CN114698549A - Tissue culture medium and tissue culture method for rapid propagation of grape rootstock stem - Google Patents
Tissue culture medium and tissue culture method for rapid propagation of grape rootstock stem Download PDFInfo
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- CN114698549A CN114698549A CN202210405789.3A CN202210405789A CN114698549A CN 114698549 A CN114698549 A CN 114698549A CN 202210405789 A CN202210405789 A CN 202210405789A CN 114698549 A CN114698549 A CN 114698549A
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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Abstract
The invention belongs to the technical field of plant cultivation, and particularly relates to a tissue culture medium and a tissue culture method for rapid propagation of grape rootstock stems. The invention provides a tissue culture medium for rapid propagation of grape rootstock stem segments, which comprises a primary culture medium, a secondary culture medium and a rooting culture medium; the primary culture medium takes an MS culture medium as a basic culture medium, and further comprises: 2.0mg/L6-BA, 0.2mg/L IBA, 0.2mg/L GA3(ii) a The subculture medium takes an MS culture medium as a basic culture medium, and further comprises: 1.25-2.5 mg/L6-BA, 0.2-1.0 mg/L IBA and 0.2-0.5 mg/L GA3. The invention solves the technical problem of low proliferation coefficient and rooting rate of grape tissue culture seedling culture by matching the components in the primary culture medium, the secondary culture medium and the rooting culture medium and using amount and relative proportion of the components.
Description
Technical Field
The invention belongs to the technical field of plant cultivation, and particularly relates to a tissue culture medium and a tissue culture method for rapid propagation of grape rootstock stems.
Background
Grapes (Vitis vinifera L.) are woody vines of the genus Vitis of the family Vitaceae, are one of the oldest fruit tree species in the world, are native to the western Asia and are now widely planted around the world, and the planting mode mainly comprises branch self-rooted seedling propagation. In the 60 s of the 19 th century, the california of the United states discovered a broad-spectrum pest, namely the grape phylloxera, which has serious influence on the worldwide grape industry, so that the world grape cultivation system is fundamentally transformed from the Eurasian self-root cultivation to the resistant rootstock grafting production. Therefore, researches find that the breeding and the utilization of the resistant rootstocks become key factors for effectively solving the problem of grape production.
The traditional grape planting process mainly adopts a self-rooted seedling breeding mode with cuttage and layering as main modes, cannot avoid various soil-borne diseases, and has a long period and easy accumulation of diseases in the planting process. The plant tissue culture technology has the characteristics of convenient material acquisition, difficult pollution and high propagation coefficient, can realize the detoxification of seedlings through the generation of organs such as roots, stems, leaves and the like of the grape test-tube seedlings, quickly propagate high-quality seedlings, reduce the production cost, and play an active role in the aspects of production, application and the like of grape stocks. However, the proliferation coefficient and rooting rate of the current grape tissue culture technology are not ideal.
Disclosure of Invention
The tissue culture medium provided by the invention is used for rapid propagation of the grape rootstock stem, the obtained cluster buds have high multiplication coefficient and the rooting rate of tissue culture seedlings is high, a large number of grape tissue culture seedlings can be obtained, and the technical support is provided for producing good variety seedling.
In order to solve the above problems, the present invention provides the following technical solutions:
the invention provides a tissue culture medium for rapid propagation of grape rootstock stem segments, which is characterized by comprising a primary culture medium, a secondary culture medium and a rooting culture medium;
the primary culture medium takes an MS culture medium as a basic culture medium, and further comprises: 2.0mg/L6-BA, 0.2mg/L IBA and 0.2mg/L GA3；
The subculture medium takes an MS culture medium as a basic culture medium, and further comprises: 1.25-2.5 mg/L6-BA, 0.2-1.0 mg/LIBA and 0.2-0.5 mg/L GA3；
The rooting culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 1.0-1.5 mg/L IBA and 0.1-0.2 g/LAc.
Preferably, the primary culture medium, the secondary culture medium and the rooting culture medium further comprise 30g/L of sucrose and 6-8 g/L of agar.
Preferably, the pH values of the primary culture medium, the secondary culture medium and the rooting culture medium are all 5.8.
The invention also provides a tissue culture method for rapid propagation of the stem segments of the grape stocks, which adopts the tissue culture medium of the technical scheme and comprises the following steps:
inoculating a sterile explant on a primary culture medium for primary culture to obtain primary cluster buds with the length of 5-6 cm;
transferring the primary cluster buds into a subculture medium for subculture to obtain subcultured cluster buds;
and carrying out rooting culture on the secondary cluster buds.
Preferably, the explant is a single shoot segment.
Preferably, the temperature of the primary culture, the temperature of the secondary culture and the temperature of the rooting culture are respectively 24-26 ℃, and the humidity is respectively 20-50%.
Preferably, the primary culture and the secondary culture are carried out under the illumination condition, the illumination time is 12-16 h/d, and the illumination intensity is 2000-2500 lx.
Preferably, the time of the primary culture and the time of the secondary culture are respectively 28-32 d.
Preferably, the rooting culture comprises dark culture and illumination culture, and the culture time of the dark culture is 3-7 d; the illumination intensity of the illumination culture is 2000-2500 lx, the illumination time is 12h/d, and the total time of the dark culture and the illumination culture of the rooting culture is 28-40 d.
Preferably, the sterilized explant is sterilized with an explant disinfectant prior to inoculation on the primary medium; the disinfection mode comprises the following steps: after washing with running water, soaking with 75% ethanol for 30s, washing with sterile water for 3-5 times, soaking with 0.1% mercuric chloride solution for 8min, and washing with sterile water for 3-5 times.
The invention has the beneficial effects that: the invention provides a tissue culture medium for rapid propagation of grape rootstock stem segments, which comprises a primary culture medium, a secondary culture medium and a rooting culture medium; the primary culture medium takes an MS culture medium as a basic culture medium, and further comprises: 2.0mg/L6-BA, 0.2mg/L IBA, 0.2mg/L GA3(ii) a The subculture medium takes an MS culture medium as a basic culture medium, and further comprises: 1.25-2.5 mg/L6-BA, 0.2-1.0 mg/L IBA and 0.2-0.5 mg/L GA3(ii) a The rooting culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 1.0-1.5 mg/L IBA and 0.1-0.2 g/LAc; in the tissue culture medium provided by the invention, IBA (indolebutyric acid) promotes differentiation and division of stem cells of grape stocks, thereby being beneficial to generation of new roots; GA3(gibberellin) promotes stem germination, plant cell elongation and cell division; 6-BA (6-benzylaminopurine) stimulates cell division to promote plant growth and development and promote the formation of callus; IBA, GA3And 6-BA under the combined action, the single-bud stem section of the grape rootstock stem section explant is favorably induced to germinate primary cluster buds, and the multiplication coefficient of the cluster buds is improved. Ac (activated carbon) can change pH in a culture medium similar to soil conditions and provide a dark environment, and can reduce inhibiting substances such as ethylene, phenols and the like through adsorption so as to improve plant development, IBA is used in rooting culture and is assisted by Ac so as to promote the growth of test-tube plantlet root systems, the rooting rate can be improved under the combined action of IBA and Ac, the rooting culture is secondary culture, the early dark environment is favorable for inducing rooting and improving the rooting rate, and the later culture is favorable for the growth of the test-tube plantlets in an illumination environment. The results of the examples show that: in the rapid propagation seedling raising of the stem section of the grape rootstock, the multiplication coefficient of the subculture multiple shoots is 2.89, and the rooting rate of the tissue culture seedling obtained by the rooting culture of the subculture multiple shoots is 100%. According to the invention, the technical problems of low proliferation coefficient and rooting rate during grape tissue culture seedling raising are solved by matching the components in the primary culture medium, the secondary culture medium and the rooting culture medium and the using amount and relative proportion of the components.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.
FIG. 1 shows the growth of multiple shoots obtained by subculture in example 1;
FIG. 2 is a drawing showing the rooting of the tissue culture seedling obtained after the rooting culture of the secondary multiple shoots in example 1;
FIG. 3 shows the growth of multiple shoots obtained by subculture in example 2;
FIG. 4 is a drawing showing the rooting of the tissue culture seedling obtained after the rooting culture of the secondary multiple shoots in example 2;
FIG. 5 shows the growth of multiple shoots obtained by subculture in example 3;
FIG. 6 is the rooting of the tissue culture plantlets obtained in example 3 after rooting culture of the secondary multiple shoots;
FIG. 7 shows the growth of multiple shoots obtained by primary culture of single shoot segments of example 1;
FIG. 8 shows the growth of multiple shoots obtained by primary culture of single shoot segments in comparative example 7;
FIG. 9 shows the growth of multiple shoots obtained by primary culture of single shoot segments in comparative example 8;
FIG. 10 shows the growth of multiple shoots obtained by primary culturing of single shoot segments of example 2;
FIG. 11 shows the growth of multiple shoots obtained by primary culture of single shoot segments in comparative example 9;
FIG. 12 shows the growth of multiple shoots obtained by primary culturing of single shoot segments in comparative example 10;
FIG. 13 shows the growth of multiple shoots obtained by primary culture of single shoot segments in example 3;
FIG. 14 shows the growth of multiple shoots obtained by primary culture of single shoot segments in comparative example 11;
FIG. 15 shows the growth of multiple shoots obtained by primary culture of single shoot segments in comparative example 12.
Detailed Description
The invention provides a tissue culture medium for rapid propagation of grape rootstock stem segments, which comprises a primary culture medium, a secondary culture medium and a rooting culture medium.
In the invention, the primary culture medium takes an MS culture medium as a basic culture medium, and further comprises: 2.0mg/L6-BA, 0.2mg/L IBA and 0.2mg/L GA3. It is further preferable that the culture medium is MS culture medium as a minimal medium, and contains only 2.0mg/L of 6-BA, 0.2mg/L of IBA and 0.2mg/L of GA3。
In the invention, the subculture medium takes an MS culture medium as a basic culture medium, and also comprises 1.25-2.5 mg/L6-BA, 0.2-1.0 mg/L IBA and 0.2-0.5 mg/L GA3(ii) a More preferably, the culture medium is an MS culture medium, and further comprises only: 1.25-2.5 mg/L6-BA, 0.2-1.0 mg/L IBA and 0.2-0.5 mg/L GA3. In the present invention, the subculture medium uses an MS medium as a minimal medium, and further includes: 1.25-2.5 mg/L6-BA, preferably 1.5-2.5 mg/L6-BA, more preferably 2.0-2.5 mg/L6-BA; the composition also comprises 0.2-1.0 mg/L IBA, preferably 0.3-1.0 mg/L IBA, and more preferably 0.5mg/L IBA; further comprises 0.2-0.5 mg/L GA3Preferably 0.3 to 0.5mg/L GA3More preferably 0.5mg/L GA3。
In the invention, the rooting medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 1.0-1.5 mg/L IBA and 0.1-0.2 g/LAc; more preferably, the medium is 1/2MS medium as a minimal medium, and only comprises: 1.0-1.5 mg/L IBA and 0.1-0.2 g/LAc. In the invention, the rooting medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 1.0-1.5 mg/L IBA, preferably 1.2-1.5 mg/L IBA, more preferably 1.5 mg/LIBA; also comprises 0.1-0.2 g/LAc, preferably 0.1 g/LAc.
In the invention, IBA (indolebutyric acid) promotes the differentiation and division of stem cells of grape stocks, thereby being beneficial to the generation of new roots; GA3(gibberellin) promotes stem germination, plant cell elongation and cell division; 6-BA (6-benzylaminopurine) stimulates cell division to promote plant growth and development and promote the formation of callus; IBA, GA36-BA under the combined action, the single-bud stem-segment bud of the explant of the stem segment of the grape stock is favorably inducedThe initial generation cluster buds are generated, and the multiplication coefficient of the secondary generation cluster buds is improved. The subculture medium of the invention uses 6-BA, IBA and GA simultaneously3Three plant growth regulators. The invention uses a plurality of growth regulators together, and combines the reasonable dosage of each plant growth regulator, which is the key point of the invention that the multiplication coefficient is improved and the rooting rate is higher. And in addition, Ac (activated carbon) in the rooting culture medium can change the pH value in the culture medium under the similar soil condition and provide a dark environment, and can reduce inhibiting substances such as ethylene, phenols and the like through adsorption so as to improve the plant development.
Plant growth regulators IBA, 6-BA and GA added into primary culture medium, secondary culture medium and rooting culture medium3The mutual matching realizes the improvement of the proliferation coefficient of the cluster buds after the subculture and the improvement of the rooting rate of the rooting culture under the specific dosage and relative proportion.
In the present invention, the IBA and GA are applied3The sources of 6-BA and Ac are not particularly limited, and conventional commercial products can be used.
In the invention, the primary culture medium, the secondary culture medium and the rooting culture medium preferably further comprise 30g/L of sucrose and 6-8 g/L of agar, and more preferably comprise 30g/L of sucrose and 7g/L of agar.
In the present invention, the primary medium is more preferably MS medium as the minimal medium, and further contains only 2.0mg/L6-BA, 0.2mg/L IBA, and 0.2mg/L GA330g/L sucrose and 7g/L agar.
In the present invention, the secondary culture medium more preferably uses MS medium as the minimal medium, and further contains only 2.5mg/L6-BA, 0.5mg/L IBA and 0.5mg/L GA330g/L sucrose and 7g/L agar.
In the present invention, the rooting medium is more preferably MS medium as the minimal medium, and further contains only 1.5mg/L IBA and 0.1g/LAc, 30g/L sucrose and 7g/L agar.
The sources of the sucrose and the agar are not particularly limited, and the conventional commercial products can be adopted.
In the present invention, the pH values of the primary medium, the secondary medium and the rooting medium are preferably 5.8. According to the invention, the pH value of the culture medium is preferably adjusted by using 1mol/L sodium hydroxide and 1mol/L hydrochloric acid.
The invention also provides a tissue culture method for rapid propagation of the stem segments of the grape stocks, which adopts the tissue culture medium of the technical scheme and comprises the following steps:
inoculating a sterile explant on a primary culture medium for primary culture to obtain primary cluster buds with the length of 5-6 cm;
transferring the primary cluster buds to a subculture medium for subculture to obtain subcultured cluster buds;
and carrying out rooting culture on the secondary cluster buds.
In the present invention, the sterilized explant is preferably sterilized with an explant disinfectant before being inoculated on the primary medium. The disinfection method is not particularly limited, and preferably includes one or more of sodium hypochlorite, washing powder and detergent, 75% ethanol and 0.1% mercuric chloride, more preferably one or more of sodium hypochlorite, 75% ethanol and 0.1% mercuric chloride, and even more preferably 75% ethanol and mercuric chloride. In an embodiment of the present invention, the sterilization method includes: after washing with running water, soaking with 75% ethanol for 30s, washing with sterile water for 3-5 times, soaking with mercuric chloride solution for 8min, and washing with sterile water for 3-5 times. The invention has no special limitation on the sterile water, and is preferably obtained after the distilled water is sterilized; the sterile water, 75% ethanol, and mercuric chloride are preferably used in amounts to submerge the explant. The 0.1% mercury liter of the invention is preferably obtained by metering to 1000mL per 1g mercury liter.
In the present invention, the sterile explant preferably comprises one or more of a single shoot stem segment, leaf, stem tip and petiole, more preferably one or more of a single shoot stem segment, leaf, stem tip, and even more preferably a single shoot stem segment. The tissue culture medium and the tissue culture method provided by the invention are particularly suitable for taking single-bud stems as explants. In the embodiment of the invention, the explant is preferably a single-bud stem segment, the single-bud stem segment is preferably obtained by taking a young sprout as the explant and shearing off two ends of the sprout after disinfection. The upper end of the stem section is preferably kept 2 cm above the bud, and the lower end of the stem section is preferably kept 2-3 cm below the bud.
In the invention, the young shoots are derived from grape rootstock seedlings. In the present invention, the variety of the grape rootstock is not particularly limited, and in the embodiment of the present invention, the variety of the grape rootstock is preferably selected from three varieties, i.e., 5BB, 1103P and SO 4.
In the present invention, the young shoots are preferably obtained from indoor potted seedlings. The invention obtains the explant material from the indoor potted seedling, effectively reduces the pollution rate of stem section rapid propagation of seedling taking from the field, can culture a large amount of explant material, and is convenient for taking materials.
In the invention, the young shoots are preferably shoots as long as 6-10 knots, more preferably shoots as long as 8-10 knots, and even more preferably shoots as long as 10 knots. According to the invention, young shoots growing to 6-10 knots and having the thickness of 1-2 mm are selected as explants, and the growth to 10 knots can ensure that a large amount of explant materials are obtained, at the moment, the stem sections have strong meristematic capacity, carry less virus, are beneficial to improving the survival rate of subsequent culture, and can ensure that the subsequent materials can continue to grow while taking materials.
In the invention, the temperatures of the primary culture, the secondary culture and the rooting culture are preferably 24-26 ℃ respectively, and more preferably 25 ℃; the humidity is preferably 20 to 50%, more preferably 25 to 45%, and still more preferably 40%.
In the invention, the primary culture and the secondary culture are preferably carried out under illumination conditions, and the illumination time is preferably 12-16 h/d, more preferably 13-15 h/d, and more preferably 12 h/d; the intensity of the illumination is preferably 2000-2500 lx, more preferably 2100-2400 lx, and even more preferably 2300 lx.
In the present invention, the time for the primary culture and the secondary culture is preferably 28 to 32 days, and more preferably 30 days.
In the invention, the rooting culture comprises dark culture and illumination culture, wherein the culture time of the dark culture is preferably 5-7 d, more preferably 6-7 d, and more preferably 7 d; the illumination intensity of the illumination culture is preferably 2000-2500 lx, more preferably 2100-2400 lx, and even more preferably 2300 lx; the illumination time is preferably 12h/d, and the total time of dark culture and illumination culture of rooting culture is preferably 28-40 d, further preferably 30-38 d, and more preferably 35 d.
In the invention, the rooting culture is secondary culture, the early dark environment is favorable for inducing rooting, the rooting rate is improved, and the later culture is carried out in the illumination environment. During the rooting culture, the test-tube plantlet subjected to subculture is inoculated into a rooting culture medium, the whole test-tube plantlet is in a dark environment, and the rooting culture medium is also added with active carbon Ac, so that the dark environment can be provided.
According to the invention, under the combined action of the tissue culture medium and the culture conditions, the propagation coefficient of the cluster buds and the rooting rate of the tissue culture seedlings during rapid propagation of the stem segments of the grape rootstocks are improved, a large number of grape tissue culture seedlings can be obtained, and a technical support is provided for producing improved variety seedlings.
For further illustration of the present invention, the tissue culture medium and tissue culture method for rapid propagation of grape rootstock stem segments provided by the present invention are described in detail below with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Example 1
Annual 5BB stock seedlings from the three-lawn teaching practice base of Xinjiang university of agriculture are used as materials, and the parents are winter grapes multiplied by riparian grapes. Planted on the indoor balcony of the research center of special fruit trees of the gardening academy of Sinkiang university for potted seedling culture. After about 60 days, the young shoots which grow to 10 knots and have the thickness of 1-2 mm are subjected to experimental treatment.
(1) And (3) disinfection of explants: cutting the young shoots into single-bud stem segments with the length of 2-3 cm, putting the single-bud stem segments into a beaker, covering the beaker with gauze, washing the beaker for 1 hour by using running water, and removing surface stains. Then soaking the seeds in 75% ethanol for 30s on a super-clean workbench, washing with sterile water for 5 times, sterilizing with 0.1% mercuric chloride solution for 8min, washing with sterile water for 5 times, and placing in a sterilized inoculation tray for later use.
(2) Primary culture: cutting off the disinfectant-impregnated parts at two ends of the single-bud stem, and collecting 1.0cm long single-bud stem, wherein MS culture medium is used as basic culture medium, and simultaneously 2.0mg/L6-BA, 0.2mg/L IBA, and 0.2mg/L GA are added330g/L sucrose and 7g/L agar. The temperature of the primary culture is 25 ℃, the humidity is 40%, the culture time is 30d, the illumination intensity is 2300lx, and the illumination time is 12 h/d.
(3) Subculturing: inoculating the primary cluster buds with the length of 5-6 cm obtained by primary culture into a subculture medium for culture, wherein the subculture medium takes an MS culture medium as a basic culture medium, and simultaneously adding 2.5mg/L6-BA, 0.5mg/L IBA and 0.5mg/L GA330g/L sucrose and 7g/L agar. The temperature of the subculture was 25 ℃ and the humidity was 40%. The culture time is 30d, the illumination intensity is 2300lx, and the illumination time is 12 h/d. Example 1 the growth of the resulting cluster buds after subculture is shown in FIG. 1.
(4) Rooting culture: after subculture, carrying out rooting culture on the obtained subculture cluster buds, taking 1/2MS culture medium as a basic culture medium, adding 1.5mg/LIBA, 0.1g/LAc, 30g/L sucrose and 7g/L agar, carrying out the total rooting culture for 35d, carrying out the dark rooting culture for 7 days, and carrying out the illumination culture for 28 d. The illumination time is 12h/d during illumination culture, and the illumination intensity is 2300 lx. The temperature for rooting culture is 25 ℃, and the humidity is 40%. And (4) carrying out rooting culture on the subculture clustered shoots to obtain tissue culture seedlings. Example 1 rooting of tissue culture seedlings obtained after rooting culture of the secondary multiple shoots is shown in FIG. 2.
Example 2
Annual 1103P rootstock seedlings from the three-lawn teaching practice base of Xinjiang university of agriculture are used as materials, and the parents are winter grapes multiplied by sandy grapes. Planted on the indoor balcony of the research center of special fruit trees of the gardening academy of Sinkiang university for potted seedling culture. After about 60 days, the seedlings grow to 10 sections, and the young shoots are taken for experimental treatment.
The culture was carried out in the same manner as in example 1 except that the MS medium was used as the minimal medium in the subculture, and 30g/L sucrose, 7g/L agar, 1.25 mg/L6-BA, 1.0mg/L IBA and 0.5mg/L GA were added simultaneously3. Culturing with 1/2MS in rooting cultureThe culture medium is a basic culture medium, 1.0mg/L IBA, 0.1g/LAc, 30g/L sucrose and 7g/L agar are added, and dark culture is carried out for 7 days. The growth of the cluster buds after subculture in example 2 is shown in FIG. 3. Example 2 rooting of the tissue culture plantlets obtained after rooting culture of the secondary multiple shoots is shown in FIG. 4.
Example 3
Annual SO4 rootstock seedlings from three-plateau teaching practice base of Xinjiang university of agriculture are used as materials, and parents are winter grapes multiplied by riparian grapes. Planted on the indoor balcony of the research center of special fruit trees of the gardening academy of Sinkiang university for potted seedling culture. After about 60 days, the young shoots were grown to 10 knots and taken for experimental treatment.
The procedure was as in example 1, except that the MS medium was used as the minimal medium in the subculture, and 30g/L sucrose, 7g/L agar, 2.0mg/L6-BA, 0.2mg/L IBA and 0.2mg/L GA were added simultaneously330g/L sucrose and 7g/L agar. In rooting culture, 1/2MS culture medium is used as a basic culture medium, 1.5mg/L IBA, 0.1g/LAc, 30g/L sucrose and 7g/L agar are added, and dark culture is carried out for 7 days. Example 3 the growth of the resulting cluster buds after subculture is shown in FIG. 5. Example 3 rooting of tissue culture seedlings obtained after rooting culture of the secondary multiple shoots is shown in FIG. 6.
Comparative example 1
The culture was performed in the same manner as in example 1 except that the culture medium was different.
(2) Primary culture: MS culture medium is used as basic culture medium, and 2.0mg/L6-BA, 0.2mg/L IBA and 0.20 mg.L are added at the same time-1GA330g/L sucrose and 7g/L agar.
(3) Subculturing: MS culture medium is used as basic culture medium, and 1.25 mg/L6-BA and 0.5mg/L GA are added at the same time330g/L sucrose and 7g/L agar.
(4) Rooting culture: 1/2MS culture medium is used as basic culture medium, 1.5mg/L IBA, 30g/L sucrose and 7g/L agar are added at the same time, the addition amount of Ac is 0, and dark culture is carried out for 7 days.
Comparative example 2
The culture was performed in the same manner as in example 1 except that the culture medium was different.
(3) Subculturing: MS culture medium is used as basic culture medium, and 1.25 mg/L6-BA, 0.5mg/L IBA, 30g/L sucrose and 7g/L agar are added.
(4) Rooting culture: 1/2MS culture medium is used as basic culture medium, 1.5mg/L IBA, 0.1g/LAc, 30g/L sucrose and 7g/L agar are added at the same time, and dark culture is carried out for 3 days.
Comparative example 3
The cultivation was performed in the same manner as in example 2 except that the subculture medium and rooting medium were different.
(3) Subculturing: wherein MS culture medium is used as basic culture medium, and 1.25 mg/L6-BA and 0.5mg/L GA are added simultaneously330g/L sucrose and 7g/L agar;
(4) rooting culture: and (3) carrying out rooting culture on the subcultured cluster buds, carrying out dark culture for 7 days by taking 1/2MS culture medium as a basic culture medium, wherein the addition amount of IBA is 1.0mg/L and the addition amount of Ac is 0.
Comparative example 4-1
The culture was performed in the same manner as in example 2 except that the subculture medium and the rooting medium were different.
(3) Subculturing: wherein MS culture medium is used as basic culture medium, and 1.25 mg/L6-BA, 0.5mg/L IBA, 30g/L sucrose and 7g/L agar are added.
(4) Rooting culture: carrying out rooting culture on the subcultured cluster buds, taking 1/2MS culture medium as a basic culture medium, adding 1.0mg/L IBA, 0.1g/LAc, 30g/L sucrose and 7g/L agar, and carrying out dark culture for 3 days.
Comparative examples 4 to 2
The cultivation was carried out in the manner of comparative example 4-1 except that the dark cultivation time for rooting cultivation was 0 day.
Comparative example 5
The culture was performed in the same manner as in example 3 except that the subculture medium and the rooting medium were different.
(3) Subculturing: MS culture medium is used as basic culture medium, and 1.25 mg/L6-BA and 0.5mg/L GA are added at the same time330g/L sucrose and 7g/L agar;
(4) rooting culture: using 1/2MS culture medium as basic culture medium, adding 1.5mg/L IBA, 30g/L sucrose and 7g/L agar, adding 0 Ac, and dark culturing for 7 days.
Comparative example 6-1
The culture was performed in the same manner as in example 3 except that the subculture medium and the rooting medium were different.
(3) Subculturing: MS culture medium is used as basic culture medium, and 1.25 mg/L6-BA, 0.5mg/L IBA, 30g/L sucrose and 7g/L agar are added.
(4) Rooting culture: 1/2MS culture medium was used as a minimal medium, and 1.5mg/L IBA, 0.1g/L Ac, 30g/L sucrose and 7g/L agar were added thereto, followed by dark culture for 3 days.
Comparative examples 6 to 2
The cultivation was carried out in the manner of comparative example 6-1 except that the dark cultivation time for rooting cultivation was 0 day.
Comparative example 7
The MS culture medium is changed to B5 culture medium, and the rest steps are the same as example 1.
Comparative example 8
The MS medium was changed to WPM medium, and the procedure was as in example 1.
Comparative example 9
The MS culture medium is changed to B5 culture medium, and the rest steps are the same as the example 2.
Comparative example 10
The MS medium was changed to WPM medium, and the procedure was as in example 2.
Comparative example 11
The MS medium was changed to B5 medium, and the procedure was as in example 3.
Comparative example 12
The MS medium was changed to WPM medium, and the procedure was as in example 3.
The germination rate, death rate, pollution rate and average plant height in the primary culture process of examples 1-3 and comparative examples 7-12 were determined by the following specific methods: the results are shown in Table 1.
The contamination rate (%) (number of explant contamination/total number of explants) x 100%;
mortality (%) — mortality (number of explant deaths/number of explant inoculations) × 100%;
germination (%) — (number of shoots of explant/total number of inoculations of explant) × 100%.
Average plant height: direct measurement with vernier caliper
Example 1 the growth of multiple shoots obtained by primary culture of single shoot segments is shown in FIG. 7. Comparative example 7 the growth of the multiple shoots obtained by primary culture of the single shoot was shown in FIG. 8. Comparative example 8 the growth of multiple shoots obtained by primary culture of single shoot stem was shown in FIG. 9.
Example 2 the growth of multiple shoots obtained by primary culture of single shoot segments is shown in FIG. 10. Comparative example 9 the growth of multiple shoots obtained by primary culture of single shoot stem was shown in FIG. 11. Comparative example 10 the growth of the multiple shoots obtained by primary culturing of the single shoot was shown in FIG. 12.
Example 3 the growth of multiple shoots obtained by primary culture of single shoot segments is shown in FIG. 13. Comparative example 11 the growth of multiple shoots obtained by primary culture of single shoot stem was shown in FIG. 14. Comparative example 12 the growth of multiple shoots obtained by primary culture of single shoot stem was shown in FIG. 15.
TABLE 1 germination, mortality, pollution, average plant height results for examples 1-3 and comparative examples 7-12
The proliferation coefficient of the subculture multiple shoots and the rooting rate of the tissue culture seedlings obtained by the subculture multiple shoots in examples 1 to 3 and comparative examples 1 to 6 were calculated by the following formula: the results are shown in Table 2.
The multiplication coefficient is total bud number/inoculation bud number multiplied by 100%
The rooting rate is equal to the number of rooted seedlings/total inoculated seedlings multiplied by 100 percent
TABLE 2 proliferation coefficient of subculture multiple shoots and rooting rate of tissue culture seedlings of examples 1 to 3 and comparative examples 1 to 6
| Group of | Multiplication coefficient of subculture cluster buds | Rooting percentage of tissue culture seedling (%) |
| Example 1 | 2.89 | 100 |
| Example 2 | 2.22 | 100 |
| Example 3 | 2.22 | 100 |
| Comparative example 1 | 1.78 | 100 |
| Comparative example 2 | 1.56 | 0 |
| Comparative example 3 | 1.22 | 0 |
| Comparative example 4-1 | 1.67 | 100 |
| Comparative examples 4 and 2 | 1.67 | 97.67 |
| Comparative example 5 | 1.11 | 70 |
| Comparative example 6-1 | 1.78 | 100 |
| Comparative examples 6 to 2 | 1.78 | 87.67 |
As can be seen from Table 1, the proliferation coefficient of the secondary multiple shoots and the rooting rate of the tissue culture seedlings obtained by rooting culture of the secondary multiple shoots in examples 1 to 3 of the invention are higher than those in comparative examples 1 to 6. The proliferation coefficients of examples 1 to 3 were 2.22 to 2.89, which were significantly higher than those of comparative examples 1 to 6, i.e., 1.11 to 1.78, the subculture media of comparative examples 1, 3 and 5 had no IBA, and the subculture media of comparative examples 2, 4 and 6 had no GA3The multiplication coefficient of the secondary cluster buds is reduced. In rooting culture, the rooting rate of tissue culture seedlings without adding Ac in comparative examples 3 and 5 is reduced. In comparative examples 4-2 and 6-2, Ac was added, and the reduction in the rooting rate of the tissue culture seedlings was not observed in the dark culture.
Therefore, the subculture medium and the rooting culture medium can simultaneously improve the proliferation coefficient of the subculture multiple shoots and the rooting rate of the tissue culture seedling during rapid propagation of the stem section of the grape stock, and the rooting rate of the tissue culture seedling can also be improved under the dark culture condition, so that a large amount of grape tissue culture seedlings can be obtained, and technical support is provided for producing improved variety seedling.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. A tissue culture medium for rapid propagation of grape rootstock stem segments is characterized by comprising a primary culture medium, a secondary culture medium and a rooting culture medium;
the primary culture medium uses an MS culture medium as a basic culture medium, and further comprises: 2.0mg/L6-BA, 0.2mg/L IBA and 0.2mg/L GA3；
The subculture medium takes an MS culture medium as a basic culture medium, and further comprises: 1.25-2.5 mg/L6-BA, 0.2-1.0 mg/LIBA and 0.2-0.5 mg/L GA3；
The rooting culture medium takes 1/2MS culture medium as a basic culture medium, and further comprises: 1.0-1.5 mg/LIBA and 0.1-0.2 g/LAc.
2. The tissue culture medium of claim 1, wherein the primary culture medium, the secondary culture medium and the rooting culture medium further comprise 30g/L sucrose and 6-8 g/L agar.
3. The tissue culture medium of claim 2, wherein the primary medium, the secondary medium and the rooting medium each have a pH of 5.8.
4. A tissue culture method for rapid propagation of grape rootstock stem segments is characterized in that the tissue culture method adopts the tissue culture medium of any one of claims 1 to 3, and comprises the following steps:
inoculating a sterile explant on a primary culture medium for primary culture to obtain primary cluster buds with the length of 5-6 cm;
transferring the primary cluster buds to a subculture medium for subculture to obtain subcultured cluster buds;
and carrying out rooting culture on the secondary cluster buds.
5. The tissue culture method of claim 4, wherein the explant is a single shoot stem segment.
6. The tissue culture method according to claim 4, wherein the temperature of the primary culture, the temperature of the secondary culture and the temperature of the rooting culture are respectively 24 to 26 ℃ and the humidity is respectively 20 to 50%.
7. The tissue culture method according to claim 4, wherein the primary culture and the secondary culture are performed under illumination conditions, the illumination time is 12-16 h/d, and the illumination intensity is 2000-2500 lx.
8. The tissue culture method according to claim 4, wherein the primary culture time and the secondary culture time are respectively 28-32 d.
9. The tissue culture method according to claim 4, wherein the rooting culture comprises dark culture and illumination culture, and the culture time of the dark culture is 3-7 days; the illumination intensity of the illumination culture is 2000-2500 lx, the illumination time is 12h/d, and the total time of the dark culture and the illumination culture of the rooting culture is 28-40 d.
10. The tissue culture method of claim 4, wherein the sterile explant is sterilized with an explant disinfectant prior to being inoculated onto the primary medium; the disinfection mode comprises the following steps: after washing with running water, soaking with 75% ethanol for 30s, washing with sterile water for 3-5 times, soaking with 0.1% mercuric chloride solution for 8min, and washing with sterile water for 3-5 times.
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| CN102144547A (en) * | 2011-01-14 | 2011-08-10 | 四川农业大学 | Method for quickly breeding and transplanting grape stock unit |
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| CN104938339A (en) * | 2015-06-29 | 2015-09-30 | 大新县科学技术情报研究所 | Rapid propagation method for tissue culture of grape |
| CN112438202A (en) * | 2019-08-28 | 2021-03-05 | 中国农业大学 | Tissue culture and rapid propagation method of grape rootstock |
| CN112438201A (en) * | 2019-08-28 | 2021-03-05 | 中国农业大学 | Tissue culture rapid propagation method of wine grapes |
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| CN102144547A (en) * | 2011-01-14 | 2011-08-10 | 四川农业大学 | Method for quickly breeding and transplanting grape stock unit |
| CN104542298A (en) * | 2015-01-26 | 2015-04-29 | 武汉市林业果树科学研究所 | Grape tissue regeneration culture method |
| CN104938339A (en) * | 2015-06-29 | 2015-09-30 | 大新县科学技术情报研究所 | Rapid propagation method for tissue culture of grape |
| CN112438202A (en) * | 2019-08-28 | 2021-03-05 | 中国农业大学 | Tissue culture and rapid propagation method of grape rootstock |
| CN112438201A (en) * | 2019-08-28 | 2021-03-05 | 中国农业大学 | Tissue culture rapid propagation method of wine grapes |
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| CN116530414A (en) * | 2023-05-22 | 2023-08-04 | 西北农林科技大学 | Method for creating polyploid seedless germplasm by utilizing clustered bud cluster stem tips |
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