Cynanchum glaucescens tissue culture seedling and cultivation method and application

Abstract

The invention belongs to the technical field of traditional Chinese medicine seedling in-vitro rapid propagation biology, and particularly relates to a cynanchum glaucescens tissue culture seedling and cultivation method and application thereof, and the method comprises the following steps: (1) performing induced budding culture on a cynanchum glaucescens explant to obtain cynanchum glaucescens bud; (2) performing rooting culture on the cynanchum glaucescens buds to obtain rooted cynanchum glaucescens seedlings; and (3) planting the cynanchum glaucescens seedlings in a water culture manner. The two kinds of cynanchum glaucescens explants adopted by the method can be subjected to 100% induction to obtain cynanchum glaucescens latent buds to obtain regenerated plants, an efficient and stable tissue culture system is successfully established, the tissue culture breeding period is shortened to 40 d, and favorable conditions are created for industrial production of excellent cynanchum glaucescens seedlings. The method has the advantages of fast propagation, no limitation of seasons, simple and feasible implementation steps and very obvious effect.

Description

Technical Field

[0001] The invention belongs to the field of in vitro rapid propagation biotechnology of traditional Chinese medicine seedlings, and in particular relates to a method and application of tissue culture seedling formation and cultivation of Psoralea corylifolia. Background Art

[0002] Baiqian, Baiqian was first recorded in the "Famous Doctors' Records" of the Han and Wei Dynasties and was listed as a medium-grade product. It includes the Apocynaceae plant Cynanchum stauntonii (Decne.) Schltr. ex Lévl or Cynanchum genkwa leaf Baiqian [Cynanchum mglaucescens (Decne.) Hand.-Mazz.], which has the effects of lowering qi, eliminating phlegm and relieving cough. It is used for lung qi congestion, cough with phlegm, chest fullness and shortness of breath. For a long time, Baiqian has been in a wild state, and there are few reports on Baiqian germplasm resources, breeding and cultivation. Wild Baiqian grows in low mountains and hills, river beach sand stains, lakes, streams, ditches or valleys on the plains. At present, Baiqian is mainly collected by collecting mature siliques and propagated by seed sowing. The low reproduction coefficient, long operation cycle and relatively low efficiency have seriously restricted the development of artificial cultivation of Baiqian. Therefore, it is necessary to explore efficient rapid propagation industrialization technology of willow-leaf Baiqian.

[0003] At present, there are few reports on the rapid propagation of high-quality seedlings of white forsythia through tissue culture technology at home and abroad, and there is no complete cultivation and planting technology support. The establishment of a rapid propagation system for white forsythia and excellent cultivation methods will lay the foundation for the further establishment of factory breeding of white forsythia and the realization of cultivation and physiological and biochemical research on white forsythia. Summary of the invention

[0004] The invention aims to provide a method and application of tissue culture seedling formation and cultivation of white forsythia, which has the advantages of fast reproduction and no restriction of seasons, simple and easy implementation steps and very significant effect.

[0005] In order to achieve the above object, the technical solution of the present invention is:

[0006] In one aspect, the present invention provides a method for tissue culture and seedling formation of white front, comprising the following steps:

[0007] (1) inducing budding culture of the explants of the white stalk to obtain white stalk buds;

[0008] (2) culturing the white front buds to root and obtain rooted white front seedlings;

[0009] (3) Planting the white forsythia seedlings in water.

[0010] In some other embodiments, in step (1), the whitehead is whitehead of willow leaf and / or whitehead of genkwa leaf;

[0011] The explant is a young stem segment, an adventitious bud, a leaf, a petiole or a hypocotyl; preferably, the explant is a young stem segment.

[0012] In some other embodiments, the culture medium used for the budding induction culture is: MS basic medium supplemented with 0.8-2.0 mg / L 6-benzylaminoadenine, 0.1-0.3 mg / L α-naphthaleneacetic acid, 0-0.5 mg / L indole-3-acetic acid, 30-35 g / L sucrose, 7-10 g / L agar, pH 5.0-6.0;

[0013] Preferably, the culture medium used for the budding induction culture is: MS basic medium supplemented with 0.5 mg / L 6-benzylaminoadenine, 0.1 mg / L α-naphthylacetic acid, 30 g / L sucrose, 7 g / L agar, pH 5.8;

[0014] The conditions for the budding induction culture are: a culture temperature of 25° C., 16 h light / 8 h dark culture for 10-15 days.

[0015] In some other embodiments, the white front bud is 3-4 cm, and the culture medium used for the rooting culture is 1 / 2MS basic culture medium supplemented with 0.1-0.3 mg / L α-naphthylacetic acid, 0-0.1 mg / L indole-3-acetic acid, 30-35 g / L sucrose, 7-10 g / L agar, and pH 5.0-6.0;

[0016] Preferably, the culture medium used for the rooting culture is 1 / 2MS basic culture medium supplemented with 0.1 mg / L NAA, 30 g / L sucrose, 7 g / L agar, and pH 5.8.

[0017] In some other embodiments, the rooting culture conditions are: culture temperature is 25°C, 16h light / 8h dark culture for 10-15d.

[0018] In some other embodiments, the nutrient solution used in the hydroponic cultivation is a modified Hoagland nutrient solution;

[0019] The improved Hoagland nutrient solution is composed of: calcium nitrate 0.8-2.0 g / L, potassium nitrate 0.5-1.0 g / L, magnesium sulfate 0.5-1.0 g / L, potassium dihydrogen phosphate 0.25-5.0 g / L, iron sulfate 0.05-0.1 g / L, chelated iron 2.5-5.0 mg / L, boric acid 2.86-6.0 mg / L, manganese chloride 1.81-4.0 mg / L, zinc sulfate 0.86-2.0 mg / L, copper sulfate 0.08-0.2 mg / L, sodium molybdate 0.02-0.05 mg / L, and the pH value is adjusted to 5.5-6.5.

[0020] In some other embodiments, the composition of the improved Hoagland nutrient solution is: 1.6 g / L calcium nitrate, 1.0 g / L potassium nitrate, 1.0 g / L magnesium sulfate, 5.0 g / L potassium dihydrogen phosphate, 0.1 g / L iron sulfate, 5.0 mg / L chelated iron, 5.72 mg / L boric acid, 3.62 mg / L manganese chloride, 1.72 mg / L zinc sulfate, 0.16 mg / L copper sulfate, 0.04 mg / L sodium molybdate, and the pH value is adjusted to 5.5-6.5;

[0021] Alternatively, the hydroponic cultivation is carried out for 10-60 days, with ventilation every 3-5 days.

[0022] In some other embodiments, the method further includes sterilizing the explants before inducing budding of the explants, wherein the sterilization is first performed with 70-75 vol% ethanol for 30-60 s, and then with 2.0 -3.0 wt% NaClO for 5-20 min;

[0023] Preferably, the disinfection treatment is to first disinfect with 75 vol% ethanol for 60 seconds and then disinfect with 2.5 wt% NaClO for 15-20 minutes.

[0024] In another aspect, the present invention provides an application of the method for forming seedlings and cultivating white formosanus according to the first aspect in in vitro rapid propagation of white formosanus.

[0025] Beneficial effects of the present invention:

[0026] The present invention uses two kinds of white formosan ginseng to obtain 100% induction of white formosan ginseng latent buds to obtain regenerated plants, successfully establishes an efficient and stable tissue culture system, and shortens the tissue culture breeding cycle to 40 days, creating favorable conditions for the industrial production of high-quality white formosan ginseng seedlings. At the same time, the tissue culture seedlings are propagated through hydroponic culture, and grow rapidly, have dark green leaves, and have a well-developed root system. The method has fast reproduction and is not restricted by seasons, and the implementation steps are simple and easy to implement, and the effect is very significant. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0028] Figure 1 The state of promoting axillary bud germination by different culture media in the embodiments of the present invention;

[0029] Figure 2 The tissue culture seedlings in the embodiment of the present invention;

[0030] Figure 3 This is the hydroponic system of Dipterocarpus salicifolius in the embodiment of the present invention. DETAILED DESCRIPTION

[0031] In the specific embodiments of the present invention, the reagents or culture media used are all commercially available products unless otherwise specified.

[0032] Example 1

[0033] Collection and preparation of explants of P. difficile

[0034] The test materials are stems of Rhizoma Cibotii and Rhizoma Cibotii, which are collected from the medicinal plant garden of Hubei University of Chinese Medicine from March to June each year. The healthy stems of Rhizoma Cibotii that were grown in the current year were cut, the leaves were removed, and the relatively sturdy stems (2-3mm) with the axils of the leaves were cut into 1.0-2.0cm. In the preparation process of the explants, all of them must be disinfected. Although the disinfection process in the prior art is to use 75% ethanol disinfection and mercuric chloride or sodium hypochlorite for disinfection and sterilization again, different explants need to be examined for the influence of different disinfection times and types of disinfectants.

[0035] The invention puts the explant into a sterile bottle of a clean bench, sterilizes the stem segment with 75% ethanol for 30s and 60s, and then transfers to 2% sodium hypochlorite (NaClO) for secondary disinfection, respectively disinfecting for 5min, 10min, 15min, 20min, and 25min. The explant after disinfection is washed three times in sterile water, and the explant is transferred to a sterile filter paper to absorb excess water. The stem segment is cut off from about 0.3cm parts at both ends, and the explant material is inoculated into a culture medium MS solid culture medium, and cultured at 25°C for 14d, and the contamination rate, whitening rate and germination rate of the explants with different disinfection treatments are counted, and the best disinfection method is selected.

[0036] The results showed that the effects of different disinfectants and disinfection time combinations of two kinds of white front were investigated on the white front stem segments, and the pollution rate, whitening rate and axillary bud germination rate were investigated as shown in Table 1. The pollution and whitening of the stem segments and leaves were observed after 14 days. Analysis of Table 1 showed that the combination of different disinfectants and time had a significant effect on the disinfection effect of white front explants. For the white front stem segments; although the whitening rate of treatments A1, A2 and A6 was low, their pollution rate was also high, so they could not be used as the most suitable disinfection method; although the pollution rate of treatments A5 and A10 was low, their whitening rate was also high, and they could not be used as the most suitable disinfection method; combined with various treatments, it can be seen that A8 and A9 treatments are significantly better than other treatments, so it can be concluded that the best disinfection method for white front stem segments is 75% ethanol for 1.0 min, and then 2% NaClO disinfection time for 15-20 minutes for the best effect.

[0037] Table 1 Effects of different disinfectants and disinfection time combinations on the stem segments of white scutellaria

[0038]

[0039] a,b,c,d,eItems marked with different letters indicate significant differences (P<0.05)

[0040] Example 2

[0041] Different hormone culture media induce elongation growth of white front shoots

[0042] The sterile and healthy material obtained in Example 1 (with the white sedge) was inoculated on different culture media to investigate the effects of different hormone ratios on the material. MS was used as the basic culture medium and the culture medium formula in Table 2 was used to prepare the culture medium. The plate was inverted on the clean bench, and 6 stem segments were inoculated in each group, and repeated for 5 groups. The germination was observed 14 days after inoculation.

[0043] Table 2 Culture medium formula with different hormone ratios

[0044]

[0045] Through the induction of culture media with different hormone formulas in Table 2, the added hormones are 6-benzylaminoadenine (6-BA), α-naphthaleneacetic acid (NAA), and indole-3-acetic acid (IAA). It can be seen that after the explants are successfully sterilized, various ratios can successfully induce the germination of leaf buds before the white leaves of willow leaves turn white, as shown in Table 3. Through observation, the growth status between the formulas is not the same. First of all, the germination rate, taking the germination growth of blank A0 as the normal germination speed (14-16d), the germination speeds of A1, A2, A3 and A6 are significantly improved (within 10d), among which the materials induced by A2 formula have larger leaves, dark green leaves, and thicker stems. The germination status induced by other formulas is significantly lower than that of A0, so it is not used as an alternative culture medium. After the buds of A4 and A5 germinate and elongate, adventitious roots also grow at the base of the stem segment. As a reference for inducing root differentiation into seedlings, the status of different culture media promoting axillary bud germination is shown in the following figure. Figure 1 As shown. Figure 1 It was found that the hormone formula with MS as the basic culture medium, 0.5-1.0mg / L 6-BA, 0.1mg / L NAA, 30g sucrose per liter, and 7g agar per liter had the best effect.

[0046] Table 3 Effects of different hormone ratios on the growth of willow leaf white front stem segments

[0047]

[0048]

[0049] Example 3

[0050] Different hormone culture media induce the growth of white front roots

[0051] The rooting induction of P. caespitosa was studied by using MS and 1 / 2MS basal medium plus different combinations of plant regulating hormones. The results are shown in Table 4. The rooting rate and rooting situation of each group were observed and compared at 7d and 21d, and the best rooting medium for P. caespitosa was obtained. 1 / 2MS is more suitable for rooting induction of P. caespitosa than MS. P. caespitosa plants cultivated with 1 / 2MS medium grew well and did not fail to root. 6-BA (6-benzyladenine), NAA (naphthaleneacetic acid) and IAA (indoleacetic acid) are plant growth regulators that play an important role in plant tissue culture. These compounds are plant hormones that can affect the growth, differentiation and development of plant cells. In tissue culture, they are usually used to induce dedifferentiation (dedifferentiation) and redifferentiation (redifferentiation) processes, as well as to regulate plant growth and development. 6-BA belongs to the cytokinin plant growth regulator, and its main function is to promote cell division and proliferation. In tissue culture, 6-BA is often used to promote the formation and maintenance of callus tissue and to increase the induction rate of buds. It can work synergistically with auxins (such as IAA and NAA) to affect cell differentiation and organ formation.

[0052] NAA has a significant effect on the growth of white sedge. Appropriate concentrations can make white sedge grow well. Too high a concentration not only inhibits the rooting of white sedge, but also may cause the plant to show abnormal color. IAA may have a synergistic effect on NAA, which has a positive effect on the growth of white sedge plants. If the plant grows well, Figure 2 As shown. 1 / 2MS is used as the basic medium, 0.3mg / L NAA, or 0.1mg / L NAA and 0.1mg / L IAA, 30g sucrose per liter, and 7g agar per liter.

[0053] Table 4 Rooting of white scutellaria baicalensis induced by different culture media

[0054]

[0055] Note: * represents the type of basal medium used in the modified formula.

[0056] Example 4

[0057] Hydroponics Planting

[0058] We found that the survival rate of hydroponic planting was high during cultivation, but the overall growth of the seedlings of white qian was slow in long-term clear water cultivation, and the leaves and stems turned yellow. Therefore, we tried to use nutrient solution and improved nutrient solution to explore the best way to hydroponic white qian. Figure 3shown. No treatment was used as the blank control: water; Treatment 1: calcium nitrate (Ca(NO3)2): 0.8g, potassium nitrate (KNO3): 0.5g, magnesium sulfate (MgSO4·7H2O): 0.5g, potassium dihydrogen phosphate (KH2PO4): 0.25g, iron sulfate (FeSO4·7H2O): 0.05g, chelated iron (EDTA iron): 2.5mg, boric acid (H3BO3): 2.86mg, manganese chloride (MnCl2·4H2O): 1.81mg, zinc sulfate (ZnSO4·7H2O): 0.86mg, copper sulfate (CuSO4·5H2O): 0.08mg, sodium molybdate (Na2MoO4·2H2O): 0.02mg, and the pH value was adjusted to 5.5-6.5; Treatment 2: each component in Treatment 1 was doubled per liter, and the pH value was adjusted to 5.5-6.5.

[0059] Table 5 shows the effects of different cultivation methods and treatments on the growth of white front. It can be seen from Table 5 that there is no significant difference between the white front tissue culture and the cultivated species in terms of leaf number and length, seedling height, stem node and length, while the number of tillers of the white front tissue culture is much greater than that of the cultivated species. At 30 days, it can be observed that the stem nodes of the white front plant have green calyx, purple corolla, and five deep lobes. At 60 days, it can be observed that the plant has a well-developed root system and a large number of white adventitious roots have grown; some young and tender side branches can be observed growing at the stem nodes of the plant.

[0060] Table 5 Effects of different cultivation methods and treatments on plant growth of Psoralea corylifolia

[0061]

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for tissue culture and cultivation of white radish, characterized in that: The following steps are involved: (1) inducing budding culture of the explants of the white stalk to obtain white stalk buds; (2) culturing the white front buds to root and obtain rooted white front seedlings; (3) Planting the white forsythia seedlings in water.

2. The method for tissue culture and seedling formation and cultivation of white front according to claim 1, characterized in that: In step (1), the whitehead is whitehead willow leaf whitehead and / or whitehead genkwa leaf whitehead; The explant is a young stem segment, an adventitious bud, a leaf, a petiole or a hypocotyl; preferably, the explant is a young stem segment.

3. The method for tissue culture and seedling formation and cultivation of white front according to claim 1, characterized in that: In step (1), the culture medium used for the budding induction culture is: MS basic medium supplemented with 0.8-2.0 mg / L 6-benzylaminoadenine, 0.1-0.3 mg / L α-naphthylacetic acid, 0-0.5 mg / L indole-3-acetic acid, 30-35 g / L sucrose, 7-10 g / L agar, and pH 5.0-6.0; Preferably, the culture medium used for the budding induction culture is: MS basic medium supplemented with 0.5 mg / L 6-benzylaminoadenine, 0.1 mg / L α-naphthylacetic acid, 30 g / L sucrose, 7 g / L agar, pH 5.8; The conditions for the budding induction culture are: a culture temperature of 25° C., 16 h light / 8 h dark culture for 10-15 days.

4. The method for tissue culture and seedling formation and cultivation of white scutellaria according to claim 1, characterized in that: In step (2), the white front bud is 3-4 cm, and the culture medium used for the rooting culture is 1 / 2MS basic culture medium supplemented with 0.1-0.3 mg / L α-naphthylacetic acid, 0-0.1 mg / L indole-3-acetic acid, 30-35 g / L sucrose, 7-10 g / L agar, and pH 5.0-6.0; Preferably, the culture medium used for the rooting culture is 1 / 2MS basic culture medium supplemented with 0.1 mg / L NAA, 30 g / L sucrose, 7 g / L agar, and pH 5.

8.

5. The method for tissue culture and seedling formation and cultivation of Psoralea corylifolia according to claim 1, characterized in that: In step (2), the rooting culture conditions are: culture temperature is 25°C, 16h light / 8h dark culture for 10-15d.

6. The method for tissue culture and seedling formation and cultivation of Psoralea corylifolia according to claim 1, characterized in that: In step (3), the nutrient solution used for hydroponic cultivation is improved Hoagland nutrient solution; The improved Hoagland nutrient solution is composed of: calcium nitrate 0.8-2.0 g / L, potassium nitrate 0.5-1.0 g / L, magnesium sulfate 0.5-1.0 g / L, potassium dihydrogen phosphate 0.25-5.0 g / L, iron sulfate 0.05-0.1 g / L, chelated iron 2.5-5.0 mg / L, boric acid 2.86-6.0 mg / L, manganese chloride 1.81-4.0 mg / L, zinc sulfate 0.86-2.0 mg / L, copper sulfate 0.08-0.2 mg / L, sodium molybdate 0.02-0.05 mg / L, and the pH value is adjusted to 5.5-6.

5.

7. The method for tissue culture and seedling formation and cultivation of Psoralea corylifolia according to claim 6, characterized in that: In step (3), the composition of the improved Hoagland nutrient solution is: 1.6 g / L calcium nitrate, 1.0 g / L potassium nitrate, 1.0 g / L magnesium sulfate, 5.0 g / L potassium dihydrogen phosphate, 0.1 g / L iron sulfate, 5.0 mg / L chelated iron, 5.72 mg / L boric acid, 3.62 mg / L manganese chloride, 1.72 mg / L zinc sulfate, 0.16 mg / L copper sulfate, 0.04 mg / L sodium molybdate, and the pH value is adjusted to 5.5-6.5; Alternatively, the hydroponic cultivation is carried out for 10-60 days, with ventilation every 3-5 days.

8. The method for tissue culture and seedling formation and cultivation of Psoralea corylifolia according to claim 1, characterized in that: Step (1) also includes disinfecting the explants before inducing budding culture, wherein the disinfection is first disinfected with 70-75 vol% ethanol for 30-60 seconds and then disinfected with 2.0-3.0 wt% NaClO for 5-20 minutes.

9. The method for tissue culture and seedling formation and cultivation of Psoralea corylifolia according to claim 8, characterized in that: In step (1), the disinfection treatment is first disinfected with 75 vol% ethanol for 60 seconds, and then disinfected with 2.5 wt% NaClO for 15-20 minutes.

10. Use of the method for tissue culture seedling formation and cultivation of Psoralea corylifolia according to any one of claims 1 to 9 in in vitro rapid propagation of Psoralea corylifolia.

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