CN111512962B - Prunus humilis tissue culture method - Google Patents
Prunus humilis tissue culture method Download PDFInfo
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- CN111512962B CN111512962B CN202010382685.6A CN202010382685A CN111512962B CN 111512962 B CN111512962 B CN 111512962B CN 202010382685 A CN202010382685 A CN 202010382685A CN 111512962 B CN111512962 B CN 111512962B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- 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
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- 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
- A01H4/001—Culture apparatus for tissue culture
Abstract
The present application provides a method for culturing cerasus humilis tissue, comprising: (1) explant collection and disinfection: selecting an Prunus humilis Bunge plant with good growth condition, taking the tender branch germinated in spring and the Prunus humilis Bunge stem segment with axillary buds as an explant, disinfecting the explant by a disinfectant, (2) inducing the axillary buds: inoculating the cut explants into a bottle of a starting culture medium to obtain cerasus humilis tissue culture seedlings, (3) carrying out proliferation culture: transferring the cerasus humilis tissue culture seedlings into a bottle of a proliferation culture medium for proliferation culture to obtain cerasus humilis proliferation seedlings, (4) rooting culture: the method for culturing the cerasus humilis tissues has the advantages that the steps are simple, callus induction is not needed, stems with axillary buds are directly used for inducing and generating buds, then proliferation culture and rooting culture are carried out, a large amount of high-quality cerasus humilis rooting tissue culture seedlings can be rapidly obtained, the propagation speed is high, and the tissue culture period is short.
Description
Technical Field
The application relates to the technical field of planting, in particular to a cerasus humilis tissue culture method.
Background
Cerasus humilis (cerasus humilis) is perennial deciduous shrub of the genus prunus in the family of rosaceae, is a special wild fruit tree in China, has high content of mineral elements iron and calcium in the fruit, and is commonly called as 'calcium fruit'. The cerasus humilis is a tree species with strong adaptability, has the characteristics of drought resistance, barren resistance, cold resistance, saline-alkali resistance and the like, and is a good ecological tree species. The cerasus humilis fruit is bright in color and unique in flavor, contains abundant vitamins, mineral elements, organic acids, tannin and other bioactive substances, can be eaten fresh, and can be processed into various products such as fruit juice, fruit wine, jam and the like, and the cerasus humilis kernel can be used as a medicine. It has wide application and high economic value.
In the prior art, prunus humilis is generally bred by seedling breeding methods such as seed and cuttage, but the seed breeding and germination are difficult, and the cuttage breeding and rooting are difficult, so the conventional seedling breeding coefficient is low. The tissue culture method can be used for large-scale production, and has low variation rate.
In the existing cerasus humilis tissue culture technology, leaves or stem segments are mostly adopted for callus induction, then the callus is used for inducing to generate buds, and then propagation culture and rooting culture are carried out.
Disclosure of Invention
In view of this, the embodiments of the present application provide a method for culturing cerasus humilis tissue to solve the technical defects in the prior art.
The embodiment of the application provides a cerasus humilis tissue culture method, which comprises the following steps:
(1) explant collection and disinfection: selecting an Prunus humilis Bunge plant with good growth condition, taking Prunus humilis Bunge stem segments with axillary buds and shoots germinated in spring as explants, sterilizing the explants by using a disinfectant, and cutting the sterilized explants into segments, wherein each segment has 1-2 axillary buds;
(2) axillary bud induction: inoculating the cut explants into a bottle of a starting culture medium to obtain cerasus humilis tissue culture seedlings, wherein the starting culture medium comprises: 1 part by mass of a basic culture medium, 0.3-0.7 part by mass of 6-benzylaminopurine, 0.03-0.06 part by mass of naphthylacetic acid, 35-45 parts by mass of sugar, 6-6.5 parts by mass of agar, and the pH is 6-6.2;
(3) and (3) proliferation culture: transferring the cerasus humilis tissue culture seedlings into a bottle of a proliferation culture medium for proliferation culture to obtain cerasus humilis proliferation seedlings, wherein the proliferation culture medium comprises: 1 part by mass of a basic culture medium, 0.3-0.7 part by mass of 6-benzylaminopurine, 0.03-0.06 part by mass of naphthylacetic acid, 35-45 parts by mass of sugar, 6-6.5 parts by mass of agar, and the pH is 6-6.2;
(4) rooting culture: transferring the propagation seedlings of the cerasus humilis to a rooting culture medium to obtain root seedlings of the cerasus humilis, wherein the rooting culture medium comprises: 0.4-0.6 parts by mass of a minimal medium, 1.3-1.7 parts by mass of indole-3-acetic acid, 25-35 parts by mass of sugar, 6-6.5 parts by mass of agar, and the pH value is 6-6.1.
Optionally, the axillary bud induction, proliferation culture and rooting culture are all performed under the following conditions: culturing in an illumination culture room at 25 ℃ for 14 hours each day with illumination intensity of 2000-.
Optionally, sterilizing the explant with a sterilizing solution, and then cutting the sterilized explant, comprising:
cutting off leaves on branches of the explants, and washing the cut leaves clean by tap water;
putting the explant with the cut leaves into 75% ethanol solution for disinfection for 30-60 seconds, and then putting the explant into 2% sodium hypochlorite solution for disinfection for 15 minutes;
soaking in sterile water for 4-6 times, and drying with sterile filter paper;
and cutting the sterilized explants into sections according to the principle of cutting 1 section of the sterilized explants.
Optionally, inoculating the cut explant into a bottle of a start medium to obtain a cerasus humilis tissue culture seedling, comprising: inoculating the cut explants into bottles of a starting culture medium, inoculating 1 explant into each bottle, and carrying out starting culture for 20-25 days to obtain Prunus humilis tissue culture seedlings.
Optionally, transferring the cerasus humilis tissue culture seedling into a bottle of a proliferation culture medium for proliferation culture to obtain a cerasus humilis proliferation seedling, including: transferring the Prunus humilis tissue culture seedlings into a bottle of a proliferation culture medium for proliferation culture, inoculating 7-10 Prunus humilis tissue culture seedlings into each bottle, and culturing for 25-45 days to obtain Prunus humilis proliferation seedlings, wherein each Prunus humilis proliferation seedling comprises multiple sections, each section is proliferated to obtain 8-12 clumpy branches, and the length of each clumpy branch is 3-4 cm.
Optionally, the method for transferring the propagation seedlings of cerasus humilis to a rooting medium to obtain the rooted seedlings of cerasus humilis comprises:
obliquely cutting the propagation seedlings of cerasus humilis from the stretched internodes to form incisions, and keeping stem tips without keeping dense leaf clusters of bases;
and transferring the cut end of each section of the propagation seedling of the cerasus humilis into the rooting culture medium, and transferring for more than 20 days to obtain the root-growing seedling of the cerasus humilis, wherein the height of each section of the propagation seedling of the cerasus humilis is more than 2 cm.
Optionally, the priming medium comprises: 1 part by mass of a basic culture medium, 0.6 part by mass of 6-benzylaminopurine, 0.05 part by mass of naphthylacetic acid, 40 parts by mass of sugar and 6 parts by mass of agar.
Optionally, the proliferation medium comprises: 1 part by mass of a basic culture medium, 0.6 part by mass of 6-benzylaminopurine, 0.05 part by mass of naphthylacetic acid, 40 parts by mass of sugar and 6 parts by mass of agar.
Optionally, the rooting medium comprises: 0.5 part by mass of a minimal medium, 1.5 parts by mass of indole-3-acetic acid, 30 parts by mass of sugar and 6 parts by mass of agar.
Optionally, the sugar is one of white sugar or sucrose.
The method for culturing the cerasus humilis tissues is simple in steps, callus does not need to be induced, stems with axillary buds are directly used for inducing and generating buds, then propagation culture and rooting culture are carried out, a large number of high-quality cerasus humilis rooting tissue culture seedlings can be quickly obtained, the propagation speed is high, and the tissue culture period is short.
Drawings
FIG. 1 is a schematic flow chart of a Prunus humilis tissue culture method according to the first embodiment of the present application;
FIGS. 2 and 3 are graphs showing the effect of the explants of example II of the present application after axillary bud induction;
FIGS. 4 and 5 are graphs showing the effect of the propagation culture step performed on cerasus humilis tissue culture seedlings according to the second embodiment of the present application;
FIG. 6 is a diagram showing the effect of the rooting culture step in the second embodiment of the present invention.
Detailed Description
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. This application is capable of implementation in many different ways than those herein set forth and of similar import by those skilled in the art without departing from the spirit of this application and is therefore not limited to the specific implementations disclosed below.
The terminology used in the description of the one or more embodiments is for the purpose of describing the particular embodiments only and is not intended to be limiting of the description of the one or more embodiments. As used in one or more embodiments of the present specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used in one or more embodiments of the present specification refers to and encompasses any and all possible combinations of one or more of the associated listed items.
It will be understood that, although the terms first, second, etc. may be used herein in one or more embodiments to describe various information, these information should not be limited by these terms. These terms are only used to distinguish one type of information from another. For example, a first can also be referred to as a second and, similarly, a second can also be referred to as a first without departing from the scope of one or more embodiments of the present description. The word "if" as used herein may be interpreted as "at … …" or "when … …" or "in response to a determination", depending on the context.
First, one or more terms referred to in the present application are schematically explained.
(1) Minimal Medium (MS): the plant tissue culture is used as a basic culture medium of a culture medium for rapid propagation. The minimal media for different microorganisms are not identical, for example:
complete medium: all natural or semi-natural media that meet the nutritional requirements of all auxotrophic strains become Complete Media (CM), sometimes denoted by the "+". The complete culture medium is rich and comprehensive in nutrition, and can be prepared by adding natural substances such as amino acid, vitamin and basic group in a basic culture medium.
Supplementing a culture medium: any combination medium that can only meet the growth requirements of the corresponding auxotrophs becomes a Supplemental Medium (SM). It is formed by adding nutrient factors which cannot be synthesized by the strain into a minimal medium.
Wild type strains: the original strain before mutation of the microorganism isolated from nature becomes a wild type strain.
Auxotrophy: the wild type strain loses the ability to synthesize certain nutrients (amino acids, vitamins, nucleic acids, etc.) through artificial mutagenesis or natural mutation, and can grow only by supplementing deficient nutritional factors in a minimal medium to become auxotrophs (auxotroph). Auxotrophy is a biochemical mutant, the occurrence of which is caused by gene mutation. The carrier of genetic information is a series of nucleic acid series coded by enzyme protein, if a certain base in the nucleic acid series is mutated, the enzyme synthesis controlled by said gene is blocked, so that said strain can not synthesize a certain nutrient factor, and its normal metabolism is out of balance.
(2) 6-benzylaminopurine (6-BA): can promote the division and growth of plant cells, inhibit the degradation of plant chlorophyll, increase the content of amino acid, and delay leaf senescence.
(3) Naphthylacetic acid (NAA): promote cell division and expansion, and induce the formation of adventitious roots.
(4) Indole-3-acetic acid (IAA): promoting rooting.
In order to solve the technical defects of complicated steps, difficult operation, low propagation efficiency and long tissue culture period in the prior art, the application provides a cerasus humilis tissue culture method, and the method is explained in detail in the following embodiments one by one.
The embodiment of the application discloses a cerasus humilis tissue culture method, which is shown in figure 1 and comprises the following steps of 101-104:
101. explant collection and disinfection: selecting Prunus humilis Bunge plants with good growth conditions, taking Prunus humilis Bunge stem segments with axillary buds and shoots germinated in spring as explants, sterilizing the explants by using a disinfectant, and cutting the sterilized explants into segments, wherein each segment has 1-2 axillary buds.
Specifically, in step 101, the step of disinfecting the explant with a disinfectant solution, and then cutting the disinfected explant, includes:
s11, cutting leaves on the branches of the explants, and washing the cut leaves clean with tap water.
Specifically, Prunus humilis Bunge plants with good growth conditions can be selected, and stem segments with axillary buds and germinating twigs in spring are selected as explants.
S12, putting the explant with the cut leaves into 75% ethanol solution for disinfection for 30-60 seconds, and then putting the explant into 2% sodium hypochlorite solution for disinfection for 15 minutes.
S13, after the disinfection, the tea is soaked in sterile water for 4-6 times, and the water is absorbed by sterilized filter paper.
And S14, cutting the sterilized explants into segments according to the principle of cutting 1 segment into 1 segment.
And after cutting, taking each section as 1 section, and reserving 1-2 axillary buds in each section.
102. Axillary bud induction: inoculating the cut explants into a bottle of a starting culture medium to obtain cerasus humilis tissue culture seedlings, wherein the starting culture medium comprises: 1 part by mass of a basic culture medium, 0.3-0.7 part by mass of 6-benzylaminopurine, 0.03-0.06 part by mass of naphthylacetic acid, 35-45 parts by mass of sugar, 6-6.5 parts by mass of agar and the pH value of the mixture is 6-6.2.
Specifically, step 102 includes: the explants after cutting were inoculated into flasks of priming medium, 1 explant per flask.
And performing start culture for 20-25 days to obtain cerasus humilis tissue culture seedlings.
In particular, the components of the start-up medium may include a variety of, for example:
one is as follows: 1 part by mass of a minimal medium, 0.3 part by mass of 6-benzylaminopurine, 0.06 part by mass of naphthylacetic acid, 35 parts by mass of sugar, 6 parts by mass of agar, and pH6.
The other mixture ratio is as follows: 1 part by mass of a minimal medium, 0.7 part by mass of 6-benzylaminopurine, 0.03 part by mass of naphthylacetic acid, 45 parts by mass of sugar, 6.5 parts by mass of agar, and pH 6.1.
The other one is as follows: 1 part by mass of a minimal medium, 0.6 part by mass of 6-benzylaminopurine, 0.05 part by mass of naphthylacetic acid, 40 parts by mass of sugar, 6.2 parts by mass of agar, and pH 6.2.
Wherein the sugar is one of white sugar or sucrose.
103. And (3) proliferation culture: transferring the cerasus humilis tissue culture seedlings into a bottle of a proliferation culture medium for proliferation culture to obtain cerasus humilis proliferation seedlings, wherein the proliferation culture medium comprises: 1 part by mass of a basic culture medium, 0.3-0.7 part by mass of 6-benzylaminopurine, 0.03-0.06 part by mass of naphthylacetic acid, 35-45 parts by mass of sugar, 6-6.5 parts by mass of agar and the pH value of the mixture is 6-6.2.
Specifically, step 103 includes: transferring the Prunus humilis tissue culture seedlings into a bottle of a proliferation culture medium for proliferation culture, inoculating 7-10 Prunus humilis tissue culture seedlings into each bottle, and culturing for 25-45 days to obtain Prunus humilis proliferation seedlings, wherein each Prunus humilis proliferation seedling comprises multiple sections, each section is proliferated to obtain 8-12 clumpy branches, and the length of each clumpy branch is 3-4 cm.
Wherein the multiplication medium comprises:
one is as follows: 1 part by mass of a minimal medium, 0.3 part by mass of 6-benzylaminopurine, 0.06 part by mass of naphthylacetic acid, 35 parts by mass of sugar, 6 parts by mass of agar, and pH6.
The other mixture ratio is as follows: 1 part by mass of a minimal medium, 0.7 part by mass of 6-benzylaminopurine, 0.03 part by mass of naphthylacetic acid, 45 parts by mass of sugar, 6.5 parts by mass of agar, and pH 6.1.
The other one is as follows: 1 part by mass of a minimal medium, 0.6 part by mass of 6-benzylaminopurine, 0.05 part by mass of naphthylacetic acid, 40 parts by mass of sugar, 6.2 parts by mass of agar, and pH 6.2.
Wherein the sugar is one of white sugar or sucrose.
104. Rooting culture: transferring the propagation seedlings of the cerasus humilis to a rooting culture medium to obtain root seedlings of the cerasus humilis, wherein the rooting culture medium comprises: 0.4-0.6 parts by mass of a minimal medium, 1.3-1.7 parts by mass of indole-3-acetic acid, 25-35 parts by mass of sugar, 6-6.5 parts by mass of agar, and the pH value is 6-6.1.
In particular, the components of the rooting medium may include a variety of, for example:
one is as follows: 0.5 parts by mass of a minimal medium, 1.5 parts by mass of indole-3-acetic acid, 30 parts by mass of sugar, 6 parts by mass of agar, pH6.
The other mixture ratio is as follows: 0.4 part by mass of a minimal medium, 1.7 parts by mass of indole-3-acetic acid, 25 parts by mass of sugar, 6.3 parts by mass of agar, and pH 6.1.
The other one is as follows: 0.6 part by mass of a minimal medium, 1.3 parts by mass of indole-3-acetic acid, 35 parts by mass of sugar, 6.5 parts by mass of agar, and pH 6.1.
Wherein the sugar is one of white sugar or sucrose.
Specifically, in step 104, the propagation seedlings of cerasus humilis are transferred to a rooting medium to obtain cerasus humilis rooted seedlings, which includes:
obliquely cutting the propagation seedlings of cerasus humilis from the stretched internodes to form incisions, and keeping stem tips without keeping dense leaf clusters of bases;
and transferring the cut end of each section of the propagation seedling of the cerasus humilis into the rooting culture medium, and transferring for more than 20 days to obtain the root-growing seedling of the cerasus humilis, wherein the height of each section of the propagation seedling of the cerasus humilis is more than 2 cm.
Wherein, the steps 102-104 are all carried out under the following conditions: culturing in an illumination culture room at 25 ℃ for 14 hours each day with illumination intensity of 2000-.
The method for culturing the cerasus humilis tissues provided by the embodiment has simple steps, does not need to induce callus, directly utilizes the stem sections with axillary buds to induce and generate axillary buds, and then performs proliferation culture and rooting culture, can quickly obtain a large amount of high-quality cerasus humilis rooting tissue culture seedlings, and has high propagation speed and short tissue culture period.
Secondly, the purpose of the cutting includes: obtaining more explant materials, wherein each stem segment with axillary buds can induce to generate cluster buds; the cut sections are convenient to disinfect, and the germs carried by the explants can be killed to the maximum extent; is convenient for explant inoculation.
Compared with the prior art which mainly adopts the induced callus, the method generates embryoid through the induction of the callus, and the method directly induces the axillary bud to generate the cluster bud which is closer to the culture in the natural state and has low aberration rate. Compared with the method for generating embryoid by callus induction in the prior art, the method has the advantages of high axillary bud survival rate and high propagation speed.
The second embodiment of the present application provides a cerasus humilis tissue culture method, including:
(1) explant collection and disinfection
Selecting an Prunus humilis Bunge plant with good growth condition, selecting a stem segment with axillary buds and sprouting in spring as an explant, cutting leaves on the branch, washing the leaf segment with tap water, putting the leaf segment into 75% ethanol solution for disinfection for 30 seconds to 1 minute, putting the leaf segment into 2% sodium hypochlorite solution for disinfection for 15 minutes, soaking the leaf segment with sterile water for 4-6 times after disinfection, sucking water by using sterile filter paper, cutting the disinfected branch into segments according to the principle of cutting 1 segment into 1 segment, wherein each segment has 1-2 buds.
(2) Axillary bud induction
The cut prune explants were inoculated into the start medium, 1 explant per flask. Starting a culture medium formula: MS +6-BA0.6mg/L + NAA0.05mg/L + sugar 40g/L + agar 6-6.5g/L, pH 6-6.2. Axillary buds begin to sprout on the 6 th day after inoculation, the pollution rate is 2 percent, and the survival rate is 98 percent.
FIGS. 2 and 3 are graphs showing the effect of the explants of this example after axillary bud induction.
(3) Proliferation culture
Starting culture for 25 days, transferring Prunus humilis tissue culture seedling to proliferation culture medium for proliferation culture, inoculating 7-10 strains in each bottle, the proliferation culture medium formula is the same as that of the starting culture medium, and MS +6-BA0.6mg/L
+ NAA0.05mg/L + sugar 40g/L + agar 6-6.5g/L, pH 6-6.2. The cerasus humilis tissue culture seedlings in the multiplication culture medium grow strongly, are long and obvious in segmentation, have large leaves, and can multiply about 10 cluster branches per material section after being cultured for 30 days, wherein each branch can grow to 3-4cm, and the statistical multiplication coefficient is 10.61 (the multiplication coefficient is the number of generated new seedlings/the number of inoculated seedlings (the effective bud length is more than 0.5 cm)).
FIGS. 4 and 5 are graphs showing the effect of the propagation culture step performed on the cerasus humilis tissue culture seedlings of the present example.
(4) Rooting culture
After culturing in the proliferation culture medium for 25-45 days, transferring the propagation seedlings of the cerasus humilis to a rooting culture medium, wherein the formula of the rooting culture medium is as follows: 1/2MS + IAA1.5mg/L + sugar 30g/L + agar 6g/L, pH6.1. When the stem tip is needed to be reserved during transferring, the height of the proliferated seedling is ensured to be more than 2cm, the internode is obviously stretched, the proliferated seedling is obliquely cut from the stretched internode, then the cut end is inserted into a culture medium, the length of the cutting is about 2cm, and the cutting cannot be carried with the dense leaf cluster of the base. Rooting starts about 12 days after the transfer, the rooting rate is about 95% in 20 days after the transfer (the rooting rate (%) -the number of rooted seedlings/the number of inoculated seedlings multiplied by 100%), the average rooting number is 7 (the average root number (one): the total root number/the number of rooted seedlings), and the average root length is 3.57cm (the average root length (cm): the total root length/the total root number).
FIG. 6 is a diagram showing the effect of the step of performing rooting culture according to the present embodiment.
(5) Culture conditions
Culturing in an illumination culture room at 25 ℃ for 14 hours each day with illumination intensity of 2000-.
It should be noted that, for the sake of simplicity, the above-mentioned method embodiments are described as a series of acts or combinations, but those skilled in the art should understand that the present application is not limited by the described order of acts, as some steps may be performed in other orders or simultaneously according to the present application. Further, those skilled in the art should also appreciate that the embodiments described in the specification are preferred embodiments and that the acts and modules referred to are not necessarily required in this application.
In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.
The preferred embodiments of the present application disclosed above are intended only to aid in the explanation of the application. Alternative embodiments are not exhaustive and do not limit the invention to the precise embodiments described. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the application and the practical application, to thereby enable others skilled in the art to best understand and utilize the application. The application is limited only by the claims and their full scope and equivalents.
Claims (5)
1. A method of culturing prunus humilis tissue, comprising:
(1) explant collection and disinfection: selecting an Prunus humilis Bunge plant with good growth condition, taking Prunus humilis Bunge stem segments with axillary buds and germinating in spring as explants, cutting leaves on the branches, washing the leaves clean with tap water, sterilizing the leaves in 75% ethanol solution for 30 seconds to 1 minute, sterilizing the leaves in 2% sodium hypochlorite solution for 15 minutes, brewing the leaves in sterile water for 4-6 times after the sterilization is finished, absorbing water by using sterilizing filter paper, and cutting the sterilized explants into 1 section according to the principle that 1 section is cut into 1 section, wherein each section has 1-2 axillary buds;
(2) axillary bud induction: inoculating the cut explants into a bottle of a starting culture medium to obtain cerasus humilis tissue culture seedlings, wherein the starting culture medium comprises:
MS +6-BA0.6mg/L + NAA0.05mg/L + sugar 40g/L + agar 6g/L, pH 6-6.2;
(3) and (3) proliferation culture: transferring the cerasus humilis tissue culture seedlings into a bottle of a proliferation culture medium for proliferation culture to obtain cerasus humilis proliferation seedlings, wherein the proliferation culture medium comprises:
MS +6-BA0.6mg/L + NAA0.05mg/L + sugar 40g/L + agar 6g/L, pH 6-6.2;
(4) rooting culture: transferring the propagation seedlings of the cerasus humilis to a rooting culture medium to obtain the root seedlings of the cerasus humilis, wherein the method comprises the following steps:
obliquely cutting the propagation seedlings of cerasus humilis from the stretched internodes to form incisions, and keeping stem tips without keeping dense leaf clusters of bases; transferring and inserting the cut end of each section of the propagation seedling of cerasus humilis into the rooting culture medium, and transferring for more than 20 days to obtain the root-growing seedling of cerasus humilis, wherein the height of each section of the propagation seedling of cerasus humilis is more than 2 cm;
wherein, the rooting culture medium comprises:
1/2MS + IAA1.5mg/L + sugar 30g/L + agar 6g/L, pH6.1.
2. The method of claim 1, wherein the axillary bud induction, the proliferation culture, and the rooting culture are performed under the following conditions: culturing in an illumination culture room at 25 ℃ for 14 hours each day with illumination intensity of 2000-.
3. The method of claim 1, wherein the explants after cutting are inoculated into flasks of start-up medium to obtain Prunus humilis tissue culture seedlings, comprising:
inoculating the cut explants into bottles of a starting culture medium, inoculating 1 explant into each bottle, and carrying out starting culture for 20-25 days to obtain Prunus humilis tissue culture seedlings.
4. The method of claim 1, wherein transferring the Prunus humilis tissue culture plantlets into a bottle of multiplication medium for multiplication culture to obtain Prunus humilis multiplied plantlets comprises:
transferring the Prunus humilis tissue culture seedlings into a bottle of a proliferation culture medium for proliferation culture, inoculating 7-10 Prunus humilis tissue culture seedlings into each bottle, and culturing for 25-45 days to obtain Prunus humilis proliferation seedlings, wherein each Prunus humilis proliferation seedling comprises multiple sections, each section is proliferated to obtain 8-12 clumpy branches, and the length of each clumpy branch is 3-4 cm.
5. The method of claim 1, wherein the sugar is one of white sugar or sucrose.
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