CN112400584A

CN112400584A - Asexual rapid propagation method for dicotyledons

Info

Publication number: CN112400584A
Application number: CN201910764148.5A
Authority: CN
Inventors: 陈芳淼; 李艺
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-08-19
Filing date: 2019-08-19
Publication date: 2021-02-26

Abstract

The invention provides a dicotyledon asexual rapid propagation method, which comprises the following steps: 1) cutting leaves with complete axillary buds from the newly born branches of dicotyledon plants in the current year; 2) removing xylem tissue at the base of the petiole, then putting the leaf into sterile water for cleaning, and taking the trimmed leaf as an explant; 3) spraying the rooting powder solution on the leaf surfaces of the explants once, and then putting the leaf explants into a seed germination box for water culture until petioles root; 4) transplanting the rooted leaf explants to soil culture. The invention establishes a method for rapidly propagating and nursing dicotyledon (such as tea trees, peanuts and cotton) materials by using leaf explants for the first time, and the method is reliable and stable. The dicotyledonous plant leaves used for propagation have sufficient sources, the required materials for experiments are few, the survival rate of the new plants is high, the experimental period is short, and the method has the potential of industrial large-scale production. The invention has great application value in the fields of rapid propagation, popularization, specific material preservation and the like of the fine variety of the tea tree. The invention also has certain reference value for gymnosperms and monocotyledons.

Description

Asexual rapid propagation method for dicotyledons

Technical Field

The invention relates to the field of plant asexual propagation, in particular to a dicotyledonous plant asexual rapid propagation method.

Background

Tea is one of the most important beverage crops in the world, the demand of tea is vigorous day by day along with the improvement of the living standard of people, and the contradiction between supply and demand of high-quality tea seedlings is increasingly prominent. The tea tree propagation includes sexual propagation and asexual propagation. The (sexual) propagation of tea tree seeds refers to a propagation mode of generating seeds by combining male and female gametes of tea trees and utilizing the tea seeds to sow and grow seedlings to propagate offspring, and is also called seed propagation. The vegetative propagation of tea tree is also called vegetative propagation, and is characterized by that it utilizes the root and stem of tea tree to form a new tea seedling, such as cutting and layering, under the proper condition created by artificial cultivation. Asexual propagation is the main means of tea plant propagation, and cuttage is the most common among the asexual propagation.

However, both sexual and asexual methods have major disadvantages. The defects of the sexual reproduction method: 1. because the seed has the genetic characteristics of two parents, the progeny of the seed is often poor in purity, large in plant growth difference, different in growth period and inconvenient to manage. 2. The raw material of the fresh leaves has uneven thickness and tender degree, which is not beneficial to the mastering of processing technology and the production and development of famous tea. 3. For good tea varieties with low dry-out or loose roots, seed propagation is difficult. The defects of the vegetative propagation method: 1. a large amount of pruning and scion shearing have great influence on the growth of the parent trees and the yield of tea leaves; 2. the technical requirements for propagation and planting are high, the labor consumption is high, and the cost is high; 3. the adaptability of tea seedlings is worse than that of seedlings.

Because the demand of high-quality tea seedlings is gradually increased, the existing tea tree propagation method has a plurality of defects, and therefore, a novel tea tree propagation technology needs to be established urgently to achieve the purposes of rapidness, effectiveness, convenience and economy.

Disclosure of Invention

The invention aims to provide a dicotyledon asexual rapid propagation method.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a dicotyledonous plant leaf hydroponic liquid comprising the following components: 1-1.55mg/L boric acid, 2-2.15mg/L zinc sulfate heptahydrate, 0.005-0.00625mg/L copper sulfate pentahydrate, 5-5.58mg/L manganese sulfate, 0.005-0.0625mg/L ammonium molybdate tetrahydrate, 0.5-0.695mg/L EDTA iron sodium salt, 200-236.25mg/L calcium nitrate tetrahydrate, 120-123.25mg/L magnesium sulfate heptahydrate, 15-18.695mg/L ammonium phosphate and 5.8-6.3 of pH value.

Preferably, the hydroponic liquid comprises the following components: 1.55mg/L boric acid, 2.15mg/L zinc sulfate heptahydrate, 0.00625mg/L copper sulfate pentahydrate, 5.58mg/L manganese sulfate, 0.0625mg/L ammonium molybdate tetrahydrate, 0.695mg/L EDTA iron sodium salt, 236.25mg/L calcium nitrate tetrahydrate, 123.25mg/L magnesium sulfate heptahydrate, 18.65mg/L ammonium phosphate and pH value of 6.0.

In a second aspect, the present invention provides a method for rapid asexual propagation of dicotyledons, comprising the steps of:

1) cutting leaves with complete axillary buds from the newly born branches of dicotyledon plants in the current year;

2) removing xylem tissue at the base of the petiole, then putting the leaf into sterile water (distilled water) for cleaning, and taking the trimmed leaf as an explant;

3) spraying the rooting powder solution on the leaf surfaces of the explants once, and then putting the leaf explants into a seed germination box for water culture until petioles root;

4) transplanting the rooted leaf explants to soil culture;

wherein, the dicotyledonous plant leaf hydroponic solution is adopted in the step 3) water culture; the rooting powder solution comprises the following components: 8-10mL/L (preferably 10mL/L), and the solvent is distilled water. The rooting powder (indbutyl-naphthylacetic acid, 5% soluble agent) is shown in the registration certificate number of the pesticide: PD20110559, available from Guanghong agriculture chemical Co., Ltd, Sichuan.

In the present invention, the dicot includes, but is not limited to, tea, peanut (dicotyledonous herbaceous) and cotton (dicotyledonous woody).

The hydroponic method in step 3) is as follows: the seed germination box contains water culture solution with the volume of 2/3, a foam plate special for water culture is placed in the seed germination box, a leaf explant is placed on foam, a petiole is covered by sterile absorbent cotton (medical absorbent cotton), and the other end of the medical absorbent cotton is immersed in the water culture solution; then, the seed germination box is placed into a proper self-sealing bag, and after 2 days of light-shielding culture at 20-30 ℃, the seed germination box is cultured under the condition of weak light. In the whole water culture process, ultraviolet treatment is avoided for the leaves.

In the foregoing method, the low light condition is: low light irradiation or artificial fluorescent lamp irradiation with the light intensity of 200 mu mol^-2s^-1The culture time is 10-12 h.

In the method, during the water culture period in the step 3), the rooting powder solution is sprayed on the leaf surfaces once according to the actual requirement.

In the method, in the step 4), when the rooting length of the petiole reaches more than 3 cm, soil culture is carried out. Strong light irradiation is avoided as much as possible within one week after the soil culture, and meanwhile, water spraying treatment is carried out on the leaves.

In the method, during the water culture in the step 3), a proper amount of sterile water is timely supplemented into the seed germination box to the initial volume of the water culture solution according to the evaporation condition of the water culture solution. To compensate for losses due to evaporation. And the hydroponic solution is replaced irregularly according to the requirement.

According to actual needs, ultraviolet disinfection is carried out on the water culture chamber at irregular intervals, and the water culture chamber is cleaned by absolute ethyl alcohol. And (3) carrying out ultraviolet sterilization on the self-sealing bag, the seed germination box, the foam board, the water culture solution and the medical absorbent cotton for 1 hour at irregular intervals. The ultraviolet irradiation intensity is 90 mu W/cm²。

In the method, the substrate used in the soil culture in the step 4) is not limited, and can be from a field or a mixture of red soil and vermiculite according to a volume ratio of 5: 1. After transplanting, the seedlings are protected from light for a week, and the overground parts are sprayed with clear water in time.

In the method, the spraying amount of the rooting powder solution in the step 3) is 150 μ L per leaf according to the area of the leaf.

Preferably, the dicotyledonous plant is tea plant 'Xinyang Maojian', peanut 'Yuanza 9102', cotton 'Yinshan No. 7'.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the invention firstly utilizes the leaf explant to establish the method for rapidly propagating and nursing tea plant materials, and the method is reliable and stable.

The explant material used by the invention is leaves, the tea tree is perennial shrub or small tree, and each tea tree has extremely many leaves. Therefore, the source of the blade is sufficient.

The invention adopts the strategy of water culture, induced rooting and soil culture, thereby greatly improving the survival rate of the regenerated seedlings. The statistical results show that: the rooting rate exceeds 85%, the transplanting survival rate exceeds 80%, and the seedling rate exceeds 60%.

Fourthly, the asexual rapid propagation method of the dicotyledons provided by the invention has low cost, seed germination boxes, special foam plates for water culture and transparent valve bags can be purchased from the market, and the materials can be recycled; the water culture solution can be prepared by self; rooting powders are also commercially available.

Compared with the traditional cuttage technology, the method has obvious advantages. a. Branches are needed for cuttage, and pruning can cause great influence on the growth and development of the parent plant of the tea tree, particularly rare tea tree varieties. And (4) taking the leaves, and having relatively small influence on the normal growth of branches. b. The cut branches require a disinfection treatment in advance. The material selected by the invention does not need disinfection treatment. The cost is further saved while the operation flow is simplified. c. The branch of cuttage usually has a plurality of leaves, but the invention only needs the leaves, has saved the material effectively. d. The method has short period, and the water culture rooting of the leaves only needs about 38 days. The conventional branch cutting rooting needs 60-90 days.

And (VI) compared with the traditional tissue culture technology, the invention also has obvious advantages. a. Tissue culture techniques require a strict sterile environment. Special pre-treatment room, tissue culture room and seedling hardening room are needed to be established. The invention does not need to establish a special pretreatment room, a tissue culture room and a seedling hardening room. b. The tissue culture technology has a complex operation flow, and operators need to have a high technical level. The method is simple to operate, and has low requirements on the technical level of operators. c. The cost required for tissue culture techniques is high. The invention simplifies the operation flow and further saves the cost. d. The method has short period, and the water culture rooting of the leaves only needs about 38 days. Conventional tissue culture takes about 90 days.

The method has wide application range, and is suitable for tea trees and some dicotyledonous plants (such as peanuts and cotton).

Drawings

FIG. 1 shows leaves of tea tree which are hydroponically cultured for about 10 days in example 1 of the present invention.

FIG. 2 shows leaves of tea tree which are hydroponically cultured for about 33 days in example 1 of the present invention.

FIG. 3 shows leaves of tea tree which are hydroponically cultured for about 52 days in example 1 of the present invention.

FIG. 4 shows peanut leaves hydroponically cultured for about 14 days in example 2 of the present invention.

FIG. 5 shows leaves of cotton cultivated in water for about 12 days in example 3 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Example 1 Rapid propagation of tea plants Using leaf explants

Test materials: tea trees from Xinyang Luoshan county, Henan Xinyang Maojian, newly grown branches in the same year, no diseases and insects, semi-ligneous branches, full axillary buds, yellow green with purple and hardened branches.

The rapid propagation of the tea tree leaves comprises the following steps:

1. preparing a hydroponic solution and a rooting powder solution:

water culture solution: 1.55mg/L boric acid, 2.15mg/L zinc sulfate heptahydrate, 0.00625mg/L copper sulfate pentahydrate, 5.58mg/L manganese sulfate, 0.0625mg/L ammonium molybdate tetrahydrate, 0.695mg/L EDTA iron sodium salt, 236.25mg/L calcium nitrate tetrahydrate, 123.25mg/L magnesium sulfate heptahydrate, 18.65mg/L ammonium phosphate and pH value of 6.0. Prepared with sterilized distilled water.

Preparing a rooting powder solution: the concentration of the solution was 10 mL/L. The preparation method comprises the following steps: 10mL of rooting powder solution (Inutan-naphthylacetic acid, 5% soluble solution, registration No.: PD20110559, available from Sichuan national photo-agriculture Co., Ltd.) was mixed with sterilized distilled water to a total volume of 1L.

2. Preparation before water culture:

the hydroponic chamber was uv-sterilized for 1 hour and then thoroughly cleaned once with absolute alcohol.

And (3) washing the seed germination box, the special foam plate for water culture and the transparent self-sealing bag with distilled water for 2 times, and then carrying out ultraviolet sterilization for 1 hour together with the water culture solution. Then the water culture solution is poured into the seed germination box till the volume of the germination box is two thirds. Then foam is put in, and the foam floats on the water culture solution.

3. Cutting leaves (with complete axillary buds) on branches of tea plants;

the requirement of branches: the new growth in the same year, no diseases and pests, half-wood, full axillary buds, yellow green with purple and hard branches.

And (4) selecting complete, good-growth-potential and healthy leaves, and quickly cutting off the leaves from bottom to top (keeping the axillary buds intact) by using a sharp blade.

4. And (5) trimming the petiole. And (3) trimming the in vitro leaf blade by using a dissecting microscope to remove xylem tissues at the base of the petiole as much as possible. The leaves were then washed 3-5 times in distilled water.

5. And (3) putting the trimmed leaves on a foam board in order, covering the base parts of the petioles with wet medical absorbent cotton, and putting the other end of cotton wool into the water culture solution.

6. After the water on the leaves is evaporated, spraying the rooting powder solution of 10mL/L on the leaves once. The seed germination boxes were then placed in transparent self-sealing bags. The first 2 days were incubated in the dark and subsequently under low light conditions. The illumination intensity is 200 mu mol^-2s^-1。

7. In the water culture process, sterilized distilled water needs to be added in time to make up for the loss of evaporation. And the hydroponic solution is replaced irregularly according to the requirement.

8. During the water culture process, the 10mL/L rooting powder solution can be sprayed on the leaf surface once according to the actual requirement.

9. The hydroponic chamber was uv sterilized at irregular intervals and cleaned with anhydrous alcohol as required. And (3) carrying out ultraviolet sterilization on the self-sealing bag, the seed germination box, the foam board, the water culture solution and the medical absorbent cotton for 1 hour at irregular intervals.

10. In the whole water culture process, ultraviolet treatment is avoided for the leaves.

11. Transplanting when the leaves root and the adventitious root grows to be more than 3 cm, and carrying out soil culture. The soil culture medium is not limited. Can come from field, or red soil: vermiculite (5:1 volume ratio). After transplanting, the seedlings are protected from light for a week, and the overground parts are sprayed with clear water in time.

Tea plant leaves cultured by water for about 10 days are shown in figure 1, tea plant leaves cultured by water for about 33 days are shown in figure 2, and tea plant leaves cultured by water for about 52 days are shown in figure 3. The statistical results show that: the rooting rate exceeds 85%, the transplanting survival rate exceeds 80%, and the seedling rate exceeds 60%.

The invention establishes a rapid propagation system which is completely different from the traditional asexual propagation means of tea trees. The traditional asexual propagation means of tea trees are two types: first, tissue culture. The method has the defects of long period, high cost, strict aseptic environment, and the need of establishing a special pretreatment room, a tissue culture room and a seedling hardening room. The operator needs to have a high level of skill. And secondly, branch cuttage. The disadvantages of this method are long period and low survival rate. It requires a high consumption of raw materials. The invention better overcomes the defects of the two modes.

The dicotyledon asexual rapid propagation method provided by the invention has the main advantages that: the tea leaves used for propagation are abundant in source; the material needed by the experiment is less; the survival rate of the new plant is high; strict external environment is not required in the experiment; the experimental period is short; has the potential of industrial large-scale production. The invention has great application value in the fields of rapid propagation, popularization, specific material preservation and the like of the fine variety of the tea tree.

Example 2 Rapid propagation of peanuts Using leaf explants

Test materials: peanut variety 'Yuanza 9102', the seedling is newly grown in the same year.

The peanut leaf rapid propagation method comprises the following steps:

1. preparing a hydroponic solution and a rooting powder solution:

2. Preparation before water culture:

3. Cutting leaves (with complete axillary buds) of peanut seedlings;

and (4) selecting complete and healthy leaves (with the leaf age of 8-35 days), and quickly cutting off the leaves from bottom to top by using a sharp blade (keeping axillary buds intact).

11. Transplanting when the leaves root and the adventitious root grows to be more than 3 cm, and carrying out soil culture. The soil culture medium is not limited. Can come from field, or red soil: vermiculite (5:1 volume ratio). After transplanting, the seedlings are protected from light for a week, and the overground parts are sprayed with clear water in time. Peanut leaves cultured in water for about 14 days are shown in figure 4. The statistical results show that: the rooting rate exceeds 92%, the transplanting survival rate exceeds 87%, and the seedling rate exceeds 85%.

Example 3 Rapid propagation of Cotton Using leaf explants

Test materials: the cotton variety 'Yinshan No. 7', which is a new branch in the current year, has no diseases and pests, is semi-woody, has plump axillary buds and is slightly hardened green.

The cotton leaf rapid propagation method comprises the following steps:

1. preparing a hydroponic solution and a rooting powder solution:

2. Preparation before water culture:

3. Cutting leaves (with complete axillary buds) on peanut branches;

and (4) selecting complete and healthy leaves (with the leaf age of 5-35 days), and quickly cutting off the leaves from bottom to top by using a sharp blade (keeping axillary buds intact).

The leaves of cotton cultured in water for about 12 days are shown in FIG. 5. The statistical results show that: the rooting rate exceeds 95%, the transplanting survival rate exceeds 92%, and the seedling rate exceeds 88%.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A dicotyledonous plant leaf hydroponic fluid, which is characterized by comprising the following components: 1-1.55mg/L boric acid, 2-2.15mg/L zinc sulfate heptahydrate, 0.005-0.00625mg/L copper sulfate pentahydrate, 5-5.58mg/L manganese sulfate, 0.005-0.0625mg/L ammonium molybdate tetrahydrate, 0.5-0.695mg/L EDTA iron sodium salt, 200-236.25mg/L calcium nitrate tetrahydrate, 120-123.25mg/L magnesium sulfate heptahydrate, 15-18.695mg/L ammonium phosphate and 5.8-6.3 of pH value;

2. The dicotyledon asexual rapid propagation method is characterized by comprising the following steps:

2) removing xylem tissue at the base of the petiole, then putting the leaf into sterile water for cleaning, and taking the trimmed leaf as an explant;

4) transplanting the rooted leaf explants to soil culture;

wherein, the step 3) water culture adopts the water culture solution of claim 1; the concentration of the rooting powder solution is 8-10mL/L, and the solvent is distilled water;

the rooting powder is shown in a pesticide registration certificate number: PD20110559, available from Guanghong agro-chemical Co., Ltd, Sichuan;

the dicotyledonous plants are tea trees, peanuts and cotton.

3. The method according to claim 2, wherein the hydroponic method in step 3) is as follows: the seed germination box contains water culture solution with the volume of 2/3, a foam plate special for water culture is placed in the seed germination box, a leaf explant is placed on the foam, a petiole is covered by sterile absorbent cotton, and the other end of medical absorbent cotton is immersed in the water culture solution; then, the seed germination box is placed into a proper self-sealing bag, and after 2 days of light-shielding culture at 20-30 ℃, the seed germination box is cultured under the condition of weak light.

4. The method according to claim 3, wherein the low light condition in step 3) is: the illumination intensity is 200 mu mol^-2s^-1The culture time is 10-12 h.

5. The method as claimed in claim 2, wherein during the water culture in step 3), the rooting powder solution is sprayed on the leaf surface once according to actual needs.

6. The method according to claim 2, wherein in step 4), when the rooting length of the petiole reaches more than 3 cm, soil culture is performed.

7. The method of claim 3, wherein during hydroponic culture in step 3), the seed germination boxes are replenished with an appropriate amount of sterile water to the original volume of hydroponic liquid in time, depending on the depletion of hydroponic liquid.

8. The method according to claim 2, wherein the substrate used in the step 4) of soil culture is a mixture of red soil and vermiculite in a volume ratio of 5: 1.

9. The method as claimed in any one of claims 2 to 8, wherein the amount of the rooting powder solution sprayed in step 3) is 100 μ L/leaf area.

10. The method according to any one of claims 2 to 8, wherein the dicotyledonous plant is tea plant species 'Xinyang Maojian', peanut species 'Yuanza 9102', cotton species 'Yinshan No. 7'.