CN109302984B

CN109302984B - Periploca sepium rapid propagation method

Info

Publication number: CN109302984B
Application number: CN201811100507.9A
Authority: CN
Inventors: 黄大庄; 牛小云; 魏佳慧; 周健; 苏筱雨; 闫海霞
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2018-09-20
Filing date: 2018-09-20
Publication date: 2022-03-01
Anticipated expiration: 2038-09-20
Also published as: CN109302984A

Abstract

The invention relates to a rapid breeding method of periploca sepium, belonging to the technical field of plant breeding. The method comprises the step of carrying out tissue culture on stem segments with buds of periploca sepium as explant materials, wherein the tissue culture comprises disinfection treatment, induction culture, proliferation culture, rooting culture and domestication and transplantation. The rapid breeding method of periploca sepium solves the problems of low breeding speed, limited breeding quantity, low survival rate and the like of periploca sepium, can obtain a large number of excellent seedlings in a short time, expands the breeding to meet the market demand, and has high social and economic significance.

Description

Periploca sepium rapid propagation method

Technical Field

The invention relates to a rapid propagation method of plants, in particular to a rapid propagation method of periploca sepium, belonging to the technical field of plant propagation.

Background

Periploca sepium Bunge (Periploca sepium Bunge) also called goat milk strips and goat horn peach, has root bark also called cortex acanthopanacis and cortex periplocae, belongs to Periploca of Asclepiadaceae, and is a shrub widely distributed in northeast, northwest and North China, and provinces such as Henan, Hunan, Hubei and Jiangxi. Periploca sepium has the characteristics of drought resistance, salt and alkali resistance, water and soil conservation, no ingestion by livestock and the like, and can grow on mountain tops, hillsides, loess hilly areas and cutover lands, so that the Periploca sepium is often used as a pioneer tree species for artificial afforestation in difficult afforestation areas and vegetation degradation areas. In addition, the root bark of Periploca sepium can be used as medicine for treating arthritis, and can also be used as pesticide. As the pharmacological action and economic value of periploca sepium are continuously explored by people, the market demand of periploca sepium is increasing. However, due to excessive mining and digging, the forest destruction is wastefully realized, the habitat of the periploca sepium is damaged, and the quantity is sharply reduced. Under natural conditions, the periploca sepium has few fruits, the number of the sprout tiller seedlings is limited, the plant division propagation speed is low, the influence of time is large, and the mass rapid propagation of the periploca sepium is limited.

The breeding method of periploca sepium is various, and seed breeding, cuttage breeding and plant division breeding are common. In recent years, our country has paid more attention to the research on the chemical components of roots and stems of Periploca sepium and the physiological and ecological strategies of Periploca sepium under environmental stress, namely the research on the pharmacological action, economic value and ecological action of Periploca sepium, and the research on the systematic propagation technology of Periploca sepium is less. Such as: the accumulation change of active ingredients of each part of Periploca sepium in different growth periods (Zhang Chunyang, Lizhonghui, etc. Chinese herbal medicines 2012,43(12): 2508-; experimental research on in-vitro inhibition of proliferation of liver cancer cells and breast cancer cells by periplocin (Merffi, Zhangjing, Dunhancer such as drug evaluation research, 2014,37(1):30-33) proves that the periplocin has an inhibition effect on proliferation of human breast cancer MDA-MB-468 cells and liver cancer HepG2 cells through experimental research; the chemical components contained in the periploca sepium root bark are initially researched and extracted in Hiroshima, the insecticidal activity of the chemical components is explored, the primary research on the chemical components and the insecticidal activity of the periploca sepium root bark (Shiqing, Mayanmin, Qinhongqiang, West North agricultural science and newspaper 2005,14(6):141-144) and the further separation of the insecticidal active components of the periploca sepium (Li-ren-Feng-northwest agriculture and forestry science and technology university, 2016) are used for further separating the periploca sepium components, and the result shows that the periploca sepium root bark extract has certain stomach toxicity activity on armyworm.

The periploca sepium has few fruits, so that seeds are not easy to collect; the plant division breeding speed is low, the seed and plant division breeding of the periploca sepium is only simply explained in the propagation and application survey of the periploca sepium (Zhang Zhongguo, Zongyuxiang, Hebei forestry science and technology, 2011, (6):30-32), the cutting propagation test is mainly carried out, the result shows that the spring cutting survival rate and the rooting rate are low, the rainy cutting survival rate is higher and can reach more than 90%, and the cutting propagation of the periploca sepium has seasonal limitation. Research on the breeding technology of periploca sepium and research on the influence of the breeding technology on the physicochemical properties of soil (the university of agriculture in Hebei of Li Wen Juan, 2013) conducted scientific and systematic research on the breeding technology of periploca sepium, and the research result shows that the highest survival rate is the breeding method for seedling by plant, but the method has the disadvantages of limited breeding quantity, waste of materials under the condition of limited resources and unsuitability for mass breeding. The survival rate of open-air cuttage is higher, but the method has strict requirements on cuttage materials and has extremely strict requirements on maintenance management. The requirements of sowing and seedling raising on the thickness of the covering soil and the humidity of the soil are very strict, and the highest survival rate is only about 60 percent. A research result of the initial report of Periploca sepium seedling culture test (Korean sages, Korean steel, Liu satellite. Shanxi forestry science and technology, 2003, (2):19-20) shows that Periploca sepium can germinate and come out of soil after seeding and seedling culture for 5-6 days. But the thickness of the covering soil exceeds 1cm, and the germination rate is obviously reduced.

At present, few research reports on tissue culture of periploca sepium are reported. Only Zhang Jian et al have carried out systematic studies, such as "investigation of sucrose concentration and nitrogen source in Periploca sepium cell suspension culture system" (Zhang Jian, Gao Wen Yuan, Wang Juan. Chinese medicine J.2011, 46(2):98-101), "study of influence of elicitor Ag +, La3+ on growth of adventitious roots of Periploca sepium and accumulation of Periploca sepium glycoside" (Zhang Jian, Gao Wen Yuan, Wang Juan, Xiaopegen. Chinese medicine J.2011, 36(1):11-15), "establishment of Periploca sepium cell suspension culture system and determination of contents of periplocin and 4-methoxysalicylaldehyde (Zhang Jian, Gao Yuan, Wang Juan, etc. Tianjin Chinese medicine, 2010,27(2): 163) 165)," study of induction of Periploca sepia tissue culture vaccine and dynamic accumulation of secondary metabolite of Epiproca glycoside (Zhang Jian, Gao Jiang Ling, etc.; Chinese medicine J. Chinese medicine, 2010,35(18): 2392-; the influence of exogenous hormone on the rapid propagation of the embryonic axis of the periploca sepium (Zhangjia, Gaowen, Li Xinglin, China journal of pharmacy 2010,45(20):1539-1543) researches the influence of the exogenous hormone on the rapid propagation of the embryonic axis of the periploca sepium and establishes a tissue culture rapid propagation system taking the embryonic axis of the periploca sepium as an explant. Research on culture of adventitious roots of pseudostellaria heterophylla, periploca sepium and liquorice (university of yishuang tianjin, 2013) conducted research on induction and proliferation of adventitious roots of periploca sepium by using a tissue culture technology, and a periploca sepium adventitious root liquid culture system was established. The research reported in the previous researches aims to obtain secondary metabolites such as periplocin which is an effective component in the root system of periploca sepium, so that systematic research on the disinfection mode of explant materials of the periploca sepium is not carried out, and factors which possibly influence the growth of tissue culture seedlings in the induction and multiplication culture processes, such as the type of culture medium, the concentration of sucrose, the illumination condition and the like, are not researched. And in all related documents, domestication and transplantation of rooted tissue culture seedlings of periploca sepium are not studied.

A stem section and terminal bud rapid propagation system of Periploca forrestii Schlt is established in research on stem section and terminal bud tissue culture and plant regeneration of Periploca forrestii (Lei Shi Fei, Gaojie, Niaohui, Shi Lei, Chentao. Chinese medicinal materials, No. 34, No. 11, 2011, 11, 1656, 1660), so that feasibility is provided for popularization and planting of Periploca forrestii Schlt. The method comprises the following steps: by using plant tissue culture technology, stem segments and terminal buds of periploca forrestii are inserted into MS culture mediums containing 6-BA (6-benzyladenine), NAA (naphthylacetic acid) and 2, 4-D (2, 4-dichlorophenoxyacetic acid) with different concentrations for culture, and the stems and the terminal buds are induced to become complete plants. The result and conclusion are that the optimal culture medium for inducing the terminal bud is MS +6-BA1.0mg/L + NAA0.3mg/L, wherein the induction rate of the bud can reach 86.29%; the optimal culture medium for stem segment induction is MS +6-BA2.0mg/L + NAA0.5mg/L, and the induction rate of the bud can reach 56.67 percent; the optimal culture medium formula for proliferation is MS +6-BA2.0mg/L + NAA0.1mg/L, and the proliferation coefficient reaches 2.10; the rooting culture medium is preferably 1/2MS + IBA (indolebutyric acid) 0.5mg/L, and the rooting rate reaches 53.33%.

As Periploca sepium Bunge and Periploca forrestii Schlt belong to different species of Periploca and have great difference in biological habits (edited by the Chinese plant record editorial Committee of Chinese academy of sciences, Beijing: scientific Press, 1977,63: 272), the tissue culture and plant regeneration technology of stem segments and terminal buds of Periploca sepium cannot be applied to mass propagation of Periploca. Therefore, a special breeding system research must be carried out on periploca sepium to ensure that the mass breeding of periploca sepium is feasible.

Disclosure of Invention

The invention aims to solve the technical problem of providing a rapid breeding method of periploca sepium, which overcomes the defects in the prior art. The rapid propagation method of periploca sepium has the advantages of high propagation efficiency, high amplification coefficient and the like.

The purpose of the invention is realized by the following technical scheme.

A method for quickly reproducing periploca ploca is characterized by that the stem segment with bud of periploca ploca is used as explant material to make tissue culture.

According to the rapid propagation method of periploca sepium, the tissue culture comprises disinfection treatment, induction culture, proliferation culture, rooting culture and domestication and transplantation.

According to the rapid breeding method of periploca sepium, the stem section with buds of periploca sepium is obtained from 5 months, 7 months or 9 months, preferably 5 months.

According to the method for rapid propagation of periploca sepium, the stem section with buds of the periploca sepium, which is obtained in 5 months, is sterilized by 1min of 75% alcohol and 15min of 8% sodium hypochlorite; or the disinfection mode of the stem section with the buds of the periploca sepium, which is obtained in 7 months, is as follows: 75% alcohol 30s + 10% sodium hypochlorite 20 min; or, the disinfection mode of the stem section with the buds of the periploca sepium, which is obtained in 9 months, is as follows: 75% alcohol 30s + 0.1% mercuric chloride 10 min.

According to the rapid propagation method of periploca sepium, the formula of the induction culture medium is MS +6-BA 1.5mg/L + NAA0.1mg/L + sucrose 30g/L, the illumination intensity is 2500lx, and the illumination time is 16 h/d.

According to the rapid propagation method of periploca sepium, the propagation medium is MS +6-BA2mg/L + IBA0.3 mg/+ sucrose 30g/L, and the illumination intensity is 3000 lx.

According to the rapid propagation method of periploca sepium, the rooting culture is carried out, and the rooting culture medium is 1/2MS, 0.5mg/L IBA and 25g/L sucrose.

The rapid breeding method of periploca sepium comprises the steps of domesticating, transplanting and hardening the seedling for 5d by closing the bottle and hardening the seedling for 5d by opening the bottle, wherein the transplanting medium is 1/2 peat and 1/2 vermiculite.

In order to obtain a large number of excellent seedlings in a short time and expand propagation to meet market demands, the invention utilizes a plant tissue culture technology, takes a stem section with buds of the periploca pulchra as an explant, and carries out systematic research on the tissue culture and rapid propagation technology of the stem section of the periploca pulchra, wherein the research comprises five main stages of disinfection treatment, induction culture, propagation culture, rooting culture and domestication and transplantation of in-vitro rapid propagation of plants, and the main research results are as follows: (1) the tissue culture method adopts 75% alcohol and mercuric chloride and sodium hypochlorite combined disinfectants with different concentrations for disinfection, the best material-obtaining period of tissue culture by taking stems with buds of periploca sepium as explant materials is 5 months, the pollution rate and the death rate are both the lowest, and the survival rate is the highest and reaches 84.44%. (2) In the induction culture, the formula of a proper culture medium is MS +6-BA 1.5mg/L + NAA0.1mg/L + sucrose 30mg/L, the proper illumination intensity is 2500lx, the illumination time is 16h/d, and the induction rate under the culture condition reaches 93.33%. In the proliferation culture, the formula of a culture medium suitable for proliferation of the stem section of the periploca sepium is MS +6-BA2mg/L + IBA0.3mg/L + sucrose 30mg/L, the suitable illumination intensity is 3000lx, the proliferation rate under the culture condition reaches 96.56%, and the proliferation coefficient reaches 6.28. (3) The rooting culture medium formula suitable for the periploca sepium tissue culture seedling is 1/2MS, 0.5mg/L IBA and 25g/L sucrose, the rooting rate under the culture condition reaches 91.67%, and the direct rooting effect by using the induced seedling is better. (4)1/2 peat and 1/2 vermiculite medium is suitable for the transplanting medium combination of the periploca sepium tissue culture seedling, and the survival rate of the transplanting medium reaches 78.89 percent. The effect of closed bottle hardening 5d + opened bottle hardening 5d is ideal. The rapid breeding method of periploca sepium solves the problems of low breeding speed, limited breeding quantity, low survival rate and the like of periploca sepium, can obtain a large number of excellent seedlings in a short time, expands the breeding to meet the market demand, and has high social and economic significance.

Drawings

FIG. 1 shows the disinfection effect of 5-month-old Periploca sepium stem.

FIG. 2 shows the disinfection effect of mercuric chloride on stem of Periploca sepium taken in 7 months.

FIG. 3 shows the disinfection effect of mercuric chloride on stem of Periploca sepium with 9 months of material.

FIG. 4 shows the effect of sodium hypochlorite on the disinfection of 5-month-old stem segments of Periploca sepium.

FIG. 5 shows the effect of sodium hypochlorite on the disinfection of stem segments of Periploca sepium taken in 7 months.

FIG. 6 shows the effect of sodium hypochlorite on the disinfection of stems of Periploca sepium (9 months old).

FIG. 7 effect of MS medium concentration on the rooting rate of tissue culture seedlings.

FIG. 81/4 MS tissue culture seedling status.

FIG. 9 effect of IBA concentration on rooting of periploca sepium tissue culture seedlings.

FIG. 10 shows the survival rate of tissue culture seedling transplantation of periploca sepium under different substrates.

FIG. 11 shows the technical route of the rapid breeding method of Periploca sepium

Detailed Description

The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.

Test example research on the rapid propagation method of Periploca sepium

1 materials and methods

1.1 general description of the test

Periploca sepium is adopted as an explant material in the experiment, and the experiment material is adopted in a test field in the western school district of Hebei agriculture university in the lotus pool area of Baoding province in Hebei province. The land belongs to a plain area, the terrain is flat, and the geographic coordinates are 115 degrees and 30 degrees of east longitude and 38 degrees and 50 degrees of north latitude. The local area is windy and dry in spring; in summer, the rainwater is more in hot days; cooling and raining in autumn; the frost-free period of the whole year is about 6 months (4 middle of the month to 10 middle of the month). The soil type of the experimental field is calcareous tidal brown soil, and the soil quality mainly comprises clay sandy soil and sandy clay.

1.2 test materials

1.2.1 Disinfection test Material for Stem segment of Periploca sepium at different sampling periods

The branches of the periploca sepium growing robustly and having no diseases, pests and scars are the basic conditions for the explant of the tissue culture. On the basis, branches with similar growth potential and about 40cm long are selected, leaves, tender top ends and over-mature base parts of the branches are removed, and the rest parts are cut into small stem sections with buds about 2 cm.

Material taking time: the materials were obtained in 2016, 5, 7, and 9 middle of the month.

1.2.2 Induction culture Material

After the stem section with the buds of the common clubmoss herb is subjected to disinfection treatment, the stem section which is free of pollution and good in growth is taken as a test material and is transferred to an induction culture medium under aseptic operation for direct induction culture, namely: the stem with buds can directly germinate into seedlings from axillary buds without inducing the process that explant materials are dedifferentiated to form callus.

1.2.3 enrichment culture Material

The stem segment is used for direct multiplication, namely, after the stem segment with the buds of the periploca sepium is subjected to disinfection treatment, the stem segment which is free of pollution and good in growth is taken as a test material and is transferred to a multiplication culture medium for culture under aseptic operation, and axillary buds (or lateral buds) of axillary parts of leaves are used for culture to directly obtain sprouts or cluster seedlings, namely: the stem segment of the periploca forrestii with buds does not need to undergo the process of callus differentiation and seedling emergence, so that a direct and efficient rapid propagation way of the stem segment of the periploca forrestii with buds is established.

1.2.4 rooting culture Material

And when the test-tube plantlet grows to 4-5 cm high, selecting a new plantlet with good growth potential, cutting the new plantlet from the base part, and transferring the new plantlet to a rooting culture medium for culture under the aseptic operation.

1.2.5 acclimatization and transplantation material

Periploca sepium tissue culture seedlings which grow well and have developed root systems after rooting culture are selected as transplanting materials and are transplanted into a nutrition pot filled with a substrate (sterilized) for transplanting test.

1.3 test design and method

1.3.1 Disinfection test of Periploca sepium Stem segments at different sampling periods

1.3.1.1 explant treatment

The operation method comprises the following steps: (1) washing the collected stem segments with tap water for about 30min to remove impurities such as dust; adding detergent, brushing with soft brush, and rinsing with tap water for 60 min; (2) placing the culture material into 75% ethanol solution under aseptic condition (in sterilized clean bench), and sterilizing for about 30 s; note that: the step is suitable for mercury bichloride and sodium hypochlorite disinfection effect tests and disinfection treatment needing ethanol in orthogonal tests; (3) washing the explant material sterilized by ethanol with sterile water; (4) and (3) sterilizing according to a test design mode, washing the explant for about 5 times by using sterile water after the sterilization is finished until residues are washed away, inoculating the explant into an autoclaved MS culture medium (containing 30g of sucrose and 7g of agar per liter of culture medium, and the pH value of the explant is 6.8 +/-0.1) after water is absorbed on a sterile paper, and then putting the explant into a culture room for culture. The culture temperature is 23 +/-2 ℃, the illumination intensity is 2500lx, the illumination time is 16h/d, and the relative humidity is 70-80%.

1.3.1.2 mercuric chloride disinfection effect test

Mercuric chloride is used as a disinfectant to respectively disinfect the periploca sepium stem sections with buds, which are obtained in the middle ten days of 5 months, 7 months and 9 months. Mercury rise concentration setting: 0.05%, 0.1% and 0.2%, and the disinfection time is divided into 5min, 10min and 15 min.

The experiment was performed with 9 treatments, each of which was inoculated with 60 stem segments of periploca sepium with buds, and repeated 3 times. After 3 weeks of inoculation, the contamination rate, mortality rate and survival rate were counted and calculated as follows.

1.3.1.3 sodium hypochlorite Disinfection Effect test

And (3) disinfecting stem sections with buds of periploca sepium, which are obtained from 5 months, 7 months and 9 months, respectively by using sodium hypochlorite as a disinfectant. The concentration of sodium hypochlorite is set as follows: 6%, 8% and 10%, and the disinfection time is 10min, 15min and 20 min.

1.3.1.4 orthogonal test

And 3-factor and 3-level orthogonal test design is adopted to screen out the optimal disinfection mode of the stem section with the buds of the periploca sepium in different periods. The factors 3 are: 0.1% mercuric chloride Disinfection time, 8% sodium hypochlorite Disinfection time and 10% Hydrogen peroxide Disinfection time, according to L9 (3)⁴) The design alignment was performed (see table 1). The experiment was repeated 3 times for a total of 9 treatments, each treatment inoculated with 30 explants. After 3 weeks of inoculation, the contamination rate, mortality rate and survival rate of the stem segments with buds of periploca sepium in the test mode are counted.

TABLE 1 design of the experiments

1.3.2 Induction and enrichment culture

1.3.2.1 Effect of different sucrose concentrations on the induction of lateral buds of Periploca sepium

Inoculating sterilized stem segments with buds of the periploca sepium into MS basic culture medium containing 20, 25, 30, 35 and 40g/L of sucrose under aseptic operation, treating 5 groups, inoculating 30 explants in each group, observing and recording the induction rate, the bud color and the robustness of the stem segments of the periploca sepium under different sucrose concentrations after culturing for 15 days, and comparing which sucrose concentration in a short period can promote the stem segments to rapidly bud and ensure the bud quality, thereby screening the appropriate sucrose concentration for inducing and culturing the stem segments with the periploca sepium buds.

1.3.2.2 Effect of different illumination conditions on the induction of lateral buds of Periploca sepium

Inoculating sterile-processed stem segments with buds to an MS basic culture medium, and culturing under different illumination conditions, wherein the illumination conditions are 3 in total: 2000, 2500 and 3000lx, 3 sets of illumination time per illumination intensity, 13, 16 and 19h/d respectively, for 9 treatments, each set of treatments inoculating 30 explants. Observing and recording the induction rate of the stem section of the periploca sepium after culturing for 15 days, and analyzing the influence of the illumination condition on the induction of the lateral bud of the periploca sepium.

1.3.2.3 Effect of different hormone combinations on the Induction and proliferation of lateral buds of Periploca sepium

Generally, when the stem with buds is used as an explant for induction culture, no exogenous auxin is required to be added, and a good induction effect can be obtained. In the test, sterile stem segments are transferred into an MS minimal medium under sterile operation, a growth regulator adopts 6-BA, IBA and NAA combinations with different concentrations, an orthogonal test with 3 factors and 3 levels is designed (the experimental design is shown in table 2), and a minimal MS medium without hormone is additionally arranged as control treatment.

Each treatment was inoculated with 30 explants, repeated 3 times (same below). After 3 weeks of inoculation, the induction rate, net plant height, number of new leaves, leaf color (the leaf color is divided into three grades of emerald green, fresh green and yellow green, and represented by + + +, and +) and callus are observed and recorded, so that the stem segment with the buds of the periploca sepium is screened out and cultured with appropriate hormone and concentration combination.

In the proliferation culture, the presence, type and concentration of exogenous hormones have a great influence on the proliferation effect of explants. Proliferation culture is carried out on the disinfected pollution-free stem with buds according to the table 3, proliferation conditions under different treatments are observed and recorded after 4 weeks of inoculation, and the influence of the types and concentrations of hormones and the combination of the hormones with different concentrations on the proliferation of the stem of the periploca sepium is researched.

TABLE 2 Induction culture test design

TABLE 3 proliferation culture test design

1.3.2.4 Effect of different sucrose concentrations on proliferation of lateral buds of Periploca sepium

In this experiment, a culture medium (MS +6-BA1.0mg/L + NAA0.1 mg/L) having a high growth effect on lateral buds of periploca sepium in experiment 1.3.2.3 was used as the growth medium in this sucrose concentration experiment. Inoculating sterilized stem segments of periploca sepium with buds to the culture medium containing 10, 20, 30 and 40g/L of sucrose under aseptic operation, and repeating the steps for 3 times, wherein the treatment of 5 groups comprises 30 explants inoculated in each group. Observing and recording after 4 weeks of inoculation, and researching the influence of different sucrose concentrations on the proliferation rate and the proliferation coefficient of the stem segments of the periploca sepium.

1.3.2.5 Effect of different illumination conditions on proliferation of lateral buds of Periploca sepium

The stem segments with buds after disinfection are inoculated into a proliferation culture medium (a culture medium formula and a test of the influence of sucrose concentration on proliferation) through aseptic operation, and then cultured under different illumination intensities, wherein 4 illumination intensities are set under the illumination conditions: 1000, 2000, 3000 and 4000lx, and the illumination time is 16 h/d. Observing and recording the growth condition of the periploca sepium tissue culture seedlings after culturing for 4 weeks, and analyzing the influence of the illumination conditions (including illumination time and illumination intensity) on the growth condition of the periploca sepium tissue culture seedlings.

1.3.3 rooting culture

1.3.3.1 MS concentration test

And respectively inoculating the strong aseptic seedlings with the height of about 5-6 cm into MS, 3/4MS, 1/2MS and 1/4MS minimal medium with the concentration of macroelements changed only for rooting culture, wherein each medium contains 0.5mg/L of IBA, and other components are unchanged. And (3) repeating the steps for 3 times after each treatment and inoculation of 20 periploca sepium tissue culture seedlings, counting the rooting rate after 30 days of inoculation, and analyzing the influence of the concentration change of the MS culture medium (macroelements) on the rooting rate.

1.3.3.2 influence of IBA concentration on rooting of periploca sepium tissue culture seedlings

1/2MS culture medium is used as basic culture medium for rooting culture, IBA concentrations of 0.0, 0.3, 0.5, 0.7 and 1.0mg/L are designed, and other components of the culture medium are unchanged. The experiment was run for a total of 5 treatments, each treatment inoculated 20 tissue culture seedlings, repeated 3 times. And (3) calculating the rooting rate, the average root number and the root length after 30 days of inoculation, and analyzing the influence of different IBA concentrations on the rooting rate and the root growth condition of the periploca sepium tissue culture seedlings.

1.3.3.3 influence of different sucrose concentrations on rooting of Periploca sepium tissue culture seedling

The tube plantlets were aseptically inoculated into 1/2MS media containing 15, 20, 25, and 30g/L sucrose, each containing 0.5mg/L IBA, with the other ingredients unchanged. And inoculating 20 periploca sepium tissue culture seedlings in each treatment, and counting the rooting rate after 30 days of inoculation.

1.3.3.4 Effect of tissue culture seedling status on rooting

The tissue culture seedling for rooting is divided into two states of directly rooting after induction and rooting after proliferation (namely single seedling and cluster seedling), 1/2MS + IBA 0.5mg/L + sucrose 25g/L is used as a rooting culture medium for culture, and 20 periploca sepium tissue culture seedlings are inoculated in each treatment. And (4) after 30d of inoculation, counting the rooting rate, carrying out visual analysis, and analyzing the influence of the two seedlings on the rooting rate and the growth condition of the tissue culture seedlings when the two seedlings are used for rooting culture.

1.3.4 acclimatization and transplantation

1.3.4.1 acclimation time screening

The domestication of the periploca sepium tissue culture seedlings is divided into two stages of closed bottle domestication and open bottle domestication. Transferring the periploca sepium tissue culture seedlings from the tissue culture room to a seedling hardening room, closing the mouth and placing for 5 days under natural illumination, and then gradually opening the bottle caps to perform bottle opening domestication. The bottle opening acclimation time is set to 4 treatments, which are respectively 0d, 3d, 5d and 7 d. 30 rooted tissue culture seedlings were transplanted per treatment, and the experiment was repeated 3 times. And (5) counting the transplanting survival rate under different domestication time treatments after 3 weeks of transplanting.

1.3.4.2 screening of the transplantation substrate

The transplanting medium is the different combination of peat, vermiculite and garden soil, totally sets up 4 and handles, respectively: 1/2 peat +1/2 vermiculite; 1/2 peat +1/2 garden soil; 1/2 garden soil +1/2 vermiculite; 1/3 peat +1/3 garden soil +1/3 vermiculite. 30 transplants were done for each treatment, which was repeated 3 times. And (5) counting the transplanting survival rate of the periploca sepium tissue culture seedlings under different treatments after transplanting for 3 weeks.

1.4 culture conditions

In the induction, proliferation and rooting culture of periploca ferox stem segments, as mentioned below, the basic culture media are all MS (rooting 1/2MS) culture media containing 7g of agar and 30g of sucrose per liter and having a pH value of 6.8 + -0.1, the culture temperature is (23 + -2) ° C, the culture is carried out for 16h/d under the illumination intensity of 2500lx, and the relative humidity is 70% -80%, as not specifically mentioned. The culture conditions for domestication and transplantation are as follows: the illumination intensity is not too high after transplanting for 1-3 days, the illumination intensity is controlled to be about 5000-8000 lx, and the illumination intensity is gradually increased in the later period. The humidity is controlled to be about 80%, the growth condition of the transplanted seedlings is observed at any time, and the temperature and the humidity in the incubator are regulated and controlled in time.

1.5 data statistics and analysis

In an explant disinfection test, Microsoft Excel is adopted to count the pollution rate, the death rate and the survival rate; in the induction, proliferation, rooting culture and domestication and transplantation tests of the periploca forrestii stem segments, Microsoft Excel is adopted to count the induction rate, the proliferation coefficient, the rooting rate and the transplantation survival rate of the periploca forrestii stem segments under different test treatments, DPS statistical analysis software is utilized to carry out variance analysis on the data, and the Duncan method is utilized to carry out multiple comparisons. The specific index statistical formula is as follows:

contamination rate (%) ═ (number of contaminated explants/total number of inoculated explants) × 100% (1)

Mortality (%) ═ number of dead uncontaminated explants/total number of inoculated explants x 100% (2)

Survival (%). ratio (number of explants not contaminated by sprouts/total number of inoculated explants). times.100% (3)

Induction rate (%). ratio (number of bud-induced explants/total number of inoculated explants) × 100% (4)

The stem growth rate (%). ratio (number of successfully grown stem/total number of inoculated stem) X100% (5)

Stem growth factor (%). x 100% (6) (total number of buds obtained by growth/total number of successfully grown stem segments)

Rooting rate (%) - (number of rooted explants/total number of inoculated explants) × 100% (7)

Transplanting survival rate (%) (number of transplanted survival seedlings/total number of transplanted seedlings) x 100% (8)

2 results and analysis

2.1 Disinfection test of Periploca sepium Stem segments at different sampling periods

2.1.1L Hg for sterilizing stem of Periploca sepium of 5 months, 7 months and 9 months

Periploca sepium stem segment disinfection effect of 2.1.1.1L Hg on 5-month-taken materials

The effect of mercuric chloride on the disinfection of 5-month-old stem segments of Periploca sepium is shown in FIG. 1. Wherein a, b and c represent the trend of the survival rate, the pollution rate and the death rate of explants respectively after different times of sterilization by using mercury bichloride with different concentrations. It can be seen from a that the survival rate at the concentration of 0.05 percent is in an increasing trend along with the increase of the disinfection time, and the survival rate is obviously increased after 5-10 min; the survival rate at the concentration of 0.1% is in an increasing trend at 5-10 min and in a decreasing trend at 10-15 min; the survival rate at the concentration of 0.2 percent is in the overall descending trend; the survival rate of 10min of disinfection under the concentration of 0.1 percent is the highest, and is 83.33 percent. From b, it can be seen that the survival rate at each concentration decreases with increasing disinfection time, with a contamination rate of 0.2% < 0.1% < 0.05%, wherein the 0.2% concentration disinfection for 15min is the lowest contamination rate, 3.89%. It can be seen from c that the mortality rates at 0.1% and 0.2% both increased with increasing disinfection time, with the highest mortality rate at 0.2% and increasing mortality rate at 0.05% decreasing first.

2.1.1.2 Mercury-mercuric chloride stem sterilizing effect on 7-month-taken periploca sepium

The effect of mercuric chloride on the disinfection of the stem segments of the periploca sepium which are obtained from 7 months is shown in figure 2. As can be seen from a, b and c, the survival rates at 0.05% and 0.1% both showed an upward trend with increasing disinfection time, while the survival rate at 0.2% did not change significantly; the contamination rate was high at each concentration, and 0.05% > 0.1% > 0.2%. This is probably due to the fact that in 7 months, the concentration of 0.05% and 0.1% cannot kill the fungus contained in the explant in a short time, resulting in a high contamination rate, and as the disinfection time increases, the shortage of concentration is compensated, resulting in a decrease in the contamination rate. The mortality rate at 0.2% concentration showed an overall increasing trend with increasing disinfection time, which is probably due to the increased mortality rate caused by the damage of explants due to the high concentration of long-term disinfection treatment; the mortality rates at 0.1% and 0.05% both decreased and increased, with the mortality rate at 0.05% being the lowest at 10min of sterilization, 3.33%. From the survival rate index, the sterilizing effect of mercuric chloride with the concentration of 0.1% and 0.2% for 10min on the stem segments of the periploca sepium buds obtained in 7 months is good, and the survival rate is 57.78%.

Periploca sepium stem segment disinfection effect of 2.1.1.3L Hg on 9-month-taken materials

The effect of mercuric chloride on disinfection of 9-month-old stem segments of Periploca sepium is shown in FIG. 3. a. b and c represent survival, contamination and mortality, respectively. As can be seen from fig. 3, the survival rates at 0.05% and 0.1% concentration reached the highest at 10min disinfection time, 66.11% and 67.22%, respectively; the survival rate of 0.2% mercuric chloride is the highest when the disinfection time is 5 min. The contamination rate at each concentration decreased with increasing disinfection time, 0.05% < 0.1% < 0.2%. The mortality rate in October is larger than that in 5 months and 7 months, the concentration of 0.1 percent and 0.2 percent are both obviously improved along with the increase of the disinfection time, wherein the mortality rate in 15min of disinfection with the concentration of 0.2 percent is as high as 32.78 percent; the mortality rate at 0.05% concentration has the same trend as that of month 5 and month 7, and has a trend of descending first and then ascending, 10min <5min <15min, wherein the mortality rate is 7.22% when the disinfection time is 10 min.

2.1.2 sodium hypochlorite Disinfection effect on stem of Periploca sepium (Thunb.) Makino obtained in 5 months, 7 months and 9 months

2.1.2.1 Disinfection effect of sodium hypochlorite on stem of Periploca sepium (Batal.) Craib obtained in 5 months

The effect of sodium hypochlorite on the disinfection of 5-month-old stem segments of periploca sepium is shown in fig. 4. From a, b and c, it can be seen that the results of disinfection treatment of stems of periploca sepium buds for 5 months with different concentrations of sodium hypochlorite are very different. Along with the increase of the disinfection time, the pollution rate of the disinfection treatment with each concentration is in a descending trend, and the mortality rate is opposite to the descending trend, wherein the pollution rate of the sodium hypochlorite with the concentration of 10% is the lowest in 20min and is 3.89%, and the pollution rate of the sodium hypochlorite with the concentration of 6% in 10min is the highest and is 42.22%. Under different concentrations, with the increase of the disinfection time, the survival rate of the stem segments of the periploca sepium reaches the highest when the disinfection time is 15min, wherein the survival rate with the concentration of 8% is the highest and is 83.89%. When the disinfection time is prolonged to 20min, the survival rate is reduced. Sodium hypochlorite concentrations of 6%, 8% and 10% all reached the highest survival rate and lowest contamination rate at 15min of disinfection time. This suggests that although the prolonged disinfection time is beneficial to reduce the contamination rate of the explants, the prolonged disinfection time may also cause toxicity or damage to the explants, increase the mortality rate and thus affect the survival rate.

2.1.2.2 Disinfection effect of sodium hypochlorite on stem of Periploca sepium (Thunb.) Makino of 7 months old

The effect of sodium hypochlorite on the disinfection of stem segments of periploca sepium obtained in 7 months is shown in figure 5. It can be seen from a, b and c that, unlike the survival rate of periploca sepium obtained in 5 months, the explants obtained in July all reach the maximum value when the disinfection time is 20min under the disinfection treatment of sodium hypochlorite with different concentrations. Wherein the survival rate of the disinfection treatment is highest under the concentration of 10 percent and is 64.74 percent; the contamination rate was the lowest at 23.33%, but the mortality rate was also the highest at 20.56%. With the increase of the disinfection time, the pollution rate at each concentration is in a descending trend, and the survival rate is in an ascending trend, and the death rate is in an ascending trend. The disinfection time is definite, the survival rate is 10% > 8% > 6%, the pollution rate is 6% > 8% > 10%, and the death rate is 10% > 8% > 6%. This indicates that the explant obtained in July has the phenomenon of difficult disinfection, and the increase of the concentration of the disinfectant and the increase of the disinfection time can not ensure the improvement of the survival rate. The best disinfection effect is achieved by using 10% sodium hypochlorite for 20min, and the survival rate is taken as a standard.

2.1.2.3 Disinfection effect of sodium hypochlorite on stem of Periploca sepium (Batal.) Craib of 9 months old

The effect of sodium hypochlorite on the disinfection of stems of Periploca sepium obtained in 9 months is shown in FIG. 6. The survival rate of each concentration reaches the maximum value when the disinfection time is 15min, and the death rate is the lowest, wherein the disinfection effect is better when the concentration of sodium hypochlorite is 10%, and the survival rate is the highest 61.11%. The survival rate of 6 percent of sodium hypochlorite concentration is in an increasing trend along with the increase of the disinfection time; when the disinfection time is increased from 15min to 20min, the survival rate at the concentration of 8% is basically kept unchanged, and the survival rate at the concentration of 10% is obviously reduced. The cause of the decreased survival rate is the increased mortality rate. The mortality rate at each concentration tended to decline slowly and then to rise as the disinfection time increased, and increased overall compared to

months

4 and 7. The mortality rate at 10% concentration reached a maximum of 51.11% at a disinfection time of 20 min.

2.1.3 Quadrature test Disinfection Effect

The results of the effect of different disinfection treatments on the contamination rate at different sampling periods are shown in table 4. As can be seen from Table 4, the disinfection effect was significantly different in different periods of material selection under the same disinfection treatment. The overall contamination rate for the material taken in 7 months is higher, while the contamination rate for the material taken in 5 months is lower. Treatment 3 and treatment 4 contamination rates in different months were treatment 4< treatment 3, indicating that the contamination rate for the two agents mixed for sterilization is lower than using a single disinfectant.

The contamination rate of explants taken in 5 months after disinfection by treatment 7 and treatment 9 is significantly lower than that of other treatments, 1.11% and 3.33% respectively, while the disinfection effect of treatment 1 is the worst, and is significantly higher than that of other treatments and the contamination rate reaches 62.22%. Treatment 7 the contamination rate after sterilization of explants taken from 7 months was significantly lower than other treatments, 17.78%. Compared with July, the 9-month material-taking pollution rate is reduced, and the pollution rates after the treatment 4 and the treatment 7 are respectively 12.22 percent and 10.00 percent.

TABLE 4 contamination Rate at different sampling periods

Note: different lower case letters after the same column of data indicate significant differences (p < 0.05).

The results of the effect of different disinfection treatments on the survival rates at different time periods are shown in table 5. As can be seen from Table 5, the disinfection effect was significantly different in different periods of material selection under the same disinfection treatment. Overall, survival rates are 5 months >9 months >7 months.

Treatments

3 and 4 disinfected explants taken at 5 months best, significantly more than the other disinfection treatments, with survival rates of 84.44% and 83.33%, respectively. The survival rate of the explants obtained in 7 months after the disinfection treatment is obviously lower than 5 months, wherein the survival rate of the explants obtained in 7 months after the disinfection treatment of 4 is higher than that of the explants obtained in other disinfection treatments, and the survival rate is 51.11%. The survival rate of the explants taken in 9 months after the treatment 4 is obviously higher than that of the explants treated by the treatment 5, and the survival rate is 61.11%.

TABLE 5 survival rates at different stages of material selection

2.2 Induction and enrichment culture

2.2.1 Effect of different sucrose concentrations on the Induction of lateral buds of Periploca sepium

As can be seen from Table 6, the germination percentage and the sprout mass of the stem with buds of Periploca sepium were different 15 days after the inoculation at different sucrose concentrations. In the induction period of the stem section of the periploca sepium, the germination rate of the stem section with the buds of the periploca sepium is in an increasing trend along with the increase of the sucrose concentration, the germination rate is the highest and is 80.00 percent under the concentration of 30g/L of the sucrose concentration, and the bud quality of the induced stem section of the periploca sepium is the best. The germination rate was lowest at a sucrose concentration of 20g/L, probably because in tissue culture the explants were not autotrophic and required to absorb energy from the medium, and the germination rate was very low because the lower sucrose concentration did not meet the energy required for germination of the periploca sepium stem segments. When the sucrose concentration is increased to 35 g/L and 40g/L, the germination rate of the stems of periploca sepium is in a downward trend, which is probably because the too high sucrose concentration can influence the nutrient absorption of plant cells, so that the germination rate of the stems of periploca sepium is reduced when the sucrose concentration is 35 g/L and 40 g/L. The quality of the bud is the worst at 40g/L, which is not beneficial to the later culture. The comparison proves that when the concentration of sucrose in the culture medium is 30g/L, the germination of buds can be promoted and the buds can grow strongly, and the culture effect of the buds of the stem segments of the periploca sepium at the bud induction stage is ideal.

TABLE 6 Effect of different sucrose concentrations on shoot Induction

2.2.2 Effect of different illumination conditions on the induction of lateral buds of Periploca sepium

As can be seen from Table 7, the influence of different illumination conditions on the induction rate of the stem segment with buds of Periploca sepium is very different, when the illumination intensity is 2000lx, the induction rate of the stem segment with buds of Periploca sepium increases with the increase of the illumination time, and under the illumination conditions of the illumination intensity of 2500lx and the illumination time of 16h/d, the induction rate of the stem segment with buds of Periploca sepium reaches the highest induction rate of 80.00%, while under the illumination conditions of the illumination intensity of 2000lx and the illumination time of 13h/d, the induction rate is the lowest induction rate of 46.67%. When the illumination intensity is 2500lx and 3000lx, the induction rate reaches the maximum value at 16h/d of illumination time along with the increase of the illumination time, and is 83.33 percent and 75.00 percent respectively, and when the illumination time is increased to 19h/d, the induction rate is reduced on the contrary. This is probably because when the illumination intensity is too low, the deficiency of the illumination intensity can be compensated to a certain extent by prolonging the illumination time, so that the induction rate is increased, and when the illumination intensity can meet the requirements of plant growth and development, the growth of the plant is affected by too long illumination time, and the induction rate is reduced.

TABLE 7 induction rate of stem with buds of Periploca sepium under different illumination conditions

2.2.3 Effect of different concentrations of hormone combination on shoot Induction

As can be seen from Table 8, the induction of periploca ferox stem segments was very different with different concentrations of the hormone combination. The induction rates under different treatments have significant differences, wherein the induction rate of treatment 5 is the highest and is 93.33 percent, and is significantly higher than that of other treatments; the net plant height of the induced buds is also obviously higher than that of other treatments and is 7.46 cm; the number of newly added leaves is 15.27 leaves, the color of the leaves is emerald green, and no callus is formed. The induction rate of the treatment 1 is 86.67 percent, which is obviously higher than that of the treatments except the treatment 5, the net plant height is 7.38cm, the leaf color is bright green, and no callus is generated. Treatment 9 had the lowest induction rate of 25.56% and was significantly higher than the other treatments; the static plant height is obviously lower than that of other treatments except for the treatment 3, is 3.25cm, and has callus; treatment 3 induction was only 33.33% higher than treatment 3 and significantly lower than treatments in groups other than treatment 3; the static plant height and the number of newly added leaves are respectively 2.58cm and 3.53, the leaves are yellow green, and callus is formed. The induction rate of the control group (MS) is only second to that of the treatment 5, and is 87.78% of net plant height of 6.31cm, which is obviously higher than that of the

treatments

2, 3, 8 and 9; the number of newly added leaves is 9.90, the color of the leaves is bright green, and no callus is formed. Experiments show that in the direct induction process of the periploca lever bud stem segment, the induction rate of the stem segment generating the callus is obviously lower than that of the stem segment without the callus. This may be due to the production of callus which inhibits axillary bud germination, or may be due to the callus's production in relation to a combination of hormone concentrations, i.e. hormone levels affect bud induction, whereas callus is only an extrinsic manifestation.

The comparison shows that a proper amount of 6-BA has a positive effect on the induction of the periploca sepium stem segments with buds, the high-concentration NAA has an inhibition effect on the induction of the periploca sepium buds, and the induction rate is reduced along with the increase of the concentration of the NAA. This may be due to the ratio of auxin to cytokinin, and may also be affected by hormones endogenous to periploca sepium itself.

TABLE 8 Effect of different hormone combinations on shoot Induction

2.2.4 Effect of combinations of hormones at different concentrations on proliferation of lateral buds of Periploca sepium

As can be seen from Table 9, when the cytokinin (6-BA) concentration was constant and the auxin NAA and IBA concentrations were the same, the proliferation factor treatment 2 > treatment 1, treatment 4 > treatment 3, treatment 6 > treatment 5, i.e.: the multiplication coefficients of the lateral buds of the periploca sepium inoculated in the culture medium added with IBA are all higher than those of NAA, and the growth condition of the tissue culture seedlings is better. This shows that the same concentration of auxin has IBA (epibatic acid) superior to NAA on the proliferation effect of lateral buds of periploca sepium. In addition, the ratio of cytokinin to auxin is different, and the proliferation effect is also different. When the ratio of cytokinin to auxin is 10 (

treatments

1, 2, 5 and 6), the proliferation coefficient is low, and the tissue culture seedling is weak and grows slowly; when the ratio was 7.5 (treatments 3 and 4) and 6.7 (treatment 7), the proliferation coefficients were high, 4.46, 5.79 and 6.29, respectively, and the growth potential of the tissue culture seedlings was good. This indicates that the ratio of cytokinin to auxin suitable for proliferation of lateral buds of periploca sepium is 6.5-7.5. Wherein MS +6-BA2mg/L + IBA0.3mg/L is the best multiplication medium, the multiplication coefficient is 6.29, and the tissue culture seedling has stout branches and grows faster.

TABLE 9 Effect of different hormone combinations on shoot proliferation

2.2.5 Effect of different sucrose concentrations on proliferation of lateral buds of Periploca sepium

As can be seen from Table 10, when the sucrose concentration is in the range of 10-30 g/L, the proliferation rate and the proliferation coefficient of lateral buds of periploca sepium gradually increase with the increase of the sucrose concentration. From the proliferation rate, the highest sucrose concentration is 30g/L, and is 96.56%; when the concentration of the sucrose is lower than 30g/L, the proliferation rate is reduced, the proliferation rate is respectively 17.78 percent and 43.33 percent under the concentration of 10g/L and 20g/L, and the proliferation rate is reduced to 75.56 percent when the concentration of 40g/L is higher than that of 30 g/L. From the proliferation coefficient, when the concentration of sucrose was 30g/L the highest, 10g/L the lowest, 6.28 and 3.57 respectively. Although the proliferation rate is still high at a sucrose concentration of 40g/L, the proliferation coefficient is low and is 3.86. This indicates that too low a concentration of sucrose may not satisfy the energy required for proliferation of lateral buds of periploca sepium, but too high a concentration may also adversely affect its proliferation.

TABLE 10 influence of sucrose concentration on proliferation of lateral buds of Periploca sepium

2.2.6 Effect of different illumination conditions on proliferation of lateral buds of Periploca sepium

As can be seen from Table 11, the growth conditions of the periploca sepium tissue culture seedlings under different illumination intensities are greatly different. The tissue culture seedlings grow best under 3000lx illumination intensity, which is reflected in better proliferation, robust seedlings and no vitrification. When the illumination intensity is 4000lx, the tissue culture seedlings have no vitrification phenomenon, but the overall growth potential is reduced, the stem nodes of the proliferated seedlings are shortened, and the growth is slow in later period. The illumination intensity of less than 2000lx is not beneficial to the proliferation of lateral buds of periploca sepium, no proliferation or less proliferation, and the seedlings are fragile and vitrified. The proper illumination intensity is necessary for the normal growth of the multiplication seedlings of the lateral buds of the periploca sepium, the normal multiplication and growth cannot be completed due to insufficient illumination intensity, and the growth of the multiplication seedlings is also adversely affected due to overhigh illumination intensity.

TABLE 11 growth conditions of Periploca sepium tissue culture seedlings under different illumination intensities

2.3 rooting culture

2.3.1 influence of MS culture medium concentration on rooting rate of Periploca sepium tissue culture seedlings

As can be seen from FIG. 7, the influence of different MS medium concentrations on rooting of periploca sepium tissue culture seedlings is different. When the concentration of MS culture medium (macroelements) is 1-1/2, the rooting rate of the tissue culture seedling is in an increasing trend along with the reduction of the concentration, and reaches the highest value at 1/2MS, namely 90%; when the concentration of MS is continuously reduced to 1/4, the rooting rate is reduced to 25 percent; the rooting rates at MS and 3/4MS were 50% and 60%, respectively. And in the experiment, the phenomenon that the leaf color of the plant is yellow and falls off is found in 1/4MS treatment, as shown in figure 8. This shows that in the rooting culture of periploca sepium tissue culture seedling, the rooting capacity of tissue culture seedling can be improved by properly reducing the concentration of macroelements, and when the concentration of macroelements in MS culture medium is 1/2, the highest or too low rooting rate of periploca sepium tissue culture seedling is not favorable for the growth of root system.

2.3.2 Effect of IBA concentration on rooting of Periploca sepium tissue culture seedlings

As can be seen from Table 12 and FIG. 9, the change in IBA concentration has a great influence on the rooting of the periploca sepium tissue culture seedlings. When the IBA concentration is increased from 0.0mg/L to 0.5mg/L, the rooting rate of the periploca sepium tissue culture seedling is in an increasing trend, and reaches the highest value of 91.67 percent when the concentration is 0.5 mg/L; when the IBA concentration is increased from 0.5mg/L to 1.0mg/L, the rooting rate is reduced on the contrary, and the rooting rate reaches the lowest value of 23.33% when the IBA concentration is 1.0mg/L, which indicates that the rooting is not facilitated when the IBA concentration is too high or too low, and the rooting rate cannot be increased by simply increasing the IBA concentration. When the IBA concentration is 0.5mg/L, the number of roots of the periploca sepium tissue culture seedlings is the largest, the average number is 11.5, and the treatment is obviously higher than that of other concentrations; the root number is 4.3 pieces at a concentration of 0.0mg/L, which is the least. When the IBA concentration is 0.0mg/L, the root system is slender and weak, and almost no lateral root (such as a) exists, so that the IBA hardly survives even if transplanted; when the concentration of IBA is 0.5mg/L, the length of the root system is 6.2cm, but the root system is thick and strong, and lateral roots are developed (such as c); at concentrations of IBA of 0.7 (as d) and 1.0mg/L (as e), the root lengths were 2.5 and 2.3cm, respectively, and no lateral roots were present, indicating that too high a concentration of IBA may inhibit the growth of the root system. In conclusion, when the IBA concentration is 0.5mg/L, the growth of the periploca sepium root system is most suitable.

TABLE 12 influence of IBA concentration on rooting of Periploca sepium tissue culture seedlings

2.3.3 Effect of different sucrose concentrations on rooting of Periploca sepium tissue culture seedlings

As can be seen from Table 13, when the sucrose concentration is 25g/L, the rooting rate of the tissue culture seedling of periploca sepium is the highest and is 90.00%, and the root growth condition under the concentration is also good, the rooting number is large, the lateral roots are large, the elongation growth is good, and the root is vigorous; when the concentration is increased to 30g/L, the rooting rate is reduced to 85 percent, the number of the roots is large, the roots are thick and strong, but the length of the roots is short; when the concentration of the sucrose is 20g/L, the rooting rate is 70%, and the root number is small and the root length is medium under the concentration. The root number is small under the concentration of 15g/L of sucrose, the root is slender and weak, and the rooting condition is not ideal.

TABLE 13 influence of sucrose concentration on rooting of tissue culture seedlings

2.3.4 Effect of tissue culture seedling status on rooting

As can be seen from Table 14, the rooting rates of the single seedlings and the cluster seedlings are different but not much from the rooting rate, the rooting rate of the single seedlings is 95%, and the rooting rate of the cluster seedlings is 85%. The inoculated seedlings are in different states, and the growth conditions of the tissue culture seedlings are also different. When the clump seedlings are cut into single plants for rooting culture, callus can still be formed on the base part, a few propagation seedlings are generated, and the number of the roots is large and the roots are thick; and when the single seedling is subjected to rooting culture, almost no callus and no propagation seedling are generated, the number of roots is 4-6, the length is long, and the number of lateral roots is large. The reasons for these differences may be related to the composition and content of phytohormones accumulated in plants.

TABLE 14 rooting percentage of single and cluster seedlings

2.4 acclimatization and transplantation

2.4.1 Effect of different acclimation times on the transplanting survival rate of the Periploca sepium tissue culture seedlings

As can be seen from table 15, on the basis of a certain closed-bottle acclimation time, the open-bottle acclimation time is different, and the survival rate of the tissue culture seedling transplantation of periploca sepium has significant differences in each treatment. The survival rate of the tissue culture seedlings of the periploca sepium directly transplanted without the bottle opening domestication is extremely lower than that of the tissue culture seedlings subjected to the bottle opening domestication, the survival rate is 24.44%, and with the increase of the number of days of the bottle opening domestication, the transplanting survival rate is highest under the treatment that the time of the bottle opening domestication is 5 days, is 77.78%, and is obviously higher than that of other treatments. This indicates that the tissue culture seedling can gradually adapt to new environment by properly increasing the bottle opening domestication time, so as to achieve the purpose of strengthening the seedling. When the bottle opening domestication time is continuously increased to 7d, the transplanting survival rate is reduced compared with 5d, which may be because the bottle opening domestication time is too long, the tissue culture seedling is greatly influenced by external environment, or nutrient components of the culture medium are excessively consumed, the viability of the tissue culture seedling is reduced, and the transplanting survival rate is reduced.

TABLE 15 influence of different acclimation times on the survival rate of tissue culture seedling transplantation

2.4.2 Effect of different transplanting substrates on the transplanting survival rate of the periploca sepium tissue culture seedlings

FIG. 10 shows the survival rate of tissue culture seedling transplantation of periploca sepium under different media. As can be seen from fig. 10, survival rates under different matrices: treatments 1 > 4 > 3 > 2. The survival rate of the processed 1(1/2 peat +1/2 vermiculite) is the highest, namely 78.89%, and the survival rate of the processed 2(1/2 peat +1/2 garden soil) is the lowest, namely 41.11%, which is probably because the garden soil and the peat are mixed together, so that the air permeability is poor, the root system is difficult to absorb water and nutrient or the root system is rotten, and the survival rate is reduced. Therefore, the proper culture medium has the nutrition required by the growth of the tissue culture seedling and good ventilation.

3 final technical scheme

In order to obtain a large number of excellent seedlings in a short time and expand propagation to meet market demands, the research utilizes a plant tissue culture technology, takes stems with buds of periploca sepium as explants, and systematically researches the tissue culture and rapid propagation technology of the stems of periploca sepium, wherein the main stages comprise five plant in-vitro rapid propagation stages of disinfection treatment, induction culture, propagation culture, rooting culture and domestication and transplantation, and the rapid propagation method of the exclusive periploca sepium is determined, namely:

(1) a systematic disinfection test is carried out on stem sections with buds of Periploca sepium in different material taking periods (5 months, 7 months and 9 months) by using disinfectants with different concentrations and different types, and a single-factor test and an orthogonal test are adopted in the method. The explant obtained in 5 months is sterilized by using 75% alcohol for 1min and 8% sodium hypochlorite for 15min, and the survival rate is 84.44%. The explant obtained in 7 months is suitable for disinfection in the following way: the survival rate of the sodium hypochlorite solution is 64.74 percent after 20min by using 30s of 75 percent alcohol and 10 percent sodium hypochlorite. The explant obtained in 9 months is sterilized properly as follows: the survival rate of the product is 67.22% by mixing 75% alcohol 30s and 0.1% mercury bichloride for 10 min. The best material taking period of tissue culture by taking the stem section with the buds of the periploca sepium as the explant material is 5 months, at the moment, the material pollution is easy to control, and the survival rate is high.

(2) Inoculating the disinfected stem segment of periploca sepium into an induction culture medium for induction culture. The influence of different sucrose concentrations and illumination conditions, and different concentrations and different types of plant growth regulator combinations on the induction of the stem segments with buds of the periploca sepium is researched. The formula of a proper culture medium is MS +6-BA 1.5mg/L + NAA0.1mg/L + sucrose 30 g/L; the appropriate illumination intensity is 2500lx, the illumination time is 16h/d, the excessive illumination time can influence the growth of plants and reduce the induction rate; the inductivity under the culture condition reaches 93.33 percent.

(3) Inoculating the disinfected stem segment of periploca sepium into a proliferation culture medium for proliferation culture. The influence of different sucrose concentrations and illumination conditions, and different concentrations and different types of plant growth regulator combinations on the proliferation of the stem segments with buds of the periploca sepium is researched. The appropriate culture medium is MS +6-BA2mg/L + IBA0.3 mg/+ sucrose 30g/L, and the concentration is too low to be beneficial to proliferation; the suitable illumination intensity is 3000 lx; the proliferation effect is better under the culture condition, and the proliferation coefficient reaches 5.79.

(4) Selecting a strong aseptic seedling for rooting culture, and researching the influence of different MS concentrations, IBA concentrations, sucrose concentrations and tissue culture seedling states on the rooting of the tissue culture seedling. The suitable rooting culture medium is 1/2MS + IBA 0.5mg/L + sucrose 25g/L, and under the condition of the culture, the seedling end is robust and the root system is developed. The direct rooting effect of the induced seedlings is better than that of the proliferated seedlings, and the rooting rate reaches 91.67%.

(5) The transplanting medium test adopts different combinations of peat, vermiculite and garden soil, and the transplanting survival rate is higher under the combination of the transplanting medium of 1/2 peat and 1/2 vermiculite, and reaches 78.89%. The effect of closed bottle hardening 5d + opened bottle hardening 5d is ideal.

From the above research contents, the rapid propagation method of periploca forrestii of the present invention is completely different from the method of stem segment and terminal bud tissue culture and plant regeneration research of periploca forrestii, such as Zhang Qian Liu Shi, and the like, and is specifically shown in table 16. The induction method is completely different from that of the tissue culture seedling of periploca sepium by Zhang Jian and the like, and is shown in Table 17.

TABLE 16 different points of the method of the present invention and the induction method of Liufei et al on Periploca sepium tissue culture seedling

TABLE 17 difference of the method of the present invention from the induction method of Zhang Jian et al for Periploca sepium tissue culture seedling

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. A rapid propagation method of periploca sepium comprises performing tissue culture on stem segments with buds of periploca sepium as explant material; the tissue culture comprises disinfection treatment, induction culture, proliferation culture, rooting culture and domestication and transplantation; the stem section with the buds of the periploca sepium is obtained from 5 months; the disinfection treatment is carried out, wherein the disinfection mode of the stem section with the periploca sepium bud, which is obtained in 5 months, is 1min of 75% alcohol and 15min of 8% sodium hypochlorite; the formula of the induction culture medium is MS +6-BA 1.5mg/L + NAA0.1mg/L + sucrose 30g/L, the illumination intensity is 2500lx, and the illumination time is 16 h/d; the enrichment culture is carried out, wherein the enrichment culture medium is MS +6-BA2mg/L + IBA0.3 mg/+ sucrose 30g/L, and the illumination intensity is 3000 lx; the rooting culture is carried out, wherein a rooting culture medium is 1/2MS, 0.5mg/L IBA and 25g/L sucrose; and (3) domesticating and transplanting, wherein a transplanting medium is 1/2 peat and 1/2 vermiculite, closing bottles and hardening seedlings for 5d, and opening bottles and hardening seedlings for 5 d.