CN113133408A

CN113133408A - Method for rapidly breeding ginkgo biloba

Info

Publication number: CN113133408A
Application number: CN202110601482.6A
Authority: CN
Inventors: 李艺; 张蕊; 王健; 陈芳淼; 魏常敏; 石娜; 何莉
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-05-31
Filing date: 2021-05-31
Publication date: 2021-07-20
Anticipated expiration: 2041-05-31
Also published as: CN113133408B

Abstract

The invention relates to a method for rapidly breeding ginkgo, which utilizes the characteristic that specific tissue cells of ginkgo have totipotency, and uses special water culture solution to induce specific parts of ginkgo leaves to split and differentiate under non-sterile environment to generate re-rooting and regeneration buds to finally form plants. The rapid propagation method for ginkgo leaves does not need a sterile environment, is convenient to obtain materials, is simple and convenient to operate, has low cost, short period and high survival rate, and has the advantages of high efficiency, economy, easiness in large-scale industrialization and the like; has great application value in the fields of rapid seedling culture of ginkgo, preservation and propagation of specific materials and the like.

Description

Method for rapidly breeding ginkgo biloba

Technical Field

The invention relates to a method for rapidly breeding ginkgo biloba, and belongs to the technical field of rapid plant breeding.

Background

Gingko is a rare tree species of middle-age writings and is a special Chinese product. Ginkgo biloba is a fast-growing precious tree species, and seeds are used for food and medicine. The leaves can be used as medicinal and pesticide preparation, and also as fertilizer. In addition, the ginkgo tree form is beautiful, the leaf color is light green in spring and summer, the leaf color becomes yellow in autumn, and the ginkgo tree form is quite beautiful and can be used as garden trees and street trees. Because of its wide use, ginkgo biloba has been in great demand.

Currently, the reproduction of ginkgo biloba is divided into sexual reproduction and asexual reproduction. And (4) sexual propagation, namely sowing and seedling raising. Vegetative propagation usually uses hardwood cutting, tillering shoot division and tissue culture.

For sexual reproduction, ginkgo trees are divided into male and female plants, the male plants do not bear fruit, and the female plants generally grow to 20 years before setting. In addition, after the seeds are mature in 9-10 months, a series of treatments are carried out, sowing and propagation can be carried out in the next 4-5 months when the temperature is stable about 20 ℃, and the germination rate is only about 60%.

For vegetative propagation, methods such as shoot cutting, tillering, and tissue culture are commonly used. The branches need to be robust and 10-15 cm long in one year in cuttage, a large amount of stalks are consumed, and a large amount of pruning has great influence on the growth and development of the mother plants. Furthermore, there is a strict seasonality. As for the tillering bud division method, there are disadvantages of low propagation coefficient, low survival rate, etc. For tissue culture, strict sterile environment, strict genotype limitation, higher economic cost and seedling hardening treatment are required. In order to meet the great demand of the socioeconomic development on the gingko and overcome the defects of the propagation mode, a rapid propagation mode of the gingko, which is rapid, effective, simple and convenient, needs to be developed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for rapidly breeding gingko, which utilizes the characteristic that specific tissue cells of the gingko have totipotency, uses a special water culture solution to induce the specific parts of gingko leaves to divide and differentiate under a non-sterile environment, generates re-rooting and regeneration buds, and finally forms plants. The invention has the advantages of no need of sterile environment, convenient material taking, simple and convenient operation, low cost, short period, high survival rate, easy large-scale industrialization and the like.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for rapidly breeding gingko comprises the following process steps:

(1) cutting the plant from the stock plant for 10-40 days, which is healthy and has no obvious plant diseases and insect pests and comes from main stem or lateral branch ginkgo leaves; the mode of cutting the blade is as follows: quickly cutting off the blades from top to bottom by using a sharp double-sided blade, wherein the cutting point is positioned at the joint of the base of the petiole and the stem; the base of the isolated ginkgo leaf contains tissues of complete axillary buds and partial stems;

(2) treating in-vitro ginkgo leaves: after cleaning, trimming the petiole to only leave the tissues at the base of the axillary bud;

(3) culturing the treated isolated ginkgo leaves by adopting a two-step method: culturing the treated isolated ginkgo leaf leaves in the special hydroponic liquid 1 for 5 days, and then transferring the isolated ginkgo leaf leaves to the special hydroponic liquid 2 for continuous culture for 25 days;

(4) and transplanting the leaves into an acid matrix after the rooting length of the leaves is equal to or more than 3cm, and performing conventional management.

The specific operation of treating the isolated ginkgo leaves comprises the following steps:

1) cleaning: carefully cleaning the whole leaf with clean tap water for 2-3 times, scrubbing the whole leaf with medical absorbent cotton wetted by distilled water for 2-3 times, and finally cleaning the whole leaf with distilled water for 2-3 times;

2) trimming: rapidly removing tissues of most axillary buds and partial stems by using a sharp double-sided blade, and only leaving tissues at the bases of the axillary buds; exposing the trimmed petioles to the air for 3-4 minutes in a shade; the whole process is to avoid the damage to the blade as much as possible.

And (3) if the leaves treated in the step (2) cannot be immediately subjected to water culture, wrapping the leaves with clean and wet filter paper, wrapping petioles with wet medical absorbent cotton, putting the leaves into a seed germination box, and storing the leaves in a dark place for 2-3 days.

The specific operation of culturing the treated isolated ginkgo leaf by adopting a two-step method is as follows:

1) pouring 2/3 volumes of special hydroponic solution 1 into the seed germination box, and placing a foam plate on the liquid level of the hydroponic solution;

2) placing the treated isolated ginkgo leaf part on a foam plate, and soaking the petiole part in a water culture solution; the seed germination box is covered by a transparent film; the water culture temperature is 20-30 deg.C, and sunlight irradiation or artificial light supplement is carried out at illumination intensity of 3000lux-5000lux for 10 h/d.

3) Culturing in the special hydroponic solution 1 for 5 days, transferring the isolated ginkgo leaves into the special hydroponic solution 2, and continuously culturing for 25 days, wherein the specific operation steps are the same as those of the special hydroponic solution 1; wherein the hydroponic solution is replaced every three days.

The pH of the special hydroponic liquid 1 is 8.0, and the special hydroponic liquid comprises the following components: 5.12mg/LCaCl₂·4H₂O、1.25mg/LFeCl₃·6H₂O、0.0001mg/LH₃BO₃、0.0001mg/LMnSO₄、0.0001mg/L(NH₄)₆Mo₇O₂₄·4H₂O。

The special hydroponic liquid 1 also comprises carbendazim powder with the mass volume ratio of 0.01%.

The pH of the special hydroponic liquid 2 is 5.5, and the special hydroponic liquid comprises the following components: 1.87mg/L potassium nitrate, 3.11mg/L magnesium sulfate, 0.52mg/L monopotassium phosphate and 0.02mg/L ammonium nitrate.

The acidic substrate is acidic pine needle soil and perlite according to the weight ratio of 3: 1, the pH value of the acidic pine needle soil is 5.5-6.5; when transplanting, the distance between the leaf stalk part and the matrix layer is 1.4-1.6 cm.

The invention has the beneficial effects that:

1. at present, the main propagation modes of ginkgo are sowing and cutting. For seeding, the ginkgo is a male and female heteroplant, and the setting rate is not high under natural conditions. In order to increase the maturing rate, pollination needs to be carried out manually, and the target tree is a plant with the age of more than 20 years, which is time-consuming and labor-consuming. More than half a year is required from pollination to maturing of the fruit. The ginkgo seeds also need to be subjected to pregermination treatment. For cuttage, the target material is a semi-woody or fully lignified shoot, and the tender part at the upper part of the shoot is removed. A large amount of materials are consumed, and the growth and development of the stock plant are greatly influenced by a large amount of pruning. The cutting period of the ginkgo is long, and generally more than three months is needed.

The rapid propagation method of ginkgo leaves overcomes the defects of the propagation method, and based on the fact that a local area at the base of the ginkgo leaves has strong regeneration capacity, and the characteristic that specific tissue cells of ginkgo have totipotency is utilized, under the non-sterile environment, the special water culture solution for ginkgo is utilized to induce the specific parts of the ginkgo leaves to split and differentiate, so as to generate re-rooting and regeneration buds, and finally, plants are formed. The invention has the advantages of no need of sterile environment, convenient material acquisition, wide raw material source, simple and convenient operation, low cost, short period, high survival rate, small influence on the growth and development of the stock plant, easy large-scale industrialization and the like. The success rate of forming regeneration seedlings can reach 90%, and the transplanting survival rate of the formed regeneration plants approaches 100%.

2. Regardless of cuttage or tissue culture, the phenomenon of obvious water shortage and withering usually occurs when the gingko explant is just separated from the body, the water culture is beneficial to providing sufficient water for the leaf explant in the early stage of culture and maintaining the normal physiological function of the gingko explant, and the acid environment is a more suitable growth environment for the gingko. Therefore, the invention adopts a two-stage method of water culture and acid matrix culture (PH is 5.5-6.5), which is beneficial to the adaptation of water culture seedlings from water culture to soil culture and obviously improves the survival rate.

3. In open culture, contamination is inevitable. Ginkgo species are rich in polyphenols compared to angiosperms, which cause the growth of certain fungi. Therefore, the proper bacteriostatic agent is added into the hydroponic liquid, so that the pollution in the hydroponic liquid is effectively inhibited, and the normal growth and development of the leaf explant are not influenced. Moreover, the concentration of the hydroponic liquid adopted in the rapid propagation of the existing angiosperm leaves is high, and salt ion stress phenomenon can be generated for gingko, so that the experiment fails. Aiming at the characteristic of the thick leaves of the ginkgo biloba, the selected hydroponic liquid has low component concentration, thereby greatly reducing the salt ion stress of the plant explants and improving the success rate.

4. Due to some protective substances that may be generated by explants that have just been isolated, these substances are concentrated at the wound site. While protecting tissues, the activity of cells can be inhibited, which is one of the main problems in cuttage and tissue culture. Therefore, the present application selects the more alkaline hydroponic liquid 1, which not only helps to dilute the substances, but also reduces the activity of the substances. After three days of water culture, the water culture solution is replaced once, and the inhibition effect of the substances on tissue division and differentiation can be reduced to the minimum. And the combination of the water culture solution 2 with subacidity can further maintain the cell activity and promote division and differentiation.

5. According to the characteristics of gingko species, the invention originally creates a two-step culture method. The hydroponic solution 1 contains ingredients that are missing at the base of ginkgo leaf blades. Without these elements, differentiation behavior of basal cells of ginkgo leaves is not normally initiated. The pH value of the water culture solution 1 is about 8.0, and the environment is favorable for starting the differentiation of basal cells of ginkgo leaves. However, the long-term exposure to such an environment inhibits the regeneration of ginkgo leaves. Therefore, after the ginkgo leaves are cultured in the hydroponic liquid 1 for a period of time, the ginkgo leaves need to be transferred to the hydroponic liquid 2 for culture.

The components contained in the hydroponic solution 2 are essential for cell division at the base of the leaf. The pH value of the hydroponic solution 2 is about 5.5, which is a suitable environment for the division of cells at the basal part of the ginkgo leaves. In summary, for ginkgo material, the hydroponic fluid 1 facilitates the opening of the differentiation of the basal cells of ginkgo leaves, and the hydroponic fluid 2 facilitates the opening of the differentiation of the basal cells of ginkgo leaves.

6. The ginkgo in this application belongs to gymnosperms, which are substantially different in plant structural composition and many physiological properties compared to angiosperms. Ginkgo biloba has been an endangered species in the natural environment for a long time, which indicates that the regeneration capacity of ginkgo biloba is very weak. The method for breeding dicotyledonous angiosperm leaves in the prior art is not suitable for gymnosperms. The rapid propagation method for ginkgo leaves has the advantages of high efficiency, economy, suitability for factory production and the like; has great application value in the fields of rapid seedling culture of ginkgo, preservation and propagation of specific materials and the like.

Drawings

FIG. 1 is a schematic diagram of the state of ginkgo leaves before hydroponic cultivation according to the present invention;

FIG. 2 is a schematic diagram showing the state of ginkgo leaves hydroponically cultured for about 15 days according to the present invention;

FIG. 3 is a schematic diagram showing the state of ginkgo leaves hydroponically cultured for about 30 days according to the present invention;

FIG. 4 is a schematic view showing the leaf status of ginkgo biloba of the present invention after soil culture for about 12 days.

Detailed Description

The following examples further illustrate the embodiments of the present invention in detail.

Example 1

In the embodiment, a ginkgo variety, namely Dongting Huang, is taken as an example, and leaves of the Dongting Huang are utilized to breed ginkgo plants, and the method comprises the following process steps:

(1) cutting folium Ginkgo of Ginkgo variety-Dongting Huang for 10-40 days, which is healthy and has no obvious diseases and insect pests, and is derived from main stem or side branch folium Ginkgo; the mode of cutting the blade is as follows: quickly cutting off the blades from top to bottom by using a sharp double-sided blade, wherein the cutting point is positioned at the joint of the base of the petiole and the stem; the isolated ginkgo leaf base contains tissues of complete axillary buds and partial stems. After sampling, the leaves of the ginkgo biloba are wrapped with wet filter paper and can be stored for 2 to 3 days in the shade if the experiment cannot be started immediately.

(2) The method is used for treating the isolated ginkgo leaves, and comprises the following specific operations:

2) trimming the petiole: rapidly removing tissues of most axillary buds and partial stems by using a sharp double-sided blade, and only leaving tissues at the bases of the axillary buds; exposing the trimmed petioles to the air for 3-4 minutes in a shade; the whole process needs to be careful, and the damage to the blade is avoided as much as possible.

If the treated leaves cannot be immediately subjected to water culture, the leaves can be wrapped by clean and wet filter paper, and the petioles are wrapped by wet medical absorbent cotton and placed in a seed germination box to be stored for 2-3 days in the shade.

(3) Culturing the treated isolated ginkgo leaves by adopting a two-step method, which comprises the following specific operations:

2) placing the treated isolated ginkgo leaf part on a foam plate, and soaking the petiole part in a water culture solution; the seed germination box is covered by a transparent film; the water culture place is not limited, the ventilation of the water culture environment is good, the illumination is good, the water culture is clean, the water culture temperature is 20-30 ℃ (25 ℃ is optimal), sunlight irradiation or artificial light supplement is carried out, the illumination intensity is 3000lux-5000lux, and the illumination time is 10 h/d.

The size of the seed germination box is not limited, and the ginkgo leaves can be flatly laid on the foam board. The container for water culture can be a seed germination box but is not limited to the seed germination box, and any container capable of containing water can be used as the water culture container, and only a layer of light-transmitting film needs to be covered.

3) Culturing in the special hydroponic solution 1 for 5 days, transferring the isolated ginkgo leaves into the special hydroponic solution 2, and continuously culturing, wherein the specific operation steps are the same as those of the special hydroponic solution 1; wherein the hydroponic solution is replaced every three days.

The pH of the special hydroponic liquid 1 is 8.0, and comprises the following steps: 5.12mg/L calcium chloride tetrahydrate (CaCl)₂·4H₂O), 1.25mg/L iron salt solution (FeCl)₃·6H₂O)、0.0001mg/LH₃BO₃、0.0001mg/LMnSO₄、0.0001mg/L(NH₄)₆Mo₇O₂₄·4H₂O; carbendazim powder with the mass volume ratio of 0.01 percent.

The pH of the special hydroponic liquid 2 is 5.5, including: 1.87mg/L potassium nitrate, 3.11mg/L magnesium sulfate, 0.52mg/L monopotassium phosphate and 0.02mg/L ammonium nitrate.

(4) And transplanting the leaves into an acid matrix after the rooting length of the leaves is equal to or more than 3cm, and performing conventional management. The acidic substrate is acidic pine needle soil and perlite according to the weight ratio of 3: 1, the pH value of the acidic pine needle soil is 5.5-6.5; when transplanting, the distance between the leaf stalk part and the matrix layer is 1.4-1.6 cm.

The observation of the whole cultivation process finds that: after about 15 days of water culture, the basal part of the ginkgo leaves is obviously expanded, and a root-shaped object is formed preliminarily. The water culture is carried out for about 30 days, obvious regeneration roots are formed at the basal parts of the ginkgo leaves, and the root length is about 3 cm. After 30 days of water culture and 12 days of soil culture, the basal part of the ginkgo leaf forms obvious regeneration buds, and the regeneration roots develop further (as shown in figures 1-4).

The results show that: by adopting the method, during water culture, the formation of the regenerated roots is quicker, and the formation of the regenerated buds takes longer time. The success rate of forming regeneration seedlings by the method can reach 90 percent. The transplanting survival rate of the regenerated plant formed by the method is close to 100 percent. The invention can quickly and efficiently breed the ginkgo variety Dongting emperor.

Claims

1. A method for rapidly breeding gingko is characterized in that: the method comprises the following process steps:

2. The method of claim 1, wherein the ex vivo ginkgo biloba leaves are treated by:

3. The method of claim 1, wherein if the leaves treated in step (2) cannot be immediately hydroponically cultured, the leaves are wrapped with clean and wet filter paper, and the petioles are wrapped with wet medical absorbent cotton, and placed in a seed germination box and stored in the shade for 2-3 days.

4. The method of claim 1, wherein the culturing the treated isolated ginkgo biloba leaves in a two-step process comprises:

5. The method as claimed in claim 1, wherein the pH of the special hydroponic liquid 1 is 8.0, comprising: 5.12mg/LCaCl₂·4H₂O、1.25mg/LFeCl₃·6H₂O、0.0001mg/LH₃BO₃、0.0001mg/LMnSO₄、0.0001mg/L(NH₄)₆Mo₇O₂₄·4H₂O。

6. The method as claimed in claim 1, wherein the special hydroponic liquid 1 further comprises carbendazim powder with a mass-volume ratio of 0.01%.

7. The method as claimed in claim 1, wherein the pH of the special hydroponic liquid 2 is 5.5, comprising: 1.87mg/L potassium nitrate, 3.11mg/L magnesium sulfate, 0.52mg/L monopotassium phosphate and 0.02mg/L ammonium nitrate.

8. The method of claim 1, wherein the acidic matrix is acidic pine needle soil and perlite in a ratio of 3: 1, the pH value of the acidic pine needle soil is 5.5-6.5; when transplanting, the distance between the leaf stalk part and the matrix layer is 1.4-1.6 cm.