Tissue rapid propagation method of sandalwood

Abstract

The invention discloses a tissue rapid propagation method of sandalwood, the method is as follows: sandalwood stem segments are inserted into a sandalwood germination culture medium for germination culture, then inserted into a sandalwood rooting culture medium for rooting culture to obtain sandalwood plants with cluster buds and roots, the sandalwood germination culture medium comprises an MS basal culture medium, 5-9g / L agar, 25-35g / L sucrose, 40-60ml / L coconut juice and 0.18-0.20 mg / L 6-BA, the sandalwood rooting culture medium comprises a 1 / 2 MS basal culture medium, 5 to 9 g / L of agar, 10 to 20 g / L of sucrose, 40 to 60 ml / L of coconut juice, 0.9 to 1.1 mg / L of IAA, 0.07 to 0.09 mg / L of KT and 0.00018 to 0.00022 mg / L of TDZ. According to the method, regenerated seedlings of sandalwood can be efficiently obtained and can be used for forestry production.

Description

[0001] This application is a divisional application of a patent application with an application date of August 8, 2023, application number 202310992839.7, and invention name “A method for rapid tissue propagation of sandalwood”. Technical Field

[0002] The invention belongs to the field of plant tissue culture and relates to a rapid tissue propagation method of sandalwood. Background Art

[0003] Sandalwood (Santalum album) is a type of wood in the genus Santalum. It is an evergreen small tree, 6 to 9 meters tall, with parasitic roots. It is distributed in India, Malaysia, Australia, Indonesia and other places. It is also cultivated in Taiwan, China. It can be harvested all year round. The sandalwood tree is called the "golden tree" because almost its entire body is a treasure. After harvesting, it is cut into small pieces and the sapwood is removed (the remaining scraps when making sandalwood utensils can also be used). Sandalwood also has important medicinal value.

[0004] Artificial propagation of sandalwood will be beneficial to quickly replenish sandalwood seedlings, but the efficiency of sandalwood propagation in existing technologies is low and cannot fully meet the needs of forest reproduction. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the invention provides a method for rapid tissue propagation of sandalwood. The method comprises the following steps: inserting a sandalwood stem segment into a sandalwood budding medium for budding culture to obtain a sandalwood plant with clustered buds; inserting the sandalwood plant with clustered buds into a sandalwood rooting medium for rooting culture to obtain a sandalwood plant with clustered buds and roots. The sandalwood budding medium comprises an MS basic medium, 5-9 g / L agar, 25-35 g / L sucrose, 40-60 ml / L coconut milk and 0.18-0.20 mg / L 6-BA; and the sandalwood rooting medium comprises 1 / 2 MS basic medium, 5-9 g / L agar, 10-20 g / L sucrose, 40-60 ml / L coconut milk, 0.9-1.1 mg / L IAA, 0.07-0.09 mg / L KT and 0.00018-0.00022 mg / LTDZ.

[0006] In some embodiments, the sandalwood budding medium includes MS basal medium, 7g / L agar, 30g / L sucrose, 50ml / L coconut milk and 0.2mg / L 6-BA, and the sandalwood rooting medium includes 1 / 2MS basal medium, 7g / L agar, 15g / L sucrose, 50ml / L coconut milk, 1.0mg / L IAA, 0.08mg / L KT and 0.0002mg / L TDZ.

[0007] In some embodiments, the pH of the sandalwood budding medium is 5.6-6.0, and the pH of the sandalwood rooting medium is 5.6-6.0.

[0008] In some embodiments, the sandalwood stem segment contains 1, 2 or 3 axillary buds.

[0009] In some embodiments, the sandalwood stem segment is from a young branch of Santalum album.

[0010] In some embodiments, the sandalwood stem segment is a sterilized sandalwood stem segment.

[0011] In some embodiments, the disinfection steps of the sandalwood stem segment are: rinse with clean water for 20-60 minutes, disinfect in 70-80v / v% alcohol for 15-25 seconds, wash with deionized water for 1-3 times, disinfect in 1.8-2.2w / v% sodium hypochlorite aqueous solution for 4-6 minutes, and wash with deionized water for 3-5 times.

[0012] In some embodiments, the culture conditions for the bud culture are: culture temperature 20-28°C, culture light time 14-18 hours per day, culture light intensity 1500-3000lx, and culture time 15-20 days; the culture conditions for the root culture are: culture temperature 21-25°C, culture light time 14-18 hours per day, light intensity 2000-3000lx, and culture time 15-20 days.

[0013] In some embodiments, the method further comprises transplanting the sandalwood plant having clustered shoots and roots into soil to obtain a sandalwood plant growing in soil.

[0014] In some embodiments, a culture solution is irrigated into the soil to maintain the soil at 60-80% humidity for 7-10 days, wherein the culture solution includes 1 / 2MS basal medium, 10-20 g / L sucrose, 40-60 ml / L coconut water, 0.9-1.1 mg / L IAA, 0.07-0.09 mg / L KT and 0.00018-0.00022 mg / LTDZ.

[0015] In some embodiments, the MS basal medium includes 600 mg / L ammonium nitrate, 530 mg / L calcium nitrate tetrahydrate, 1900 mg / L potassium nitrate, 370 mg / L magnesium sulfate heptahydrate, 200 mg / L potassium dihydrogen phosphate, 0.025 mg / L copper sulfate pentahydrate, 6.2 mg / L boric acid, 18.9 mg / L manganese sulfate monohydrate, 0.25 mg / L sodium molybdate dihydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.83 mg / L potassium iodide, 27.8 mg / L ferrous sulfate heptahydrate, 37.26 mg / L Na2-EDTA, 2.0 mg / L glycine, 100 mg / L inositol, 0.50 mg / L niacin, 0.1 mg / L vitamin B1 and 0.5 mg / L vitamin B6.

[0016] In some embodiments, the 1 / 2MS basal medium includes: 950 mg / L potassium nitrate, 166.1 mg / L calcium chloride, 825 mg / L ammonium nitrate, 90.35 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 0.025 mg / L copper sulfate, 6.2 mg / L boric acid, 16.9 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 8.6 mg / L zinc sulfate, 0.025 mg / L cobalt chloride, 0.83 mg / L potassium iodide, 27.8 mg / LFeSO4·7H2O, 37.26 mg / LNa2-EDTA·2H2O, 2.0 mg / L glycine, 100 mg / L inositol, 0.50 mg / L niacin, 0.1 mg / L vitamin B1 and 0.5 mg / L vitamin B6. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Photo of the rapid propagation plant of Sandalwood.

[0018] Figure 2 Photo of the rapid propagation plant of Sandalwood. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present invention more clear, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0020] Example 1: Rapid tissue propagation of sandalwood

[0021] 1. Preparation of culture medium

[0022] (1) Basic culture medium

[0023] MS basal culture medium includes macroelements: 600mg / L ammonium nitrate, 530mg / L calcium nitrate tetrahydrate, 1900mg / L potassium nitrate, 370mg / L magnesium sulfate heptahydrate, 200mg / L potassium dihydrogen phosphate; trace elements: 0.025mg / L copper sulfate pentahydrate, 6.2mg / L boric acid, 18.9mg / L manganese sulfate monohydrate, 0.25mg / L sodium molybdate dihydrate, 8.6mg / L zinc sulfate heptahydrate, 0.025mg / L cobalt chloride hexahydrate, 0.83mg / L potassium iodide; iron salts: 27.8mg / L ferrous sulfate heptahydrate, 37.26mg / L Na2-EDTA; organic components: 2.0mg / L glycine, 100mg / L inositol, 0.50mg / L niacin, 0.1mg / L vitamin B1, 0.5mg / L vitamin B6.

[0024] 1 / 2MS basal culture medium includes macroelements: 950mg / L potassium nitrate, 166.1mg / L calcium chloride, 825mg / L ammonium nitrate, 90.35mg / L magnesium sulfate, 85mg / L potassium dihydrogen phosphate; trace elements: 0.025mg / L copper sulfate, 6.2mg / L boric acid, 16.9mg / L manganese sulfate, 0.25mg / L sodium molybdate, 8.6mg / L zinc sulfate, 0.025mg / L cobalt chloride, 0.83mg / L potassium iodide; iron salts: 27.8mg / LFeSO4·7H2O, 37.26mg / L Na2-EDTA·2H2O; organic components: 2.0mg / L glycine, 100mg / L inositol, 0.50mg / L niacin, 0.1mg / L vitamin B1, 0.5mg / L vitamin B6.

[0025] (2) Sandalwood Bud Culture Medium

[0026] MS medium was used as the basic medium, and the formula also included 7 g / L agar, 30 g / L sucrose, and 50 ml / L coconut milk. The hormone components and contents in the medium are shown in Table 1. The pH was 5.8.

[0027] Coconut juice was purchased from Blue Ridge Beverage Company, Inc.

[0028] (3) Sandalwood rooting medium

[0029] The basal medium is 1 / 2MS medium, and the formula also includes 7g / L agar, 15g / L sucrose, and 50ml / L coconut milk. The hormone components and contents in the medium are shown in Table 2. The pH is 5.8.

[0030] 2. Obtaining and sterilizing explants

[0031] (I) Selection and treatment of explants

[0032] Take young branches of sandalwood grown in the field, remove the leaves, clean the dust, soil and other impurities on the surface of the branches, and rinse them under tap water for 30-40 minutes.

[0033] (II) Disinfection of explants

[0034] 1. Prepare 100 ml of 75 v / v% alcohol, add 1-2 drops of Tween-20, shake well and set aside; prepare 100 ml of 2 w / v% (2 g / 100 mL) sodium hypochlorite aqueous solution, add 1-2 drops of Tween-20 and set aside;

[0035] 2. Cut the branches into segments of moderate length, ensuring that each branch contains an axillary bud, and the axillary bud is at least 0.5 cm away from the lower end of the stem segment.

[0036] 3. Place the cut stem segments into the 75v / v% alcohol prepared in advance for disinfection for 20 seconds, and shake slowly.

[0037] 4. Pour out the alcohol and add sterilized deionized water to rinse twice.

[0038] 5. Add the prepared sodium hypochlorite solution and disinfect for 5 minutes, shaking slowly to achieve better disinfection effect.

[0039] 6. Pour out the sodium hypochlorite solution and wash with sterile deionized water for 4-5 times until the smell of sodium hypochlorite is gone. Place the stem segments on sterilized filter paper to absorb excess water and set aside.

[0040] 3. Cluster bud induction

[0041] For each culture dish of the sandalwood sprouting medium corresponding to the hormone in Table 1, 2-3 sterilized stem segments were inoculated for induction culture. Culture conditions: temperature 24°C, photoperiod 16 hours light / 8 hours dark, light intensity 1500-3000lx, culture time 15-20d. The culture results are shown in the last two columns of Table 1, and 10 culture dishes were cultured in each culture medium.

[0042] Table 1

[0043]

[0044]

[0045] It can be seen that the culture medium No. 3 in Table 1 is the best and is called the optimal culture medium. Figure 1 and Figure 2 The figures show the sandalwood plants with clustered shoots obtained by culture on medium No. 3.

[0046] 4. Rooting Induction

[0047] Transplant the vigorous sandalwood plants with clustered buds cultured in medium No. 3 in step 3 into the sandalwood rooting medium with hormones shown in Table 2, 1-2 plants per bottle, and culture for 15-20 days under the conditions of culture temperature 23±2°C, light / dark cycle 16 / 8h / d, and light intensity 2000-3000lx, and observe the rooting condition of the seedlings.

[0048] Table 2

[0049] serial number IAA(mg / L) KT(mg / L) TDZ(mg / L) Root situation 1 0.5 0.02 0.005 Almost no rooting 2 0.8 0.06 0.001 Slow rooting 3 1.0 0.08 0.0002 Fastest rooting 4 1.2 1.0 0.0003 Slow rooting 5 1.4 1.2 0.0004 Slowest rooting 6 1.6 1.5 0.0005 The production of rootlessness

[0050] 5. Transplanting

[0051] The formula of sandalwood rooting medium No. 3 without agar was used as the culture medium for transplanting.

[0052] The sandalwood plants with clustered buds and roots obtained by culture in No. 3 sandalwood rooting medium were transplanted into soil, the soil composition was: vermiculite: nutrient soil: moss (1:1:1 weight ratio), the aforementioned culture solution was added to the soil, and the amount added was to keep the soil humidity at 70%. The temperature was maintained at 20-28°C, the light and dark cycle was 16h light / 8h dark, the light intensity was 1500-3000lx, and the culture solution was stopped from being irrigated after 7-10 days of culture, and ordinary clean water was used for irrigation, and the light and temperature and humidity control were stopped, and routine management was entered.

[0053] Therefore, the method of the present invention can achieve the rapid propagation of sandalwood.

[0054] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

Claims

1. A method for rapid tissue propagation of sandalwood, comprising: inserting a sandalwood stem segment into a sandalwood budding medium for budding culture to obtain a sandalwood plant with clustered buds; inserting the sandalwood plant with clustered buds into a sandalwood rooting medium for rooting culture to obtain a sandalwood plant with clustered buds and roots, wherein the sandalwood budding medium is composed of MS basal medium, 5-9g / L agar, 25-35g / L sucrose, 40-60ml / L coconut milk and 0.18-0.20mg / L 6-BA; and the sandalwood rooting medium is composed of 1 / 2MS basal medium, 5-9g / L agar, 10-20g / L sucrose, 40-60ml / L coconut milk, 0.9-1.1mg / L IAA, 0.07-0.09mg / L KT and 0.00018-0.00022mg / L TDZ; the pH of the sandalwood bud culture medium is 5.6-6.0, and the pH of the sandalwood root culture medium is 5.6-6.0; the culture conditions of the bud culture are: culture temperature 20-28°C, culture light time 14-18 hours per day, culture light intensity 1500-3000lx, and culture time 15-20 days; the culture conditions of the root culture are: culture temperature 21-25°C, culture light time 14-18 hours per day, light intensity 2000-3000lx, and culture time 15-20 days.

2. The method according to claim 1, characterized in that The sandalwood budding medium consists of an MS basal medium, 7 g / L agar, 30 g / L sucrose, 50 ml / L coconut milk and 0.2 mg / L 6-BA, and the sandalwood rooting medium consists of a 1 / 2 MS basal medium, 7 g / L agar, 15 g / L sucrose, 50 ml / L coconut milk, 1.0 mg / L IAA, 0.08 mg / L KT and 0.0002 mg / L TDZ.

3. The method according to claim 1 or 2, characterized in that The sandalwood stem segment contains 1, 2 or 3 axillary buds.

4. The method according to any one of claims 1 to 3, characterized in that The sandalwood stem segments are from sandalwood twigs.

5. The method according to claim 4, characterized in that The sandalwood stem segments are sterilized sandalwood stem segments.

6. The method according to claim 5, characterized in that The disinfection steps of sandalwood stem segments are as follows: rinse with clean water for 20-60 minutes, disinfect in 70-80v / v% alcohol for 15-25s, wash with deionized water 1-3 times, disinfect in 1.8-2.2w / v% sodium hypochlorite aqueous solution for 4-6 minutes, and wash with deionized water 3-5 times.

7. The method according to any one of claims 1 to 6, characterized in that The method further comprises transplanting the sandalwood plant with clustered buds and roots into soil to obtain a sandalwood plant grown in soil.

8. The method according to claim 7, characterized in that The soil was irrigated with a culture solution to maintain the soil at 60-80% humidity for 7-10 days. The culture solution consisted of 1 / 2MS basal medium, 10-20 g / L sucrose, 40-60 ml / L coconut milk, 0.9-1.1 mg / L IAA, 0.07-0.09 mg / L KT and 0.00018-0.00022 mg / L TDZ.

9. The method according to any one of claims 1 to 8, characterized in that The MS basal medium includes 600 mg / L ammonium nitrate, 530 mg / L calcium nitrate tetrahydrate, 1900 mg / L potassium nitrate, 370 mg / L magnesium sulfate heptahydrate, 200 mg / L potassium dihydrogen phosphate, 0.025 mg / L copper sulfate pentahydrate, 6.2 mg / L boric acid, 18.9 mg / L manganese sulfate monohydrate, 0.25 mg / L sodium molybdate dihydrate, 8.6 mg / L zinc sulfate heptahydrate, 0.025 mg / L cobalt chloride hexahydrate, 0.83 mg / L potassium iodide, 27.8 mg / L ferrous sulfate heptahydrate, 37.26 mg / L Na2-EDTA, 2.0 mg / L glycine, 100 mg / L inositol, 0.50 mg / L nicotinic acid, 0.1 mg / L vitamin B1 and 0.5 mg / L vitamin B6.

10. The method according to any one of claims 1 to 8, characterized in that The 1 / 2MS basic culture medium includes 950 mg / L potassium nitrate, 166.1 mg / L calcium chloride, 825 mg / L ammonium nitrate, 90.35 mg / L magnesium sulfate, 85 mg / L potassium dihydrogen phosphate, 0.025 mg / L copper sulfate, 6.2 mg / L boric acid, 16.9 mg / L manganese sulfate, 0.25 mg / L sodium molybdate, 8.6 mg / L zinc sulfate, 0.025 mg / L cobalt chloride, 0.83 mg / L potassium iodide, 27.8 mg / L FeSO4·7H2O, 37.26 mg / L Na2-EDTA·2H2O, 2.0 mg / L glycine, 100 mg / L inositol, 0.50 mg / L nicotinic acid, 0.1 mg / L vitamin B1 and 0.5 mg / L vitamin B6.

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