CN113598059A - Method for rapidly regenerating plants by inducing adventitious buds of mature embryos of juglans mandshurica maxim - Google Patents
Method for rapidly regenerating plants by inducing adventitious buds of mature embryos of juglans mandshurica maxim Download PDFInfo
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- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 9
- 229910004619 Na2MoO4 Inorganic materials 0.000 claims description 9
- 229930006000 Sucrose Natural products 0.000 claims description 9
- 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 9
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- 239000011159 matrix material Substances 0.000 claims description 8
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- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 3
- 229960000643 adenine Drugs 0.000 claims description 3
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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
- A01H4/002—Culture media for tissue culture
-
- 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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Abstract
The invention discloses a method for inducing adventitious buds of mature embryos of juglans mandshurica maxim to rapidly regenerate plants, which belongs to the technical field of plant tissue culture, wherein the endosperm of the juglans mandshurica maxim with cotyledons is cultured into seedlings in a tissue rapid propagation mode through the steps of pretreatment of the mature embryos, induction of the adventitious buds of the mature embryos, growth culture of the adventitious buds, rooting culture of the adventitious buds, seedling hardening, transplanting and the like; the method has the advantages of simple, rapid and efficient operation steps, not only breaks through the technical bottlenecks of serious browning of the tissue culture explant of the juglans mandshurica maxim and difficult rooting caused by the browning, but also greatly shortens the plant regeneration time and the seedling period, has important significance for tissue culture rapid propagation and industrial seedling culture of the juglans mandshurica maxim, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for rapidly regenerating a plant by inducing adventitious buds of mature embryos of juglans mandshurica maxim.
Background
Juglans mandshurica Maxim is a Juglaceae Juglans deciduous tree, is a precious medicinal material tree in northeast China, is an economic forest tree of China which is also used as an important woody, grain, oil and fruit material, and integrates economic value, nutritional value and medicinal value. Therefore, the juglans mandshurica maxim has wide development and utilization prospects. The juglans mandshurica maxim is intensively distributed in the east mountain region of the northeast of China, has sporadic distribution in the northeast China, and extends to the great Khingan mountain and the far east Russia region towards the north, and reaches the north of the south east to the south of the Korea and Japan. However, due to the long term excessive harvesting, the natural forest resources of juglans mandshurica have been in near-endangered. At present, the research on the juglans mandshurica is mainly focused on the aspects of distribution, site afforestation, fostering renewal, material property and the like, and the research on the propagation aspect is relatively less.
In production, the propagation of the juglans mandshurica maxim mainly adopts seed propagation, but the juglans mandshurica maxim seeds belong to dormant seeds, germination accelerating treatment is needed before propagation, the period is long, the emergence rate is low, the quality of seedlings is uneven, the seedling propagation speed is slow, the requirements of the actual market are difficult to meet, and the development of forestry production is seriously hindered. Researchers also aim at the tissue culture of the juglans mandshurica, however, the juglans mandshurica plant is rich in polysaccharide polyphenol substances, and a brown necrotic layer is easily generated at the cut of an explant in the tissue culture process, so that the juglans mandshurica explant is difficult to germinate, and an isolated culture is seriously browned and degenerated, thereby becoming a main factor for hindering the tissue culture of the juglans mandshurica. Therefore, the invention provides a method for inducing the adventitious bud of the mature embryo of the juglans mandshurica maxim to rapidly regenerate the plant.
Disclosure of Invention
The invention provides a method for inducing adventitious buds of mature embryos of juglans mandshurica maxim to rapidly regenerate plants, which effectively solves the problems of low seed reproduction germination rate, low emergence rate and uneven seedling quality; the tissue propagation explant incision is easy to generate a brown necrotic layer, which causes the technical problem that the germination of the juglans mandshurica maxim explant is difficult, and the method for quickly obtaining the juglans mandshurica maxim tissue culture seedling with better effect is provided.
The invention provides a method for inducing adventitious buds of mature embryos of juglans mandshurica maxim to rapidly regenerate plants, which is characterized by comprising the following steps:
s1 pretreatment of Juglans mandshurica fruit
Selecting single-plant juglans mandshurica maxim fruits, removing green husks, crushing, soaking endosperm with cotyledons in water for 2-3 h, washing with running water for 0.5-1 h, and disinfecting to obtain pretreated endosperm.
S2, inducing adventitious buds of mature embryos
Inoculating the pretreated endosperm obtained in the step S1 into a basic culture medium, adjusting the pH value to 5.6-5.8, culturing for 5 days at 23-25 ℃ under a dark condition, culturing for 3 days under a weak light condition, and culturing for 10-15 days under 1500-2000 Lx illumination conditions for 12-14 hours per day to obtain germinated adventitious buds;
s3, growth culture of adventitious bud
Inoculating the germinated adventitious buds obtained in the step S2 into a subculture medium, adjusting the pH value to 5.6-5.8, and culturing for 10-15 days at the temperature of 23-25 ℃ and under the illumination condition of 1500-2000 Lx for 12-14 h every day to obtain grown adventitious buds;
s4, rooting culture of adventitious bud
Inoculating the grown adventitious bud obtained in the step S3 into a rooting culture medium, controlling the pH value to be 5.6-5.8, and culturing for 15-25 d at the temperature of 23-25 ℃ and under the illumination condition of 1500-2000 Lx for 12-14 h per day to obtain rooted adventitious buds;
s5, hardening and transplanting seedlings
And (3) culturing the rooted adventitious bud obtained in the step (S4) in an environment with the temperature of 23-25 ℃ and the humidity of 65-75% for 2-3 days, transplanting the bud into a matrix, and culturing in a seedling growing room to obtain a juglans mandshurica regenerated plant.
Preferably, in S1, the specific method of the sterilization treatment is: washing the cotyledon-containing endosperm with sterile water, soaking in 75% ethanol for 30s, repeating the above steps, and adding 0.1% HgCl2Soaking in the solution for 10min, washing with sterile water for 5 times, drying, peeling off seed coat of mature embryo, and removing oxidized endosperm.
Preferably, in S2, the minimal medium is composed of the following raw materials in parts by mass: 1416 parts of NH4NO34.8 parts of H3BO3112.5 parts of CaCl2·H2O, 1367.0 parts of Ca (NO)3)20.25 portion of CuSO4·5H2O, 45.4 parts of a2-EDTA, 33.8 parts of FeSO4·7H2O, 361.49 parts of MgSO4·7H2O, 33.5 parts of MnSO4·H2O, 0.39 part of Na2MoO40.005 part of NiSO4·6H2O, 265.0 parts of KH2PO41559.0 parts of K2SO417.0 part of N2O6Zn·6H2O, 5.22 parts of thiamine hydrochloride and 1000 parts of Na2S2O32.5 parts of 6-benzylaminopurine, 0.1 part of indolebutyric acid, 4500 parts of agar, 2000 parts of cane sugar, 1000 parts of adenine and 950-1000 parts of distilled water.
Preferably, in S3, the subculture medium is composed of the following raw materials in parts by mass: 1416.0 parts of NH4NO34.8 parts of H3BO3112.5 parts of CaCl2·H2O, 1367.0 parts of Ca (NO)3)20.25 portion of CuSO4·5H2O, 45.4 parts of a2EDTA, 33.8 parts of FeSO4·7H2O, 361.49 parts of MgSO4·7H2O, 33.5 parts of MnSO4·H2O, 0.39 part of Na2MoO40.005 part of NiSO4·6H2O, 265.0 parts of KH2PO41559.0 parts of K2SO417.0 part of N2O6Zn·6H2O, 5.22 parts of thiamine hydrochloride and 1000 parts of Na2S2O32.5 parts of 6-benzylaminopurine, 0.01-0.1 part of indolebutyric acid, 0.2-0.25 part of gibberellin, 4500 parts of agar, 2000 parts of cane sugar and 950-1000 parts of distilled water.
Preferably, in S4, the rooting medium is composed of the following raw materials in parts by mass: 1416.0 parts of NH4NO34.8 parts of H3BO3112.5 parts of CaCl2·H2O, 1367.0 parts of Ca (NO)3)20.25 portion of CuSO4·5H2O, 45.4 parts of a2-EDTA, 33.8 parts of FeSO4·7H2O, 361.49 parts of MgSO4·7H2O, 33.5 parts of MnSO4·H2O, 0.39 part of Na2MoO40.005 part of NiSO4·6H2O, 265.0 parts of KH2PO41559.0 parts of K2SO417.0 part of N2O6Zn·6H2O, 5.22 parts of thiamine hydrochloride and 1000 parts of Na2S2O32.5 parts of 6-benzylaminopurine, 0.1 part of indolebutyric acid, 0.2 part of gibberellin, 0.2 part of activated carbon, 30000 parts of cane sugar, 4500 parts of agar and 950-1000 parts of distilled water.
Preferably, in S4, the length of the root of the rooted adventitious bud is 2-5 cm.
Preferably, in S5, the matrix is composed of turfy soil and vermiculite according to a ratio of 3:1, and the humidity of the matrix is 65-75%.
In the preferable S5, the temperature of the seedling raising room is 25-28 ℃.
Compared with the prior art, the invention has the beneficial effects that:
(1) according to the method, endosperm of mature embryos of the juglans mandshurica maxim with cotyledons is used as an explant for adventitious bud induction and plant regeneration, the consistency of excellent quality of seedlings is kept through tissue culture in vitro rapid propagation, the culture time is short, the adventitious bud induction lasts for 10-15 days, the adventitious bud grows for 15-25 days, and only 35-55 days are used from the induction culture of the mature embryos to the seedling; the adventitious bud inductivity is up to 100%, the browning rate is only 5.56%, the adventitious bud rooting rate is up to 100%, and the transplanting survival rate is up to more than 95%.
(2) The invention has the advantages of rapid regeneration of the mature embryo adventitious bud induction plant of the juglans mandshurica, simple, rapid and efficient operation steps, breakthrough of the technical bottlenecks of serious browning of the tissue culture explant of the juglans mandshurica and difficult rooting caused by the browning, greatly shortened plant regeneration time and seedling period, great significance for tissue culture rapid propagation and industrialized seedling of the juglans mandshurica and wide application prospect.
Drawings
FIG. 1 is a diagram of adventitious bud induction of a mature embryo; panel A shows a first example of the induction of adventitious buds by a mature embryo; FIG. B is a second example of the induction of adventitious buds by a mature embryo;
FIG. 2 is a graph of adventitious bud growth; panel A shows a first example of adventitious bud growth; panel B shows a second example of adventitious bud growth;
FIG. 3 is a graph of adventitious bud rooting; FIG. A is a first example of rooting of adventitious buds; b is a second example of rooting adventitious buds;
FIG. 4 is a diagram of a plant of a tissue culture seedling of Juglans mandshurica; FIG. A is a first example of a plant of a tissue culture seedling of Juglans mandshurica; b is a second example of a Juglans mandshurica tissue culture seedling plant; FIG. C shows a third example of a plant of a tissue culture seedling of Juglans mandshurica.
Detailed Description
In order to make the technical solutions of the present invention better understood and enable those skilled in the art to practice the present invention, the following embodiments are further described, but the present invention is not limited to the following embodiments. The following test methods and detection methods, unless otherwise specified, are conventional methods; the reagents and starting materials are all commercially available, unless otherwise specified.
The invention provides a method for inducing adventitious buds of mature embryos of juglans mandshurica maxim to rapidly regenerate plants, which is characterized by comprising the following steps:
s1 pretreatment of Juglans mandshurica fruit
Selecting single-plant juglans mandshurica maxim fruits, removing green husks, crushing, soaking endosperm with cotyledons in water for 2-3 h, washing with running water for 0.5-1 h, and disinfecting to obtain pretreated endosperm.
S2, inducing adventitious buds of mature embryos
Inoculating the pretreated endosperm obtained in the step S1 into a basic culture medium, adjusting the pH value to 5.6-5.8, culturing for 5 days at 23-25 ℃ under a dark condition, culturing for 3 days under a weak light condition, and culturing for 10-15 days under 1500-2000 Lx illumination conditions for 12-14 hours per day to obtain germinated adventitious buds;
s3, growth culture of adventitious bud
Inoculating the germinated adventitious buds obtained in the step S2 into a subculture medium, adjusting the pH value to 5.6-5.8, and culturing for 10-15 days at the temperature of 23-25 ℃ and under the illumination condition of 1500-2000 Lx for 12-14 h every day to obtain grown adventitious buds;
s4, rooting culture of adventitious bud
Inoculating the grown adventitious bud obtained in the step S3 into a rooting culture medium, controlling the pH value to be 5.6-5.8, and culturing for 15-25 d at the temperature of 23-25 ℃ and under the illumination condition of 1500-2000 Lx for 12-14 h per day to obtain rooted adventitious buds;
s5, hardening and transplanting seedlings
And (3) culturing the rooted adventitious bud obtained in the step (S4) in an environment with the temperature of 23-25 ℃ and the humidity of 65-75% for 2-3 days, transplanting the bud into a matrix, and culturing in a seedling growing room to obtain a juglans mandshurica regenerated plant.
The basic culture medium is composed of the following raw materials in parts by mass: 1416 parts of NH4NO34.8 parts of H3BO3112.5 parts of CaCl2·H2O, 1367.0 parts of Ca (NO)3)20.25 portion of CuSO4·5H2O, 45.4 parts of a2-EDTA, 33.8 parts of FeSO4·7H2O, 361.49 parts of MgSO4·7H2O, 33.5 parts of MnSO4·H2O, 0.39 part of Na2MoO40.005 part of NiSO4·6H2O, 265.0 parts of KH2PO41559.0 parts of K2SO417.0 part of N2O6Zn·6H2O, 5.22 parts of thiamine hydrochloride and 1000 parts of Na2S2O32.5 parts of 6-benzylaminopurine, 0.1 part of indolebutyric acid, 4500 parts of agar, 2000 parts of cane sugar, 1000 parts of adenine and 950-1000 parts of distilled water.
The subculture medium comprises the following raw materials in parts by mass: 1416.0 parts of NH4NO34.8 parts of H3BO3112.5 parts of CaCl2·H2O, 1367.0 parts of Ca (NO)3)20.25 portion of CuSO4·5H2O, 45.4 parts of a2-EDTA33.8 portions of FeSO4·7H2O, 361.49 parts of MgSO4·7H2O, 33.5 parts of MnSO4·H2O, 0.39 part of Na2MoO40.005 part of NiSO4·6H2O, 265.0 parts of KH2PO41559.0 parts of K2SO417.0 part of N2O6Zn·6H2O, 5.22 parts of thiamine hydrochloride and 1000 parts of Na2S2O32.5 parts of 6-benzylaminopurine, 0.01-0.1 part of indolebutyric acid, 0.2-0.25 part of gibberellin, 4500 parts of agar, 2000 parts of cane sugar and 950-1000 parts of distilled water.
The rooting medium is composed of the following raw materials in parts by mass: 1416.0 parts of NH4NO34.8 parts of H3BO3112.5 parts of CaCl2·H2O, 1367.0 parts of Ca (NO)3)20.25 portion of CuSO4·5H2O, 45.4 parts of a2-EDTA, 33.8 parts of FeSO4·7H2O, 361.49 parts of MgSO4·7H2O, 33.5 parts of MnSO4·H2O, 0.39 part of Na2MoO40.005 part of NiSO4·6H2O, 265.0 parts of KH2PO41559.0 parts of K2SO417.0 part of N2O6Zn·6H2O, 5.22 parts of thiamine hydrochloride and 1000 parts of Na2S2O32.5 parts of 6-benzylaminopurine, 0.1 part of indolebutyric acid, 0.2 part of gibberellin, 0.2 part of activated carbon, 30000 parts of cane sugar, 4500 parts of agar and 950-1000 parts of distilled water.
Example 1
A method for inducing adventitious buds of mature embryos of juglans mandshurica maxim to rapidly regenerate plants comprises the following steps:
s1 pretreatment of Juglans mandshurica fruit
Selecting mature fruits of good single plant of Manchurian walnut, removing green peel, crushing with small fruit shell crusher, soaking the endosperm with cotyledon in water for 2 hr, washing with running water for 0.5 hr, washing with sterile water on sterile super clean bench, soaking the endosperm with cotyledon in 75% alcohol for 30s, repeating twice, and adding 0.1% HgCl2Soaking in the solution for 10min, washing with sterile water for 5 times, drying to remove water on seed surface, peeling mature embryo seed coat, and removing oxidized endosperm to obtain pretreated endosperm.
S2, inducing adventitious buds of mature embryos
Inoculating the pretreated endosperm obtained in the step S1 into a basic culture medium, adjusting the pH value to 5.6, culturing for 5 days at 23 ℃ under a non-light condition, then culturing for 3 days under a weak light condition, and then culturing for 10 days under a 1500Lx illumination condition and illumination for 12h every day to obtain germinated adventitious buds;
s3, growth culture of adventitious bud
Inoculating the germinated adventitious bud obtained in the step S2 into a subculture medium, adjusting the pH value to 5.6, and culturing at 23 ℃ under 1500Lx illumination for 12h every day for 10d to obtain a growing adventitious bud;
s4, rooting culture of adventitious bud
Inoculating the grown adventitious bud obtained in S3 into rooting culture medium, controlling pH to 5.6, and culturing at 23 deg.C under 1500Lx illumination for 12 hr per day for 15d to obtain rooted adventitious bud;
s5, hardening and transplanting seedlings
And (3) culturing the rooted adventitious bud obtained in the step (S4) in an environment with the temperature of 23 ℃ and the humidity of 65% for 2 days, transplanting the bud into a matrix consisting of turfy soil and vermiculite according to the ratio of 3:1, and culturing in a seedling room with the humidity of 65% to obtain a juglans mandshurica regenerated plant.
Example 2
A method for inducing adventitious buds of mature embryos of juglans mandshurica maxim to rapidly regenerate plants comprises the following steps:
s1 pretreatment of Juglans mandshurica fruit
Selecting mature fruits of good single plant of Manchurian walnut, removing green peel, crushing with small fruit shell crusher, soaking the endosperm with cotyledon in water for 3 hr, washing with running water for 1 hr, washing the washed endosperm with cotyledon with sterile water on a sterile super clean bench, soaking the endosperm with cotyledon in 75% alcohol for 30s, repeating the above steps twice, and soaking in 0.1% HgCl2Soaking in the solution for 10min, washing with sterile water for 5 times, and suckingAfter the surface of the dry seed is hydrated, the mature embryo seed coat is peeled off and the oxidized endosperm is removed, so that the pretreated endosperm is obtained.
S2, inducing adventitious buds of mature embryos
Inoculating the pretreated endosperm obtained in S1 into a basic culture medium, adjusting the pH value to 5.8, culturing for 5 days at 25 ℃ under a non-light condition, then culturing for 3 days under a weak light condition, and then culturing for 15 days under 2000Lx illumination conditions and illumination for 14h every day to obtain germinated adventitious buds;
s3, growth culture of adventitious bud
Inoculating the germinated adventitious bud obtained in the step S2 into a subculture medium, adjusting the pH value to 5.8, and culturing for 15d at 25 ℃ under 2000Lx illumination for 14h every day to obtain a growing adventitious bud;
s4, rooting culture of adventitious bud
Inoculating the grown adventitious bud obtained in the step S3 into a rooting culture medium, controlling the pH value to be 5.8, and culturing for 25d under the conditions of 25 ℃ and 2000Lx illumination and illumination for 14h every day to obtain a rooted adventitious bud;
s5, hardening and transplanting seedlings
And (3) culturing the rooted adventitious bud obtained in the step (S4) in an environment with the temperature of 25 ℃ and the humidity of 75% for 3 days, transplanting the rooted adventitious bud into a matrix consisting of turfy soil and vermiculite according to the ratio of 3:1, and culturing in a seedling room with the humidity of 75% to obtain a juglans mandshurica regenerated plant.
Example 3
A method for inducing adventitious buds of mature embryos of juglans mandshurica maxim to rapidly regenerate plants comprises the following steps:
s1 pretreatment of Juglans mandshurica fruit
Selecting mature fruits of good single plant of Manchurian walnut, removing green peel, crushing with small fruit shell crusher, soaking the endosperm with cotyledon in water for 2.5h, washing with running water for 45min, washing the washed endosperm with cotyledon with sterile water on a sterile ultraclean workbench, soaking the endosperm with cotyledon in 75% alcohol for 30s, repeating the above steps twice, and adding 0.1% HgCl2Soaking in the solution for 10min, washing with sterile water for 5 times, drying, and peeling off mature embryoThe peel is removed and the oxidized endosperm is removed, resulting in a pretreated endosperm.
S2, inducing adventitious buds of mature embryos
Inoculating the pretreated endosperm obtained in S1 into a basic culture medium, adjusting the pH value to 5.7, culturing for 5d at 24 ℃ under a non-light condition, then culturing for 3d under a weak light condition, and then culturing for 13d under 1750Lx light condition and light for 13h every day to obtain germinated adventitious buds;
s3, growth culture of adventitious bud
Inoculating the germinated adventitious bud obtained in the step S2 into a subculture medium, adjusting the pH value to 5.7, and culturing for 13d in light for 13h every day at 24 ℃ under 1750Lx light to obtain a growing adventitious bud;
s4, rooting culture of adventitious bud
Inoculating the grown adventitious bud obtained in S3 into rooting culture medium, controlling pH to 5.7, and culturing at 24 deg.C under 1750Lx light for 13 hr per day for 20d to obtain rooted adventitious bud;
s5, hardening and transplanting seedlings
And (3) culturing the rooted adventitious bud obtained in the step (S4) in an environment with the temperature of 24 ℃ and the humidity of 70% for 2.5 days, transplanting the bud into a matrix consisting of turfy soil and vermiculite according to the proportion of 3:1, and culturing in a seedling room with the humidity of 70% to obtain a juglans mandshurica regenerated plant.
The method for rapidly regenerating the mature embryo adventitious bud induced plant of the juglans mandshurica as set forth in the above embodiments 1 to 3 can obtain a juglans mandshurica regenerated plant with good growth vigor, the cultivation time is short, the adventitious bud is induced for 10 to 15 days, the adventitious bud is induced for 15 to 25 days, and only 35 to 55 days are used from the induction culture of the mature embryo to the seedling; the adventitious bud inductivity is up to 100%, the browning rate is only 5.56%, the adventitious bud rooting rate is up to 100%, and the transplanting survival rate is up to more than 95%.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (8)
1. A method for inducing the adventitious bud of a mature embryo of a juglans mandshurica maxim to rapidly regenerate a plant is characterized by comprising the following steps:
s1 pretreatment of Juglans mandshurica fruit
Selecting single-plant juglans mandshurica maxim fruits, removing green husks, crushing, soaking endosperm with cotyledons in water for 2-3 h, washing with running water for 0.5-1 h, and disinfecting to obtain pretreated endosperm.
S2, inducing adventitious buds of mature embryos
Inoculating the pretreated endosperm obtained in the step S1 into a basic culture medium, adjusting the pH value to 5.6-5.8, culturing for 5 days at 23-25 ℃ under a dark condition, culturing for 3 days under a weak light condition, and culturing for 10-15 days under 1500-2000 Lx illumination conditions for 12-14 hours per day to obtain germinated adventitious buds;
s3, growth culture of adventitious bud
Inoculating the germinated adventitious buds obtained in the step S2 into a subculture medium, adjusting the pH value to 5.6-5.8, and culturing for 10-15 days at the temperature of 23-25 ℃ and under the illumination condition of 1500-2000 Lx for 12-14 h every day to obtain grown adventitious buds;
s4, rooting culture of adventitious bud
Inoculating the grown adventitious bud obtained in the step S3 into a rooting culture medium, controlling the pH value to be 5.6-5.8, and culturing for 15-25 d at the temperature of 23-25 ℃ and under the illumination condition of 1500-2000 Lx for 12-14 h per day to obtain rooted adventitious buds;
s5, hardening and transplanting seedlings
And (3) culturing the rooted adventitious bud obtained in the step (S4) in an environment with the temperature of 23-25 ℃ and the humidity of 65-75% for 2-3 days, transplanting the bud into a matrix, and culturing in a seedling growing room to obtain a juglans mandshurica regenerated plant.
2. The method for inducing rapid regeneration of an adventitious bud of a mature embryo of a juglans mandshurica maxim as claimed in claim 1, wherein the specific method of the sterilization treatment in S1 is: washing the washed endosperm with cotyledons with sterile waterSoaking the cotyledon-containing endosperm in 75% ethanol for 30s, repeating the soaking for two times, and adding 0.1% HgCl2Soaking in the solution for 10min, washing with sterile water for 5 times, drying, peeling off seed coat of mature embryo, and removing oxidized endosperm.
3. The method for inducing the rapid regeneration of the adventitious bud of a mature embryo of juglans mandshurica maxim as claimed in claim 1, wherein in S2, the minimal medium comprises the following raw materials in parts by mass: 1416 parts of NH4NO34.8 parts of H3BO3112.5 parts of CaCl2·H2O, 1367.0 parts of Ca (NO)3)20.25 portion of CuSO4·5H2O, 45.4 parts of a2-EDTA, 33.8 parts of FeSO4·7H2O, 361.49 parts of MgSO4·7H2O, 33.5 parts of MnSO4·H2O, 0.39 part of Na2MoO40.005 part of NiSO4·6H2O, 265.0 parts of KH2PO41559.0 parts of K2SO417.0 part of N2O6Zn·6H2O, 5.22 parts of thiamine hydrochloride and 1000 parts of Na2S2O32.5 parts of 6-benzylaminopurine, 0.1 part of indolebutyric acid, 4500 parts of agar, 2000 parts of cane sugar, 1000 parts of adenine and 950-1000 parts of distilled water.
4. The method for inducing the rapid regeneration of the adventitious bud of a mature embryo of a juglans mandshurica maxim as claimed in claim 1, wherein in S3, the subculture medium comprises the following raw materials in parts by mass: 1416.0 parts of NH4NO34.8 parts of H3BO3112.5 parts of CaCl2·H2O, 1367.0 parts of Ca (NO)3)20.25 portion of CuSO4·5H2O, 45.4 parts of a2EDTA, 33.8 parts of FeSO4·7H2O, 361.49 parts of MgSO4·7H2O, 33.5 parts of MnSO4·H2O, 0.39 part of Na2MoO40.005 part of NiSO4·6H2O, 265.0 parts of KH2PO41559.0 parts of K2SO417.0 part of N2O6Zn·6H2O, 5.22 parts of thiamine hydrochloride and 1000 parts of Na2S2O32.5 parts of 6-benzylaminopurine, 0.01-0.1 part of indolebutyric acid, 0.2-0.25 part of gibberellin, 4500 parts of agar, 2000 parts of cane sugar and 950-1000 parts of distilled water.
5. The method for inducing the rapid regeneration of the adventitious bud of the mature embryo of juglans mandshurica maxim as claimed in claim 1, wherein in S4, the rooting medium comprises the following raw materials in parts by mass: 1416.0 parts of NH4NO34.8 parts of H3BO3112.5 parts of CaCl2·H2O, 1367.0 parts of Ca (NO)3)20.25 portion of CuSO4·5H2O, 45.4 parts of a2-EDTA, 33.8 parts of FeSO4·7H2O, 361.49 parts of MgSO4·7H2O, 33.5 parts of MnSO4·H2O, 0.39 part of Na2MoO40.005 part of NiSO4·6H2O, 265.0 parts of KH2PO41559.0 parts of K2SO417.0 part of N2O6Zn·6H2O, 5.22 parts of thiamine hydrochloride and 1000 parts of Na2S2O32.5 parts of 6-benzylaminopurine, 0.1 part of indolebutyric acid, 0.2 part of gibberellin, 0.2 part of activated carbon, 30000 parts of cane sugar, 4500 parts of agar and 950-1000 parts of distilled water.
6. The method for inducing rapid regeneration of an adventitious bud of a mature juglans mandshurica embryo according to claim 1, wherein in S4, the length of the root of the rooted adventitious bud is 2-5 cm.
7. The method for inducing the rapid regeneration of the adventitious bud of a mature embryo of juglans mandshurica maxim as claimed in claim 1, wherein in S5, the substrate is composed of turfy soil and vermiculite according to a ratio of 3:1, and the humidity of the substrate is 65% -75%.
8. The method for inducing the rapid regeneration of plants by adventitious buds of mature embryos of juglans mandshurica maxim as claimed in claim 1, wherein the temperature of the seedling raising room in S5 is 25-28 ℃.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103960130A (en) * | 2014-05-09 | 2014-08-06 | 黑龙江省林业科学研究所 | Adventitious bud induction method for Juglans mandshurica Maxim. |
| CN106472317A (en) * | 2016-10-19 | 2017-03-08 | 黑龙江省林业科学研究所 | The method of the isolated culture adventive bud evoked plant regeneration of Juglans mandshurica |
| CN111213584A (en) * | 2020-02-03 | 2020-06-02 | 鲁东大学 | Method for regenerating plants by high-frequency induction of kalopanax septemlobus leaves |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103960130A (en) * | 2014-05-09 | 2014-08-06 | 黑龙江省林业科学研究所 | Adventitious bud induction method for Juglans mandshurica Maxim. |
| CN106472317A (en) * | 2016-10-19 | 2017-03-08 | 黑龙江省林业科学研究所 | The method of the isolated culture adventive bud evoked plant regeneration of Juglans mandshurica |
| CN111213584A (en) * | 2020-02-03 | 2020-06-02 | 鲁东大学 | Method for regenerating plants by high-frequency induction of kalopanax septemlobus leaves |
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