CN104115751B - A kind of cultural method utilizing Chinese cabbage ball leaf to obtain regeneration plant - Google Patents
A kind of cultural method utilizing Chinese cabbage ball leaf to obtain regeneration plant Download PDFInfo
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Abstract
A kind of facility of drawing materials, low cost, reproducible and without deformity bud utilize Chinese cabbage ball leaf blade obtain regeneration plant method.Its feature comprises the following steps: (1) chooses heading stage Chinese cabbage external ball leaf, aseptically sterilization.(2) face of blade after sterilizing is upwards flat in bud inducement culture medium cultivates.(3), after outer implant cultivates 28d in bud inducement culture medium, proceed to MS minimal medium continue cultivate.The regenerated adventitious bud more than 1.5cm of the length in outer implant is cut, proceeds to root media root induction.(4) after regeneration bud base portion forms a large amount of Rhizoma Imperatae, the root media of regrowth root is cleaned, plant in the plastic flowerpot filling solid matrix.This method is directly drawn materials from field growing plant, it is not necessary to through Seed Germination, to preserving in time, excellent precious Chinese cabbage material sense is great, provides stable receptor new material for Chinese cabbage transgenic breeding research simultaneously.
Description
Technical field
The present invention relates to Chinese cabbage technical field of tissue culture, draw materials facility, low cost, reproducible particularly to one
And the Chinese cabbage ball leaf that utilizes without deformity bud obtains the cultural method of regeneration plant.
Background technology
Chinese cabbage (Brassica campestris L.ssp.pekinensis (Lour) Olsson) is that face is cultivated by China
Long-pending maximum Cruciferae brassica plant, is constantly in critical role in the vegetable production and consumption of the whole nation.The most biological skill
Art effect in improving Chinese cabbage breeding efficiency highlights day by day.Tissue culture as one of most important animal nutrition,
To the preservation of Chinese cabbage germplasm materials and the most numerous, and in genetic engineering breeding, obtain the most stable transfer-gen plant have
Significance.Chinese cabbage belongs to Brassica genus AA genome, carry suppression shoot regeneration gene, relatively belong in other genome types from
Body regeneration difficulty is big.Between different cultivars, regeneration frequency there is also larger difference.
Additionally, the commodity property of Chinese cabbage often could sufficiently show in the balling later stage, comprehensive to field material
Shape investigation also focuses primarily upon this period.If can at this moment the excellent material found be preserved and expanding propagation in time, then can carry
Front to excellent Chinese cabbage material expansion breeding utilization.And current renovation process often cannot preserve precious excellent kind matter in time
Material, and the material succeeded for transgenic can not expanding propagation in time, it is impossible to provide numerous studies material for follow-up research
Material, constrains the development of Chinese cabbage biotechnology breeding.
Forefathers use cotyledon, cotyledon petiole-cotyledon, hypocotyl, floral organ (Pernell etc., 2002；Sun Baojuan etc., 2005)
Chinese cabbage regeneration plant is obtained by evoking adventive bud Deng outer implant；Use sporidiole, flower pesticide, Protoplast cuhnre by induction
Embryo obtains regeneration plant.In the regenerating system set up at present, the most frequently used outer implant is cotyledon petiole-cotyledon, on this basis,
Different researcheres regenerate with the outer implant of the cotyledon petiole-cotyledon retaining different proportion cotyledon, and result of study differs greatly.And it is sub
The outer implant of petiole-cotyledon can only disposably be drawn materials, the outer implant limited amount every time obtained.
Chinese cabbage true leaf regeneration research both at home and abroad is less.Become (2001) such as thin China first with the aseptic seedling that seedling age is 3 weeks
Top 2-3 sheet tender leaf is outer implant, is cut into 2-4mm square, obtains adventitious bud by callus induction.At Liu Xuecheng etc.
(2011) in research, when Chinese cabbage aseptic seedling grows the 2nd true leaf, culture medium is inserted, from leaf with the petiole of the 1st true leaf
The induction of handle base portion obtains adventitious bud；And it is square that true leaf sheet is cut into 5mm, it is laid on inducing culture and then divides without adventitious bud
Change.Have not yet to see the report utilizing strain Chinese cabbage balling blade to carry out plant regeneration.
Summary of the invention
It is an object of the invention to provide a kind of facility of drawing materials, low cost, reproducible and without deformity bud utilize DABAI
Dish ball leaf blade obtains the method for regeneration plant.Save the Aseptic seedling culture link in conventional Chinese cabbage tissue culture procedures, make
Elite plant and the precious material of collection that field finds can be preserved and expanding propagation in time, and grind for Chinese cabbage genetic engineering
Study carefully the receptor providing new.
The technical scheme is that a kind of method utilizing Chinese cabbage ball leaf to obtain regeneration plant, it is characterised in that bag
Include following steps:
1) choose heading stage Chinese cabbage external ball leaf, remove petiole.Aseptically, first with the wine of volume fraction 70%
Essence surface sterilization 30s, rinsed with sterile water one time.Pouring the NaClO solution sterilization 10min of mass fraction 5% again into, sterilized water is gently
Rinsing 15min, period changes water 5-6 time.Sterilize complete, with sterilizing absorbent paper, blade residual moisture is blotted.
2) by the leaf block that blade cutting is length of side 1.5-2.0cm after sterilizing, avoiding thicker vein, leaf faces up horizontal
In bud inducement culture medium.At intensity of illumination 1500-2000Lux, photoperiod 16h/d, train under the conditions of temperature 24 ± 1 DEG C
Support.
3), after outer implant cultivates 28d in bud inducement culture medium, proceed to MS minimal medium (table 2) continues training afterwards
Support.The regenerated adventitious bud more than 1.5cm of the length in outer implant is cut, proceeds to root media root induction.More than cultivate bar
Part is: intensity of illumination 1500-2000Lux, photoperiod 16h/d, temperature 24 ± 1 DEG C.
4), after regeneration bud base portion forms a large amount of Rhizoma Imperatae, cultivation bottle cap seedling exercising 4-5d is opened, by the training of taking root of regrowth root
Support base to clean, plant in the plastic flowerpot filling solid matrix.Substrate uses Vermiculitum and peat composed of rotten mosses volume ratio 1: 1 to mix.Basin
Mouth antistaling film covers moisturizing.After 3d, beating a small amount of aperture at film surface, later stage punching number is gradually increased, and hole gradually adds
Greatly.After overlay film cultivates 10d, throw off antistaling film, regeneration plant is put and cultivates at room temperature.During substrate culture, all give
Natural lighting.
Step 2) described in bud inducement culture medium be MS+4mg/L6-BA+1mg/LNAA+4mg/LAgNO3+ 30g/L sugarcane
Sugar+0.7mg/L agar, pH is 5.8.
Step 3) described in root media be MS+0.5mg/L NAA+30g/L sucrose+0.7mg/L agar, pH is
5.8。
The invention has the beneficial effects as follows: the present invention is outer implant with Chinese cabbage ball leaf blade, to 6-BA in inducing culture
It is optimized with NAA concentration proportioning.More traditional Chinese cabbage tissue culture procedures simplifies, and eliminates Aseptic seedling culture link, joint
Save the time.Incubation is simple, and outer implant, after the inducing culture adding hormone cultivates 28d, can be placed directly on without any
Continue evoking adventive bud on the MS minimal medium of hormone, save hormone dosage, reduce tissue culture's cost.Draw materials just
Profit, outer planting body preparation amount is big, has significant application value to preserving precious material in time.The present invention is Chinese cabbage transgenic simultaneously
Breeding provides new transformation receptor, and also the rapid expanding propagation for transfer-gen plant provides efficient Regeneration Ways, widely should have
Use prospect.
The present invention is described further with embodiment below in conjunction with the accompanying drawings.
Accompanying drawing explanation
Fig. 1 is that the ball leaf blade inoculation after the sterilization of ' New Beijing three ' Chinese cabbage is in bud inducement culture medium picture；
Fig. 2 cultivates the picture of 7d for ' New Beijing three ' cabbage leaves in bud inducement culture medium, and blade edge expands,
Form adventitious root；
Fig. 3 cultivates the picture of 14d for ' New Beijing three ' cabbage leaves in bud inducement culture medium, sprouts adventitious root
Callus is formed at leaf margin；
Fig. 4 cultivates the picture (bottom) of 14d, adventitious root for ' New Beijing three ' cabbage leaves in bud inducement culture medium
Base portion forms bigger wound healing group；
Fig. 5 cultivates the picture of 22d for ' New Beijing three ' cabbage leaves in bud inducement culture medium, by leaf margin wound healing group
Knit place's some adventitious buds of induced synthesis；
Fig. 6 cultivates the picture of 15d for ' New Beijing three ' cabbage leaves in MS minimal medium, and adventitious bud blade is stretched
Long, increase；
Fig. 7 cultivates the picture of 10d for ' New Beijing three ' cabbage leaves regeneration bud in root media；
Fig. 8 cultivates the picture of 15d for ' Chengyang is blue or green ' cabbage leaves regeneration bud in root media；
Fig. 9 transfers to the picture after cultivating 10d in cultivation matrix for ' New Beijing three ' Chinese cabbage ball leaf regeneration plant；
Figure 10 transfers to the picture after cultivating 10d in cultivation matrix for ' Chengyang is blue or green ' Chinese cabbage ball leaf regeneration plant.
Detailed description of the invention
A kind of method utilizing Chinese cabbage ball leaf to obtain regeneration plant, comprises the following steps:
1) choose heading stage Chinese cabbage external ball leaf, remove petiole.Aseptically, first with the wine of volume fraction 70%
Essence surface sterilization 30s, rinsed with sterile water one time.Pouring the NaClO solution sterilization 10min of mass fraction 5% again into, sterilized water is gently
Rinsing 15min, period changes water 5-6 time.Sterilize complete, with sterilizing absorbent paper, blade residual moisture is blotted.
2) by the leaf block that blade cutting is length of side 1.5-2.0cm after sterilizing, avoiding thicker vein, leaf faces up horizontal
In bud inducement culture medium.At intensity of illumination 1500-2000Lux, photoperiod 16h/d, train under the conditions of temperature 24 ± 1 DEG C
Support.
3), after outer implant cultivates 28d in bud inducement culture medium, proceed to MS minimal medium (table 2) continues training afterwards
Support.The regenerated adventitious bud more than 1.5cm of the length in outer implant is cut, proceeds to root media root induction.More than cultivate bar
Part is: intensity of illumination 1500-2000Lux, photoperiod 16h/d, temperature 24 ± 1 DEG C.
4), after regeneration bud base portion forms a large amount of Rhizoma Imperatae, cultivation bottle cap seedling exercising 4-5d is opened, by the training of taking root of regrowth root
Support base to clean, plant in the plastic flowerpot filling solid matrix.Substrate uses Vermiculitum and peat composed of rotten mosses volume ratio 1: 1 to mix.Basin
Mouth antistaling film covers moisturizing.After 3d, beating a small amount of aperture at film surface, later stage punching number is gradually increased, and hole gradually adds
Greatly.After overlay film cultivates 10d, throw off antistaling film, regeneration plant is put and cultivates at room temperature.During substrate culture, all give
Natural lighting.
Step 2) described in bud inducement culture medium be MS+4mg/L6-BA+1mg/L NAA+4mg/LAgNO3+ 30g/L sugarcane
Sugar+0.7mg/L agar, pH is 5.8.
Step 3) described in root media be MS+0.5mg/L NAA+30g/L sucrose+0.7mg/L agar, pH is
5.8。
Experiment 1:
1) choose heading stage Chinese cabbage ' New Beijing three ' and ' the external ball leaf of blue or green ' two kind in Chengyang, removes petiole.
Aseptically, first by the ethanol surface sterilization 30s of volume fraction 70%, rinsed with sterile water one time.Pour mass fraction again into
The NaClO solution sterilization 10min of 5%, sterilized water rinses 15min gently, and period changes water 5-6 time.Sterilize complete, absorb water with sterilizing
Blade residual moisture is blotted by paper.
2) by the leaf block that blade cutting is length of side 1.5-2.0cm after sterilizing, thicker vein is avoided.Leaf faces up horizontal
In different 6-BA/NAA proportionings (table 1), additional 4mg/L AgNO3Bud inducement culture medium on.In intensity of illumination 1500-
2000Lux, photoperiod 16h/d, cultivate under the conditions of temperature 24 ± 1 DEG C.Each process 30-35 outer implant, in triplicate.
Adventitious shoot regeneration situation is added up after 4 weeks.
Different 6-BA Yu the NAA concentration proportioning impact on Chinese cabbage adventitious shoot regeneration of table 1
Note: use LSD method to carry out multiple comparisons, represent that difference does not shows in 5% level with identical person alphabetical after column data
Write.
Table 2 MS minimal medium formula
Culture medium is MS minimal medium+4mg/L AgNO3+ 30g/L sucrose+0.7mg/L agar, pH is 5.8.Substantially
Culture medium prescription is shown in Table 2.
The outer implant number of adventitious shoot regeneration rate (%)=have adventitious shoot regeneration/inoculation outer implant sum × 100%
The regenerated adventitious bud number of bud/outer implant (individual)=have independent growths point/inoculation outer implant sum
As shown in Table 1, in culture medium in the presence of only 6-BA, it is impossible to induce adventitious bud.In culture medium, 6-BA is 4mg/
When L, NAA are 1mg/L, ' New Beijing three ' and ' blue or green ' two the kind blade adventitious shoot regeneration rate in Chengyang all reaches the highest, respectively
It is 88.46% and 73.77%.Under this concentration combination, ' New Beijing three ' each outer implant average regeneration bud number is the highest, significantly
Higher than other concentration combination；' Chengyang is blue or green ' each outer implant average regeneration bud number is 1.64, less than 6-BA4mg/L, NAA0.5mg/
Average regeneration bud number 1.72 during L, but both are without significant difference.In this experiment, two kinds are at MS+4mg/L6-BA+1mg/L
NAA+4mg/LAgNO3In the culture medium of+30g/L sucrose+0.7mg/L agar, regeneration effect is best.This culture medium is set to this
Bud inducement culture medium in bright subsequent experimental.
It was experimentally observed that, first outer implant induces a large amount of adventitious root in the culture medium adding NAA.Cultivate 12-14d
After, at adventitious root base portion generation light green color callus.The visible substantially leaf one-tenth of adventitious bud after cultivating 20 days.And without NAA
Culture medium in, an induced synthesis green densification wound healing, without Adventitious root initiation, the later stage is also without adventitious bud formation.
3) outer implant cultivates 28d in bud inducement culture medium, proceeds to afterwards continue in MS minimal medium to cultivate.By outer planting
On body, the length regenerated adventitious bud more than 1.5cm cuts, and proceeds to MS+0.5mg/L NAA+30g/L sucrose+0.7mg/L agar
Root media root induction.Above condition of culture is: intensity of illumination 1500-2000Lux, photoperiod 16h/d, and temperature 24 ±
1℃。
In this experiment, if outer implant continuous subculture in bud inducement culture medium, the adventitious bud blade of induction increases, but abnormal
Shape leaf large percentage, and vitrification phenomenon occurs.Therefore, outer implant is induced 28 days (4 in the culture medium containing hormone by we
Week) after, proceed to the MS minimal medium without hormone, the existing more adventitious bud formation of outer implant, and from induction training
Support in base and absorb a certain amount of 6-BA and NAA, MS minimal medium is able to maintain that the growth in adventitious bud later stage, and remains to
The adventitious bud that induced synthesis is new.Adventitious bud grows normally in MS cultivates substantially, and growing point is obvious, few vitrification, and elongation
Rapider.This approach simplify tissue culture's cost, simplify operation, and the normal growth of beneficially adventitious bud.
4), after regeneration bud base portion forms a large amount of Rhizoma Imperatae, cultivation bottle cap seedling exercising 4-5d is opened, by the training of taking root of regrowth root
Support base to clean, plant in the plastic flowerpot filling solid matrix.Substrate uses Vermiculitum and peat composed of rotten mosses volume ratio 1: 1 to mix.Basin
Mouth antistaling film covers moisturizing, after 3d, beats a small amount of aperture at film surface, and later stage punching number is gradually increased, and hole gradually adds
Greatly.After overlay film cultivates 10d, throw off antistaling film, regeneration plant is put and cultivates at room temperature.During substrate culture, all give
Natural lighting.
Experiment 2:
1) obtain the leaf-head of heading stage Chinese cabbage ' New Beijing three ' from field, plastic sheeting wraps up.Transfer 4 DEG C of conditions
Put 30h.Remove the ball leaf of petiole, aseptically, first with the ethanol surface sterilization 30s of volume fraction 70%, sterilized water
Rinse one time.Pouring the NaClO solution sterilization 10min of mass fraction 5% again into, sterilized water rinses 15min gently, and period changes water 5-
6 times.Sterilize complete, with sterilizing absorbent paper, blade residual moisture is blotted.
2) by the leaf block that blade cutting is length of side 1.5-2.0cm after sterilizing, avoiding thicker vein, leaf faces up horizontal
In bud inducement culture medium.At intensity of illumination 1500-2000Lux, photoperiod 16h/d, train under the conditions of temperature 24 ± 1 DEG C
Support.In triplicate.
3), after outer implant cultivates 28d in bud inducement culture medium, proceed to afterwards MS minimal medium continue cultivate.Will outward
In implant, the length regenerated adventitious bud more than 1.5cm cuts, and proceeds to root media root induction.Above condition of culture is:
Intensity of illumination 1500-2000Lux, photoperiod 16h/d, temperature 24 ± 1 DEG C.
4), after regeneration bud base portion forms a large amount of Rhizoma Imperatae, cultivation bottle cap seedling exercising 4-5d is opened, by the training of taking root of regrowth root
Support base to clean, plant in the plastic flowerpot filling solid matrix.Substrate uses Vermiculitum and peat composed of rotten mosses volume ratio 1: 1 to mix.Basin
Mouth antistaling film covers moisturizing.After 3d, beating a small amount of aperture at film surface, later stage punching number is gradually increased, and hole gradually adds
Greatly.After overlay film cultivates 10d, throw off antistaling film, regeneration plant is put and cultivates at room temperature.During substrate culture, all give
Natural lighting.
5) interpretation
As shown in Table 3, after K cryogenic treatment 30h, ' New Beijing three ' average adventitious shoot regeneration rate is 79.34%, does not locates
(test 1) before reason to be declined slightly, but still maintain higher level.
Table 3 low temperature (4 DEG C) processes ' New Beijing three ' ball leaf adventitious shoot regeneration situation after 30h
This experiment proof Chinese cabbage leaf-head is after short time cryopreservation, and leaf regeneration effect is little with room temperature phase difference.
For can not sterilize in time inoculation material can short-term cryopreservation, add the flexibility ratio of experimental implementation.
Experiment 3:
1) choose early Chinese cabbage kind ' strong spring ', ' chrysanthemum brocade ' heading stage external ball leaf, remove petiole.Aseptically,
First by the ethanol surface sterilization 30s of volume fraction 70%, rinsed with sterile water one time.Pour the NaClO solution of mass fraction 5% again into
Sterilizing 10min, sterilized water rinses 15min gently, and period changes water 5-6 time.Sterilize complete, with sterilizing absorbent paper by blade residual water
Divide and blot.
2) by the leaf block that blade cutting is length of side 1.5-2.0cm after sterilizing, avoiding thicker vein, leaf faces up horizontal
In bud inducement culture medium.At intensity of illumination 1500-2000Lux, photoperiod 16h/d, train under the conditions of temperature 24 ± 1 DEG C
Support.In triplicate.
3), after outer implant cultivates 28d in bud inducement culture medium, proceed to afterwards MS minimal medium continue cultivate.Will outward
In implant, the length regenerated adventitious bud more than 1.5cm cuts, and proceeds to root media root induction.Above condition of culture is:
Intensity of illumination 1500-2000Lux, photoperiod 16h/d, temperature 24 ± 1 DEG C.
4), after regeneration bud base portion forms a large amount of Rhizoma Imperatae, cultivation bottle cap seedling exercising 4-5d is opened, by the training of taking root of regrowth root
Support base to clean, plant in the plastic flowerpot filling solid matrix.Substrate uses Vermiculitum and peat composed of rotten mosses volume ratio 1: 1 to mix.Basin
Mouth antistaling film covers moisturizing.After 3d, beating a small amount of aperture at film surface, later stage punching number is gradually increased, and hole gradually adds
Greatly.After overlay film cultivates 10d, throw off antistaling film, regeneration plant is put and cultivates at room temperature.During substrate culture, all give
Natural lighting.
5) interpretation
From table 4, table 5, ' strong spring ', average adventitious shoot regeneration rate was 71.61%, average each outer implant regeneration bud number
It it is 1.44.' chrysanthemum brocade ' average adventitious shoot regeneration rate is 57.82%, and average each outer implant regeneration bud number is 1.57.
Table 4 ' strong spring ' ball leaf adventitious shoot regeneration situation
Table 5 ' chrysanthemum brocade ' ball leaf adventitious shoot regeneration situation
Claims (1)
1. one kind utilizes the cultural method that Chinese cabbage ball leaf obtains regeneration plant, it is characterised in that comprise the following steps:
(1) choose heading stage Chinese cabbage external ball leaf, remove petiole, aseptically, first with the ethanol of volume fraction 70%
Surface sterilization 30s, rinsed with sterile water one time, then pour the NaClO solution sterilization 10min of mass fraction 5% into, sterilized water floats gently
Washing 15min, period changes water 5-6 time, sterilizes complete, is then blotted by blade residual moisture with sterilizing absorbent paper；
(2) by the leaf block that blade cutting is length of side 1.5-2.0cm after sterilizing, avoiding thicker vein, leaf faces up and is flat on
In bud inducement culture medium, at intensity of illumination 1500-2000Lux, photoperiod 16h/d, cultivate under the conditions of temperature 24 ± 1 DEG C；
(3) outer implant cultivates 28d in bud inducement culture medium, proceeds to afterwards continue in MS minimal medium to cultivate, by outer implant
The upper length regenerated adventitious bud more than 1.5cm cuts, and proceeds to root media root induction, and above condition of culture is: illumination
Intensity 1500-2000Lux, photoperiod 16h/d, temperature 24 ± 1 DEG C；
(4), after regeneration bud base portion forms a large amount of Rhizoma Imperatae, cultivation bottle cap seedling exercising 4-5d is opened, by the root media of regrowth root
Cleaning, plant in the plastic flowerpot filling solid matrix, substrate uses Vermiculitum and peat composed of rotten mosses volume ratio 1: 1 to mix, and basin mouth is used
Antistaling film covers moisturizing, after 3d, beats a small amount of aperture at film surface, and later stage punching number is gradually increased, and hole is gradually increased, and covers
After Membrance cuiture 10d, throw off antistaling film, regeneration plant is put and cultivates at room temperature, during substrate culture, all give nature light
According to；
Bud inducement culture medium in described step (2) is by MS, 6-BA, NAA, AgNO3, sucrose and agar composition, wherein in every liter of MS
Add 6-BA 4mg, NAA 1mg, AgNO34mg, sucrose 30g, agar 0.7mg, pH is 5.8；
MS minimal medium in described step (3) is the regular MS media without hormone；
Root media in described step (3) is made up of MS, NAA, sucrose and agar, wherein, adds NAA in every liter of MS
0.5mg, sucrose 30g and agar 0.7mg, pH is 5.8.
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| CN109392697B (en) * | 2018-11-16 | 2021-09-17 | 东北农业大学 | Method for cultivating new purple Chinese cabbage germplasm by utilizing distant hybridization technology |
| CN109392682A (en) * | 2018-12-13 | 2019-03-01 | 江苏徐淮地区徐州农业科学研究所(江苏徐州甘薯研究中心) | A kind of sweet potato tissue culture method for transplanting of high-survival rate |
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