阅读说明：本技术 一种粉皮冬瓜组培条件下加倍试验方法 (Doubling test method for white gourd with white gourd peel under tissue culture condition ) 是由 乔燕春 李志芳 彭家柱 韩向峰 吴宇军 刘玉平 张素平 夏秀娴 于 2021-08-20 设计创作，主要内容包括：本发明涉及植物组培技术领域,公开了一种粉皮冬瓜组培条件下加倍试验方法,包括种子消毒处理、催芽处理、配置秋水仙素、芽体处理、继代扩繁、生根和驯化。本发明方法简单,可获得多倍体粉皮冬瓜,可丰富粉皮冬瓜资源创新途径,加快粉皮冬瓜倍性育种研究,加速资源纯化速度,选育出三倍体等非整倍体冬瓜,丰富冬瓜育种的多样性；本发明0.2％处理的芽体变化最为明显,刚长出侧芽的第一片叶基部膨大明显,常规苗在基部接触到培养基的部分会有膨大现象,但基部很快出现表皮撕裂,产生愈伤组织等现象,而茎段中上部分诱导10天后长出的芽起初是第一片膨大,然后整个芽体变大变光滑,继续生长不会愈伤开裂。(The invention relates to the technical field of plant tissue culture, and discloses a doubling test method under the tissue culture condition of a white gourd with sheet jelly. The method is simple, the polyploid white gourd with sheet jelly can be obtained, the innovation way of the white gourd with sheet jelly resource can be enriched, the research on ploidy breeding of the white gourd with sheet jelly can be accelerated, the resource purification speed can be accelerated, the aneuploid white gourd with triploid and the like can be bred, and the diversity of white gourd breeding can be enriched; the bud body processed by 0.2 percent of the invention has the most obvious change, the first leaf base part of the lateral bud which just grows out is obviously expanded, the part of the base part of the conventional seedling which is contacted with the culture medium can be expanded, but the epidermis of the base part is torn quickly, the phenomena of callus and the like are generated, the bud which grows out after the upper part of the stem section is induced for 10 days is initially expanded by the first leaf, then the whole bud body is enlarged and becomes smooth, and the bud body can not grow continuously and can not be callus and crack.)

1. A double test method of white gourd with white gourd peel under tissue culture condition is characterized by comprising the following steps:

s1: seed disinfection treatment

Cleaning the white gourd seeds, soaking for 4-6h, draining, transferring to an ultra-clean workbench, treating with 75% alcohol for 1min, sterilizing with 0.1% mercuric chloride for 20min, and cleaning with double distilled water for 3 times;

s2: pregermination treatment

Putting the seeds in the step S1 into a sterilized plastic bottle, wherein the plastic bottle is filled with absorbent cotton containing water, the water content of the absorbent cotton is suitable for meeting the humidity required by seed germination, and the plastic bottle is put into a sunlight digital box for dark culture and germination acceleration, wherein the temperature in the sunlight digital box is 35 ℃, and the air humidity is 70%;

s3: preparing colchicine

Preparing 0.2% colchicine solution with sterile water, and vacuum filtering and sterilizing the colchicine solution for 2 times on a super clean bench with 0.22um special filter screen and 500 ml sterile needle tube;

s4: bud processing

When the sprouts of the seeds in the step S2 grow to stems 4-5cm high, cutting the sprouts into stem segments 0.8-1.0cm, removing 1-2 cut leaves at the base of each stem segment, stably inserting the stem segments into a subculture medium, adding 2-3ml of sterile colchicine solution processed in the step S3 on the surface of the subculture medium, carrying out low-speed oscillation culture on a shaking table for 7 days, fully contacting the colchicine with the stem segments, increasing the effect of the colchicine, and then selecting the expanded and stout sprouts for subculture;

s5: and (5) carrying out subculture propagation, rooting and domestication on the buds selected in the step S4.

2. The doubling test method of white gourd with white gourd peel under tissue culture conditions as claimed in claim 1, wherein the formula of the subculture medium for subculture propagation in step S5 is as follows: MS +6-BA0.2mg/L + NAA0.05mg/L + 3% white granulated sugar + 0.65% agar powder.

3. The doubling test method of the pink-skinned wax gourd under the tissue culture condition according to claim 1, wherein the formula of the rooting medium used for rooting in the step S5 is as follows: 1/2MS + IAA0.5mg/L + 2% white granulated sugar + 0.65% agar powder.

4. The doubling test method under tissue culture conditions of white gourd with white gourd peel powder as claimed in claim 1, wherein the operations of step S1 to step S4 are all performed in a tissue culture chamber, and the culture conditions of the tissue culture chamber are as follows: the temperature is 25 +/-2 ℃, the illumination time is 14h, and the relative humidity is 70%.

Technical Field

The invention relates to the technical field of plant tissue culture, and particularly relates to a doubling test method of a white gourd with pink skin under tissue culture conditions.

Background

The white gourd with sheet jelly has a long planting history in China, has the advantages of high quality, wide adaptability, high yield, heat resistance, storage and transportation resistance and the like, is a vegetable which is popular among consumers in midsummer and is one of main varieties for adjusting the vegetables in summer and autumn. The innovative white gourd resources are the basis for breeding new varieties with good quality, disease resistance and stress resistance, the white gourd resources in production are mainly diploid materials, the production and consumption markets are mainly large in the types of large white gourd, the large white gourd has high yield and good benefit and is deeply favored by growers, the white gourd with white gourd is mainly planted in areas of south China, southwest China and Central China in a large area, but the white gourd with white gourd per mu yield is not high in yield per mu, the white gourd with white gourd selling price is higher than the white gourd with black white gourd by one yuan on average in farmer markets, the white gourd with white gourd is cultivated in a larger number, the innovation of ploidy breeding materials is accelerated, and the method is a way for breeding the large white gourd with white gourd.

In the traditional doubling method, colchicine is mostly used for stimulating terminal buds or exposed white germs under field conditions, the success rate is low, the colchicine dosage is large, the seed dosage is also large, and soil pollution is easily caused. Therefore, the inventor develops a double test method of the pink-skin wax gourd under the tissue culture condition, which has less colchicine consumption and is environment-friendly.

Disclosure of Invention

Based on the problems, the invention provides a doubling test method under the tissue culture condition of the white gourd with sheet jelly, the method is simple, polyploid white gourd with sheet jelly can be obtained, innovation ways of white gourd with sheet jelly resources are enriched, and the research on ploidy breeding of white gourd with sheet jelly is accelerated.

In order to solve the technical problems, the invention provides a doubling test method of the sheet jelly wax gourd under the tissue culture condition, which comprises the following specific steps:

s1: seed disinfection treatment

Cleaning the white gourd seeds, soaking for 4-6h, draining, transferring to an ultra-clean workbench, treating with 75% alcohol for 1min, sterilizing with 0.1% mercuric chloride for 20min, and cleaning with double distilled water for 3 times;

s2: pregermination treatment

Putting the seeds in the step S1 into a sterilized plastic bottle, wherein the plastic bottle is filled with absorbent cotton containing water, the water content of the absorbent cotton is suitable for meeting the humidity required by seed germination, and the plastic bottle is put into a sunlight digital box for dark culture and germination acceleration, wherein the temperature in the sunlight digital box is 35 ℃, and the air humidity is 70%;

s3: preparing colchicine

Preparing 0.2% colchicine solution with sterile water, and vacuum filtering and sterilizing the colchicine solution for 2 times on a super clean bench with 0.22um special filter screen and 500 ml sterile needle tube;

s4: bud processing

When the sprouts of the seeds in the step S2 grow to stems 4-5cm high, cutting the sprouts into stem segments 0.8-1.0cm, removing 1-2 cut leaves at the base of each stem segment, stably inserting the stem segments into a subculture medium, adding 2-3ml of sterile colchicine solution processed in the step S3 on the surface of the subculture medium, carrying out low-speed oscillation culture on a shaking table for 7 days, fully contacting the colchicine with the stem segments, increasing the effect of the colchicine, and then selecting the expanded and stout sprouts for subculture;

s5: and (5) carrying out subculture propagation, rooting and domestication on the buds selected in the step S4.

Further, the formula of the subculture medium for subculture propagation in step S5 is as follows: MS +6-BA0.2mg/L + NAA0.05mg/L + 3% white granulated sugar + 0.65% agar powder.

Further, the formulation of the rooting medium used for rooting in step S5 is as follows: 1/2MS + IAA0.5mg/L + 2% white granulated sugar + 0.65% agar powder.

Further, the operations of step S1 to step S4 are all performed in a tissue culture chamber, and the culture conditions of the tissue culture chamber are as follows: the temperature is 25 +/-2 ℃, the illumination time is 14h, and the relative humidity is 70%.

Compared with the prior art, the invention has the beneficial effects that: the invention ensures that the tissue culture process is not influenced by external hybrid pollination, mosquito bite, bad weather and the like through a tissue culture mode, not only can maintain the excellent characteristics of the female parent, but also can stabilize the doubling condition, greatly increases the doubling probability, and increases the number of doubled tissue culture seedlings in geometric quantity which is far more than that of the seeding seedlings; in the invention, a proper amount of colchicine is added in the bud stage, and the expanded and thickened bud is selected for subculture, so that the effects of rapid propagation and seedling purification are achieved, the success rate is higher than that of field treatment, the colchicine consumption is water, the direct contact between the colchicine and soil is reduced, and the method is green and environment-friendly; the invention can enrich the innovative ways of the white gourd with sheet jelly resources, accelerate the research on ploidy breeding of the white gourd with sheet jelly, accelerate the purification speed of resources, breed aneuploid white gourd with triploid and the like, and enrich the diversity of white gourd breeding.

Drawings

FIG. 1 is a comparison of the bud growth before and after doubling for an example of the present invention, where A and C are conventional bud growth, and B and D (0.2% colchicine treated buds) are doubled bud growth;

FIG. 2 is a diagram showing the swelling growth of the sprout after doubling according to the embodiment of the present invention;

FIG. 3 is a state diagram of the colchicine-acting secondary seedlings in the embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.

Example (b):

in this example, colchicine was prepared into treatment solutions with concentrations of 0.2% and 0.3% respectively using sterile water, and the influence of colchicine solutions at two concentrations on the bud of white gourd with sheet jelly was studied, and the specific method is as follows.

Seed disinfection treatment: dividing the white gourd seeds into two groups (0.2% colchicine treatment group and 0.3% colchicine treatment group), cleaning the seeds, soaking the seeds for 4-6h, draining water, transferring to an ultra-clean workbench, treating with 75% alcohol for 1min, sterilizing with 0.1% mercuric chloride for 20min, and cleaning with double distilled water for 3 times.

Accelerating germination: and respectively placing the two groups of sterilized seeds into two groups of sterilized plastic bottles, wherein water-containing absorbent cotton is filled in the plastic bottles, the water content of the absorbent cotton is suitable for meeting the humidity required by seed germination, placing the plastic bottles into a sunlight digital box for dark culture and germination acceleration, wherein the temperature in the sunlight digital box is 35 ℃, and the air humidity is 70%.

And (3) sterilizing colchicine: the colchicine solution with the concentration of 0.2 percent and 0.3 percent is taken and filtered and sterilized for 2 times on a super clean bench by a special filter screen with the unit of 0.22um and a 500 ml sterile needle tube for standby.

And (3) bud body treatment: when the bud induced by the accelerating germination grows to a stem with a height of 4-5cm (about 2-3 node positions), cutting the stem into stem sections with a height of 0.8-1.0cm, removing 1-2 cut leaves at the base of each stem section, stably inserting the stem sections into a subculture medium, adding 2-3ml of the sterile colchicine solution after the treatment on the surface of the subculture medium, dividing the subculture medium into two groups, respectively adding colchicine solutions with concentrations of 0.2% and 0.3%, and using sterile water as a control group, carrying out low-speed shaking culture on a shaking table for 7 days to ensure that the colchicine is fully contacted with the stem sections to increase the effect of the colchicine, and observing that a new bud growing on the node positions expands, shortens and thickens leaf veins. Then selecting the expanded and stout bud for subculture propagation, rooting and domestication.

In the culture process, newly-grown buds are found to change greatly from the 11 th day, the change of 0.2 percent treatment is most obvious, the first leaf base part of the side bud which just grows out is obviously expanded, the part of the conventional seedling, which is contacted with a culture medium, of the base part can be expanded, but the epidermis of the base part is torn quickly to generate callus and the like, the bud which grows out after the upper part of the stem section is induced for 10 days is expanded firstly, then the whole bud body becomes large and smooth, and the bud body can continue to grow without callus and crack.

In this example, the other operations from step S1 to step S4 were performed in a tissue culture chamber under the following culture conditions, except that the training of tissue culture seedlings was performed in a greenhouse: the temperature is 25 +/-2 ℃, the illumination time is 14h, and the relative humidity is 70%; basic culture medium: MS + 3% white granulated sugar + 0.65% agar powder. The formula of the subculture medium for subculture propagation in the present embodiment is as follows: MS +6-BA0.2mg/L + NAA0.05mg/L + 3% white granulated sugar + 0.65% agar powder; the formula of the rooting culture medium used for rooting is as follows: 1/2MS + IAA0.5mg/L + 2% white granulated sugar + 0.65% agar powder.

The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the process of verifying the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all the equivalent structural changes made by applying the content of the specification of the invention should be covered by the scope of the invention.