CN109220791A - A kind of tissue culture method using bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait - Google Patents
A kind of tissue culture method using bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait Download PDFInfo
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- CN109220791A CN109220791A CN201811060924.5A CN201811060924A CN109220791A CN 109220791 A CN109220791 A CN 109220791A CN 201811060924 A CN201811060924 A CN 201811060924A CN 109220791 A CN109220791 A CN 109220791A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
A kind of tissue culture method using bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait, selection and disinfection including explant, Fiber differentiation, shoot proliferation culture, it takes root and transplant step, the present invention is with the clove explant of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait, conducive to reduction pollution rate, improve inductivity, it is not required to by callus phase, it just can induce out Multiple Buds, Multiple Buds directly induce and obtain bulb, shorten induction time, Multiplying culture can be put into, it takes root to the test tube ball after proliferation, a step balling-up for present invention realization Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait clove, save the time of tissue culture needs, reduce operating procedure, maintain the good strains of seeds of protocorm, effective way is provided for the efficient industrial breeding of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait, technical support is provided for Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait industry development.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to a kind of tissue culture side using bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait
Method.
Background technique
Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait is Hybrid group's general name that Amaryllidaceae belladonna lily belongs to, and has large flower and brilliant color, form abundant etc. obvious excellent
Gesture has very high ornamental values and the economic values.China has introduced part kind, and market acceptance is higher, and Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait becomes
One of emerging flower variety, still, the natural propagation rate of hippeastrum bulb are very low, wherein most kinds and cenospecies hardly produce
Raw bulbec.
Although also there is the production of hippeastrum bulb in Asia, production facility falls behind relatively, domestic Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait kind phase
To single, bulb production is less, and only domestic bulb is second-rate, and excellent bulb is largely relied on from countries such as Holland, South Africa
Import, expensive bulb price strongly limit this bulb root flowers in the development in China.
The common propagation method of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait mainly has: natural bulb separation, cuttage and sowing etc., still, reproduction speed is very slow, very
Difficulty meets the needs of market.
In recent years, it has been widely used both at home and abroad and has hurt method at bulb cuttage, tissue cultures and quarter to breed, to accelerate its breeding speed
Degree.It is cumbersome to hurt method operation sequence at bulb cuttage and quarter, it is at high cost, it is influenced by external condition.
Tissue culture technique is one of current plant fast propagation most efficient method, has breeding coefficient height, seedling time short
With the advantage convenient for commercialization and industrialization production.
Currently, the tissue culture explant selection of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait is relatively more, there are big bulb, blade, scape, bennet etc., still, only
When double scales in bulb are as explant, Multiple Buds can be just induced, the induction of other explants all can only obtain callus,
And when bulb is as explant greatly, disinfection efficiency is low, and pollution rate reaches as high as 90%, and after general induction, growth coefficient only has 2
~3, and after obtaining Multiple Buds, culture of rootage is directly carried out to Multiple Buds, or carry out strong ball culture again, finally transplanted,
Transplanting survival rate 70~90%.
As it can be seen that existing Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait tissue culture explant disinfection efficiency is low, growth coefficient is lower, transplanting survival rate is limited, proliferation
The problems such as process also needs strong ball culture, complex for operation step.
Summary of the invention
The purpose of the present invention is to provide a kind of tissue culture methods using bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait, using clove as explant
Body, inoculation pollution rate is low, and fast breeding goes out test tube clove, and easy to operate effective, inductivity is high, the test tube bulbs of a step balling-up
Rooting rate is high, and transplanting survival rate is high, shortens induction and breeding time, saves the tissue culture time, reduce operating procedure, maintain
The good strains of seeds of protocorm overcomes the problems, such as that operating procedure is more in Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait tissue culture and survival rate is lower, sends out for Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait industry
Exhibition provides technical support.
In order to achieve the above object, the invention provides the following technical scheme:
A kind of tissue culture method using bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait, comprising the following steps:
1) selection and disinfection of explant
It chooses health, the Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait clove that diameter is 0.5~1cm, is placed in 4~10 DEG C of cold treatments 4~6 weeks, goes scaling
Film, water rinse 1~3h, sterilize, and rinse, spare after suck dry moisture；
2) Fiber differentiation
The hippeastrum bulb disinfected is cut off into top, the plateau with 1/2~2/3 scale base portion is stayed, by plateau
Cross is averagely longitudinal sectional, and at 2~4 parts, base portion is inoculated with cultivates on the induction medium downward, and illumination cultivation induces after 30~40 days
Multiple Buds, Multiple Buds base portion expand, and continue culture 20~30 days, obtain bulb of growing thickly；
22~27 DEG C of cultivation temperature, humidity 60~80%；Illumination cultivation condition: 10~14h/d of light application time, intensity of illumination
For 1500~2500LX；
3) shoot proliferation
The bulb that will grow thickly is cut into single bulb, cuts off top, retains 0.3~0.5cm of base portion, is seeded to proliferated culture medium
On, it cultivates 40~60 days, obtains proliferation bulb；
Condition of culture: 22~27 DEG C of temperature, humidity 60~80%, 10~14h/d of light application time, intensity of illumination be 1500~
2500LX；
4) culture of rootage
It is the single bulb with base portion by proliferation bulb cutting, is inoculated in root media, carries out culture of rootage, culture
15~20 days, obtain the Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait seedling to take root；
Condition of culture are as follows: 22~27 DEG C, humidity 60~80%, 10~14h/d of light application time, intensity of illumination 1500~
2500LX；
5) it transplants
The Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait seedling to take root is taken out, root system agar is cleaned, is transplanted into matrix, regeneration Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait is obtained after hardening and is planted
Strain.
Further, the step 2) induced medium includes: MS minimal medium, 1~5mg/L of 6-benzyl aminopurine, naphthalene
0.5~3mg/L of acetic acid, 30~40g/L of sucrose, 4~6g/L of agar, pH5.8~6.0.
Also, the step 3) proliferated culture medium are as follows: MS minimal medium, 1~5mg/L of 6-benzyl aminopurine, methyl α-naphthyl acetate 0.5
~2mg/L, 4~6g/L of agar, sucrose 30~60g/L, pH 5.8~6.0.
Further, the root media includes: 1/2MS minimal medium, 0.05~1.0mg/L of methyl α-naphthyl acetate, active carbon
0.5~1.0g/L, 30~40g/L of sucrose, 4~6g/L of agar powder, pH5.8~6.0.
Preferably, in step 5) transplanting into matrix before, carry out hardening, hardening process are as follows: under greenhouse experiment, keep
Air and matrix are wet, 20~25 DEG C of temperature, shade 7~15 days, then full exposure, a water are poured within every 2~3 days, after 20~30 days
Transplanting, survival rate 100%.
Also, the matrix is peat: perlite=7:3~5:5, volume ratio.
Further, in step 3), the diameter of single bulb is longitudinally averagely cut into 2~4 pieces in 0.5cm or more, by it,
It inoculates to proliferated culture medium, when the diameter of single bulb is less than 0.5cm, it is directly seeded on proliferated culture medium.
MS minimal medium of the invention refers to: Murashige and Skoog was tobacco cell Training Design in 1962
, it is the culture medium being commonly used.
1/2MS minimal medium of the invention refers to: a great number of elements, microelement in MS minimal medium, have molysite
Machine material concentration halves.
The present invention directly induces test tube clove using Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait clove as explant, and to the small squama of test tube after proliferation
Stem is taken root, and is saved the time of tissue culture needs, is reduced operating procedure, maintain the good strains of seeds of protocorm, is Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait
Industry development provides technical support.
Compared with prior art, the invention has the following beneficial effects:
1) for the present invention using the clove of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait as explant material, clove growth year is short, and contained endophyte is few,
So that pollution rate is reduced to 6~10%, overcomes the problems such as pollution rate is high after disinfection when the big bulb of utilization is explant material in the past.
2) inductivity of test tube clove is increased to 99.6% by the present invention, meanwhile, present invention reduces induction time,
It just can induce out Multiple Buds in 1 month or so explant, grow into test tube clove within 2 months or so, proliferation training can be put into
It supports, growth coefficient is increased to 4~6 from existing 2~3.
3) the present invention in vitro directly induces Multiple Buds, no replacement is required culture medium, is implemented as on former culture medium
Ball, an one-step inducing go out clove, eliminate after inducing callus in the past, by the process for being divided into Multiple Buds again.
4) after the present invention induces test tube ball, take root through fast breeding, transplanting survival rate up to 100%, and make entirely from
Explant is inoculated into the tissue culture process of transplant survival, from original 5~6 months, shortens to 3~4 months or so, is greatly shortened
Breeding process is accelerated, it can be achieved that efficient industrial breeding, solves the problems, such as that the domestic middle bulb of Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait is rare breeding cycle.
Detailed description of the invention
Fig. 1 is the clove after being proliferated in the embodiment of the present invention.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment
A kind of tissue culture method using bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait, comprising the following steps:
1, the selection and disinfection of explant
After taking bulb, the clove (diameter < 1cm) of health, 4 DEG C or so the Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Aits after cold treatment 4 weeks is chosen, is gone
Fall outer layer brown epithelium, tap water rinses 1h, on superclean bench, is impregnating 30s, sterile water wash with 70% alcohol, then use
0.1% mercuric chloride impregnates 8~12min, then uses aseptic water washing 5 times, spare after suck dry moisture.
2, Fiber differentiation
By the clove cross disinfected it is average it is longitudinal sectional at 4 parts, base portion is inoculated on the induction medium downward, after inoculation,
Pollution rate is lower than 8%.
Wherein, the induced medium is MS+6-BA2mg/L+NAA1mg/L+ sucrose 30g/L+ agar 4g/L, PH5.8.
Inducing culturing condition are as follows: condition of culture is 25 DEG C of temperature, humidity 70%.12h/d illumination cultivation, intensity of illumination are
2000LX。
Multiple Buds are induced after about 1 month, continue to cultivate on former culture medium, it is small that Multiple Buds base portion gradually enlarges into test tube
Bulb；If culture 2 months, the explant after each cutting can produce 3.5 cloves.
3, shoot proliferation culture
Will test tube bulbs (diameter be greater than 0.5cm) that induction generates are single separates, cut at the top of stem, it is average longitudinal sectional at 2 half,
It is both needed to band plateau.After cutting, it is seeded on proliferated culture medium and cultivates 1 month, growth coefficient 4.8, after Multiplying culture, small squama
Stem nematode is healthy and strong, and clove diameter is 8~12cm up to 0.3~1.0cm, plant height, and single clove has 2~3, blade, and companion
With taking root on a small quantity, referring to Fig. 1.
Wherein, the proliferated culture medium are as follows: MS+6-BA2mg/L+NAA0.5mg/L+ sucrose 40g/L+ agar 4g/L
(PH5.8)；Multiplying culture condition are as follows: 25 DEG C, humidity 70%, intensity of illumination 2000LX, light application time: 10h/d.
4, test tube bulbs are taken root
The test tube bulbs that shoot proliferation is generated individually separate, and each band plateau is inoculated on root media, trains
It supports 20 days, rooting rate reaches 100%；
Wherein, the root media are as follows: 1/2MS+NAA0.5mg/L+AC0.5g/L+ sucrose 40g/L+ agar 4g/L
(PH5.8)；Condition of culture are as follows: 25 DEG C, humidity 70%, light application time 10h/d, intensity of illumination 2000LX.
5, test tube bulbs are transplanted
The test tube bulbs taken root are taken out from culture bottle, clean the agar on root system, root system dips a small amount of pentachloro- nitre
Base benzene wettable powder is transplanted in 32 hole hole trays, and cultivation matrix is peat: perlite=7:3.Under greenhouse experiment, shade, keeps
Air and matrix are wet, and 20~25 DEG C of temperature or so.After 10 days, full exposure pours a water in every 2~3 days, after 1 month transplanting at
Motility rate 100%.
Claims (7)
1. a kind of tissue culture method using bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait, comprising the following steps:
1) selection and disinfection of explant
The Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait clove for choosing health, 0.5~1cm of diameter, is placed in 4~10 DEG C of cold treatments 4~6 weeks, removes epithelium, water punching
1~3h is washed, is sterilized, is rinsed, it is spare after suck dry moisture；
2) Fiber differentiation
The hippeastrum bulb disinfected is cut off into top, the plateau with 1/2~2/3 scale base portion is stayed, by plateau cross
Averagely longitudinal sectional at 2~4 parts, base portion is inoculated with cultivates on the induction medium downward, and illumination cultivation induces after 30~40 days grows thickly
Bud, Multiple Buds base portion expand, and continue culture 20~30 days, obtain bulb of growing thickly；
22~27 DEG C of cultivation temperature, humidity 60~80%；Illumination cultivation condition: 10~14h/d of light application time, intensity of illumination are
1500~2500LX；
3) shoot proliferation
The bulb that will grow thickly is cut into single bulb, cuts off top, retains 0.3~0.5cm of base portion, is seeded on proliferated culture medium, trains
It supports 40~60 days, obtains proliferation bulb；
Condition of culture: 22~27 DEG C of temperature, humidity 60~80%, 10~14h/d of light application time, intensity of illumination be 1500~
2500LX；
4) culture of rootage
It is the single bulb with base portion by proliferation bulb cutting, is inoculated in root media, progress culture of rootage, culture 15~
20 days, obtain the Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait seedling to take root；
Condition of culture are as follows: 22~27 DEG C, humidity 60~80%, 10~14h/d of light application time, 1500~2500LX of intensity of illumination；
5) it transplants
The Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait seedling to take root is taken out, root system agar is cleaned, is transplanted into matrix, regeneration Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait plant is obtained after hardening.
2. utilizing the tissue culture method of bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait according to claim 1, which is characterized in that the step 2) induction
Culture medium includes: MS minimal medium, 1~5mg/L of 6-benzyl aminopurine, 0.5~3mg/L of methyl α-naphthyl acetate, 30~40g/L of sucrose,
Agar 4~6g/L, pH 5.8~6.0.
3. utilizing the tissue culture method of bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait according to claim 1, which is characterized in that the step 3) proliferation
Culture medium are as follows: MS minimal medium, 1~5mg/L of 6-benzyl aminopurine, 0.5~2mg/L of methyl α-naphthyl acetate, 4~6g/L of agar, sucrose
30~60g/L, pH 5.8~6.0.
4. utilizing the tissue culture method of bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait according to claim 1, which is characterized in that the root media
Include: 1/2MS minimal medium, 0.05~1.0mg/L of methyl α-naphthyl acetate, 0.5~1.0g/L of active carbon, 30~40g/L of sucrose, agar
Powder 4~6g/L, pH 5.8~6.0.
5. utilizing the tissue culture method of bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait according to claim 1, which is characterized in that moving in step 5)
Before planting into matrix, hardening, hardening process are carried out are as follows: under greenhouse experiment, keep air and matrix wet, 20~25 DEG C of temperature,
Shading 7~15 days, then full exposure, pour a water in every 2~3 days, transplanting survival rate 100% after 20~30 days.
6. utilizing the tissue culture method of bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait according to claim 1, which is characterized in that the matrix is mud
Charcoal: perlite=7:3~5:5, volume ratio.
7. any one of -6 tissue culture method using bulb breeding Hipeastrum vittalum (L Her.) Herb.- Amaryllisvittata Ait according to claim 1, which is characterized in that step 3)
In, the diameter of single bulb is longitudinally averagely cut into 2~4 pieces in 0.5cm or more, by it, it inoculates to proliferated culture medium,
When the diameter of single bulb is less than 0.5cm, it is directly seeded on proliferated culture medium.
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Cited By (7)
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| CN110447537A (en) * | 2019-08-23 | 2019-11-15 | 江苏省中国科学院植物研究所 | A kind of tissue culture method obtaining regeneration plant using hippeastrum bulb disk as explant |
| CN112690213A (en) * | 2021-01-14 | 2021-04-23 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality seedlings of hippeastrum rutilum |
| CN112931210A (en) * | 2021-03-18 | 2021-06-11 | 四川迪菲特药业有限公司 | Bulb proliferation culture method taking fritillaria paniculata bulb discs as explants |
| CN113243295A (en) * | 2021-05-25 | 2021-08-13 | 广东省林业科学研究院 | Hippeastrum rutilum tissue culture breeding method |
| CN113348806A (en) * | 2021-07-01 | 2021-09-07 | 浙江省农业科学院 | Method for promoting lateral bud of pseudobulb of orchid to sprout in advance |
| CN114557278A (en) * | 2022-03-17 | 2022-05-31 | 重庆三峡职业学院 | Tissue culture and rapid propagation method and culture medium for hippeastrum rutilum |
| CN116369208A (en) * | 2023-06-07 | 2023-07-04 | 包头市农牧科学技术研究所 | Tissue culture and rapid propagation method for hippeastrum henryi and bulb pretreatment device thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110447537A (en) * | 2019-08-23 | 2019-11-15 | 江苏省中国科学院植物研究所 | A kind of tissue culture method obtaining regeneration plant using hippeastrum bulb disk as explant |
| CN112690213A (en) * | 2021-01-14 | 2021-04-23 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality seedlings of hippeastrum rutilum |
| CN112690213B (en) * | 2021-01-14 | 2022-07-12 | 中国科学院华南植物园 | Tissue culture and rapid propagation method for high-quality seedlings of hippeastrum rutilum |
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| CN112931210B (en) * | 2021-03-18 | 2022-06-21 | 四川迪菲特药业有限公司 | Bulb proliferation culture method taking fritillaria paniculata bulb discs as explants |
| CN112931210A (en) * | 2021-03-18 | 2021-06-11 | 四川迪菲特药业有限公司 | Bulb proliferation culture method taking fritillaria paniculata bulb discs as explants |
| CN113243295A (en) * | 2021-05-25 | 2021-08-13 | 广东省林业科学研究院 | Hippeastrum rutilum tissue culture breeding method |
| CN113243295B (en) * | 2021-05-25 | 2022-06-07 | 广东省林业科学研究院 | Hippeastrum rutilum tissue culture breeding method |
| CN113348806A (en) * | 2021-07-01 | 2021-09-07 | 浙江省农业科学院 | Method for promoting lateral bud of pseudobulb of orchid to sprout in advance |
| CN114557278A (en) * | 2022-03-17 | 2022-05-31 | 重庆三峡职业学院 | Tissue culture and rapid propagation method and culture medium for hippeastrum rutilum |
| CN114557278B (en) * | 2022-03-17 | 2023-01-17 | 重庆三峡职业学院 | Rapid propagation method and culture medium for hippeastrum rutilum tissue culture |
| CN116369208A (en) * | 2023-06-07 | 2023-07-04 | 包头市农牧科学技术研究所 | Tissue culture and rapid propagation method for hippeastrum henryi and bulb pretreatment device thereof |
| CN116369208B (en) * | 2023-06-07 | 2024-04-26 | 包头市农牧科学技术研究所 | Tissue culture and rapid propagation method for hippeastrum henryi and bulb pretreatment device thereof |
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