CN114375839A - Ginkgo regeneration method based on leaf induction - Google Patents
Ginkgo regeneration method based on leaf induction Download PDFInfo
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- CN114375839A CN114375839A CN202210113345.2A CN202210113345A CN114375839A CN 114375839 A CN114375839 A CN 114375839A CN 202210113345 A CN202210113345 A CN 202210113345A CN 114375839 A CN114375839 A CN 114375839A
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Abstract
The invention discloses a culture medium composition for gingko regeneration, which comprises a gingko leaf induction callus culture medium, a gingko adventitious bud elongation culture medium and a gingko rooting culture medium; the callus induction culture medium of ginkgo leaf comprises MS culture medium, 4-8g/L agar, 25-32g/L sucrose, 0.08-0.15mg/L IAA and 0.06-0.10mg/L IBA; the ginkgo adventitious bud elongation culture medium comprises an MS culture medium, 4-8g/L agar, 25-32g/L sucrose, 0.25-0.40mg/L IBA and 0.06-0.12mg/L zeatin; the rooting culture medium for ginkgo comprises 1/2MS culture medium, 4-8g/L agar, 25-32 sucrose, 0.20-0.30mg/L IBA and 0.2-0.4mg/L NAA. The invention also discloses a method for regenerating ginkgo biloba by using the culture medium combination.
Description
Technical Field
The invention belongs to the field of establishment of plant leaf induction and regeneration systems, and relates to a method and a culture medium combination for a ginkgo leaf induction and regeneration system.
Background
Ginkgo (with the scientific name of Ginkgo biloba L.) is a tree of Ginkgo of Ginkgoaceae. Ginkgo biloba appears hundreds of millions of years ago and is the oldest wiggler in gymnosperms left after the movement of the fourth glacier, the existing gingko biloba is rare and scattered, old trees of hundreds of years old are not common, and all other plants in the same class are killed, so the gingko biloba has the name of activated stone. The varieties and varieties comprise 26 kinds of ginkgo biloba including yellow leaf ginkgo, tower-shaped ginkgo, cracked ginkgo, weeping ginkgo, spotted leaf ginkgo and the like.
The fruit of the ginkgo tree is commonly called ginkgo, and therefore ginkgo is also known as ginkgo tree. The ginkgo tree grows slowly and has extremely long service life, and a great amount of fruits can be obtained after more than twenty years from planting to bearing the ginkgo fruits under natural conditions and forty years later, so the ginkgo tree is called as a Gong Sun Tree and has the meaning of 'male species and sun getting food', is a longevity star in the tree and has ornamental, economic and medicinal values.
Ginkgo was first found in the carbolic age 3.45 million years ago. Once widely distributed in europe, asia and america in the northern hemisphere, the middle generation of Jurassic ginkgo has been widely distributed in the northern hemisphere, and the later period of the Chalkbrood period begins to decline. By 50 ten thousand years ago, the movement of the fourth glacier occurs, the earth becomes cold suddenly, most of the ginkgo plants are endangered by the dead and extinct in europe, north america and most of asia, and the ginkgo plants are stored wonderfully only if the natural conditions of china are superior. So, it is called "activating stone" by scientists, panda in the plant kingdom. The wild ginkgo leaves are remained in the mountain area in the west of Zhejiang province in the south of Shandong province (Tan city) in the north of Xuzhou (Tezhou city) in Jiangsu province of China.
The wild and semi-wild ginkgo communities are found in small surrounding villages and towns, Dabie mountains, Shennongjia and the like, such as Tianmu mountains in Zhejiang, Yichang city in Hubei, Anluo city in Hubei, Guilin Lingchuan county, sea village to Gaoshan town in Xingan county. Because of the rare individuals, the remaining forest will be replaced by hermaphrodite plants, if not strictly protected and natural renewal promoted. Ginkgo biloba distribution is largely in the field of artificial cultivation, mainly in China, France and the United states of America south Carolina. There is no doubt that the foreign ginkgo biloba leaves are imported directly or indirectly from china.
The establishment of the regeneration system of the ginkgo accelerates the basic scientific research of the ginkgo, promotes the research and application of the biotechnology of gene editing assistance and the like for cultivating new ginkgo varieties, and saves endangered ginkgo varieties and germplasm resources.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides a culture medium combination for gingko regeneration, which comprises a gingko leaf induction callus culture medium, a gingko adventitious bud elongation culture medium and a gingko rooting culture medium;
the ginkgo leaf induction callus culture medium comprises an MS culture medium, 4-8g/L agar, 25-32g/L sucrose, 0.08-0.15mg/L IAA and 0.06-0.10mg/L IBA;
the elongation culture medium of the adventitious bud of the ginkgo comprises an MS culture medium, 4-8g/L agar, 25-32g/L sucrose, 0.25-0.40mg/L IBA and 0.06-0.12mg/L zeatin;
the rooting culture medium for ginkgo biloba comprises 1/2MS culture medium, 4-8g/L agar, 25-32 sucrose, 0.20-0.30mg/L IBA and 0.2-0.4mg/L NAA.
In some embodiments, the variety of ginkgo biloba is a large potato.
In some embodiments, the ginkgo leaf induction callus medium comprises MS medium, 5.6g/L agar, 28.5g/L sucrose, 0.1mg/L IAA, and 0.08mg/L IBA.
In some embodiments, the Ginkgo biloba adventitious bud elongation medium comprises MS medium, 5.6g/L agar, 28.5g/L sucrose, 0.3mg/L IBA, and 0.08mg/L zeatin.
In some embodiments, the rooting media for ginkgo biloba comprises 1/2MS medium, 5.6g/L agar, 28.5g/L sucrose, 0.25mg/L IBA, and 0.3mg/L NAA.
In some embodiments, the ginkgo leaf induction callus medium has a pH of 5.5 to 6.2.
In some embodiments, the pH of the adventitious bud elongation medium of ginkgo biloba is 5.5 to 6.2.
In some embodiments, the ginkgo rooting medium has a pH of 5.5 to 6.2.
In some embodiments, the MS medium comprises: 1900mg/L potassium nitrate, 332.2mg/L calcium chloride, 1650mg/L ammonium nitrate, 180.7mg/L magnesium sulfate and 170mg/L phosphorusPotassium dihydrogen acid; 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 and 0.83mg/L potassium iodide; 27.8mg/LFeSO4·7H2O、37.26mg/L Na2-EDTA·2H2O; 2.0mg/L glycine, 100mg/L inositol, 0.50mg/L nicotinic acid, 0.1mg/L thiamine VB1 and 0.5mg/L pyridoxine VB 6.
In some embodiments, the 1/2MS medium includes: 950mg/L potassium nitrate, 166.1mg/L calcium chloride, 825mg/L ammonium nitrate, 90.35mg/L magnesium sulfate, 85mg/L monopotassium phosphate; 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 and 0.83mg/L potassium iodide; 27.8mg/LFeSO4·7H2O、37.26mg/L Na2-EDTA·2H2O; 2.0mg/L glycine, 100mg/L inositol, 0.50mg/L nicotinic acid, 0.1mg/L thiamine VB1 and 0.5mg/L pyridoxine VB 6.
In a second aspect, the present invention provides a method for regenerating ginkgo biloba, comprising the steps of:
s1: callus induction
Inoculating the ginkgo leaf explant to a ginkgo leaf induction callus culture medium for induction culture to form a ginkgo callus with adventitious buds;
the ginkgo leaf induction callus culture medium is the ginkgo leaf induction callus culture medium in the culture medium combination of the first aspect of the invention;
s2: bud elongation induction
Inoculating the callus clusters of the callus with the adventitious buds to a ginkgo adventitious bud elongation culture medium for bud elongation culture to form ginkgo plants with adventitious buds elongated;
the ginkgo adventitious bud elongation culture medium is the ginkgo adventitious bud elongation culture medium in the culture medium combination of the first aspect of the invention;
s3: root induction
Inoculating the gingko plant with the extended adventitious bud into a gingko rooting culture medium for rooting culture to form a gingko plant with the adventitious bud and a root;
the gingko rooting culture medium is the gingko rooting culture medium in the culture medium combination of the first aspect of the invention.
In some embodiments, in step S1, the ginkgo biloba leaves are washed clean with water, sterilized with a disinfectant, and washed clean with sterile water to obtain the ginkgo biloba leaf explant.
In some embodiments, in step S1, the ginkgo biloba leaf explant is obtained by soaking ginkgo biloba leaves in 70-80% aqueous ethanol for 25-45S, washing ginkgo biloba leaves with sterile water for 1-5 times, soaking ginkgo biloba leaves in 1.0-2.0 w/v% aqueous sodium hypochlorite for 15-25min, and washing ginkgo biloba leaves with sterile water for 5-20 times.
In some embodiments, in step S1, the ginkgo leaf explant is divided into 1-2cm2Inoculating the leaf tissue into the ginkgo leaf induction callus culture medium for the induction culture.
In some embodiments, in step S1, the inducing culture is a dark culture at a temperature of 22-25 ℃ for a period of 2-4 days.
In some embodiments, in step S2, the incubation temperature is 22-25 deg.C, the daily illumination is 14-18 hours, and the illumination intensity is 150-2And/s, the culture time is 21-28 days.
In some embodiments, in step S3, the incubation temperature is 22-25 ℃, the daily illumination time is 14-18 hours, and the illumination intensity is 150-2And/s, the culture time is 30-40 days.
In some embodiments, the method further comprises the steps of:
s4: hardening off seedlings
And (3) hardening and culturing the ginkgo plant with the adventitious bud and the root.
In some embodiments, in step S4, the incubation temperature is 22-25 deg.C, the daily illumination is 14-18 hours, and the illumination intensity is 150-2The relative humidity is 80-90%, and the culture time is 30-40 days.
In some embodiments, the method further comprises the steps of:
s5: transplanting
And taking out the hardened ginkgo seedlings from the bottle, and transplanting the ginkgo seedlings into soil.
In some embodiments, in step S5, the temperature is 22-25 ℃ and the relative humidity is 50-60%.
In the method for regenerating ginkgo biloba based on leaf induction, the callus induction rate can reach 96.8%, the germination rate can reach 98.2%, and the rooting rate can reach 98.6%, so that the method can be effectively applied to scientific research and forestry production.
Drawings
FIG. 1 is a photograph showing callus growth conditions in example 1 of the present invention
FIG. 2 is a photograph showing the process from initial callus to gradual germination.
FIG. 3 is a photograph showing the conditions of shoot culture in example 1 of the present invention.
FIG. 4 is a photograph showing the root growth in example 1 of the present invention (in the direction of the body).
FIG. 5 is a photograph showing the root growth in example 1 of the present invention (flat bottom direction).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
The steps not described in detail in the present invention are all the operations conventional in the art, and the materials not described in detail are all the materials conventional in the art.
The ginkgo varieties used in the invention are: large potatoes.
Example 1
Preparation of culture Medium
(1) Ginkgo leaf induction callus culture medium
The basic culture medium is an MS culture medium, and specifically comprises macroelements: 1900mg/L potassium nitrate, 332.2mg/L calcium chloride, 1650mg/L ammonium nitrate, 180.7mg/L magnesium sulfate and 170mg/L monopotassium 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 iodidePotassium; iron salt: 27.8mg/LFeSO4·7H2O、37.26mg/L Na2-EDTA·2H2O; organic components: 2.0mg/L glycine, 100mg/L inositol, 0.50mg/L nicotinic acid, 0.1mg/L thiamine VB1, 0.5mg/L pyridoxine VB 6. The formula also comprises 5.6g/L agar and 28.5g/L sucrose, and the pH value is 5.85 plus or minus 0.2. The hormones in the medium were IAA and IBA, and the hormone content was shown in table 1.
(2) Ginkgo adventitious bud elongation culture medium
The basic culture medium is an MS culture medium, and specifically comprises macroelements: 1900mg/L potassium nitrate, 332.2mg/L calcium chloride, 1650mg/L ammonium nitrate, 180.7mg/L magnesium sulfate and 170mg/L monopotassium 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 and 0.83mg/L potassium iodide; iron salt: 27.8mg/LFeSO4·7H2O、37.26mg/L Na2-EDTA·2H2O; organic components: 2.0mg/L glycine, 100mg/L inositol, 0.50mg/L nicotinic acid, 0.1mg/L thiamine VB1, 0.5mg/L pyridoxine VB 6. The formula also comprises 5.6g/L agar and 28.5g/L sucrose, and the pH value is 5.85 plus or minus 0.1. The hormones in the medium were IBA and zeatin, and the hormone contents are shown in table 2, respectively.
(3) Ginkgo rooting culture medium
The basic culture medium is 1/2MS culture medium, and specifically comprises macroelements: 950mg/L potassium nitrate, 166.1mg/L calcium chloride, 825mg/L ammonium nitrate, 90.35mg/L magnesium sulfate, 85mg/L monopotassium 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 and 0.83mg/L potassium iodide; iron salt: 27.8mg/LFeSO4·7H2O、37.26mg/L Na2-EDTA·2H2O; organic components: 2.0mg/L glycine, 100mg/L inositol, 0.50mg/L nicotinic acid, 0.1mg/L thiamine VB1, 0.5mg/L pyridoxine VB 6. The formulation also comprises 5.6g/L agar 28.5g/L sucrose, pH5.85 plus or minus 0.2. The hormones in the medium were IBA and NAA, and the hormone contents are shown in table 3, respectively.
(II) callus induction culture:
selection of explants: selecting healthy ginkgo biloba with 3 years old and good growth state, selecting healthy ginkgo biloba with tender green leaf and good growth state, and cutting off the ginkgo biloba leaves with scissors.
Pretreatment of explants: washing the blades for 8 hours by flowing clear water, placing the washed blades on sterile absorbent paper to absorb water on a superclean workbench of a drying chamber, and carrying out sterile operation on the superclean workbench in the following operation.
And (3) disinfection of explants: soaking the leaves in 75% ethanol water solution for 30s, washing with sterile water for 3 times, soaking the leaves in 1.5 w/v% (each 100mL of water solution contains 1.5g of sodium hypochlorite) sodium hypochlorite water solution for 18min, sterilizing, washing with sterile water for 12 times, removing water with sterile paper, and cutting into 1.5cm pieces with sterile scissors2The left and right leaf tissue small blocks are ready for use.
The sterilized explants were inoculated into 6 kinds of callus induction culture medium of Ginkgo biloba as described in Table 1, 10 bottles of each culture medium were prepared, and 3-10 leaf tissue pieces were added to each bottle.
Culturing the explant in a dark room for 3 days at 22-25 deg.C, and observing callus induction and growth of the explant, i.e. leaf tissue, wherein a bottle of the explant is shown in FIG. 1. The photographs of 1 callus taken from another 3 flasks are shown in FIG. 2, wherein A to C are the process from the initial callus to the gradual budding (adventitious bud) of the ginkgo leaf callus, and the callus induction rate is shown in Table 1.
Therefore, the method can effectively induce the ginkgo biloba explants to generate callus and sprout.
(III) bud elongation Induction
Selecting the callus with good growth condition in the step (II) to an aseptic super clean workbench, clamping the callus groups by using aseptic forceps, respectively transferring the callus groups to 6 ginkgo bud elongation culture mediums shown in the table 2, wherein 3-10 callus groups are placed in each bottle, and the callus groups are cultured at the culture temperature of 22-25 ℃, the light-dark period of 16/8h/d and the illumination intensity of 150-2Culturing for about 21-28 days under the condition of/s, observing and recording the germination rate and the growth vigor of seedlings after germination, wherein the number of bottles of each culture medium is 10, the picture of one bottle is shown in figure 3, and the germination rate is shown in table 2.
Therefore, the method can effectively induce the adventitious bud elongation of the callus of the ginkgo to grow.
(IV) root induction
Cutting the bud plantlets which grow vigorously in the step (III), transferring the cut bud plantlets into 6 gingko rooting culture media shown in the table 3, wherein 1 plant is cultured in each bottle at the culture temperature of 22-25 ℃, the light-dark period of 16/8h/d and the illumination intensity of 150-2Culturing for 35 days under the condition of/s, observing the rooting condition of the plantlet and recording the rooting rate. The picture of one bottle body is shown in figure 4, the picture of the bottle bottom is shown in figure 5, and the rooting rate is shown in table 3.
Therefore, the method can effectively induce the gingko adventitious bud plant to root.
(V) hardening off the seedlings
Performing seedling hardening culture on the ginkgo plant with the adventitious bud and the root at the culture temperature of 22-25 ℃, with the light-dark period of 16/8h/d (namely 16h of light irradiation and 8h of dark culture every day), and with the light intensity of 150-2The relative humidity is 80-90%, and the culture time is 35 days.
(VI) transplanting
Taking out the ginkgo plant after 35 days of hardening, transplanting to the soil in a greenhouse, controlling the temperature at 22-25 ℃ and the relative humidity at 50-60%.
TABLE 1 Ginkgo biloba induction callus culture Medium and culture results
| Numbering | IAA(mg/L) | IBA(mg/L) | Callus induction rate | Callus growth state |
| 1 | 0.1 | 0.04 | 38.9％ | + |
| 2 | 0.1 | 0.06 | 59.8％ | ++ |
| 3 | 0.1 | 0.08 | 96.8％ | ++++ |
| 4 | 0.2 | 0.10 | 40.6％ | + |
| 5 | 0.2 | 0.12 | 66.5％ | ++ |
| 6 | 0.2 | 0.16 | 35.6％ | + |
Note: "+" indicates callus growth status.
The best growth state is shown as ++++, the healing time is short, the growth is fast, the callus mass is large, granular, loose in structure and light yellow;
"+ + + +" indicates better callus growth status, short callus growth time, faster callus growth, granular, light yellow;
"+ +" indicates that the callus is in a normal growth state, slow in growth, and in a block shape, yellow green;
"+" indicates that the callus has poor growth state, long healing time, slow growth, block shape, compact structure, hard texture and yellow green.
TABLE 2 Ginkgo bud elongation Medium and culture results
| Numbering | IBA(mg/L) | Zeatin (mg/L) | Percentage of sprouting (%) | Growth state |
| 1 | 0.3 | 0.04 | 45.6％ | + |
| 2 | 0.3 | 0.06 | 65.4％ | ++ |
| 3 | 0.3 | 0.08 | 98.2％ | ++++ |
| 4 | 0.4 | 0.12 | 59.8％ | ++ |
| 5 | 0.4 | 0.16 | 65.2％ | ++ |
| 6 | 0.4 | 0.18 | 39.6％ | + |
Note: "+" indicates the bud growth state.
The growth state is best, the bud is fast, the bud is many, the growth is strong, and the leaves are green;
"+ + + +" indicates better growth status, more buds, strong growth and green leaves;
"+ +" indicates the general growth state of the bud, general growth vigor and green leaves;
"+" indicates poor growth state of buds, weak growth and green leaves.
TABLE 3 rooting media and results for Ginkgo biloba
| Numbering | IBA(mg/L) | NAA(mg/L) | Rooting percentage (%) | Growth state |
| 1 | 0.25 | 0.10 | 39.3％ | + |
| 2 | 0.25 | 0.20 | 56.7 | ++ |
| 3 | 0.25 | 0.30 | 98.6 | ++++ |
| 4 | 0.30 | 0.40 | 63.2％ | ++ |
| 5 | 0.30 | 0.50 | 33.6％ | + |
| 6 | 0.30 | 0.60 | 37.8％ | + |
Note: "+" indicates the rooting status.
The root taking time of +++ "is shortest and the adventitious roots are more;
the root growing time of the table is short and the number of adventitious roots is large;
"+ +" indicates slow rooting, few adventitious roots;
"+" indicates very slow rooting with few adventitious roots.
Therefore, in the method for regenerating ginkgo biloba based on leaf induction, the callus induction rate can reach 96.8%, the germination rate can reach 98.2%, and the rooting rate can reach 98.6%, so that the method can be effectively applied to scientific research and forestry production.
It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.
Claims (10)
1. A culture medium composition for regenerating ginkgo biloba comprises a ginkgo biloba leaf induction callus culture medium, a ginkgo biloba adventitious bud elongation culture medium and a ginkgo biloba rooting culture medium;
the ginkgo leaf induction callus culture medium comprises an MS culture medium, 4-8g/L agar, 25-32g/L sucrose, 0.08-0.15mg/L IAA and 0.06-0.10mg/L IBA;
the elongation culture medium of the adventitious bud of the ginkgo comprises an MS culture medium, 4-8g/L agar, 25-32g/L sucrose, 0.25-0.40mg/L IBA and 0.06-0.12mg/L zeatin;
the rooting culture medium for ginkgo biloba comprises 1/2MS culture medium, 4-8g/L agar, 25-32 sucrose, 0.20-0.30mg/L IBA and 0.2-0.4mg/L NAA.
2. The culture medium composition of claim 1, wherein the variety of Ginkgo biloba is a large potato.
3. The culture medium combination of claim 1, wherein the ginkgo leaf induction callus medium comprises MS medium, 5.6g/L agar, 28.5g/L sucrose, 0.1mg/L IAA, and 0.08mg/L IBA;
preferably, the gingko adventitious bud elongation culture medium comprises MS culture medium, 5.6g/L agar, 28.5g/L sucrose, 0.3mg/L IBA and 0.08mg/L zeatin;
preferably, the ginkgo rooting medium comprises 1/2MS medium, 5.6g/L agar, 28.5g/L sucrose, 0.25mg/L IBA and 0.3mg/L NAA;
preferably, the pH value of the ginkgo leaf blade induction callus culture medium is 5.5-6.2;
preferably, the pH value of the adventitious bud elongation culture medium of the ginkgo is 5.5-6.2;
preferably, the pH value of the gingko rooting medium is 5.5-6.2.
4. The culture medium combination of claim 1, wherein the MS medium comprises: 1900mg/L potassium nitrate, 332.2mg/L calcium chloride, 1650mg/L ammonium nitrate, 180.7mg/L magnesium sulfate and 170mg/L monopotassium phosphate; 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 and 0.83mg/L potassium iodide; 27.8mg/LFeSO4·7H2O、37.26mg/L Na2-EDTA·2H2O; 2.0mg/L Glycine, 100mg/L GlycineInositol, 0.50mg/L nicotinic acid, 0.1mg/L thiamine VB1 and 0.5mg/L pyridoxine VB 6;
preferably, the 1/2MS medium includes: 950mg/L potassium nitrate, 166.1mg/L calcium chloride, 825mg/L ammonium nitrate, 90.35mg/L magnesium sulfate, 85mg/L monopotassium phosphate; 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 and 0.83mg/L potassium iodide; 27.8mg/LFeSO4·7H2O、37.26mg/L Na2-EDTA·2H2O; 2.0mg/L glycine, 100mg/L inositol, 0.50mg/L nicotinic acid, 0.1mg/L thiamine VB1 and 0.5mg/L pyridoxine VB 6.
5. A method of regenerating ginkgo biloba, said method comprising the steps of:
s1: callus induction
Inoculating the ginkgo leaf explant to a ginkgo leaf induction callus culture medium for induction culture to form a ginkgo callus with adventitious buds;
the ginkgo leaf induction callus medium is the ginkgo leaf induction callus medium in the culture medium combination of any one of claims 1 to 4;
s2: bud elongation induction
Inoculating the callus clusters of the callus with the adventitious buds to a ginkgo adventitious bud elongation culture medium for bud elongation culture to form ginkgo plants with adventitious buds elongated;
the ginkgo adventitious bud elongation culture medium is the ginkgo adventitious bud elongation culture medium in the culture medium combination of any one of claims 1 to 4;
s3: root induction
Inoculating the gingko plant with the extended adventitious bud into a gingko rooting culture medium for rooting culture to form a gingko plant with the adventitious bud and a root;
the rooting culture medium for ginkgo biloba is the rooting culture medium for ginkgo biloba in the culture medium combination of any one of claims 1 to 4.
6. The method of claim 5, wherein in step S1, the ginkgo biloba leaves are washed clean with water, sterilized with a disinfectant, and washed clean with sterile water to obtain the ginkgo biloba leaf explant;
preferably, in step S1, the ginkgo biloba leaf blade is soaked in 70-80% ethanol aqueous solution for 25-45S, washed with sterile water for 1-5 times, soaked with 1.0-2.0 w/v% sodium hypochlorite aqueous solution for 15-25min, and washed with sterile water for 5-20 times to obtain the ginkgo biloba leaf explant;
preferably, in step S1, the ginkgo leaf explant is divided into 1-2cm2Inoculating the leaf tissue into the ginkgo leaf induction callus culture medium for the induction culture;
preferably, in step S1, the induction culture is dark culture, the culture temperature is 22-25 ℃, and the culture time is 2-4 days;
preferably, in step S2, the cultivation temperature is 22-25 ℃, the daily illumination is 14-18 hours, and the illumination intensity is 150-2The culture time is 21-28 days;
preferably, in step S3, the culture temperature is 22-25 ℃, the daily illumination is 14-18 hours, and the illumination intensity is 150-2And/s, the culture time is 30-40 days.
7. The method of claim 5, further comprising the steps of:
s4: hardening off seedlings
And (3) hardening and culturing the ginkgo plant with the adventitious bud and the root.
8. The method as claimed in claim 7, wherein the cultivation temperature is 22-25 ℃ and the light irradiation is carried out for 14-18 hours per day at an intensity of 150 μmol/m and 160 μmol/m in step S42The relative humidity is 80-90%, and the culture time is 30-40 days.
9. The method of claim 7, further comprising the steps of:
s5: transplanting
And taking out the hardened ginkgo seedlings from the bottle, and transplanting the ginkgo seedlings into soil.
10. The method of claim 9, wherein the temperature is 22-25 ℃ and the relative humidity is 50-60% in step S5.
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| WO1993002204A1 (en) * | 1991-07-18 | 1993-02-04 | Han-Dok Remedia Ind. Co., Ltd. | Production of ginkgolides in cell culture |
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