CN104304032B - Watermelon somatic embryo efficient induction and plant regeneration method suitable for multiple genotypes - Google Patents

Abstract

The invention discloses a watermelon somatic embryo high-efficiency induction and plant regeneration method suitable for multi-genotypes. The method includes pretreatment, callus induction, somatic embryo induction, complete plant formation, seedling hardening and transplanting step. In the pretreatment process, the plant growth regulator used was TDZ with a concentration of 0.01-0.1 mg/L; in the process of callus induction and somatic embryo induction, the growth regulators used were 6?BA and IAA, 6 The concentration of ?BA is 2?4mg/L, and the concentration of IAA is 0.01?0.5mg/L. The present invention is not only applicable to multi-genotype watermelon somatic cell induction, but also has the advantages of high callus induction rate, high somatic embryo induction rate, large number of somatic embryos per explant, simple implementation steps and less stringent implementation conditions. advantage.

Description

Watermelon somatic embryo efficient induction and plant regeneration method suitable for multiple genotypes

technical field

The invention relates to the field of biotechnology, in particular to a method for efficiently inducing somatic embryos of watermelon and regenerating plants suitable for multi-genotypes.

Background technique

Watermelon is an important vegetable crop, ranking fifth among the top 10 fruits in the world. As a high-value economic crop, it is widely planted all over the world. With the improvement of people's living standards, people have higher and higher requirements on the types and quality of watermelons, and the breeding goals of watermelons also tend to be diversified. However, traditional genetic breeding has deficiencies such as lack of germplasm resources, long breeding cycle, and unstable genetic traits displayed by offspring; however, obtaining high-yield and high-quality varieties through in vitro culture, genetic transformation and other biotechnologies has become an important means of plant breeding one.

The embryoid structure formed after embryogenesis and embryonic development without fertilization in vitro is called somatic embryo. Somatic embryos can not only improve the ideal experimental system and technical basis for the study of cell differentiation, cell totipotency mechanism and artificial seeds, but also have the advantages of long-term storage, high genetic stability, and high regeneration frequency, and are also conducive to the selection of mutants. It can also be used as a material for the preparation of protoplasts. Specifically, the regeneration of plants through the somatic embryo route has very important theoretical significance and application value for the rapid propagation of watermelon, the preservation of germplasm resources, the cultivation of excellent traits, and the optimization of genetic engineering receptor systems.

However, there are few successful reports on watermelon somatic embryo induction and plant regeneration in the prior art, and the tissue culture conditions of different watermelon varieties are significantly different, and the successful reports have low repeatability, and can only target one to two genotype. In 1993, Compton and Gray successfully induced somatic embryo regeneration by using cotyledons of watermelon young embryos as explants. The highest induction rate was only about 7%, and the difference between different genotypes was very large. (2007) used cotyledons of watermelon seedlings as explants and reported the highest somatic embryo induction rate of 25% and 27.5%, but the quality of induced somatic embryos and the number of somatic embryos per explant were also low. Or undocumented. In the process of tissue culture, the choice of medium and the addition of hormones are especially important, which have an important impact on the quality of sterile seedlings, induction of callus and somatic embryogenesis, and are the key factors that determine the quality of explants. A key link in embryogenesis and plant regeneration. However, the performance of different media and hormones on different genotypes is very different, and the prior art has not yet found a method for efficient induction and plant regeneration of watermelon somatic embryos suitable for multiple genotypes.

Therefore, it is of great significance to establish a watermelon high-frequency somatic embryogenesis and plant regeneration method suitable for multiple genotypes, which can provide a good receptor regeneration system for excellent breeding of watermelon and transgenic research and other related research.

Contents of the invention

The object of the present invention is to provide a watermelon high-frequency somatic embryogenesis and plant regeneration method suitable for multi-genotypes in view of the deficiencies in the prior art. The method includes the following steps:

1) Pre-treatment: Peel off the seed coat of watermelon seeds and then sterilize them, then inoculate the embryos in MS medium containing TDZ for culture, inoculate 8 seeds per bottle, and culture them in the dark for 3 days after inoculation. The culture temperature was 23-27°C, and then transferred to the conditions with light time of 16 h/d for 3 days. The culture temperature was 23-27°C, the light intensity was 3000 lx, and the concentration of TDZ was 0-0.1 mg/L. 30 g/L of sucrose and 6.0 g/L of agar were added to the base, and the pH value was adjusted to 6;

2) Induction of callus: cut out the cotyledon explants, put them into 1/2MS medium containing 6-BA and IAA, culture them in the dark for 7 days, and then transfer them to light with 16 h/d Conditioned culture for 14 days, the culture temperature is 23-27℃, the light intensity is 3000 lx, the concentration of 6-BA is 2-4mg/L, the concentration of IAA is 0.01-0.5mg/L, and 30 g/L The sucrose of L and the agar of 6.0 g/L, the pH value is adjusted to 6;

3) Induction of somatic embryos: put the calli obtained in step 2) into 1/2MS medium containing 6-BA and IAA, and culture them for 28 days under the condition that the light time was 16 h/d to obtain somatic embryos. The culture temperature is 23-27°C, the light intensity is 3000lx, the concentration of 6-BA is 2-4mg/L, the concentration of IAA is 0.01-0.5mg/L, 30 g/L sucrose and 6.0g/L L of agar, the pH value was adjusted to 6;

4) Formation of complete plants: Put the somatic embryos obtained in step 3) into a hormone-free culture bottle filled with 1/2 MS medium for 28 days, at a temperature of 23-27°C, under light The intensity was 3000 lx, the light time was 16 h/d, 30 g/L sucrose and 6.0 g/L agar were added to the medium, and the pH value was adjusted to 6;

5) Seedling hardening: Select the robust seedlings with more than 3 leaves and 5-6cm strong tap roots after step 4), loosen the lid of the culture bottle and place it for 2 days, then half open it for 2 days, and then fully open it 2 days, during half-open and fully-open lids need to constantly replenish water;

6) Transplanting: Pull up the plants after hardening, wash the root medium with water, and transplant them into a nutrient bowl filled with peat soil, perlite, plant ash and humus soil matrix, at 23-27°C , put on a fresh-keeping bag and place it in an artificial climate box for moisture cultivation, and then move it outside for cultivation after 1-2 weeks.

The sterilization process is as follows: peel off the seed coat of the watermelon seeds, first soak and disinfect with 75% alcohol for 1 minute, then soak and disinfect with 0.1% mercury liter for 5 minutes, and finally rinse with sterile water for 5 times.

The mass ratio of peat soil, perlite, plant ash and humus soil matrix in the nutrient bowl is 1:1:1:1.

Preferably, the TDZ concentration in step 1) is 0.01 mg/L. When the concentration of TDZ was 0.01mg/L, the optimal induction rate of somatic embryos and the number of somatic embryos produced per explant could be obtained.

Preferably, the concentration of 6-BA in step 2) and step 3) is 3 mg/L, and the concentration of IAA is 0.05 mg/L. When the concentration of IAA was 0.05mg/L, the induction rate of somatic embryos and the number of somatic embryos per explant reached the optimum.

The present invention has following beneficial effect:

1. Suitable for multi-genotype watermelon somatic embryo induction;

2. The callus induction rate is high, the somatic embryo induction rate is high, and the number of somatic embryos per explant is large;

3. The implementation steps are simple and the implementation conditions are not harsh.

Description of drawings

Figure 1: Callus induced on watermelon cotyledons;

Figure 2: Somatic embryos produced on callus;

Figure 3: Germination of somatic embryos;

Figure 4: Rooting of somatic embryos;

Figure 5: Regenerated seedlings;

Figure 6: Transplanting seedlings.

detailed description

The present invention is described in detail by the following examples, it is necessary to point out that the following examples are only used to further illustrate the present invention, and can not be interpreted as limiting the scope of the present invention, those skilled in the art according to the content of the above invention Some non-essential improvements and adjustments still belong to the protection scope of the present invention.

Example 1

Select plump watermelon seeds of uniform size, peel off the seed coat on an ultra-clean workbench, soak and disinfect with 75% alcohol for 1 minute, then soak and disinfect with 0.1% mercury liter for 6 minutes, then rinse with sterile water for 5 times, and then Place them on sterile filter paper in a petri dish, use sterilized tweezers to inoculate the seed embryos in MS medium containing TDZ for cultivation, and inoculate 8 seeds per bottle. After inoculation, culture in dark conditions for 3 days, then transfer to 16 h/d photoperiod conditions and culture for 3 days. The culture temperature is 23-27°C, the light intensity is 3000 lx, and the concentration of TDZ is 0-0.1 mg/L , 30 g/L sucrose and 6.0 g/L agar were added to the medium, and the pH value was adjusted to 6. During induction, three groups of experimental groups were established, the concentration of 6-BA in the first group was 2.0mg/L, the concentration of IAA was 0.01mg/L; the concentration of 6-BA in the second group was 4.0mg/L, and the concentration of IAA 0.5mg/L; the concentration of 6-BA in the third group was 3.0mg/L, and the concentration of IAA was 0.1mg/L; the experimental results were the average value of the three groups of experimental results.

The effects of TDZ pretreatment on callus and somatic embryo induction are shown in Table 1.

It can be seen from Table 1 that there was no significant difference in callus induction rate when 0-0.1 mg/L TDZ was used for pretreatment. However, TDZ had a significant effect on the induction rate of somatic embryos and the number of somatic embryos produced per explant. The somatic embryo induction rate reached the maximum at 0.01 mg/L TDZ concentration, which was 52.7%. When the TDZ concentration increased to 0.1 mg/L, the somatic embryo induction rate decreased again. The number of somatic embryos produced by each explant also reached the maximum when the concentration of TDZ was 0.01 mg/L, which was 7.3. In summary, adding 0.01 mg/L TDZ can obtain the most ideal induction rate of somatic embryos and the number of somatic embryos produced per explant.

Example 2

The concentration of TDZ in the pretreatment was 0.01 mg/L. After the pretreatment, the cotyledon explants were cut out and cultured in 1/2 MS medium containing 6-BA and IAA, and cultured in the dark for 7 days. , and then transferred to 16 h/d photoperiod conditions for 14 d. Then the induced callus was transferred to the fresh medium with the same formula, and continued to culture for 28 days under the condition of 16 h/d photoperiod. The culture temperature is 23-27°C, the light intensity is 3000 lx, the concentration of 6-BA is 2-4mg/L, the concentration of IAA is 0.01-0.5mg/L, 30 g/L of sucrose and 6.0 g of agar are added to the medium /L, the pH value was adjusted to 6.

The effects of the combination of 6-BA and IAA on the induction of callus and somatic embryos are shown in Table 2.

It can be seen from Table 2 that there are differences in the incidence of callus induced by different concentrations of 6-BA and IAA, but the difference is not obvious, and the overall is high, above 81.7%. The occurrence rate and quality of somatic embryos were significantly different due to the different concentrations of 6-BA and IAA, the induction rate ranged from 25.9-60.7%, and the number of somatic embryos per explant ranged from 3.2-8.3. When the concentration of IAA was constant, the callus induction rate decreased slowly with the increase of 6-BA concentration, but the induction rate of somatic embryos and the number of somatic embryos per explant showed an upward trend, and the concentration of 6-BA was 3.0 mg /L, the somatic embryo induction rate reached the maximum, and the number of somatic embryos per explant also reached the maximum. When the concentration of 6-BA was constant, the induction rate of somatic embryos showed a downward trend with the increase of IAA concentration, and the highest induction rate of somatic embryos appeared when IAA was 0.05 mg/L. When the concentration of 6-BA was 3.0 mg/L and the concentration of IAA was 0.05 mg/L, the induction rate of somatic embryos and the number of somatic embryos per explant reached the optimum.

Example 3

Formation of complete plants: put the somatic embryos into hormone-free 1/2 MS medium for culture, the culture time is 28 days, the culture temperature is 23-27°C, the light intensity is 3000 lx, and the light time is 16 h/day , add 15 g/L sucrose and 6.0 g/L agar to the medium, and adjust the pH value to 6. As shown in accompanying drawings 3 and 4 of the description, the method provided by the present invention enables somatic embryos to form complete plants.

Example 4

Seedling hardening and transplanting: After pretreatment, callus induction, somatic embryo induction and complete plant formation steps, select the formed complete plants in a hormone-free culture flask filled with 1/2 MS medium For robust seedlings with more than 3 leaves and 5-6 cm strong taproots, first unscrew the lid of the culture bottle and place it for 2 days, then half-open it for 2 days, and then fully open it for 2 days. Keep hydrated. Then, pull up the plants after hardening, wash the root culture medium with water, transplant it into a nutrient bowl filled with peat soil, perlite, plant ash and leaf humus soil substrate, and cover it with The fresh-keeping bag is placed in an artificial climate box for moisture cultivation, and then moved outside for cultivation after 1-2 weeks. The mass ratio of peat soil, perlite, plant ash and leaf humus matrix is 1:1:1:1. The effects of seedling hardening and transplanting are shown in accompanying drawings 5 and 6 of the description respectively.

Example 5

Watermelon seeds of four genotypes, Zaojia, Texiaofeng, Jingxin and Zaochunhongyu, were used to induce somatic embryos. The pretreatment process is as follows: peel off the seed coat of the watermelon seeds and then sterilize them, then inoculate the seed embryos in MS medium containing TDZ for culture, inoculate 8 seeds in each bottle, and culture them in the dark for 3 days after inoculation , the culture temperature was 23-27°C, and then transferred to the conditions with light time of 16 h/d for 3 days, the culture temperature was 23-27°C, the light intensity was 3000 lx, the concentration of TDZ was 0.01mg/L, and the Add 30 g/L sucrose and 6.0 g/L agar, adjust the pH value to 6TDZ concentration of 3 mg/L; when inducing callus and somatic embryos, cut cotyledon explants and put them in cultured with 1/2 MS medium of IAA, cultured in dark conditions for 7 d, and then transferred to 16 h/d photoperiod conditions for 14 d; then the induced callus was transferred to fresh In the culture medium, culture was continued for 28 days under the condition of 16 h/d photoperiod. The culture temperature is 23-27°C, the light intensity is 3000 lx, the concentration of 6-BA is 3 mg/L, the concentration of IAA is 0.05 mg/L, 30 g/L of sucrose, 6.0 g/L of agar, pH Adjust the value to 6.

The effect of genotype on callus and somatic embryo induction is shown in Table 3.

As can be seen from Table 3, the method provided by the present invention can successfully induce somatic embryos for different genotypes, and the induction rate of somatic embryos is above 45%, and the number of somatic embryos induced on each explant is 7.6-8.5 between. All genotypes in Table 3 can be used as transgenic recipient regeneration systems by using this technical system.

Claims (3)

1. the Citrullus vulgaris somatic embryo being applicable to polygene type is efficiently induced and plant regeneration method, it is characterised in that: include following Step:

1) pre-treatment: carry out sterilizing after watermelon seed is peelled off seed coat, then embryo is seeded in only MS culture medium containing TDZ carries out Cultivating, every bottle graft kind 8, be first placed under dark condition cultivation 3d after inoculation, cultivation temperature is 23-27 DEG C, is then transferred to illumination Time is the CMC model 3d of 16h/d, and cultivation temperature is 23-27 DEG C, intensity of illumination be the concentration of 3000lx, TDZ be 0- 0.1mg/L, adds the sucrose of 30g/L and the agar of 6.0g/L in culture medium, pH value is adjusted to 6；

2) induction of callus: cut the outer implant of cotyledon, put in the 1/2MS culture medium only containing 6-BA and IAA, be first placed in black Cultivating 7d under dark condition, be then transferred to the CMC model 14d that light application time is 16h/d, cultivation temperature is 23-27 DEG C, and illumination is strong Degree is 2-4mg/L for the concentration of 3000lx, 6-BA, and the concentration of IAA is 0.01-0.5mg/L, adds the sugarcane of 30g/L in culture medium Sugar and the agar of 6.0g/L, pH value is adjusted to 6；

3) induction of somatic embryo: by step 2) gained callus put into only containing 6-BA and IAA 1/2MS culture medium in, Light application time is that the CMC model 28d of 16h/d obtains somatic embryo, and cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000lx, The concentration of 6-BA be the concentration of 2-4mg/L, IAA be 0.01-0.5mg/L, culture medium is added the sucrose of 30g/L and 6.0g/L Agar, pH value is adjusted to 6；

4) formation of whole plant: by step 3) somatic embryo of gained puts into the training equipped with the 1/2MS culture medium without hormone Supporting in bottle and cultivate, incubation time is 28d, and cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000lx, and light application time is 16h/d, adds the sucrose of 30g/L and the agar of 6.0g/L in culture medium, pH value is adjusted to 6；

5) seedling exercising: choose through step 4) cultivate after there are 3 healthy and strong seedlings with tetanic of blade and 5-6cm, will cultivate The lid of bottle first unscrews placement 2d, more half-open 2d, standard-sized sheet 2d the most again, needs to be continuously replenished water during half-open and standard-sized sheet lid Point；

6) transplant: the plant after being completed by seedling exercising pulls up, and washes root culture medium, be transplanted to equipped with peat soil, perlite, In the nutritive cube of plant ash and leaf mould substrate, at 23-27 DEG C, put freshness protection package and be placed in moisturizing cultivation in growth cabinet, 1-2 Zhou Houzai moves on to outdoor and cultivates.

Method the most according to claim 1, it is characterised in that: step 1) described in sterilization process be: peel off watermelon seed Seed coat, first with 75% alcohol-pickled sterilization 1min, then with 0.1% mercuric chloride soaking disinfection 5min, finally with aseptic water washing 5 Secondary.

Method the most according to claim 1, it is characterised in that: step 6) peat soil, perlite, vegetation in described nutritive cube The mass ratio of ash and leaf mould substrate is 1:1:1:1.