CN104381130B - It is applicable to the method for building up of the Semen vignae sinensis high-efficiency regeneration system of polygene type - Google Patents
It is applicable to the method for building up of the Semen vignae sinensis high-efficiency regeneration system of polygene type Download PDFInfo
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Abstract
The invention discloses the method for building up of the Semen vignae sinensis high-efficiency regeneration system being applicable to polygene type, described method includes pre-treatment, the induction of adventitious bud, successive transfer culture, the elongation of adventitious bud, root culture, seedling exercising and transplant step.In pretreatment process, plant growth regulator used be concentration be 6 BA or TDZ that concentration is 0.001 0.1mg/L of 1 4mg/L；In the Induction Process of adventitious bud, plant growth regulator used is 6 BA and KT combinations or KT and IBA combination, and the concentration of 6 BA is 0.8 1.2mg/L, and the concentration of KT is 0.02 2mg/L, and the concentration of IBA is 0.15 0.25mg/L；In the elongation process of adventitious bud, plant growth regulator used is 6 BA and IBA, the concentration of 6 BA be the concentration of 0.5mg/L, IBA be 0.1 0.3mg/L.The present invention is not only adapted to the regeneration of polygene type Semen vignae sinensis and cultivates, and has the advantage that inductivity is high, Induce aerosor number is many, adventitious bud length, adventitious bud dry weight high and implementation condition is the harshest, has good promotion prospect.
Description
Technical field
The present invention relates to biological technical field, particularly to the foundation of the Semen vignae sinensis high-efficiency regeneration system being applicable to polygene type
Method.
Background technology
Semen vignae sinensis is important vegetable crop, and in China, cultivation history is long, all has plantation in addition to the extremely frigid zones such as Tibet, with
The cultivation of each province and city, south is more.Semen vignae sinensis nutritive value is the highest, its seed rich in proteins (accounting for dry substance mixture 25%), vitamin,
The mineral elements such as folic acid and potassium, magnesium, ferrum, and also containing lysine and tryptophan necessary to human body.
The yield of tradition Semen vignae sinensis cultigen is the lowest, and susceptible to insect pest and fungal disease height is cause it to yield poorly one
Principal element.Semen vignae sinensis has 31 kinds of insect pests and more than 20 kinds of diseases, only insect pest every year during field growing and during storage
May result in the loss of 20%-60%.For the preventing and treating of pest and disease damage, people mainly take to use chemical agent, reinforcement agricultural management to arrange
Execute and cultivate the modes such as disease and insect resistance new varieties.But life-time service chemical agent, produces a series of serious consequence: disease pest produces
Drug resistance, environment is caused serious harm by using in a large number of medicament, threatens human security, and " poison Semen vignae sinensis " event is startling.Agriculture
Industry preventing and treating expends substantial amounts of manpower and materials and but produces little effect.Owing to nature lacks effective Resistant germ plasm resource and hybridizes not
Affinity etc., the most difficult by conventional breeding methods selection-breeding resistant variety.
In recent years, along with reaching its maturity of developing rapidly of biotechnology, especially plant gene engineering technology, for plant
Breed improvement provide new approach.Since first transgenic plant appearance of nineteen eighty-three, some natural anti insect genes
(such as Bt gene, protease inhibitor and agglutinin etc.) are successfully proceeded in some crops.Improve Semen vignae sinensis yield, strengthen pest-resistant
The maximally effective approach of property is through genetic engineering and anti insect gene proceeds to from other material Semen vignae sinensis cultivar, carries out it
Genetic improvement.In numerous transgenic technologys, agrobacterium-mediated transformation is low with its expense, copy number is low, the transformation cycle is short and energy
Convert the particular advantages such as larger piece section to be widely used.And be intended to set up with the premise of agrobacterium-mediated transformation acquisition transgenic line
The reconstituted receptors system of one efficient stable.Therefore, setting up Semen vignae sinensis high-efficiency regeneration system can be Semen vignae sinensis genetic conversion system
Foundation lays the foundation.
Utilize adventitious organogenesis to set up regeneration system for Vigna unguiculata and have multiple, mainly have cotyledonary node, cotyledon, true leaf, epicotyl,
Hypocotyl, stem apex, the thin cellular layer of cotyledonary node, shoot apical meristem and embryo point etc..Wherein, cotyledonary node regeneration capacity research is compared
Many, as Li Xiaomei utilizes cotyledonary node to obtain ideal adventitious bud induction frequency as outer implant.But, both at home and abroad to Semen vignae sinensis
Tissue culture there is also many problems with plant regeneration research.There is gene dependency in Semen vignae sinensis regeneration capacity, different genotype it
Between regeneration capacity there is significant difference, be the most also not set up the regeneration system for Vigna unguiculata of an applicable different genotype.Meanwhile, main
The growth of bud is in general more preferable, excellent most important for regenerating system of the dry weight of adventitious bud.But, at present to how
The quantity improving main bud and the dry weight improving adventitious bud are also rarely reported.
Therefore, a kind of be applicable to Multi-genotype, can improve main bud quantity and improve adventitious bud dry weight Semen vignae sinensis regeneration body
The construction method of system is urgently developed.
Summary of the invention
Present invention aims to the deficiencies in the prior art, it is provided that a kind of Semen vignae sinensis being applicable to polygene type is the most again
The method for building up of raw system.Construction method comprises the following steps:
1) pre-treatment: cowpea seed is carried out sterilizing, is then placed in the MSB containing 6-BA or TDZ5Culture medium is cultivated,
Incubation time is 5 days, and cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000 lx, and light application time is 16 h/ days, and 6-BA's is dense
Degree is 0.001-0.1mg/L for the concentration of 1-4mg/L, TDZ；
2) induction of adventitious bud: cut the cotyledonary node with a cotyledon, is then placed in containing 6-BA and KT or containing KT and IBA
MSB5Culture medium is cultivated, and incubation time is 7 days, and cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000 lx, during illumination
Between be 16 h/ days, the concentration of 6-BA be the concentration that concentration is 0.02-0.1mg/L, IBA of 0.8-1.2mg/L, KT be 0.15-
0.25mg/L；
3) successive transfer culture: by step 2) axillalry bud of adventitious bud of gains cuts, discards, be then inoculated in by residue and contain
6-BA and KT or the MSB containing KT and IBA5Culture medium is cultivated, and incubation time is 7 days, and cultivation temperature is 23-27 DEG C,
Intensity of illumination is 3000 lx, and light application time is 16 h/ days；
4) elongation of adventitious bud: step 3) gains are put into the MSB containing 6-BA and IBA5Culture medium is cultivated, and cultivates
Time is 14 days, and cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000 lx, and light application time is 16 h/ days, and the concentration of 6-BA is
The concentration of 0.5mg/L, IBA is 0.1-0.3mg/L；
5) root culture: cut by the adventitious bud of step 4) gains, is seeded on the culture bottle containing root media training
Supporting, incubation time is 14 days, and cultivation temperature is for being maintained at 23-27 DEG C, and intensity of illumination is 3000 lx, and light application time is 16 h/
My god, described root media is that MS culture medium adds 30g/L sucrose and 7g/L agar, pH=5.8；
6) seedling exercising: choose the healthy and strong seedling that root length is 4-5cm after step 5) is cultivated, the lid of culture bottle is first unscrewed
Place 2 days, more half-open 2 days, standard-sized sheet 2 d the most again, need to be continuously replenished moisture during half-open and standard-sized sheet lid；
7) transplant: the plant after being completed by seedling exercising pulls up, and washes root culture medium, is transplanted to equipped with peat soil, treasure
In the nutritive cube of Zhu Yan, plant ash and leaf mould substrate, at 23-27 DEG C, put freshness protection package and be placed in moisturizing in growth cabinet
Cultivate 2 d, 1-2 Zhou Houzai to move on to outdoor and cultivate.
Described sterilization process is: on superclean bench, to cowpea seed first with 75% alcohol-pickled sterilization 1min,
Again with 0.1% mercuric chloride soaking disinfection 5min, finally use aseptic water washing 5 times.
When cutting the cotyledonary node with a cotyledon, nodal point separation epicotyl 0.5 mm, away from hypocotyl 5 mm.
In described nutritive cube, the mass ratio of peat soil, perlite, plant ash and leaf mould substrate is 1:1:1:1.
Preferably, the MSB in step 1)5The concentration that culture medium contains 6-BA, 6-BA is 3mg/L.When in step 1)
When the concentration of 6-BA is 3mg/L, the inductivity of adventitious bud is 100%；When 6-BA concentration is less than 3.0 mg/L, Induce aerosor
Number raises with the concentration of 6-BA and increases, and when 6-BA concentration is more than 3.0mg/L, under Induce aerosor number there will be slightly
Fall, selecting 6-BA concentration is that 3.0mg/L can obtain best inducing effect.
Preferably, step 2) in MSB5The concentration that culture medium contains 6-BA and KT, 6-BA is the dense of 1.0mg/L, KT
Degree is 0.06mg/L.When 6-BA is 1.0 mg/L, when KT concentration is less than 0.06 mg/L, adventitious bud quantity and adventitious bud main bud
Quantity raises with KT concentration and increases, when the concentration of 6-BA is 1.0mg/L, when the concentration of KT is 0.06mg/L, and adventitious bud quantity
And adventitious bud main bud quantity reaches most.
Preferably, the MSB in step 4)5In culture medium, the concentration of 6-BA be the concentration of 0.5mg/L, IBA be 0.2mg/L.
When 6-BA the concentration that concentration is 0.5mg/L, IBA less than 0.2mg/L time, adventitious bud length, main bud length, adventitious bud fresh weight and
Adventitious bud dry weight raises with the concentration of IBA and increases accordingly and increase.When the concentration that concentration is 0.5mg/L, IBA of 6-BA is
During 0.2mg/L, adventitious bud length and main bud length reach the longest, and adventitious bud fresh weight and adventitious bud dry weight reach the highest.
There is advantages that
1, it is applicable to multiple genotype, has bigger promotion prospect；
2, adventitious bud induction frequency is up to more than 95%, and Induce aerosor number is many, reaches as high as 12.91；
3, adventitious bud fresh weight and adventitious bud dry weight are high, well-grown adventitious bud be conducive to after the building of genetic conversion system
Vertical；
4, implementing step simple, implementation condition is the harshest.
Accompanying drawing explanation
Fig. 1 aseptic seedling after pre-treatment；
The cotyledonary node of one cotyledon of Fig. 2 band；
Adventitious bud after Fig. 3 inducing culture；
Fig. 4 cuts away the outer implant of axillalry bud；
Fig. 5 extends the adventitious bud after cultivation；
Adventitious root after Fig. 6 root culture；
Fig. 7 seedling exercising terminate after regrowth；
Fig. 8 transplanted seedling.
Detailed description of the invention
Below by embodiment, the present invention is specifically described, it is necessary to it is pointed out here that be that following example are simply used
In the present invention is further detailed, it is impossible to be interpreted as limiting the scope of the invention, being skilled in technique of this field
Some nonessential improvement and adjustment that personnel are made according to foregoing invention content, still fall within protection scope of the present invention.
Embodiment 1
Choose the cowpea seed that size is uniform, full, on superclean bench first with 75% alcohol-pickled sterilization 1min,
Again with 0.1% mercuric chloride soaking disinfection 5min, then with aseptic water washing 5 times, it is subsequently placed on the aseptic filter paper in culture dish, with disappearing
Seed is seeded in culture medium by the tweezers after poison, 10 seeds of every bottle graft kind.Cowpea seed is put into containing 6-BA's or TDZ
MSB5Culture medium is cultivated, and incubation time is 5 days, and cultivation temperature is 25 DEG C, and intensity of illumination is 3000 lx, and light application time is
16 h/ days.Then at containing 6-BA and KT or the MSB containing KT and IBA5Culture medium is cultivated, and incubation time is 7 days, cultivates
Temperature is 23-27 DEG C, and intensity of illumination is 3000 lx, and light application time is 16 h/ days, if 6 groups of experimental grouies, in the first experimental group, adopts
With 6-BA Yu KT combine, the concentration of 6-BA be the concentration of 0.8mg/L, KT be 0.02mg/L, in the second experimental group, use 6-BA with
KT combine, the concentration of 6-BA be the concentration of 1.0mg/L, KT be 0.06mg/L；In 3rd experimental group, use 6-BA Yu KT combination,
The concentration of 6-BA be the concentration of 1.2mg/L, KT be 0.1mg/L；In 4th experimental group, use KT and IBA combination, the concentration of KT
Concentration for 0.02mg/L, IBA is 0.15mg/L；In 5th experimental group, using KT and IBA combination, the concentration of KT is
The concentration of 0.06mg/L, IBA is 0.20mg/L；In 6th experimental group, using KT and IBA combination, the concentration of KT is 0.1mg/
The concentration of L, IBA is 0.25mg/L；Experimental result takes six groups of experimental group statistical average.
6-BA and TDZ pre-treatment is shown in Table 1 to the impact of Induce aerosor.
Table 1
When pretreatment stage is without hormone, adventitious bud induction frequency is 100%.With 6-BA (1.0,2.0,3.0,4.0
Mg/L), TDZ (0.001,0.005,0.01,0.05,0.1 mg/L) as the hormone of pre-treatment time, adventitious bud induction frequency is also
100%, add the suitable basic element of cell division in pretreatment stage and adventitious bud induction frequency is had no significant effect.Can be seen by table 1
Go out, add the suitable basic element of cell division in pretreatment stage and can significantly improve Induce aerosor quantity, when 6-BA concentration is less than 3.0
During mg/L, Induce aerosor number raises with the concentration of 6-BA and increases.When TDZ concentration is less than 0.01 mg/L or 0.05 mg/L
Time, Induce aerosor number raises with the concentration of TDZ and increases.6-BA inducing effect is better than TDZ.
Embodiment 2
When the concentration of currently processed middle 6-BA is 3mg/L, after pre-treatment, the aseptic seedling after pre-treatment cuts band one
The cotyledonary node of individual cotyledon, nodal point separation epicotyl 1 mm, away from hypocotyl 5 mm, it is then seeded on adventitious bud induction culture base, often
The outer implant of bottle graft kind 5-6, is then placed in containing 6-BA and KT combination or the MSB containing KT and IBA combination5Culture medium is trained
Supporting, incubation time is 7 days, and cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000 lx, and light application time is 16 h/ days, 6-BA's
Concentration be the concentration that concentration is 0.02-2mg/L, IBA of 0.8-1.2mg/L, KT be 0.15-0.25mg/L.
When the concentration of currently processed middle 6-BA is 3mg/L, adventitious bud is lured by 6-BA and KT combination and KT and IBA combination
The impact led is shown in Table 2.
Table 2
As can be seen from Table 2, the concentration of pre-treatment 6-BA is 3.0 mg/L, when the concentration of KT is less than 0.08 mg/L, indefinite
Bud inducement quantity raises with KT concentration and increases.When KT concentration is less than 0.06 mg/L, adventitious bud main bud quantity is with KT concentration liter
High and increase.
When 6-BA concentration be 1.0mg/L, KT concentration be 0.06 time, adventitious bud number, adventitious bud bud length and main bud bud length combine
Conjunction situation is optimum.
Embodiment 3
When the concentration of currently processed middle TDZ is 0.01mg/L, after pre-treatment, the aseptic seedling after pre-treatment cuts band
The cotyledonary node of one cotyledon, nodal point separation epicotyl 0.5 mm, away from hypocotyl 5 mm, it is then seeded into adventitious bud induction culture base
On, the outer implant of every bottle graft kind 5-6, it is then placed in containing 6-BA and KT combination or the MSB containing KT and IBA combination5Culture medium
Cultivating, incubation time is 7 days, and cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000 lx, and light application time is 16 h/ days,
The concentration of 6-BA be the concentration that concentration is 0.02-2mg/L, IBA of 0.8-1.2mg/L, KT be 0.15-0.25mg/L.
When the concentration of currently processed middle TDZ is 0.01mg/L, 6-BA and KT combination and KT and IBA combine adventitious bud
The impact of induction is shown in Table 3.
Table 3
When pre-treatment is TDZ 0.01 mg/L, research IBA (0.15-0.25mg/L) and KT (0.05,0.1,0.5,
1.0,2.0 mg/L) on Induce aerosor when affecting, outer implant all has adventitious root to generate, and adventitious root quantity is with KT concentration
Raising and reduce, adventitious root length shortens with the rising of KT concentration.
As can be seen from Table 3, the concentration of pre-treatment TDZ 0.01 be mg/L, KT concentration less than 0.04 mg/L time, no
Normal bud inducing amount raises with KT concentration and increases, when KT concentration is more than 0.06 mg/L and less than 0.1mg/L, and Induce aerosor
Quantity raises with KT concentration and increases.
When pre-treatment TDZ concentration is 0.01 mg/L, Induce aerosor effect is 3.0 less than pre-treatment 6-BA concentration
Effect during mg/L.
Embodiment 4
Adventitious bud through pre-treatment, Induce aerosor and successive transfer culture is put into the MSB containing 6-BA and IBA5Culture medium
Cultivating, incubation time is 14 days, and cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000 lx, and light application time is 16 h/
My god, the concentration of 6-BA be the concentration of 0.5mg/L, IBA be 0.1-0.3mg/L；MSB in pre-treatment5Culture medium contains 6-BA, 6-
The concentration of BA is 3mg/L；MSB in Induce aerosor5The concentration that culture medium contains 6-BA and KT, 6-BA is 1.0mg/L, KT
Concentration be 0.06mg/L.Before successive transfer culture, the axillalry bud of the adventitious bud gone out by Induce aerosor cuts, and discards.
The impact that Elongation of adventitious bud is induced by variable concentrations 6-BA, IBA/ and KT is shown in Table 4.
Table 4
As can be seen from Table 4, variable concentrations 6-BA and IBA has significant difference to main bud elongation induction, but stretches clump bud
Long induction there was no significant difference.When Elongation of adventitious bud inducing culture concentration is 6-BA 0.5mg/L+IBA 0.2mg/L, right
Adventitious bud length and main bud length inducing effect are best, and adventitious bud fresh weight and dry weight increments to whole outer implant are most.
6-BA 0.5mg/L+IBA 0.2mg/L is best to Elongation of adventitious bud inducing effect, selects 6-BA 0.5mg/L+IBA
0.2mg/L is as optimal Elongation of adventitious bud derivant.
Embodiment 5
Take into respectively cowpea 7, cowpea 28-2, blue or green cowpea 901, magnificent and summer precious 5 genotype cowpea seed carry out regeneration training
Support.When regeneration is cultivated, the concentration of pre-treatment 6-BA is the 6-of the culture medium interpolation of 3.0mg/L, Induce aerosor and successive transfer culture
The concentration of BA is 1.0 mg, and the concentration of KT is 0.06 mg/L, studies the different genotype impact on Induce aerosor, and subculture is trained
Data are recorded at the end of Yanging.When pre-treatment, Induce aerosor and successive transfer culture, cultivation temperature is 23-27 DEG C, and intensity of illumination is
3000 lx, light application time is 16 h/ days, and the incubation time of pre-treatment is 5 days, and the incubation time of Induce aerosor is 7 days, continues
The incubation time of culture is 7 days.
Different genotype is shown in Table 5 to the impact of Induce aerosor
Table 5
As can be seen from Table 5, the regeneration system for Vigna unguiculata that this time experiment is set up can induce adventitious bud to different genotype,
And adventitious bud induction frequency is more than 95%, adventitious bud number, main bud number and clump bud number are the most considerable, and adventitious bud quantity exists
Between 6.38～12.91.In table 5, various genotype utilize this regeneration system for Vigna unguiculata to can serve as agriculture bacillus mediated regeneration to be subject to
System is united.
Claims (5)
1. it is applicable to the method for building up of the Semen vignae sinensis high-efficiency regeneration system of polygene type, it is characterised in that: comprise the steps:
1) pre-treatment: cowpea seed is carried out sterilizing, is then placed in the MSB containing 6-BA or TDZ5Culture medium is cultivated, during cultivation
Between be 5 days, cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000lx, and light application time is 16h/ days, and the concentration of 6-BA is 1-
The concentration of 4mg/L, TDZ is 0.001-0.1mg/L；
2) induction of adventitious bud: cut the cotyledonary node with a cotyledon, is then placed in containing 6-BA and KT or the MSB containing KT and IBA5
Culture medium is cultivated, and incubation time is 7 days, and cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000lx, and light application time is
16h/ days, the concentration of 6-BA be the concentration that concentration is 0.02-0.1mg/L, IBA of 0.8-1.2mg/L, KT be 0.15-0.25mg/
L；
3) successive transfer culture: by step 2) axillalry bud of adventitious bud of gains cuts, discards, be then inoculated in residue containing 6-BA
With KT or the MSB containing KT and IBA5Culture medium is cultivated, and incubation time is 7 days, and cultivation temperature is 23-27 DEG C, intensity of illumination
For 3000lx, light application time is 16h/ days, the concentration of 6-BA be the concentration of 0.8-1.2mg/L, KT be 0.02-0.1mg/L, IBA
Concentration be 0.15-0.25mg/L；
4) elongation of adventitious bud: by step 3) gains put into the MSB containing 6-BA and IBA5Culture medium is cultivated, incubation time
Being 14 days, cultivation temperature is 23-27 DEG C, and intensity of illumination is 3000lx, and light application time is 16h/ days, and the concentration of 6-BA is 0.5mg/
The concentration of L, IBA is 0.1-0.3mg/L；
5) root culture: by step 4) adventitious bud of gains cuts, and is seeded in the culture bottle containing root media cultivation, training
The foster time is 14 days, and cultivation temperature is for being maintained at 23-27 DEG C, and intensity of illumination is 3000lx, and light application time is 16h/ days, described life
Root culture medium is that MS culture medium adds 30g/L sucrose and 7g/L agar, pH=5.8；
6) seedling exercising: choose through step 5) cultivate after the healthy and strong seedling that root length is 4-5cm, the lid of culture bottle is first unscrewed placement
2 days, more half-open 2 days, standard-sized sheet 2d the most again, need to be continuously replenished moisture during half-open and standard-sized sheet lid；
7) transplant: the plant after being completed by seedling exercising pulls up, and washes root culture medium, be transplanted to equipped with peat soil, perlite,
In the nutritive cube of plant ash and leaf mould substrate, at 23-27 DEG C, put freshness protection package and be placed in moisturizing cultivation in growth cabinet
2d, 1-2 Zhou Houzai moves on to outdoor and cultivates.
Method the most according to claim 1, it is characterised in that: step 1) described in sterilization process be: at superclean bench
On, to cowpea seed first with 75% alcohol-pickled sterilization 1min, then with 0.1% mercuric chloride soaking disinfection 5min, finally with aseptic
Water rinses 5 times.
Method the most according to claim 1, it is characterised in that: step 2) in, when cutting the cotyledonary node with a cotyledon,
Nodal point separation epicotyl 0.5mm, away from hypocotyl 5mm.
Method the most according to claim 1, it is characterised in that: step 7) peat soil, perlite, vegetation in described nutritive cube
The mass ratio of ash and leaf mould substrate is 1:1:1:1.
Method the most according to claim 1, it is characterised in that: step 1) in MSB5Culture medium contains the dense of 6-BA, 6-BA
Degree is 3mg/L；Step 2) in MSB5The concentration that concentration is 1.0mg/L, KT that culture medium contains 6-BA and KT, 6-BA is
0.06mg/L；Step 4) in MSB5In culture medium, the concentration of 6-BA be the concentration of 0.5mg/L, IBA be 0.2mg/L.
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Non-Patent Citations (5)
| Title |
|---|
| Development of a rapid, highly efficient system of organogenesis in cowpea Vigna unguiculata (L.) Walp;S. Raveendar等;《South African Journal of Botany》;20091231;第75卷;第17-21页 * |
| 扁豆整个子叶节离体再生体系的建立;袁娟等;《中国园艺文摘》;20111231(第7期);第3，7-9页 * |
| 菜用大豆再生与转化体系的研究;钟灿;《中国优秀硕士学位论文全文数据库 农业科技辑》;20130115(第1期);第D048-32页 * |
| 豇豆子叶节与幼胚再生体系建立与优化;罗倩;《四川农业大学硕士学位论文》;20130601;第i，7-9,页 * |
| 豇豆组织培养繁殖研究;商宏俐;《中国蔬菜》;19921231(第1期);第8-11页 * |
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