CN116235783B - Culture medium and culture method for in-vitro regeneration of balsam pear - Google Patents

Abstract

The invention relates to the technical field of plant tissue culture, and particularly provides a culture medium and a culture method for in-vitro regeneration of balsam pear. The culture medium comprises an adventitious bud induction culture medium, a proliferation culture medium and a rooting culture medium, wherein growth hormone is added into an MS culture medium, and the culture medium comprises the following concrete steps: the adventitious bud induction culture medium is characterized in that IBA and 6-BA are added into each liter of MS culture medium; the proliferation culture medium is obtained by adding IBA and 6-BA into each liter of MS culture medium; rooting medium is to add NAA and IBA in every liter of MS medium. The culture method comprises the following steps: selecting fresh and active balsam pear seeds, removing seed coats and sterilizing; sowing in a seed germination culture medium; selecting an explant when the hypocotyl of the seed is elongated by about 2-3 cm; inoculating to adventitious bud induction culture medium, treating in dark for 3d, and culturing for 18-25d; elongation of adventitious buds; rooting culture; hardening seedlings. The advantages are that: the tissue culture method is easy to operate, shortens the period and has high regeneration rate.

Description

Culture medium and culture method for in-vitro regeneration of balsam pear

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a culture medium and a culture method for in-vitro regeneration of balsam pear.

Background

The balsam pear is an annual herb plant of balsam pear in cucurbitaceae, is rich in nutrition, can be used as a medicine, has the effects of clearing heat and detoxicating, tonifying kidney yang, dispelling cold dampness, reducing blood sugar and the like, is a plant for both food and medicine, and is popular with consumers. The plant is widely planted in tropical and subtropical areas, and becomes one of the pillar vegetable types in Hainan province, and simultaneously planted in large areas in Hunan, hubei, fujian, sichuan, guangdong, guangxi, shandong and other provinces in China, so that the method has great significance in the breeding of high-yield high-quality and wide-adaptability new varieties.

Compared with the traditional breeding technology, the transgenic breeding is a more efficient and accurate modern breeding technology, can effectively accelerate the breeding process, and a stable and efficient genetic transformation system is a key step for cultivating new varieties of transgenic crops, and is also an important method for researching gene functions and innovating germplasm. However, at present, balsam pear callus is difficult to induce, the differentiation rate of adventitious buds is low, the regeneration system is low in efficiency, and instability becomes a bottleneck for limiting genetic transformation. In addition, due to low propagation coefficient of the balsam pear, the price of seeds is high, and if industrialized seedling culture can be realized through tissue culture, the problem of high production cost of the balsam pear can be effectively solved. As described above, the establishment of the efficient regeneration system is the basis for realizing the works, breaks through the difficult problem, helps to accelerate the breeding process, realize directional breeding, enrich the germplasm resources of the balsam pear, and promote the research of the functional genomics of the balsam pear so as to promote the rapid development of the balsam pear industry.

Disclosure of Invention

The invention provides a culture medium and a culture method for in-vitro regeneration of balsam pear, which are used for solving the problems of unstable regeneration system and low regeneration rate of the balsam pear at present.

The first aim of the invention is to provide a culture medium for balsam pear in vitro regeneration, which comprises an adventitious bud induction culture medium, a proliferation culture medium and a rooting culture medium; the pH=5.8-6.0 of the adventitious bud induction culture medium, the proliferation culture medium and the rooting culture medium is obtained by adding growth hormone into an MS culture medium, and the specific steps are as follows:

the adventitious bud induction culture medium is prepared by adding 0.01-0.05mg IBA and 2-3mg 6-BA into each liter of MS culture medium;

the proliferation culture medium is prepared by adding 0.001-0.01mg IBA and 0.5-1.5mg 6-BA into each liter of MS culture medium;

the rooting culture medium is prepared by adding 0.1-0.2mg NAA and 0.1mg IBA into each liter MS culture medium.

Preferably, each liter of MS culture medium comprises 4.4-4.5g of MS powder, 25-35g of sucrose, 2.5-3.5g of plant gel and the balance of water.

Preferably, the MS powder is added in an amount of 4.42g, the sucrose is added in an amount of 30g, and the plant gel is added in an amount of 3g.

Preferably, IBA is 0.01mg and 6-BA is 2.5mg in adventitious bud induction medium; IBA in the proliferation culture medium is 0.005mg, 6-BA is 1mg; NAA in the rooting medium is 0.15mg.

The second aim of the invention is to provide a culture method for in-vitro regeneration of balsam pear, which adopts the culture medium for in-vitro regeneration of balsam pear and comprises the following steps:

s1, acquiring a sterile material: selecting fresh and active balsam pear seeds, removing seed coats and sterilizing; sowing on seed germination culture medium, and dark-treating at 25-30deg.C for 6-8 d; the seed germination culture medium is an MS culture medium;

s2, selecting an explant: selecting an explant when the hypocotyl of the seed stretches 2-3 cm;

s3, induction of adventitious buds: inoculating the explant onto adventitious bud induction culture medium, and performing dark treatment at 25-30deg.C for 2-4d; taking out, placing in an environment with illumination intensity of 1800-2200lx and illumination intensity of 16h, darkness of 8h every day, and culturing for 18-25d to obtain adventitious buds;

s4, elongation of adventitious buds: inoculating adventitious bud into proliferation culture medium, alternately performing light irradiation for 16h and dark for 8h every day, wherein the light irradiation intensity is 1800-2200lx, the temperature is 25-30deg.C, and culturing for 18-25d; during the period, the growth speed, the extension length, the presence or absence of vitrification browning and the number of adventitious buds are observed;

s5, rooting culture: separating adventitious buds into single plants, respectively inoculating into rooting culture medium, alternately performing illumination for 16h and darkness for 8h every day according to the light period, wherein the illumination intensity is 1800-2200lx, the temperature is 25-30 ℃, and culturing for 13-17d;

s6, hardening seedling treatment: when the root grows to 2.5-3cm, transferring to an incubator for culturing for 2-3d, transferring to sterile culture soil for culturing for 3-4d in dark place, and transferring to outdoor under normal light for culturing.

Preferably, the sterilization in step S1 specifically includes: sterilizing with 75% ethanol solution for 1-2min, soaking in 3-5% sodium hypochlorite solution for 8-10 min, and washing with sterile water for 4-6 times.

Preferably, the concentration of sodium hypochlorite solution is 3.6%.

Preferably, the explant selected in step S2 is a cotyledon with a handle.

Preferably, the cotyledon with a handle is obtained by cutting off one third of the cotyledon at the far axial end of the hypocotyl of the seed, removing the hypocotyl and the growing point, and longitudinally cutting into two parts.

Preferably, the dark treatment time in step S2 is 3d.

Preferably, the temperature of the dark treatment is 28 ℃; the illumination intensity is 2000lx.

Preferably, the sterile culture soil comprises a matrix, perlite and fertilizer, wherein the mass ratio is 6:3:1.

the invention has the beneficial effects that:

the operation method has high regeneration rate, the tissue culture method is easy to operate, the consumption of germplasm materials and the experimental cost can be reduced, the growth period is shortened, the treated medicines are easy to purchase, and the damage to human bodies is small. The in vitro regeneration can shorten the growth period of plants, fix heterosis, facilitate the realization of industrial seedling culture, and play an important role in the production of balsam pear and the research of gene functions.

Drawings

FIG. 1 is a schematic diagram of a process of inducing regeneration seedlings of adventitious bud organogenesis of balsam pear according to the embodiment of the invention; a is a schematic drawing of cotyledon with handle as explant; b is an adventitious bud differentiation schematic diagram; c is a schematic diagram of adventitious buds cultured in an elongation mode; d is an elongated adventitious bud induction rooting chart; e is a rooted regenerated seedling; f is the transplanted regenerated seedling.

FIG. 2 shows the growth of regenerated seedlings in different rooting media according to an embodiment of the present invention; S1-S6 are sequentially schematic diagrams of the influence of 0, 0.05, 0.1, 0.15, 0.2 and 0.25 mg/L NAA added with 0.1mg/L IBA on the rooting condition of the regenerated seedlings.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention.

Example 1

A culture method for in-vitro regeneration of balsam pear comprises the following steps:

s1, acquiring a sterile material: selecting fresh and active balsam pear seeds, removing seed coats, sterilizing with 75% ethanol solution for 1 min, soaking in 3.6% sodium hypochlorite solution for 10min, and washing with sterile water for 4-6 times; sowing on seed germination culture medium, and dark processing at 28deg.C for 6-8 d; each liter of seed germination medium (MS medium) comprises 4.42g of MS powder, 30g of sucrose, 3g of plant gel and the balance of water;

s2, selecting an explant: cutting off one third of cotyledons at the far shaft end when the hypocotyl of the seed extends by about 2-3cm, removing the hypocotyl and the growing point, and longitudinally cutting into two pieces to obtain an explant;

s3, induction of adventitious buds: inoculating the explant onto adventitious bud induction culture medium, performing dark treatment at 28deg.C for 3d, and culturing for 20d to obtain adventitious bud with photoperiod of 16h light/8 h darkness (alternately) and light intensity of 2000 lx; the adventitious bud induction culture medium (per liter) comprises 4.42g of MS powder, 30g of sucrose, 3g of plant gel, 0.01mg of IBA, 2.5mg of 6-BA, and the balance of water, wherein the pH value is 5.8-6.0;

s4, elongation of adventitious buds: inoculating adventitious bud (cluster bud) into proliferation culture medium, alternately performing light with light period of 16 h/dark for 8h every day, and culturing at 28deg.C for 20d at light intensity of 2000 lx; observing the growth speed, the extension length, the presence or absence of vitrification browning and the number of adventitious buds of the adventitious buds; the proliferation medium (per liter) comprises 4.42g of MS powder, 30g of sucrose, 3g of plant gel, 0.005mg of IBA, 1mg of 6-BA, and the balance of water, wherein the pH value is 5.8-6.0;

s5, rooting culture: separating adventitious buds (cluster buds) into single plants, respectively inoculating the single plants into a rooting culture medium, alternately carrying out light irradiation for 16h and darkness for 8h every day according to a light period, wherein the light intensity is 2000lx, the temperature is 28 ℃, and culturing for 15 d, and rooting the adventitious buds; the rooting culture medium (per liter) comprises 4.42g of MS powder, 30g of sucrose, 3g of plant gel, 0.15mg of NAA, 0.1mg of IBA and the balance of water, and the pH value is 5.8-6.0;

s6, hardening seedling treatment: when the root grows to 2.5-3cm, opening the bottle cap of the culture bottle, and placing the culture bottle in an incubator for 2-3d; after 2-3d of cultivation, transferring the regenerated plantlets into sterile cultivation soil (wherein the ratio of the matrix, the perlite and the fertilizer is 6:3:1) for 3d of light-proof cultivation, and then removing the regenerated plantlets to outdoor normal light for cultivation.

Wherein IBA is indole-3-butyric acid, 6-BA is 6-benzylaminoadenine, and NAA is naphthylacetic acid.

Example 2

The specific steps of the culture method for in-vitro regeneration of balsam pear are the same as in example 1, and the addition amount of substances in each culture medium is only adjusted as follows:

(1) The seed germination medium (per liter) comprises 5g MS powder, 32g sucrose, 2.5g plant gel and the balance of water;

(2) The adventitious bud induction culture medium (per liter) comprises 5g MS powder, 32g sucrose, 2.5g plant gel, 0.5mg IBA, 3mg 6-BA, and water with pH of 5.8-6.0;

(3) The proliferation medium (per liter) comprises 5g MS powder, 32g sucrose, 2.5g plant gel, 0.01mg IBA+0.8mg 6-BA, and water with pH of 5.8-6.0;

(4) The rooting medium (per liter) comprises 5g of MS powder, 32g of sucrose, 2.5g of plant gel, 0.15mg of NAA, 0.1mg of IBA, the balance being water and the pH value being 5.8-6.0.

Example 3

1. Experimental method

1. Culturing was performed in the same manner as in example 1;

2. selecting fresh and active balsam pear seeds in the step S1, removing seed coats, soaking for 15min, sterilizing for 1 min by using 75% ethanol solution, soaking for 8min in 4% sodium hypochlorite solution, and washing for 4-6 times by using sterile water; the rest of the procedure is the same as in example 1;

3. selecting fresh and active balsam pear seeds in the step S1, carrying out water bath for 15min at 55 ℃, soaking overnight at room temperature, and removing shells of the seeds; sterilizing with 75% ethanol solution for 1 min, soaking in 4.5% sodium hypochlorite solution for 10min, and washing with sterile water for 4-6 times; the rest of the procedure is the same as in example 1.

2. Results

Culturing by the three methods, and exploring the influence of different disinfection treatment methods on seeds;

TABLE 1 Effect of different disinfection treatments on seeds

As can be seen from Table 1, the seed contamination rate increases as the soaking time is too long during seed pretreatment; seed contamination rate was 21% by seed sterilization method in comparative example 1; the seed contamination rate was 76% by the seed sterilization method of comparative example 2; the seed contamination rate was 7% and the seed germination rate was 98% by the seed sterilization method of example 1.

Example 4

Exploring the induction conditions of adventitious buds of different explants of different strains, wherein the hypocotyls, the cotyledons with handles and the cotyledons with handles are respectively selected as the explants according to the adjustment of the step S2 of the embodiment 1, and the rest steps are the same;

TABLE 2 adventitious bud Induction of different explants of different lines

As can be seen from the results in Table 2, the present invention selects 5 different strain cotyledons with stalks as explants, the lower part of the explants expands, adventitious buds are induced near the incision, the differentiation rate of the adventitious buds is 19.23% at the minimum, and 77.56% at the maximum; the average bud number of the adventitious buds of each explant of different strains is at least 4.25 and at most 21.05; the 5 lines of hypocotyls and cotyledons from which the cotyledons were removed were used as explants, and no adventitious buds were induced, so that the optimal explants in this technical condition were cotyledons with the stalk.

Example 5

Researching the influence of different hormone ratios in an adventitious bud induction culture medium on the differentiation rate of the adventitious buds of the explant; 6-8d seedling-age cotyledons with handles are selected and respectively inoculated into the induction culture mediums described in table 5 for observation; the MS culture medium in the table is a seed germination culture medium and comprises 4.42g/L MS powder, 30g/L sucrose and 3g/L plant gel;

TABLE 3 Effect of different hormone ratios on regeneration Rate of explants

The result shows that the effect is relatively good when IBA and 6-BA are added into the MS culture medium; wherein, the callus induction rate and the average adventitious bud differentiation rate of each strain of the Y1 induction culture medium are the highest, so that MS+0. mg/L IBA+2.5 mg/L6-BA is the optimal induction culture medium.

Example 6

The effect of the time of the dark treatment on the differentiation of adventitious buds of the explants was studied. After sterilization in the sterilization manner of step S1 of example 1, 5 lines (25-15, 07-11, 03-8, 11-8, 04-17-4) were treated with cotyledons with handles as explants, and then subjected to grouping treatment (3 groups each including 5 lines) for dark treatment of 0d, 3d and 7d, respectively;

TABLE 4 influence of the time of the dark treatment on the differentiation of adventitious buds of an explant

As shown in Table 4, the differentiation rate of adventitious buds of each strain of explant and the average bud number of adventitious buds of each explant were both at high level at the time of dark treatment for three days. From this, it was concluded that the optimal number of days for dark treatment in the technical run was 3d.

Example 7

The effect of different hormone concentrations in rooting medium on root induction was studied. NAA with different concentrations is set, rooting time is observed, and rooting rate is calculated;

TABLE 5 Effect of different rooting media on root induction

The results are shown in FIG. 2 and Table 5, and the rooting rate reaches the peak value and the rooting time is early when the NAA concentration is 0.15 mg/L; the rooting rate is reduced when the NAA concentration is lower than or higher than 0.15 mg/L, and the rooting time is prolonged. Comprehensive knowledge shows that MS+0.15 mg/L NAA+0.1 mg/L IBA is the optimal rooting medium.

In summary, the invention comprises the steps of obtaining sterile materials of a plurality of strains of balsam pears, inducing the formation of callus, inducing adventitious buds, carrying out proliferation culture, rooting culture, hardening seedlings and the like, firstly provides a sterile material with good growth and low pollution rate, selects less cotyledons with stems as explants in the aspect of balsam pears regeneration, improves the differentiation rate of the adventitious buds of the plurality of strains of balsam pears compared with the prior related research, and improves the rooting condition of regenerated seedlings, thereby obtaining a high-quality and robust balsam pear regenerated plant.

The culture method can obtain regenerated seedlings quickly and has low pollution rate, and the adventitious buds can be induced by selecting the most suitable explant cotyledon with the handle, and the differentiation rate of the adventitious buds can reach 77.56% at most by 8-12 d; the adventitious buds can induce and send out root systems in a rooting culture medium for 9 days, and the rooting rate is 100%; transplanting the regenerated seedlings after hardening seedlings into the prepared matrix, culturing, and obtaining good growth conditions.

The method for obtaining the balsam pear regenerated plant is not limited by natural environment, has higher regeneration rate and lays a foundation for the construction of a balsam pear genetic transformation system.

While embodiments of the present invention have been illustrated and described above, it will be appreciated that the above described embodiments are illustrative and should not be construed as limiting the invention. Variations, modifications, alternatives and variations of the above-described embodiments may be made by those of ordinary skill in the art within the scope of the present invention.

The above embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made in accordance with the technical idea of the present invention shall be included in the scope of the claims of the present invention.

Claims (9)

1. A culture method for in-vitro regeneration of balsam pear is characterized in that: the method comprises the following steps:

s1, acquiring a sterile material: selecting fresh and active balsam pear seeds, removing seed coats and sterilizing; sowing on seed germination culture medium, and dark-treating at 25-30deg.C for 6-8 d; the seed germination culture medium is an MS culture medium;

s2, selecting an explant: when the hypocotyl of the seed stretches 2-3cm, cutting off one third of cotyledons at the far shaft end, removing the hypocotyl and the growing point, and longitudinally cutting into two parts to obtain an explant; s3, induction of adventitious buds: inoculating the explant onto adventitious bud induction culture medium, and performing dark treatment at 25-30deg.C for 2-4d; taking out, placing in an environment with illumination intensity of 1800-2200lx and illumination intensity of 16h, darkness of 8h every day, and culturing for 18-25d to obtain adventitious buds;

s4, elongation of adventitious buds: inoculating adventitious bud into proliferation culture medium, alternately performing light irradiation for 16h and dark for 8h every day, wherein the light irradiation intensity is 1800-2200lx, the temperature is 25-30deg.C, and culturing for 18-25d; during the period, the growth speed, the extension length, the presence or absence of vitrification browning and the number of adventitious buds are observed;

s5, rooting culture: separating adventitious buds into single plants, respectively inoculating into rooting culture medium, alternately performing illumination for 16h and darkness for 8h every day according to the light period, wherein the illumination intensity is 1800-2200lx, the temperature is 25-30 ℃, and culturing for 13-17d;

s6, hardening seedling treatment: when the root grows to 2.5-3cm, transferring to an incubator for culturing for 2-3d, transferring to sterile culture soil for culturing for 3-4d in dark place, and transferring to outdoor under normal light for culturing;

the pH=5.8-6.0 of the adventitious bud induction culture medium, the proliferation culture medium and the rooting culture medium is obtained by adding growth hormone into an MS culture medium, and the specific steps are as follows: the adventitious bud induction culture medium is prepared by adding 0.01-0.05mg IBA and 2-3mg 6-BA into each liter of MS culture medium; the proliferation culture medium is prepared by adding 0.001-0.01mg IBA and 0.5-1.5mg 6-BA into each liter of MS culture medium; the rooting culture medium is prepared by adding 0.1-0.2mg NAA and 0.1mg IBA into each liter MS culture medium.

2. The culture method for in vitro regeneration of balsam pear according to claim 1, which is characterized in that: the MS culture medium comprises 4.4-4.5g of MS powder, 25-35g of sucrose, 2.5-3.5g of plant gel and the balance of water.

3. The culture method for in vitro regeneration of balsam pear according to claim 2, which is characterized in that: the MS powder is added in an amount of 4.42g, the sucrose is added in an amount of 30g, and the plant gel is added in an amount of 3g.

4. A culture method for in vitro regeneration of balsam pear according to any one of claims 1 to 3, characterized in that: IBA is 0.01mg and 6-BA is 2.5mg in the adventitious bud induction culture medium; IBA in the proliferation culture medium is 0.005mg, 6-BA is 1mg; NAA in the rooting medium is 0.15mg.

5. The culture method for in vitro regeneration of balsam pear according to claim 4, which is characterized in that: the sterilization in step S1 specifically includes: sterilizing with 75% ethanol solution for 1-2min, soaking in 3-5% sodium hypochlorite solution for 8-10 min, and washing with sterile water for 4-6 times.

6. The culture method for in vitro regeneration of balsam pear according to claim 5, which is characterized in that: the concentration of the sodium hypochlorite solution was 3.6%.

7. The culture method for in-vitro regeneration of balsam pear according to claim 6, which is characterized in that: the dark treatment time in the step S3 is 3d.

8. The culture method for in vitro regeneration of balsam pear according to claim 7, which is characterized in that: the temperature of the dark treatment is 28 ℃; the illumination intensity is 2000lx.

9. The culture method for in-vitro regeneration of balsam pear according to claim 8, which is characterized in that: the sterile culture soil comprises a matrix, perlite and fertilizer, wherein the mass ratio is 6:3:1.