CN106665367A - Tabebuia chrysantha tissue culture and rapid propagation method - Google Patents
Tabebuia chrysantha tissue culture and rapid propagation method Download PDFInfo
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- CN106665367A CN106665367A CN201710170198.1A CN201710170198A CN106665367A CN 106665367 A CN106665367 A CN 106665367A CN 201710170198 A CN201710170198 A CN 201710170198A CN 106665367 A CN106665367 A CN 106665367A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a tabebuia chrysantha tissue culture and rapid propagation method. The tabebuia chrysantha tissue culture and rapid propagation method comprises the steps of treatment and disinfection of an explants, acquisition of aseptic seedlings, differentiation and proliferation of aseptic buds and culture and transplanting of rooting seedlings. A propagation method and a culture medium formula of the invention are used for carrying out tabebuia chrysantha tissue culture, the annual growth coefficient of the aseptic buds reaches 3.012, and the rooting rate reaches 97 percent. The tabebuia chrysantha tissue culture and rapid propagation method is provided by using tabebuia chrysantha seeds as the explants, an effective path is provided for seedling breeding of tabebuia chrysantha, and large-scale production requirements of the seedling breeding are met.
Description
Technical field
The invention belongs to nursery stock vegetative propagation technique field, more particularly to a kind of Golden Bell Tree tissue-culturing quick-propagation
Method.
Background technology
Golden Bell Tree (Tabebuia chrysantha) be Bignoniaceae windbell wood category deciduous tree, originate in Mexico,
Central America, South America.Its color is various, is a kind of tree that style and features can be changed with seasonal variations, with spring flower, Xia Shi, autumn
Withered unique graceful bearing of green, winter.In spring at florescence, first leaf is spent to open, corolla windbell shape completely sets golden yellow when in full bloom, rather grand, has
Splendid ornamental value, introduces a fine variety for over ten years, and South China gardens, courtyard greening have been widely used in, is excellent gardens
Greening and avenue tree planting.In addition, Golden Bell Tree strong adaptability, extensive management, plantation profit height, are received by the market very much.
The Sterile culture mode of Golden Bell Tree is seeding and seedling raising, but its seed longeivity is extremely short, it is easy to devitalization, is needed
To adopt and broadcast, its seed maturity in the 4-5 months, therefore, breeding limited by seasonality, it is difficult to meet the demand in market.And lead to
The mode for crossing tissue cultures is bred, and its reproduction speed is fast, and seedling quality is good, can reach the purpose of large-scale production.It is domestic
Using tissue culture technique the research of Golden Bell Tree is bred there is not yet report.
The content of the invention
The purpose of the present invention is to set up a kind of Golden Bell Tree quick breeding method for tissue culture, improves seedling propagation speed
Degree, shortens the breeding cycle, and solves seedling propagation by seasonal restricted problem.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of Golden Bell Tree quick breeding method for tissue culture, including process and sterilization, initial incubation, the bud of explant
Differentiation and propagation, culture of rootage, take root transplantation of seedlings the step of, specifically include following steps：
Step 1, the process and sterilization of explant：With Golden Bell Tree seed as explant, outer layer kind shell is peelled off, take kind
Benevolence, sterilizes on superclean bench, then with sterile water wash, it is stand-by after draining；
Step 2, initial incubation：The explant for disinfecting is seeded in Initial culture base, light culture then moves to natural light
Cultivate according under the conditions of；
Step 3, the differentiation of bud and propagation：The aseptic seedling obtained in step 2 is cut into into band axillary bud sections, bud point is inoculated into
Change with proliferated culture medium, will be placed under the indoor low light condition of culture and cultivate with bud sections oblique cutting on culture medium during inoculation,
Budlet is differentiated at axillary bud, bud clump is cut, be forwarded to the Multiplying culture that fresh bud breaks up and bud is carried out on proliferated culture medium,
Timing is transferred；
Step 4, culture of rootage：The sterile bud of clip robust growth is seeded on root media, is placed on culture indoor weak
Cultivate under optical condition, treat that the base portion incision of seedling starts to expand and grow callus, from callus director after continuing to cultivate
Go out radicula；
Step 5, transplantation of seedlings of taking root：Culture of rootage treats that the root system of seedling is grown together substantially, and bottle seedling of taking root moves to hardening canopy and enters
Row hardening, is transplanted through the seedling of taking root of hardening, and the seedling that will take root during transplanting takes out from blake bottle, and with clear water seedling root is washed away
The culture medium of system, then by little transplantation of seedlings to the Seedling bag equipped with matrix, transplanting is carried out in seedling growth greenhouse, to be irrigated after transplanting
Water, with film covering and heat insulating moisturizing, progressively divulges information, and finally removes film；
Culture medium prescription used in the present invention is as follows：
Initial incubation culture medium：MS+6-BA0.4mg/L+NAA0.2mg/L+ sucrose 30g/L, agar powder 4.2g/L, pH=
5.8；
Bud breaks up and proliferated culture medium：MS+6-BA1.2-1.6mg/L+IAA0.3-0.5mg/L+L- Cys2 5mg/L
+ sucrose 30g/L, agar powder 4.2g/L, pH=5.8；
Root media：1/2MS+IBA0.8-1.0mg/L+ activated carbon 0.2g/L+ sucrose 20g/L, agar powder 4.5g/L,
PH=5.8.
Sterilizing on superclean bench described in step 1 of the present invention, then with sterile water wash, preferably on superclean bench
With 0.1%HgCl2Solution disinfection 8-10min, then with sterile water wash 4-5 time.
Light culture described in step 2, then moves to be cultivated under the conditions of natural lighting, and preferred light culture 5 days then moves to natural light
Cultivate 20d according under the conditions of, cultivation temperature is 25 ± 2 DEG C, light culture after 5 days seed start to sprout, under the conditions of natural lighting after
Continuous culture can grow seedling for 5 days, and 20 days aseptic seedlings can be grown high to about 4cm.
The differentiation of the bud described in step 3 and propagation, the aseptic seedling obtained in step 2 is preferably cut into the band for being about 2cm
Axillary bud sections, is inoculated on bud differentiation and proliferated culture medium, and culturing room will be placed on bud sections oblique cutting on culture medium during inoculation
Cultivate under interior low light condition, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 1000-1500lx, daily illumination 12-14h, 20d is left for culture
The right side can differentiate budlet at axillary bud；When the budlet for differentiating grows to more than 1cm, then bud clump is cut, be forwarded to fresh
Bud breaks up and the Multiplying culture that bud is carried out on proliferated culture medium, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 2000-3000lx, daily
Illumination 12-14h, transfers once every 30d or so.
Culture of rootage described in step 4, preferred clip robust growth, the sterile bud of long more than 3cm is seeded to culture of rootage
On base, it is placed under the indoor low light condition of culture and cultivates, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 1000-1500lx, daily illumination
The base portion incision of seedling starts to expand and grows callus after 12-14h, 12d, continues to cultivate and grows from callus after 5d
Radicula.
Transplantation of seedlings of taking root described in step 5, after preferred culture of rootage 25d, the root system for treating seedling is grown together, by bottle of taking root substantially
Seedling moves to hardening canopy and carries out hardening, and the hardening time is 10d, can be carried out transplanting through the seedling of taking root of hardening, will be raw during transplanting
Offspring takes out from blake bottle, and with clear water the culture medium of seedling root system is washed away, then by little transplantation of seedlings to the nursery equipped with matrix
In bag, transplanting medium selects yellow soil 50%+ peat soils 50%, transplanting to carry out in seedling growth greenhouse, to pour permeable after transplanting, with thin
Film covering and heat insulating moisturizing, progressively divulges information after 20d, and 25d removes film.
Compared with prior art, the invention has the advantages that：
1st, the present invention carries out seedling propagation to Golden Bell Tree using the method for tissue cultures first, and the method ratio is by broadcasting
Plant nursery to breed faster, can reach the requirement of large-scale production, and can effectively keep maternal excellent inhereditary feature, it is ensured that
The quality and quality of seedling, for Golden Bell Tree seedling propagation new effective way is provided, and is solved and is planted with seeding and seedling raising
The problem that sub- vigor and season limit affect.
2nd, explant material used in the present invention is seed, and material is conveniently easy to get, easily sterilization, and rudiment is fast, only need to lack
Amount material just can rapidly obtain sterilizable material, and application cost is relatively low.
3rd, the method for the present invention, benevolence can directly induce acquisition aseptic seedling on initial medium, with the nothing of Seed inducement
Vaccine is passed through with the numerous bud Multiplying culture of bud, and sterile bud year, breeding coefficient was up to 3.012, aseptic seedling rooting rate cultivated 10 days up to 97.0%
Left and right can go out root, well developed root system, and averaging out root amount has 4, can transplant within 30 days, and transplanting survival rate can reach scale up to 95.0%
The requirement that metaplasia is produced.
Specific embodiment
Below with embodiment, the invention will be further described, but the invention is not limited in these embodiments.
Embodiment 1：
(1) preparation of culture medium：Including the constituent and its per liter of institute of minimal medium and each cultivation stage culture medium
The weight for containing, wherein：
1st, minimal medium：MS culture mediums；
2nd, initial incubation culture medium：MS+6-BA0.4mg/L+NAA0.2mg/L+ sucrose 30g/L, agar powder 4.2g/L, pH
=5.8；
3rd, bud differentiation and proliferated culture medium：MS+6-BA1.2mg/L+IAA0.3-0.5mg/L+L- Cys2 5mg/L+
Sucrose 30g/L, agar powder 4.2g/L, pH=5.8；
4th, root media：1/2MS+IBA0.8mg/L+ activated carbon 0.2g/L+ sucrose 20g/L, agar powder 4.5g/L, pH
=5.8；
(2) process and sterilization of explant：With Golden Bell Tree seed as explant, outer layer kind shell is peelled off, takes benevolence,
With 0.1%HgCl on superclean bench2Solution disinfection 8min, then with sterile water wash 4-5 time, it is stand-by after draining；
(3) initial incubation：The explant for disinfecting is seeded in Initial culture base, light culture 5 days then moves to nature
Cultivate 20d under illumination condition, cultivation temperature is 25 ± 2 DEG C, light culture after 5 days seed start to sprout, under the conditions of natural lighting
Continue to cultivate 5 days and can grow seedling, aseptic seedling can be grown to about 4cm within 20 days；
(4) differentiation of bud and propagation：The aseptic seedling obtained in step 2 is cut into the band axillary bud sections for being about 2cm, is connect
Plant in bud differentiation and proliferated culture medium, the indoor low light condition of culture will be placed on bud sections oblique cutting on culture medium during inoculation
Lower culture, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 1000-1500lx, daily illumination 12-14h, culture 20d or so are at axillary bud
Budlet can be differentiated；When the budlet for differentiating grows to more than 1cm, then bud clump is cut, be forwarded to fresh bud differentiation and increasing
Growing carries out the Multiplying culture of bud on culture medium, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 2000-3000lx, daily illumination 12-
14h, transfers once every 30d or so；
(5) culture of rootage：Clip robust growth, the sterile bud of long more than 3cm is seeded on root media, is placed on training
Cultivate under the conditions of foster indoor weak light, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 1000-1500lx, daily illumination 12-14h, after 12d
The base portion incision of seedling starts to expand and grows callus, continues to cultivate and radicula is grown from callus after 5d；
(6) take root transplantation of seedlings：After culture of rootage 25d, the root system for treating seedling is grown together substantially, and bottle seedling of taking root moves to hardening
Canopy carries out hardening, and the hardening time is 10d；Can be carried out transplanting through the seedling of taking root of hardening, the seedling that will take root during transplanting is from culture
Take out in bottle, with clear water the culture medium of seedling root system is washed away, then by little transplantation of seedlings to the Seedling bag equipped with matrix, transplant base
Matter selects yellow soil 50%+ peat soils 50%, transplanting to carry out in seedling growth greenhouse, to pour permeable after transplanting, uses film covering and heat insulating
Moisturizing, progressively divulges information after 20d, and 25d removes film.
Embodiment 2：
(1) preparation of culture medium：Including the constituent and its per liter of institute of minimal medium and each cultivation stage culture medium
The weight for containing, wherein：
1st, minimal medium：MS culture mediums；
2nd, initial incubation culture medium：MS+6-BA0.4mg/L+NAA0.2mg/L+ sucrose 30g/L, agar powder 4.2g/L, pH
=5.8；
3rd, bud differentiation and proliferated culture medium：MS+6-BA1.6mg/L+IAA0.3-0.5mg/L+L- Cys2 5mg/L+
Sucrose 30g/L, agar powder 4.2g/L, pH=5.8；
4th, root media：1/2MS+IBA1.0mg/L+ activated carbon 0.2g/L+ sucrose 20g/L, agar powder 4.5g/L, pH
=5.8；
(2) process and sterilization of explant：With Golden Bell Tree seed as explant, outer layer kind shell is peelled off, takes benevolence,
With 0.1%HgCl on superclean bench2Solution disinfection 10min, then with sterile water wash 4-5 time, it is stand-by after draining；
(3) initial incubation：The explant for disinfecting is seeded in Initial culture base, light culture 5 days then moves to nature
Cultivate 20d under illumination condition, cultivation temperature is 25 ± 2 DEG C, light culture after 5 days seed start to sprout, under the conditions of natural lighting
Continue to cultivate 5 days and can grow seedling, aseptic seedling can be grown to about 4cm within 20 days；
(4) differentiation of bud and propagation：The aseptic seedling obtained in step 2 is cut into the band axillary bud sections for being about 2cm, is connect
Plant in bud differentiation and proliferated culture medium, the indoor low light condition of culture will be placed on bud sections oblique cutting on culture medium during inoculation
Lower culture, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 1000-1500lx, daily illumination 12-14h, culture 20d or so are at axillary bud
Budlet can be differentiated；When the budlet for differentiating grows to more than 1cm, then bud clump is cut, be forwarded to fresh bud differentiation and increasing
Growing carries out the Multiplying culture of bud on culture medium, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 2000-3000lx, daily illumination 12-
14h, transfers once every 30d or so；
(5) culture of rootage：Clip robust growth, the sterile bud of long more than 3cm is seeded on root media, is placed on training
Cultivate under the conditions of foster indoor weak light, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 1000-1500lx, daily illumination 12-14h, after 12d
The base portion incision of seedling starts to expand and grows callus, continues to cultivate and radicula is grown from callus after 5d；
(6) take root transplantation of seedlings：After culture of rootage 25d, the root system for treating seedling is grown together substantially, and bottle seedling of taking root moves to hardening
Canopy carries out hardening, and the hardening time is 10d；Can be carried out transplanting through the seedling of taking root of hardening, the seedling that will take root during transplanting is from culture
Take out in bottle, with clear water the culture medium of seedling root system is washed away, then by little transplantation of seedlings to the Seedling bag equipped with matrix, transplant base
Matter selects yellow soil 50%+ peat soils 50%, transplanting to carry out in seedling growth greenhouse, to pour permeable after transplanting, uses film covering and heat insulating
Moisturizing, progressively divulges information after 20d, and 25d removes film.
Tissue culture culture is carried out to Golden Bell Tree by above example, plantlet in vitro can be successfully cultivated, it is aseptic after
Dai Miaonian breeding coefficients are up to 3.012, Regenerated plant Bud Differentiation is more, and bud is healthy and strong, and growth is neat, and leaf color is dark green；Aseptic seedling rooting rate reaches
97.0%, cultivating can go out root for 10 days or so, well developed root system, and averaging out root amount has 4, can transplant within 30 days, and transplanting survival rate reaches
95.0%.The technology ratio is bred faster by seeding and seedling raising, can reach the requirement of large-scale production, and can effectively keep maternal
Original excellent inhereditary feature, it is ensured that the quality and quality of seedling.
Claims (6)
1. a kind of Golden Bell Tree quick breeding method for tissue culture, it is characterised in that specifically include following steps：
Step 1, the process and sterilization of explant：With Golden Bell Tree seed as explant, outer layer kind shell is peelled off, take benevolence,
Sterilize on superclean bench, then with sterile water wash, it is stand-by after draining；
Step 2, initial incubation：The explant for disinfecting is seeded in Initial culture base, light culture, then moves to natural lighting bar
Cultivate under part；
Step 3, the differentiation of bud and propagation：By in step 2 obtain aseptic seedling cut into band axillary bud sections, be inoculated into bud differentiation and
On proliferated culture medium, will be placed under the indoor low light condition of culture and cultivate, in axillary bud with bud sections oblique cutting on culture medium during inoculation
Place differentiates budlet, and bud clump is cut, and is forwarded to the Multiplying culture that fresh bud breaks up and bud is carried out on proliferated culture medium, timing
Switching；
Step 4, culture of rootage：The sterile bud of clip robust growth is seeded on root media, is placed on culture indoor weak light bar
Cultivate under part, treat that the base portion incision of seedling starts to expand and grow callus, continue to grow from callus after cultivating carefully
Root；
Step 5, transplantation of seedlings of taking root：Culture of rootage treats that the root system of seedling is grown together substantially, and bottle seedling of taking root moves to hardening canopy and refined
Seedling, is transplanted through the seedling of taking root of hardening, and the seedling that will take root during transplanting takes out from blake bottle, and with clear water seedling root system is washed away
Culture medium, then by little transplantation of seedlings to the Seedling bag equipped with matrix, transplanting is carried out in seedling growth greenhouse, to pour permeable after transplanting,
With film covering and heat insulating moisturizing, progressively divulge information, finally remove film；
The culture medium prescription for being used is as follows：
Initial incubation culture medium：MS+6-BA0.4mg/L+NAA0.2mg/L+ sucrose 30g/L, agar powder 4.2g/L, pH=5.8；
Bud breaks up and proliferated culture medium：MS+6-BA1.2-1.6mg/L+IAA0.3-0.5mg/L+L- Cys2 5mg/L+ sugarcanes
Sugared 30g/L, agar powder 4.2g/L, pH=5.8；
Root media：1/2MS+IBA0.8-1.0mg/L+ activated carbon 0.2g/L+ sucrose 20g/L, agar powder 4.5g/L, pH=
5.8。
2. a kind of Golden Bell Tree quick breeding method for tissue culture according to claim 1, it is characterised in that：Step 1
Described sterilizes on superclean bench, then with sterile water wash, refers on superclean bench with 0.1%HgCl2Solution disappears
Malicious 8-10min, then with sterile water wash 4-5 time.
3. a kind of Golden Bell Tree quick breeding method for tissue culture according to claim 1, it is characterised in that：Step 2
Described light culture, then moves to be cultivated under the conditions of natural lighting, refers to light culture 5 days, then moves to be cultivated under the conditions of natural lighting
20d, cultivation temperature is 25 ± 2 DEG C.
4. a kind of Golden Bell Tree quick breeding method for tissue culture according to claim 1, it is characterised in that：Step 3
The differentiation of described bud and propagation, refer to that the aseptic seedling that will be obtained in step 2 shears the band axillary bud sections of growth 2cm, are inoculated into
Bud breaks up with proliferated culture medium, will be placed under the indoor low light condition of culture and train with bud sections oblique cutting on culture medium during inoculation
Support, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 1000-1500lx, daily illumination 12-14h, culture 20d differentiates little at axillary bud
Bud, when the budlet for differentiating grows to 1cm, then bud clump is cut, and is forwarded to fresh bud and is broken up and carry out on proliferated culture medium
The Multiplying culture of bud, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 2000-3000lx, daily illumination 12-14h, every 30d switchings one
It is secondary.
5. a kind of Golden Bell Tree quick breeding method for tissue culture according to claim 1, it is characterised in that：Step 4
Described culture of rootage, refers to clip robust growth, and the sterile bud of long 3cm is seeded on root media, is placed on culture indoor
Cultivate under low light condition, 25 ± 2 DEG C of cultivation temperature, intensity of illumination 1000-1500lx, daily illumination 12-14h, seedling after 12d
Base portion incision starts to expand and grows callus, continues to cultivate and radicula is grown from callus after 5d.
6. a kind of Golden Bell Tree quick breeding method for tissue culture according to claim 1, it is characterised in that：Step 5
Described transplantation of seedlings of taking root, after referring to culture of rootage 25d, the root system of seedling is grown together substantially, and bottle seedling of taking root moves to hardening canopy and enters
Row hardening, the hardening time is 10d, is just transplanted through the seedling of taking root of hardening, and the seedling that will take root during transplanting takes out from blake bottle,
The culture medium of seedling root system is washed away with clear water, then by little transplantation of seedlings to the Seedling bag equipped with matrix, transplanting medium is from yellow
Cubsoil 50%+ peat soils 50%, transplanting is carried out in seedling growth greenhouse, to pour permeable after transplanting, with film covering and heat insulating moisturizing, 20d
Progressively divulge information afterwards, 25d removes film.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108391591A (en) * | 2018-01-26 | 2018-08-14 | 长江大学 | A kind of Golden Bell Tree tissue cultivation rapid breeding method |
| CN110100687A (en) * | 2019-04-28 | 2019-08-09 | 广东美景园林建设有限公司 | A kind of breeding method of growing thickly of Golden Bell Tree |
| CN116784234A (en) * | 2023-06-27 | 2023-09-22 | 华南农业大学 | Method for obtaining regenerated complete plants by inducing in-vitro hypocotyls of aeolia annua |
-
2017
- 2017-03-21 CN CN201710170198.1A patent/CN106665367B/en active Active
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108391591A (en) * | 2018-01-26 | 2018-08-14 | 长江大学 | A kind of Golden Bell Tree tissue cultivation rapid breeding method |
| CN108391591B (en) * | 2018-01-26 | 2021-07-06 | 长江大学 | Tissue culture and rapid propagation method for tabebuia flavedo |
| CN110100687A (en) * | 2019-04-28 | 2019-08-09 | 广东美景园林建设有限公司 | A kind of breeding method of growing thickly of Golden Bell Tree |
| CN116784234A (en) * | 2023-06-27 | 2023-09-22 | 华南农业大学 | Method for obtaining regenerated complete plants by inducing in-vitro hypocotyls of aeolia annua |
| CN116784234B (en) * | 2023-06-27 | 2023-12-19 | 华南农业大学 | Method for obtaining regenerated complete plants by inducing in-vitro hypocotyls of aeolia annua |
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| CN106665367B (en) | 2018-07-27 |
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