction culture medium.

The key steps (2) and (3) are implemented in the following specific steps:

the 30% trichloroisocyanuric acid aqueous solution with the concentration of 1% can effectively promote the germination of seeds. Soaking indica rice seeds such as 93-11 and Mudgo for 0-72h by using 1% 30% trichloroisocyanuric acid aqueous solution, sterilizing mature embryos after pretreatment, and inoculating 10-12 seeds in each induction culture medium plate and 3 dishes in each treatment. Taking water soaking or not as a reference, and counting the healing rate of the mature embryos soaked in different concentrations for different times after 21 days of inoculation. The results show that the cure rate is higher for treatment 12h, and the specific results are shown in table 1 and fig. 1.

The test result of the output rate calculation method is counted according to the following formula:

the survival rate (%) is the number of seeds growing out callus/the number of inoculated seeds multiplied by 100%

TABLE 1 cure rate of trichloroisocyanuric acid solution with 1% mass concentration after pretreatment for 12h

From the results in table 1, it is understood that the germination rate and the healing rate increase with time in the first 4 hours after the pretreatment with the trichloroisocyanuric acid solution having a mass concentration of 1%, but the germination rate and the healing rate decrease with time at the 8 th hour, and the germination rate and the healing rate are the highest and then gradually decrease after 12 hours. And the germination rate and the recovery rate when reaching 12h are obviously higher than the time points of 1h, 4h, 8h, 24h, 36h, 48h and 72h, and the difference has statistical significance (P is less than 0.05).

Taking 1% as a base point, preparing 30% trichloroisocyanuric acid aqueous solutions with mass concentrations of 0.5% and 1.5%, and respectively soaking indica rice seeds of 93-11, Mudgo and the like for 8h, 12h and 24 h. After the pretreatment is finished, the mature embryo is disinfected, 10-12 seeds are inoculated to each induction culture medium plate, and 3 plates are inoculated to each treatment. After 21 days of inoculation, the callus rate of the mature embryos soaked in different concentrations for different times is counted. The result shows that no callus is obtained when 30% trichloroisocyanuric acid aqueous solution with the mass concentration of 1.5% is treated for 8 hours, 12 hours and 24 hours; callus is obtained when 30% trichloroisocyanuric acid water solution with the mass concentration of 0.5% is processed for 8 hours, 12 hours and 24 hours, so that the healing rate is higher when the callus is processed for 12 hours, and specific results are shown in table 2.

TABLE 2 cure rate of trichloroisocyanuric acid solution pre-treatment at 0.5% and 1.5% by mass for 8, 12 and 24h

0.1% is used as a variation to prepare 30% trichloroisocyanuric acid aqueous solution with mass concentration of 0.6%, 0.7%, 0.8%, 0.9%, 1.1%, 1.2%, 1.3% and 1.4%. Soaking indica rice seeds of 93-11, Mudgo, etc. for 8, 12 and 24h respectively. After the pretreatment is finished, the mature embryo is disinfected, 10-12 seeds are inoculated to each induction culture medium plate, and 3 plates are inoculated to each treatment. After 21 days of inoculation, the callus rate of the mature embryos soaked in different concentrations for different times is counted. The results show that the higher healing rate can be obtained by treating 0.6-0.9% of 30% trichloroisocyanuric acid aqueous solution for 8-12h, and the specific results are shown in Table 3.

TABLE 3 cure rate at 8, 12 and 24h pre-treatment with trichloroisocyanuric acid solution with mass concentration of 0.5% -1.5%

According to the results of comparison of the healing rate of 0-72h of indica rice seeds treated by 30% trichloroisocyanuric acid aqueous solution with the concentration gradient, the indica rice seeds treated by 0.5-1% of 30% trichloroisocyanuric acid aqueous solution for 8-12h can obtain higher healing rate.

Indica rice seeds are treated by 30 percent trichloroisocyanuric acid aqueous solution with the mass concentration of 1 percent for 12 hours. After the pretreatment is finished, the mature embryo is disinfected, 10-12 seeds are inoculated to each induction culture medium plate, and 3 plates are inoculated to each treatment. The callus was weighed 21 days after inoculation with or without water soaking as a control, and it was analyzed whether there was a significant difference between 12h treatment of indica rice seeds with 1% aqueous 30% trichloroisocyanuric acid and water soaking. The results show that there is a significant difference between 12h of indica rice seeds treated with 30% trichloroisocyanuric acid aqueous solution with mass concentration of 1% and the quality of callus soaked with water, as shown in table 4 and fig. 2.

TABLE 4 callus weight pretreated with and without trichloroisocyanuric acid solution for 12h

Categories	Using trichloroisocyanuric acid	Without using trichloroisocyanuric acid
			Weight (g)	0.4667±0.1413^**	0.1191±0.0505

Note: p < 0.01.

The results show that for most indica rice seeds, 30 percent trichloroisocyanuric acid solution with the mass concentration of 0.5 to 1 percent is used for soaking for 8 to 12 hours, the healing rate of the indica rice seeds is obviously improved compared with that of the indica rice seeds soaked by water, and the quality of callus is also obviously improved compared with that of the indica rice seeds soaked by water.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Claims

1. A pretreatment method for improving callus induction of mature embryos of indica rice comprises the following steps:

hulling mature embryos of the indica rice;

preparing a pretreatment reagent;

pre-treating the mature embryo; and

and (5) sterilizing the mature embryo.

2. The pretreatment method according to claim 1, wherein:

the pretreatment reagent is a trichloroisocyanuric acid solution with the mass concentration of 0.5-1% prepared from 30% of trichloroisocyanuric acid compound and sterilized water.

3. The pretreatment method according to claim 1, wherein:

the pretreatment of the mature embryo comprises the following steps:

washing the hulled rice seeds, and rinsing with sterile water for 3-5 times;

soaking the hulled rice seeds in the pretreatment reagent for 8-12h at 25-37 ℃, and rinsing with sterile water for 3-5 times;

finally, the seeds are blotted with sterile filter paper for later use.

4. The pretreatment method according to claim 1, wherein:

the sterilization of the mature embryo comprises:

treating the rice seeds sucked by sterile filter paper with 75% alcohol for 2min, and rinsing with sterile water for 2-3 times;

sterilizing with 5-10% sodium hypochlorite for 15-20min, or sterilizing with 0.15% mercuric chloride for 12min, and rinsing with sterile water for 2-3 times;

the seeds were blotted dry with sterile filter paper and inoculated into callus induction medium.

5. The pretreatment method according to claim 4, wherein:

the callus induction culture medium is prepared by the following method:

to NB minimal medium was added 4mg/L of 2,4-D, 30g/L of sucrose, 2.7g/L of plant gel phytagel, and the pH was adjusted to 5.8.

6. The pretreatment method according to claim 2, wherein:

the preparation of the pretreatment reagent comprises the following steps: 30 percent trichloroisocyanuric acid aqueous solution with the mass concentration of 0.5 to 1 percent.

7. The pretreatment method according to claim 3, wherein:

the pretreatment of the mature embryo comprises the following steps: 30 percent trichloroisocyanuric acid aqueous solution with the mass concentration of 0.5 to 1 percent is used for soaking the seeds for 8 to 12 hours.

8. The pretreatment method according to claim 1, wherein:

after the pretreatment is finished, the mature embryos are disinfected, 10-12 seeds are inoculated on each induction culture medium plate, 3 dishes are inoculated on each treatment, water soaking or no water soaking is used as a control, inoculation is carried out for 21 days, the recovery rate of the mature embryos soaked in different concentrations for different time is counted, and the quality of the callus is weighed.