CN114080980B

CN114080980B - Tissue culture method of Ottelia cordifolia Jackfost

Info

Publication number: CN114080980B
Application number: CN202111328119.8A
Authority: CN
Inventors: 高丽; 辛培培; 杨聪儿; 石俊; 刘小妹; 王芳; 钱蕾
Original assignee: Beijing Huaxiang Flower Science And Technology Research Institute Co ltd; Beijing Green Garden Group Co ltd
Current assignee: Beijing Huaxiang Flower Science And Technology Research Institute Co ltd; Beijing Green Garden Group Co ltd
Priority date: 2021-11-10
Filing date: 2021-11-10
Publication date: 2022-06-24
Anticipated expiration: 2041-11-10
Also published as: CN114080980A

Abstract

The application relates to the technical field of in-vitro plant culture, and particularly discloses a heart leafAll-grass of Bull tongueJack FrostThe tissue culture method of (1). The method specifically comprises the following steps: obtaining and disinfecting an explant, inducing the explant to form a callus and an adventitious bud, proliferating the adventitious bud and inducing the adventitious bud to root; wherein in the step of obtaining and disinfecting the explant, the explant selects a new leaf of a mother plant which grows vigorously and has no plant diseases and insect pests; and provides a culture medium formula used in inducing the explant to form callus and adventitious buds, proliferating the adventitious buds and inducing the adventitious buds to root. The application can effectively improve the herba Hedyotidis DiffusaeJack FrostThe breeding efficiency of the Chinese medicinal herb is improvedJack FrostThe use of (1).

Description

Tissue culture method of Ottelia cordifolia Jack Frost

Technical Field

The application relates to the technical field of in vitro plant culture, in particular to a tissue culture method of Ottelia cordifolia Jack Frost.

Background

Herba Hedyotis Auriculariae Jack Frost (Brunnera macrophylla Jack Frost), belonging to family Boraginaceae, genus Hedyotis, is perennial herb, clumpy, and hemispheric; the leaves are oval heart-shaped, and the color of the leaves is frosty silver with green veins; the leaves and the leaf backs are dense, white and soft. The plant of the variety has higher ornamental value, is suitable for the arrangement of the floriculture prospect in the shade land, the matching planting of the stream waterscape, the cluster planting on the roadside, the planting in the garden of the private garden and the potted plant ornamental, has wide application range and good market prospect.

The herba Hedyotidis Diffusae Jack Frost is pleased with cool and humid climate, is pleased with loose sandy loam with good drainage, is resistant to half-shade, and can grow well under tree shade condition. At present, the breeding mode of the Ottelia cordifolia Jack Frost is mainly seeding breeding and plant division breeding, the breeding mode is more traditional, and the breeding efficiency is lower. And the ardisia gracilis Jack Frost is forbidden to be high temperature and high humidity and is forbidden to be directly irradiated by strong light for a long time, so the breeding efficiency of the ardisia gracilis Jack Frost is influenced by seasonal alternation, and the breeding efficiency of the ardisia gracilis Jack Frost in a high-temperature season is lower.

For the related technologies, the inventor considers that, on one hand, the traditional propagation technology depended on by the current herba hedyotis diffusae Jack Frost makes the propagation efficiency of the herba hedyotis diffusae Jack Frost lower, so that the application of the herba hedyotis diffusae Jack Frost is obviously limited by depending on the current propagation technology of the herba hedyotis diffusae Jack Frost; on the other hand, under the influence of seasonal variation, the reproductive efficiency of the Okins cardinalis Jack Frost becomes lower, thereby further limiting the application of the Okins cardinalis Jack Frost.

Disclosure of Invention

In order to improve the propagation efficiency of the Ottelia cordifolia Jack Frost and promote the application of the Ottelia cordifolia Jack Frost, the application provides a tissue culture method of the Ottelia cordifolia Jack Frost.

The tissue culture method of Ottelia cordifolia Jack Frost provided by the application adopts the following technical scheme:

a tissue culture method of Ottelia cordifolia Jack Frost, the tissue culture method comprises the following steps: obtaining and disinfecting an explant, inducing the explant to form a callus and an adventitious bud, proliferating the adventitious bud and inducing the adventitious bud to root; wherein the content of the first and second substances,

in the step of obtaining and disinfecting the explant, selecting new leaves of a mother plant which grows strongly and has no plant diseases and insect pests from the explant;

in the step of inducing the explant to form callus and adventitious buds, the formula of the used culture medium is as follows: MS culture medium, 6-BA 1.0-2.0mg/L, NAA 0.1-0.5mg/L, sucrose 25-30g/L, agar 4.5-5g/L, pH value is 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-;

in the step of adventitious bud proliferation, the formula of the culture medium is as follows: MS culture medium, 6-BA 1.0-2.0mg/L, IBA 0.1-0.5mg/L, sucrose 25-30g/L, agar 4-4.5g/L, pH value is 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-1800Lux, and the illumination time is 8-10 h/d;

in the step of inducing adventitious buds to grow roots, the formula of the used culture medium is as follows: 1/2MS culture medium, AC 0-0.5g/L, sucrose 25-30g/L, agar 4-4.5g/L, pH 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-1800Lux, and the illumination time is 8-10 h/d.

By adopting the technical scheme, in the tissue culture of the Ottelia cardiocarpa Jack front, the new leaf of the stock plant which grows robustly and has no plant diseases and insect pests is selected as the explant, and the culture medium formula used in the steps of inducing the explant to form callus and adventitious buds, proliferating the adventitious buds and inducing the adventitious buds to root is provided. The tissue culture method of the Ottelia cordifolia Jack Frost provided by the application can improve the propagation efficiency of the Ottelia cordifolia Jack Frost, reduce the limitation of seasons on the production of the Ottelia cordifolia Jack Frost, and provide a new attempt for the blank Ottelia cordifolia Jack Frost plant tissue culture field at present.

In the tissue culture of the ardisia cordifolia Jack Frost provided by the application, after the disinfection treatment of the new leaves taken from the robust and disease and pest free ardisia cordifolia Jack Frost stock plant, the induction of the new leaves is carried out by utilizing the culture medium for inducing the explants to form callus and adventitious buds, under the combined action of the culture medium for inducing the explants to form callus and adventitious buds and the proper culture environment, the new leaves taken as the explants are dedifferentiated and redifferentiated to form callus, the callus is the cell with meristematic capacity, and the redifferentiation of the callus is further induced to form adventitious buds. And (2) separating the adventitious buds on the callus into single adventitious buds, inducing the single adventitious buds by using a culture medium for inducing the proliferation of the adventitious buds, and proliferating the single adventitious buds to form cluster adventitious buds under the combined action of the culture medium for inducing the proliferation of the adventitious buds and a proper culture environment. And (3) separating the cluster adventitious buds into single plants again, inducing the single plants by using a culture medium for inducing the adventitious buds to root, and forming rooted plantlets by the single plants under the combined action of the culture medium for inducing the adventitious buds to root and a proper culture environment.

The selection of a proper mother plant part as an explant for plant tissue culture can directly influence the final result of the Jack Frost tissue culture of the Ottelia cordifolia. This application selects new leaf of heart leaf spring Jack Frost to carry out tissue culture as the explant, on the one hand, other positions of selecting heart leaf spring Jack Frost carry out tissue culture as the explant, new leaf is drawn materials more easily, and the injury of drawing materials to the stock plant of new leaf is less, is favorable to the protection to the stock plant more, and reuse to the stock plant in tissue culture, thereby improve tissue culture's reproductive efficiency, reduce tissue culture's cost. On the other hand, the new leaves have vigorous cell division activity, can improve the induction rate and the success rate of tissue culture to a certain extent, can effectively shorten the induction period in the process of tissue culture, and can improve the multiplication coefficient and the rooting rate. Therefore, the new leaves of the mother plant which grows robustly and has no plant diseases and insect pests are selected as the explant to carry out tissue culture to breed the Ottelia cordifolia Jack Frost, the breeding efficiency of the Ottelia cordifolia Jack Frost can be effectively improved, the limitation of seasons on the production of the Ottelia cordifolia Jack Frost is reduced, and the application of the Ottelia cordifolia Jack Frost is promoted.

Explants were induced to form callus and adventitious shoots in medium using 6-BA and NAA as phytohormones. 6-BA is a common cytokinin, the main function of which is to promote cell division, and NAA is a broad-spectrum plant growth regulator which can promote cell growth elongation and cell division and induce the formation of callus. At the stage of callus and adventitious bud induction, 1.0-2.06-BA and 0.1-0.5mg/L NAA are added into the culture medium to promote cell proliferation and expansion and induce explant to dedifferentiate to form callus and adventitious bud. Compared with a culture medium which utilizes 2,4-D or IBA to replace NAA, the culture medium provided by the application can effectively improve the quality of the obtained callus and the differentiation condition of adventitious buds, so that the propagation efficiency of Jack Frost of heart-leaf hedyotis diffusa is improved.

In the culture medium for inducing the explant to form callus and adventitious buds, the addition amount of 6-BA is 1.0-2.0 mg/L.

By adopting the technical scheme, when the addition amount of the 6-BA is less than the concentration, the differentiated seedling obtained by final differentiation has general growth vigor and a plant is weak; when the addition amount of 6-BA is less than the above concentration, the proliferation rate of Jack Frost of the herba Hedyotidis Diffusae is low. Therefore, the amount of 6-BA added to the medium for inducing the formation of callus and adventitious buds from explants is preferably controlled to be 1.0 to 2.0 mg/L.

6-BA and IBA are used as plant hormones in a culture medium for inducing the proliferation of adventitious buds. The addition of cytokinin 6-BA to the culture medium for inducing the proliferation of adventitious buds can promote the division and proliferation of plant cells, and the addition of IBA can stimulate the growth of plants and promote the proliferation and enlargement of cells, so that the addition of 6-BA and IBA to the culture medium for the proliferation of adventitious buds can promote the proliferation of adventitious buds. In the culture medium for inducing the adventitious bud to root, no phytohormone is additionally added. Under the combined action of the rooting culture medium and various hormones synthesized by the adventitious buds, the proliferated adventitious buds are promoted to grow roots and form seedlings. Activated carbon is also added into the culture medium for inducing the adventitious bud to root. The activated carbon can adsorb the metabolite of the adventitious bud and enhance the permeability of the culture medium.

Preferably, the new leaves are new leaves of which the growing period is 20-50 days on the mother plant.

In a specific embodiment, the new leaf blade may have a growth period of 20 days, 35 days, 50 days on the mother plant.

In a specific embodiment, the new leaf blade may have a growth period of 20 to 35 days on the mother plant.

In a specific embodiment, the new leaf blade may have a growth period of 35 to 50 days on the mother plant.

Preferably, the new leaves are new leaves of which the growing period is 20 to 35 days on the mother plant.

By adopting the technical scheme, in the tissue culture of the Ottelia cardiocarpa Jack Frost, a new leaf with a short growth period is selected as an explant. The tissue cell division activity of the new leaves with short growth period is vigorous and well developed, so that the induction success rate of Jack Frost of the heart-leaf hedyotis diffusa can be effectively improved. In addition, compared with the plant organs with longer growth time, the new leaves with shorter growth period have shorter time and are less interfered by the outside, so the pollution rate of the Jack Frost tissue culture of the orius cordifolius can be effectively reduced.

New leaves with a growth period of 20-50 days are selected as explants and the new leaves are completely unfolded. Compared with the incompletely unfolded young leaves with the growth period of less than 20 days, the completely unfolded new leaves have thick tissue and strong tolerance to various disinfectants. Therefore, the contamination rate of the explants can be reduced by prolonging the disinfection time, and the negative effect of the prolonged disinfection time on new leaves is small, and the survival rate is obviously improved. Compared with old leaves with the growth period of more than 50 days, the old leaves have increased fibrosis, the metabolic activity of leaf cells is weakened, the induction success rate is obviously reduced, and cytokinin and auxin with higher concentration need to be added into a culture medium in the induction process, so that the physiological disorder of plants is easily caused, and the production cost is increased. The new leaf cells with the growth period of 20-50 days have vigorous metabolic activity and strong division capability, the concentration of various hormones in the leaves is higher, the callus and adventitious buds can be successfully induced by using the cytokinin and the auxin with lower concentrations, and vitrification and other adverse reactions caused by overhigh various hormones can be effectively avoided.

In summary, the present application selects new leaves with a growth period of 20-50 days as explants for tissue culture of Jack Frost of Ottelia cordifolia.

Preferably, in the step of obtaining and sterilizing the explant, the sterilization treatment comprises: washing, alcohol disinfection and mercuric chloride solution treatment.

Preferably, the mercuric chloride solution treatment is carried out by using a 0.1% mercuric chloride solution.

Preferably, the treatment time of the mercuric chloride solution is 5-9 min.

Preferably, the treatment time of the mercuric chloride solution is 7 min.

By adopting the technical scheme, after the explant is soaked and disinfected by alcohol, a small amount of pathogens attached to the surface of the explant can be killed, the interference of possible attached pollution sources on the surface of the explant can be effectively eliminated, the permeability of the disinfectant on the surface of the explant material can be effectively increased, and the success rate of explant disinfection is further improved. The mercury chloride can kill propagules, lipophilic viruses and the like, and when the explants are cultured in the plant tissue culture for disinfection, the mercury is a high-efficiency disinfectant, so that the disinfectant has a strong killing effect on pathogens and the like carried by the explants, and the success rate of explant disinfection is further improved. The explant is disinfected by 75% alcohol and then disinfected by the disinfectant, the alcohol has a strong infiltration effect, and air on the surface of the explant can be removed, so that the infiltration of the disinfectant is facilitated, the permeability of the disinfectant on the surface of the explant material is improved, the disinfection effect of the disinfectant is improved, and the success rate of explant disinfection is improved under the matching of alcohol disinfection and disinfectant disinfection. The mercuric chloride has a large killing effect on plants, if the soaking time is long, the growth of plant organs can be influenced and even killed, so that the soaking time needs to be strictly controlled when the mercuric chloride is used for disinfecting the explants, and through tests, the time for soaking the explants in the mercuric chloride solution is controlled within the range of 7-9 min.

In summary, the present application has the following beneficial effects:

this application selects new leaf of growing on the ox tongue grass Jack Frost mother plant robust and no plant diseases and insect pests as the explant, carries out the tissue culture of ox tongue grass Jack Frost, selects other positions of ox tongue grass Jack Frost as the explant and carries out tissue culture, and the induction cycle in the tissue culture process can effectively be shortened in this application, improves the multiplication coefficient and the rooting rate of ox tongue grass Jack Frost, thereby improve the reproductive efficiency of ox tongue grass Jack Frost, reduce the restriction of season to this variety production, thereby promote the application of ox tongue grass Jack Frost.

6-BA and NAA were used as phytohormones in the medium inducing explants to form callus and adventitious shoots. Compared with a culture medium which utilizes 2,4-D or IBA to replace NAA, the culture medium provided by the application can effectively improve the quality of the obtained callus and the differentiation condition of adventitious buds, so that the propagation efficiency of Jack Frost of heart-leaf hedyotis diffusa is improved.

The method selects new leaves with the growth period of 20-50 days on a mother plant as explants to perform the tissue culture of the Jack Frost of the Ottelia cordifolia, the tissue cell division activity is vigorous, the development is good, and the induction success rate of the Ottelia cordifolia can be improved; and the new blade has short growth time and small interference from the outside, and can reduce the pollution rate of the tissue culture of the oldenlandia diffusa.

Drawings

FIG. 1 is a schematic representation of the induction of explants to form callus and adventitious buds as described herein.

FIG. 2 is a schematic diagram showing the proliferation and differentiation of adventitious buds to form clumpy adventitious buds according to the present application.

FIG. 3 is a schematic illustration of rooting of adventitious buds in the present application.

Detailed Description

The alcohol used in the application is 500mL of Europe 75% disinfection alcohol, purchased from Hangzhou Europop biotechnology, Inc.; the mercuric chloride is a mercuric chloride standard solution purchased from Guangdi Fine chemical research institute in Tianjin; the preventive disinfectant is 50% carbendazim, which is purchased from Sichuan Runle technologies, Inc.

The Okinra cardinalis Jack Frost parent strain used in the application is selected from Beijing Huaxiang flower science and technology research institute, Inc.

The application provides a tissue culture method of Ottelia cordifolia Jack Frost, which specifically comprises the following steps:

1. explant harvest and sterilization

(1) Obtaining an explant: culturing the stock plant in a greenhouse for 30-60 days, removing withered and diseased leaves in the process, and spraying a preventive bactericide once every 15 days; selecting a stock plant which is robust in growth, good in performance and free of diseases and insect pests, and selecting new leaves which are completely unfolded on the stock plant and free of diseases and insect pests;

(2) and (3) disinfection of explants:

washing: washing the new leaves obtained in the step (1) for 10min in flowing tap water, and airing;

alcohol disinfection: air drying, soaking in 75% alcohol, shaking for 30-60s, and washing with sterile water for 1-2 times (each time for 1-2 min);

0.1% mercuric chloride solution: soaking the new leaves in 0.1% mercuric chloride solution, shaking for 5-9min, and washing with sterile water for 1-2min each time for 3-5 times;

cutting: and cutting the edges of the disinfected leaves, and cutting the leaves into squares with the side length of 1cm to obtain explants to be induced.

2. Inducing explants to form callus and adventitious buds

And (3) the explant to be induced obtained in the step (1) is tiled and inoculated into a culture medium for inducing the explant to form callus and adventitious buds, and the explant is induced to form callus and adventitious buds, so that the adventitious buds integrated with the callus are obtained.

Wherein the formula of the culture medium used in the step is as follows: MS culture medium, 6-BA (6-benzylamino adenine) 1.0-2.0mg/L, NAA (naphthalene acetic acid) 0.1-0.5mg/L, sucrose 25-30g/L, agar 4-4.5g/L, pH value is 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-1800Lux, and the illumination time is 8-10 h/d.

3. Proliferation of adventitious buds

And (3) separating the adventitious buds integrated with the callus tissue obtained in the step (2) from the callus tissue, stripping the separated adventitious buds into single adventitious buds, and respectively inoculating the single adventitious buds into a culture medium for inducing the adventitious buds to proliferate to obtain clustered adventitious buds after proliferation.

Wherein the formula of the culture medium used in the step is as follows: MS culture medium, 6-BA 1.0-2.0mg/L, IBA 0.1-0.5mg/L, sucrose 25-30g/L, agar 4-4.5g/L, and pH value of 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-1800Lux, and the illumination time is 8-10 h/d.

4. Inducing adventitious bud to root

And (3) mutually separating the proliferated clustered adventitious buds obtained in the step (3) to obtain independent adventitious buds, and inoculating the independent adventitious buds into a culture for inducing the adventitious buds to root.

Wherein the formula of the culture medium used in the step is as follows: 1/2MS culture medium, AC (activated carbon) 0-0.5g/L, sucrose 25-30g/L, agar 4-4.5g/L, pH value 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-.

The present application is described in further detail below with reference to examples 1-5, comparative examples 1-7, FIGS. 1-3, and the test assays.

Examples

Example 1

The embodiment provides a tissue culture method of herba Hedyotidis Diffusae Jack Frost, which comprises the following steps:

1. explant harvesting and Sterilization

(1) Obtaining an explant: culturing the stock plant in a greenhouse for 35 days, removing withered and diseased leaves in the process, and spraying a preventive bactericide once every 15 days; selecting a stock plant which is robust in growth, good in performance and free of diseases and insect pests, and selecting new leaves which are completely unfolded on the stock plant and free of diseases and insect pests;

(2) and (3) disinfection of explants:

washing: washing the new blade obtained in the step (1) in flowing tap water for 10min, and airing;

alcohol disinfection: after drying, placing the new leaves in 75% alcohol for soaking, shaking and sterilizing for 30s, and then washing for 1 time with sterile water for 1-2min each time;

0.1% mercuric chloride treatment: soaking and shaking the new leaves in 0.1% mercuric chloride solution for 5min, and shaking and cleaning with sterile water for 3 times, each time for 1-2 min;

2. Inducing explants to form callus and adventitious buds

And (2) tiling and inoculating the explant to be induced obtained in the step (1) into a culture medium for inducing the explant to form callus and adventitious buds, and inducing the explant to form callus and adventitious buds to obtain the adventitious buds integrated with the callus.

3. Proliferation of adventitious buds

4. Inducing adventitious bud to root

Refer to fig. 1-3.

FIG. 1 is a schematic representation of the induction of explants to form callus and adventitious buds.

FIG. 2 is a schematic view showing the proliferation and differentiation of adventitious buds to form clumpy adventitious buds.

FIG. 3 is a schematic representation of rooting of adventitious buds.

The parameters involved in each step and the formulation of the medium used are shown in Table 1.

Table 1 parameters relating to the various steps and the formulation of the culture medium used

Examples 2 to 3

Examples 2-3 each provide a tissue culture method of Jack Frost of Bluegum-type heart, which is different from example 1 in the parameters of each step and the formula of the used culture medium, as shown in Table 1.

Comparative example

Comparative examples 1 to 4

Comparative examples 1-4, which differ from example 2 in the parameters and medium formulations used in the callus and adventitious bud induction steps of the explants, are provided for tissue culture in Jack Frost, respectively, of Ottelia cordifolia, as shown in Table 1.

Examples 4 to 5

Examples 4-5 each provide a tissue culture method of Jack Frost of Ottelia cordifolia, which is different from example 2 in the growth period of new leaves as explants on the mother plant, as shown in Table 2.

Comparative examples 5 to 7

Comparative examples 5-7, which are different from example 2 in the selection of explants and the steps of explant harvesting and sterilization, are provided for a tissue culture method of Ottelia cordifolia Jack Frost, respectively, as shown in Table 2.

TABLE 2 selection of explants and harvesting and Sterilization of explants of example 2, examples 4-5, and comparative examples 5-7

Detection test

The method for culturing the plant tissues provided by the examples 1-5 and the comparative examples 1-7 is used for culturing the Jack Frost of the heart-leaf bulltongue, the investigation parameters of the cultures in the corresponding culture processes at all stages are recorded, and the pollution rate and the death rate of the Jack Frost of the heart-leaf bulltongue are calculated, wherein the specific investigation parameters and the recorded results are shown in the table 3. Meanwhile, the disinfection effect of the explant, the quality of the callus and the condition of forming adventitious buds in the culture process of the Ottelia cordifolia Jack Frost are recorded, and the specific recording results are shown in table 4.

TABLE 3 results of tissue culture of Ottelia cordifolia Jack Frost (I) in examples 1 to 5 and comparative examples 1 to 7

TABLE 4 results of tissue culture of Jack Frost of Okinra cordifolia, examples 1 to 5 and comparative examples 1 to 7 (II)

By combining the tissue culture methods of examples 1-5 and comparative examples 1-7 and the contents in tables 3 and 4, the application selects new leaves which are fully developed and have a growth period of 20-50 days on a mother plant as explants, and selects proper culture medium formulas and culture conditions at each tissue culture stage to perform tissue culture of the Ottelia cordifolia Jack Frost, so that the disinfection success rate of the Ottelia cordifolia explants is improved, the induction period is shortened, the multiplication rate of adventitious buds is improved, the rooting effect of the Ottelia cordifolia is better, the propagation efficiency of the Ottelia cordifolia Jack Frost is improved, the limitation of seasons on the production of the variety is reduced, and the application of the Ottelia cordifolia Jack Frost is promoted.

Firstly, the tissue culture of the Jack Frost of the heart-leaf Oncorhynchus blume is carried out by selecting a proper mother plant part as an explant, and the final result of the Jack Frost of the heart-leaf Oncorhynchus blume can be directly influenced. Comparing the culture results of example 2, examples 4-5 and comparative examples 5-7, under the same tissue culture conditions and no pest and disease damage requirements, the explants selected in the example 2 of the present application are new leaves which grow for 35d and are completely unfolded on the mother plant, while the explants selected in the comparative examples 5-7 are petioles of new leaves which grow for 35d on the mother plant, young leaves which grow for 8d and are not completely unfolded on the mother plant, and old leaves which grow for 55d on the mother plant, respectively. As can be seen from tables 3 and 4, the tissue culture methods of examples 2 and 4 to 5 resulted in contamination rates of 5%, 5% and 15% and mortality rates of 10%, 15% and 5% respectively, and induced adventitious buds with a large number of adventitious buds that grow rapidly without vitrification, and differentiated seedlings with good growth vigor, strong plants and normal proliferation rates; the pollution rates obtained by the tissue culture methods of the comparative examples 5-7 are 5%, 0%, 20% and the death rates are 20%, 100% and 0% respectively, but the comparative example 5 has fluffy callus formed, the callus is not differentiated into adventitious buds, the leaves are completely browned and dead after disinfection of the comparative example 6, no callus and adventitious buds are formed, the comparative example 7 has a small amount of adventitious buds which are differentiated, the growth rate of the adventitious buds is slightly slow, and no vitrification phenomenon exists, although the differentiated seedlings are good in growth vigor and strong in plants, the proliferation rate is low, the proliferation times are only 2-3 times, the induction rate is low, and the induction time is long.

From the above, it can be seen that the selection of the explant has a great influence on the effect of plant tissue culture, and the selection of an appropriate mother plant part as the explant for plant tissue culture can improve the effect of Jack Frost tissue culture of Ottelia cordifolia. The method selects new leaves which are fully unfolded and have the growth period of 20-50d on a mother plant as explants for tissue culture of the Ottelia cordifolia Jack Frost, can effectively reduce the pollution rate and the death rate of the Ottelia cordifolia Jack Frost in the process of plant tissue culture, improves the induction rate, the multiplication coefficient and the rooting rate of the Ottelia cordifolia Jack Frost, and shortens the induction time for the Ottelia cordifolia Jack Frost to form callus and adventitious buds.

In addition, the culture medium formula and culture conditions selected at each stage of the Ottelia cordifolia Jack Frost tissue culture can also influence the result of the Ottelia cordifolia Jack Frost tissue culture, especially the selection of phytohormones in the steps of inducing explants to form callus and adventitious buds. Comparing the culture results of example 2 and comparative examples 1-2, the phytohormone used in example 2 to induce the explants to form callus and adventitious buds was 6-BA and NAA in combination, while the phytohormone used in comparative examples 1-2 to induce the explants to form callus and adventitious buds was 6-BA and 2,4-D in combination, 6-BA and IBA in combination, respectively. As can be seen from tables 3 and 4, the tissue culture method of example 2 resulted in a contamination rate and a mortality rate of 5% and 10%, respectively, induced the formation of a large number of adventitious buds, which grew rapidly without vitrification, and the differentiated seedlings grew well, the plants were strong, and the proliferation rate was normal; while the contamination rates obtained by the tissue culture methods of comparative examples 1-2 were 5% and 5% respectively, and the mortality rates were 10% and 10% respectively, but the callus of comparative example 1 was compact and dark green, and the callus was not differentiated into adventitious buds, the callus of comparative example 2 was slightly differentiated into adventitious buds, and the growth vigor was weak, the differentiated seedling was also poor, the plant was delicate, the proliferation rate was low, the proliferation multiple was only 2-3 times, and at the same time, the induction rate was low, and the induction time was long.

According to the method, the selection of the plant hormone in the step of inducing the explant to form the callus and the adventitious bud also has great influence on the effect of plant tissue culture, the application selects the combination of 6-BA and NAA as the plant hormone of the culture medium used for inducing the explant to form the callus and the adventitious bud, can improve the quality of the callus induced and formed in the tissue culture of the Ottelia cordifolia Jack Frost, can induce a large number of adventitious buds, simultaneously improves the induction rate and the multiplication coefficient of the Ottelia cordifolia Jack Frost, and shortens the induction time of the Ottelia cordifolia Jack Frost for forming the callus and the adventitious bud. Therefore, in the step of inducing the explant to form callus and adventitious buds, NAA plays a role that 2,4-D, IBA cannot be replaced, the reproduction efficiency of the Ottelia cordifolia Jack Frost is improved, and the limitation of seasons on the production of the Ottelia cordifolia is reduced.

Moreover, in the steps of obtaining and disinfecting the explants, the disinfection time has a great influence on the disinfection effect of the explants, and the selection of the proper disinfection time of the explants has a great influence on the tissue culture effect of the Ottelia acuminata Jack Frost. As a result of comparing the culture results of examples 1 to 3, the sterilization time periods of examples 1 to 3 were 5min, 7min and 9min, respectively. As can be seen from tables 3 and 4, the contamination rates of examples 1 to 3 were 20%, 5%, and 0%, and the mortality rates were 0%, 10%, and 20%, respectively, and example 1 had a large number of adventitious buds formed, the adventitious buds grew rapidly, no vitrification occurred, the differentiated seedlings grew well, and the plants grew well, but the proliferation rate was slightly low, example 3 had a large number of adventitious buds formed, the adventitious buds grew rapidly, but about 10% of the adventitious buds had slight vitrification, the differentiated seedlings grew well, the plants grew slightly weakly, and example 2 had a large number of adventitious buds formed, the adventitious buds grew rapidly, no vitrification, and the differentiated seedlings grew well, the plants grew well, and the proliferation rate was normal.

According to the method, when the explants are disinfected by 0.1% of mercuric chloride, the disinfection time is controlled within 5-9min, and the disinfected explants show normal appearance. When the disinfection time is long, the lower the pollution rate in the tissue culture process is, the better the disinfection effect is, but the death rate of the explant is increased, which indicates that the explant cannot be in the mercuric chloride solution for a long time; when the sterilization time is shorter, the explant is not dead basically, but the contamination rate of the explant is remarkably increased, which is obviously lower than the sterilization effect when the sterilization time is longer. Therefore, the present application finally controls the sterilization duration of the explants to be in the range of 5-9min, with the optimal sterilization duration being 7 min.

Further, the amount of 6-BA added in the step of inducing the explant to form callus and adventitious buds also has a large influence on the effect of plant tissue culture. As a result of comparing the culture results of example 2 and comparative examples 3 to 4, the amount of 6-BA added in example 2 was 1.5mg/L, while the amount of 6-BA added in comparative examples 3 to 4 was 0.5mg/L and 4.0mg/L, respectively. As can be seen from tables 3 and 4, the induction rate obtained by the tissue culture method of example 2 is 100%, the induction time is 15 to 18 days, a large number of adventitious buds are formed by induction, the adventitious buds grow rapidly, no vitrification occurs, the growth vigor of differentiated seedlings is good, the plants are strong, and the proliferation rate is normal; whereas the induction rates obtained by the tissue culture methods of comparative examples 3-4 were 100% and 80%, respectively, and the induction times were 15-18d and 20-25d, respectively; although comparative example 3 has a large number of adventitious buds formed, the adventitious buds grow rapidly, no vitrification phenomenon exists, and the proliferation rate is high, the differentiated seedlings of comparative example 3 grow normally, and the plants are weak; although comparative example 3 had a large number of adventitious buds formed, the adventitious buds grew rapidly, no vitrification occurred, the differentiated seedlings grew well, and the plants were vigorous, comparative example 4 had a low proliferation rate, and the proliferation coefficient was only 2 to 3 times.

As is clear from the above, the amount of 6-BA added in the step of inducing the formation of callus and adventitious buds from explants also has a large influence on the effect of plant tissue culture. According to the application, the addition amount of NAA is 1.0-2.0mg/L, so that the induction rate in the tissue culture of the Ottelia cordifolia Jack Frost can be improved, the induction time for forming callus and adventitious buds of the Ottelia cordifolia Jack Frost is shortened, and healthy differentiated seedlings are cultured.

In summary, according to the tissue culture method of the herba hedyotis diffusae Jack Frost provided by the application, the explant selects new leaves which are fully unfolded and have the growth period of 20-50d on the mother plant, the new leaves are easy to obtain, are not limited by seasons, and are less harmful to the mother plant. The explant has high induction success rate, can directly induce callus and adventitious buds, shortens the induction period, improves the multiplication coefficient of the adventitious buds, has good rooting effect and hundred percent rooting rate, improves the transplanting rate of the Ottelia cordifolia Jack Frost, and promotes the application of the Ottelia cordifolia Jack Frost.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Claims

1. All-grass of Bulbophyllum caudatumJack FrostThe tissue culture method of (1), wherein the tissue culture method comprises the steps of: obtaining and disinfecting an explant, inducing the explant to form a callus and an adventitious bud, proliferating the adventitious bud and inducing the adventitious bud to root; wherein the content of the first and second substances,

in the step of obtaining and disinfecting the explant, selecting a new leaf of a mother plant which grows vigorously and has no plant diseases and insect pests from the explant; the new leaf is a new leaf of the parent plant with the growing period of 20-50 days;

in the step of adventitious bud proliferation, the formula of the used culture medium is as follows: MS culture medium, 6-BA 1.0-2.0mg/L, IBA 0.1-0.5mg/L, sucrose 25-30g/L, agar 4-4.5g/L, pH value is 5.8-6.0; the culture temperature is 23-27 ℃, the illumination intensity is 1500-;

2. The dens lemongrass of claim 1Jack FrostThe tissue culture method of (1), wherein: in the step of obtaining and disinfecting the explant, the disinfection treatment comprises the following steps: washing, alcohol disinfection and mercuric chloride solution treatment.

3. The dens cardinalis according to claim 2Jack FrostThe tissue culture method of (1), wherein: the mercuric chloride solution treatment is carried out by using a 0.1% mercuric chloride solution.

4. The dens edulis ker of claim 3Jack FrostThe tissue culture method of (1), characterized in that: the treatment time of the mercuric chloride solution is 5-9 min.