阅读说明：本技术 一种变叶榕组织培养与快速繁殖的方法 (Tissue culture and rapid propagation method for ficus variabilis ) 是由 徐爱春 郭瑞 章书声 孙骏威 于 2019-11-01 设计创作，主要内容包括：本发明公开了一种变叶榕组织培养与快速繁殖的方法,依次包括以下步骤：(1)变叶榕茎段的消毒和接种：取当年抽出的变叶榕新茎,剪成带腋芽茎段进行接种；(2)丛生芽的诱导；(3)不定芽的增殖；(4)再生植株的生根培养；(5)炼苗移栽。在上述步骤中采用了不同处理、不同成分及配比的培养基。采用本发明的方法,能够快速地获得变叶榕植株,利于产业化生产。采用本发明方法,变叶榕丛生芽的不定芽诱导率高达87.5%,不定芽数6.8,不定芽增殖倍数可达6.5,生根率达100%,植株移栽成活率可达99%以上。(The invention discloses a method for tissue culture and rapid propagation of ficus variabilis, which sequentially comprises the following steps: (1) sterilizing and inoculating the ficus variabilis stem section: taking new stems of ficus variabilis which are extracted in the current year, shearing the new stems into stem segments with axillary buds, and inoculating; (2) inducing cluster buds; (3) multiplication of adventitious buds; (4) rooting culture of the regenerated plant; (5) hardening and transplanting the seedlings. The culture mediums with different treatments, different components and proportions are adopted in the steps. By adopting the method, the ficus variabilis plant can be quickly obtained, and the industrial production is facilitated. By adopting the method, the adventitious bud induction rate of the ficus variabilis cluster buds is up to 87.5 percent, the number of the adventitious buds is 6.8, the multiplication multiple of the adventitious buds can reach 6.5, the rooting rate can reach 100 percent, and the plant transplanting survival rate can reach more than 99 percent.)

1. A method for tissue culture and rapid propagation of Ficus variabilis is characterized by sequentially comprising the following steps:

(1) disinfection of Ficus variabilis stems

Soaking a proper amount of detergent solution in the stem section of the current-year Ficus variabilis for 2 minutes, washing with running water for 0.5 hour, draining, placing in a refrigerator for low-temperature refrigeration at 4 ℃ for 0-48 hours, taking out, soaking and disinfecting in 0.1% by mass mercuric chloride solution dropwise added with 1 drop of Tween-80 in an ultra-clean bench for 10-12 minutes, then fully soaking and washing with sterile water for 3 times, cutting off the head and the tail on the sterile filter paper, cutting into stem sections with axillary buds, soaking in 1% ascorbic acid solution for 0-60 minutes, and fully soaking and washing with sterile water for 3 times to obtain a disinfected explant material;

(2) induction of clumpy buds

Inoculating the explant material obtained in the last step into a WPM culture medium according to growth polarity for culture, wherein the culture medium is added with ZT not more than 2.0 mg/L, 6-BA not more than 5.0 mg/L, PVP not more than 5.0 g/L, CH not more than 1.0 g/L, sucrose 30 g/L and agar 9.0 g/L, the pH value is 5.8-6.0, the culture medium inoculated with the explant is placed in the dark for 1 day, and the culture temperature is 21-25 ℃; then transferring to light culture, culturingThe conditions are as follows: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m²Second;

(3) proliferation of adventitious buds

Cutting the adventitious bud cluster obtained by the previous step into bud blocks with 2-3 adventitious buds, inoculating the bud blocks into a WPM culture medium for culture, wherein the culture medium is added with 6-BA of not more than 5.0 mg/L, CH of not more than 1.0 g/L, PVP of not more than 5.0 g/L, sucrose of 30 g/L, agar of 9.0 g/L and a pH value of 5.8-6.0, and the culture conditions are as follows: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m²Second;

(4) rooting culture of regenerated plants

Cutting adventitious buds with tender green leaves, vigorous growth and 3-4 leaves, inserting the adventitious buds into a WPM culture medium with the concentration of 12.5% -100% for rooting culture, wherein the culture medium is added with NAA not more than 2.0 mg/L, AC not more than 5.0 g/L, sucrose 20 g/L and agar 8.0 g/L, the pH value is 5.8-6.0, and the culture conditions are as follows: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m²Second;

(5) hardening off and transplanting

The height of the tissue culture seedling to be rooted is more than 3 cm, when the root length is more than 5 cm, the bottle cap of the tissue culture bottle is loosened, a small amount of sterile water is injected into the bottle to prevent the culture medium from drying and cracking, the bottle cap is removed after 6 hours, the sterile water is added, the bottle cap is removed after 18 hours, and the seedling is grown at 30-40 micromoles/m²Directly irradiating the regenerated plant with light of second light intensity for 2 days, adding sterile water for a plurality of times, carefully mashing the culture medium, taking out the regenerated plant, carefully washing the residual agar with running water, planting the plant in the disinfected vegetable garden soil with vermiculite added, wherein the plant spacing is more than 2 cm, watering thoroughly, covering with a transparent polyethylene plastic film for heat preservation and moisture preservation, controlling the indoor temperature at 18-22 ℃, uncovering four corners of the polyethylene plastic film after 3 days, uncovering all the polyethylene plastic film after the next day, and transplanting the plant to the field with soil after new leaves are uncovered.

2. The method for tissue culture and rapid propagation of ficus variabilis according to claim 1, wherein the method comprises the following steps: and (2) cleaning the stem segments in the step (1), refrigerating the stem segments at a low temperature of 4 ℃ for 24 hours in a refrigerator, disinfecting and cleaning the stem segments in a mercuric chloride solution, and soaking the stem segments in a 1% ascorbic acid solution for 30 minutes.

3. The method for tissue culture and rapid propagation of ficus variabilis according to claim 1, wherein the method comprises the following steps: the WPM minimal medium in the step (2) is added with 0.5 mg/L ZT, 2.0 mg/L6-BA of 6-BA, 0.2 g/L CH and 0.5-1.0 g/L PVP.

4. The method for tissue culture and rapid propagation of ficus variabilis according to claim 1, wherein the method comprises the following steps: in the step (3), the WPM minimal medium is added with 2.0 mg/L of 6-BA, 0.2 g/L of CH and 0.5-1.0 g/L of PVP.

5. The method for tissue culture and rapid propagation of ficus variabilis according to claim 1, wherein the method comprises the following steps: the WPM with the rooting medium concentration of 50% in the step (4) is added with NAA of 0.2 mg/L and AC of 1.0 g/L.

6. The method for tissue culture and rapid propagation of ficus variabilis according to claim 1, which is characterized in that: the volume ratio of the vermiculite added into the sterilized vegetable garden soil in the step (5) is 1/20.

Technical Field

The invention belongs to the technical field of plant culture, and particularly relates to a method for tissue culture and rapid propagation of ficus variabilis.

Background

Ficus variabilis lindl. ex Benth, Ficus, a shrub or small tree of Ficus, and the provinces of zhejiang, jiangxi, fujian, guangdong (and coastal islands), guangxi, hunan, Guizhou, southeast and south of Yunnan. It is usually in the wet area under the forest. Its root is named as Ficus variabilis, also called Jinbuhuan. It is warm, bitter and pungent in flavor, has the effects of dispelling pathogenic wind, removing dampness, promoting blood circulation, relieving pain and inducing lactation, and can be used for treating rheumatalgia, gastralgia, furuncle, traumatic injury and galactostasis. The stalk bark fiber can be used as raw material for artificial cotton, sacks and paper making. At present, ficus variabilis is basically in a wild state, and the ficus variabilis in the wild state are distributed in a sporadic shape, which indicates that the efficiency of breeding by seeds is not high. At present, no research report on the rapid propagation of ficus variabilis exists.

At present, the research reports of tissue culture and rapid propagation of ficus plants are mostly concentrated on garden plants such as ficus elastica, linden, ficus elastica and the like and fruit trees such as figs and the like, used explants are mostly stem sections, stem tips and leaves, but the induction rate and the proliferation rate of adventitious buds are generally low, and a basic culture medium used for experiments generally adopts MS with high ion concentration. The Moraceae plant, especially Ficus plant, has a large amount of milk tubes distributed in stems and leaves, and the milk in the milk tubes contains a large amount of polyphenols, and is easy to flow out and be oxidized into brown quinones in the air during injury or in vitro culture, so as to cause browning of the culture medium, thereby influencing the smooth operation of tissue culture. And how to prevent browning is not substantially concerned in the literature.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: aiming at the defects in the prior art, the method for tissue culture and rapid propagation of ficus variabilis, which has high survival rate and is beneficial to realizing industrialized production, is provided.

In order to realize the purpose of the invention, the following technical scheme is adopted for realizing the purpose:

a method for tissue culture and rapid propagation of Ficus variabilis sequentially comprises the following steps:

(1) disinfection of Ficus variabilis stems

Soaking the stem section of the current year Ficus variabilis in a proper amount of detergent solution for 2 minutes, washing with running water for 0.5 hour, draining, placing in a refrigerator for low-temperature refrigeration at 4 ℃ for 0-48 hours, taking out, soaking and disinfecting in 0.1% by mass mercuric chloride solution dropwise added with 1 drop of Tween-80 in an ultra-clean bench for 10-12 minutes, then fully soaking and washing with sterile water for 3 times, cutting off the head and the tail on sterile filter paper and cutting into stem sections with axillary buds, transferring into 1% ascorbic acid solution for filtration and sterilization, soaking for 0-60 minutes, and fully soaking and washing with sterile water for 3 times to obtain a disinfected explant material;

(2) induction of clumpy buds

Inoculating the explant material obtained in the last step into a WPM (woody plant medium) basic culture medium according to the growth polarity for culture, wherein the culture medium is added with ZT not more than 2.0 mg/L, 6-BA not more than 5.0 mg/L, CH not more than 1.0 g/L, PVP not more than 5.0 g/L, sucrose 30 g/L and agar 9.0 g/L, the pH value is 5.8-6.0, the culture medium inoculated with the explant is placed in the dark for 1 day, and the culture temperature is 21-25 ℃; then, the culture is transferred to illumination culture under the culture conditions that: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m²Second;

(3) proliferation of adventitious buds

Cutting the adventitious bud cluster obtained by the previous step induction into bud blocks with 2-3 adventitious buds, inoculating the bud blocks into a WPM culture medium for culture, wherein the culture medium is added with 6-BA not more than 5.0 mg/L, CH not more than 1.0 mg/L, PVP not more than 5.0 g/L, sucrose 30 g/L and agar 9.0 g/L, the pH value is 5.8-6.0, and the culture conditions are as follows: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m²Second;

(4) rooting culture of regenerated plants

Cutting adventitious buds with tender green leaves, vigorous growth and 3-4 leaves, inserting the adventitious buds into a WPM culture medium with the concentration of 12.5% -100% for rooting culture, wherein NAA with the concentration not more than 2.0 mg/L, AC with the concentration not more than 5.0 g/L, cane sugar with the concentration of 20 g/L and agar with the concentration of 8.0 g/L are added into the culture medium, the pH value is 5.8-6.0, and the culture conditions are as follows: culturing at 21-25 deg.C, with illumination time of 14 hr/day and illumination intensity of 30-40 micromoles/m²Second;

(5) hardening off and transplanting

The height of the tissue culture seedling to be rooted is more than 3 cm, when the root length is more than 5 cm, the bottle cap of the tissue culture bottle is loosened, a small amount of sterile water is injected into the bottle to prevent the culture medium from drying and cracking, the bottle cap is removed after 6 hours, the sterile water is added, the bottle cap is removed after 18 hours, and the seedling is grown at 30-40 micromoles/meter²Second light intensity of the light directly irradiated the regenerated plants for 2 days, during which sterile water was added several times. Then carefully triturating the medium, taking out the regenerated plants, and using running waterCarefully washing the residual agar, planting the plants in disinfected vegetable garden soil added with vermiculite at a plant spacing of more than 2 cm, thoroughly watering, covering with a transparent polyethylene plastic film for heat preservation and moisture preservation, controlling the indoor temperature at 18-22 ℃, uncovering the four corners of the polyethylene plastic film after 3 days, uncovering the polyethylene plastic film on the next day, and transplanting the plants to the field with soil after new leaves are unfolded.

As a preferable scheme: and (2) cleaning the stem segments inoculated in the step (1), refrigerating at a low temperature of 4 ℃ for 24 hours in a refrigerator, disinfecting and cleaning the stem segments by using a mercuric chloride solution, and soaking the stem segments in a 1% ascorbic acid solution for 30 minutes.

As a preferable scheme: the WPM minimal medium in the step (2) is added with ZT of 0.5 mg/L, 6-BA of 2.0 mg/L, CH of 0.2 g/L and PVP of 0.5-1.0 g/L.

As a preferable scheme: in the step (3), the WPM minimal medium is added with 0.2 mg/L NAA, 1.0 mg/L6-BA, 0.2 g/L CH and 0.5-1.0 g/L PVP.

As a preferable scheme: and (3) adding 0.2 mg/L NAA and 1.0 g/L AC into the WPM with the rooting medium formula concentration of 50% in the step (4).

As a preferable scheme: the volume ratio of the vermiculite added into the sterilized vegetable garden soil in the step (5) is 1/20.

Compared with the prior art, the invention has the beneficial effects that: by adopting the method, the browning can be reduced as much as possible, the multiplication coefficient can be improved, the ficus variabilis regenerated plant can be quickly obtained, and the industrial production is facilitated. By adopting the method, the induction rate of the cluster buds of the ficus variabilis stem section is up to 87.5 percent, the number of the adventitious buds is 6.8, the multiplication multiple of the adventitious buds can reach 6.5, the rooting rate can reach 100 percent, and the plant transplanting survival rate can reach more than 99 percent.

Detailed Description

The abbreviations are annotated as: HgCl₂Mercuric chloride, AC activated carbon, 6-BA 6-benzylamino adenine, CH hydrolyzed casein, NAA naphthylacetic acid, ZT zeatin and PVP polyvinylpyrrolidone.