CN117084172B - Tissue culture and rapid propagation method of rhododendron serrulata - Google Patents
Tissue culture and rapid propagation method of rhododendron serrulata Download PDFInfo
- Publication number
- CN117084172B CN117084172B CN202311159262.8A CN202311159262A CN117084172B CN 117084172 B CN117084172 B CN 117084172B CN 202311159262 A CN202311159262 A CN 202311159262A CN 117084172 B CN117084172 B CN 117084172B
- Authority
- CN
- China
- Prior art keywords
- culture
- tissue culture
- culture medium
- propagation method
- rhododendron
- 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.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 33
- 241000245165 Rhododendron ponticum Species 0.000 title 1
- 230000006698 induction Effects 0.000 claims abstract description 58
- 241000208422 Rhododendron Species 0.000 claims abstract description 55
- 230000001954 sterilising effect Effects 0.000 claims abstract description 20
- 238000005728 strengthening Methods 0.000 claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims description 88
- 230000035755 proliferation Effects 0.000 claims description 44
- 239000002689 soil Substances 0.000 claims description 23
- 239000011159 matrix material Substances 0.000 claims description 18
- 238000005286 illumination Methods 0.000 claims description 16
- 244000083724 Rhododendron simsii Species 0.000 claims description 13
- 239000004576 sand Substances 0.000 claims description 13
- 239000003415 peat Substances 0.000 claims description 12
- 235000019362 perlite Nutrition 0.000 claims description 12
- 239000010451 perlite Substances 0.000 claims description 12
- 239000005708 Sodium hypochlorite Substances 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
- 229930006000 Sucrose Natural products 0.000 claims description 9
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 8
- 239000000758 substrate Substances 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 20
- 241000405965 Scomberomorus brasiliensis Species 0.000 abstract 2
- 240000007967 Rhododendron lapponicum Species 0.000 abstract 1
- 235000008296 Rhododendron lapponicum Nutrition 0.000 abstract 1
- 238000012258 culturing Methods 0.000 description 24
- 239000002609 medium Substances 0.000 description 20
- 230000000694 effects Effects 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 10
- 230000035784 germination Effects 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 5
- 230000012010 growth Effects 0.000 description 4
- 229930192334 Auxin Natural products 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 239000002363 auxin Substances 0.000 description 3
- 230000008635 plant growth Effects 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 2
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 2
- 239000004062 cytokinin Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000000762 glandular Effects 0.000 description 2
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 238000009331 sowing Methods 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241001573881 Corolla Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241001183967 Isodon Species 0.000 description 1
- 241001065871 Rhododendron rivulare Species 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 241000212749 Zesius chrysomallus Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000431 effect on proliferation Effects 0.000 description 1
- 230000001700 effect on tissue Effects 0.000 description 1
- 210000000720 eyelash Anatomy 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000010413 gardening Methods 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000002786 root growth Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
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
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
- A01G24/28—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material containing peat, moss or sphagnum
-
- 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
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/36—Ericaceae, e.g. azalea, cranberry or blueberry
- A01H6/364—Rhododendron, e.g. Azalea
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Inorganic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Botany (AREA)
- Soil Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Physiology (AREA)
- Cell Biology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a tissue culture and rapid propagation method of rhododendron serra, and belongs to the technical field of propagation of rhododendron serra. The invention effectively solves the technical problems of difficult sterilization and easy browning of explants by intercepting a series of steps of fresh rhododendron parvifolium stem disinfection, axillary bud induction, subculture multiplication culture, seedling strengthening and seedling hardening transplanting, and can effectively solve the problems of large influence of propagation seasons and low propagation speed of the existing propagation method while retaining excellent properties.
Description
Technical Field
The invention relates to the technical field of breeding of rhododendrons at the brookfield, in particular to a tissue culture and rapid propagation method of rhododendrons at the brookfield.
Background
The rhododendron (Rhododendron rivulare hand. -Mazz.) is a evergreen small arbor or shrub of the genus Rhododendron of the family Rhododendron, 1-3 m high. The leaves are egg-shaped, needle-shaped or oval, the corolla is funnel-shaped, the light white color has red spots, the single plant flowering period is up to 30d, and the number of flowers in a single inflorescence can be up to 18. The rhododendron is beautiful in tree form and long in flowering period, is a new garden greening tree species integrating flower, leaf and shape observation, and has higher gardening value. As a good garden landscape tree species, the rhododendron has the problems of low natural hybridization setting rate, small seeds, difficult sowing survival, slow growth of seed propagation seedlings and the like, at least 3-4 years, some even more than 8 years are needed from sowing to flowering, and in addition, the cutting seedlings have the problems of difficult survival, slow growth vigor and more diseases and insect pests.
In addition, the exophyte of the stem of the rhododendron is collected outdoors, the short glandular hair is densely covered, the disinfection is difficult, and the pollution rate is high. The most tender stem segments with lateral buds are used when selecting the explant material, but the tender stem segments are limited by seasons. When the seeds are used as the explants, the explants are easy to sterilize, but the offspring have separation phenomenon, and the flower buds are limited by seasons. Although the leaf is used as an explant and is not limited by seasons, the leaf is closely covered by the glandular eyelash, and sterilization is difficult.
Disclosure of Invention
In order to solve the technical problems, the invention aims to take explants such as rhododendron stems at the brook side as materials for tissue culture and rapid propagation research, establish an efficient cultivation mode and management technology, and effectively solve the problems of large influence on propagation seasons and low propagation speed of the existing propagation method while retaining excellent properties.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the tissue culture and rapid propagation method of the rhododendron serrulata is characterized by comprising the following steps of:
step 1: intercepting fresh stems of rhododendron serrulata and sterilizing to obtain a sterile explant;
step 2: inoculating the sterile explant to an axillary bud induction culture medium, and carrying out axillary bud induction culture to obtain axillary buds;
Step 3: respectively inoculating the axillary buds into a secondary proliferation culture medium for secondary proliferation culture, and growing adventitious buds at the stem nodes after the secondary culture;
step 4: transferring the stem node growing with adventitious bud into seedling strengthening culture medium for seedling strengthening culture to obtain aseptic seedling;
Step 5: transferring the aseptic seedlings into a rooting induction culture medium for culture, and then performing seedling hardening and transplanting;
the axillary bud induction culture medium in the step 2 is WPM+ZT (1.0-3.0) mg.L -1+NAA(0.1～1.0)mg·L-1;
The secondary proliferation culture medium in the step 3 is WPM+ZT (1.0-4.0) mg.L -1+GA3(0.0～1.0)mg·L-1+NAA(0.05～0.50)mg·L-1;
The strong seedling culture medium in the step 4 is WPM+ZT (0.1-1.0) mg.L -1+NAA(0.1～0.5)mg·L-1;
The rooting induction medium in the step 5 is WPM+sucrose (15-25) g.L -1+NAA(0.5～1.5)mg·L-1+IBA(0.5～1.5)mg·L-1+AC(0.1～2.0)g·L-1.
Preferably, in the step 1, sodium hypochlorite with the concentration of 2% is used for sterilization, and the sterilization time is 8-12min.
Preferably, the axillary bud induction culture period in the step 2 is 20d.
Preferably, the period of the secondary proliferation culture in the step 3 is 50d.
Preferably, the cultivation period of the strong seedlings in the step 4 is 50d.
Preferably, the rooting induction culture period in the step 5 is 60d.
Preferably, the seedling hardening and transplanting step in the step 5 specifically includes: hardening off the tissue culture seedlings after being cultured by the rooting induction culture medium in an indoor space for 5-6 d, taking out the hardened-off tissue culture seedlings, cleaning the culture medium of the root system, transplanting the tissue culture seedlings on the disinfected substrate, transplanting the rhododendron simsii into soil, watering thoroughly once, watering once a week, and carrying out conventional management.
Preferably, the matrix is peat soil, perlite and river sand according to the mass ratio of 2:1:1.
Preferably, in the steps 1 to 5, the culture temperature is 23 to 27 ℃, the illumination is 12h/d, and the illumination intensity is 2000lx.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention can rapidly obtain a large number of tissue culture seedlings of the rhododendron serrulata through a series of procedures of sterilization, basic culture, proliferation culture, rooting culture and the like of the rhododendron serrulata, and sodium hypochlorite with proper concentration is selected as a disinfectant in the sterilization process, so that the tissue culture seedlings of the rhododendron serrulata are favorable for absorption and browning prevention.
(2) When the WPM is used as a basic culture medium, the culture effect is obviously higher than that of other culture media, the influence on the germination time of the axillary buds reaches a remarkable level, and the influence on rooting induction reaches a remarkable level. The cytokinin ZT and the auxin NAA with proper concentrations are added into the culture medium, and the cytokinin ZT and the auxin NAA act synergistically, so that the proliferation speed of the cluster buds is increased, and the proliferation time is shortened. In the proliferation culture of azalea in the brook, GA 3 is matched with ZT for use, so that a better proliferation effect can be achieved; in root growth induction, proper amount of AC can promote the seedlings to root more, grow fast and luxuriantly.
(3) During transplanting, the ratio of peat soil to perlite to river sand in the matrix is 2:1:1, so that the transplanting survival rate can be remarkably increased, and the peat soil has rich nutrition and good water retention; the perlite has unique capillary action, can improve the porosity of the matrix, improve the ventilation and drainage properties of the matrix, and is beneficial to oxygen diffusion; the river sand particles are larger, the air content in the soil is higher, and the respiration and the growth of plants are facilitated. In addition, the river sand can also increase the stability of soil and avoid soil loss. Therefore, the peat soil, perlite and river sand can have good rooting effect by the cooperation, and particularly the growth of root length is obviously promoted.
Drawings
FIG. 1 is a graph showing the germination effect of the axillary bud induced plants in example 1 of the present invention;
FIG. 2 is a graph showing the effect of plant growth after the secondary proliferation culture in example 1 of the present invention;
FIG. 3 is a graph showing the effect of plant growth after seedling strengthening in example 1 of the present invention;
FIG. 4 is a graph showing rooting effect of the plants after rooting in example 1 of the present invention.
Detailed Description
Example 1
A tissue culture and rapid propagation method of rhododendron serrulata, which comprises the following steps:
1. Configuration of the culture medium:
(1) The formula of the axillary bud induction culture medium is WPM+ZT3.0mg.L -1+NAA0.5mg·L-1;
(2) The formula of the secondary proliferation culture medium is WPM+ZT2.0mg.L -1ZT+GA30.1mg·L-1+NAA0.1mg·L-1;
(3) The formula of the seedling strengthening culture medium is WPM+ZT0.5mg.L -1+NAA0.5mg·L-1;
(4) The rooting induction medium formula is WPM+sucrose 20g.L -1+1NAA1.0mg·L-1+IBA1.5mg·L-1+AC1.0g·L-1.
2. Tissue culture and rapid propagation method for rhododendron simsii
Step 1: intercepting fresh stems of rhododendron serrulata and sterilizing to obtain a sterile explant;
step 2: inoculating the aseptic explant to an axillary bud induction culture medium, and carrying out axillary bud induction culture to obtain axillary buds, and culturing for 20d;
Step 3: respectively inoculating the axillary buds into a secondary proliferation culture medium for secondary proliferation culture, and culturing for 50d after secondary culture, wherein adventitious buds grow at the stem nodes;
step 4: transferring the stem node growing with adventitious bud into strong seedling culture medium for strong seedling culture to obtain aseptic seedling, and culturing for 50d;
Step 5: transferring the aseptic seedlings into a rooting induction culture medium, culturing for 60 days, and performing seedling hardening and transplanting;
And in the step 1, sodium hypochlorite with the concentration of 2% is used for sterilization, and the sterilization time is 8min.
The seedling hardening and transplanting step in the step 5 specifically comprises the following steps: hardening off the tissue culture seedlings after being cultured by the rooting induction culture medium in a room for 14d, taking out the hardened-off tissue culture seedlings, cleaning the culture medium of the root system, transplanting the tissue culture seedlings on the sterilized matrix, transplanting the azalea of the brook side into the soil, watering thoroughly once, watering once a week, and carrying out conventional management.
The matrix is prepared from peat soil, perlite and river sand according to the mass ratio of 2:1:1.
In the steps 1 to 5, the culture temperature is 25 ℃, the illumination is 12h/d, and the illumination intensity is 2000lx.
Example 2
A tissue culture and rapid propagation method of rhododendron serrulata, which comprises the following steps:
1. Configuration of the culture medium:
(1) The formula of the axillary bud induction culture medium is WPM+ZT2.0mg.L -1+NAA1.0mg·L-1;
(2) The formula of the secondary proliferation culture medium is WPM+ZT2.0mg.L -1ZT+GA30.5mg·L-1+NAA0.5mg·L-1;
(3) The formula of the seedling strengthening culture medium is WPM+ZT1.0mg.L -1+NAA0.5mg·L-1;
(4) The rooting induction medium formula is WPM+sucrose 15 g.L -1+1NAA1.5mg·L-1+IBA0.5mg·L-1+AC2.0g·L-1.
2. Tissue culture and rapid propagation method for rhododendron simsii
Step 1: intercepting fresh stems of rhododendron serrulata and sterilizing to obtain a sterile explant;
step 2: inoculating the aseptic explant to an axillary bud induction culture medium, and carrying out axillary bud induction culture to obtain axillary buds, and culturing for 20d;
Step 3: respectively inoculating the axillary buds into a secondary proliferation culture medium for secondary proliferation culture, and culturing for 50d after secondary culture, wherein adventitious buds grow at the stem nodes;
step 4: transferring the stem node growing with adventitious bud into strong seedling culture medium for strong seedling culture to obtain aseptic seedling, and culturing for 50d;
Step 5: transferring the aseptic seedlings into a rooting induction culture medium, culturing for 60 days, and performing seedling hardening and transplanting;
And in the step 1, sodium hypochlorite with the concentration of 2% is used for disinfection, and the disinfection time is 9min.
The seedling hardening and transplanting step in the step 5 specifically comprises the following steps: hardening off the tissue culture seedlings after being cultured by the rooting induction culture medium in a room for 14d, taking out the hardened-off tissue culture seedlings, cleaning the culture medium of the root system, transplanting the tissue culture seedlings on the sterilized matrix, transplanting the azalea of the brook side into the soil, watering thoroughly once, watering once a week, and carrying out conventional management. The matrix is prepared from peat soil, perlite and river sand according to the mass ratio of 2:1:1.
In the steps 1 to 5, the culture temperature is 27 ℃, the illumination is 12h/d, and the illumination intensity is 2000lx.
Example 3
A tissue culture and rapid propagation method of rhododendron serrulata, which comprises the following steps:
1. Configuration of the culture medium:
(1) The formula of the axillary bud induction culture medium is WPM+ZT1.0mg.L -1+NAA0.1mg·L-1;
(2) The formula of the secondary proliferation culture medium is WPM+ZT2.0mg.L -1ZT+GA30.0mg·L-1+NAA0.1mg·L-1;
(3) The formula of the seedling strengthening culture medium is WPM+ZT0.1mg.L -1+NAA0.1mg·L-1;
(4) The rooting induction medium formula is WPM+sucrose 20g.L -1+1NAA0.1mg·L-1+IBA1.0mg·L-1+AC2.0g·L-1.
2. Tissue culture and rapid propagation method for rhododendron simsii
Step 1: intercepting fresh stems of rhododendron serrulata and sterilizing to obtain a sterile explant;
step 2: inoculating the aseptic explant to an axillary bud induction culture medium, and carrying out axillary bud induction culture to obtain axillary buds, and culturing for 20d;
Step 3: respectively inoculating the axillary buds into a secondary proliferation culture medium for secondary proliferation culture, and culturing for 50d after secondary culture, wherein adventitious buds grow at the stem nodes;
step 4: transferring the stem node growing with adventitious bud into strong seedling culture medium for strong seedling culture to obtain aseptic seedling, and culturing for 50d;
Step 5: transferring the aseptic seedlings into a rooting induction culture medium, culturing for 60 days, and performing seedling hardening and transplanting;
And in the step 1, sodium hypochlorite with the concentration of 2% is used for sterilization, and the sterilization time is 10min.
The seedling hardening and transplanting step in the step 5 specifically comprises the following steps: hardening off the tissue culture seedlings after being cultured by the rooting induction culture medium in a room for 14d, taking out the hardened-off tissue culture seedlings, cleaning the culture medium of the root system, transplanting the tissue culture seedlings on the sterilized matrix, transplanting the azalea of the brook side into the soil, watering thoroughly once, watering once a week, and carrying out conventional management.
The matrix is prepared from peat soil, perlite and river sand according to the mass ratio of 2:1:1.
In the steps 1 to 5, the culture temperature is 23 ℃, the illumination is 12h/d, and the illumination intensity is 2000lx.
Comparative example 1
A tissue culture and rapid propagation method of rhododendron serrulata, which comprises the following steps:
1. Configuration of the culture medium:
(1) The formula of the axillary bud induction culture medium is 1/4MS
+ZT3.0mg·L-1+NAA0.5mg·L-1；
(2) The formula of the secondary proliferation culture medium is WPM+ZT0.5mg.L -1+GA30.1mg·L-1+NAA0.1mg·L-1;
(3) The formula of the seedling strengthening culture medium is WPM+ZT0.5mg.L -1+NAA0.0mg·L-1;
(4) The rooting induction medium formula is 1/2 MS+sucrose 20g.L -1+1NAA1.0mg·L-1+IBA1.5mg·L-1+AC1.0g·L-1.
2. Tissue culture and rapid propagation method for rhododendron simsii
Step 1: intercepting fresh stems of rhododendron serrulata and sterilizing to obtain a sterile explant;
step 2: inoculating the aseptic explant to an axillary bud induction culture medium, and carrying out axillary bud induction culture to obtain axillary buds, and culturing for 20d;
Step 3: respectively inoculating the axillary buds into a secondary proliferation culture medium for secondary proliferation culture, and culturing for 50d after secondary culture, wherein adventitious buds grow at the stem nodes;
step 4: transferring the stem node growing with adventitious bud into strong seedling culture medium for strong seedling culture to obtain aseptic seedling, and culturing for 50d;
Step 5: transferring the aseptic seedlings into a rooting induction culture medium, culturing for 60 days, and performing seedling hardening and transplanting;
In the step 1, sodium hypochlorite with the concentration of 2% is used for sterilization, and the sterilization time is 11min.
The seedling hardening and transplanting step in the step 5 specifically comprises the following steps: hardening off the tissue culture seedlings after being cultured by the rooting induction culture medium in a room for 14d, taking out the hardened-off tissue culture seedlings, cleaning the culture medium of the root system, transplanting the tissue culture seedlings on the sterilized matrix, transplanting the azalea of the brook side into the soil, watering thoroughly once, watering once a week, and carrying out conventional management.
The matrix is prepared from peat soil, perlite and river sand according to the mass ratio of 2:1:1.
In the steps 1 to 5, the culture temperature is 24 ℃, the illumination is 12h/d, and the illumination intensity is 2000lx.
The difference between this comparative example and example 1 is that in step 2, the minimal medium is 1/4MS medium; in the step 3, the concentration of ZT is 0.5 mg.L -1; in the step 4, NAA concentration is 0.00 mg.L -1; in step 5, the minimal medium was 1/2MS medium.
Comparative example 2
A tissue culture and rapid propagation method of rhododendron serrulata, which comprises the following steps:
1. Configuration of the culture medium:
(1) The formula of the axillary bud induction culture medium is 1/2MS+ZT3.0mg.L -1+NAA0.5mg·L-1;
(2) The formula of the secondary proliferation culture medium is WPM+ZT5.0mg.L -1ZT+GA30.1mg·L-1+NAA0.1mg·L-1;
(3) The formula of the seedling strengthening culture medium is WPM+ZT1.5mg.L -1+NAA0.5mg·L-1;
(4) The rooting induction medium formula is 1/4 MS+sucrose 15 g.L -1+1NAA1.5mg·L-1+IBA0.5mg·L-1+AC2.0g·L-1.
2. Tissue culture and rapid propagation method for rhododendron simsii
Step 1: intercepting fresh stems of rhododendron serrulata and sterilizing to obtain a sterile explant;
step 2: inoculating the aseptic explant to an axillary bud induction culture medium, and carrying out axillary bud induction culture to obtain axillary buds, and culturing for 20d;
Step 3: respectively inoculating the axillary buds into a secondary proliferation culture medium for secondary proliferation culture, and culturing for 50d after secondary culture, wherein adventitious buds grow at the stem nodes;
step 4: transferring the stem node growing with adventitious bud into strong seedling culture medium for strong seedling culture to obtain aseptic seedling, and culturing for 50d;
Step 5: transferring the aseptic seedlings into a rooting induction culture medium, culturing for 60 days, and performing seedling hardening and transplanting;
And in the step 1, sodium hypochlorite with the concentration of 2% is used for disinfection, and the disinfection time is 12min.
The seedling hardening and transplanting step in the step 5 specifically comprises the following steps: hardening off the tissue culture seedlings after being cultured by the rooting induction culture medium in a room for 14d, taking out the hardened-off tissue culture seedlings, cleaning the culture medium of the root system, transplanting the tissue culture seedlings on the sterilized matrix, transplanting the azalea of the brook side into the soil, watering thoroughly once, watering once a week, and carrying out conventional management.
The matrix is prepared from peat soil, perlite and river sand according to the mass ratio of 2:1:1.
In the steps 1 to 5, the culture temperature is 26 ℃, the illumination is 12h/d, and the illumination intensity is 2000lx.
The difference between this comparative example and example 1 is that in step 2, the minimal medium is 1/2MS medium; in the step 3, the ZT concentration is 5.0 mg.L -1; in the step 4, the ZT concentration is 1.5 mg.L -1; the difference from example 2 is that in step 5, the minimal medium is 1/4MS medium.
Comparative example 3
A tissue culture and rapid propagation method of rhododendron serrulata, which comprises the following steps:
1. Configuration of the culture medium:
(1) The formula of the axillary bud induction culture medium is WPM+ZT0.5mg.L -1+NAA0.5mg·L-1;
(2) The formula of the secondary proliferation culture medium is WPM+ZT2.0mg.L -1ZT+GA31.5mg·L-1+NAA0.1mg·L-1;
(3) The formula of the seedling strengthening culture medium is WPM+ZT0.5mg.L -1+NAA1.0mg·L-1;
(4) The rooting induction medium formula is WPM+sucrose 20g.L -1+1NAA1.0mg·L-1+IBA1.5mg·L-1+AC0.0g·L-1.
2. Tissue culture and rapid propagation method for rhododendron simsii
Step 1: intercepting fresh stems of rhododendron serrulata and sterilizing to obtain a sterile explant;
step 2: inoculating the aseptic explant to an axillary bud induction culture medium, and carrying out axillary bud induction culture to obtain axillary buds, and culturing for 20d;
Step 3: respectively inoculating the axillary buds into a secondary proliferation culture medium for secondary proliferation culture, and culturing for 50d after secondary culture, wherein adventitious buds grow at the stem nodes;
step 4: transferring the stem node growing with adventitious bud into strong seedling culture medium for strong seedling culture to obtain aseptic seedling, and culturing for 50d;
Step 5: transferring the aseptic seedlings into a rooting induction culture medium, culturing for 60 days, and performing seedling hardening and transplanting;
And in the step 1, sodium hypochlorite with the concentration of 2% is used for disinfection, and the disinfection time is 12min.
The seedling hardening and transplanting step in the step 5 specifically comprises the following steps: hardening off the tissue culture seedlings after being cultured by the rooting induction culture medium in a room for 14d, taking out the hardened-off tissue culture seedlings, cleaning the culture medium of the root system, transplanting the tissue culture seedlings on the sterilized matrix, transplanting the azalea of the brook side into the soil, watering thoroughly once, watering once a week, and carrying out conventional management.
The matrix is prepared from peat soil, perlite and river sand according to the mass ratio of 2:1:1.
In the steps 1 to 5, the culture temperature is 23 ℃, the illumination is 12h/d, and the illumination intensity is 2000lx.
The difference between this comparative example and example 1 is that the ZT concentration in step 2 is 0.5 mg.L -1; in the step 3, the concentration of GA 3 is 1.5mg.L -1; NAA concentration in the step 4 is 1.0mg.L -1; in step 5, the concentration of AC was 0.0 mg.L -1.
Propagation was performed in the same manner as in examples 1 to 3 and comparative examples 1 to 3, and the results were recorded, and specific test results are shown in tables 1 and 2.
Table 1 comparative experiment of results of the induced culture of axillary buds of Rhododendron simsii at different Rabdosia
According to the experiment, the culture medium formula for improving the germination time and germination rate of axillary buds of the rhododendron serrulata and carrying out secondary proliferation is searched through hormone ratios of different basic culture mediums and different concentrations; selecting ZT and NAA auxins, and screening the axillary bud induction culture medium formula with optimal axillary bud germination time and germination rate.
As can be seen from Table 1, the basal medium germinated higher than 1/4MS and 1/2MS medium in WPM medium. Therefore, the axillary bud induction culture medium of the rhododendron serrulata is WPM+ZT3.0mg.L -1+NAA0.5mg·L-1, the germination time is shortest and the germination rate is highest.
Table 2 comparative experiment of proliferation and culture results of Rhododendron simsii
As can be seen from Table 2, each combination had an effect on proliferation of rhododendron serrulata in the same minimal medium, but the proliferation effect was also significantly different depending on the concentration of the plant growth regulator. With the increase of ZT concentration, the proliferation coefficient is in a trend of rising and then falling, when the ZT concentration is 2.0mg.L -1, the proliferation effect is optimal, the proliferation coefficient is 9.11 at the maximum, and at the moment, the proliferation seedlings grow well and grow healthily; with the increase of GA 3 concentration, the proliferation coefficient is correspondingly increased, but when GA 3 is more than 1.0mg.L -1, the proliferation seedlings start to be dwarfed and the leaves are aged, and when GA 3 with low concentration is matched with ZT, the proliferation effect is better. In addition, when NAA with a lower concentration is used in combination with ZT with a high concentration, the situation that the trunk of the propagation seedling is slim occurs. Therefore, the most suitable treatment for the secondary proliferation culture of rhododendron at the side of the study is WPM+ZT2.0mg.L -1+GA30.1mg·L-1+NAA0.1mg·L-1.
Table 3 comparative experiment of the results of the cultivation of the Rhododendron simsii Maxim
The number of the cluster buds obtained by the multiplication culture is large, but some of the cluster buds are relatively short, small and weak, so that the quality of the part of aseptic seedlings needs to be improved by the strong seedling culture.
As is clear from Table 3, the growth of the tissue culture seedlings was improved with the increase in ZT concentration, and the stems began to become thicker, but at 1.0 mg.L -1, the tissue culture seedlings began to proliferate. Tissue culture Miao Jiejian began to elongate when NAA concentration was increased, and tissue culture Miao Jiejian did not elongate when NAA was not added. In addition, when only ZT is added, the blade width is large, and as the NAA concentration increases, the blade becomes smaller as the difference between the two concentrations is large. Therefore, the suitable strong seedling culture medium formula for the study is WPM+ZT0.5mg.L -1+NAA0.5mg·L-1.
Table 4 experiment of rooting induction culture results of azalea at brook side
As is clear from Table 4, when the minimal medium was WPM, the average root number and rooting rate were higher than those of the 1/2MS and 1/4MS medium, and the rooting effect was good. At the concentration of 20 g.L -1 and 15 g.L -1, the rooting effect on the azalea from the side of the stream is better than 25 g.L -1. When AC is not added, the rooting rate of the tissue culture seedlings is low; after AC is added, the rooting effect is remarkable, and the rooting rate is in a trend of rising and falling along with the increase of the concentration. The rooting effect is best when the AC is 1.0 g.L -1. In addition, the rooting rate of the azalea in the brook side is not changed along with the concentration of NAA and IBA, which indicates that the NAA and IBA have less influence on the rooting effect. Therefore, the formula of the culture medium suitable for rooting culture of the rhododendron serrulata is WPM+20g.L -1 sucrose+NAA1.0mg.L -1+IBA1.5mg·L-1+AC1.0g·L-1.
According to the special culture medium and the tissue culture rapid propagation method thereof, disclosed by the embodiment of the invention, the tissue culture rapid propagation method has complete set, special property, special effect and comprehensive effect on tissue culture rapid propagation of the rhododendron simsii varieties, the germination rate can reach more than 50%, the rooting rate can reach more than 80%, the proliferation rate and the seedling strengthening rate of cluster seedlings are also improved, theoretical basis and matched technical means are provided for large-scale industrial development of the rhododendron simsii, a high-efficiency cultivation mode and management technology are established, excellent characters are reserved, and meanwhile, the seedling raising cost is reduced.
The foregoing description is only of the preferred embodiments of the invention, and all changes and modifications that come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. The tissue culture and rapid propagation method of the rhododendron serrulata is characterized by comprising the following steps of:
step 1: intercepting fresh stems of rhododendron serrulata and sterilizing to obtain a sterile explant;
step 2: inoculating the sterile explant to an axillary bud induction culture medium, and carrying out axillary bud induction culture to obtain axillary buds;
Step 3: respectively inoculating the axillary buds into a secondary proliferation culture medium for secondary proliferation culture, and growing adventitious buds at the stem nodes after the secondary culture;
step 4: transferring the stem node growing with adventitious bud into seedling strengthening culture medium for seedling strengthening culture to obtain aseptic seedling;
Step 5: transferring the aseptic seedlings into a rooting induction culture medium for culture, and then performing seedling hardening and transplanting;
The axillary bud induction culture medium in the step 2 is WPM+ZT1.0-3.0 mg.L -1+NAA0.1～1.0mg•L-1;
the secondary proliferation culture medium in the step 3 is WPM+ZT1.0-4.0 mg.L -1+GA30.0～1.0mg•L-1+NAA0.05～0.50mg•L-1;
The strong seedling culture medium in the step4 is WPM+ZT0.1-1.0 mg.L -1+NAA0.1～0.5mg•L-1;
The rooting induction culture medium in the step 5 is WPM+sucrose 15-25 g.L -1+NAA0.5～1.5mg•L-1+IBA0.5～1.5mg•L-1+AC0.1～2.0g•L-1.
2. The tissue culture rapid propagation method of azalea as claimed in claim 1, wherein the step 1 is performed with sodium hypochlorite with concentration of 2% for 8-12min.
3. The tissue culture rapid propagation method of the stream side azalea as claimed in claim 1, wherein the axillary bud induction culture period in the step 2 is 20d.
4. The tissue culture rapid propagation method of the stream side azalea as claimed in claim 1, wherein the period of the secondary proliferation culture in the step 3 is 50d.
5. The tissue culture rapid propagation method of the stream side azalea as claimed in claim 1, wherein the strong seedling cultivation period in the step 4 is 50d.
6. The tissue culture rapid propagation method of the stream side azalea as claimed in claim 1, wherein the rooting induction culture period in the step 5 is 60d.
7. The tissue culture rapid propagation method of the stream side azalea as claimed in claim 1, wherein the seedling hardening and transplanting step in the step 5 is specifically: hardening off the tissue culture seedlings after being cultured by the rooting induction culture medium in an indoor space for 5-6 d, taking out the hardened-off tissue culture seedlings, cleaning the culture medium of the root system, transplanting the tissue culture seedlings on the disinfected substrate, transplanting the rhododendron simsii into soil, watering thoroughly once, watering once a week, and carrying out conventional management.
8. The tissue culture rapid propagation method of azalea as claimed in claim 7, wherein the matrix is peat soil, perlite and river sand according to the mass ratio of 2-4:1-2:1-2.
9. The tissue culture rapid propagation method of azalea as claimed in claim 7, wherein the matrix is peat soil, perlite and river sand in a mass ratio of 2:1:1.
10. The tissue culture rapid propagation method of the stream side azalea as claimed in claim 1, wherein in the step 1-5, the culture temperature is 23-27 ℃, the illumination is 12h/d, and the illumination intensity is 2000 lx.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311159262.8A CN117084172B (en) | 2023-09-09 | 2023-09-09 | Tissue culture and rapid propagation method of rhododendron serrulata |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311159262.8A CN117084172B (en) | 2023-09-09 | 2023-09-09 | Tissue culture and rapid propagation method of rhododendron serrulata |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117084172A CN117084172A (en) | 2023-11-21 |
| CN117084172B true CN117084172B (en) | 2024-08-02 |
Family
ID=88781777
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311159262.8A Active CN117084172B (en) | 2023-09-09 | 2023-09-09 | Tissue culture and rapid propagation method of rhododendron serrulata |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117084172B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117546780B (en) * | 2023-12-22 | 2024-09-24 | 华中农业大学 | Formula and preparation method of rhododendron grandiflorum bud seedling strengthening culture medium |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2970277B2 (en) * | 1992-12-25 | 1999-11-02 | 王子製紙株式会社 | A method for promoting rooting of cultured shoots in Rhododendron plants |
| JPH06189643A (en) * | 1992-12-25 | 1994-07-12 | New Oji Paper Co Ltd | Large-scale multiplication of rhododendron |
| CN101647392B (en) * | 2009-09-14 | 2011-07-27 | 云南绿大地生物科技股份有限公司 | Tissue-culture rapid-propagation method of double-happiness cuckoo variety and special culture medium thereof |
| CN105475138A (en) * | 2015-12-18 | 2016-04-13 | 深圳职业技术学院 | Rapid propagation method of Rhododendron moulmainense Hook. f. |
-
2023
- 2023-09-09 CN CN202311159262.8A patent/CN117084172B/en active Active
Non-Patent Citations (1)
| Title |
|---|
| 杜鹃组培快繁技术研究;谢嘉浇;中国优秀硕士学位论文全文数据库 农业科技辑;20240415(第4期);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117084172A (en) | 2023-11-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103380730B (en) | Tissue-culture rapid propagation method for pyrus betulaefolia bunge | |
| CN108293878B (en) | Tissue culture seedling raising method for trichosanthes kirilowii Maxim tender leaves | |
| CN103931497B (en) | A kind of method improving dragon fruit plantlet in vitro planting percent | |
| CN103988777B (en) | A kind of in-vitro culture method for tender stem segments of the wide yulan of leaflet dwarf form | |
| CN111616052A (en) | Rapid propagation and sugar-free rooting culture method and application of apple rootstock catalpa bungei | |
| CN101946703A (en) | Method for regenerating plants of Chinese rose by using leaves as explants | |
| CN117084172B (en) | Tissue culture and rapid propagation method of rhododendron serrulata | |
| CN100998314B (en) | Method for tissue culture quick breeding of sea-buckthorn | |
| CN103155868B (en) | Rapid seeding raising method of cherry rootstock ZY-1 tissue culture | |
| CN106165648B (en) | A kind of cercis tissue culture culture medium and cultural method | |
| CN101743908A (en) | Tissue culture, rapid propagation and cultivation method of grevillea banksii | |
| CN102792889B (en) | Chuanminshen violaceum tissue culture rapid propagation technology | |
| CN111480578A (en) | Tissue culture and rapid propagation method for seed embryo of Epimedium sagittatum | |
| CN112931226B (en) | Tissue culture rapid propagation method for alnus ferox | |
| CN111919751B (en) | Tissue culture method for murraya paniculata seeds | |
| CN112106664B (en) | Sterile germination and rapid propagation method for michelia spectabilis seeds | |
| CN111480579A (en) | Tissue culture and rapid propagation method for immature embryos of epimedium dauricum | |
| KR20110064365A (en) | The mass producing method of regenerated plant from the leaf segment of calanthe discolor | |
| CN111448985A (en) | Tissue culture method of rosa tenuifolia | |
| CN111699971B (en) | Method for culturing cymbidium sinense tissue | |
| CN110402816B (en) | System for increasing transplanting seedling emergence amount of test-tube plantlets of grapes and application method thereof | |
| CN114208665B (en) | Rapid propagation method of bulbus brueckii oil spot lily | |
| CN110432151B (en) | Tissue culture and rapid propagation method of Chongyang wood | |
| CN112690215B (en) | Tissue culture method for Pixumu | |
| CN111264393B (en) | Method for rapidly breeding epimedium test-tube plantlets |
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 |