A culture medium combination for inducing regeneration of camphor tree plants and its application

Abstract

The present invention belongs to the technical field of plant tissue culture, and specifically relates to a culture medium combination for inducing the regeneration of camphor trees and its application. The induction culture medium in the culture medium combination can effectively induce callus tissue, and the induction rate is as high as 100%; the obtained callus tissue regenerates adventitious buds on the differentiation culture medium of the culture medium combination, and the germination rate can reach 90%, the new buds are young and tender, and the monthly average proliferation coefficient of the adventitious buds can reach up to 2.8 at most; the obtained adventitious buds are cultured on the rooting culture medium of the culture medium combination, and the rooting rate can be as high as 80%, and the ability of sprouting after transplanting is strong, which is conducive to the cultivation of clumped dwarf camphor trees, and provides high-quality seedlings for the efficient and high-quality production of camphor trees. The present invention adopts sterile seedlings germinated from sterile seeds as explants, and takes its microstem segments as culture materials, which overcomes the technical problems of high contamination rate, easy browning, and difficult regeneration in the existing tissue culture technology.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium combination for inducing the regeneration of camphor trees and an application thereof. Background Art

[0002] Traditional methods of propagating camphor tree seedlings mainly include seed propagation and branch cutting propagation. Seed propagation mainly uses fruits that mature in September. After steps such as composting, peeling, drying and sand storage, sowing can be carried out. After one year, the seedling height can reach 1.5 to 2 meters, and the breast diameter can reach 1.5 to 2 centimeters. If dwarfed seedlings are needed, the stems need to be cut and cultivated after one year, and the main stem should be kept at a height of about 20 centimeters. This method has high requirements on the seed collection period and the handling techniques. Improper handling can easily cause problems such as seed mildew, water loss and oiling, resulting in a lower germination rate. If dwarfed seedlings are needed, the stems need to be cut and cultivated again after one year, which adds one year to the cultivation time. Branch cutting propagation mainly uses semi-lignified branches of Cinnamomum camphora in spring, cuts into 15 cm long cuttings, leaves 2 to 3 half leaves on the top of the cuttings, cuts the bottom into a horseshoe shape, soaks in rooting drugs and then propagates cuttings. This method mainly relies on the healing of the incisions, and the comprehensive effect of cuttings is easily affected by several aspects, especially the cutting time, the age of the mother tree and the cultivation conditions, resulting in unstable cutting survival rate. In general, the traditional breeding technology of Cinnamomum camphora is mainly limited by the breeding time, and the breeding efficiency is low. The stability of breeding cannot be effectively controlled, and improper management is prone to large-scale deaths.

[0003] Based on the technical problems faced by the above-mentioned seedling propagation method, there have been relevant reports on camphor tree tissue culture. In the existing reports on camphor tree tissue culture, the explants selected are the young and tender branches of the current year outside the crown of healthy camphor trees, including the steps of primary bud induction, proliferation culture and rooting culture. However, there are still problems such as easy browning, high contamination rate and difficulty in inducing transformation. Summary of the invention

[0004] The purpose of the present invention is to provide a culture medium combination for inducing the regeneration of camphor trees and its application. The culture medium combination can quickly induce the differentiation and regeneration of camphor trees to obtain rooted camphor tree seedlings, thus overcoming the problems of high contamination rate, severe browning, difficult regeneration and slow reproduction speed of existing camphor tree tissue culture.

[0005] The present invention provides a culture medium combination, which includes an induction culture medium, a differentiation culture medium and a rooting culture medium;

[0006] The induction medium is based on MS medium and also includes plant hormones, 30 g / L sucrose and 3.5 g / L agar, with a pH value of 5.8; the plant hormones include 0.5-5 mg / L 6-BA or 0.01-1 mg / L LTDZ;

[0007] The differentiation medium is based on WPM medium or 2WPM medium, and further includes 0.1-0.3 mg / L IBA, 0.1-1 mg / L NAA, 1-5 mg / L 6-BA, 30 g / L sucrose and 3.5 g / L agar; the pH value is 5.8;

[0008] The rooting medium comprises a first rooting medium or a second rooting medium, wherein the first rooting medium is based on 1 / 2MS medium and further comprises 0.1-1 mg / L NAA, 30 g / L sucrose and 3.5 g / L agar; and the pH value is 5.8;

[0009] The second rooting medium is based on 1 / 2WPM medium and further includes 0.1-1 mg / L NAA, 30 g / L sucrose and 3.5 g / L agar; and the pH value is 5.8.

[0010] The present invention also provides application of the culture medium combination described in the above technical solution in the propagation of camphor tree seedlings.

[0011] Preferably, the camphor tree seedling propagation includes camphor tree tissue culture.

[0012] The present invention also provides a method for inducing plant regeneration by using micro-stem segments of camphor trees, wherein the camphor tree stem segments are cultured using the culture medium combination described in the above technical solution to obtain rooted camphor tree seedlings.

[0013] Preferably, the culturing comprises the following steps:

[0014] Inducing and culturing the microstem segments cut from the sterile seedlings of Cinnamomum camphora in an induction medium to obtain callus tissue;

[0015] The callus tissue is differentiated and cultured in a differentiation medium to obtain clustered adventitious buds;

[0016] The clustered adventitious buds are subjected to rooting culture in a rooting medium to obtain rooted Cinnamomum camphora seedlings.

[0017] Preferably, the micro-stem segment comprises a stem segment with nodes, and the length of the micro-stem segment is 4 to 6 mm.

[0018] Preferably, the induction culture temperature is 24°C, the light intensity is 1000-2000LX, the light cycle is 16L:8D, and the culture time is 3-5 weeks;

[0019] Preferably, the differentiation culture temperature is 24°C, the light intensity is 2000-3000LX, the light cycle is 16L:8D, and the culture time is 2-4 weeks;

[0020] Preferably, the rooting culture temperature is 24° C., the light intensity is 2000-3000 LX, the light cycle is 16L:8D, and the culture time is 1-4 weeks.

[0021] Preferably, the method for cultivating the sterile camphor tree seedlings comprises: removing the seed shells of sterilized camphor tree seeds, and sequentially performing dark culture and light culture on a growth medium to obtain the sterile camphor tree seedlings.

[0022] Preferably, the dark culture time is 12 to 48 hours; the light cycle of the light culture is 16L:8D, the light intensity is 1000 to 2000LX, and the culture time is 15 days; the temperature of the light culture and dark culture is 22 to 26°C.

[0023] Preferably, the growth medium uses MS medium as the basic medium, further includes 30 g / L sucrose and 3.5 g / L agar, and has a pH value of 5.8.

[0024] Beneficial effects:

[0025] The invention provides a culture medium combination, which comprises an induction culture medium, a differentiation culture medium and a rooting culture medium; the culture medium combination can quickly induce the differentiation and regeneration of camphor trees to obtain rooted camphor tree seedlings.

[0026] The invention also provides an application of the culture medium combination in camphor tree seedling propagation. The invention uses sterile seedlings germinated from sterile seeds as explants, and applies the culture medium combination to camphor tree tissue culture for camphor tree seedling propagation. The camphor tree microstem segments have a wide source and a strong regeneration ability, save space and reduce land use compared with traditional propagation methods, and overcome the problems of easy browning, high contamination rate and difficulty in inducing transformation in in vitro propagation of mature stem segments in the existing tissue culture technology. The induction culture medium in the culture medium combination can effectively induce callus tissue, and the induction rate is as high as 100%. The obtained callus tissue regenerates adventitious buds on the differentiation culture medium of the culture medium combination, and the germination rate can reach 90%, the new buds are young and tender, and the monthly average proliferation coefficient of the adventitious buds can reach 2.8 at most. The obtained adventitious buds are cultured on the rooting culture medium of the culture medium combination, and the rooting rate can reach 80%. The sprouting ability is strong after transplanting, which is conducive to the cultivation of clumped dwarf camphor tree, provides high-quality seedlings for efficient and high-quality production of camphor tree, and overcomes the technical problems of high contamination rate and difficult regeneration in the existing tissue culture technology.

[0027] In addition, the culture medium combination of the present invention can be used to propagate camphor tree seedlings throughout the year, without relying on seasonal growth. Compared with the traditional method of germinating seeds, cutting them off after one year, cultivating clustered buds and germinating seedlings, which takes two years, the cultivation cycle is greatly shortened and is not restricted by seasons. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 The diagram shows the various growth stages of the method for inducing plant regeneration from camphora microstem segments in Example 1. DETAILED DESCRIPTION

[0030] The present invention provides a culture medium combination, which includes an induction culture medium, a differentiation culture medium and a rooting culture medium;

[0031] The induction medium is based on MS medium and also includes plant hormones, 30 g / L sucrose and 3.5 g / L agar, with a pH value of 5.8; the plant hormones include 0.5-5 mg / L 6-BA or 0.01-1 mg / L LTDZ;

[0032] The differentiation medium is based on WPM medium or 2WPM medium, and further includes 0.1-0.3 mg / L IBA, 0.1-1 mg / L NAA, 1-5 mg / L 6-BA, 30 g / L sucrose and 3.5 g / L agar; the pH value is 5.8;

[0033] The rooting medium comprises a first rooting medium or a second rooting medium, wherein the first rooting medium is based on 1 / 2MS medium and further comprises 0.1-1 mg / L NAA, 30 g / L sucrose and 3.5 g / L agar; and the pH value is 5.8;

[0034] The second rooting medium is based on 1 / 2WPM medium and further includes 0.1-1 mg / L NAA, 30 g / L sucrose and 3.5 g / L agar; and the pH value is 5.8.

[0035] In the present invention, the induction medium is based on MS medium and further includes plant hormones, 30 g / L sucrose and 3.5 g / L agar. The plant hormones of the present invention include 0.5-5 mg / L 6-BA or 0.01-1 mg / L TDZ. The concentration of 6-BA in the induction medium of the present invention is preferably 1-4 mg / L, more preferably 3 mg / L. The concentration of TDZ in the induction medium of the present invention is preferably 0.02-0.08 mg / L, more preferably 0.05 mg / L.

[0036] In the present invention, the differentiation medium is based on WPM medium or 2WPM medium, and further includes 0.1-0.3 mg / L IBA, 0.1-1 mg / L NAA, 1-5 mg / L 6-BA, 30 g / L sucrose and 3.5 g / L agar. The basal medium of the differentiation medium of the present invention is preferably 2WPM medium. The concentration of IBA in the differentiation medium of the present invention is preferably any value in the range of 0.1-0.3 mg / L, such as 0.1, 0.15, 0.2, 0.25 or 0.3 mg / L, further preferably 0.1 or 0.3 mg / L, and more preferably 0.1 mg / L. The concentration of NAA in the differentiation medium of the present invention is preferably any value in the range of 0.1-1 mg / L, such as 0.3, 0.55, 0.6 mg / L or 0.7 mg / L, and more preferably 0.5 mg / L. The concentration of 6-BA in the differentiation medium of the present invention is preferably any value between 1 and 5 mg / L, such as 1, 2.5, 3.5, 3.8 or 4 mg / L, and more preferably 3 mg / L.

[0037] In the present invention, the rooting medium includes a first rooting medium or a second rooting medium, more preferably a second rooting medium.

[0038] The present invention also provides the application of the culture medium combination described in the above technical solution in the propagation of camphor tree seedlings. The seedling propagation described in the present invention preferably includes camphor tree tissue culture. The induction culture medium in the culture medium combination described in the present invention can effectively induce callus tissue, and the obtained callus tissue regenerates adventitious buds on the differentiation culture medium of the culture medium combination, and the new buds are young and tender, and the monthly average proliferation coefficient of the adventitious buds can reach up to 2.8 at most; the obtained adventitious buds are cultured on the rooting culture medium of the culture medium combination, and the rooting rate can be as high as 80%, and the ability to sprout after transplanting is strong, which is conducive to the cultivation of clumped dwarf camphor tree, and provides high-quality seedlings for the efficient and high-quality production of camphor tree. The present invention adopts sterile seedlings germinated from sterile seeds as explants, and takes its microstem segments as culture materials, which overcomes the technical problems of high contamination rate, easy browning, and difficult regeneration in existing tissue culture technology.

[0039] The present invention also provides a method for inducing plant regeneration by using micro-stem segments of camphor trees, wherein the camphor tree stem segments are cultured using the culture medium combination described in the above technical solution to obtain rooted camphor tree seedlings.

[0040] The camphor tree stem segments of the present invention are preferably obtained by cutting from sterile seedlings germinated from sterile seeds. The method for cultivating the sterile camphor tree seedlings preferably comprises: removing the seed shells of the sterilized camphor tree seeds, and sequentially performing dark culture and light culture on a growth medium to obtain the sterile camphor tree seedlings.

[0041] In the present invention, the ripe camphor tree fruit is preferably cleaned after the pulp is removed. The cleaning in the present invention preferably includes first rinsing with detergent once, and then rinsing with clean water for 2 hours. The present invention does not specifically limit the specific process of the cleaning, and the conventional cleaning process in the art can be used.

[0042] After the cleaning, the present invention preferably disinfects the camphor seeds obtained after the cleaning. The disinfection method of the present invention preferably includes: after the camphor seeds are first soaked in an alcohol solution, they are second soaked in a sodium hypochlorite solution. The volume percentage concentration of the alcohol solution of the present invention is 75%; the time of the first soaking is preferably 20 to 40 seconds, and more preferably 30 seconds. The sodium hypochlorite solution of the present invention is preferably an 8-fold sodium hypochlorite solution, that is, the volume ratio of the sodium hypochlorite stock solution and water in the sodium hypochlorite solution is 1:7; the time of the second soaking is preferably 6 to 10 minutes, and more preferably 8 minutes.

[0043] After the disinfection, the present invention preferably further comprises rinsing the disinfected camphor seeds with sterile water, and the number of rinsing is preferably 5 times. The disinfection of the present invention is preferably carried out on a clean bench.

[0044] After the washing, the present invention preferably uses sterile absorbent paper to absorb the water on the surface of the washed camphor seeds to obtain sterilized camphor seeds.

[0045] After obtaining the sterilized camphor seeds, the present invention preferably removes the seed shells of the sterilized camphor seeds, and then sequentially performs dark culture and light culture on a growth medium to obtain the sterile camphor seedlings. The present invention does not specifically limit the method of removing the seed shells, and the conventional method of removing the seed shells in the art can be used.

[0046] The growth medium of the present invention is preferably based on MS medium, and also includes 30g / L sucrose and 3.5g / L agar. The pH value of the growth medium of the present invention is preferably 5.8. The dark culture time of the present invention is preferably 12 to 48h, more preferably 24h; the temperature is preferably 22 to 26°C, more preferably 24°C. The photoperiod of the light culture of the present invention is preferably 16L:8D; the light intensity is preferably 1000 to 2000LX, more preferably 1500LX; the culture time is preferably 10 to 20d, more preferably 15d; the temperature is preferably 22 to 26°C, more preferably 24°C.

[0047] After obtaining the aseptic seedlings of camphor, the present invention preferably cuts the camphor microstem segments from the aseptic seedlings of camphor, and the camphor microstem segments are sterile camphor stem segments. The camphor microstem segments of the present invention preferably include camphor stem segments with nodes. The length of the camphor stem segments is preferably 4 to 6 mm, more preferably 5 mm.

[0048] After obtaining the sterile camphor stem segment, the present invention performs induction culture on the sterile camphor stem segment in an induction culture medium to obtain callus tissue. The induction culture temperature of the present invention is preferably 24°C; the light intensity is preferably 1000-2000LX, more preferably 1500LX; the light cycle is preferably 16L:8D; the culture time is preferably 3-5 weeks, more preferably 4 weeks.

[0049] After obtaining the callus tissue, the present invention performs differentiation culture on the callus tissue in a differentiation medium to obtain clustered adventitious buds. The temperature of the differentiation culture of the present invention is preferably 24°C; the light intensity is preferably 2000-3000LX, more preferably 2500LX; the light cycle is preferably 16L:8D; the culture time is 2-4 weeks, more preferably 3 weeks.

[0050] After obtaining the clustered adventitious buds, the present invention performs rooting culture on the clustered adventitious buds in a rooting medium to obtain rooted camphor tree seedlings. The rooting culture temperature of the present invention is preferably 24°C; the light intensity is preferably 2000-3000LX, more preferably 2500LX; the light cycle is preferably 16L:8D; the culture time is 1-4 weeks, more preferably 2 weeks.

[0051] The rooted camphor tree seedlings obtained by the method of the present invention can be directly used for the propagation of camphor tree seedlings including dwarfed clumping seedlings, thereby shortening the breeding cycle.

[0052] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] Example 1

[0054] A method for inducing plant regeneration using camphor microstem segments, comprising the following steps:

[0055] 1) Aseptic sowing of mature embryos

[0056] Select the mature camphor fruits of the year, remove the pulp and rinse them with detergent once, then rinse with clean water for 2 hours, disinfect them with 75% alcohol solution by volume for 30 seconds on the clean bench, then disinfect them with 8 times sodium hypochlorite solution for 8 minutes, and rinse them with sterile water for 5 times. Then use sterile absorbent paper to dry the surface moisture, peel off the seed shell and inoculate them into MS medium with 30g / L sucrose, 3.5g / L agar, and pH 5.8, first culture them in the dark for 24 hours, and then culture them for 15 days under the environment of light intensity of 1500LX, temperature of 24℃, and light time of 16h / d.

[0057] 2) Callus induction from Cinnamomum camphora stem segments

[0058] The sterile seedlings were obtained by aseptic sowing, and the stem segments with nodes and length of about 5 mm were cut as test materials. Figure 1 As shown in A, the induction medium (denoted as induction medium 1) was used at a temperature of 24°C, a light intensity of 1500LX, and a light cycle of 16L:8D for 4 weeks to obtain callus tissue, as shown in FIG. Figure 1 As shown in B.

[0059] The components of induction medium 1 are: MS as the basic culture medium (M519 powder dissolved in water, the concentration is 4.43g / L, purchased from Sigma, the same below), which is also contained 30g / L sucrose, 3.5g / L agar and 3mg / L 6-BA, and the pH value is 5.8.

[0060] 3) Callus differentiation into clusters of adventitious buds

[0061] The callus obtained in step 2) was used as the test material and cultured on a differentiation medium (denoted as differentiation medium 1) under the conditions of a temperature of 24°C, a light intensity of 2500LX, a light cycle of 16L:8D, and a total culture of 3 weeks to obtain clustered adventitious buds. Figure 1 As shown in C to D.

[0062] The components of differentiation medium 1 are: 2WPM medium as the basic medium, and also contain 30 g / L sucrose, 3.5 g / L agar, 0.1 mg / L IBA, 0.5 mg / L NAA and 3 mg / L 6-BA, and the pH value is 5.8.

[0063] 4) Clustered adventitious buds take root

[0064] The clustered adventitious buds obtained in step 3) were used as test materials and cultured on a rooting medium (referred to as rooting medium 1). The culture conditions were a temperature of 24°C, a light intensity of 2500LX, a light cycle of 16L:8D, and a total culture of 2 weeks to obtain rooted clustered adventitious buds. Figure 1 As shown in E to G.

[0065] The composition of the rooting medium 1 is: 1 / 2WPM medium as the basic medium, and also contains 30 g / L sucrose, 3.5 g / L agar and 1 mg / L NAA, and the pH value is 5.8.

[0066] Example 2

[0067] Example 1 was repeated, except that only step 1) and step 2) were performed, and the induction medium used was induction medium 2, to obtain callus tissue.

[0068] The components of induction medium 2 are MS as the basic medium (M519 powder dissolved in water, the concentration is 4.43g / L, purchased from Sigma, the same below), which is also composed of 30g / L sucrose, 3.5g / L agar and 0.5mg / L 6-BA, with a pH of 5.8.

[0069] Example 3

[0070] Example 1 was repeated, except that only step 1) and step 2) were performed, and the induction medium used was induction medium 3, to obtain callus tissue.

[0071] The components of induction medium 3 are MS as the basic medium (M519 powder dissolved in water, the concentration is 4.43g / L, purchased from Sigma, the same below), which is also contained 30g / L sucrose, 3.5g / L agar and 1mg / L 6-BA, and the pH value is 5.8.

[0072] Example 4

[0073] Example 1 was repeated, except that only step 1) and step 2) were performed, and the induction medium used was induction medium 4, to obtain callus tissue.

[0074] The components of induction medium 4 are MS as the basic medium (M519 powder dissolved in water, the concentration is 4.43g / L, purchased from Sigma, the same below), which is also 30g / L of sucrose, 3.5g / L of agar and 5mg / L of 6-BA, with a pH of 5.8.

[0075] Example 5

[0076] Example 1 was repeated, except that only step 1) and step 2) were performed, and the induction medium used was induction medium 5, to obtain callus tissue.

[0077] The components of induction medium 5 are MS as the basic medium (M519 powder dissolved in water, the concentration is 4.43g / L, purchased from Sigma, the same below), which is also 30g / L sucrose, 3.5g / L agar and 0.01mg / L TDZ, and the pH value is 5.8.

[0078] Example 6

[0079] Example 1 was repeated, except that only step 1) and step 2) were performed, and the induction medium used was induction medium 6, to obtain callus tissue.

[0080] The components of induction medium 6 are MS as the basic medium (M519 powder dissolved in water, the concentration is 4.43g / L, purchased from Sigma, the same below), which is also 30g / L sucrose, 3.5g / L agar and 0.05mg / L TDZ, and the pH value is 5.8.

[0081] Example 7

[0082] Example 1 was repeated, except that only step 1) and step 2) were performed, and the induction medium used was induction medium 7, to obtain callus tissue.

[0083] The components of induction medium 7 are MS as the basic medium (M519 powder dissolved in water, the concentration is 4.43g / L, purchased from Sigma, the same below), which also contains 30g / L sucrose, 3.5g / L agar and 1mg / L TDZ, and the pH value is 5.8.

[0084] The induction rates of callus obtained in Examples 1 to 6 were statistically analyzed. Three biological replicates were set in each example. When the statistical data were analyzed, the average value of the three biological replicates was calculated. The results are shown in Table 1:

[0085] Table 1 Effect of induction medium in Examples 1 to 6 on callus induction of Cinnamomum camphora microstem segments

[0086]

[0087] It can be concluded from Table 1 that both 6-BA and TDZ can induce callus tissue in Cinnamomum camphora microstem segments, especially when the 6-BA concentration is 3 mg / L (Example 1) or the TDZ concentration is 0.05 mg / L (Example 6), the callus induction rate can reach 100%, which is significantly higher than that of other examples.

[0088] Example 8

[0089] Example 1 was repeated, except that only steps 1) to 3) were performed, and the differentiation medium used was differentiation medium 2, to obtain clustered adventitious buds.

[0090] The components of differentiation medium 2 are: 2WPM medium as the basic medium, and also contain 30 g / L sucrose, 3.5 g / L agar, 0.3 mg / L IBA, 0.5 mg / L NAA and 3 mg / L 6-BA, and the pH value is 5.8.

[0091] Example 9

[0092] Example 1 was repeated, except that only steps 1) to 3) were performed, and the differentiation medium used was differentiation medium 3, to obtain clustered adventitious buds.

[0093] The components of differentiation medium 3 are: WPM medium as the basic medium, and also contain 30 g / L sucrose, 3.5 g / L agar, 0.1 mg / L IBA, 0.5 mg / L NAA and 3 mg / L 6-BA, and the pH value is 5.8.

[0094] Example 10

[0095] Example 1 was repeated, except that only steps 1) to 3) were performed, and the differentiation medium used was differentiation medium 4, to obtain clustered adventitious buds.

[0096] The components of differentiation medium 4 are: WPM medium as the basic medium, and also contain 30 g / L sucrose, 3.5 g / L agar, 0.3 mg / L IBA, 0.5 mg / L NAA and 3 mg / L 6-BA, and the pH value is 5.8.

[0097] The average number of proliferated buds and the length of buds obtained in Example 1 and Examples 8 to 10 were statistically analyzed, and the results are shown in Table 2:

[0098] Table 2 Effect of differentiation medium on induction of buds in Cinnamomum camphora in Examples 1 and 8-10

[0099]

[0100] It can be concluded from Table 2 that using WPM medium or 2WPM medium as the base medium, adding IBA, NAA and 6-BA can make callus differentiate into clustered adventitious buds, especially using 2WPM medium as the base culture, adding IBA 0.1 mg / L, NAA 0.5 mg / L and 6-BA 3 mg / L (Example 1), the monthly average number of proliferating buds of clustered adventitious buds can be as high as 2.8, which is significantly higher than other examples, and the bud length is 1.12 cm, which is similar to Example 2 and significantly higher than Examples 3 and 4.

[0101] Embodiment 11

[0102] Example 1 was repeated, except that the rooting medium used was rooting medium 2, so that the clustered adventitious buds could take root.

[0103] The composition of rooting medium 2 is: 1 / 2MS medium as the basic medium, and also contains 30 g / L sucrose, 3.5 g / L agar and 0.5 mg / L NAA, and the pH value is 5.8.

[0104] Comparative Example 1

[0105] Example 1 was repeated, except that the rooting medium used was rooting medium 3, so that the clustered adventitious buds could take root.

[0106] The composition of rooting medium 3 is: 1 / 2MS medium as the basic medium, and also contains 30 g / L sucrose, 3.5 g / L agar and 0.1 mg / L IBA, and the pH value is 5.8.

[0107] Comparative Example 2

[0108] Example 1 was repeated, except that the rooting medium used was rooting medium 4, so that the clustered adventitious buds could take root.

[0109] The components of rooting medium 4 are: 1 / 2MS medium as the basic medium, 30 g / L sucrose, 3.5 g / L agar and 0.5 mg / L IBA, and the pH value is 5.8.

[0110] Comparative Example 3

[0111] Example 1 was repeated, except that the rooting medium used was rooting medium 5, so that the clustered adventitious buds could take root.

[0112] The composition of rooting medium 5 is: 1 / 2MS medium as the basic medium, and also contains 30 g / L sucrose, 3.5 g / L agar and 1 mg / L IBA, and the pH value is 5.8.

[0113] Comparative Example 4

[0114] Example 1 was repeated, except that the rooting medium used was rooting medium 6, so that the clustered adventitious buds could take root.

[0115] The composition of rooting medium 6 is: 1 / 2MS medium as the basic medium, and also contains 30 g / L sucrose, 3.5 g / L agar and 0.1 mg / L NAA, and the pH value is 5.8.

[0116] Comparative Example 5

[0117] Example 1 was repeated, except that the rooting medium used was rooting medium 7, so that the clustered adventitious buds could take root.

[0118] The composition of rooting medium 7 is: 1 / 2MS medium as the basic medium, and also contains 30 g / L sucrose, 3.5 g / L agar and 1 mg / L NAA, and the pH value is 5.8.

[0119] Comparative Example 6

[0120] Example 1 was repeated, except that the rooting medium used was rooting medium 8, so that the clustered adventitious buds could take root.

[0121] The composition of rooting medium 8 is: 1 / 2WPM medium as the basic medium, and also contains 30 g / L sucrose, 3.5 g / L agar and 0.1 mg / L NAA, and the pH value is 5.8.

[0122] Comparative Example 7

[0123] Example 1 was repeated, except that the rooting medium used was rooting medium 9, so that the clustered adventitious buds could take root.

[0124] The composition of rooting medium 9 is: 1 / 2WPM medium as the basic medium, and also contains 30 g / L sucrose, 3.5 g / L agar and 0.5 mg / L NAA, and the pH value is 5.8.

[0125] Comparative Example 8

[0126] Example 1 was repeated, except that the rooting medium used was rooting medium 10, so that the clustered adventitious buds could take root.

[0127] The components of the rooting medium 10 are: 1 / 2WPM medium as the basic medium, 30 g / L sucrose, 3.5 g / L agar and 0.1 mg / L IBA, and the pH value is 5.8.

[0128] Comparative Example 9

[0129] Example 1 was repeated, except that the rooting medium used was rooting medium 11, so that the clustered adventitious buds could take root.

[0130] The components of the rooting medium 11 are: 1 / 2WPM medium as the basic medium, 30 g / L sucrose, 3.5 g / L agar and 0.5 mg / L IBA, and the pH value is 5.8.

[0131] Comparative Example 10

[0132] Example 1 was repeated, except that the rooting medium used was rooting medium 12, so that the clustered adventitious buds could take root.

[0133] The components of the rooting medium 12 are: 1 / 2WPM medium as the basic medium, 30 g / L sucrose, 3.5 g / L agar and 1 mg / L IBA, and the pH value is 5.8.

[0134] The rooting conditions of the clustered adventitious buds in Example 1, Example 11 and Comparative Examples 1 to 10 were statistically analyzed, including the average number of roots, average root length and rooting rate. The results are shown in Table 3:

[0135] Table 3 Rooting of clustered adventitious buds in Example 1, Example 11 and Comparative Examples 1 to 10

[0136]

[0137]

[0138] It can be concluded from Table 3 that: under 1 / 2MS basic culture medium, when the NAA concentration is 0.5 mg / L (Example 11), the rooting rate can reach up to 70%, the average number of roots can reach 2.2 roots, and the average root length is 1.43 cm; under 1 / 2WPM basic culture medium, when the NAA concentration is 1 mg / L (Example 1), the rooting rate can reach up to 80%, the average number of roots can reach 1.8 roots, and the average root length is 0.95 cm; which is significantly higher than Comparative Examples 1 to 10.

[0139] From the above examples, it can be concluded that the culture medium combination and method of the present invention can significantly and rapidly induce the differentiation and regeneration of Cinnamomum camphora to obtain rooted Cinnamomum camphora seedlings, and can reduce the contamination rate and browning rate ( Figure 1 shown).

[0140] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An application of a culture medium combination in the propagation of camphor tree seedlings, characterized in that: The culture medium combination consists of an induction medium, a differentiation medium and a rooting medium; The induction medium is based on MS medium, and the remaining components are: plant hormones, 30 g / L sucrose and 3.5 g / L agar, with a pH value of 5.8; the plant hormones are 0.5-5 mg / L 6-BA or 0.01-1 mg / L TDZ; The differentiation medium is based on WPM medium or 2WPM medium, and the remaining components are: 0.1-0.3 mg / L IBA, 0.1-1 mg / L NAA, 1-5 mg / L 6-BA, 30 g / L sucrose and 3.5 g / L agar; the pH value is 5.8; The rooting medium is a first rooting medium or a second rooting medium, wherein the first rooting medium uses 1 / 2MS medium as a basic medium, and the remaining components are: 0.1-1 mg / L NAA, 30 g / L sucrose and 3.5 g / L agar; and the pH value is 5.8; and the second rooting medium uses 1 / 2WPM medium as a basic medium, and the remaining components are: 0.1-1 mg / L NAA, 30 g / L sucrose and 3.5 g / L agar; and the pH value is 5.

8.

2. The use according to claim 1, characterized in that: The camphor tree seedling propagation includes camphor tree tissue culture.

3. A method for inducing plant regeneration from camphora microstem segments, characterized in that: Cinnamomum camphora stem segments are cultured using the culture medium combination described in claim 1 to obtain rooted Cinnamomum camphora seedlings.

4. The method according to claim 3, characterized in that The culturing comprises the following steps: Inducing and culturing the microstem segments cut from the sterile seedlings of Cinnamomum camphora in an induction medium to obtain callus tissue; The callus tissue is differentiated and cultured in a differentiation medium to obtain clustered adventitious buds; The clustered adventitious buds are subjected to rooting culture in a rooting medium to obtain rooted Cinnamomum camphora seedlings.

5. The method according to claim 4, characterized in that The micro-stem segment includes a stem segment with a node, and the length of the micro-stem segment is 4-6 mm.

6. The method according to claim 5, characterized in that The induction culture temperature is 24°C, the light intensity is 1000-2000LX, the light cycle is 16L:8D, and the culture time is 3-5 weeks; The differentiation culture temperature is 24°C, the light intensity is 2000-3000LX, the light cycle is 16L:8D, and the culture time is 2-4 weeks; The rooting culture temperature is 24°C, the light intensity is 2000-3000LX, the light cycle is 16L:8D, and the culture time is 1-4 weeks.

7. The method according to claim 4, characterized in that The method for cultivating the aseptic seedlings of camphor trees comprises: removing the seed shells of sterilized camphor trees seeds, and sequentially performing dark culture and light culture on a growth medium to obtain the aseptic seedlings of camphor trees.

8. The method according to claim 7, characterized in that The dark culture time is 12-48 hours; the light cycle of the light culture is 16L:8D, the light intensity is 1000-2000LX, and the culture time is 15d; the temperature of the light culture and the dark culture is 22-26°C.

9. The method according to claim 7 or 8, characterized in that: The growth medium is based on MS medium and further includes 30 g / L sucrose and 3.5 g / L agar, and the pH value is 5.8.

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