Bamboo reed test-tube plantlet cluster bud induction culture medium and rapid propagation seedling method

Abstract

The invention discloses a bamboo reed test-tube plantlet cluster bud induction culture medium and a tissue culture and rapid propagation seedling culture method. A primary induction culture medium in the culture medium is prepared from Murashige; the culture medium is prepared from 4.4 g / L of Skoog Medium, 30 g / L of cane sugar, 5 g / L of agar, 4 mg / L of 6-BA (6-Butyl Acetate) and 4 mg / L of a pH value of 7.7-8.0. The subculture multiplication culture medium is prepared from the following components: Murashige; the culture medium is prepared from 4.4 g / L of Skoog Medium, 30 g / L of cane sugar, 5 g / L of agar, 6 mg / L of 6-BA and 0.8 mg / L of IBA, wherein the pH value of the culture medium is 7.7-8.0. By means of the seedling raising method, the cultivation time of the bamboo reed can be shortened, the breeding speed of the bamboo reed can be effectively increased, and the efficient tissue culture breeding capacity is achieved by properly adjusting and optimizing the hormone concentration and the components of the culture medium.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of reed cultivation, in particular to a reed test tube seedling cluster bud induction medium and rapid propagation seedling method BACKGROUND Reed is a perennial tall herb, because of its strong adaptability, rapid growth, often used for ecological restoration, biomass energy development and landscape, economic value lies in its stem contains high cellulose, is a high-quality raw material for papermaking, fiberboard, biofuels (such as ethanol, biogas).

[0002] But the propagation control is difficult, relying on the plant seed propagation is difficult, the natural seed setting rate is low, the seed germination rate is unstable, needs low temperature stratification treatment or hormone treatment. Therefore, mainly relying on asexual reproduction, relying on rhizome cutting or mechanical excavation, traditional cultivation method, long cultivation time, high labor intensity.

[0003] The patent number CN201610597056.9 discloses a reed test tube seedling cluster bud induction medium, a basic MS is added with a plant growth regulator combination to form a culture medium formula, the tissue culture and rapid propagation culture medium of the application is used, the axillary bud stem segment of the superior plant is used as an explant, the whole sterile culture process is simple and easy to operate, and even can be simplified into a culture medium (one-time seedling and tillering rapid propagation culture medium).

[0004] But the above-mentioned culture method has a long cultivation time, needs to further improve the propagation speed of reed under the premise of healthy development of reed, reduces the required culture time, therefore, a new reed test tube seedling cluster bud induction medium and culture method are urgently needed, which uses plant tissue culture technology to develop special culture medium and culture method, accelerates the propagation cycle of reed, and improves the propagation efficiency. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a reed test tube seedling cluster bud induction medium and rapid propagation seedling method, and to improve the propagation speed of reed.

[0006] The technical scheme adopted by the present application to solve the above technical problem is as follows: in the first aspect, a reed test tube seedling cluster bud induction medium comprises a primary induction medium and a subculture proliferation medium. The primary induction medium comprises: Murashige & Skoog Medium (with Vitamins) 4.4g / L, Sucrose 30g / L, Agar 5g / L, pH=7.7-8.0, 6-BA 4mg / L; Subculture medium includes: Murashige & Skoog Medium (with Vitamins) 4.4 g / L, Sucrose 30 g / L, Agar 5 g / L, pH = 7.7-8.0, 6-BA 6 mg / L, IBA 0.8 mg / L.

[0007] Preferably, a reed test tube seedling cluster bud induction medium also includes a rooting medium, a tillering medium and an induction cluster bud medium.

[0008] As an embodiment of the present application, the rooting medium includes: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2 g / L, Sucrose 20 g / L, Activated carbon 1 g / L, pH = 7.7-8.0, NAA 0.2 mg / L.

[0009] As another embodiment of the present application, the rooting medium includes: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2 g / L, Sucrose 15-20 g / L, Activated carbon or agar 1-5 g / L pH = 7.7-8.0 IBA 1 mg / L.

[0010] As an embodiment of the present application, the tillering medium includes: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2 g / L, or Murashige & Skoog Medium (with Vitamins) 4.4 g / L, NAA 0.1-0.5 mg / L, IBA 5 mg / L, Sucrose 15 g, Agar 7 g.

[0011] As an embodiment of the present application, the induction cluster bud medium includes: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2g / L, or Murashige & Skoog Medium (with Vitamins) 4.4g / L, 6-BA 1-4mg / L, NAA 0.1mg / L, IBA 5mg / L, Sucrose 15g, Agar 7g.

[0012] In a second aspect, a rapid propagation method of Arundo donax comprises the following steps: S1: collecting healthy stem segments of Arundo donax, removing bracts, selecting stem segments with buds, washing and disinfecting to obtain pre-processed explant bases; S2: cutting the disinfecting explant bases in S1 into small segments of 2-3 cm, beveling the lower end to increase the absorption area, peeling off the lateral bud sheath, and obliquely or vertically inserting into a glass bottle containing the primary induction medium of claim 1 for culture; S3: cutting the large bud bodies formed during the culture process to 2 cm or more, cutting the cut large bud bodies into several bud segments as subculture explants, inoculating into the subculture proliferation medium of claim 1, and culturing under light conditions for 25-30 days to induce the formation of multiple shoots; The explant bases with cut large buds continue to be cultured in the glass bottle containing the primary induction medium of claim 1 to induce the formation of multiple shoots.

[0013] Further, a rapid propagation method of Arundo donax further comprises the following steps: S4: selecting plants with a height of 5 cm or more in the subculture proliferation medium in step S3 for rooting, transferring to a glass bottle containing the rooting medium of claim 3 or 4 for light culture for 10-14 days to induce complete root systems, further proliferating the multiple shoots into high-quality seedlings, and uncapping the well-rooted seedlings in the tissue culture bottle for two days of acclimation.

[0014] Further, a rapid propagation method of Arundo donax further comprises the following steps: S5: selecting the bud seedlings after rooting in S4, transferring to a glass bottle containing a tillering medium for light culture, 3-4 days for axillary bud germination, 6-20 days for root germination at the base of the axillary bud, and forming complete small plants.

[0015] Further, a rapid propagation method of Arundo donax tissue culture further comprises the following steps: S6: continue to culture in the light in the induction of the cluster bud culture medium, 20-30 days grow 3-15 or so part of the root cluster buds or seedlings, realize the stem node cut piece rapid propagation and the root node tiller rapid propagation.

[0016] The cultivated seedling is transplanted: when the seedling height is 3.0-4.0 cm, the root length is more than 1.0 cm and has 4-5 roots, the seedling is hardened for 3-4 days, and then transplanted after washing the agar.

[0017] The beneficial effects of the present application are: through the optimization of the primary induction medium, the subculture proliferation medium and the rooting medium, the bamboo reed rapid propagation seedling method can shorten the cultivation time of the bamboo reed, can make the bamboo reed rapid propagation coefficient and efficiency improve 2-3 times, can effectively improve the propagation speed of the bamboo reed, reduce the required time, and at the same time ensure the healthy growth of the plant.

[0018] The present application realizes the performance-price ratio optimization of nutrients and hormones by moderately adjusting and optimizing the hormone concentration and the medium composition, the induction rate, the multiplication rate, the rooting rate and the survival rate of the present application all reach more than 85-95%, and the present application has high tissue culture propagation capacity. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a schematic diagram of the base of the bamboo reed explant inserted in the primary induction medium; Figure 2 is a schematic diagram of the axillary bud grown after the base of the bamboo reed explant is cultured in the primary induction medium for 8 days; Figure 3 is a schematic diagram of the bud segment in the subculture proliferation medium; Figure 4 is a schematic diagram of the cluster bud expansion into seedlings; Figure 5 is a schematic diagram of the bamboo reed seedling transplantation. DETAILED DESCRIPTION

[0020] The present application will be further described in detail in combination with the drawings and preferred embodiments. These drawings are all simplified schematic diagrams, and only illustrate the basic structure of the present application in a schematic manner, and therefore only show the configurations related to the present application.

[0021] Example 1, a bamboo reed test tube seedling cluster bud induction culture medium, including a primary induction culture medium, a subculture proliferation culture medium, a rooting culture medium, a tillering culture medium and an induction cluster bud culture medium.

[0022] A bamboo reed rapid propagation seedling method, comprising the following steps: S1: collecting healthy stem segments of bamboo reed, removing bracts, selecting bud-bearing stem segments, washing and disinfecting to obtain pretreated explant bases; Specifically: select the healthy and robust Arundo donax plants, cut the new tender axillary bud stem segments as explants, soak them in 75% ethanol for 50-70 seconds on the clean bench, then soak them in 0.1%-0.2% mercuric chloride solution for 8 minutes, and wash them with sterile water for 3-5 times.

[0023] S2: cut the Arundo donax explant base sterilized in S1 into 2-3 cm small segments, cut the lower end obliquely to increase the absorption area, peel off the axillary bud lateral leaf sheath, and insert it obliquely or vertically into a glass bottle containing the primary induction medium for culture, as shown in Figure 1 The primary induction medium comprises: Murashige & Skoog Medium (with Vitamins) 4.4g / L, Sucrose 30g / L, Agar 5g / L, pH=7.7-8.0, 6-BA 4mg / L; The Arundo donax explant base is cultured in the primary induction medium for 6-8 days to induce axillary buds, and an average of 1.8-2.3 effective bud points are induced from each stem segment, as shown in Figure 2 After 8 days, the Arundo donax explant base grows axillary buds in the primary induction medium; the primary induction medium enables the Arundo donax to start bud germination in a relatively short time, and usually forms a larger bud group in a relatively short time, which proves the effectiveness of the primary induction medium in inducing bud meristem. Murashige & Skoog Medium (with Vitamins) is a MS medium containing vitamins.

[0024] S3: cut the large bud body formed during the culture process to several bud segments as subculture explants, inoculate them into the subculture proliferation medium of claim 1, and culture them under light conditions for 25-30 days to induce the formation of multiple shoots; The explant base with the removed large bud is continued to be cultured in the glass bottle of the primary induction medium of claim 1 to induce the formation of multiple shoots.

[0025] ​"Large bud" refers to an individual that has elongated and is significantly larger than other buds in the initial or early culture process, with an elongated length of more than 2 cm. If such a large bud is directly retained, it will grow faster and compete for nutrients in the culture medium, which is not conducive to the germination of other latent buds and the formation of cluster buds. By cutting the large bud and dividing the large bud into segments as subculture material, on the one hand, the propagation coefficient can be effectively improved, and on the other hand, the nutrient competition of the dominant bud in the initial induction medium can be weakened, thereby promoting the germination of latent buds on the explant base in the initial induction medium, which is conducive to obtaining more and uniform cluster bud groups, and the cluster buds grow into large buds of more than 2 cm.

[0026] The bud segments of the large bud are further inoculated into the subculture proliferation medium to induce cluster buds, and are cultured under light in a glass bottle containing the subculture proliferation medium for 25-30 days; as shown in Figure 3 , the bud segments induce cluster buds in the glass bottle containing the subculture proliferation medium.

[0027] The subculture proliferation medium comprises: Murashige & Skoog Medium (with Vitamins) 4.4 g / L, Sucrose 30 g / L, Agar 5 g / L, pH = 7.7-8.0, 6-BA 6 mg / L, IBA 0.8 mg / L.

[0028] Increasing the concentration of 6-BA in the subculture proliferation medium to 6 mg / L helps to control overgrowth and abnormal buds and improve the quality of subculture buds. Under the cooperation of 6-BA (6 mg / L) and IBA (0.8 mg / L), the proliferation rate of the plant can be increased by 1.5-2 times compared with the medium without adding these hormones. This proliferation rate can achieve at least 3-4 times of bud proliferation within 30 days, and the average number of bud groups generated by each proliferation is 10-15, which significantly improves the propagation coefficient.

[0029] S4: Selecting plants with a height of more than 5 cm in the subculture proliferation medium in S3 for rooting, and transferring them to a glass bottle containing a rooting medium for light culture for 10-14 days to induce complete root systems, as shown in Figure 4 , the cluster buds are further expanded into seedlings, and the seedlings with good rooting state are acclimated in the culture bottle for two days.

[0030] The rooting medium formula 1 comprises: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2 g / L, Sucrose 20 g / L, Activated carbon 1 g / L, pH = 7.7-8.0, NAA 0.2 mg / L.

[0031] The rooting medium formula 2 comprises: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2 g / L, Sucrose 15-20 g / L, Activated carbon or agar 1-5 g / L, pH = 7.7-8.0, IBA 1 mg / L.

[0032] Either of the two formulas, the rooting medium uses 1 / 2 MS dilution of the basic salt concentration, which reduces the osmotic pressure and is conducive to root development.

[0033] In the rooting medium, using 1 / 2 MS medium (2.2 g / L), sucrose (15 g / L) and NAA (0.2 mg / L), about 90% of the plants can successfully root within 10-14 days. Reducing the sucrose concentration to 15 g / L is conducive to energy regulation during the rooting period and prevents excessive sugar from inhibiting root growth. The length of the root system can reach 3-5 cm within 20 days, and the growth rate of the root system is relatively stable.

[0034] Through the optimization of the primary induction medium, the secondary proliferation medium, and the rooting medium, the cultivation time of the bamboo cane is shortened, which can increase the rapid propagation coefficient and efficiency of the bamboo cane by 2-3 times, effectively improve the propagation speed, reduce the required time, and at the same time ensure the healthy growth of the plant.

[0035] After the cultivation is completed, the tillering rapid propagation is carried out.

[0036] S5: Select the rooted sprouts in S4 and transfer them to a glass bottle containing tillering medium for light culture. The axillary buds sprout after 3-4 days, and the roots sprout at the base of the axillary buds after 6-20 days, forming complete small plants. The tillering medium comprises: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2 g / L, or Murashige & Skoog Medium (with Vitamins) 4.4 g / L, NAA 0.1-0.5 mg / L, IBA 5 mg / L, Sucrose 15 g, Agar 7 g.

[0037] After the tillering culture is completed, 1-4 mg / L 6-BA is added to the tillering culture medium to form a cluster bud induction culture medium.

[0038] S6: Continue to culture under light in the cluster bud induction culture medium, and 3-15 partially rooted cluster buds or seedlings are grown in 20-30 days to realize stem node cutting and root node tillering rapid propagation.

[0039] Transplant the cultivated seedlings: when the seedlings are 3.0-4.0 cm high, the roots are more than 1.0 cm long and have 4-5 roots, acclimate the seedlings for 3-4 days, transplant the seedlings after washing the agar, transplant the robust seedlings to the field as shown in the formula, and grow into adult plants. Figure 5 The transplanted seedlings are acclimated in the greenhouse for 20 days, and the membrane is completely opened to complete domestication.

[0040] The survival rate of the transplanted seedlings can reach more than 95%. The seedlings resume growth after 20 days of transplantation and gradually adapt to the natural environment.

[0041] From induction to subculture, rooting, tillering, cluster bud induction, and transplantation, the formula is closely linked to realize rapid propagation of bamboo.

[0042] The present application realizes the cost-effective optimization of nutrients and hormones by moderately adjusting and optimizing the hormone concentration and medium composition. The induction rate, multiplication rate, rooting rate, and survival rate of the present application all reach more than 85-95%, and the present application has high tissue culture propagation capacity.

[0043] The above description is only a specific embodiment of the present application, and various examples do not limit the essential content of the present application. Those skilled in the art can modify or deform the above-described specific embodiments after reading the specification without departing from the essence and scope of the present application.

Claims

1. A culture medium for inducing multiple shoots of Arundo donax test-tube seedlings, characterized by comprising: The primary induction medium and the secondary proliferation medium; ​ The primary induction medium comprises: Murashige & Skoog Medium (with Vitamins) 4.4g / L, Sucrose 30g / L, Agar 5g / L, pH=7.7-8.0, 6-BA 4mg / L; The secondary proliferation medium comprises: Murashige & Skoog Medium (with Vitamins) 4.4g / L, Sucrose 30g / L, Agar 5g / L, pH=7.7-8.0, 6-BA 6mg / L, IBA 0.8 mg / L.

2. The induction medium for cluster bud of Arundo donax test-tube seedling according to claim 1, characterized in that: The rooting medium, the tillering medium and the induction of the cluster medium are also included.

3. The induction medium for cluster bud of Arundo donax test-tube seedling according to claim 2, characterized in that: the induction medium is MS medium containing 2,4-D 2.0 mg / L, 6-BA 0.5 mg / L, and GA 0.5 mg / L. The rooting medium comprises: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2g / L, Sucrose 20g / L, Activated carbon 1g / L, pH=7.7-8.0, NAA 0.2mg / L.

4. The induction medium for cluster bud of Arundo donax test-tube seedling according to claim 2, characterized in that: The rooting medium comprises: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2g / L, Sucrose 15-20g / L, Activated carbon or agar 1-5g / L pH=7.7-8.0 IBA 1mg / L.

5. The culture medium for inducing shoot proliferation in test-tube seedlings of *Arundinaria salsa* according to claim 2, characterized in that: The tillering medium comprises: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2g / L, or Murashige & Skoog Medium (with Vitamins) 4.4g / L, NAA 0.1-0.5 mg / L, IBA 5 mg / L, Sucrose 15 g, Agar 7 g.

6. The culture medium for inducing shoot proliferation in test-tube seedlings of *Arundinaria salsa* according to claim 2, characterized in that: The induction of the cluster medium comprises: 1 / 2 Murashige & Skoog Medium (with Vitamins) 2.2g / L, or Murashige & Skoog Medium (with Vitamins) 4.4g / L, 6-BA 1-4mg / L, NAA 0.1mg / L, IBA 5mg / L, Sucrose 15 g, Agar 7 g.

7. A method for rapid propagation of Arundo donax, characterized by: The method comprises the following steps: S1: collecting healthy stem segments of Arundo donax, removing the bracts, selecting the stem segments with buds, washing and disinfecting to obtain the pretreated explant base; S2: cutting the disinfecting explant base in S1 into small segments of 2-3 cm, beveling the lower end to increase the absorption area, peeling off the lateral leaf sheath of the axillary bud, and obliquely or vertically inserting into a glass bottle containing the primary induction medium of claim 1 for culture; S3: cutting the large bud body of more than 2 cm formed in the culture process, cutting the cut large bud body into several bud segments as the secondary explants, inoculating into the secondary proliferation medium of claim 1, and culturing under light conditions for 25-30 days to induce the formation of cluster buds. The explant base of the large bud that has been cut off is continuously cultured in a glass bottle of the primary induction medium of claim 1 to induce the formation of a cluster bud.

8. The rapid propagation method of Arundo donax according to claim 7, characterized in that: Further comprising the following steps: S4: Selecting the high 5 cm or more plants in the subculture medium of step S3 for rooting, and transferring to a glass bottle containing the rooting medium of claim 3 or 4 for illumination culture for 10-14 days to induce a complete root system, and further expanding the cluster bud into high seedlings, and culturing the seedlings with good rooting state in a tissue culture bottle for two days.

9. The method for rapid propagation of Arundo donax according to claim 8, characterized in that: Further comprising the following steps: S5: Selecting the bud seedlings after rooting in S4, and transferring to a glass bottle containing the tillering medium of claim 5 for illumination culture, and the axillary buds sprout after 3-4 days, and the roots sprout at the base of the axillary buds after 6-20 days to form complete small plants.

10. The rapid propagation method of Arundo donax according to claim 9, characterized in that: Further comprising the following steps: S6: Continuing illumination culture in the cluster bud induction medium of claim 6 for 20-30 days to grow 3-15 or so cluster buds or seedlings with partial roots.

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