CN112753579B - In-vitro culture and plant regeneration method for leaves of Chinese medicinal herb chlorophytum comosum - Google Patents

Abstract

The invention belongs to the technical field of in-vitro culture, and discloses an in-vitro culture and plant regeneration method of the leaves of the national drug chlorophytum comosum, wherein the callus induction culture medium MS is +1.0mg/L6-BA+0.5mg/LNAA, the pH is 5.8-6.0, the induction rate is 91.67%, the adventitious bud induction culture medium MS is +2.0mg/L6-BA, the pH is 5.8-6.0, and the bud yield is 89.54%; adventitious bud proliferation culture medium MS+1.0mg/L6-BA+0.5mg/LNAA, pH 5.8-6.0, culture 40d, proliferation coefficient 5.56; rooting culture medium MS+0.1mg/L6-BA+1.5mg/LNAA, pH 5.8-6.0, rooting rate 100%, 21.6 roots and root length about 3.5 cm; transplanting the rooting tissue culture seedlings into a seedling raising tray with a mixed matrix, wherein the survival rate is 94.96%.

Description

In-vitro culture and plant regeneration method for leaves of Chinese medicinal herb chlorophytum comosum

Technical Field

The invention belongs to the technical field of in-vitro culture, and particularly relates to an in-vitro culture and plant regeneration method of national drug chlorophytum comosum leaves.

Background

At present: herba Lysionoti Pauciflori, and Chinese medicine. Is whole herb of herba Gei Alopecuroidis Lysionotus pauciflorus Maxim. Distributed in Jiangsu, zhejiang, anhui, jiangxi, fujian, taiwan, hubei, hunan, guangdong, guangxi, sichuan, guizhou and other places. Has effects of dispelling pathogenic wind, removing dampness, eliminating phlegm, relieving cough, removing blood stasis, and dredging channels. Can be used for treating rheumatalgia, cough with excessive phlegm, menoxenia, dysmenorrhea, and traumatic injury. The root of common stonecrop is: black bone (plant Mingshi Tujia kou), dan Jiangdou (plant Mingshu Tuo Kong), herba Lycopi, semen glycines, herba Lycopi (classified herb property), semen Vignae sinensis, folium Camelliae sinensis (Guizhou folk medicine set), herba Lycopi (Guiyang folk medicine set), herba Lycopi, herba Sambuci Chiensis, lumbricus, bee pollen, melon seed grass, stone flower (Hunan medicine Zhi), sambusheng, herba Lycopi, qian hammer, and puerperal tea (Guangxi medicine set). The shrubs have stems 7-30 cm long, branch or not branch, no hair or upper sparse and short hair. 3 rotagenesis of leaves, sometimes opposite or three rotagenesis, has short handle or near no handle; the blade has the advantages of revolutionary shape, large shape change, linear inverted needle shape, long and narrow round shape or inverted oval shape, small number of narrow inverted oval shape or oblong shape, sharp or blunt tip, blunt base, wide wedge shape or nearly round shape, few teeth or small teeth at the edge above or on the middle part, sometimes nearly full edge, no hair on both sides, sinking above middle pulse, 3-5 side pulses on each side, and no obvious effect; the petioles are often short-piled. The inflorescence has 1-2 (-5) flowers; the inflorescence peduncles are fine and have no hair; the bracts are in a needle shape, so that the bracts are loose, short or nearly free; the pedicel is hairless. The calyx 5 is split or near the basal part, has no hair or is free of hair and short hair; slit triangle or linear triangle. Corolla Bai Sedai light purple streaks or light purple, hairless; a cylinder is thin and funnel-shaped; the upper lip 2 is shallow cracked and the lower lip 3 is cracked. Stamen have no fuzz, filament throat; degenerated stamen 3, hairless. The flower disc is cup-shaped and is provided with sharp teeth. Pistil is hairless. The capsules are linear and have no hair. Seed spindle shape. The flowering period is 7-10 months. On hills, mountain forests or on rocks at the yin or on trees at an altitude of 300-2000 m.

At present, scholars research the tissue culture and rapid propagation technology of the asulaca polytricha and the plants of the same genus, and the adopted technology is as follows:

stone hanging and laver hanging: (1) Callus and adventitious bud induction culture medium MS+6-BA 3.0mg/L, unpublished induction rate; (2) Proliferation and subculture medium MS+6-BA 3.0mg/L+NAA 0.1mg/L, proliferation coefficient 3-4; (3) Rooting culture medium 1/2MS+NAA 0.5mg/L, rooting rate 100% and no other rooting indexes. Adding 3% sucrose and 0.7% agar powder into the culture medium, and the pH value is 5.8-6.0; (4) Culturing tissue culture seedlings under natural light for 5d, opening a bottle cap to smelt the seedlings for 3-5 d, carefully taking out the seedlings after the seedling is smelt, washing the culture medium, transplanting the seedlings into a mixed matrix (wood chips: garden soil: sand=7:2:1), spraying and watering the seedlings once by 500 times of carbendazim, keeping the humidity of the matrix at about 60%, and keeping the temperature at 15-20 ℃ and the survival rate after one month to reach 100%.

Stone-hanging moss: (1) The induction culture medium comprises MS+6-BA 1.0mg/L+IBA 0.1mg/L+3% sucrose, wherein in the uncontaminated explant, the callus induction rate is 85.7%, and the adventitious bud induction rate is 14.3%; (2) Proliferation culture medium MS+6-BA 0.5-1.0 mg/L+IBA 0.05mg/L+3% sucrose, proliferation coefficient is 4.0; (3) Rooting culture medium MS+6-BA 0.1mg/L+IBA 0.01mg/L+2% sucrose, rooting rate over 95% and other rooting indexes not published; the above culture medium was added with 0.6% agar, and the pH was 6.0. (4) And (3) after the rooting test tube seedlings are subjected to seedling hardening for 2d in an indoor bottle cap opening, the seedlings are gently taken out from a culture bottle, a root culture medium is washed by tap water and transplanted into a matrix, and the transplanted matrix is prepared by composting and decomposing pond sludge with rich organic matters. The transplanted seedlings are cultivated in a multi-span greenhouse which is 75% of the shading greenhouse. And (5) watering regularly according to weather conditions, keeping the humidity of the seedling pot, and counting the survival rate after 40 days, wherein the survival rate is more than 90%.

As a result of culturing the leaves of Isodon japonicus with MS medium as a minimal medium, the combination of 6-BA 3.0mg/L,1.0mg/L6-BA+0.1mg/L indolebutyric acid (IBA) and 1.0mg/L6-BA+2, 4-dichlorophenoxyacetic acid (0.5, 1.0 mg/L) was added, and as a result, it was found that the explants were prone to browning and vitrification, and callus or adventitious buds could not be induced normally. Analysis of possible causes: only cytokinin with higher concentration is added in the MS+6-BA 3.0mg/L formula, so that vitrification of materials is easily caused; in addition, the aeschynanthis schneideriana is a tender leaf, the experimental material is a mature leaf, and the leaves in different development stages can cause the difference of culture results. The auxin used for the primary induction formula of the multi-tooth herba Hedyotidis Diffusae Dan Ju is IBA, the subject group also uses IBA and 6-BA with different proportions to induce the callus of herba Gei Alopecuroidis and Hemiboea Guizhou (same family plant), the callus and adventitious bud are not induced, the experimental material is from Guizhou, and the herba Hedyotidis Diffusae and the multi-tooth herba Hedyotidis Diffusae are from Yunnan and Guangxi, and can be different due to material difference.

The proliferation coefficient of adventitious buds in the 2 proliferation culture mediums is not high enough, probably because the ratio of cytokinin to auxin is not the optimal ratio, or NAA is more beneficial to in vitro culture of materials taken in the experiment than IBA.

Through the above analysis, the problems and defects existing in the prior art are as follows:

(1) In the existing tissue culture technology of the chicory, the explant is a tender leaf, and the material taking amount is limited.

(2) The induction medium disclosed in the prior art cannot induce callus and adventitious buds.

(2) The primary induction times have been published to be longer.

(3) The proliferation coefficient of the existing proliferation culture medium is not high.

The difficulty of solving the problems and the defects is as follows: the screening is carried out through different proportions of different plant growth regulators, the workload is large, and the experimental period is long.

The meaning of solving the problems and the defects is as follows: the herba Centipedae is a traditional medicinal material of the ethnic group such as Miao nationality, tujia, and Buyi, and compared with other herba Centipedae plant distribution areas, the medicine development of the plant as the main raw material in Guizhou province is mature, a plurality of Chinese patent medicines using herba Centipedae as the main raw material are produced at present, such as herba Lysionoti Pauciflori tablet, compound rock tablet of Guiyang Dechang auspicious pharmaceutical industry, YIFEIZHIKE Capsule of Guizhou Feiyuanling pharmaceutical industry, YANGGUOZHIKE liquid of Guizhou Guangzheng pharmaceutical industry, maryland cold-sensing capsule of Guizhou national pharmaceutical industry, and KEKANG buccal tablet of Guizhou Konghui pharmaceutical industry. Also, a plurality of scholars analyze medicinal components and the like of the chlorophytum comosum produced in Guizhou, and prove that the chlorophytum comosum produced in Guizhou has higher content of effective components. In recent years, commercial species have purchased herba Lysionoti Pauciflori, and the wild resources of herba Lysionoti Pauciflori in Guizhou have been greatly reduced. At present, an in-vitro culture technology for the chlorophytum comosum produced in Guizhou is not available, and the defects of limited material amount of explants, incapability of inducing callus and adventitious buds, long primary induction time, low proliferation coefficient and the like exist in the prior art, so that an in-vitro culture and plant regeneration technology system for the chlorophytum comosum produced in Guizhou is established, technical support can be provided for ensuring material supply of pharmaceutical enterprises, and scientific basis and technical reference are provided for reasonably protecting and utilizing the chlorophytum comosum.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides a method for in-vitro culture and plant regeneration of the leaves of the national drug-stone chlorophytum comosum.

The invention is realized in this way, a method for the in vitro culture and plant regeneration of the leaves of the national herb, the method for the in vitro culture and plant regeneration of the leaves of the national herb comprises the following steps:

firstly, adopting a rock chlorophytum comosum She Yu wound tissue and adventitious bud induction culture medium, and sterilizing at high temperature in an autoclave to prepare an MS solid culture medium;

secondly, selecting ripe leaves which are subjected to sterilization treatment and have no plant diseases and insect pests and no browning, cutting the leaves into small pieces, and inoculating the small pieces to a callus and adventitious bud induction culture medium;

thirdly, cutting off cluster buds with better growth vigor in primary induction and basal explants, inoculating the cluster buds and basal explants to an adventitious bud proliferation culture medium, and culturing;

fourthly, inoculating the adventitious bud proliferation strains with more consistent growth states to a rooting culture medium for induction rooting;

fifthly, after rooting the tissue culture seedlings, opening a bottle cap of a culture bottle, transplanting after hardening seedlings, carefully taking out the seedlings from the culture bottle, cleaning a root culture medium, transplanting the seedlings into a seedling tray filled with a sterilized mixed matrix, thoroughly watering the seedlings with an MS culture medium solution for the first time, and culturing the seedlings in a culture room;

further, the first step adopts the rock chlorophytum comosum She Yu wound tissue induction culture medium which is MS+1.0mg/L6-BA+0.5mg/LNAA, 3% of sucrose and 0.6-0.7% of agar powder are added, the pH is 5.8-6.0, the adventitious bud induction culture medium which is MS+2.0mg/L6-BA, 3% of sucrose and 0.6-0.7% of agar powder are added, and the pH is 5.8-6.0.

Further, the first step of high-temperature sterilization is carried out for 20min at 121 ℃ in an autoclave to prepare an MS solid culture medium, the culture temperature is 24+/-1 ℃, and the illumination time is 12 h.d ^-1 The illumination intensity is 2500lx.

Further, the second step is to select mature leaves which are sterilized and have no plant diseases and insect pests, cut into small blocks of 1cm multiplied by 1cm, and respectively inoculate the callus and the adventitious bud induction culture medium adopted in the first step.

Further, the third step is to take the cluster buds with better growth vigor in the primary induction and cut off together with the basal explants, and inoculate the cluster buds in an adventitious bud proliferation culture medium of MS+1.0mg/L6-BA+0.5mg/LNAA, and culture for 40d, wherein the pH value is 5.8-6.0.

Further, the rooting culture medium for the fourth rooting culture is MS+0.1mg/L6-BA+1.5mg/LNAA, and the pH value is 5.8-6.0;

and step four, inoculating the proliferation adventitious bud strains with more consistent growth state and 2-4 cm high to a rooting culture medium of MS+0.1mg/L6-BA+1.5mg/LNAA, and adding 3% of sucrose and 0.6-0.7% of agar powder, wherein the pH value is 5.8-6.0, so as to induce rooting.

And further, after rooting the tissue culture seedlings in the fifth step, opening the bottle cap of the culture bottle, hardening the seedlings for 3d, transplanting, carefully taking out the seedlings from the culture bottle, and cleaning the root culture medium.

Further, the fifth step of peat soil: vermiculite: perlite = 3V:1V:1V, sterilizing and cooling, and after transplanting the tissue culture seedlings, thoroughly watering the tissue culture seedlings by using 2% of MS culture medium solution for the first time, and keeping the soil moist.

Further, the fifth step is to pour thoroughly with 2% MS culture medium solution for the first time, and then to put the solution into a culture room for culture.

These parameters can shorten the induction time of callus and adventitious bud, raise the induction rate of callus and adventitious bud, and possesses good physiological state, raise proliferation coefficient of adventitious bud, and possesses high rooting rate, rooting number and transplanting survival rate.

Another object of the present invention is to provide a method for cultivating the leaves of the national herb, which uses the method for in vitro cultivation and plant regeneration of the leaves of the national herb.

By combining all the technical schemes, the invention has the advantages and positive effects that: the explant is a mature leaf, and the material is rich; the optimal culture medium for inducing the chlorophytum comosum callus is MS+1.0mg/L6-BA+0.5mg/LNAA, 3% of sucrose and 0.6-0.7% of agar powder are added, the pH is 5.8-6.0, and the induction rate is 91.67%; the optimal culture medium for inducing adventitious buds is MS+2.0mg/L6-BA, 3% sucrose and 0.6-0.7% agar powder are added, the pH is 5.8-6.0, and the bud ratio is 89.54%; the optimal culture medium for adventitious bud proliferation is MS+1.0mg/L6-BA+0.5mg/LNAA, 3% sucrose and 0.6-0.7% agar powder are added, the pH is 5.8-6.0, the culture is carried out for 40d, and the proliferation coefficient is 5.56; the optimal rooting culture medium is MS+0.1mg/L6-BA+1.5mg/LNAA, 3% of sucrose and 0.6-0.7% of agar powder are added, the pH is 5.8-6.0, the rooting rate is 100%, and the roots are thick and large in quantity; transplanting the rooting tissue culture seedlings into seedling trays filled with sterilized mixed matrixes (peat soil: perlite: vermiculite=3v:1v:1v), and watering thoroughly with 2% of MS culture medium solution for the first time, wherein after 30d, the survival rate is 94.96%.

According to the invention, cytokinins such as KT, TDZ, 6-BA and the like and auxins such as NAA, IBA, 2,4-D and the like are selected as experimental reagents, a large number of experimental formulas are used for screening out that the 6-BA and the NAA are most suitable for the in vitro culture of the chlorophytum comosum, and different proportions are adopted to induce callus and adventitious buds, proliferate the adventitious buds and induce rooting.

The callus induction culture medium is used for culturing about 10d of leaves to expand, about 15d of leaves to grow callus, the edges of the leaves grow full of compact callus after 30d, the induction formula of the pennywort herb callus is published, and after 25d of culture, the wound parts of leaf blocks expand and light yellow transparent colloid callus appears; the formula of the herba Cichorii does not clearly describe the callus induction time, but statistics are carried out after 45 days; from the viewpoint of culture time, the induction culture medium can shorten the callus induction time, and the callus induction rate is more than 90 percent, so that the physiological state is good.

According to the invention, the adventitious bud induction culture medium is cultured for about 40 days, each explant can form bud seedlings with the height of 0.3-0.8 cm, 2-4 leaves and unequal quantity, the total bud ratio can reach 89.54%, the culture medium can bud quickly, the quantity of the generated buds is large, and the seedling quality is good; the germination rate of the published induction culture medium of the herba cichorii is not described, and ideal adventitious buds are obtained after about 57 d; the induction rate of the adventitious buds of the induction culture medium of the aeschynanthia polysacharin is 14.3%, and ideal adventitious buds can be obtained after 59-66 d.

The proliferation coefficient of the adventitious buds in the adventitious bud proliferation culture medium is 5.56, which is 3-4 higher than that of the existing scheme of the Philippine rock-sochlaina and the polydentate Philippine rock-sochlaina.

The rooting rate in the rooting medium is 100%, the average root number is 21.6, the average root length is about 3.5cm, and the rooting rate is higher than 95% of that of the herba cichorii with multiple teeth; the transplanting survival rate of the tissue culture seedling is 94.96 percent, and the survival rate is high.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following description will briefly explain the drawings needed in the embodiments of the present application, and it is obvious that the drawings described below are only some embodiments of the present application, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a flowchart of a method for in vitro culture and plant regeneration of leaves of the national drug-stone chlorophytum comosum provided by the embodiment of the invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Aiming at the problems existing in the prior art, the invention provides a method for in-vitro culture and plant regeneration of the leaves of the national drug chlorophytum comosum, and the invention is described in detail below with reference to the accompanying drawings.

As shown in fig. 1, the method for in-vitro culture and plant regeneration of the national drug-stone chlorophytum comosum leaves provided by the invention comprises the following steps:

s101: the optimal culture medium for callus induction is MS+1.0mg/L6-BA+0.5mg/LNAA, the optimal culture medium for adventitious bud induction is MS+2.0mg/L6-BA, the pH is 5.8-6.0, the high temperature sterilization is carried out for 20min at 121 ℃ in an autoclave, the solid culture medium for MS is prepared, the culture temperature is 24+/-1 ℃, the illumination time is 12 h.d-1, and the illumination intensity is 2500lx;

s102: selecting mature leaves which are sterilized, have no plant diseases and insect pests and no browning, cutting the leaves into small pieces with the length of 1cm multiplied by 1cm, and inoculating the small pieces into a callus induction culture medium, wherein the steps are as follows: MS+1.0mg/L6-BA+0.5mg/LNAA, and the optimal culture medium for inducing adventitious buds is: MS+2.0mg/L6-BA;

s103: cutting off cluster buds with better growth vigor in primary induction and basal explants, inoculating the cluster buds and basal explants into an adventitious bud proliferation optimal culture medium which is MS+1.0mg/L6-BA+0.5mg/LNAA, and culturing for 40d, wherein the pH value is 5.8-6.0.

S104: the proliferation adventitious bud with a more consistent growth state and a height of 2-4 cm is inoculated in a basal culture medium taking MS as a main culture medium, and rooting culture medium with MS+0.1mg/L6-BA+1.5mg/LNAA and pH of 5.8-6.0 is added for induction rooting.

S105: after rooting of the tissue culture seedlings, opening a bottle cap of a culture bottle, transplanting after hardening seedlings for 3d, carefully taking out the seedlings from the culture bottle, cleaning a root culture medium, transplanting the seedlings into a mixed matrix (peat soil: vermiculite: perlite=3V:1V:1V) filled with bacteria, pouring the seedlings thoroughly with a 2% MS culture medium solution for the first time after transplanting the tissue culture seedlings, keeping the soil moist, and culturing the seedlings in a culture room.

The technical scheme of the invention is further described in the following in connection with experiments.

1 pretreatment of test Material

The test material, the chlorophytum comosum, is originally grown in a water-saving dry barrel dam in Guizhou province and a Pichia cover mountain, and is transplanted in a plant cultivation room of the Zunyi academy of education. The mature leaves without plant diseases and insect pests and mechanical damage are cut from the top ends of the branches of the parent plant of the chlorophytum comosum and used as explant materials, impurities on the surfaces of the leaves are washed by a hairbrush, the leaves are washed for 45-60 min by running water, the leaves are placed in a clean small beaker, redundant water is drained, and disinfection and sterilization treatment are carried out in an ultra-clean workbench. The test explant is sterilized by adopting a combined disinfectant, and the effect is optimal, namely the explant is sterilized for 15s in 75% alcohol, is washed three to five times by sterile water, is soaked in 0.1% mercuric chloride for sterilization for 5min, is washed five times by sterile water, and is gently stirred during the period. The sterilized explant is placed in a sterile bottle and ready for inoculation.

2 test method

2.1 culture conditions

MS culture medium is used as basic culture medium for inducing callus and adventitious bud of herba Centipedae, and for subculturing proliferation and rooting. Adding 3% sucrose, 0.6-0.7% agar powder, cytokinin (6-BA) and Naphthalene Acetic Acid (NAA) with different mass concentration ratios into the basic culture medium, sterilizing at 121 ℃ for 20min in an autoclave at pH of 5.8-6.0, and preparing the MS solid culture medium. The culture temperature is 24+/-1 ℃, and the illumination time is 12 hours.d ^-1 The illumination intensity is 2500lx.

2.2 Primary Induction

Selecting sterilized and browning-free leaves, cutting into small pieces of 1cm multiplied by 1cm, inoculating on MS culture medium combined with plant growth regulators with different concentrations, inoculating 20 dishes in each treatment, inoculating 3-4 leaves in each dish, and repeating for 3 times. The callus differentiation was observed periodically after 14d, and then every 4d, and the callus growth status, induction rate, etc. were recorded after 30 d. In the process of callus induction, part of the explants differentiate buds, so as to search the influence of the mixture ratio of the regulators with different concentrations on the differentiation of the explants, and further culture for 10d on the basis of callus induction for 30d, record the growth condition of buds, count the bud number, the bud yield and the like.

2.3 adventitious bud proliferation culture

Cutting off cluster buds with better growth vigor in primary induction and basal explants, inoculating 3 buds per block on a proliferation culture medium, inoculating 10 bottles, repeating for 3 times, counting the number of adventitious buds after 40d, calculating proliferation coefficients, recording bud seedling states, callus color changes and the like.

2.4 rooting culture

Inoculating the adventitious bud propagation strains with more consistent growth state and 2-4 cm high on a basal culture medium which takes MS as a base, adding 6-BA and NAA rooting culture mediums with different mass concentrations for induction rooting, inoculating 15 strains for each treatment, repeating for 3 times, recording bud seedling rooting conditions and root system states after 15 days, and counting rooting induction rate and rooting quantity after 30 days.

2.5 seedling hardening and transplanting

After rooting of the tissue culture seedlings, opening a bottle cap of a culture bottle, transplanting after hardening seedlings for 3d, carefully taking out the seedlings from the culture bottle, cleaning root culture medium, transplanting the rooting tissue culture seedlings into a seedling tray filled with sterilized mixed matrix (peat soil: perlite: vermiculite=3V:1V:1V), watering thoroughly with 2% MS culture medium solution for the first time, placing in a culture room for culture, strictly controlling moisture during the culture, periodically monitoring the growth condition of the seedlings, and counting the survival rate of the seedlings after 30 d.

The technical effects of the present invention will be described in detail with reference to experiments.

1 Primary Induction

The leaf was inoculated into the MS medium shown in Table 1, and the results are shown in Table 2. It was found that most explants will either continue to differentiate into shoots after callus induction or directly induce adventitious shoots. On the A3 culture medium, the leaves are cultivated for about 10 days to expand, the calli are started to sprout about 15 days, the edges of the leaves are full of compact and light yellow calli after 30 days, the induction rate of the calli can reach 91.67%, part of the edges of the leaves do not produce calli, but the differentiation rate of the late buds is lower, and the total germination rate is only 72.57%; on the A7 culture medium, after 20d culture, the callus is generated, the callus induction rate is only 53.33%, and the culture is continued for about 20d, each explant can form bud seedlings with the height of 0.3-0.8 cm, 2-4 leaves and unequal quantity, the total bud ratio can reach 89.54%, the culture medium is rapid in bud emergence, the quantity of the generated buds is large, and the seedling quality is better; the callus induction rate and bud differentiation rate of the A2 culture medium are low, and the generated buds have yellowing phenomenon. The experiment also cultures herba Cephalanthi Albae leaves with a combination of 3.0mg/L6-BA and 0.1mg/L indolebutyric acid (IBA), 2, 4-dichlorophenoxyacetic acid (0.5, 1.0 mg/L), and as a result, the explants were found to be prone to browning and vitrification and not grow normally.

TABLE 1 Primary Induction of different regulator ratios by Chlorophytum procumbens

TABLE 2 influence of different regulator ratios on the induction of the generation Dan Lanchu of the crane

Note that: capital english letters after the same column data represent differences to a significant level (p < 0.05).

2 adventitious bud proliferation culture

The test finds that: the chlorophytum comosum can grow and the bud number is increased in MS culture medium added with different mass concentration regulators. As can be seen from Table 3, the C5 culture medium has obvious promotion effect on the proliferation of adventitious buds, the formed buds are stronger, the leaves are large, the proliferation speed of the buds is high, the number of generated new buds is large, the proliferation coefficient reaches 5.56 after 50d of culture, the growth condition is relatively best, and the yellowing of the leaves at the later stage is possibly related to insufficient nutritional ingredients in the culture medium. Although the proliferation coefficient of the C7 culture medium is higher, the proliferated bud stem node is short, the leaf blade is not stretched, and the late stage She Bianhuang is easy to fall off.

TABLE 3 influence of different regulator ratios on proliferation culture of adventitious buds of Chlorophytum procumbens

Note that: capital english letters after the same column data represent differences to a significant level (p < 0.05).

3 rooting culture

Adventitious root induction was performed using MS medium added with 6-BA and NAA of different mass concentrations, adventitious buds were inoculated into rooting medium for cultivation, and the number of adventitious roots and rooting rate were counted after 30d, and the results are shown in Table 4. The sprouts cultured by the P1, P2, P3, P5 and P6 rooting culture mediums can root 100%, and no obvious difference exists between the treatments; the P3 culture medium added with 0.1mg/L6-BA and 1.5mg/LNAA is best in rooting, fast in rooting, large in root number and thick, the root length can reach about 3.5cm, and after rooting culture is carried out for 50 days, the plant length is obvious, and the leaves are stretched and enlarged; p1 and P2 root slowly, root short, P5 and P6 leaves turn yellow and fall off. Under the condition that the rooting rate and the average root number are close, the combined production efficiency of the low-concentration 6-BA and the relatively high-concentration NAA is higher.

In addition, experiments show that vermiculite is used as a matrix, the prepared liquid rooting culture medium is used for fully soaking the vermiculite, the pH is 5.8-6.0, indexes such as rooting rate and rooting number are not obviously different from those of culture materials in the agar culture medium, the solidification culture medium is not required to be removed during transplanting, and the damage to tissue culture seedlings can be reduced.

TABLE 4 Effect of different hormone ratios on the differentiation of Isodon japonicus

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Note that: capital english letters after the same column data represent differences to a significant level (p < 0.05).

4 hardening off and transplanting

After rooting of the tissue culture seedlings, opening a bottle cap of a culture bottle, transplanting after hardening seedlings for 3d, carefully taking out the seedlings from the culture bottle, cleaning the root culture medium, transplanting the root culture medium rooting tissue culture seedlings into a seedling raising tray filled with a sterilized mixed matrix (peat soil: perlite: vermiculite=3V:1V:1V), watering thoroughly with a 2% MS culture medium solution for the first time, and after 30d, enabling the survival rate to be 94.96%.

The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.

Claims (8)

1. The method for in-vitro culture and plant regeneration of the national herb chlorophytum comosum leaves is characterized by comprising the following steps of:

firstly, adopting a root of Chinese starwort She Yu wound tissue induction culture medium, and sterilizing at high temperature in an autoclave to prepare an MS solid culture medium;

secondly, selecting mature leaves which are subjected to sterilization treatment and have no plant diseases and insect pests and no browning, cutting the leaves into small pieces, and inoculating the small pieces to a callus induction culture medium;

thirdly, cutting off cluster buds with better growth vigor in primary induction and basal explants, inoculating the cluster buds and basal explants to an adventitious bud proliferation culture medium, and culturing; the adventitious bud proliferation culture medium is MS+1.0mg/L6-BA+0.5mg/LNAA, the pH is 5.8-6.0, and the culture is carried out for 40d;

fourthly, inoculating the adventitious bud proliferation strains with more consistent growth states to a rooting culture medium for induction rooting; the rooting culture medium is MS+0.1mg/L6-BA+1.5mg/LNAA, and the pH value is 5.8-6.0;

fifthly, after rooting the tissue culture seedlings, opening a bottle cap of a culture bottle, transplanting after hardening seedlings, carefully taking out the seedlings from the culture bottle, cleaning a root culture medium, transplanting the seedlings into a seedling tray filled with a sterilized mixed matrix, thoroughly watering the seedlings with an MS culture medium solution for the first time, and culturing the seedlings in a culture room;

in the first step, the root of Chinese holly She Yu wound tissue inducing culture medium is MS+1.0mg/L6-BA+0.5mg/LNAA, 3% of sucrose and 0.6-0.7% of agar powder are added, and the pH value is 5.8-6.0.

2. The method for in vitro culture and plant regeneration of national herb chlorophytum comosum leaves according to claim 1, wherein the first step of high-pressure sterilization is carried out in a high-temperature sterilization pot at 121 ℃ for 20min to prepare an MS solid culture medium, the culture temperature is 24+/-1 ℃, and the illumination time is 12 h.d ^-1 The illumination intensity is 2500lx.

3. The method for in vitro culture and plant regeneration of national herb chlorophytum comosum leaves according to claim 1, wherein the second step is to select mature leaves which are sterilized and have no plant diseases and insect pests, cut the mature leaves into small pieces of 1cm multiplied by 1cm, and inoculate the callus induction culture medium adopted in the first step respectively.

4. The method for in vitro culture and plant regeneration of the leaves of the national herb chlorophytum comosum as claimed in claim 1, wherein,

and step four, inoculating the proliferation adventitious bud strains with more consistent growth state and 2-4 cm high to a rooting culture medium of MS+0.1mg/L6-BA+1.5mg/LNAA, and adding 3% of sucrose and 0.6-0.7% of agar powder, wherein the pH value is 5.8-6.0, so as to induce rooting.

5. The method for in vitro culture and plant regeneration of the leaves of the national herb chlorophytum comosum as claimed in claim 1, wherein after rooting of the tissue culture seedling in the fifth step, the bottle cap of the culture bottle is opened, transplanting is carried out after hardening the seedling for 3d, the seedling is carefully taken out from the culture bottle, and the root culture medium is cleaned.

6. The method for in vitro culture and plant regeneration of the leaves of the national herb chlorophytum comosum as claimed in claim 1, wherein the mixed matrix of the fifth step is peat soil: vermiculite: perlite = 3V:1V:1V, sterilizing and cooling, and after transplanting the tissue culture seedlings, thoroughly watering the tissue culture seedlings by using 2% of MS culture medium solution for the first time, and keeping the soil moist.

7. The method for in vitro culture and plant regeneration of the leaves of the national herb chlorophytum comosum as claimed in claim 1, wherein the fifth step is to pour thoroughly with 2% of MS culture medium solution for the first time and then to culture in a culture room.

8. A method for cultivating the leaves of the national herb-stone chlorophytum comosum, which is characterized in that the method for cultivating the leaves of the national herb-stone chlorophytum comosum uses the method for in-vitro cultivation and plant regeneration of the leaves of the national herb-stone chlorophytum comosum as claimed in any one of claims 1 to 7.

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