CN107711502B - A medium kit and its application in the rapid propagation of Vitex unifolia in vitro - Google Patents

Abstract

The invention discloses a kind of culture medium box set and its applications in Vitex rotundifolia Vitro Quick Reproduction.Culture medium box set of the present invention includes for establishing the culture medium of sterile system, inducing the matrix mixed with the culture medium being proliferated, the culture medium for adventitious bud inducing, culture medium, the culture medium for taking root and the volume ratio for Elongation of adventitious bud induction for the yellow mud and peat soil of 3:1 for clump bud.Present invention firstly discovers that the adventitious bud development ways of stem with bud flattening induction and the through the invention in vitro culture of culture medium box set progress Vitex rotundifolia, clump bud breeding multiplying power reaches as high as 21.9, the adventitious bud of average each flattening explant occur quantity be up to 84.9, Elongation of adventitious bud up to 3.0cm, rooting rate with averagely take root number respectively up to 70.5% with 86.4, transplant 30 days after survival rate reach as high as 86%, have a good application prospect.

Description

A medium kit and its application in the rapid propagation of Vitex unifolia in vitro

technical field

The present invention belongs to the technical field of rapid propagation of plants in vitro, and more particularly, the present invention relates to a medium kit for rapid propagation in vitro for inducing buds of single-leaf vines and forming adventitious bud regeneration pathways on flattened stem segments and the same. method.

Background technique

Vitex rotundifolia L. is a small deciduous shrub of the genus Vitex of the Verbena family. It is widely distributed on beaches in tropical and subtropical regions, seasides, and barren islands by lakes. It has strong adaptability, does not have strict environmental requirements, and has the characteristics of alkali resistance and high temperature resistance. pioneer vegetation. Vitex unifolia also has high medicinal value. Its fruit is one of the sources of commonly used traditional Chinese medicine "Vanjingzi" in my country, which has the effect of dispersing wind-heat and clearing the head. Modern pharmacological research shows that Vitex has chemical components such as camphene, pinene, flavonoids, vitexin and luteolin, which have obvious analgesic, anti-inflammatory, antihypertensive, anti-tumor and anti-aging effects. And its stems can also be used for the weaving of baskets, flowers can extract aroma and so on.

With the development and utilization of new uses and new products of Vitex in recent years, its market demand is increasing day by day. leading to the depletion of its resources. In the "National Key Protected Wild Medicinal Species List", Vitex unifolia has been listed as a national class III endangered protected plant. At present, domestic and foreign researches on Vitex chinensis mainly focus on chemical components and pharmacological effects, and there are few studies on artificial protection and reproduction. The propagation methods of Vitex unifolia mainly include seed propagation, cutting propagation, division propagation, layering propagation, and non-in vitro vegetative organ rapid seedling breeding. At present, there is no report on the rapid propagation of Vitex unifolia in tissue culture.

SUMMARY OF THE INVENTION

The object of the present invention is to: overcome the problems such as lack of artificial propagation methods to Vitex unifolia in the prior art, provide a kind of explants with axillary bud stem section of Vitex unifolia, explore the plant growth of different concentrations and types The effects of regulators and their combinations on the induction of clustered buds, the induction of stem flattening, adventitious bud formation, adventitious bud elongation and rooting were investigated.

In order to achieve the above purpose of the invention, the present invention provides a medium for establishing a sterile system, which is based on MS medium and added with 25-35 g/L sucrose and 6.5-7.5 g/L agar.

As a preferred technical solution of the present invention for establishing a culture medium for a sterile system, the added amount of sucrose is 30 g/L, and the added amount of agar is 7 g/L.

The present invention also provides a medium for inducing and multiplying clump buds, which is based on the medium used for establishing a sterile system as a basic medium, and is supplemented with 0.5-2.5 mg/L 6-benzyl adenine.

The present invention also provides a medium for inducing adventitious buds, which is based on the medium used for establishing a sterile system as a basic medium, and is supplemented with 2.5 mg/L 6-benzyl adenine.

The present invention also provides a medium for inducing elongation of adventitious buds, which is based on the medium used for establishing a sterile system as a basic medium, and is supplemented with 1.5-2.5 mg/L naphthalene acetic acid.

The present invention also provides a medium for rooting, which is based on 1/2 MS medium and added with 3.0 mg/L indole butyric acid, 2.0-3.0 mg/L naphthalene acetic acid, 25-35 g /L sucrose and 6.5～7.5g/L agar.

BA, namely 6-benzyl adenine, is a widely used cytokinin added to plant growth medium. It can inhibit the decomposition of chlorophyll, nucleic acid and protein in plant leaves, preserve green and prevent aging; It is widely used in agriculture, fruit trees and horticultural crops at all stages from germination to harvest; it can promote cell division and differentiation from callus or organs in the rapid propagation of plants in vitro Adventitious buds, while helping to liberate axillary buds from the inhibition of apical dominance.

NAA, namely naphthalene acetic acid, is a broad-spectrum plant growth regulator, which is used to induce cell division and root differentiation during in vitro culture of plants, and can also affect stem and node elongation, tropism, and apical dominance. , leaves falling off, etc.

TDZ, namely thidiazuron, is an efficient cytokinin, which can be used to promote the growth of callus, the occurrence of lateral buds and adventitious buds, and the formation of embryoid bodies in the rapid propagation of plants in vitro.

2,4-D, or 2,4-dichlorophenoxyacetic acid, is a synthetic auxin analog. Under the condition of low concentration, it can effectively promote callus, but it can strongly inhibit the occurrence of organs. It is also a systemic herbicide.

KT, or kinetin, is a synthetic cytokinin, which can promote cell differentiation, division, growth, promote cell division, and differentiate adventitious buds on callus or organs.

ZT, namely zeatin, is a natural plant hormone extracted from the grains of sweet corn during the grain filling stage, but it can be synthesized artificially to promote callus germination.

IAA, namely indole acetic acid, is an endogenous auxin in plants, which is used to promote cell division and cell growth, and induce the formation of adventitious roots during the rapid propagation of plants in vitro.

IBA, i.e. indole butyric acid, is an endogenous auxin in plants. It is used to promote cell division and cell growth in the process of rapid propagation of plants in vitro, induce the formation of adventitious roots, increase fruit setting in agricultural production, prevent fruit drop, change female, Male flower ratio, etc.

In order to achieve the above object of the invention, the present invention also provides a medium kit, which includes the above-mentioned medium for establishing a sterile system, a medium for clump bud induction and proliferation, and a culture for adventitious bud induction. base, medium for adventitious bud elongation induction, medium for rooting, and a matrix of yellow mud and peat soil in a volume ratio of 3:1.

The above-mentioned medium kit of the present invention can be used for promoting the rapid propagation of Vitex unifolia in vitro.

In order to achieve the above-mentioned object of the invention, the present invention also provides a method for promoting the rapid propagation of Vitex unifolia in vitro, which comprises the following steps:

(1) choose the stem segment with axillary buds in the semi-lignified seedling of Vitex unifolia as explant (it is preferable to take the stem segment with a length of 1.5～3cm and with 2 lateral buds);

(2) under sterile conditions, the explants are inoculated on the described culture medium for establishing a sterile system after sterilizing with mercuric chloride to obtain sterile seedlings;

(3) cutting the sterile seedling obtained in step (2) into a bud-bearing stem segment with 1 node (2 side buds), inoculating on the described medium for clump bud induction and proliferation, to obtain clump bud;

(4) the clump buds obtained in step (3) are cut into stem segments with 1 node, inoculated on the described medium for adventitious bud induction, and the stem segments are induced to be flattened to obtain adventitious buds;

(5) inoculating the adventitious buds obtained in step (4) on the described medium for adventitious bud elongation induction to obtain adventitious buds that are elongated as normal buds;

(6) inoculate the adventitious buds obtained in step (5) as normal buds on the described medium for rooting, carry out rooting culture, obtain tissue culture seedlings, clean the root medium, and transplant The rapid propagation of Vitex unifolia in vitro was completed on the substrate mixed with yellow soil and peat soil with a volume ratio of 3:1.

As a preferred technical solution of the method for promoting the rapid propagation of Vitex unifolia in vitro according to the present invention, the culture conditions in steps (2) to (6) are: (25±1)° C., illumination time 12h/12h (L /D), light intensity 1500～2000lx.

As a preferred technical solution of the method for promoting the rapid propagation of Vitex unifolia in vitro according to the present invention, the process of disinfection in step (2) includes: disinfection in 75% alcohol for 10-15s, rinsing with sterile water once, Soak and disinfect in 0.1% mercuric chloride for 8 minutes, rinse with sterile water for 7 times, and then use sterile filter paper to absorb the surface moisture.

As a preferred technical solution of the method for promoting the rapid propagation of Vitex unifolia in vitro according to the present invention, the method is specifically as follows: taking the stem section of the healthy branch of Vitex unifolia, cutting off redundant leaves, taking a length of 1.5-3 cm and taking The stem section with 2 lateral buds is an explant, and after surface sterilization treatment, it is inoculated on the culture medium for establishing a sterile system to obtain sterile buds; the band 1 node (2) of the sterile buds is obtained. The stem segments of each side bud) were respectively inoculated on the medium for cluster bud induction and proliferation, and the appropriate cluster bud induction and proliferation medium was determined according to the index of cluster bud induction rate, bud multiplication, average plant height and growth status. Get the well-growing clump buds formed on the medium used for clump bud induction and proliferation, cut the stem nodes with axillary buds with a blade, inoculate on the adventitious bud induction medium, by the adventitious bud occurrence rate (stem segment); The flattening induction rate), the average number of adventitious buds on each explant and the growth status were used as references to determine the appropriate stem segment flattening induction and adventitious bud generation formula; The flattened stem section of the adventitious bud is formed on the top, and under aseptic conditions, the flattened stem section with the same number of adventitious buds is cut with a blade, inoculated on the inducing medium for adventitious bud elongation, and the adventitious bud elongation rate, The average length of adventitious buds and growth conditions were used to determine the appropriate formulation of adventitious bud elongation induction medium. Take the elongating adventitious buds with good growth on the medium used for the elongation induction of adventitious buds, and inoculate them on the medium used for rooting for rooting induction. As an index, the appropriate rooting induction formula was determined. The well-rooted plants, after washing off the medium attached to their roots, were transplanted into a matrix mixed with yellow mud and peat soil (volume ratio of 3:1), and cultivated for 30 days to obtain well-growing tissue culture seedlings. . The present invention establishes for the first time a rapid propagation system of in vitro stem segment cluster bud induction and adventitious bud generation on the flattened stem segment of Vitex unifolia. Occurrence, adventitious bud elongation, rooting and other treatments, after transplanting, a complete and considerable number of single-leaf Vitex chinensis tissue culture seedlings were obtained.

Compared with the prior art, the present invention has the following advantages:

(1) The present invention is used for establishing the culture medium of the aseptic system, without adding any plant growth regulator, only using MS as the basic medium to induce the germination of the axillary buds of the explants, and establishing the aseptic system of Vitex unifolia.

(2) the present invention is used in the culture medium of clump bud induction and proliferation, and excising the stem segment of the aseptic bud with a node formed in the culture medium for establishing the aseptic system is an explant, using MS basic medium , adding different mass concentrations of BA, using the cluster bud induction rate, bud multiplication multiple and plant height as indicators, screened the appropriate cluster bud induction formula, and found for the first time that the bud stem segment of Vitex unifolium can flatten and form The phenomenon of adventitious buds. Taking 30 days as the culture period, the clump bud induction rate, bud multiplication and plant height were up to 81.11%, 21.91 and 5.25 cm, respectively. The formed clump buds had bright green leaves, compact internodes and good growth.

(3) The flattening of the bud and stem segment of Vitex unifolia and the occurrence of adventitious buds were found in the medium used for the induction and proliferation of clump buds. Substrate for segment flattening induction and adventitious bud formation, used for flattening induction and adventitious bud formation of buds and stems of Vitex unifolia The in vitro bud-bearing stem segments of the clump buds were explants. MS was used as the basic medium, and high concentration (≥2.5mg/L) of BA was added. The number of adventitious buds was used as the index, and the suitable adventitious bud induction medium was screened. Taking 30 days as the culture period, the flattening induction of stem segments, the incidence of adventitious buds, and the average number of adventitious buds per explant were up to 60% and 84.9, respectively, and short and compact adventitious buds were obtained.

(4) The present invention is used in the adventitious bud elongation induction medium, and the flattened stem segment used for adventitious bud induction in the adventitious bud induction medium is excised as explants, MS is used as the basic medium, and 1.5-2.5 mg is added. /L NAA or the combination of BA and NAA, with the shoot elongation rate and the average length of adventitious buds as indicators, the suitable formula for the induction of adventitious bud elongation was screened. Taking 30 days as the culture period, the elongation rate of adventitious buds and the average length of adventitious buds were up to 58.9% and 3.0 cm, respectively. The short and compact buds could form normal buds and grow well.

(5) The elongation and robust buds obtained in the adventitious shoot elongation induction medium were rooted in 1/2 MS medium containing IBA or NAA and their combination, and finally acclimated and transplanted. The rooting rate and the average rooting number can reach 70.5% and 86.4 respectively. The highest survival rate was 86% after 30 days of transplanting.

(6) The present invention discovers for the first time the occurrence of adventitious buds induced by the flattening of the stem segments of Vitex unifolium, and establishes a tissue culture rapid propagation system of the adventitious bud pathway of clump buds, resulting in a genetic consistency that can maintain the mother plant Tissue culture seedlings with excellent characters, and solve the problems of low reproduction rate of cutting propagation and seed propagation, limited by time period and environmental restrictions.

Description of drawings

The method and beneficial effects of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

Fig. 1 is that the aseptic seedling of Vitex unifolia is established and cultivated effect diagram;

Fig. 2 is the effect diagram of clump bud induction;

Fig. 3 is the effect diagram of stem segment flattening and adventitious bud induction;

Fig. 4 is the effect diagram of adventitious bud elongation;

Figure 5 is a rooted plant;

Figure 6 shows the transplanted surviving plants.

Detailed ways

In order to make the objectives, technical solutions and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are only for explaining the present invention, not for limiting the present invention.

In the following examples, the term "ex vivo" refers to a state in which a part of an organism is removed and freed from outside the organism for various research purposes.

The term "explant" refers to a cultured tissue section that is transplanted into a new medium when subcultured. In the present invention, "explant" specifically refers to the stem segment with axillary buds of Vitex unifolia.

1/2 medium refers to the medium prepared by reducing the amount of macroelements in MS medium to 1/2 of the original.

The composition of MS medium is shown in Table 1 below:

Table 1 Composition of MS medium

Unless otherwise specified, the culture medium and substrate involved in the method for promoting the rapid propagation of Vitex unifolia in vitro in the embodiment of the present invention are prepared according to the following methods respectively (when comparing growth regulators of different types and concentrations, then replace the corresponding components):

(1) Preparation of 1L MS medium: Accurately weigh various compounds described in Table 1, add an appropriate amount of distilled water to dissolve, stir with a glass rod to promote dissolution, adjust pH to 6.0 with NaOH, and finally make up to 1L.

(2) preparing a medium for establishing a sterile system: on the basis of the MS medium prepared in step (1), 25-35 g/L sucrose and 6.5-7.5 g/L agar are also added;

(3) preparing a medium for cluster bud induction and proliferation: on the basis of the MS medium prepared in step (2), 0.5-2.5 mg/L BA is also added;

(4) preparing a medium for adventitious bud induction: on the basis of the MS medium prepared in step (2), 2.5 mg/L BA was also added;

(5) preparing a medium for inducing elongation of adventitious buds: on the basis of the MS medium prepared in step (2), 1.5-2.5 mg/L naphthalene acetic acid is also added;

(6) Preparation of a medium for rooting: 3.0 mg/L indole butyric acid, 2.0-3.0 mg/L naphthalene acetic acid, 25 mg/L indole butyric acid, 25 mg/L naphthalene acetic acid, 25 mg/L are added on the basis of 1/2 of the MS medium prepared in step (1). ～35g/L sucrose and 6.5～7.5g/L agar;

(7) Preparation of base 1: mix according to the ratio of yellow mud: peat soil=3:1 (volume ratio).

Unless otherwise specified, the following culture conditions are: (25±1)°C, light time 12h/12h (L/D), light intensity 1500-2000lx.

The establishment of embodiment 1 aseptic system

Only one group was set up in the test. In April 2017, the bud and stem sections of Vitex unifolia were taken to establish the aseptic system of Vitex unifolia. The specific steps are as follows:

Selection of explants: Take the stem segment of the healthy branch of Vitex unifolia, cut off the excess leaves, and take the stem segment with a length of 1.5-3 cm and two lateral buds as the explant.

Surface disinfection: put the explants in 75% alcohol for 10-15s on the ultra-clean workbench, rinse with sterile water once, soak and disinfect with 0.1% mercuric chloride for 8 minutes, rinse with sterile water for 7 times, and dry the surface moisture.

Inoculation: After cutting off both ends of the stem segment on sterile filter paper, inoculate it on MS blank medium without any plant growth regulators.

After culturing for 30 days, sterile buds with green, stretched leaves and good growth can be obtained (Figure 1).

Embodiments 2 to 6 test the growth regulators of different types and concentrations and add them to the above various culture mediums respectively, to bring about the effects of each period in the rapid propagation of Vitex unifolia in vitro, and to obtain better growth regulation. agent selection.

Example 2 The effect of different types and concentrations of plant growth regulators on the induction of clump buds in isolated stem segments of Vitex unifolia

The experiment was set up in groups 1-25, MS was used as the basic medium, and 0.1-2.5 mg/L IAA, 0.1-2.5 mg/L NAA, 0.1-2.5 mg/L KT, 0.1-2.5 mg/L IAA, 0.1-2.5 mg/L KT, 0.1～2.5mg/L ZT, 0.1～2.5mg/LBA and 0.1～2.5mg/L TDZ;

Specific steps are as follows:

Take the stem segment with 1 node (2 side buds) from the sterile buds in Example 1 and inoculate it in the culture medium of tests 1 to 25 respectively, each bottle is inoculated with 3 stem segments, and each formula is 10 bottles .

After culturing for 30 days, the growth conditions were observed and recorded, and the average germination rate of buds, the induction rate of clump buds, the multiplication ratio of buds and the average plant height were counted.

The experiment was repeated twice after an interval of 15 d.

Average bud germination rate = (number of explants with bud germination/number of explants at the time of inoculation) x 100%; cluster bud induction rate = number of explants producing cluster buds/number of explants at the time of inoculation; shoot proliferation Multiple = total number of shoots/total number of shoots at inoculation; average plant height = total plant height/number of explants at inoculation.

Adopt the above-mentioned method to carry out the clump bud induction of Vitex simplicifolia, and the result obtained is as shown in Table 2:

Table 2 Effects of different types and concentrations of plant growth regulators on bud induction and proliferation of Vitex unifolia

It can be seen from Table 2 that under the treatments of different types and concentrations of plant growth regulators and their combinations, there were significant differences in bud germination rate, cluster bud induction rate, multiplication multiple and plant height (P<0.5). Most of the leaves of the plants cultured on the medium containing auxin (IAA and NAA) are green and stretched, and some are yellowed. The plant grows as a single plant, and the proliferation rate is low. root;

In contrast, there is no rooting phenomenon in the medium cultured on the medium containing cytokinins (KT, ZT, BA, TDZ), but different kinds of cytokinins have significant effects on shoot proliferation and plant height. difference.

Most of the plants cultured in the medium containing KT or ZT grew as single plants, the induction rate of clump buds and the multiplication rate of buds were lower, but the average plant height was significantly higher than other treatment groups, and the leaves were green and stretched, and the growth was better.

The plants cultured in the medium containing BA had the best growth, the multiplication ratio was significantly higher than that of the other treatment groups, a large number of clump buds grew at the axillary buds near the base, and some green and dense callus appeared. The cluster bud induction rate and bud multiplication ratio were positively correlated with the concentration, and the highest were 81.11% and 21.91, respectively. When the concentration was higher than 0.5mg/L, the difference was not significant. Under the condition of high concentration (≥0.5mg/L) of BA, some stems were flattened, and adventitious buds were directly produced from the stems. With the extension of culture time, adventitious buds could grow into normal buds;

Most of the lateral shoots cultured in TDZ medium formed callus, and the germination rate was the lowest; the lateral shoots that could germinate would also dwarf, cluster, and form a large number of highly watery callus at the base, and the growth rate was the worst. Therefore, the optimal cluster bud proliferation formula is MS+0.5～2.5mg/L BA. It has high bud germination rate, cluster bud induction rate and multiplication multiple, and it grows well.

Example 3 The effect of plant growth regulators of different concentrations and types on the induction of adventitious buds in isolated stem segments of Vitex unifolia

The experiment was set up in experimental groups 1-17. MS was used as the basic medium in experimental groups 1-17, and 0.5-5.0 mg/L BA, 1.0 mg/L TDZ, 1.0 mg/L 2 , 4-D or 1.0 mg/L BA in combination with 0.1 mg/L NAA and 1.0 mg/L BA, 0.1 mg/L NAA and 0.1 mg/L TDZ;

Specific steps are as follows:

The stem nodes with axillary buds were taken from the clustered buds of Example 2 and inoculated into the culture medium of test groups 1-17 respectively, each bottle was inoculated with 3 stages, and each test group was inoculated with 10 bottles.

After culturing for 30 days, observe and record its growth and count the adventitious bud induction rate and the average number of adventitious buds per explant.

The experiment was repeated twice after an interval of 15 d.

Induction rate of adventitious buds=(number of explants with adventitious buds/number of explants at the time of inoculation)×100%; average number of adventitious buds=total number of adventitious buds/number of explants with adventitious buds.

Adopt the above method to carry out the adventitious bud formation induced by the flattening of the stem segment of Vitex unifolia, and the obtained results are shown in Table 3.

Table 3 Effects of different concentrations and types of plant growth regulators and their combinations on the induction of adventitious buds of Vitex unifolia

It can be seen from Table 3: the stem segments formed callus at the incision of the culture medium and most of the axillary buds on the medium supplemented with 1.0 mg/L 2,4-D or 1.0 mg/L TDZ, and there was no occurrence of flattening and adventitious buds. formation; however, flattening occurred with the addition of BA or high concentrations (≥1.0 mg/L) of KT or ZT, forming adventitious buds (Fig. 3). In the medium supplemented with BA, the adventitious bud occurrence rate and the average adventitious bud number were the highest, and the adventitious bud occurrence rate and the average adventitious bud number increased with the increase of BA concentration, up to 60% and 84.87, respectively. When the concentration is ≥2.5mg/L, the difference is not significant, and high concentration (≥5.0mg/L) BA can make leaves curl with slight vitrification; when 1.0mg/L BA is combined with NAA or with NAA and TDZ When BA alone was combined with NAA, the adventitious bud incidence was not significantly different from that when 1.0 mg/L BA was added alone, but the number of adventitious buds when BA was combined with NAA was significantly less than that of the other two treatment groups. It can be seen that NAA has a certain negative effect on adventitious bud formation. The optimal adventitious bud induction formula should be: MS+2.5mg/L BA.

Example 4 Effects of plant growth regulators of different concentrations and types on the elongation of adventitious buds on the flattened stem section of Vitex unifolia

The experiment was set up in groups 1-13, MS was used as the basic medium in the experimental groups 1-13, and 0-2.5mg/L KT, 0.5-2.5mg/L BA, 0.5-2.5mg/L KT, 0.5-2.5mg/L BA, 0.5-2. 2.5mg/L NAA or 0.5～2.5mg/L BA combined with 0.1mg/L NAA;

The specific test steps are as follows:

(1) get the flat stem section that forms adventitious bud from embodiment 3, cut into the adventitious bud block of the same size under sterile conditions, the adventitious bud number is about 10/block, and the adventitious bud height is roughly the same, about 0.2cm /indivual.

(2) Micro-cutting was carried out in experimental groups 1-13 under sterile conditions to induce adventitious bud elongation.

(3) 6 stem segments were inoculated in each bottle, and 10 bottles were treated each. After culturing for 30 d, observe and record its growth. Take the adventitious bud length ≥ 0.5 cm as the elongation bud, and count the elongation rate of the adventitious bud and the average length of each adventitious bud. The experiment was repeated twice after an interval of 15 d. Elongation rate=(number of adventitious buds with elongation/total number of adventitious buds at the time of inoculation)×100%; average length of each adventitious bud=total length of adventitious buds/number of adventitious buds at the time of inoculation.

Table 4 Effects of different plant growth regulators and their combinations on adventitious shoot elongation and average shoot length

It can be seen from Table 4 that the adventitious buds have the best elongation effect on the medium supplemented with NAA, the buds are slender and emerald green (Fig. 4), and the elongation rate and length of shoots are significantly higher than other treatment groups. With the increase of NAA concentration, shoot elongation rate and adventitious shoot length also increased, up to 58.9% and 3.0 cm, respectively, but the difference was not significant when the concentration was ≥ 1.5 mg/L; KT or BA induced adventitious shoot elongation The effect was not significantly different from the blank control group. In addition, the adventitious buds induced to elongate on the medium supplemented with BA have short internodes, and new adventitious buds can be formed at the same time, thus affecting the elongation rate and average bud length of adventitious buds. Although the adventitious buds induced by KT elongated leaves, the nodes are stretched. Intervals are long, but adventitious bud elongation and average length are lower. When BA was combined with NAA, the shoot elongation rate and length were higher than those when BA was added alone. It can be seen that NAA plays an important role in the induction of shoot elongation. The optimal adventitious bud induction formula is: MS+1.5～2.5mg/L NAA.

Example 5 Effects of NAA and IBA on rooting of sprouts of Vitex unifolia

The experiment was set up in experimental groups 1-16. 1/2MS was used as the basic medium in experimental groups 1-16, and experimental group 1 was a blank control group without any plant growth regulators; experimental groups 2-16 were added with 0.5-5.0 mg/L of NAA or IBA and IBA (1.0 and 3.0 mg/L) combined with NAA (1.0, 2.0, 3.0 mg/L), see Table 5 for details.

The specific test steps are as follows:

(1) From Example 4, a vigorously growing bud with a plant height of 3 to 4 cm and three nodes was taken, and excised from the base of the bud from the mother plant under aseptic conditions.

(2) The excised buds were respectively inoculated into the culture medium of test groups 1-16, 6 per bottle, and 10 bottles in each test group.

(3) After culturing for 40 days, the rooting rate, the average number of roots per plant and the average root length were counted, and the growth of the seedlings was recorded. The test was repeated 3 times. Rooting rate=(number of rooted plants/total number of plants at the time of inoculation)×100%. Average number of roots per plant = total number of roots/total number of rooted plants; average root length = total number of root lengths/total number of rooted plants.

Table 5 Effects of different concentrations of IBA and NAA and their combinations on rooting of sprouts of Vitex unifolia

It can be seen from Table 5 that the shoots could not take root in the test group 1 without any plant growth regulators, and after culturing for 10-15 days in other treatments, the shoots began to take root one after another. The rooting induction effect of sprouts in the medium supplemented with NAA alone is poor. When NAA ≥ 1.0 mg/L, rooting can be induced, and the rooting induction rate is the lowest compared with other treatment groups. However, once rooting is induced, the number of roots is very dense. , the lateral roots are developed, but the average root length is shorter (Fig. 5 left plant).

The rooting induction rate of shoots cultured on the medium supplemented with 0.5-5.0 mg/L IBA alone has a positive correlation with the concentration of IBA. Significantly. The average rooting number and average root length were not significantly different between different concentrations of IBA, both showed low rooting coefficient and underdeveloped lateral roots, but the average root length was longer (Fig. 5 right plant).

The rooting effect of shoots cultured on the medium supplemented with IBA and NAA was ideal, and both were better than those supplemented with NAA or IBA alone. When the concentration of NAA was constant, the rooting rate increased with the increase of the concentration of IBA. When the IBA concentration was constant, the average rooting number increased with the increase of NAA concentration, and the highest rooting rate and rooting coefficient were 70.48% and 86.37, respectively. The best combination of rooting induction was 1/2MS+3.0mg/L IBA+2.0～3.0mg/L NAA. It takes into account the advantages of adding the same concentration of NAA or IBA alone, not only has a higher rooting induction rate and rooting coefficient, but also has developed lateral roots, and the average root length is in the middle.

Example 6 Effects of different induction sources of plant growth regulators on transplantation of tissue culture seedlings

The experiment was set up with 1 to 3 experimental groups. In test groups 1-3, yellow mud and peat soil were mixed with a volume ratio of 3:1 as the matrix, and then the tissue culture seedlings induced by the combination of IBA, NAA and IBA and NAA were respectively hardened and transplanted into the matrix.

The specific experimental steps are as follows:

(1) The tissue culture seedlings which were added with IBA, NAA alone or combined with IBA and NAA for rooting induction were taken respectively.

(2) After the tissue culture seedlings grow to a height of 3 to 4 cm, open the cap of the culture bottle where they are located, and refine the seedlings for 3 to 5 days, so that the seedlings are initially adapted to the external environment.

(3) Wash off the solid medium attached to its root system with tap water, then transplant it into a matrix mixed with yellow mud and peat soil (volume ratio = 3:1), transplant one plant in each bag, and transplant each plant in each bag. 100 plants were transplanted for each treatment.

(4) Pour enough water to fix the roots, and use the method of spraying every morning and evening to keep the soil moist. The survival rate and growth status of seedlings were counted after 30 days.

Table 6 Effects of different induction sources of plant growth regulators on the transplanting of tissue culture seedlings

The well-rooted tissue culture seedlings induced on the medium supplemented with NAA alone or IBA and the combination of NAA and IBA, respectively, were washed with tap water to wash off the agar attached to the root system. The taproots are easy to fall off during the agar removal process, and some taproots are even completely fallen off and cannot be used for transplanting. On the other hand, although the roots of tissue culture seedlings with well-developed lateral roots cultured in NAA-containing medium may also drop off the roots, but because of the dense root system, the shedding of some roots does not affect the transplanting. It can be seen from Table 6 that after 30 days of transplanting, observation and statistics showed that the transplant survival rates of adding NAA or IBA alone and the combination of NAA and IBA were: 82%, 66.67% and 86%, respectively. Stout and growing well (Figure 6).

It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should The technical solutions of the invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the invention.

Claims (1)

1. a kind of method for promoting Vitex rotundifolia Vitro Quick Reproduction, which comprises the steps of:

(1) choosing Vitex rotundifolia semi-lignified seedling stem segment with axillary bud is explant；

(2) aseptically, it is inoculated in the culture medium for being used to establish sterile system after explant being sterilized with mercuric chloride, obtains Aseptic seedling；

(3) aseptic seedling that step (2) obtain is cut into the stem with bud of 1, band section, is inoculated in and is induced and be proliferated for clump bud Culture medium on, obtain clump bud；

(4) clump bud that step (3) obtain is cut into the stem section of 1, band section, is inoculated in the culture medium for adventitious bud inducing, Stem section flattening is induced, adventitious bud is obtained；

(5) adventitious bud that step (4) obtain is inoculated in the culture medium for Elongation of adventitious bud induction, obtaining elongation is normality The adventitious bud of bud；

(6) adventitious bud that the elongation that step (5) obtain is normality bud is inoculated in the culture medium for taking root, carries out training of taking root Support, obtain tissue-cultured seedling, by its root, culture medium is cleaned up, transplant in volume ratio be 3:1 yellow soil and peat soil mixing and At matrix on, complete the Vitro Quick Reproduction of Vitex rotundifolia；

The culture medium for being used to establish sterile system is to be added to 25~35g/L with MS culture medium for basic culture medium Sucrose and 6.5~7.5g/L agar；

Described induces the culture medium with proliferation for clump bud, is cultivated based on the culture medium for establishing sterile system Base is added to 0.5~2.5mg/L6- benzyladenine；

The culture medium for adventitious bud inducing is with the culture medium for establishing sterile system for basic culture medium, It is added to 2.5mg/L6- benzyladenine；

The culture medium for Elongation of adventitious bud induction, is cultivated based on the culture medium for establishing sterile system Base is added to 1.5~2.5mg/L methyl α-naphthyl acetate；

The culture medium for taking root is to be added to 3.0mg/L indoles fourth with 1/2MS culture medium for basic culture medium Acid, 2.0~3.0mg/L methyl α-naphthyl acetate, 25~35g/L sucrose and 6.5~7.5g/L agar；

Condition of culture in step (2)~(6) is equal are as follows: 25 ± 1 DEG C, light application time 12h/12h L/D, intensity of illumination 1500~ 2000lx。