Method for rapid propagation by utilizing sandalwood hypocotyl

Abstract

The invention discloses a method for rapid propagation by utilizing sandalwood hypocotyl, and belongs to the field of plant tissue culture. The method comprises the following steps: adventitious bud induction and subculture: cutting a germ-free seedling hypocotyl, and obliquely inoculating the germ-free seedling hypocotyl to a bud induction culture medium for bud induction; strong seedling culture: when buds are 2-3 cm long, cutting the buds and placing the buds on a strong seedling culture medium for strong seedling culture; rooting induction: transferring the rootless seedlings into a rooting culture medium for rooting culture; performing root elongation culture: when the adventitious roots grow to 2-3mm, transferring the adventitious roots to a root elongation culture medium for continuous culture; and seedling hardening and transplanting: after seedling hardening, transplanting the seedlings into a culture medium for culture to obtain sandalwood plants. According to the method, a large number of sandalwood tissue culture seedlings can be rapidly obtained through tissue culture by using sandalwood hypocotyls as explants, the bud induction number of each explant can reach 20-25, the rooting rate is up to 70% or above, and the transplanting survival rate is 100%.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for rapid propagation using sandalwood hypocotyls. Background Technology

[0002] sandalwood( Santalum album L .) belongs to the genus *Santalum* of the family Santalaceae. Santalum Sandalwood is an economically valuable forest tree, mainly distributed in India, Indonesia, Australia, and some islands in the Pacific Ocean. It is particularly famous for its wood, which can be used for carving and extracting sandalwood oil, earning it the nickname "green gold." Its main components are α- and β-santalol, which can be used as raw materials in cosmetics, perfumes, and high-end craft carvings. It also possesses antibacterial and antitumor properties and is widely used in the fragrance industry and medicine. Its economic benefits are 5-10 times that of other forest trees. However, driven by the high profits from sandalwood wood and its oil, long-term over-harvesting and ecological destruction have led to a sharp decline in the world's wild sandalwood resources. This species has been listed as an endangered species by the International Union for Conservation of Nature (IUCN).

[0003] For many years, people have hoped to develop the sandalwood industry through artificial cultivation to achieve higher economic returns. Researchers have studied the rapid propagation of sandalwood through tissue culture, mainly using young shoots and stem segments from mature sandalwood trees as explants to induce adventitious buds. Chinese Patent 200710032982.2 discloses "A method for rapid propagation of sandalwood through tissue culture," which uses young sandalwood shoots as explants for adventitious bud induction and plant regeneration. Currently, there are no reports on using sandalwood hypocotyls as explants to induce bud differentiation and plant regeneration. Summary of the Invention

[0004] The purpose of this invention is to provide a method for rapid propagation using sandalwood hypocotyls, effectively solving problems such as the lack of sandalwood resources, providing a new method for sandalwood tree breeding and seedling cultivation, and promoting the development of bio-agriculture and related industries.

[0005] A method for rapid propagation using sandalwood hypocotyls includes the following steps: Hypocotyls of sterile sandalwood seedlings are inoculated onto adventitious bud induction medium MS + 0.2-0.5 mg / L 6-BA + 20-30 g / L sucrose + 6-7 g / L agar for adventitious bud induction and subculture to obtain differentiated buds; the differentiated buds are inoculated onto seedling strengthening medium MS + 0.2-0.5 mg / L 6-BA + 0.05-0.1 mg / L NAA + 20-30 g / L sucrose + 6-7 g / L agar for seedling strengthening culture to obtain rootless seedlings; the rootless seedlings are inoculated onto rooting medium B5 + 2-5 mg / L IBA + 0.2-0.5 mg / L NAA + 20-30 g / L sucrose + 6-7 g / L agar for rooting culture to obtain rooted seedlings; the rooted seedlings are inoculated onto root elongation medium B5 + 1-5 mg / L IBA + 0.2-1 mg / L 6-BA + 0.05-0.1 mg / L NAA + 20-30 g / L sucrose + 6-7 g / L agar for rooting culture to obtain rooted seedlings. The root elongation medium substrate obtained by moistening perlite and vermiculite with mg / L gibberellin + 20-30 g / L sucrose was used to obtain tissue culture seedlings; the tissue culture seedlings were then transplanted into the substrate.

[0006] Preferably, the adventitious bud induction medium is MS + 0.2 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar.

[0007] Preferably, the seedling culture medium is MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 7 g / L agar.

[0008] Preferably, the rooting medium is B5 + 2 mg / L IBA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar.

[0009] Preferably, the root elongation culture medium is B5 + 1 mg / L IBA + 0.2 mg / L gibberellin + 30 g / L sucrose.

[0010] Preferably, the culture conditions for the adventitious bud induction and subculture, the seedling strengthening culture, the rooting culture, and the root elongation culture are all a temperature of 26 ± 2℃ and a light intensity of 40-60 µmol / m². -2 s -1 Light exposure time is 12-14 hours per day.

[0011] Preferably, the substrate to which the seedling is transplanted has a volume ratio of perlite:vermiculite = 2:1.

[0012] Preferably, the hypocotyl is cut into 0.3-0.5 cm segments and inoculated at an angle of 10-20° onto the adventitious bud induction medium with the seedlings facing upwards.

[0013] Preferably, the rootless seedling is inoculated with a rooting culture medium after the stem base is cut and infected with Agrobacterium rhizogenes.

[0014] Preferably, the rooted seedlings are inoculated into a root elongation medium when the adventitious roots are 2-3 mm long.

[0015] Advantages of this invention:

[0016] This invention provides a method for rapid propagation using sandalwood hypocotyls, comprising: (1) explant disinfection: using mature sandalwood seeds as explants for disinfection treatment; (2) obtaining sterile sandalwood seedlings: sowing seeds on MS medium and culturing to obtain sterile sandalwood seedlings; (3) adventitious bud induction and subculture: inoculating hypocotyl segments at an angle onto bud induction medium for bud induction; (4) seedling strengthening culture: when the buds are 2-3 cm long, cutting them and placing them on seedling strengthening medium for seedling strengthening culture; (5) rooting induction: transferring the rootless seedlings after seedling strengthening culture to rooting medium for rooting culture; (6) root elongation culture: when the adventitious roots grow to 2-3 mm, transferring them to root elongation medium for continued culture; (7) hardening and transplanting: after hardening, transplanting the seedlings into the culture medium for culture to obtain sandalwood plants. This invention utilizes sandalwood hypocotyls as explants to rapidly obtain a large number of sandalwood tissue culture seedlings through tissue culture. Each explant can induce 20-25 buds, with a rooting rate of over 70% and a transplant survival rate of 100%. This invention achieves rapid and abundant sandalwood seedling production through tissue culture, and is simple to operate, has a short cycle, and a high seedling yield. Attached Figure Description

[0017] Figure 1 This is a flowchart for establishing a rapid propagation technique for sandalwood. A represents induction of adventitious buds for 2 weeks; B represents induction of adventitious buds for 4 weeks; C represents seedling cultivation; D represents root induction culture; E represents root elongation culture; and F and E are indicated by red arrows on the diagram, which point to the sandalwood seedling roots. Detailed Implementation

[0018] The technical approach of this invention is as follows:

[0019] (1) Inactivation of seed dormancy and disinfection: Soak mature sandalwood seeds with pericarps in 100-200 mg / L gibberellin for 24 hours, then rinse with sterile water 2-5 times; on a clean bench, soak in 75% alcohol for 30 seconds, rinse with sterile water, then soak in 0.1% HgCl solution for about 8-10 minutes, rinse with sterile water 3-5 times, peel off the pericarp, and inoculate the remaining seeds onto sterile sandalwood seedling culture medium, and place them in a culture environment at 26 ± 2℃ and a light intensity of 40-60 µmol / m². -2 s -1 The seedlings were cultured under a light exposure of 12-14 h / d for 2 weeks. After 2 weeks, the germinated material was transferred to the same culture medium and cultured for another 3 weeks to obtain 4-5 cm tall sterile sandalwood seedlings.

[0020] (2) Adventitious bud induction: The hypocotyl of sterile seedlings was cut into 0.3-0.5 cm segments, and inoculated at an angle of 10-20° onto the adventitious bud induction medium with the seedlings facing upwards. Culture conditions: temperature 26 ± 2℃, light intensity 40-60 µmol / m². -2 s -1 Light exposure time is 12-14 h / d.

[0021] (3) Subculture of adventitious buds and cultivation of vigorous seedlings: After 3 weeks, the seedlings were transferred to the same culture medium for further cultivation. When the differentiated buds were 2-3 cm long, they were cut off and placed on vigorous seedling culture medium for 3 weeks. Culture conditions: temperature 26 ± 2℃, light intensity 40-60 µmol / m -2 s -1 Light exposure time is 12-14 h / d.

[0022] (4) Rooting culture: The base of the stems of rootless sandalwood seedlings was infected with Agrobacterium rhizogenes solution and then transferred to rooting medium for root induction. Culture conditions: temperature 26 ± 2℃, light intensity 40-60 µmol / m -2 s -1 Light exposure time is 12-14 hours per day.

[0023] (5) Root elongation culture: When the adventitious roots have grown to 2-3 mm, they are transferred to the root elongation medium for further culture. Culture conditions: temperature 26 ± 2℃, light intensity 40-60 µmol / m². -2 s -1 Light exposure time is 12-14 h / d.

[0024] (6) Hardening and transplanting: After hardening for 1 week, the seedlings were transplanted into a growth substrate with a volume ratio of perlite:vermiculite = 2:1 and placed in the sandalwood demonstration base for cultivation.

[0025] Aseptic sandalwood seedling culture medium: Each liter of the medium contains 0.5 mg / L gibberellin, 20-30 g sucrose and 6-7 g / L agar, with the remainder being MS medium, pH 5.5-5.8.

[0026] Adventitious bud induction medium: Each liter of induction medium contains 0.2-0.5 mg 6-BA, 20-30 g sucrose and 6-7 g / L agar, with the remainder being MS medium, pH 5.5-5.8.

[0027] Seedling growth medium: Each liter of medium contains 0.2-0.5 mg 6-BA and 0.05-0.1 mg NAA, 20-30 g sucrose and 6-7 g / L agar, with the remainder being MS medium, pH 5.5-5.8.

[0028] Rooting medium: Each liter of root induction medium contains 2-5 mg IBA, 0.2-0.5 mg NAA, 20-30 g sucrose, and 6-7 g / L agar, with the remainder being B5 medium, pH 5.5-5.8.

[0029] Root elongation medium substrate: Each liter of root elongation medium contains 1-5 mg IBA, 0.2-1 mg gibberellin, 20-30 g sucrose, and the remainder is B5 medium, pH 5.5-5.8. The root elongation medium substrate is obtained by wetting a solid substrate with a volume ratio of perlite:vermiculite = 2:1 with the liquid root elongation medium.

[0030] Transplanting substrate: perlite: vermiculite = 2:1.

[0031] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0032] Example 1:

[0033] A method for rapid propagation using sandalwood hypocotyls, the steps of which are as follows:

[0034] (1) Inactivation of seed dormancy and disinfection: Mature sandalwood seeds with pericarps were soaked in 200 mg / L gibberellin solution for 24 hours, then rinsed twice with sterile water; on a clean bench, the seeds were soaked in 75% alcohol for 30 s and disinfected with 0.1% HgCl for 10 min, then rinsed five times with sterile water, the pericarps were removed, and the remaining seeds were inoculated onto MS medium containing 0.5 mg / L gibberellin, 30 g / L sucrose, and 7 g / L agar (pH 5.5-5.8) and placed at a culture temperature of 26 ± 2℃ and a light intensity of 60 µmol / m². -2 s -1 The seedlings were cultured under a 14-hour light exposure per day. After 2 weeks, the germinated material was transferred to the same culture medium and cultured for another 3 weeks to obtain 4-5 cm tall sterile sandalwood seedlings.

[0035] (2) Adventitious bud induction: The hypocotyls of sterile sandalwood seedlings were cut into 0.3-0.5 cm segments, and inoculated at an angle of 10-20° onto the adventitious bud induction medium with the seedlings facing upwards. The culture conditions were: culture temperature 26 ± 2℃, light intensity 50 µmol / m². -2 s -1 The light duration was 14 h / d. The adventitious bud induction medium formula contained 0.2 mg 6-BA, 30 g sucrose and 7 g / L agar per liter, with the remainder being MS medium (MS + 0.2 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar), pH 5.5-5.8.

[0036] (3) Subculture of adventitious buds and cultivation of robust seedlings: After 3 weeks, the seedlings were transferred to the same culture medium (adventitious bud induction medium) for further culture. When the differentiated buds were 2-3 cm long, they were cut off and placed on the robust seedling culture medium for 3 weeks. Culture conditions: culture temperature 26±2℃, light intensity 60 µmol / m -2 s -1 The light duration was 14 h / d. The seedling culture medium was: MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.5-5.8.

[0037] (4) Rooting culture: The rootless sandalwood seedlings obtained in step (3) are cut off at the base of their stems and placed in Agrobacterium rhizogenes K599 bacterial solution (exponential growth phase, OD). 600 =0.6) Infected for 10 min, then transferred to rooting medium for root induction. Culture conditions: temperature 26 ± 2℃, light intensity 60 µmol m -2 s -1 The light duration was 14 h / d. The rooting medium was: B5 + 2 mg / L IBA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH 5.5-5.8.

[0038] (5) Root elongation culture: When the adventitious roots have grown to 2-3 mm, they are transferred to the root elongation medium for further culture. Culture conditions: temperature 26 ± 2℃, light intensity 60 µmol / m². -2 s -1 The light duration was 14 h / d. The root elongation medium was: B5 + 1 mg / L IBA + 0.2 mg / L gibberellin + 30 g / L sucrose, pH 5.5-5.8. The root elongation medium matrix was obtained by wetting the solid matrix with a volume ratio of perlite:vermiculite = 2:1.

[0039] (6) Hardening and transplanting: After the roots grow to 4-5 cm, open the cover to harden the seedlings for 1 week, wash off the agar on the surface of the roots, and transplant them into a growth substrate with a volume ratio of perlite:vermiculite = 2:1. Place them in the sandalwood demonstration base for natural light cultivation, and the survival rate of the plants reaches 100%.

[0040] After the above steps, the number of buds induced in each explant can reach 20-25, and the aseptic seedling root induction rate can reach over 70%; the transplant survival rate is 100%, enabling rapid propagation of sandalwood seedlings. Figure 1 ).

Claims

1. A method for rapid propagation using sandalwood hypocotyls, characterized in that, The procedure includes the following steps: Hypocotyls of aseptic sandalwood seedlings are inoculated onto MS medium for adventitious bud induction, supplemented with 0.2-0.5 mg / L 6-BA, 20-30 g / L sucrose, and 6-7 g / L agar for adventitious bud induction and subculture to obtain differentiated buds; the differentiated buds are then inoculated onto MS medium for strengthening seedlings, supplemented with 0.2-0.5 mg / L 6-BA, 0.05-0.1 mg / L NAA, 20-30 g / L sucrose, and 6-7 g / L agar for strengthening seedlings to obtain rootless seedlings; the rootless seedlings are then inoculated onto B5 medium, supplemented with 2-5 mg / L IBA, 0.2-0.5 mg / L NAA, 20-30 g / L sucrose, and 6-7 g / L agar for rooting culture to obtain rooted seedlings; finally, the rooted seedlings are inoculated onto B5 medium, supplemented with 1-5 mg / L IBA, 0.2-1 mg / L 6-BA, 0.05-0.1 mg / L NAA, 20-30 g / L sucrose, and 6-7 g / L agar for rooting culture to obtain rooted seedlings. The root elongation medium substrate obtained by moistening perlite and vermiculite with mg / L gibberellin + 20-30g / L sucrose was used to obtain tissue culture seedlings; the tissue culture seedlings were then transplanted into the substrate.

2. The method according to claim 1, characterized in that, The adventitious bud induction medium was MS + 0.2 mg / L 6-BA + 30 g / L sucrose + 7 g / L agar.

3. The method according to claim 1, characterized in that, The seedling culture medium consisted of MS + 0.5 mg / L 6-BA + 0.05 mg / L NAA + 30 g / L sucrose + 7 g / L agar.

4. The method according to claim 1, characterized in that, The rooting medium consisted of B5 + 2 mg / L IBA + 0.5 mg / L NAA + 30 g / L sucrose + 7 g / L agar.

5. The method according to claim 1, characterized in that, The root elongation medium consisted of B5 + 1 mg / L IBA + 0.2 mg / L gibberellin + 30 g / L sucrose.

6. The method according to claim 1, characterized in that, The culture conditions for adventitious bud induction and subculture, seedling strengthening culture, rooting culture, and root elongation culture were all: temperature 26 ± 2℃, light intensity 40-60 µmol / m³. -2 s -1 Light exposure time is 12-14 h / d.

7. The method according to claim 1, characterized in that, The substrate to which the seedlings are transplanted has a volume ratio of perlite to vermiculite of 2:

1.

8. The method according to claim 1, characterized in that, The hypocotyl is cut into 0.3-0.5 cm segments and inoculated at an angle of 10-20° onto the adventitious bud induction medium with the seedlings facing upwards.

9. The method according to claim 1, characterized in that, The rootless seedlings were cut at the base of their stems, infected with Agrobacterium rhizogenes solution, and then inoculated onto a rooting culture medium.

10. The method according to claim 1, characterized in that, When the adventitious roots of the rooted seedlings are 2-3 mm long, they are inoculated into root elongation medium.

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