阅读说明：本技术 一种茄子砧木试管微扦插无菌繁殖的方法 (Test tube micro-cuttage aseptic propagation method for eggplant rootstocks ) 是由 于文进 张映卿 钟川 阳燕娟 于 2019-09-12 设计创作，主要内容包括：本发明公开了一种茄子砧木试管微扦插无菌繁殖的方法,所述方法包括：无菌苗获得、芽诱导培养、继代增殖培养、生根培养、炼苗移栽。芽诱导的培养基为MS+KT0.4～0.6mg/L+IBA0.05～0.15mg/L,继代增殖培养基为MS+IBA0.35～0.45mg/L,生根培养基为1/2MS+IBA0.15～0.25mg/L。本发明以茎段为外植体,所用外植体简单易取；植物生长调节剂配比简单,所用试剂价格低廉,成本低；芽诱导率达90％,15d能长出1cm左右的侧芽；继代增殖30d,增殖系数达5～6倍,生根效果明显,移栽成活高。本发明建立了水茄整个组培快繁技术体系,整个生产过程周期短,操作简便,增殖倍数高,完全能满足大规模化种苗繁育的需求,具有良好的应用前景。(The invention discloses a test tube micro-cuttage aseptic propagation method for eggplant stocks, which comprises the following steps: obtaining aseptic seedlings, bud induction culture, subculture proliferation culture, rooting culture and hardening seedling transplantation. The bud induction culture medium is MS + KT 0.4-0.6 mg/L + IBA 0.05-0.15 mg/L, the subculture multiplication culture medium is MS + IBA 0.35-0.45 mg/L, and the rooting culture medium is 1/2MS + IBA 0.15-0.25 mg/L. The stem section is used as the explant, and the used explant is simple and easy to take; the plant growth regulator has simple proportioning, low price of the used reagent and low cost; the bud inductivity reaches 90 percent, and lateral buds about 1cm can grow after 15 days; subculture multiplication is carried out for 30 days, the multiplication coefficient reaches 5-6 times, the rooting effect is obvious, and the survival rate of transplanting is high. The invention establishes the whole tissue culture and rapid propagation technical system of the solanum torvum, has short period of the whole production process, simple and convenient operation and high multiplication factor, can completely meet the requirement of large-scale seedling propagation and has good application prospect.)

1. A method for the test tube micro-cuttage aseptic propagation of eggplant stocks is characterized by comprising the following steps: the method comprises the following steps:

(1) Obtaining aseptic seedlings: pretreating solanum torvum seeds, sowing the solanum torvum seeds on the surface of an MS culture medium, and obtaining primary sterile seedlings when the seeds germinate and grow to 3-4 true leaves;

(2) Inducing lateral buds: taking the stem segment of the aseptic seedling obtained in the step (1) as an explant, cutting the stem segment of the side bud of the aseptic seedling into 0.5cm in a superclean bench by using a sterilization blade, inoculating the stem segment of the side bud of the aseptic seedling into a bud induction culture medium in a micro-cuttage mode, and culturing until the side bud grows into 5-6 true leaves;

(3) subculture multiplication culture: micro-cuttage is carried out on the stem segments growing out the buds in the step (2) into a subculture multiplication culture medium, the buds can be continuously multiplied in the culture medium, subculture is carried out every 25-30 days, and the step is repeated to realize mass multiplication;

(4) Rooting culture: cutting the buds propagated in the step (3) when the buds grow to 1-2 cm long into a rooting culture medium for rooting culture, and obtaining tissue culture seedlings for hardening seedling and transplanting after 25-30 d of rooting;

(5) hardening and transplanting seedlings: transferring the tissue culture seedlings obtained in the step (4) from culture to natural light for hardening for 3 days, opening a bottle cover, continuing hardening for 3 days, taking out the plants, washing a root culture medium with clear water, punching a hole in the substrate, implanting the tissue culture seedlings, covering the substrate, slightly compacting to enable the plants to be upright, and keeping the substrate moist.

2. The method for test-tube micro-cutting aseptic propagation of eggplant stocks according to claim 1, characterized in that: in step (1), the pretreatment is: soaking solanum torvum seeds in 1g/L gibberellin for 3 hours, washing with sterile water on a super clean workbench for 3 times, soaking with 70% ethanol for 15 seconds, sterilizing with 0.1% HgCl2 for 10min, and washing with sterile water for 5-6 times.

3. the method for test-tube micro-cutting aseptic propagation of eggplant stocks according to claim 1, characterized in that: in the step (2), the bud induction culture medium is MS culture medium added with 0.4-0.6 mg/L, IBA 0.05.05-0.15 mg/L of KT, 25-32 g/L of sucrose, 4.5-5.0 g/L of agar, and pH is 5.8-6.0.

4. The method for test-tube micro-cutting aseptic propagation of eggplant stocks according to claim 1, characterized in that: in the step (3), the subculture multiplication medium is MS medium added with 0.35-0.45 mg/L of IBA, 25-32 g/L of sucrose, 4.5-5.0 g/L of agar and with the pH value of 5.8-6.0.

5. The method for test-tube micro-cutting aseptic propagation of eggplant stocks according to claim 1, characterized in that: in the step (4), 0.15-0.25 mg/L of IBA, 25-32 g/L of cane sugar, 4.5-5.0 g/L of agar and pH 5.8-6.0 are added into 1/2MS culture medium.

6. The method for test-tube micro-cutting aseptic propagation of eggplant stocks according to claim 1, characterized in that: in step (5), the substrate is peat: coconut husk: perlite is 3: 2: 1.

7. The method for test-tube micro-cutting aseptic propagation of eggplant stocks according to claim 1, characterized in that: in the culture process, the temperature of the culture room is set to be 25 +/-2 ℃, the relative humidity is set to be 50-60%, the illumination time is 16h/d, and the illumination intensity is 2000-3000 lx.

Technical Field

the invention relates to the technical field of plant tissue culture and rapid propagation, in particular to a test tube micro-cuttage aseptic propagation method for eggplant stocks.

background

solanum torvum (Solanum torvum), also called "Toolubam", is a closely related wild species of the cultivated species eggplant (Solanum melogena) and is native to America. The plant has high resistance to bacterial wilt, verticillium wilt, blight, root-knot nematode and other main soil-borne diseases and pests, and is a commonly used grafting stock in eggplant and tomato production.

although the growth potential of the plant is extremely strong, the solanum torvum seeds are extremely small and have low endogenous hormone content, so that the solanum torvum seeds have extremely strong dormancy after being mature, and the normally sown solanum torvum seeds bud for about one month. After the germination and the soil breaking, the seedlings grow very slowly in the initial stage, and particularly the growth speed is nearly stopped under the low-temperature condition.

Due to the problems of long seedling age, low germination rate, germination vigor and germination index, slow seedling growth speed and the like of the solanum torvum bunge, the large-scale application of the solanum torvum bunge on industrial seedling culture is limited, so that the development of other methods for improving the seedling culture efficiency of the solanum torvum bunge and reducing the seedling culture cost is urgently needed.

disclosure of Invention

The invention aims to: aiming at the problems, the method for the test tube micro-cuttage sterile propagation of the eggplant stocks is provided, the method is simple to operate, low in production cost, easy to obtain explants, high in multiplication coefficient and short in growth period, and is an effective way for obtaining a large number of solanum torvum in a short time.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a method for the test tube micro-cuttage aseptic propagation of eggplant stocks comprises the following steps:

(1) obtaining aseptic seedlings: pretreating solanum torvum seeds, sowing the solanum torvum seeds on the surface of an MS culture medium, and obtaining primary sterile seedlings when the seeds germinate and grow to 3-4 true leaves;

(2) Inducing lateral buds: taking the stem segment of the aseptic seedling obtained in the step (1) as an explant, cutting the stem segment of the side bud of the aseptic seedling into 0.5cm in a superclean bench by using a sterilization blade, inoculating the stem segment of the side bud of the aseptic seedling into a bud induction culture medium in a micro-cuttage mode, and culturing until the side bud grows into 5-6 true leaves;

(3) Subculture multiplication culture: micro-cuttage is carried out on the stem segments growing out the buds in the step (2) into a subculture multiplication culture medium, the buds can be continuously multiplied in the culture medium, subculture is carried out every 25-30 days, and the step is repeated to realize mass multiplication;

(4) rooting culture: cutting the buds propagated in the step (3) when the buds grow to 1-2 cm long into a rooting culture medium for rooting culture, and obtaining tissue culture seedlings for hardening seedling and transplanting after 25-30 d of rooting;

(5) hardening and transplanting seedlings: transferring the tissue culture seedlings obtained in the step (4) from culture to natural light for hardening for 3 days, opening a bottle cover, continuing hardening for 3 days, taking out the plants, washing a root culture medium with clear water, punching a hole in the substrate, implanting the tissue culture seedlings, covering the substrate, slightly compacting to enable the plants to be upright, and keeping the substrate moist.

preferably, in step (1), the pretreatment is: soaking solanum torvum seeds in 1g/L gibberellin for 3 hours, washing with sterile water on a super clean workbench for 3 times, soaking with 70% ethanol for 15 seconds, sterilizing with 0.1% HgCl2 for 10min, and washing with sterile water for 5-6 times.

Preferably, in step (2), the bud induction medium is MS medium added with 0.4-0.6 mg/L, IBA 0.05.05-0.15 mg/L of KT, 25-32 g/L of sucrose, 4.5-5.0 g/L of agar, and pH 5.8-6.0.

Preferably, in the step (3), the subculture multiplication medium is MS medium added with 0.35-0.45 mg/L IBA, 25-32 g/L sucrose, 4.5-5.0 g/L agar, and pH 5.8-6.0.

Preferably, in the step (4), 0.15-0.25 mg/L of IBA, 25-32 g/L of sucrose, 4.5-5.0 g/L of agar and pH 5.8-6.0 are added into the 1/2MS culture medium.

Preferably, in step (5), the substrate is peat: coconut husk: perlite is 3: 2: 1.

preferably, in the culture process, the temperature of the culture chamber is set to be 25 +/-2 ℃, the relative humidity is set to be 50-60%, the illumination time is 16h/d, and the illumination intensity is set to be 2000-3000 lx.

the pH value of the culture medium is adjusted by 1mol/L HCl or NaOH.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

(1) the stem section is used as the explant, and the used explant is simple and easy to take; the plant growth regulator has simple proportioning, low price of the used reagent and low cost; the bud inductivity reaches more than 90 percent, and side buds about 1cm can grow after 15 days; subculture multiplication is carried out for 30 days, the multiplication coefficient reaches 5-6 times, the rooting effect is obvious, and the survival rate of transplanting is high.

(2) the invention establishes the whole tissue culture and rapid propagation technical system of the solanum torvum, has short period of the whole production process, simple and convenient operation and high multiplication factor, can completely meet the requirement of large-scale seedling propagation and has good application prospect.

drawings

FIG. 1 is a schematic diagram of the induction of Solanum torvum buds;

FIG. 2 is a schematic diagram of the subculture proliferation of Solanum torvum;

FIG. 3 is another schematic diagram of the subculture proliferation of Solanum torvum;

FIG. 4 is a schematic view of the rooting effect of Solanum torvum.

Detailed Description

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below with reference to preferred embodiments. It should be noted, however, that the numerous details set forth in the description are merely for the purpose of providing the reader with a thorough understanding of one or more aspects of the present invention, which may be practiced without these specific details.