CN108935101B - Method for reducing browning rate of begonia leaf tissue culture - Google Patents
Method for reducing browning rate of begonia leaf tissue culture Download PDFInfo
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- 241000218993 Begonia Species 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000005286 illumination Methods 0.000 claims abstract description 65
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 14
- 238000003860 storage Methods 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims description 112
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 45
- 239000000123 paper Substances 0.000 claims description 38
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 26
- 230000006698 induction Effects 0.000 claims description 25
- 229920001817 Agar Polymers 0.000 claims description 23
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 23
- 229930006000 Sucrose Natural products 0.000 claims description 23
- 239000008272 agar Substances 0.000 claims description 23
- 239000005720 sucrose Substances 0.000 claims description 23
- 238000005406 washing Methods 0.000 claims description 22
- 229910052799 carbon Inorganic materials 0.000 claims description 20
- 239000002655 kraft paper Substances 0.000 claims description 17
- 239000008223 sterile water Substances 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 14
- 238000006748 scratching Methods 0.000 claims description 10
- 230000002393 scratching effect Effects 0.000 claims description 10
- 241001481296 Malus spectabilis Species 0.000 claims description 9
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 claims description 9
- 238000012258 culturing Methods 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 7
- 238000004161 plant tissue culture Methods 0.000 abstract description 4
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 abstract description 2
- 230000008635 plant growth Effects 0.000 abstract description 2
- 230000001954 sterilising effect Effects 0.000 abstract description 2
- 238000011081 inoculation Methods 0.000 abstract 1
- 229960002523 mercuric chloride Drugs 0.000 abstract 1
- 238000005057 refrigeration Methods 0.000 abstract 1
- 238000002791 soaking Methods 0.000 description 11
- 230000000052 comparative effect Effects 0.000 description 10
- 238000012136 culture method Methods 0.000 description 10
- 238000011146 sterile filtration Methods 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 244000134242 Malus prunifolia Species 0.000 description 3
- 235000005087 Malus prunifolia Nutrition 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 241000425308 Begonia fimbristipula Species 0.000 description 2
- 241000220225 Malus Species 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 244000099147 Ananas comosus Species 0.000 description 1
- 241001520688 Begonia masoniana Species 0.000 description 1
- 241000218999 Begoniaceae Species 0.000 description 1
- 241000189662 Calla Species 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 241000217407 Margaritifera Species 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 240000008254 Rosa chinensis Species 0.000 description 1
- 235000000664 Rosa chinensis Nutrition 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010352 biotechnological method Methods 0.000 description 1
- 238000012364 cultivation method Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 230000014639 sexual reproduction Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/22—Improving land use; Improving water use or availability; Controlling erosion
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Abstract
The invention relates to a method for reducing the browning rate of begonia leaf tissue culture, belonging to the technical field of plant tissue culture. According to the method, fresh young begonia marmorata leaves are subjected to refrigeration storage treatment before inoculation, and after disinfection and sterilization of alcohol and mercuric chloride, different illumination culture modes in tissue culture are combined with continuous bottle rotating treatment, namely dark culture → semi-dark culture → light culture, and the young begonia marmorata leaves are transferred once every 5-7 days, so that the begonia marmoration is effectively prevented or even eliminated, the browning rate is reduced, the inductivity is improved to a certain extent, and the growth of plants is promoted. Compared with the existing method for reducing browning, the method of the invention does not need to add special equipment, has small using concentration, low cost and simple and convenient operation, and is easy to popularize and apply.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for reducing browning rate of begonia leaf tissue culture.
Background
Begonia (begonia)Begonia masoniana) The plum-leaf crab is a perennial evergreen herb of the begonia of the begonian, the leaf is egg-shaped, the surface of the plum-leaf crab has wrinkles and burs, and the center of the yellow-green leaf surface is embedded with a red-brown horseshoe-shaped girdle band, so the plum-leaf crab is very beautiful and is a more famous and precious variety in the begonias. However, a plant belonging to the genus Malus, which is not easily obtained as a seed, has a weak sexual reproduction ability, many variations in progeny, and is difficult to obtain excellent traitsAnd (4) maintaining. As one of the modern emerging biotechnological methods, the plant tissue culture technology has the advantages of high propagation coefficient, stable genetic character, no limitation of seasons and the like, so that the plant tissue culture technology is very suitable for the popularization and propagation of excellent ornamental plants, is widely applied to the reproduction of ornamental plants such as calla, orchid, chrysanthemum, Chinese rose and the like at present, and has many varieties of successful tissue culture in malus plants. However, there are few reports about the begonia fimbristipula tissue culture, and the conventional tissue culture has a serious browning problem, which severely limits the efficiency of the begonia fimbristipula tissue culture. Therefore, whether the browning can be effectively controlled is the key point of the success of the begonia aquiculture.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a method for reducing the browning rate of begonia leaf tissue culture.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a method for reducing the browning rate of begonia leaf tissue culture comprises the following steps:
step (1), cold storage and preservation treatment: refrigerating fresh young leaves of the Malus spectabilis in a refrigerator at 4 ℃ for 1-2 days;
step (2), explant disinfection and treatment: cleaning the tender leaves treated in the step (1) with laundry powder water, soaking the tender leaves in alcohol with the volume concentration of 70-75% for 20-30 s, and then using HgCl with the mass concentration of 0.1-0.15%2Treating the solution for 5-8 min, and then rinsing the solution with sterile water for multiple times; then, absorbing water by using sterile filter paper under the aseptic condition, and then cutting the leaves into leaf disks;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the back of the leaf disc downwards into a primary culture medium, and culturing for 5 days under a dark condition at the culture temperature of 23 +/-2 ℃;
step (4), semi-dark culture: transferring the culture obtained in the step (3) to a new primary culture medium, then placing the culture into a paper box, tightly covering the paper box, and cutting a light-transmitting gap at each of two sides of the paper box to ensure that scattered light enters the paper box but avoid direct light irradiation; placing the carton in an illumination culture room, wherein the culture temperature is 23 +/-2 ℃, the illumination time is 10-12 h/d, and the illumination intensity is 2000-3000 lx; replacing a new primary culture medium every 5-7 days, and continuously transferring for 3-5 times;
step (5), light culture: inoculating the culture obtained in the step (4) into an adventitious bud induction culture medium, removing the paper box, directly placing the culture on a culture rack for illumination culture, wherein the culture temperature is 23 +/-2 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 10-12 h/d; subculturing once every 20-30 days to obtain adventitious buds;
the primary culture medium is MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the primary culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is an MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon, 80mg/L VC and pH5.8 are added into the culture medium, and the mixture is sterilized at high temperature and cooled to room temperature for later use.
Further, in the step (2), it is preferable that the rinsing with sterile water is performed 3 to 5 times.
Further, it is preferable that in the step (2), rinsing is performed for 2min each time.
Further, in the step (2), the size of the leaf disc is preferably 1.8-2.2 cm2。
Further, in the step (4), the width of each side gap is preferably 4.8-5.2 cm.
Further, it is preferable that in the step (4), the carton is a kraft carton.
Compared with the prior art, the invention has the beneficial effects that:
the fresh begonia leaves are refrigerated and stored before being inoculated, so that the inductivity of the begonia leaves is ensured, and the pollution rate and the browning rate of the begonia leaves are reduced; the alcohol and mercury bichloride are used for disinfection and sterilization, so that the pollution rate and browning rate of primary culture are reduced, and the induction rate of the primary culture is ensured; different illumination culture modes in tissue culture are combined with continuous bottle rotating treatment (namely dark culture → semi-dark culture → light culture, and the bottle is rotated once every 5-7 days), the browning rate can be reduced by 80% -90%, so that the browning of the begonia marmorata with the colored stripes is effectively prevented or even eliminated, the inductivity of the begonia marmorata is improved to a certain extent, and the growth of plants is promoted. Compared with the existing substances for reducing browning, the method of the invention does not need to add special equipment, and has the advantages of small using concentration, low cost and simple and convenient operation.
Drawings
FIG. 1 is a diagram of adventitious buds of begonia variegated obtained by the method of the present invention;
FIG. 2 is a picture of a full plant of Begonia ananas obtained by adventitious bud induction according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
Example 1
A method for reducing the browning rate of begonia leaf tissue culture comprises the following steps:
step (1), cold storage and preservation treatment: refrigerating fresh young leaves of the Malus spectabilis at 4 ℃ for 1 d;
step (2), explant disinfection and treatment: washing the tender leaves treated in the step (1) with washing powder water, soaking the tender leaves in 75% alcohol for 30s, and then using HgCl with the mass concentration of 0.1%2Solution treatment for 5min followed by 3 rinses with sterile water; then, the water was removed by sterile filtration under aseptic conditions, and the leaves were cut into 2cm pieces2A leaf disc of a size;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the back of the leaf disc downwards into a primary culture medium, and culturing for 5 days under a dark condition at the culture temperature of 23 +/-2 ℃;
step (4), semi-dark culture: transferring the culture obtained in the step (3) to a new primary culture medium, then placing the culture into a kraft paper box, tightly covering the paper box, cutting a light-transmitting gap at each side of the paper box to ensure that scattered light enters the paper box but avoid direct light irradiation, wherein the width of the gap at each side is 5 cm; placing the carton in an illumination culture room, wherein the culture temperature is 23 +/-2 ℃, the illumination time is 10-12 h/d, and the illumination intensity is 2000-3000 lx; changing new primary culture medium every 5 days, and continuously transferring for 3 times;
step (5), light culture: inoculating the culture obtained in the step (4) into an adventitious bud induction culture medium, removing a kraft paper box, directly placing the culture on a culture rack for illumination culture, wherein the culture temperature is 23 +/-2 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 10-12 h/d; subculturing every 20d to obtain adventitious bud;
the primary culture medium is MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the primary culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is an MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon, 80mg/L VC and pH5.8 are added into the culture medium, and the mixture is sterilized at high temperature and cooled to room temperature for later use.
Example 2:
a method for reducing the browning rate of begonia leaf tissue culture comprises the following steps:
step (1), refrigerating and preserving treatment: refrigerating fresh tender leaves of Begonia spectabilis in a refrigerator at 4 ℃ for 2 d;
step (2), explant disinfection and treatment: washing the tender leaves treated in the step (1) with washing powder water, soaking the tender leaves in 75% alcohol for 30s, and then using HgCl with the mass concentration of 0.1%2Treating with the solution for 8min, and rinsing with sterile water for 2min for 3 times; then, the leaves are removed by sucking water with sterile filter paper under aseptic conditionsCutting into 2cm2A leaf disc of a size;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the back of the leaf disc downwards into a primary culture medium, and culturing for 5 days under a dark condition at the culture temperature of 23 +/-2 ℃;
step (4), semi-dark culture: transferring the culture obtained in the step (3) to a new primary culture medium, then placing the culture into a kraft paper box, tightly covering the paper box, cutting a light-transmitting gap at each side of the paper box to ensure that scattered light enters the paper box but avoid direct light irradiation, wherein the width of the gap at each side is 5 cm; placing the carton in an illumination culture room, wherein the culture temperature is 23 +/-2 ℃, the illumination time is 10-12 h/d, and the illumination intensity is 2000-3000 lx; changing new primary culture medium every 5 days, and continuously transferring for 5 times;
step (5), light culture: inoculating the culture obtained in the step (4) into an adventitious bud induction culture medium, removing a kraft paper box, directly placing on a culture rack for illumination culture, wherein the culture temperature is 23 +/-2 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 10-12 h/d; subculturing every 25d to obtain adventitious bud;
the primary culture medium is MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the primary culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is an MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon and 80mg/L VC are added into the culture medium, the pH value is 5.8, and the culture medium is sterilized at high temperature and cooled to room temperature for later use.
Example 3
A method for reducing the browning rate of begonia leaf tissue culture comprises the following steps:
step (1), cold storage and preservation treatment: refrigerating fresh young leaves of the Malus spectabilis at 4 ℃ for 1 d;
step (2), explant disinfection and treatment: washing the tender leaves treated in the step (1) with washing powder water, soaking the tender leaves in 70% alcohol for 20s, and then using 0.1% HgCl2At the solutionTreating for 5min, and rinsing with sterile water for 2min for 3 times; then, the water was removed by sterile filtration under aseptic conditions, and the leaves were cut into 1.8cm pieces2A leaf disc of a size;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the back of the leaf disc downwards into a primary culture medium, and culturing for 5 days under a dark condition at the culture temperature of 23 +/-2 ℃;
step (4), semi-dark culture: transferring the culture obtained in the step (3) to a new primary culture medium, then placing the culture into a kraft paper box, tightly covering the paper box, cutting a light-transmitting gap at each side of the paper box to ensure that scattered light enters the paper box but avoid direct light irradiation, wherein the width of the gap at each side is 4.8 cm; placing the carton in a lighting culture room, wherein the culture temperature is 23 +/-2 ℃, the lighting time is 10h/d, and the lighting intensity is 2000-2200 lx; changing new primary culture medium every 5 days, and continuously transferring for 3 times;
step (5), light culture: inoculating the culture obtained in the step (4) into an adventitious bud induction culture medium, removing a kraft paper box, directly placing the culture on a culture rack for illumination culture, wherein the culture temperature is 23 +/-2 ℃, the illumination intensity is 2000-2200 lx, and the illumination time is 10 h/d; subculturing every 20d to obtain adventitious bud;
the primary culture medium is MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the primary culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is an MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon and 80mg/L VC are added into the culture medium, the pH value is 5.8, and the culture medium is sterilized at high temperature and cooled to room temperature for later use.
Example 4
A method for reducing the browning rate of begonia leaf tissue culture comprises the following steps:
step (1), cold storage and preservation treatment: refrigerating fresh young leaves of the Malus spectabilis at 4 ℃ for 2 days;
step (2), explant disinfection and treatment: the young leaves treated in the step (1) are put into a pot for storageWashing the tablet with washing powder water, soaking in 75 vol% ethanol for 30s, and adding HgCl 0.15 wt%2The solution treatment is carried out for 8min, and then the solution is rinsed for 5 times with sterile water, and each rinsing is carried out for 2 min; then, the water was removed by sterile filtration under aseptic conditions, and the leaves were cut into 2.2cm pieces2A leaf disc of a size;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the back of the leaf disc downwards into a primary culture medium, and culturing for 5 days under a dark condition at the culture temperature of 23 +/-1 ℃;
step (4), semi-dark culture: transferring the culture obtained in the step (3) into a new primary culture medium, then placing the culture into a kraft paper box, tightly covering the paper box, cutting a light-transmitting gap at each side of the paper box to ensure that scattered light enters the paper box but avoid direct light irradiation, wherein the width of the gap at each side is 4.8-5.2 cm; placing the carton in an illumination culture room, wherein the culture temperature is 23 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is 2800-3000 lx; changing new primary culture medium every 7 days, and continuously transferring for 5 times;
step (5), light culture: inoculating the culture obtained in the step (4) into an adventitious bud induction culture medium, removing a kraft paper box, directly placing on a culture rack for illumination culture, wherein the culture temperature is 23 +/-1 ℃, the illumination intensity is 2800-3000 lx, and the illumination time is 12 h/d; subculturing every 30d to obtain adventitious bud;
the primary culture medium is MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the primary culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is an MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon and 80mg/L VC are added into the culture medium, the pH value is 5.8, and the culture medium is sterilized at high temperature and cooled to room temperature for later use.
Example 5
A method for reducing the browning rate of begonia leaf tissue culture comprises the following steps:
step (1), cold storage and preservation treatment: refrigerating fresh young leaves of the Malus spectabilis at 4 ℃ for 1.5 days;
step (2), explant disinfection and treatment: washing the young leaves treated in the step (1) with washing powder water, soaking the young leaves in 72% alcohol for 25s, and then using HgCl with the mass concentration of 0.12%2Treating with the solution for 7min, and rinsing with sterile water for 2min for 4 times; then, the water was removed by sterile filtration under aseptic conditions, and the leaves were cut into 2cm pieces2A leaf disc of a size;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the back of the leaf disc downwards into a primary culture medium, and culturing for 5 days under a dark condition at the culture temperature of 23 +/-0.5 ℃;
step (4), semi-dark culture: transferring the culture obtained in the step (3) to a new primary culture medium, then placing the culture into a kraft paper box, tightly covering the paper box, cutting a light-transmitting gap at each side of the paper box to ensure that scattered light enters the paper box but avoid direct light irradiation, wherein the width of the gap at each side is 5 cm; placing the carton in an illumination culture room, wherein the culture temperature is 23 +/-0.5 ℃, the illumination time is 11h/d, and the illumination intensity is 2300-2600 lx; changing new primary culture medium every 6 days, and continuously transferring for 4 times;
step (5), light culture: inoculating the culture obtained in the step (4) into an adventitious bud induction culture medium, removing a kraft paper box, directly placing the culture on a culture rack for illumination culture, wherein the culture temperature is 23 +/-0.5 ℃, the illumination intensity is 2300-2600 lx, and the illumination time is 11 h/d; subculturing every 25d to obtain adventitious bud;
the primary culture medium is MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the primary culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is an MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon, 80mg/L VC and pH5.8 are added into the culture medium, and the mixture is sterilized at high temperature and cooled to room temperature for later use.
Comparative example 1:
cleaning fresh tender leaf of Begonia speciosa with washing powder water, and adding 75% alcoholSoaking for 30s, and adding 0.1% HgCl2The treatment is carried out for 6min, and rinsed 5 times with sterile water for 2min each time. Then, the water was removed by aseptic filter paper under aseptic conditions, and after ensuring no residual water, the leaves were cut into 2cm pieces2A large small leaf disk was scratched on the back side of the leaf disk, and the leaf disk was inoculated with primary medium (MS +0.5 mg/L6-BA +0.1mg/L NAA +30g/L sucrose +6.5g/L agar +2.0g/L activated carbon, pH 5.8) with the back side facing downward. And performing conventional illumination culture at the culture temperature of 23 +/-2 ℃, the illumination intensity of 2000-3000 lx and the illumination time of 10-12 h/d. Subculture every 5 d.
Comparative example 2:
(1) refrigerating fresh tender leaves of Begonia spectabilis in a refrigerator at 4 ℃ for 2 d;
(2) washing the tender leaves treated in the step (1) with washing powder water, soaking in 75% alcohol for 30s, and adding 0.1% HgCl2The treatment is carried out for 8min, and the solution is rinsed 3 times with sterile water for 2min each time. Then, the water was removed by aseptic filter paper under aseptic conditions, and after ensuring no residual water, the leaves were cut into 2cm pieces2A large small leaf disk was scratched on the back side of the leaf disk, and the back side of the leaf disk was inoculated downward into an adventitious bud induction medium (MS +0.5 mg/L6-BA +0.1mg/L NAA +30g/L sucrose +6.5g/L agar +2.0g/L activated carbon +80mg/L VC, pH 5.8). And performing conventional illumination culture at the culture temperature of 23 +/-2 ℃, the illumination intensity of 2000-3000 lx and the illumination time of 10-12 h/d. Subcultured every 5 d.
Comparative example 3:
cleaning fresh tender leaf of Begonia spectabilis with washing powder water, soaking in 75% alcohol for 30s, and adding 0.1% HgCl2The treatment is carried out for 8min, and the mixture is rinsed 5 times for 2min each time with sterile water. Then, the water was removed by aseptic filter paper under aseptic conditions, and after ensuring that no residual water was present, the leaves were cut into about 2cm pieces2A large small leaf disk was scratched on the back side of the leaf disk, and the leaf disk was inoculated with primary medium (MS +0.5 mg/L6-BA +0.1mg/L NAA +30g/L sucrose +6.5g/L agar +2.0g/L activated carbon, pH 5.8) with the back side facing downward. And performing conventional illumination culture at the culture temperature of 23 +/-2 ℃, the illumination intensity of 2000-3000 lx and the illumination time of 10-12 h/d. After 30d, observation was performed.
Comparative example 4:
comparative example 4 is different from example 2 in that the semi-dark culture step is not performed, and the rest is the same. The method specifically comprises the following steps:
a method for reducing the browning rate of begonia leaf tissue culture comprises the following steps:
step (1), cold storage and preservation treatment: refrigerating fresh young leaves of the Malus spectabilis at 4 ℃ for 2 days;
step (2), explant disinfection and treatment: washing the tender leaves treated in the step (1) with washing powder water, soaking the tender leaves in 75% alcohol for 30s, and then using HgCl with the mass concentration of 0.1%2Treating with the solution for 8min, and rinsing with sterile water for 2min for 3 times; then, the water was removed by sterile filtration under aseptic conditions, and the leaves were cut into 2cm pieces2A leaf disc of a size;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the back of the leaf disc downwards into a primary culture medium, and culturing for 5 days under a dark condition at the culture temperature of 23 +/-2 ℃;
step (4), light culture: inoculating the culture obtained in the step (3) into an adventitious bud induction culture medium, removing a kraft paper box, directly placing the culture on a culture rack for illumination culture, wherein the culture temperature is 23 +/-2 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 10-12 h/d; subculturing every 25d to obtain adventitious bud;
the primary culture medium is MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the primary culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is an MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon, 80mg/L VC and pH5.8 are added into the culture medium, and the mixture is sterilized at high temperature and cooled to room temperature for later use.
Comparative example 5:
comparative example 5 is different from example 2 in that there is no dark culture step, and the rest is the same. The method specifically comprises the following steps:
a method for reducing the browning rate of begonia leaf tissue culture comprises the following steps:
step (1), cold storage and preservation treatment: refrigerating fresh young leaves of the Malus spectabilis at 4 ℃ for 2 days;
step (2), explant disinfection and treatment: washing the tender leaves treated in the step (1) with washing powder water, soaking the tender leaves in 75% alcohol for 30s, and then using HgCl with the mass concentration of 0.1%2Treating with the solution for 8min, and rinsing with sterile water for 2min for 3 times; then, the water was removed by sterile filtration under aseptic conditions, and the leaves were cut into 2cm pieces2A leaf disc of a size;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the leaf disc into a primary culture medium with the back facing downwards, then placing the leaf disc into a kraft paper box, tightly covering the paper box, cutting a light-transmitting gap at each of two sides of the paper box to ensure that scattered light enters the paper box, but avoiding direct light irradiation, wherein the width of the gap at each side is 5 cm; placing the carton in an illumination culture room, wherein the culture temperature is 23 +/-2 ℃, the illumination time is 10-12 h/d, and the illumination intensity is 2000-3000 lx; changing new primary culture medium every 5 days, and continuously transferring for 5 times;
step (4), light culture: inoculating the culture obtained in the step (3) into an adventitious bud induction culture medium, removing a kraft paper box, directly placing on a culture rack for illumination culture, wherein the culture temperature is 23 +/-2 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 10-12 h/d; subculturing every 25d to obtain adventitious bud;
the primary culture medium is MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the primary culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is an MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon, 80mg/L VC and pH5.8 are added into the culture medium, and the mixture is sterilized at high temperature and cooled to room temperature for later use.
Comparative example 6:
comparative example 6 is different from example 2 in that DMEM medium was used as a basic medium, and the rest was the same. The method comprises the following specific steps:
a method for reducing the browning rate of begonia leaf tissue culture comprises the following steps:
step (1), cold storage and preservation treatment: refrigerating fresh young leaves of the Malus spectabilis at 4 ℃ for 2 days;
step (2), explant disinfection and treatment: washing the tender leaves treated in the step (1) with washing powder water, soaking the tender leaves in 75% alcohol for 30s, and then using HgCl with the mass concentration of 0.1%2Solution treatment for 8min, followed by 3 rinses with sterile water for 2min each; then, the water was removed by sterile filtration under aseptic conditions, and the leaves were cut into 2cm pieces2A leaf disc of a size;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the back of the leaf disc downwards into a primary culture medium, and culturing for 5 days under a dark condition at the culture temperature of 23 +/-2 ℃;
step (4), semi-dark culture: transferring the culture obtained in the step (3) to a new primary culture medium, then placing the culture into a kraft paper box, tightly covering the paper box, cutting a light-transmitting gap at each side of the paper box to ensure that scattered light enters the paper box but avoid direct light irradiation, wherein the width of the gap at each side is 5 cm; placing the carton in an illumination culture room, wherein the culture temperature is 23 +/-2 ℃, the illumination time is 10-12 h/d, and the illumination intensity is 2000-3000 lx; changing new primary culture medium every 5 days, and continuously transferring for 5 times;
step (5), light culture: inoculating the culture obtained in the step (4) into an adventitious bud induction culture medium, removing a kraft paper box, directly placing the culture on a culture rack for illumination culture, wherein the culture temperature is 23 +/-2 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 10-12 h/d; subculturing every 25d to obtain adventitious bud;
the primary culture medium is a DMEM culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the DMEM culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is a DMEM culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon and 80mg/L VC are added into the culture medium, the pH value is 5.8, and the culture medium is sterilized at high temperature and cooled to room temperature for later use.
The above examples and comparative examples treated 30 leaves per group, and after about 1 month of cultivation, the browning rate and the induction rate of the begonia variegate plants were observed and recorded, and the results are shown in table 1.
Table 1 influence of different cultivation methods on the browning rate and the inductivity of begonia variegates.
Different culture methods were comparatively analyzed, including: the culture method (dark culture → light culture method) and the conventional culture method (light culture method) have influence on the browning rate and the inductivity of the begonia margaritifera. The results show that: the browning rate of the culture method is obviously lower than that of a control culture method, and the induction rate is obviously higher than that of the control culture method. By comprehensive data analysis, the culture method is obviously superior to the conventional culture method.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (4)
1. A method for reducing the browning rate of begonia leaf tissue culture is characterized by comprising the following steps:
step (1), cold storage and preservation treatment: refrigerating fresh young leaves of the Malus spectabilis in a refrigerator at 4 ℃ for 1-2 days;
step (2), explant disinfection and treatment: washing the tender leaves treated in the step (1) with washing powder water, and then firstly using the washing powder water with the volume concentration ofSoaking the mixture in 70-75% alcohol for 20-30 s, and then using HgCl with the mass concentration of 0.1-0.15%2Treating the solution for 5-8 min, and then rinsing the solution with sterile water for multiple times; then, absorbing water by using sterile filter paper under the aseptic condition, and then cutting the leaves into leaf disks;
step (3), dark culture: scratching the back of the leaf disc obtained in the step (2), inoculating the back of the leaf disc downwards into a primary culture medium, and culturing for 5 days under a dark condition at the culture temperature of 23 +/-2 ℃;
step (4), semi-dark culture: transferring the culture obtained in the step (3) to a new primary culture medium, then placing the culture into a paper box, tightly covering the paper box, and cutting a light-transmitting gap at each of two sides of the paper box to ensure that scattered light enters the paper box but avoid direct light irradiation; placing the carton in an illumination culture room, wherein the culture temperature is 23 +/-2 ℃, the illumination time is 10-12 h/d, and the illumination intensity is 2000-3000 lx; replacing a new primary culture medium every 5-7 days, and continuously transferring for 3-5 times;
step (5), light culture: inoculating the culture obtained in the step (4) into an adventitious bud induction culture medium, removing the paper box, directly placing the culture on a culture rack for illumination culture, wherein the culture temperature is 23 +/-2 ℃, the illumination intensity is 2000-3000 lx, and the illumination time is 10-12 h/d; subculturing once every 20-30 days to obtain adventitious buds;
the primary culture medium is MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar and 2.0g/L active carbon are added into the culture medium, the pH value is 5.8, and the primary culture medium is sterilized at high temperature and cooled to room temperature for later use;
the adventitious bud induction culture medium is an MS culture medium, 0.5 mg/L6-BA, 0.1mg/L NAA, 30g/L sucrose, 6.5g/L agar, 2.0g/L active carbon, 80mg/L VC and pH5.8 are added into the culture medium, and the culture medium is sterilized at high temperature and cooled to room temperature for later use;
in the step (2), the size of the leaf disc is 1.8-2.2 cm2；
In the step (4), the width of the gap on each side is 4.8-5.2 cm.
2. The method for reducing the tissue culture browning rate of the begonia leaves according to claim 1, wherein in the step (2), the number of times of rinsing with sterile water is 3-5.
3. The method for reducing the tissue culture browning rate of the begonia leaves according to claim 2, wherein in the step (2), the leaves are rinsed for 2min each time.
4. The method for reducing the browning rate of begonia leaf tissue culture according to claim 2, wherein in the step (4), the paper boxes are kraft paper boxes.
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