Calli induction and rapid propagation of raphiolepis indica

Abstract

The present application belongs to the technical field of plant callus breeding, and discloses a callus induction and rapid propagation method of bamboo joint tree, which comprises the following steps: S1, inoculation of bamboo joint tree seeds: inoculating mature bamboo joint tree seeds on MS medium after sterilization treatment; S2, differentiation of seed germination and new callus breeding: placing the seeds in a differentiation medium after germination to induce callus and buds, and subculturing the callus to induce new buds and new callus; S3, rooting culture of bamboo joint tree: inoculating the differentiated new bud callus on a rooting medium to induce root system formation, and then transferring to a seedling hardening stage after culture; and S4, greenhouse domestication: hardening the rooted seedlings, and then cultivating the rooted seedlings to be directly transplanted to a field; the present application solves the problems of great breeding difficulty of the prior art bamboo joint tree and easy death of new roots of the bamboo joint tree induced by the existing plant root induction scheme, and is suitable for induction and rapid propagation of the callus of the bamboo joint tree.

Description

Technical Field

[0001] This invention relates to the field of plant callus propagation technology, specifically a method for inducing and rapidly propagating callus from bamboo trees. Background Technology

[0002] The bamboo tree (Carallia brachiata (Lour.) Merr) is an evergreen tree belonging to the Rhizophoraceae family and the Carallia genus. It can grow up to 10 meters tall, with smooth bark, rarely cracked, and a grayish-brown color. Its wood is hard and heavy, with an interlocking grain and a rather coarse texture. The heartwood is large, dark reddish-brown with a yellowish tinge, while the sapwood is lighter in color with a reddish tinge and a glossy sheen. It is excellent for making musical instruments, decorative wood, doors, windows, and utensils. Furthermore, the bamboo tree exhibits a first-class resistance to air pollutants, especially SO2, earning it the nickname "Green Diamond." It is an ideal new ornamental tree species for landscaping and a valuable pollution-resistant tree. Its bark also has medicinal uses, treating malaria, and it is a national second-class protected tree species.

[0003] However, the propagation of bamboo trees is difficult. Currently, seed propagation is commonly used, but seed collection is difficult, maturity is uneven, propagation speed is slow, the seedling cycle is long, and the variation rate of seedling sowing is high, making it difficult to maintain the excellent traits of the parent plant. This outdated production method is extremely difficult to meet market demand. Although asexual reproduction methods such as tissue culture are commonly used in this field to achieve plant propagation, there are no reports in this field on inducing callus tissue through bud propagation in the tissue culture process of bamboo trees. Secondly, the inventors found that the induced new roots of bamboo trees are prone to die back, based on the root induction schemes commonly used in this field. Summary of the Invention

[0004] The present invention aims to provide a method for callus induction and rapid propagation of bamboo trees, in order to solve the problems of high propagation difficulty of bamboo trees in the prior art and the easy death of new roots induced by existing plant root induction methods.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for callus induction and rapid propagation of *Phyllostachys edulis* includes the following steps:

[0007] S1. Inoculation of Bamboo Tree Seeds: Remove the pulp from mature Bamboo Tree fruits, leaving the seeds. After sterilization, the seeds are inoculated onto MS medium.

[0008] S2. Differentiation of shoots and new callus propagation from *Phyllostachys edulis* seeds: Seeds were planted and germinated in MS medium. When the hypocotyl reached 3-5 cm in length, sterile seedlings were removed, and the hypocotyl was cut to 0.5-2 cm in length under sterile conditions. Then, it was placed in differentiation medium for callus and shoot induction. The hypocotyl of *Phyllostachys edulis* will simultaneously produce shoots and callus. Callus is mostly formed in the section in contact with the medium. After callus formation, it was promptly separated and transferred to differentiation medium for subculture, so that the callus would differentiate again into shoots and new callus.

[0009] S3. Rooting culture of bamboo: When the new shoots differentiated in S2 grow to 0.5-1cm, the callus tissue at the base of the stem is cut off, and the stems with new shoots are inoculated into the rooting medium to induce root formation. After dark culture for one week, they are cultured under light. When 2-4 roots grow on each stem and the root system is 0.25-0.5cm long, they are transferred to MS medium to continue growing until the root system grows to 1-2cm, and then they are transferred to the hardening-off stage.

[0010] S4. Greenhouse Acclimatization: The rooted seedlings that have entered the hardening-off stage in S3 are subjected to closed-bottle hardening and open-bottle hardening. Then, the rooted seedlings are soaked in a 1000-fold dilution of carbendazim solution for 3-5 minutes, taken out and dried before transplanting. The transplanted seedlings are then sprayed with a 1000-fold dilution of carbendazim solution and then cultivated. When the seedlings are taller than 10cm or have more than 8 leaves, they are transplanted to the field for normal field water and fertilizer management. This completes the callus induction and rapid propagation of bamboo trees.

[0011] Furthermore, in S1, S2 and S3, the agar powder in the MS medium was replaced with 6 g / L plant gel, while the other components and concentrations remained unchanged, and the pH of the medium was 5.8 to 6.0.

[0012] Furthermore, in S1, the aseptic treatment steps for the seeds are as follows: first, rinse with clean tap water, then soak and clean with 75% alcohol, and then rinse twice with double-distilled water; next, soak in 1% sodium hypochlorite solution for 30 minutes, shaking clockwise once every 10 minutes for 1 minute each time, and after treatment, rinse twice with double-distilled water for no less than 3 minutes each time.

[0013] Furthermore, in S2, the germination conditions of the seeds in MS medium were: ambient temperature of 28℃±2℃, light duration of 12h / d, and light intensity of 3000lx~5000lx.

[0014] Furthermore, in S2, the differentiation medium is MS medium supplemented with 1.5 mg / L ZT zeatin and 0.3 mg / L IAA.

[0015] Furthermore, in S3, the rooting medium is MS medium supplemented with 7 mg / L indolebutyric acid.

[0016] Furthermore, in S4, the hardening-off time for rooted seedlings in closed bottles is 7 days, and the hardening-off time for rooted seedlings in open bottles is 3 days; the substrate for transplanting rooted seedlings is a 1:1 volume ratio of river sand and peat moss, and the substrate is thoroughly watered before transplanting.

[0017] Furthermore, in S4, the cultivation conditions for seedlings are: air humidity greater than 90%, ambient temperature of 28℃±2℃, light duration of 12h / d, and light intensity of 3000lx.

[0018] The beneficial effects of the technical solution are:

[0019] This invention can not only induce new shoots, but also induce callus formation in bamboo trees to achieve the purpose of propagation; it solves the problems of the difficulty in propagating bamboo trees in the existing technology, and the problem that the new roots induced by existing plant root induction methods are prone to die-off. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to the embodiments:

[0021] A method for callus induction and rapid propagation of *Phyllostachys edulis* includes the following steps:

[0022] S1. Inoculation of Bamboo Tree Seeds: Remove the pulp from mature Bamboo Tree fruits, leaving the seeds. Rinse the seeds with clean tap water, then soak them in 75% alcohol, and then rinse them twice with double-distilled water. Next, soak them in 1% sodium hypochlorite solution for 30 minutes, shaking clockwise once every 10 minutes for 1 minute each time. After treatment, rinse them twice with double-distilled water for no less than 3 minutes each time. After this aseptic treatment, inoculate them onto MS medium.

[0023] S2. Differentiation and budding of *Phyllostachys edulis* seeds and propagation of new callus: Seeds were planted and germinated in MS medium at an ambient temperature of 28℃±2℃, a photoperiod of 12h / d, and a light intensity of 3000lx~5000lx. When the hypocotyl reached 3~5 cm in length, sterile seedlings were removed, and the hypocotyl was cut to 0.5~2 cm in length under sterile conditions. Then, they were placed in differentiation medium for induction of callus and buds. The differentiation medium was MS medium supplemented with 1.5 mg / L ZT zeatin and 0.3 mg / L IAA. The hypocotyl of *Phyllostachys edulis* will simultaneously produce buds and callus. The callus mostly forms in the section in contact with the medium. After the callus forms, it is promptly separated and transferred to the differentiation medium for subculture, so that the callus will differentiate again into buds and new callus.

[0024] S3. Rooting culture of bamboo: When the new shoots differentiated in S2 grow to 0.5-1cm, the callus tissue at the base of the shoot is cut off, and the shoots with new shoots are inoculated into the rooting medium to induce root formation. The rooting medium is MS medium supplemented with 7mg / L indolebutyric acid. After dark culture for one week, it is then cultured under light. When 2-4 roots grow on each shoot and the root system is 0.25-0.5cm long, it is transferred to MS medium to continue growth until the root system grows to 1-2cm, and then it enters the hardening stage.

[0025] S4. Greenhouse Acclimation: The rooted seedlings transferred from S3 to the hardening-off stage are first subjected to 7 days of closed-bottle hardening-off, followed by 3 days of open-bottle hardening-off. Then, the rooted seedlings are soaked in a 1000-fold dilution of carbendazim solution for 3-5 minutes, removed and dried, and then transplanted into a 1:1 volume ratio of river sand and peat moss substrate. Before transplanting, the substrate is thoroughly watered. After transplanting, the seedlings are sprayed with a 1000-fold dilution of carbendazim solution. They are then cultured under conditions of air humidity greater than 90%, ambient temperature of 28℃±2℃, light duration of 12h / d, and light intensity of 3000lx. When the seedlings are taller than 10cm or have more than 8 leaves, they are transplanted to the field for normal field water and fertilizer management. This completes the callus induction and rapid propagation of bamboo trees.

[0026] In S1, S2 and S3, the agar powder in all MS media was replaced with 6 g / L plant gel, while the other components and concentrations remained unchanged, and the pH of the media was 5.8 to 6.0.

[0027] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific technical solutions or characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for callus induction and rapid propagation of *Phyllostachys edulis*, characterized in that, Includes the following steps: S1. Inoculation of Bamboo Tree Seeds: Remove the pulp from mature Bamboo Tree fruits, leaving the seeds. After sterilization, the seeds are inoculated onto MS medium. S2. Differentiation and budding of *Phyllostachys edulis* seeds and propagation of new callus: Seeds are planted and germinated in MS medium. When the hypocotyl reaches 3-5 cm in length, sterile seedlings are removed, and the hypocotyl is cut to 0.5-2 cm in length under sterile conditions. Then, it is placed in differentiation medium for callus and bud induction. The hypocotyl of *Phyllostachys edulis* will simultaneously produce buds and callus. Callus mainly forms in the section in contact with the medium. After callus formation, it is promptly separated and transferred to differentiation medium for subculture, allowing the callus to differentiate again into buds and new callus. The differentiation medium is MS medium supplemented with 1.5 mg / L ZT zeatin and 0.3 mg / L IAA. S3. Rooting culture of *Phyllostachys edulis*: When the new shoots differentiated in S2 reach 0.5–1 cm in length, the callus tissue at the base of the shoots is separated, and the shoots with new shoots are inoculated into a rooting medium to induce root formation. After one week of dark culture, they are then cultured under light. When 2–4 roots grow on each shoot and the root system reaches 0.25–0.5 cm in length, they are transferred to MS medium to continue growth until the roots reach 1–2 cm in length, at which point they enter the hardening-off stage. The rooting medium is MS medium supplemented with 7 mg / L indolebutyric acid. S4. Greenhouse Acclimatization: The rooted seedlings that have entered the hardening-off stage in S3 are subjected to closed-bottle hardening and open-bottle hardening. Then, the rooted seedlings are soaked in a 1000-fold dilution of carbendazim solution for 3-5 minutes, taken out and dried before transplanting. The transplanted seedlings are then sprayed with a 1000-fold dilution of carbendazim solution and then cultivated. When the seedlings are taller than 10cm or have more than 8 leaves, they are transplanted to the field for normal field water and fertilizer management. This completes the callus induction and rapid propagation of bamboo trees.

2. The method for callus induction and rapid propagation of bamboo trees according to claim 1, characterized in that: In S1, S2 and S3, the MS medium contains 6 g / L of plant gel, with the remaining components and concentrations unchanged, and the pH of the medium is 5.8 to 6.

0.

3. The method for callus induction and rapid propagation of bamboo trees according to claim 1, characterized in that: In S1, the aseptic treatment steps for the seeds are as follows: first, rinse with clean tap water, then soak in 75% alcohol, and then rinse twice with double-distilled water; next, soak in 1% sodium hypochlorite solution for 30 minutes, shaking clockwise once every 10 minutes for 1 minute each time, and after treatment, rinse twice with double-distilled water for no less than 3 minutes each time.

4. The method for callus induction and rapid propagation of bamboo trees according to claim 1, characterized in that: In S2, the germination conditions of seeds in MS medium were: ambient temperature of 28℃±2℃, light duration of 12h / d, and light intensity of 3000lx~5000lx.

5. The method for callus induction and rapid propagation of bamboo trees according to claim 1, characterized in that: In S4, the hardening-off time for rooted seedlings in closed bottles is 7 days, and the hardening-off time for rooted seedlings in open bottles is 3 days; the substrate for transplanting rooted seedlings is a 1:1 volume ratio of river sand and peat moss, and the substrate is thoroughly watered before transplanting.

6. The method for callus induction and rapid propagation of bamboo trees according to claim 1, characterized in that: In S4, the cultivation conditions for seedlings are: air humidity greater than 90%, ambient temperature 28℃±2℃, light duration 12h / d, and light intensity 3000lx.