CN113331059A - Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants - Google Patents
Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants Download PDFInfo
- Publication number
- CN113331059A CN113331059A CN202110824281.2A CN202110824281A CN113331059A CN 113331059 A CN113331059 A CN 113331059A CN 202110824281 A CN202110824281 A CN 202110824281A CN 113331059 A CN113331059 A CN 113331059A
- Authority
- CN
- China
- Prior art keywords
- tea
- culture
- plant
- culture medium
- king
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000008929 regeneration Effects 0.000 title claims abstract description 23
- 238000011069 regeneration method Methods 0.000 title claims abstract description 23
- 235000009024 Ceanothus sanguineus Nutrition 0.000 title claims abstract description 13
- 240000003553 Leptospermum scoparium Species 0.000 title claims abstract description 13
- 235000015459 Lycium barbarum Nutrition 0.000 title claims abstract description 13
- 244000269722 Thea sinensis Species 0.000 claims abstract description 93
- 230000006698 induction Effects 0.000 claims abstract description 61
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 55
- 239000001963 growth medium Substances 0.000 claims abstract description 47
- 241000196324 Embryophyta Species 0.000 claims abstract description 46
- 230000035784 germination Effects 0.000 claims abstract description 35
- 230000004069 differentiation Effects 0.000 claims abstract description 29
- 238000011282 treatment Methods 0.000 claims abstract description 16
- 235000006468 Thea sinensis Nutrition 0.000 claims abstract description 13
- 239000008399 tap water Substances 0.000 claims abstract description 10
- 235000020679 tap water Nutrition 0.000 claims abstract description 10
- 238000002791 soaking Methods 0.000 claims abstract description 9
- 239000008174 sterile solution Substances 0.000 claims abstract description 6
- 238000001784 detoxification Methods 0.000 claims abstract description 5
- 210000001161 mammalian embryo Anatomy 0.000 claims description 29
- 238000012258 culturing Methods 0.000 claims description 26
- 239000002609 medium Substances 0.000 claims description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 241000167835 Tyrannus Species 0.000 claims description 9
- 238000004659 sterilization and disinfection Methods 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 6
- 239000012883 rooting culture medium Substances 0.000 claims description 6
- 239000002689 soil Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000010451 perlite Substances 0.000 claims description 5
- 239000010455 vermiculite Substances 0.000 claims description 5
- 229910052902 vermiculite Inorganic materials 0.000 claims description 5
- 230000000249 desinfective effect Effects 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 4
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 3
- 239000002250 absorbent Substances 0.000 claims description 3
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 claims description 3
- 239000006013 carbendazim Substances 0.000 claims description 3
- 239000003599 detergent Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000004161 plant tissue culture Methods 0.000 claims description 3
- 239000012869 germination medium Substances 0.000 claims description 2
- 229960002523 mercuric chloride Drugs 0.000 claims description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 2
- 239000008223 sterile water Substances 0.000 claims description 2
- 239000002352 surface water Substances 0.000 claims description 2
- 210000002257 embryonic structure Anatomy 0.000 abstract description 17
- 238000000338 in vitro Methods 0.000 abstract description 8
- 239000000463 material Substances 0.000 abstract description 7
- 238000009395 breeding Methods 0.000 abstract description 5
- 230000001488 breeding effect Effects 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 4
- 230000018109 developmental process Effects 0.000 abstract description 4
- 238000004140 cleaning Methods 0.000 abstract description 3
- 230000002068 genetic effect Effects 0.000 abstract description 3
- 230000009466 transformation Effects 0.000 abstract description 3
- 238000004321 preservation Methods 0.000 abstract description 2
- 230000012010 growth Effects 0.000 description 24
- 230000004083 survival effect Effects 0.000 description 15
- 230000001965 increasing effect Effects 0.000 description 13
- 230000000694 effects Effects 0.000 description 11
- 239000003630 growth substance Substances 0.000 description 8
- 238000011081 inoculation Methods 0.000 description 7
- 239000005708 Sodium hypochlorite Substances 0.000 description 6
- 230000003247 decreasing effect Effects 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 6
- 230000035755 proliferation Effects 0.000 description 5
- 230000028446 budding cell bud growth Effects 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000006870 ms-medium Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 208000009701 Embryo Loss Diseases 0.000 description 3
- 239000007640 basal medium Substances 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 239000003205 fragrance Substances 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 229930191978 Gibberellin Natural products 0.000 description 2
- 235000011203 Origanum Nutrition 0.000 description 2
- 241001529744 Origanum Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 2
- 239000003448 gibberellin Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241001164374 Calyx Species 0.000 description 1
- 241000209507 Camellia Species 0.000 description 1
- 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 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 235000018597 common camellia Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Images
Classifications
-
- 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
-
- 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/40—Afforestation or reforestation
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for establishing an efficient regeneration system by taking an embryonic axis of a tea tree of bird king as an explant, which comprises the following steps: picking seeds in the middle ten days of 9-10 months, performing detoxification and degerming treatment, picking seed embryos, and inoculating the seed embryos to a culture medium for germination induction culture; cutting off hypocotyls of seedlings, cutting the hypocotyls into 1cm small sections of explants, inoculating the explants onto a callus induction and differentiation culture medium, cutting off the upper part of cluster buds when the cluster buds grow to 2-3 cm, obliquely cutting the lower end of the cluster buds, inoculating the cut clusters buds to a strong seedling culture medium, selecting robust seedlings with the height of 4 cm, obliquely cutting the lower end of the seedlings, soaking the obliquely cut parts with IBA sterile solution, inoculating the seedlings, and performing rooting culture; selecting a plant with a stronger root length of 4 cm, moving the whole bottle to a greenhouse, hardening the seedling for 5 d, cleaning a culture medium at the root of the plant by using tap water, and transplanting. The invention provides sterile materials for the in vitro preservation of rare tea plant germplasm resources and provides technical reference for the development of subsequent genetic transformation and molecular breeding of tea plants.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for establishing a high-efficiency regeneration system by taking an embryonic axis of a tea tree of bird king as an explant.
Background
Bird king tea, Latin's nameCamellia sinensis (L.)Kuntze var. niaowangensis Q.H.ChenThe woody plant is also called fishhook tea, Yunwu tribute tea and Yuwang tea. Is a new variety in the camellia genus of Guizhou, and has larger and thinner leaves; the lateral veins of the leaves are concave and wrinkled; the calyx sheet is slightly hairy and has limbal hair; the length of the petiole is within the range of 3-4 mm. The pollen is divided into single particles and long spheres, the polar axis length of the large-particle pollen is within the range of 50.23-52.41 mu m, the outer wall of the large-particle pollen is smooth, the germination holes are narrow, and the inner holes are not obvious. In early spring, tea leaves are green, the buds and leaves are fat and tender for a long time, and the tea made of the tea leaves has the advantages of honey fragrance, strong fragrance and dark green liquor color. The fragrance component contains 136 kinds of hydrocarbons, esters, acids, alcohols, etc., and the main components are esters, acids, alcohols.
In recent years, with the popularization and wide planting of the tea plant of bird king in various places, the tea seedlings with consistent specifications and superior quality are required in the market in a large amount, however, the seed germination rate of the traditional breeding mode of the tea plant, such as seed propagation, under natural conditions is low, and the defects of long breeding period, low cuttage efficiency, few resistance resources and the like exist in the cutting seedling raising. As a means of asexual propagation and seedling raising, the tissue culture regeneration technology can realize the industrial cultivation of excellent varieties and the improvement of germplasm resources, and can be combined with other biological technologies such as genetic engineering or protoplast fusion and the like. Therefore, the establishment of a regeneration system for the tea tree of bird king becomes a key technology which needs to be solved urgently at present.
Disclosure of Invention
In order to solve the problems, the invention provides a method for establishing a high-efficiency regeneration system by taking the embryonic axis of the tea plant of king birds as an explant, which takes the embryonic axis of the tea seed of king birds as a material, induces callus and regenerates plants, obtains the complete regeneration plants of the tea plant of king birds by applying a tissue culture technology and establishes a regeneration system, thereby providing a sterile material for the in vitro preservation of the germplasm resources of rare tea trees and also providing a technical reference for the development of the genetic transformation and the molecular breeding of the follow-up tea trees.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for establishing a high-efficiency regeneration system by taking the embryonic axis of the tea tree of bird king as an explant comprises the following steps:
s1, pretreatment and disinfection treatment: picking seeds in the middle ten days of 9-10 months, and performing detoxification and degerming treatment on the seeds;
s2, seed embryo germination induction: picking the embryo in the seed on a superclean bench, inoculating to a germination induction culture medium for germination induction culture;
s3, inducing and culturing cluster buds: cutting off hypocotyls of young seedlings of the King tea NW32 on a germination induction culture medium, cutting the young seedlings into small segments of explants of about 1cm, and then inoculating the small segments of explants onto a callus induction and differentiation culture medium to perform callus and adventitious bud differentiation induction culture;
s4, culturing cluster buds: when the cluster buds grow to about 2-3 cm, cutting the cluster buds from the callus, beveling the lower end of the cluster buds, and inoculating the cluster buds to a strong seedling culture medium for strong seedling culture;
s5, rooting: selecting a strong seedling culture medium with strong and strong sprouts with the height of about 4 cm, beveling the lower ends of the sprouts, soaking the beveled parts with IBA sterile solution, and inoculating the seedlings into a rooting culture medium for rooting culture;
s6, transplanting: selecting a plant with a robust root length of about 4 cm from the King tea NW32 in a rooting culture medium, transferring the plant into a greenhouse for hardening for 5 d, cleaning the culture medium at the root of the plant with tap water, and transplanting the plant to a matrix with a ratio VNutrient soil:VPerlite:VVermiculiteCulturing in a ratio of =2:1: 1.
Further, in step S1, the seed detoxification and degerming method includes: putting the picked seeds in tap water added with detergent for 5 min, picking out the seeds floating on the water surface, rubbing off impurities on the surfaces of the seeds, soaking for 15 min by using 5% carbendazim, taking out, washing the seeds clean by using tap water, putting the seeds in a super-clean workbench, disinfecting for 3 min by using 75% alcohol, disinfecting for 15 min by using 0.1% mercuric chloride, washing for 5 times by using sterile water, and absorbing surface water by using sterile absorbent paper.
Further, in the step S2, the germination induction medium is added with 1.5 mg.L-1MS minimal medium of GA3, pH =5.80, cultured in germination medium for 45 days.
Further, in the step S3, the wound is healedWound induction and differentiation culture medium added with 4.0 mg.L-16-BA and 1.0 mg.L-1MS minimal medium of IBA, pH =5.80, in differentiation medium for 60 days.
Further, in step S4, 2.0 mg.l of strong seedling medium is added-16-BA and 0.8 mg.L of-1The MS minimal medium of IBA (Becton Dickinson) with pH =5.80, is cultured in a strong seedling medium for 45 days.
Further, in step S5, the IBA sterile solution has a concentration of 50.0 mg.l-1The rooting culture medium is added with 1.5 mg.L-1The 1/2MS minimal medium of IBA, the hardening-seedling time of the plant is 5 days.
Furthermore, in each stage of the plant tissue culture, the illumination intensity is 2000 lx, the illumination is carried out for 12 hours every day, and the culture temperature is (26 +/-2) DEG C.
In the scheme, the embryonic axis of the bird king tea tree, which is excellent in comprehensive character and can be popularized nationwide, is used as an explant to induce the regeneration of cluster buds, so that the tissue culture regeneration system of the bird king tea tree is perfected. Through the research, a high-efficiency tissue culture regeneration system of the tea tree of the bird king is established, and a technical reference can be provided for the development of subsequent genetic transformation and molecular breeding of the tea tree.
Drawings
FIG. 1 shows the growth vigor of the germination induction of the seed embryo at different times;
in the figure: A. carrying out germination induction culture on the seed embryo for 10 days; B. carrying out germination induction culture on the seed embryo for 25 d; C. germination induction culture of the seed embryo for 45 days; the scales are all 1 cm.
FIG. 2 shows the growth of hypocotyl-induced callus and shoot differentiation culture for different periods of time;
in the figure: A. culturing hypocotyl for 0 d; B. culturing hypocotyl for 60 days; C. inducing and culturing the bud of 60 days for differentiation and growth by hypocotyl; except for the annotations, the scale bar in the figure is 1 cm.
FIG. 3 shows the growth vigor of hypocotyl callus and adventitious bud growth culture;
in the figure: A. callus and adventitious bud induced by hypocotyl explant of tea NW32 of king bird; B. the callus and adventitious bud growth culture of the bird king tea NW32 is carried out for 15 d; C. the callus and adventitious bud of the bird king tea NW32 are cultured for 30 d; the scale bars in the figure are all 1 cm.
FIG. 4 shows the strong seedling and rooting of adventitious bud and the transplanting and growing of regenerated plant;
in the figure: A. culturing adventitious bud strong seedlings for 0 d; B. culturing strong adventitious buds for 45 days; C. culturing adventitious buds for 60 days; D. culturing adventitious bud rooting for 60 d of root elongation; E. transplanting and culturing the regenerated plants for 60 days; the scale bars in the figure are all 1 cm.
FIG. 5 transplanting and surviving plants of King tea NW 32;
in the figure: A. cleaning clean rooted plants after seedling hardening; B. transplanting and culturing for 0 d; C. transplanting and culturing for 60 d; except for the annotations, the scale bar in the figure is 1 cm.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Examples
Material
The tea plant of the bird king tea NW32 used in this example was planted in the Yunwu town of Guidingxian county (107 ° 6 '35 "from east longitude, 26 ° 12' 56" from north latitude) of Guizhou province, and the stem segment of the current-year young sprout with axillary bud in 3-4 months was used as the explant to establish the in vitro rapid propagation system. Taking capsules on plants in 9-10 months as materials, selecting a seed embryo to germinate under an aseptic condition, and taking a hypocotyl of the germinated seedling of the seed embryo and a stem section and a leaf of the aseptic seedling induced by proliferation in an in vitro rapid propagation system as explants to establish a regeneration system. The research material is a gift from the tea research institute of agricultural academy of sciences of Guizhou province.
Method
Pretreatment and disinfection treatment of capsule
And (3) completely removing pericarp of a capsule on a newly picked plant of the Piwang tea NW32 in 9-10 months, then taking a part for an experiment, and preserving the rest for later use. Firstly, soaking seeds in tap water added with detergent for 5 min, picking out the seeds floating on the water surface, and rubbing off impurities such as soil on the surfaces of the seeds. Soaking in 0.50% carbendazim solution for 15 min, washing in tap water for 1 hr, treating with 75% disinfectant alcohol for 1 min, 2 min and 3 min and 20% sodium hypochlorite for 10 min, 15 min and 20 min in a super clean bench, designing orthogonal test, and sterilizing the seeds at three levels for nine treatment groups. After 20 days of culture, the survival rate, the pollution rate and the death rate of the bird king tea NW32 embryos are counted, and the survival rate, the pollution rate and the death rate are calculated according to the following calculation formula:
seed embryo survival rate (%) = (seed embryo survival number/seed embryo inoculation number) × 100%
Seed embryo contamination rate (%) = (number of seed embryo contamination/number of seed embryo inoculation) × 100%
Seed embryo death rate (%) = (number of seed embryo deaths/number of seed embryos inoculated) x 100%
Seed embryo germination induction
And (3) in a super-clean workbench, placing the disinfected seeds in a sterile empty dish, and sucking the water on the surface of the seeds by using sterile absorbent paper. Picking embryos from seeds of King tea NW32 near the alcohol burner with sterile forceps and a disposable scalpel blade, and inoculating to the seeds added with GA at different concentrations3The MS culture medium is used for germination induction culture, after 45 days of culture, the germination number of the seed embryo is counted, and the growth vigor of the germinated seedling is observed and recorded. And finally, calculating the germination rate of the seed embryo of the King tea NW32, wherein the calculation formula is as follows:
seed embryo germination rate (%) = (seed embryo germination number/seed embryo inoculation number) × 100%
Callus and adventitious bud differentiation induction
Cutting off hypocotyls of young seedlings of the King tea NW32 on a germination induction culture medium, cutting into small segments of explants of about 1cm, and then inoculating the explants to MS culture media added with 6-BA and IBA with different concentrations in proportion to perform callus and adventitious bud differentiation induction culture. Inoculating the stem and leaf of aseptic seedling induced by proliferation in the fast propagation system as explant to MS culture medium with different concentrations of 6-BA, IBA or NAA in proportion for callus and adventitious bud differentiation induction culture. The explants are first dark cultured for 10 days and then transferred to light culture. And (4) counting the callus induction rate of the explant after culturing for 30 days, and counting the adventitious bud differentiation number of the explant after continuously culturing for 60 days. According to the statistical data, the callus induction rate and the adventitious bud differentiation induction rate of the origanum tea NW32 explant are calculated according to the following calculation formulas:
explant callus induction rate (%) = (explant callus induction rate/explant inoculation number) × 100%
Adventitious bud differentiation induction rate (%) of explant = (explant bud differentiation induction rate/explant inoculation number) × 100%
Callus and adventitious bud growth culture
And (3) treating the callus with the adventitious bud induced, and inoculating the callus into the same culture medium for continuous growth culture. After 30 days of culture, the growth of adventitious buds was observed and recorded.
Strong seedling and root induction
The above adventitious bud was cut from the callus, and the lower end was beveled. Then inoculating the mixture into MS culture medium containing 6-BA and IBA with different concentrations for strong seedling culture. And after strong seedling culture for 45 days, counting the plant height of the adventitious bud, and observing and recording the growth vigor of the plant. The plant height difference of the adventitious buds of the King tea NW32 was calculated, and the calculation formula is as follows.
Selecting strong seedling culture medium with strong seedling and height of about 4 cm, and beveling the lower end of the seedling. Then soaking the beveled part with 50.00 mg/L IBA sterile solution for 5 min, and finally inoculating into 1/2MS culture medium added with different concentrations of IBA for rooting culture. And after rooting culture for 60 days, counting the rooting number of the adventitious buds, and observing and recording the growth vigor of the plants. The rooting rate of the adventitious bud of the King tea NW32 was calculated by the following calculation formula.
Plant height difference (cm) = average plant height of adventitious bud after 45 d culture-average plant height of adventitious bud at inoculation
Rooting percentage (%) = (total adventitious bud number of inoculated rooting/total adventitious bud number) x 100%
Hardening off and transplanting
And (4) selecting a plant with the robust root length of about 4 cm of the plant of the bird king tea NW32 in a rooting culture medium, and transferring the whole bottle of the plant to a greenhouse for seedling hardening for 5 d. Then washing the culture medium of the plant root with tap water, and transplanting to the medium with the ratio of VNutrient soil:VPerlite:VVermiculiteCulturing in a ratio of =2:1: 1. After culturing for 60 d, observing and recording the growth vigor of the plants.
Culture conditions
In the whole process of establishing and culturing the in vitro culture and regeneration system of the origanum tea NW32 explant, MS is used as a basic culture medium, wherein the MS powder is 4.43 g/L, the sucrose is 30.00 g/L, and the agar is 8.00 g/L. Wherein, when rooting induction culture is carried out, the MS powder is 2.22 g/L, and the plant gel is 2.40 g/L. The pH value is between 5.80 and 6.00, the illumination intensity is 2000 lx, the temperature is (24 +/-2) DEG C, and the illumination time is 12 h/d.
Results
Figure 5 The influence of the number of buds in different inoculations of Bird King Tea NW32 on the induction of sub-generation proliferation
FIG. 5 Effect of different inoculum shoot numbers of Oreoweb NW32 on the proliferation of induced progeny
FIG 5 birds inoculated Wang tea containing different numbers NW32 FIG bud proliferation induced by subculture on
Figure 5 bird graphs of vigorous tea containing varying amounts of NW 32.
Cannot load all results
Retry
Is retrying …
Is retrying …
Effect of Disinfection time on survival Rate of King tea NW32 seed embryos
The length of treatment time for different combinations of sterilization conditions directly affects the survival rate of explants. The results show that: different treatment times of the combination of 75% alcohol and 20% sodium hypochlorite had a significant effect on the survival and contamination rates of the queen bird tea NW32 embryos (table 1). With the prolonged treatment time of 75% alcohol and 20% sodium hypochlorite, the survival rate of the bird king tea NW32 seed embryos increased and then decreased, and the contamination rate decreased continuously. When the soaking treatment is carried out for 3 min by 75% alcohol, the pollution rate of the bird king tea NW32 embryos is not obviously changed along with the increase of the treatment time of 20% sodium hypochlorite, and the pollution rate is 15.00% at least. However, the survival rate of the bird king tea NW32 embryos increased first and then decreased to a maximum of 85.00%. Mortality occurred in all four treatment groups with no change in regularity, with a maximum of 1.33% and a minimum of 0.26%. This may be due to mishandling such that 75% alcohol and 20% sodium hypochlorite were harmful to the young bird king tea NW32 embryos. Therefore, when the 75% alcohol is soaked for 3 min, the 20% sodium hypochlorite is treated for 15 min, which is the most suitable disinfection combination treatment condition.
TABLE 1 Effect of different Disinfection times on the survival of NW32 seed embryos of King tea
Note that the mean. + -. standard deviation
The different letters in the table are significance at the 0.05 level
GA of different concentrations3Influence on germination of NW32 embryos of King tea
Gibberellin is a high-efficiency plant growth regulator and has the function of promoting the growth and development of plants. GA3Is a commonly used regulator in gibberellin and mainly has the function of promoting cell elongation so as to elongate and increase plant stems. The results show that: addition of different concentrations of GA to MS Medium3The germination of the seed embryo of the bird king tea NW32 can be effectively promoted, the germination time is shortened, and the influence of difference is obvious (Table 2). When the MS culture medium is not added with GA3In time, the germination time of the queen bird tea NW32 seed embryos was long and inconsistent. When different concentrations of GA were added to the MS medium3In time, the germination time of the bird king tea NW32 embryos was shortened. With GA3The increase in concentration resulted in an increase and a decrease in germination rate of the bird king tea NW32 embryos. At the same time, along with GA3The growth vigor of the germinated embryos is also deteriorated along with the increase of the concentration. According to the germination rate of the seed embryo of the tea king NW32 and the growth vigor in the culture process, the following results are obtained: adding 1.50 mg/L GA to MS culture medium3In time, the germination rate of the seed embryo is high at 86.67%, the seed embryo grows well, the hypocotyl is long, and no embryo death is seen in the germination process (fig. 1A-C). Thus, medium MS +1.50 mg/L GA3Is the most suitable germination culture medium for the germination of the NW32 embryo of the tea king bird.
TABLE 2 different concentrations of GA332 kinds of bird king tea NWEffect of embryo Germination
Note that the mean. + -. standard deviation
Significance of the different letters in the Table at the 0.05 level
Influence of different growth regulator ratios on healing of embryo axis and bud differentiation of King tea NW32
Callus and adventitious bud differentiation induction culture of woody plants are generally carried out in the same medium, and callus and adventitious bud differentiation are induced at the same time. Therefore, selection of an appropriate medium is a key condition for inducing adventitious buds. The young shoots of the superior-growing tea of king bird NW32 were selected to germinate, the hypocotyls thereof were cut off, and cut into small pieces of about 1cm in length as callus and adventitious bud differentiation-induced explants, which were then inoculated into MS medium supplemented with 6-BA and IBA at different concentrations for culture (FIG. 2A). The results show that: the average induction rate of the callus of the bird king tea NW32 by the 6-BA and the IBA with different concentrations is very high, and the difference has an insignificant effect; while the average differentiation induction rate for adventitious buds was low and there was a significant difference in the influence (table 3).
TABLE 3 Effect of 6-BA and IBA in the same concentration ratio on hypocotyl callus and adventitious bud differentiation of King tea NW32
Note that the mean. + -. standard deviation
The different letters in the table are significance at the 0.05 level
When the concentration of 6-BA added in the MS culture medium of the basic culture medium is constant, the callus induction rate of the explant is increased and then reduced along with the increase of the consumption of the IBA concentration. When the concentration of 6-BA is 4.00 mg/L and the concentration of IBA is 1.00 mg/L, the induction rate of the callus of the hypocotyl of the bird king tea NW32 is 95.28 percent at most. Therefore, the optimal culture medium for callus induction by taking hypocotyl of the King tea NW32 as an explant is MS +4.00 mg/L6-BA +1.00 mg/L IBA. Similarly, when the concentration of 6-BA added to the MS culture medium of the basal medium is constant, the adventitious bud differentiation induction rate of the hypocotyl explant of the bird king tea NW32 is increased and then decreased along with the increase of the usage amount of the IBA concentration. When the use amount of the 6-BA concentration is 4.00 mg/L and the use amount of the IBA concentration is 1.00 mg/L, the adventitious bud differentiation rate of hypocotyls of the bird king tea NW32 is 23.33 percent at most. After culturing for 45 days, some calluses begin to differentiate adventitious buds, and when culturing is continued for 60 days, the undifferentiated calluses are still undifferentiated, even partial calluses generate browning phenomena (figure 2B), the differentiated adventitious buds are weak and small, only partial leaves and no obvious stem segments (figure 2C) exist, and therefore, the continuous culturing is needed to be beneficial to the next experiment. Therefore, the optimal culture medium for adventitious bud differentiation by taking hypocotyls of the King tea NW32 as explants is MS +4.00 mg/L6-BA +1.00 mg/L IBA.
Influence of different growth regulator ratios on sterile seedling stem segment and leaf callus induction
Young bud seedlings cultured by subculture multiplication in an in-vitro rapid propagation system of the King tea NW32 are selected, tender stem segments and leaves of the young bud seedlings are cut out to be used as explants, and then the young bud seedlings are inoculated into MS culture media added with 6-BA and IBA or NAA with different concentrations for culture. The results show that: there was a significant difference in the callus induction rate of the stem segments of the queen bird tea NW32 between the 6-BA and IBA at different concentrations (table 4). When the concentration of 6-BA added in the MS culture medium of the basic culture medium is constant, along with the increase of the consumption of the IBA concentration, the callus induction rate of the explant is continuously increased, and when the consumption of the IBA concentration is increased to a certain concentration, the callus induction rate of the explant is increased and then reduced. When the concentration of 6-BA is 9.00 mg/L and the concentration of IBA is 6.00 mg/L, the induction rate of the callus of the stem of the bird king tea NW32 is 99.33 percent at most, and the callus grows well. Therefore, the optimal culture medium for callus induction by taking stem segments of the King tea NW32 as explants is MS +9.00 mg/L6-BA +6.00 mg/L IBA.
When callus induction is carried out on the leaf explants of the tissue culture plantlets of the bird king tea NW32, the difference of the influence of 6-BA and NAA with different concentrations on the callus induction rate of the explants is obviously insignificant (Table 5). When the concentration of 6-BA added in the MS culture medium of the basal medium is constant, along with the increase of the usage amount of the NAA concentration, the callus induction rate of the leaf explants of the bird king tea NW32 is continuously increased, and when the usage amount of the IBA concentration is increased to a certain concentration, the callus induction rate of the explants is increased and then decreased. When the amount of 6-BA used was 3.00 mg/L and the amount of NAA used was 9.00 mg/L, the callus induction rate of the leaf of the bird king tea NW32 was 96.00% at the highest, and the growth thereof was good. After further culturing for 60 days, some calli had started to produce brown material and no adventitious bud differentiation was observed. Therefore, the most suitable culture medium for callus induction only by using the leaves of the King tea NW32 as explants is MS +3.00 mg/L6-BA +9.00 mg/L NAA.
TABLE 4 influence of different growth regulator ratios on the callus induction rate of the stem segments
Note that the mean. + -. standard deviation
The different letters in the table are significance at the 0.05 level
TABLE 5 influence of different growth regulator ratios on the leaf callus induction rate
Note that the mean. + -. standard deviation
The different letters in the table are significance at the 0.05 level
Effect of growth culture on adventitious bud growth vigor
The hypocotyl explant of the bird king tea NW32 cultured adventitious buds through callus and adventitious bud differentiation induction is very weak and has most leaves and few stem segments. If the adventitious bud is directly inoculated into a strong seedling culture medium for strong seedling culture, the regenerated adventitious bud is easy to die. If the culture is continued on the original medium, the adventitious bud is not greatly changed, and the growth is slow, probably because the nutrients and growth regulators in the medium are depleted. Therefore, callus with adventitious buds attached thereto was picked up on a medium depleted in nutrients and growth regulators (FIG. 3A), and then inoculated into the same newly prepared medium for growth culture. After 15 days of culture, the adventitious bud was significantly long and grew well (FIG. 3B). When the culture was continued for 30 days, the adventitious bud was about 1cm long, well grown and robust (FIG. 3C). Through the growth culture of the callus and the adventitious bud, the adventitious bud is not easy to die during the culture of strong seedlings and grows rapidly. Therefore, the adventitious bud induced by the hypocotyl of the obtained bird king tea NW32 is cultured for growth first, which is beneficial to the survival and growth of the adventitious bud in strong seedling culture.
Influence of different growth regulator ratios on adventitious bud strong seedling
The strong seedling culture of the adventitious bud in the growth culture is favorable for promoting the rooting culture and simultaneously is favorable for improving the transplanting survival rate of the plant in the later period. The stem section of the adventitious bud after growth culture is obvious, the adventitious bud is easy to cut from the callus for the next experiment, the bud seedling is weak (figure 4A), the bud seedling cannot be directly subjected to rooting culture, and otherwise, the transplanting survival rate in the later period is influenced. Therefore, adventitious buds of the bird king tea NW32 were inoculated to MS basal medium containing different concentrations of 6-BA and IBA for strong shoot culture. When the amount of 6-BA used was not changed, the difference in plant height of adventitious buds was not significant but each treatment group had an effect on the growth of adventitious buds as the amount of IBA used was increased (Table 6). After 45 days of culture, when the 6-BA concentration was used at 2.00 mg/L and the IBA concentration was used at 0.80 mg/L, the plant height difference of the adventitious bud of the King tea NW32 was 1.57 cm, and cluster buds were differentiated at the base thereof, and the plants grew well, had spread leaves, and grew vigorously (FIG. 4B). Therefore, the addition of 2.00 mg/L of 6-BA and 0.80 mg/L of IBA to the MS medium is more suitable for the strong seedling culture of the adventitious bud of the bird king tea NW 32.
Table 6 influence of different growth regulator formulations on sound seedlings of adventitious buds of queen bird tea NW32
Note that the mean. + -. standard deviation
The different letters in the table are significance at the 0.05 level
Influence of IBA proportions with different concentrations on adventitious bud rooting
Rooting culture is a key step for determining whether the regenerated plants can survive after being transplanted into a matrix. The results show that different concentrations of IBA had a significantly different effect on the rooting culture of the adventitious buds of the queen bird tea NW32 (table 7). After 60 days of culture, the rooting rate of the adventitious buds of the Oriental tea NW32 increased and then decreased with increasing consumption of IBA concentration contained in the basal culture 1/2MS, and when the concentration of IBA was 1.50 mg/L, the rooting rate of the adventitious buds was 87.78% at the highest, and the stem segments of the adventitious buds of the Oriental tea NW32 began to lignify, leaves stretched, and became dark green in color, and many roots were grown from the base, including the main root and lateral roots, wherein part of the lateral roots grew out of the medium (FIGS. 4C-D). Therefore, the culture conditions suitable for rooting culture of the adventitious bud of the bird king tea NW32 are that the adventitious bud base is soaked with 50.00 mg/L IBA for 5 min, and then 1.50 mg/L IBA is added into the basic culture medium 1/2MS culture medium.
TABLE 7 Effect of IBA at different concentrations on rooting of adventitious buds of King tea NW32
Note that the mean. + -. standard deviation
The different letters in the table are significance at the 0.05 level
Effect of hardening-seedling time and transplanting matrix on regeneration plant culture
The adventitious bud regenerated in vitro by the bird king tea NW32 is weak and small, and the selection of proper seedling hardening time and transplanting matrix after strong seedling and rooting induction culture is of great importance. In addition, due to the small number of regenerated sprouts and the limited number of experiments, the selection of appropriate acclimatization time and transplanting matrix is the key point for the survival of adventitious buds of the queen bird tea NW 32. The research obtains proper hardening-off time in the in-vitro rapid propagation system of the bird king tea NW32 and the transplanting matrix is better in the aspects of survival rate and growth vigor of the transplanted plants. Therefore, the better conditions are directly adopted to carry out the hardening-seedling and transplanting treatment on the regenerated adventitious bud of the bird king tea NW 32. Hardening off for 5 d, transplanting adventitious buds regenerated from bird king tea NW32 to VNutrient soil: VPerlite: VVermiculiteThe culture was carried out in 2:1:1 medium. After hardening off for 5 d, the regenerated plant of the bird king tea NW32 has no mixed bacteria pollution and intact root, and is transplanted to VNutrient soil:VPerlite:VVermiculiteAfter culturing for 60 days in 2:1:1 medium, the plants grew well, and the leaves were spread, 5-6 pieces (fig. 4E).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (7)
1. A method for establishing a high-efficiency regeneration system by taking the embryonic axis of the tea tree of bird king as an explant is characterized by comprising the following steps: the method comprises the following steps:
s1, pretreatment and disinfection treatment: picking seeds in the middle ten days of 9-10 months, and performing detoxification and degerming treatment on the seeds;
s2, seed embryo germination induction: picking the embryo in the seed on a superclean bench, inoculating to a germination induction culture medium for germination induction culture;
s3, inducing and culturing cluster buds: cutting off hypocotyls of the young seedlings of the tea king NW32 on the germination induction culture medium, cutting into 1cm small segments of explants, and then inoculating the explants onto a callus induction and differentiation culture medium to perform callus and adventitious bud differentiation induction culture;
s4, culturing cluster buds: when the cluster buds grow to 2-3 cm, cutting the cluster buds from the callus, obliquely cutting the lower end of the cluster buds, and inoculating the cluster buds to a strong seedling culture medium for strong seedling culture;
s5, rooting: selecting a strong seedling culture medium with strong and 4 cm high bud seedling, beveling the lower end of the bud seedling, soaking the beveled part with IBA sterile solution, and inoculating the bud seedling into a rooting culture medium for rooting culture;
s6, transplanting: selecting rootIn the culture medium, the plant with strong root length of about 4 cm of the King tea NW32 plant is transferred to a greenhouse for hardening for 5 d, then the culture medium of the plant root is cleaned by tap water, and the plant is transplanted to the medium with the mixture ratio of VNutrient soil:VPerlite:VVermiculiteCulturing in a ratio of =2:1: 1.
2. The method for establishing the high-efficiency regeneration system by using the embryonic axis of the tea plant of king birds as the explant, as claimed in claim 1, wherein the method comprises the following steps: in the step S1, the seed detoxification and degerming treatment method includes: putting the picked seeds in tap water added with detergent for 5 min, picking out the seeds floating on the water surface, rubbing off impurities on the surfaces of the seeds, soaking for 15 min by using 5% carbendazim, taking out, washing the seeds clean by using tap water, putting the seeds in a super-clean workbench, disinfecting for 3 min by using 75% alcohol, disinfecting for 15 min by using 0.1% mercuric chloride, washing for 5 times by using sterile water, and absorbing surface water by using sterile absorbent paper.
3. The method for establishing the high-efficiency regeneration system by using the embryonic axis of the tea plant of king birds as the explant, as claimed in claim 1, wherein the method comprises the following steps: in the step S2, the germination induction culture medium is added with 1.5 mg.L-1MS minimal medium of GA3, pH =5.80, cultured in germination medium for 45 days.
4. The method for establishing the high-efficiency regeneration system by using the embryonic axis of the tea plant of king birds as the explant, as claimed in claim 1, wherein the method comprises the following steps: in step S3, 4.0 mg.L of callus induction and differentiation medium is added-16-BA and 1.0 mg.L-1MS minimal medium of IBA, pH =5.80, in differentiation medium for 60 days.
5. The method for establishing the high-efficiency regeneration system by using the embryonic axis of the tea plant of king birds as the explant, as claimed in claim 1, wherein the method comprises the following steps: in the step S4, 2.0 mg.L of strong seedling culture medium is added-16-BA and 0.8 mg.L of-1The MS minimal medium of IBA (Becton Dickinson) with pH =5.80, is cultured in a strong seedling medium for 45 days.
6. The method for establishing the high-efficiency regeneration system by using the embryonic axis of the tea plant of king birds as the explant, as claimed in claim 1, wherein the method comprises the following steps: in the step S5, the concentration of the IBA sterile solution is 50.0 mg.L-1The rooting culture medium is added with 1.5 mg.L-1The 1/2MS minimal medium of IBA, the hardening-seedling time of the plant is 5 days.
7. The method for establishing the high-efficiency regeneration system by using the embryonic axis of the tea plant of king birds as the explant, as claimed in claim 1, wherein the method comprises the following steps: in each stage of the plant tissue culture, the illumination intensity is 2000 lx, the illumination is 12 hours per day, and the culture temperature is (26 +/-2) DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110824281.2A CN113331059B (en) | 2021-07-21 | 2021-07-21 | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110824281.2A CN113331059B (en) | 2021-07-21 | 2021-07-21 | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113331059A true CN113331059A (en) | 2021-09-03 |
| CN113331059B CN113331059B (en) | 2022-09-09 |
Family
ID=77480232
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110824281.2A Active CN113331059B (en) | 2021-07-21 | 2021-07-21 | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113331059B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115885855A (en) * | 2023-01-03 | 2023-04-04 | 贵州大学 | Method for establishing regeneration system by taking hypocotyl of Zikui tea tree as explant |
| CN115918542A (en) * | 2023-01-03 | 2023-04-07 | 贵州大学 | Method for quickly establishing propagation system by taking stem segments with axillary buds of Zikui tea trees as explants |
| CN117546778A (en) * | 2023-12-15 | 2024-02-13 | 安徽农业大学 | Tea tree leaf adventitious bud induction method |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5294549A (en) * | 1989-03-09 | 1994-03-15 | Weyerhaeuser Company | Method for reproducing conifers by somatic embryogenesis using mixed growth hormones for embryo culture |
| US5589621A (en) * | 1991-09-18 | 1996-12-31 | Bayer Aktiengesellschaft | Pinosylvine synthase genes |
| CN102726296A (en) * | 2012-07-04 | 2012-10-17 | 安徽农业大学 | Method for establishing tissue culture regeneration system of tea tree |
| CN104686332A (en) * | 2015-02-22 | 2015-06-10 | 刘木娇 | Method for establishing tissue culture regeneration system of camellia sinensis L. |
| CN104938335A (en) * | 2015-06-08 | 2015-09-30 | 中南林业科技大学 | Method for obtaining regeneration plants by use of camellia oleifera hypocotyl |
| CN105900845A (en) * | 2016-06-16 | 2016-08-31 | 南京晓庄学院 | Somatic embryo rapid propagation seedling raising method for camellia chekiangoleosa |
| CN106416487A (en) * | 2016-08-31 | 2017-02-22 | 贵州省生物研究所 | Soil improvement method for Niaowang tea |
| CN106613975A (en) * | 2016-12-15 | 2017-05-10 | 中国热带农业科学院热带作物品种资源研究所 | Mallotus oblongifolius callus culture method and culture medium thereof |
| CN107529551A (en) * | 2017-09-06 | 2018-01-02 | 安徽农业大学 | A kind of highly effective revulsion induction method of the mixed type tea tree with transgenosis root of hair |
| CN112662686A (en) * | 2021-01-26 | 2021-04-16 | 贵州大学 | Cloning method and application of Yunwu tribute tea CsPPO gene |
-
2021
- 2021-07-21 CN CN202110824281.2A patent/CN113331059B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5294549A (en) * | 1989-03-09 | 1994-03-15 | Weyerhaeuser Company | Method for reproducing conifers by somatic embryogenesis using mixed growth hormones for embryo culture |
| US5589621A (en) * | 1991-09-18 | 1996-12-31 | Bayer Aktiengesellschaft | Pinosylvine synthase genes |
| CN102726296A (en) * | 2012-07-04 | 2012-10-17 | 安徽农业大学 | Method for establishing tissue culture regeneration system of tea tree |
| CN104686332A (en) * | 2015-02-22 | 2015-06-10 | 刘木娇 | Method for establishing tissue culture regeneration system of camellia sinensis L. |
| CN104938335A (en) * | 2015-06-08 | 2015-09-30 | 中南林业科技大学 | Method for obtaining regeneration plants by use of camellia oleifera hypocotyl |
| CN105900845A (en) * | 2016-06-16 | 2016-08-31 | 南京晓庄学院 | Somatic embryo rapid propagation seedling raising method for camellia chekiangoleosa |
| CN106416487A (en) * | 2016-08-31 | 2017-02-22 | 贵州省生物研究所 | Soil improvement method for Niaowang tea |
| CN106613975A (en) * | 2016-12-15 | 2017-05-10 | 中国热带农业科学院热带作物品种资源研究所 | Mallotus oblongifolius callus culture method and culture medium thereof |
| CN107529551A (en) * | 2017-09-06 | 2018-01-02 | 安徽农业大学 | A kind of highly effective revulsion induction method of the mixed type tea tree with transgenosis root of hair |
| CN112662686A (en) * | 2021-01-26 | 2021-04-16 | 贵州大学 | Cloning method and application of Yunwu tribute tea CsPPO gene |
Non-Patent Citations (6)
| Title |
|---|
| 刘德华等: "茶子子叶柄下胚轴组织培养的研究", 《茶叶通讯》 * |
| 王莹等: "云雾贡茶组培繁殖技术研究初探", 《贵州科学》 * |
| 田维丽: "鸟王茶NW32离体快繁与再生体系建立", 《万方学位论文》 * |
| 田维丽等: "鸟王茶优良品系NW32组培快繁体系的建立", 《分子植物育种》 * |
| 蔡玲等: "油茶胚组织离体培养实验", 《西部林业科学》 * |
| 陈平等: "大叶茶的组织培养", 《植物生理学通讯》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115885855A (en) * | 2023-01-03 | 2023-04-04 | 贵州大学 | Method for establishing regeneration system by taking hypocotyl of Zikui tea tree as explant |
| CN115918542A (en) * | 2023-01-03 | 2023-04-07 | 贵州大学 | Method for quickly establishing propagation system by taking stem segments with axillary buds of Zikui tea trees as explants |
| CN115918542B (en) * | 2023-01-03 | 2023-10-24 | 贵州大学 | Method for rapidly establishing propagation system by taking stem segments with axillary buds of purple tea tree as explants |
| CN115885855B (en) * | 2023-01-03 | 2023-12-01 | 贵州大学 | Method for establishing regeneration system by taking hypocotyl of tea tree kui as explant |
| CN117546778A (en) * | 2023-12-15 | 2024-02-13 | 安徽农业大学 | Tea tree leaf adventitious bud induction method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113331059B (en) | 2022-09-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Godbole et al. | Somatic embryogenesis and its conversion into plantlets in a multipurpose bamboo, Dendrocalamus hamiltonii Nees et Arn. Ex Munro | |
| CN113331059B (en) | Method for establishing efficient regeneration system by taking bird king tea tree hypocotyls as explants | |
| CN106106163A (en) | A kind of iris cultivates propagation method | |
| Debnath | A scale-up system for lowbush blueberry micropropagation using a bioreactor | |
| CN104855292B (en) | A kind of method of Cinnamomum kanahirai hay stem segment tissue culture fast breeding | |
| CN110574682B (en) | Method for inducing regeneration seedlings by using paprika explants | |
| CN105613288B (en) | A kind of construction method of Phnom Penh Chinese littleleaf box rapid propagation system | |
| CN107223566B (en) | A kind of Wulian poplar method for tissue culture | |
| CN113080063B (en) | Rapid rooting method for tissue culture of coarse chaff tree | |
| CN109258469A (en) | A kind of method of Chinese Capsicum Cotyledons with petiole regeneration induction plant | |
| CN102232359B (en) | In-vitro rapid propagation method of double-petal Jasminum sambac | |
| CN115885855B (en) | Method for establishing regeneration system by taking hypocotyl of tea tree kui as explant | |
| CN106577280A (en) | Rapid propagation aseptic seedling by means of tender stem segments and blades of merrillanthus hainanensis | |
| CN113475402B (en) | Method for in vitro culture of test-tube plantlet by using tender stem segment of rubber tree | |
| KR101887221B1 (en) | Method of mass propagation of bamboo by in vitro culture | |
| CN110583481A (en) | Method for inducing somatic embryogenesis and plant regeneration of Aralia elata | |
| CN110301352B (en) | Tissue culture method of sasanqua | |
| CN105123522B (en) | A kind of quick breeding method for tissue culture of kwangsi mayten herb | |
| CN106665358A (en) | Rapid shoot induction and tissue culture propagation method for platycodon grandiflorum leaves | |
| CN114586684A (en) | Tissue culture rapid propagation method of triploid eucalyptus new variety' Jinggui eucalyptus I | |
| Naing et al. | In Vitro Micropropagation and Conservation of Rhynchostylis retusa BL. | |
| CN112438202A (en) | Tissue culture and rapid propagation method of grape rootstock | |
| CN117158320B (en) | Construction method of eustoma grandiflorum multi-variety somatic embryo efficient regeneration system | |
| CN112438200A (en) | Tissue rapid propagation method of fresh grapes | |
| CN112640782B (en) | Tissue culture method of orange yellow jade phoenix flower |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240726 Address after: 665622 Xiaoxinzhai Group 1, Mangyun Village, Huimin Town, Lancang Laku Autonomous County, Pu'er City, Yunnan Province Patentee after: Pumao Hao (Pu'er) Country or region after: China Patentee after: Tea Industry Development Co.,Ltd. Address before: 550025 Jiaxiu South Road, Huaxi District, Guiyang City, Guizhou Province Patentee before: Guizhou University Country or region before: China |
|
| TR01 | Transfer of patent right |