A method for in vitro seedling propagation of ginkgo sprouts

Abstract

The present invention relates to the field of rapid plant propagation technology, and specifically discloses a method for propagating ginkgo sucker buds in vitro seedlings. The method comprises the following steps: (1) preparing explant material; (2) inducing fixed buds; (3) inducing adventitious buds; (4) elongating the adventitious buds; and (5) rooting culture to obtain complete plants. The method of the present invention can effectively propagate ginkgo sucker buds in vitro seedlings, thereby reducing resource waste in the breeding process and stably providing the market with high-quality seedlings with stable traits and uniform quality.

Description

Technical Field

[0001] The invention relates to the technical field of rapid plant propagation, and particularly discloses a method for in vitro seedling propagation of ginkgo sucker buds. Background Art

[0002] Ginkgo biloba L., a member of the Ginkgoaceae family and genus Ginkgo, is considered a "living fossil" of the plant kingdom, the oldest remaining relict plant from the Quaternary glacial catastrophe. Ginkgo not only has diverse uses, including ornamental, timber, and ecological protection, but is also an ancient dual-use medicinal and edible plant, playing a crucial role in the production of drugs with antibacterial, antioxidant, anti-cardiovascular, and other biological activities. Ginkgo has broad market application development prospects and is of high research value.

[0003] Ginkgo biloba originated in China and was later introduced to Japan, South Korea and other countries. It is currently mainly distributed in China's temperate and subtropical climate zones, throughout 22 provinces (autonomous regions) in my country, mainly in Zhejiang, Jiangsu and other provinces.

[0004] Ginkgo biloba is a dioecious plant with slow growth. The harvesting of its leaves and fruits is seasonally restricted, and seedlings take 20-30 years to bear fruit, making it difficult to meet market demand. Traditional methods of growing ginkgo seedlings include seedling propagation, grafting, and cuttings, but these methods suffer from low reproduction coefficients, poor genetic stability, and long production cycles.

[0005] Using plant tissue culture technology to propagate Ginkgo biloba seedlings in vitro can shorten the breeding cycle, increase the reproduction coefficient, and maintain the original excellent traits of the variety. However, the in vitro regeneration technology of the above-mentioned tree species is not yet mature. The technical difficulties are: (1) difficulty in obtaining sterile materials; (2) low reproduction coefficient; (3) low survival rate. Summary of the Invention

[0006] In response to actual needs and existing problems, the present invention establishes an in vitro seedling propagation system for ginkgo sucker buds through research on in vitro culture technologies such as establishment of sterile lines, direct adventitious bud induction, and adventitious root induction, in order to quantitatively produce ginkgo seedlings rich in medicinal active ingredients, with a short growth cycle and raw material harvesting not restricted by season.

[0007] The present invention provides a method for in vitro seedling propagation of ginkgo tiller buds, which comprises the following steps:

[0008] (1) Preparation of explant material: 4-6 cm young shoots of ginkgo biloba sprouts were taken as starting explants and sterilized;

[0009] (2) Bud induction: Cut 3-4 cm stem nodes from the sterile explants obtained in step (1), dip the base of the stem nodes in a plant hormone solution of 1-3 mg / L 6-BA + 0.1-0.3 mg / L NAA + 0.5-2 mg / L IBA at an appropriate concentration, and inoculate them into a bud induction medium containing WPM0, 1 / 2MS, YX + 0-1 mg / L 6-BA + 0-0.1 mg / L NAA;

[0010] (3) Adventitious bud induction: Cut a stem node from the elongated adventitious bud obtained in step (2) and inoculate it into adventitious bud induction medium, which is YX + 0.5-3 mg / L 6-BA + 0.01-0.05 mg / L KT + 0.2 g / L proline, and culture for 8-10 weeks, transferring every 3-5 weeks;

[0011] (4) Adventitious bud elongation: The adventitious buds obtained in step (3) were transferred to an adventitious bud elongation medium containing YX + 0.5-2 mg / L 6-BA + 0.05-0.2 mg / L NAA + 0.01-0.05 mg / L GA3 for 3-5 weeks;

[0012] (5) Rooting culture: The buds elongated in step (4) are inoculated into a rooting medium and cultured until roots are formed to obtain complete plants;

[0013] The culture medium in the above steps was added with 30 g / L sucrose and 5.0 g / L agar powder, and the pH value of the culture medium was adjusted to 5.80.

[0014] In specific implementation, the culture conditions are 25±2°C, light intensity 2000-3000lx, and light 16h / d.

[0015] Furthermore, the method further includes the step (6) of hardening and transplanting: selecting the bottle seedlings that have taken root and are growing vigorously in step (5), hardening them and then transplanting them.

[0016] In a preferred embodiment, the disinfection in step (1) is specifically as follows: cleaning the surface with detergent, and then rinsing under running water for 2 hours, placing the washed material in a sterile conical flask, transferring it to a clean bench, treating it with 75% ethanol solution for 30-60 seconds, treating it with PB disinfectant containing a mixture of 50% plant tissue culture antibacterial agent PPM and 1% benzalkonium bromide for 6-10 minutes, and finally rinsing it with sterile water for 3-5 times.

[0017] In another preferred embodiment, the bud induction medium in step (2) is WPM0, 1 / 2MS, YX+0-0.5 mg / L 6-BA+0-0.05 mg / L NAA.

[0018] In a further preferred embodiment, the adventitious bud induction medium in step (3) is YX+0.5-2 mg / L 6-BA+0.01-0.03 mg / L KT+0.2 g / L proline.

[0019] More preferably, the elongation medium in step (4) is YX+0.5-1 mg / L 6-BA+0.05-0.1 mg / L NAA+0.01-0.03 mg / L GA3.

[0020] In one embodiment, the rooting medium in step (5) is WPM+0.1-2 mg / L IBA+0.2% AC; preferably, the rooting medium in step (5) is WPM+0.5-1.5 mg / L IBA+0.2% AC.

[0021] In one embodiment, the seedling hardening method in step (6) is as follows: open the culture bottle cap, inject 0.5-1 cm of clean water, place it at room temperature and under natural light, let it stand for 2-3 days, take out the bottle seedlings, wash the culture medium attached to the base, and prepare for transplanting;

[0022] In another embodiment, the transplanting matrix in step (6) is peat soil: perlite: vermiculite = 3:1:1. Before transplanting, the matrix and the pot are immersed in water to make it completely moistened. After transplanting, the matrix is ​​bagged to keep it moist. When the humidity is not enough, use a spray bottle to spray the surface of the plant. After the seedlings grow strong, remove the bag and maintain it normally.

[0023] The preparation method of the Ginkgo biloba culture medium (YX) is as follows: prepare the basic culture medium according to Table 1, pH 5.80, and sterilize at 121° C. for 15-25 minutes.

[0024] Table 1 List of components of Ginkgo biloba culture medium (YX)

[0025] serial number Element Concentration (mg / l) 1 <![CDATA[KNO3]]> 1425 2 <![CDATA[NH4NO3]]> 1237.5 3 MgSO4·7H2O 370 4 <![CDATA[CaCl2]]> 249.2 5 <![CDATA[KH2PO4]]> 170 6 <![CDATA[MnSO4]]> 16.9 7 <![CDATA[ZnSO4·7H2O]]> 8.6 8 <![CDATA[H3BO3]]> 6.2 9 KI 0.83 10 <![CDATA[CuSO4·5H2O]]> 0.25 11 <![CDATA[Na2MoO4·2H2O]]> 0.025 12 <![CDATA[CoCl2·6H2O]]> 0.025 13 <![CDATA[Na2·EDTA·H2O]]> 37.3 14 <![CDATA[FeSO4·7H2O]]> 27.9 15 Glycine 2 16 Thiamine hydrochloride 0.1 17 Pyridoxine hydrochloride 0.5 18 niacin 0.5 19 Inositol 100

[0026] The present invention, through research and exploration, determines the key links of in vitro seedling propagation of ginkgo sucker buds and obtains an effective propagation method, thereby reducing the waste of resources in the breeding process and stably providing the market with high-quality seedlings with stable traits and uniform quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Ginkgo biloba sprouts and young branches.

[0028] Figure 2 Ginkgo buds are formed.

[0029] Figure 3 Adventitious bud induction stage I (30 days).

[0030] Figure 4 Adventitious bud induction stage II (70 days).

[0031] Figure 5 Induction of adventitious bud elongation.

[0032] Figure 6 Front view of rooting ginkgo seedlings.

[0033] Figure 7 Bottom view of a rooting ginkgo seedling. DETAILED DESCRIPTION

[0034] The present invention is further illustrated below through detailed descriptions of specific embodiments, which, however, are not intended to limit the present invention and are merely illustrative.

[0035] (1) Preparation of Ginkgo biloba culture medium (YX)

[0036] Prepare the basic culture medium according to Table 1, pH 5.8, and sterilize at 121°C for 15-25 minutes. Use this medium for both bud induction and adventitious bud elongation.

[0037] Table 1 List of components of Ginkgo biloba culture medium (YX)

[0038]

[0039]

[0040] (2) Preparation of explant materials

[0041] Take 4-6cm young shoots of ginkgo as starting explants ( Figure 1 ), clean the surface with detergent, then rinse under running water for 2 hours. The cleaned material was placed in a sterile Erlenmeyer flask and transferred to a clean bench. The material was treated with 75% ethanol solution for 30-60 seconds, followed by PB disinfectant (a mixture of 50% plant preservative mixture (PPM) and 1% benzyldodecyldimethylammonium bromide) for 4-12 minutes, and finally rinsed 3-5 times with sterile water. The experimental results (Table 2) showed that too short a PB disinfectant treatment time resulted in a high contamination rate, while too long a treatment time resulted in a low contamination rate but also a reduced survival rate. A PB disinfectant treatment time of 6-10 minutes was most effective, with the combination of 75% ethanol disinfection for 40 seconds and PB disinfectant treatment for 7 minutes achieving the highest disinfection effect, with a survival rate of 88.3%.

[0042] Table 2 Effects of different disinfection methods on the vigor of Ginkgo biloba explants

[0043] Disinfection plan Pollution rate (%) Survival rate (%) 75% ethanol 30 seconds + PB 12 minutes 3.3 78.3 75% ethanol 30s + PB 10min 5.0 85.0 75% ethanol 40s + PB 7min 6.7 88.3 75% ethanol 60s + PB 6min 18.3 55.0 75% ethanol 60s + PB 4min 41.7 48.3

[0044] (3) Bud induction

[0045] The obtained sterile explants were cut into 3-4 cm stem nodes, and the base was dipped in a plant hormone solution of 1-3 mg / L 6-BA + 0.1-0.3 mg / L NAA + 0.5-2 mg / L IBA at an appropriate concentration, and inoculated into a bud induction medium of WPM0, 1 / 2MS, YX + 0-1 mg / L 6-BA + 0-0.1 mg / L NAA. The experimental results (Table 3) showed that the bud induction effect of the medium without hormone addition was higher than that of the medium with hormone addition, and the bud induction rate was higher than that of the medium without hormone addition. WPM0, 1 / 2MS, YX + 0-0.5 mg / L 6-BA + 0-0.05 mg / L NAA were all able to induce buds, and the highest bud induction rate reached 100%. Axillary buds began to sprout about 2 days after inoculation ( Figure 2 ).

[0046] Table 3 Effects of different culture media and hormones on bud induction

[0047]

[0048]

[0049] (4) Adventitious bud induction

[0050] The obtained fixed buds were cut into a stem node and inoculated into a fixed bud induction medium containing YX+0.5-3mg / L 6-BA+0.01-0.05mg / L KT+0.2g / L proline. The culture was continued for 8-10 weeks and the transplant was performed every 3-5 weeks. The experimental results showed (Table 4) that fixed buds did not differentiate into adventitious buds in YX medium without hormone addition. The addition of different cytokinins to YX medium was beneficial to the induction of Ginkgo adventitious buds. After about 3 weeks of induction, the adventitious buds began to differentiate, and after about 4 weeks of culture, a certain number of adventitious buds were induced ( Figure 3 ), after 8-10 weeks of culture, a large number of adventitious buds were induced ( Figure 4 ), wherein the culture medium supplemented with 0.5-2 mg / L 6-BA + 0.01-0.03 mg / L KT + 0.2 g / L proline is preferred, the adventitious bud differentiation rate reaches 93.33%, and the average number of buds reaches 23.67 per explant.

[0051] Table 4 Effects of different plant hormone combinations on adventitious bud induction

[0052]

[0053] (5) Adventitious bud elongation

[0054] The induced adventitious buds were transferred to the adventitious bud elongation medium. The culture medium used YX as the basic culture medium, and added 0.5-2mg / L 6-BA+0.05-0.2mg / L NAA+0.01-0.05mg / LGA3, and cultured for 3-5 weeks. The results showed (Table 5): Different combinations of plant hormones can induce the elongation of ginkgo adventitious buds, but the induction effects of different combinations are different. Among them, the adventitious bud elongation medium with the combination of 0.5-1mg / L6-BA+0.05-0.1mg / L NAA+0.01-0.03mg / LGA3 is preferred, and the adventitious bud elongation rate reaches 85.71% ( Figure 5 ).

[0055] Table 5 Effects of different plant hormone combinations on adventitious bud elongation

[0056]

[0057] (6) Rooting culture

[0058] The elongated shoots were inoculated into rooting medium. The medium used WPM as the basic medium and was supplemented with 0.1-2 mg / L IBA + 0.2% AC. The results showed (Table 6) that adventitious roots began to sprout after 7-10 days of addition of 0.5-1.5 mg / L IBA + 0.2% AC, with an adventitious root induction rate of 91.67% and an average number of rooted shoots of 5 ( Figure 6 、 Figure 7 ).

[0059] Table 6 Effect of IBA on adventitious root induction

[0060]

[0061] (7) Hardening and transplanting

[0062] Select the rooted and strong ginkgo seedlings from (6) for hardening. The hardening method is as follows: open the culture bottle cap, inject 0.5-1cm of clean water, place it at room temperature and natural light, and let it stand for 2-3 days. Then take out the seedlings, wash the culture medium attached to the base, and prepare for transplanting. The transplanting medium is peat soil: perlite: vermiculite = 3:1:1. Before transplanting, immerse the medium and the pot in water to make it completely wet. After transplanting, bag the plant to keep it moist. If the humidity is insufficient, use a spray bottle to spray the plant surface. After the seedlings grow strong, remove the bag and maintain normal maintenance.

Claims

1. A method for in vitro seedling propagation of Ginkgo biloba L. tiller buds, comprising the following steps: (1) Preparation of explant material: 4-6 cm young shoots of ginkgo biloba were taken as starting explants and disinfected; the disinfection method was: cleaning the surface with detergent, then rinsing under running water for 2 hours, transferring the washed material to a clean bench, treating with 75% ethanol solution for 30-60 seconds, treating with PB disinfectant containing a mixture of 50% plant tissue culture antibacterial agent PPM and 1% benzalkonium bromide for 7-12 minutes, and finally rinsing with sterile water 3-5 times; (2) Bud induction: Cut 3-4 cm stem nodes from the sterile explants obtained in step (1), dip the base of the stem nodes in a suitable concentration of 1-3 mg / L 6-BA + 0.1-0.3 mg / L NAA + 0.5-2 mg / L IBA plant hormone solution, and inoculate them into a bud induction medium, which is a bud induction medium of YX + 0-0.5 mg / L 6-BA + 0-0.03 mg / L NAA; (3) Adventitious bud induction: Cut a stem node from the elongated adventitious bud obtained in step (2) and inoculate it into adventitious bud induction medium, which is YX + 0.5-2 mg / L 6-BA + 0.01-0.03 mg / L KT + 0.2 g / L proline, and culture for 8-10 weeks; (4) Adventitious bud elongation: The adventitious buds obtained in step (3) were transferred to an adventitious bud elongation medium containing YX + 0.5-1 mg / L 6-BA + 0.05-0.1 mg / L NAA + 0.01-0.03 mg / L GA3 for 3-5 weeks; (5) Rooting culture: The elongated buds in step (4) are inoculated into a rooting medium and cultured until roots form to obtain complete plants; the rooting medium is WPM + 0.5-1.5 mg / LIBA + 0.2% AC; The culture medium in the above steps was supplemented with 30 g / L sucrose and 5.0 g / L agar powder, and the pH value of the culture medium was adjusted to 5.80; The components of the YX culture medium are as follows:

2. The in vitro seedling propagation method of ginkgo tiller buds according to claim 1, wherein: The culture conditions are 25±2° C., light intensity 2000-3000 lx, and light intensity 16 h / d.

3. The in vitro seedling propagation method of ginkgo tiller buds according to claim 1, wherein: The method also includes step (6) of hardening and transplanting the seedlings: selecting the bottle seedlings that have taken root and are growing vigorously in step (5) to harden them and then transplant them.

4. The in vitro seedling propagation method of ginkgo tiller buds according to claim 1, wherein: The bud induction medium in step (2) is YX+0-0.5mg / L 6-BA+0-0.05mg / L NAA, and the inoculation is carried out until the axillary buds begin to sprout.

5. The in vitro seedling propagation method of ginkgo tiller buds according to claim 1, wherein: The adventitious bud induction medium in step (3) is YX+0.5-2 mg / L 6-BA+0.01-0.03 mg / L KT+0.2 g / L proline.

6. The in vitro seedling propagation method of ginkgo sucker buds according to claim 1, wherein: During the culture in step (4), the cells are transferred every 3-5 weeks.

7. The in vitro seedling propagation method of ginkgo sucker buds according to claim 1, wherein: The elongation medium in step (4) is YX+0.5-1 mg / L6-BA+0.05-0.1 mg / LNAA+0.01-0.03 mg / L GA3.

8. The in vitro seedling propagation method of ginkgo sucker buds according to claim 1, wherein: The rooting medium in step (5) is WPM+0.5-1.5 mg / LIBA+0.2% AC.

9. The in vitro seedling propagation method of ginkgo tiller buds according to claim 2, wherein: The method for hardening the seedlings in step (6) is as follows: open the culture bottle cap, inject 0.5-1cm of clean water, place it at room temperature and under natural light, let it stand for 2-3 days, take out the seedlings from the bottle, wash the culture medium attached to the base, and prepare for transplanting.

10. The in vitro seedling propagation method of ginkgo sucker buds according to claim 2, wherein: The transplanting medium in step (6) is peat soil: perlite: vermiculite = 3:1:

1. Before transplanting, immerse the medium and the pot in water to make it completely moist. After transplanting, bag the plant to keep it moist. If the humidity is not enough, use a spray bottle to spray the plant surface. After the seedlings grow strong, remove the bag and maintain them normally.

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