Method for breaking dormancy cycle of top bud of tissue culture seedling and keeping ginkgo biloba tissue culture seedling

Abstract

The application relates to a method for breaking the dormancy cycle of the top bud of a tissue culture seedling and keeping the ginkgo biloba tissue culture seedling, belonging to the technical field of plant tissue culture, which combines exogenous application and medium addition, solves the problems of top bud dormancy and slow growth in the ginkgo biloba tissue culture rapid propagation by applying GA3 to the top bud which no longer elongates, realizes the cycle culture of the ginkgo biloba tissue culture seedling, and provides the tissue culture seedling continuously without repeated explant disinfection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant culture, and particularly relates to a method for breaking dormancy cycle of top buds of tissue culture seedlings and keeping ginkgo biloba tissue culture seedlings. BACKGROUND

[0002] Ginkgo biloba is a rare tree species of Mesozoic relic, is a special product of China, is the only species of ancient ginkgo plants surviving on the earth, and appeared earliest in the Carboniferous period 3.45 billion years ago, and thus is regarded as the "world's first living fossil" and "the giant panda in the plant kingdom". Ginkgo biloba leaf tastes bitter and is cool in nature, and ginkgo leaf is flat in nature and tastes sweet and bitter, can activate blood stasis, dredge meridians, condense lung qi, relieve asthma and cough, stop leucorrhea and reduce blood lipids. Ginkgo nut is flat in nature, tastes sweet, slightly bitter and astringent, has small toxicity, and belongs to the lung meridian. It has the functions of condensing lung and relieving asthma, stopping leucorrhea and reducing urine, and can "disinfect and kill insects" when used externally.

[0003] With the increasing demand for ginkgo biloba in the market, the contradiction between the demand and the decreasing resources of ginkgo biloba is gradually intensified. Ginkgo biloba belongs to gymnosperms and grows slowly, and currently there are some ginkgo biloba rapid propagation technologies, but one of the problems to be solved is the dormancy of top buds in the process of tissue culture, which is one of the reasons why ginkgo biloba tissue culture is not as fast as other trees such as poplar and phyllostachys edulis. In the process of ginkgo biloba tissue culture and rapid propagation, whether through the method of callus induction regeneration or the method of adventitious bud induction, the ginkgo biloba seedlings regenerated will have problems such as top bud dormancy, stem unable to elongate, and leaf abscission, so that the tissue culture seedlings cannot be preserved for a long time, and if the tissue culture and rapid propagation is to be continued, the ginkgo biloba seedlings need to be obtained from the natural environment, and the external explants need to be disinfected again, which greatly limits the market promotion and application of ginkgo biloba tissue culture and rapid propagation technology. SUMMARY

[0004] The present application provides a method for breaking dormancy cycle of top buds of tissue culture seedlings and keeping ginkgo biloba tissue culture seedlings, which combines exogenous application and medium addition, solves the problems of top bud dormancy and slow growth in ginkgo biloba tissue culture and rapid propagation, realizes the cycle culture of ginkgo biloba tissue culture seedlings, and provides ginkgo biloba tissue culture seedlings without repeated disinfection of external explants.

[0005] The present application is realized by the following technical scheme:

[0006] A method for breaking dormancy cycle of top buds of tissue culture seedlings and keeping ginkgo biloba tissue culture seedlings, and the specific steps are as follows:

[0007] First step, external explant disinfection: select ginkgo biloba seedlings growing healthily and free of diseases in March-April, take the young branches as external explants, and disinfect the external explants;

[0008] Second step, adventitious bud induction: divide the disinfected external explants into 3-5 cm, containing 2-3 nodes, and subculture to adventitious bud induction medium;

[0009] The third step is to take the new induced adventitious buds from the mother plant and subculture them to the elongation medium for culture;

[0010] The fourth step is to break the dormancy of the terminal bud by applying GA3 to the terminal bud which has stopped elongation. The application method is to peel the scales of the Ginkgo terminal bud and add 11 mM of GA3 to the terminal bud. 20 uL is added each time and the addition is performed once every 3 days for 4 times. After 20 days, the terminal bud breaks dormancy and starts to grow leaves and resume growth.

[0011] The fifth step is to continue the induction of adventitious buds by repeating the second to fourth steps described above.

[0012] The sixth step is to root by subculturing the 5-10 cm Ginkgo stem segments to the rooting medium.

[0013] As one of the preferred embodiments, the adventitious bud induction medium formula is MS+WPM+0.02 mg / L NAA+0.2 mg / L 6BA+0.5 g / L enzymatic hydrolysis casein+30 g / L sucrose+2 g / L activated carbon+7.8 g / L agar, pH 5.8-6.0.

[0014] As one of the preferred embodiments, the elongation culture formula is MS+WPM+0.2 mg / L 6BA+0.5 g / L enzymatic hydrolysis casein+30 g / L sucrose+2 g / L activated carbon+7.8 g / L agar, pH 5.8-6.0.

[0015] As one of the preferred embodiments, the rooting medium formula is 1 / 2MS+1 / 2WPM+5 mg / L IBA+0.5 mg / L NAA+20 g / L sucrose+2 g / L activated carbon+7.8 g / L agar, pH 5.8-6.0.

[0016] As one of the preferred embodiments, the culture conditions of the Ginkgo seedlings after rooting are as follows: the whole propagation process is performed under the culture conditions of temperature 25-28℃, light intensity 6000-9000 Lx, and photoperiod 16 h light and 8 h darkness.

[0017] Compared with the prior art, the method of the present application solves the problems of terminal bud dormancy and slow growth in Ginkgo tissue culture and rapid propagation, realizes the cycle culture of Ginkgo tissue culture seedlings, and provides the tissue culture seedlings continuously without repeated external sterilization. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The efficiency of inducing adventitious buds from Ginkgo branches of different lengths is counted;

[0019] Figure 2 Induction of adventitious buds from different length of Ginkgo branches; A-D: induction of adventitious buds from fresh branches. A, C: branches cut into 5-10 cm, containing 4-5 nodes, no induction of adventitious buds after 20 days of induction, A is the overall picture, C is the detail picture. B, D: branches cut into 3-5 cm, containing 2-3 nodes, induction of adventitious buds after 20 days of induction, B is the overall picture, D is the detail picture.

[0020] Figure 3 Induction of adventitious buds from different length of Ginkgo branches; A-D: induction of adventitious buds from fresh branches. A, C: branches cut into 5-10 cm, containing 4-5 nodes, no induction of adventitious buds after 20 days of induction, A is the overall picture, C is the detail picture. B, D: branches cut into 3-5 cm, containing 2-3 nodes, induction of adventitious buds after 20 days of induction, B is the overall picture, D is the detail picture.

[0021] Figure 4 Statistics of induction of adventitious buds from different length of Ginkgo branches;

[0022] Figure 5 Induction of adventitious buds from different length of Ginkgo branches; A-D: induction of adventitious buds from fresh branches. A, C: branches cut into 5-10 cm, containing 4-5 nodes, no induction of adventitious buds after 20 days of induction, A is the overall picture, C is the detail picture. B, D: branches cut into 3-5 cm, containing 2-3 nodes, induction of adventitious buds after 20 days of induction, B is the overall picture, D is the detail picture.

[0023] Figure 6 Induction of adventitious buds from different length of Ginkgo branches; A-D: induction of adventitious buds from fresh branches. A, C: branches cut into 5-10 cm, containing 4-5 nodes, no induction of adventitious buds after 20 days of induction, A is the overall picture, C is the detail picture. B, D: branches cut into 3-5 cm, containing 2-3 nodes, induction of adventitious buds after 20 days of induction, B is the overall picture, D is the detail picture.

[0024] Figure 7 Induction of adventitious buds from different length of Ginkgo branches; A-D: induction of adventitious buds from fresh branches. A, C: branches cut into 5-10 cm, containing 4-5 nodes, no induction of adventitious buds after 20 days of induction, A is the overall picture, C is the detail picture. B, D: branches cut into 3-5 cm, containing 2-3 nodes, induction of adventitious buds after 20 days of induction, B is the overall picture, D is the detail picture.

[0025] Figure 8 Induction of adventitious buds from different length of Ginkgo branches; A-D: induction of adventitious buds from fresh branches. A, C: branches cut into 5-10 cm, containing 4-5 nodes, no induction of adventitious buds after 20 days of induction, A is the overall picture, C is the detail picture. B, D: branches cut into 3-5 cm, containing 2-3 nodes, induction of adventitious buds after 20 days of induction, B is the overall picture, D is the detail picture.

[0026] Figure 9 Induction of adventitious buds from different length of Ginkgo branches; A-D: induction of adventitious buds from fresh branches. A, C: branches cut into 5-10 cm, containing 4-5 nodes, no induction of adventitious buds after 20 days of induction, A is the overall picture, C is the detail picture. B, D: branches cut into 3-5 cm, containing 2-3 nodes, induction of adventitious buds after 20 days of induction, B is the overall picture, D is the detail picture. DETAILED DESCRIPTION

[0027] The technical solutions of the present application are further explained below through examples, but the protection scope of the present application is not limited in any form by the examples.

[0028] Example 1

[0029] A method for breaking the dormancy cycle of the top bud of tissue culture seedlings and maintaining the tissue culture seedlings of Ginkgo biloba, the specific steps of which are as follows:

[0030] Step 1: Disinfecting the explants: Select healthy Ginkgo biloba seedlings growing in March-April, and take their young branches as explants for disinfection treatment.

[0031] Step 2: Inducing adventitious buds: Divide the disinfected explants into 3-5 cm, containing 2-3 nodes, and subculture to the adventitious bud induction medium.

[0032] Step 3: Single bud culture of adventitious buds: Take the newly induced adventitious buds from the mother plant and subculture to the elongation medium for culture.

[0033] Step 4: Breaking the dormancy of the top bud: Apply GA3 to the top bud that no longer elongates; the application method is to peel off the scales of the Ginkgo biloba top bud, and add 11 mM GA3 on the top bud with a pipette gun; add 20 uL each time, add once every 3 days, and add 4 times; after 20 days, the top bud breaks dormancy and starts to grow leaves and resumes growth.

[0034] Step 5: Continue to induce adventitious buds: Repeat the above-mentioned Step 2 for the elongated Ginkgo biloba branches.

[0035] Step 6: Rooting: Subculture the 5-10 cm Ginkgo biloba stem segments to the rooting medium for rooting.

[0036] In the second step, the length of the adventitious bud induction should be 3-5 cm, containing 2-3 nodes, and too long branches are not easy to induce. The verification process is as follows: cut the newly taken branches into 5-10 cm, containing 4-5 nodes for the first group, and 3-5 cm, containing 2-3 nodes for the second group, and subculture to the adventitious bud induction medium, and observe after 20 days. The experiment is repeated three times, and the number of explants for each condition is not less than 10 each time. As shown in Table 1, none of the first group has induced adventitious buds, and the induction rate of the second group is more than 70%. Figure 1 、 Figure 2

[0037] ​The fourth step is to add GA3 on the top bud, not in the culture medium. The specific verification process is as follows: add 11 mM GA3 in the adventitious bud induction medium (MS+WPM+0.02 mg / L NAA+0.2 mg / L 6BA+0.5 g / L enzymatic hydrolysis casein+30 g / L sucrose+2 g / L activated carbon+7.8 g / L agar, pH 5.8-6.0) as the first group, and subculture the fresh ginkgo branches. The results are observed after 20 days; the second group uses the adventitious bud induction medium, and adds GA3 on the top bud with a pipette. The concentration of GA3 is 11 mM, 20 uL each time, once every 3 days, and 4 times. The experiment is repeated three times, and the number of explants in each condition is not less than 10 each time. The results are counted after 20 days.

[0038] As shown in Figure 3 , the top buds in the first group of medium with GA3 do not germinate. The top buds of the branches added with GA3 from the top bud by pipette have 65% of the starting phenomenon.

[0039] The concentration of GA3 added in the fourth step is 11 mM, and too high or too low concentration cannot break the dormancy of the top bud. The specific verification process is as follows: add 3 mM, 11 mM, and 30 mM GA3 solution on the top of the dormant bud, 20 uL each time, once every 3 days, and 4 times. The top bud is observed after 20 days. The experiment is repeated three times, and the number of explants in each condition is not less than 10 each time. Figure 4 Figure 5 As shown in Figure 4 , 3 mM and 30 mM GA3 cannot break the dormancy of ginkgo top bud, only 11 mM GA3 can break the dormancy of more than 65% of the top bud.

[0040] Example 2

[0041] A method for breaking the dormancy cycle of tissue culture seedlings and maintaining ginkgo tissue culture seedlings, the specific steps of which are as follows:

[0042] First step, explant disinfection: select 4-month-old healthy and disease-free ginkgo seedlings, take their young branches as explants, and disinfect them;

[0043] Second step, adventitious bud induction: divide the disinfected explants into 3 cm, containing 2 nodes, and subculture them to the adventitious bud induction medium;

[0044] Third step, single bud culture of adventitious bud: take the newly induced adventitious bud from the mother plant and subculture it to the elongation medium for culture;

[0045] Step 4, breaking the dormancy of the terminal bud: applying GA3 to the terminal bud which is no longer elongating; the method of application: peeling the scales of the terminal bud of Ginkgo biloba, and adding 11 mM GA3 on the terminal bud with a pipette gun; adding 20 uL each time, adding once every 3 days, and adding 4 times; after 20 days, the terminal bud breaks dormancy, starts to grow leaves, and restores growth;

[0046] Step 5, continuing the induction of adventitious buds: repeating the above-mentioned steps 2 to 4 to the elongated Ginkgo biloba branches;

[0047] Step 6, rooting: subculturing the 5-10 cm Ginkgo biloba stem segments into the rooting medium to root;

[0048] As one of the preferred embodiments, the rooting medium formula is: 1 / 2MS+1 / 2WPM+5mg / L IBA+0.5mg / L NAA+20g / L sucrose+2g / L activated carbon+7.8g / L agar, pH 5.8-6.0.

[0049] As one of the preferred embodiments, the adventitious bud induction medium formula is MS+WPM+0.02mg / L NAA+0.2mg / L 6BA+0.5g / L enzymatic hydrolysis casein+30g / L sucrose+2g / L activated carbon+7.8g / L agar, pH 5.8-6.0.

[0050] As one of the preferred embodiments, the elongation culture formula is MS+WPM+0.2mg / L 6BA+0.5g / L enzymatic hydrolysis casein+30g / L sucrose+2g / L activated carbon+7.8g / L agar, pH 5.8-6.0;

[0051] As one of the preferred embodiments, the culture conditions of Ginkgo biloba after rooting: the entire propagation process is carried out under the culture conditions of temperature 25-28℃, light intensity 5000-9000Lx, and photoperiod 16h light, 8h dark.

[0052] The entire Ginkgo biloba tissue culture seedling culture process needs to be carried out under high light intensity, and the light intensity cannot reach 6000Lx, which will affect the development of Ginkgo biloba leaves. The specific verification process is as follows: the tissue culture bottles containing Ginkgo biloba branches are placed under 3000Lx-5000Lx culture conditions as the first group, and placed under 6000-9000Lx as the second group. After 20 days, observe the growth conditions. The results show that, as shown in Figure 6 the first group under low light, the terminal bud of Ginkgo biloba can germinate, but the leaves cannot fully develop and expand. Only under the high light of the second group, the Ginkgo biloba leaves can grow and develop healthily.

[0053] Example 3

[0054] First step, disinfection of explants: select 3 months of healthy growth without disease of Ginkgo biloba seedlings, take its young branches as explants, and disinfect them;

[0055] Second step, induction of adventitious buds: divide the disinfected explants into 5 cm, containing 3 nodes, and subculture to the adventitious bud induction medium. The formula of the adventitious bud induction medium is MS+WPM+0.02 mg / L NAA+0.2 mg / L 6-BA+0.5 g / L enzymatic hydrolysis casein+30 g / L sucrose+2 g / L activated carbon+7.8 g / L agar, pH 5.8-6.0. The induced adventitious buds are as shown in Figure 7 .

[0056] Third step, single bud culture of adventitious buds: take the newly induced adventitious buds from the mother plant, as shown in Figure 8 , subculture to the elongation medium for culture. The formula of the elongation medium is MS+WPM+0.2 mg / L 6BA+0.5 g / L enzymatic hydrolysis casein+30 g / L sucrose+2 g / L activated carbon+7.8 g / L agar, pH 5.8-6.0.

[0057] Fourth step, breaking dormancy of terminal buds: apply GA3 to the terminal buds that no longer elongate. Application method: peel the scales of the Ginkgo terminal buds, and add 11 mM GA3 on the terminal buds with a pipette gun. Add 20 uL each time, add once every 3 days, and add 4 times. After 20 days, the terminal buds break dormancy, start to grow leaves, and resume growth.

[0058] Fifth step, continue to induce adventitious buds: repeat the second to fourth steps described above for the elongated Ginkgo branches.

[0059] Sixth step, rooting: subculture 5-10 cm Ginkgo stem segments to the rooting medium for rooting. The formula of the rooting medium is 1 / 2MS+1 / 2WPM+5 mg / L IBA+0.5 mg / L NAA+20 g / L sucrose+2 g / L activated carbon+7.8 g / L agar, pH 5.8-6.0.

[0060] Seventh step, culture conditions of Ginkgo: the entire propagation process is carried out under the culture conditions of temperature 25-28℃, light intensity 5000-9000 Lx, and photoperiod 16 h light, 8 h dark, as shown in Figure 9 .

Claims

1. A method for breaking the dormancy cycle of the terminal bud of tissue culture seedlings and maintaining the tissue culture seedlings of Ginkgo biloba, characterized in that, The whole Ginkgo biloba L. tissue culture process needs to be carried out under the illumination intensity of 6000 Lx-9000 Lx, and the specific steps are as follows: The first step, disinfection of explants: select 3-4 months old healthy Ginkgo biloba L. seedlings, take the young branches as explants, and carry out disinfection treatment; The second step, adventitious bud induction: the disinfected explants are divided into 3-5 cm, containing 2-3 nodes, and subcultured to adventitious bud induction medium; The third step, single bud culture of adventitious bud: the newly induced adventitious bud is taken off from the mother plant and subcultured to the elongation medium for culture; The fourth step, breaking dormancy of terminal bud: GA3 is applied to the terminal bud which no longer elongates; the application method is: peeling the scales of the terminal bud of Ginkgo biloba L., adding 11 mM GA3 on the terminal bud; 20 µL each time, adding once every 3 days, adding 4 times; after 20 days, the terminal bud breaks dormancy and starts to grow leaves and restore growth; The fifth step, continue to induce adventitious buds: repeat the second step to the fourth step for the elongated Ginkgo biloba L. branches; The sixth step, rooting: subculture 5-10 cm Ginkgo biloba L. stem segments to rooting medium for rooting.

2. The method for breaking the dormancy cycle of the apical bud of the tissue culture seedling and maintaining the tissue culture seedling of Ginkgo biloba according to claim 1, characterized in that, The adventitious bud induction medium formula is MS+WPM+0.02 mg / L NAA+0.2 mg / L 6BA+0.5 g / L enzymatic hydrolysis casein+30 g / L sucrose+2 g / L activated carbon+7.8 g / L agar, pH 5.8-6.

0.

3. The method for breaking the dormancy cycle of the apical bud of the tissue culture seedling and maintaining the tissue culture seedling of Ginkgo biloba according to claim 1, characterized in that, The elongation culture formula is MS+WPM+0.2 mg / L 6BA+0.5 g / L enzymatic hydrolysis casein+30 g / L sucrose+2 g / L activated carbon+7.8 g / L agar, pH 5.8-6.

0.

4. The method for breaking the dormancy cycle of the apical bud of the tissue culture seedling and maintaining the tissue culture seedling of Ginkgo biloba according to claim 1, characterized in that, The rooting medium formula is 1 / 2MS+1 / 2WPM+5 mg / L IBA+0.5 mg / L NAA+20 g / L sucrose+2 g / L activated carbon+agar 7.8 g / L, pH 5.8-6.

0.

5. The method for breaking the dormancy cycle of the apical bud of the tissue culture seedling and maintaining the tissue culture seedling of Ginkgo biloba according to claim 1, characterized in that, The entire propagation process was carried out at a temperature of 25-28 o C, under a photoperiod of 16 h light, 8 h darkness, and culture conditions.

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