CN102754597B - Method for inducing adventitious bud regeneration of hypocotyl explant of soybean - Google Patents
Method for inducing adventitious bud regeneration of hypocotyl explant of soybean Download PDFInfo
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- CN102754597B CN102754597B CN2012102070724A CN201210207072A CN102754597B CN 102754597 B CN102754597 B CN 102754597B CN 2012102070724 A CN2012102070724 A CN 2012102070724A CN 201210207072 A CN201210207072 A CN 201210207072A CN 102754597 B CN102754597 B CN 102754597B
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Abstract
The invention belongs to the technical field of plant biology. When soybean hypocotyl is taken as an explant to induce the adventitious bud regeneration, cytokinins are added in a soybean seed germination culture medium or/and a hypocotyl explant adventitious bud regeneration culture medium generally to cause that the hypocotyl explant has low adventitious bud regeneration rate and poorer bud quality under the condition. According to the method disclosed by the invention, no BA (Butyl Acrylate) is added in the seed germination culture medium and the adventitious bud regeneration culture medium, after the explant is obtained, a BA solution with remarkable high concentration is prepared to carry out the short-time soaking treatment on a part near the notch at the morphological upper end of the hypocotyl explant of the soybean, and a stronger adventitious bud differential stimulus effect is served on cells with the differentiation ability at the notch, therefore, a large amount of adventitious buds with better quality can be obtained. Through the application of the method, the efficiency and the quality of the adventitious bud regeneration of the hypocotyl explant of the soybean can be improved remarkably, and the hypocotyl becomes an ideal receptor for soybean genetic transformation.
Description
Technical field
The present invention relates to plant biotechnology field, be specifically related to a kind of method of adventitious shoot regeneration of inducing soybean Hypocotyl Explants.
Background technology
Plant Biotechnology is a kind of important and advanced technology, and it is the important component part of biotechnology that tissue is cultivated.Because the plant regeneration technique of soybean callus tissue and blade etc. is not also established, hypocotyl becomes unique explant material that can regenerated adventitious bud.But hypocotylar adventitious shoot regeneration efficiency is always unsatisfactory up to now, the quality of indefinite bud is also poor, has seriously affected thus the process of bio-technology improvement soybean varieties.
Usining soybean hypocotyl during as the explant induction adventitious shoot regeneration, normally at the Germination of Soybean Seed medium or/and add the basic element of cell division in Hypocotyl Explants adventitious shoot regeneration medium, and the most frequently used basic element of cell division is benzyladenine (BA), concentration is 1-3 mg/L.Under this condition, the adventitious shoot regeneration efficiency of explant is very low, and the quality of sprout is also poor.At first the reason that causes this situation is can not use the basic element of cell division of high concentration due to long-time cultivation explant, because long-term High concentration auxin is cultivated explant, will cause explant injured even dead, and the effect of the stimulation adventitious shoot regeneration of the basic element of cell division of low concentration is strong not.Next is to remain in the basic element of cell division in medium and explantation tissue at adventitious bud formation the further growth of indefinite bud is had to very adverse influence later.
Summary of the invention
The objective of the invention is to overcome the defect of above-mentioned prior art, a kind of method of adventitious shoot regeneration of efficient inducing soybean Hypocotyl Explants is provided.
Method provided by the invention comprises following concrete steps:
(1) after soybean seedling is cultivated on the MSB5 medium, cut and obtain Hypocotyl Explants;
(2) to the morphology of Hypocotyl Explants benzyladenine (BA) solution immersion treatment for upper part;
(3) Hypocotyl Explants after step (2) processing is seeded on the MSB5 medium and cultivates.
Preferably, in step (1) and the described MSB5 medium of step (3), the concentration of BA is 0.Do not add the Hypocotyl Explants regeneration effect the best that obtains on the germination medium of BA.
Preferably, the immersion treatment time of step (2) is 20-40 min, and now the adventitious shoot regeneration effect of explant is best.
Preferably, the benzyladenine solution concentration of step (2) is 30mg/L, can obtain higher adventitious shoot regeneration rate and individual plant bud number under this concentration, and the indefinite bud of regeneration can continued growth.
Preferably, remove the benzyladenine solution that remains in the Hypocotyl Explants surface after step (2) immersion treatment, further to reduce the impact on the indefinite bud growth.
The BA solution of step (2) adopts conventional method to be mixed with required getting final product, and particularly, can adopt following method preparation: take a certain amount of BA powder, fully dissolve with 1mol/L NaOH, then use the deionized water constant volume, be mixed with the BA solution of 30mg/L; Adopt 1mol/L HCl to adjust the pH to 5.8-6.0 of BA solution.
The condition of culture of step (1) and step (2) is: room temperature is that 25 ℃, intensity of illumination are that 2000lx, light application time are 12 hours every days.
In the MSB5 medium of step (1) and step (3), contain: organic principle, 25 g/L sucrose, 100 mg/L inositols, 300 mg/L peptones, the 7 g/L agar of the inorganic constituents of MS culture medium prescription, B5 medium formula, pH is 5.8-6.0.Specifically, the inorganic constituents of MS culture medium prescription comprises 16.5 g/L ammonium nitrate, 19 g/L potassium nitrate, 1.7 g/L potassium dihydrogen phosphates, 3.7 g/L epsom salts, 4.4 g/L calcium chloride dihydrates, 16.9 mg/L manganese sulfate monohydrates, 8.6 mg/L white vitriols, 6.2 mg/L boric acid, 0.83 mg/L potassium iodide, 0.25 mg/L sulfate dihydrate molybdenum disodium, 0.025 mg/L cupric sulfate pentahydrate, 0.025 mg/L CoCL2 6H2O, 37.3 mg/L bis-water sodium ethylene diamine tetracetates, 27.8 mg/L ferrous sulfate heptahydrates; The organic principle of B5 medium formula comprises 10 mg/L thiamine hydrochlorides, 1 mg/L puridoxine hydrochloride, 1 mg/L nicotinic acid, 100 mg/L inositols.
The present invention also provides a kind of suitable treating apparatus, for easily Hypocotyl Explants being carried out to the short-term HORMONE TREATMENT, for the immersion treatment of step (2): adopt 4 cm that are generally used for the nucleotide fragments amplification * 6 cm PCR plates, the neat tubule that cuts away PCR plate bottom, the tubule retained on four angles is made support, is placed in the culture dish of lid.This device can be placed in high-pressure steam sterilizing pan and carry out conventional sterilizing.
The present invention innovates common soybean hypocotyl explant adventitious shoot regeneration cultural method, do not add BA at the germination medium for obtaining Hypocotyl Explants with in inducing the regeneration culture medium of hypocotyl indefinite bud, the substitute is near the BA solution that the utilizes high concentration upper end otch to the Hypocotyl Explants obtained from seedling the immersion treatment of carrying out short-term, give to there is the medium stronger stimulation of the cell of differentiation capability with respect to common interpolation BA near the otch of explant upper end, induce and form more indefinite bud.Simultaneously, owing to being only local immersion treatment in short-term, the concentration of BA in cell reduces subsequently very soon, reduced the negative effect to the growth of formed indefinite bud in late stage of culture, can not affect the adventive root regeneration of Hypocotyl Explants lower end otch yet, can reach and once cultivate the efficient purpose that operation just can obtain whole plant.
Beneficial effect of the present invention: 1) method is easy, can significantly shorten whole cultivation cycle; 2) obtain indefinite bud more, better quality, existing methodical indefinite bud pick-up rate is generally 10 %-30 %, and adopt the indefinite bud pick-up rate of method of the present invention, is 31.94 %-54.17 %; 3) the easier hardening of regeneration plant and the transplant survival that by the Hypocotyl Explants that has formed up bud and below and formed root, are produced.
The accompanying drawing explanation
Fig. 1: the device of processing explant;
Fig. 2: cutting part and explant form; A: cutting part (black line means together by the cutting of seedling cotyledon crotch base portion); B: explant morphology upper end morphosis;
Fig. 3: the regeneration effect of BA solution-treated hypocotyl after 20 minutes of variable concentrations; A:0 mg/L; B:15 mg/L; C:30 mg/L; D:60 mg/L; E:120 mg/L;
Fig. 4: the transplanting growth of hypocotyl regeneration plant; A: the rooting culture of hypocotyl regeneration plant; B: ripe plant.
Embodiment
Embodiment 1
A kind of method of adventitious shoot regeneration of inducing soybean Hypocotyl Explants, the solution wherein used is as follows:
(1) BA solution: take a certain amount of BA powder, fully dissolve with 1 mg/L NaOH, then use the deionized water constant volume, be mixed with the BA solution of 30 mg/L; Adopt 1 mg/L HCl to adjust the pH to 5.8-6.0 of BA solution; Use high-pressure steam sterilizing pan, under 121 ℃, the condition of 0.1 MPa, the BA solution prepared is carried out to 20 min sterilizings.
(2) MSB5 medium: by the inorganic constituents of MS culture medium prescription (16.5 g/L ammonium nitrate, 19 g/L potassium nitrate, 1.7 g/L potassium dihydrogen phosphate, 3.7 g/L epsom salt, 4.4 g/L calcium chloride dihydrate, 16.9 mg/L manganese sulfate monohydrate, 8.6 mg/L white vitriol, 6.2 mg/L boric acid, 0.83 mg/L potassium iodide, 0.25 mg/L sulfate dihydrate molybdenum disodium, 0.025 mg/L cupric sulfate pentahydrate, 0.025 mg/L CoCL2 6H2O, 37.3 mg/L bis-water sodium ethylene diamine tetracetates, 27.8 the mg/L ferrous sulfate heptahydrate), organic principle (the 10 mg/L thiamine hydrochlorides of B5 medium formula, 1 mg/L puridoxine hydrochloride, 1 mg/L nicotinic acid, 100 mg/L inositols), 25 g/L sucrose, 300 mg/L peptones, 7 g/L agar join successively in the plastics large container that volume is 2 L (deionized water that has added wherein 200 mL in advance) and fully dissolve, use the deionized water constant volume, adopt 1 mol/L NaOH solution to adjust pH to 5.8-6.0, finally medium is divided and is filled in the glass blake bottle that volume is 200 mL, every bottle adds 20 mL medium.Medium is sterilizing 20 min under 121 ℃, the condition of 0.1 MPa.
Previously prepared a kind for the treatment of apparatus, for easily Hypocotyl Explants being carried out to the short-term HORMONE TREATMENT: adopt 4 cm that are generally used for the nucleotide fragments amplification * 6 cm PCR plates, the neat tubule that cuts away PCR plate bottom, the tubule retained on four angles is made support, is placed in the culture dish of lid.This device can be placed in high-pressure steam sterilizing pan and carry out conventional sterilizing.The device prepared as shown in Figure 1.
The method of the present embodiment comprises following concrete steps:
(1) after soybean seedling is cultivated on the MSB5 medium, cut and obtain Hypocotyl Explants;
(2) to the morphology upper part of Hypocotyl Explants BA solution immersion treatment;
(3) Hypocotyl Explants after step (2) processing is seeded on the MSB5 medium and cultivates.
Wherein: the method for obtaining soybean hypocotyl explant donor material in step (1) is: choose uniform ripe soya seeds, after conventional sterilizing, be seeded to without on hormone MSB5 medium, be that 25 ℃, intensity of illumination are to cultivate 5 days under 2000lx, the light application time condition that is 12 hours every days in room temperature, obtain soybean seedling as the Hypocotyl Explants donor material.
The method of obtaining Hypocotyl Explants in step (1) is: choose that to have the soybean seedling that length is greater than the plumular axis of 1 cm be donor material, neat, by seedling cotyledon crotch base portion, cut, the hypocotyl segment that acquisition length is 1 cm left and right is explant, and otch is concordant.Cutting part and explant form are shown in Fig. 2.
In step (2), the method for BA solution-treated Hypocotyl Explants is: in superclean bench, the Hypocotyl Explants cut is inserted one by one in each hole of above-mentioned treating apparatus, make the morphology of explant upside down, then pour the extremely approximately 1 mm degree of depth of above-mentioned BA Treatment Solution in the culture dish of this installation composition part, cover the culture dish lid, standing 20 min.Outwell BA solution, take out Hypocotyl Explants, be placed on aseptic blotting paper, suck the BA solution of explant remained on surface.
The regeneration and cultivation method of Hypocotyl Explants is in step (3): Hypocotyl Explants after treatment is seeded to without on hormone MSB5 medium to erect the mode of inserting, and is that 25 ℃, intensity of illumination are cultivation 20 days under 2000 lx, the light application time condition that is 12 hours every days in room temperature.
After said method is processed, re-use bottle outlet hardening and the implantation methods of following regrowth: take out upper end and there is the hypocotyl that regeneration bud and lower end have regenerated root from blake bottle, after cleaning the medium on root, in the fine sand matrix that can plant clean, carry out conventional moisturizing hardening.After several days, can be planted in regrowth in general soil and carry out Routine Management.Fig. 4 is shown in the transplanting growth of hypocotyl regeneration plant.
Embodiment 2
Add the impact of the BA of variable concentrations on Hypocotyl Explants regeneration in the seed germination medium
In the present embodiment, adopt and cultivate the soybean seedling after 5 days on the MSB5 medium that adds variable concentrations BA, it is cut and obtains Hypocotyl Explants; The morphology upper part of Hypocotyl Explants, with after 30 mg/L BA solution immersion treatment 20 min, is seeded to without cultivating 20 days on hormone MSB5 medium.Experimental result is as shown in table 1.
The impact of different B A concentration on Hypocotyl Explants regeneration in table 1 germination medium
| BA（mg/L） | Adventitious shoot regeneration rate/% | Individual plant bud number/ | Adventive root formation rate/% |
| 0 | 31.94a | 1.56a | 100a |
| 1 | 13.06b | 0.44b | 47.22b |
| 2 | 13.89b | 0.56b | 13.89c |
Annotate 1: the data SPSS Statistics 17.0 statistical analysis softwares carry out variance analysis and Duncan multiple ratio (P<=0.05), the different significant differences that mean of letter after data.
Annotate 2: adventitious shoot regeneration rate (%)=(the explant number of the indefinite bud of regenerating/total explant number) * 100%; Individual plant bud number=regenerated adventitious bud sum/total explant number; Adventive root forms (%)=(the explant number of regeneration adventive root/total explant number) * 100 %.
From the data of table 1, along with the increase of BA concentration in germination medium, the hypocotyl adventitious shoot regeneration rate obtained, individual plant bud number, adventive root formation rate all decrease.Do not add the Hypocotyl Explants regeneration effect the best that obtains on the germination medium of BA.
Embodiment 3
The impact of BA solution-treated time on the Hypocotyl Explants adventitious shoot regeneration
In the present embodiment, the soybean seedling after 5 days is cultivated in employing on without hormone MSB5 medium, it is cut and obtains Hypocotyl Explants, after adopting 30 mg/L BA solution immersion treatment explant morphology upper end certain hours, the explant after processing is seeded to without cultivating 20 days on hormone MSB5 medium.Experimental result is as shown in table 2.
The impact of table 2 different disposal time on hypocotyl regeneration
| Processing time/min | Adventitious shoot regeneration rate/% | Individual plant bud number/ | Adventive root formation rate/% |
| 0 | 0d | 0d | 100a |
| 10 | 25.00b | 0.83b | 100a |
| 20 | 31.94a | 1.56a | 100a |
| 40 | 32.64a | 1.54a | 100a |
| 80 | 7.64c | 0.35c | 97.82a |
Annotate: the data SPSS Statistics 17.0 statistical analysis softwares carry out variance analysis and Duncan multiple ratio (P<=0.05), the different significant differences that mean of letter after data.
As known as table 2, processing time is in the scope of 0-40 min, the adventitious shoot regeneration rate of explant and individual plant bud number increase along with the prolongation in processing time, arrive 80 min, the adventitious shoot regeneration rate of explant and individual plant bud number reduce again, when wherein the processing time is 20-40 min, the adventitious shoot regeneration effect of explant is better; The generation almost not impact of processing time on the explant adventive root.
Embodiment 4
The impact of the concentration of BA Treatment Solution on the Hypocotyl Explants regeneration effect
In the present embodiment, the soybean seedling after 5 days is cultivated in employing on without hormone MSB5 medium, it is cut and obtains Hypocotyl Explants, BA solution-treated through 5 kinds of concentration, the time of the morphology upper end of BA solution immersion treatment explant is all 20 min, explant after processing, be seeded on without hormone MSB5 medium and cultivate 20 days.Experimental result is as shown in Fig. 3, table 3.
The impact of the BA solution of table 3 variable concentrations on hypocotyl regeneration
| BA solution concentration (mg/L) | Adventitious shoot regeneration rate/% | Individual plant bud number/ | Adventive root formation rate/% | Individual plant radical/ |
| 0 | 0d | 0d | 100 | 5.22 |
| 15 | 12.50c | 0.71c | 100 | 4.88a |
| 30 | 31.94b | 1.56b | 100 | 5.44a |
| 60 | 47.22a | 1.69ab | 100 | 4.78a |
| 120 | 54.17a | 1.92a | 100 | 4.75a |
Annotate: the data SPSS Statistics 17.0 statistical analysis softwares carry out variance analysis and Duncan multiple ratio (P<=0.05), the different significant differences that mean of letter after data; Individual plant radical=adventive root the sum of regenerating/total explant number.
As shown in Table 3, when short-term is processed, BA concentration is 0 mg/L, there is no the hypocotyl regenerated adventitious bud; The BA concentration of processing along with short-term raises, and hypocotylar adventitious shoot regeneration rate and individual plant bud number also increase thereupon, but the regeneration of adventive root is not had to obviously impact; Make discovery from observation, the explant after 60 mg/L and 120 mg/L BA solution-treated, the indefinite bud of its regeneration mostly is the budlet that grows thickly that is difficult to continued growth.
Claims (5)
1. the method for the adventitious shoot regeneration of an inducing soybean Hypocotyl Explants is characterized in that comprising the following steps:
(1) after soybean seedling is cultivated on the MSB5 medium, cut and obtain Hypocotyl Explants;
(2) to the morphology upper part of Hypocotyl Explants benzyladenine solution immersion treatment;
(3) Hypocotyl Explants after step (2) processing is seeded on the MSB5 medium and cultivates;
In step (1) and the described MSB5 medium of step (3), the concentration of benzyladenine is 0;
The immersion treatment time of step (2) is 20-40 min;
The benzyladenine solution concentration of step (2) is 30mg/L;
Be to contain in the MSB5 medium of step (1) and step (3): organic principle, 25 g/L sucrose, 100 mg/L inositols, 300 mg/L peptones, the 7 g/L agar of the inorganic constituents of MS culture medium prescription, B5 medium formula, pH is 5.8-6.0.
2. the method for the adventitious shoot regeneration of inducing soybean Hypocotyl Explants as claimed in claim 1, is characterized in that removing the benzyladenine solution that remains in the Hypocotyl Explants surface after step (2) immersion treatment.
3. the method for the adventitious shoot regeneration of inducing soybean Hypocotyl Explants as claimed in claim 1, the benzyladenine solution preparation method that it is characterized in that step (2) is: take a certain amount of benzyladenine powder, with 1 mol/L NaOH, dissolve, use again the deionized water constant volume, be mixed with the benzyladenine solution of 30 mg/L; Adopt 1 mol/L HCl to adjust the pH to 5.8-6.0 of benzyladenine solution.
4. the method for the adventitious shoot regeneration of inducing soybean Hypocotyl Explants as claimed in claim 1, the incubation time that it is characterized in that step (1) is 5 days.
5. the method for the adventitious shoot regeneration of inducing soybean Hypocotyl Explants as claimed in claim 1, it is characterized in that the device that in step (2), immersion treatment is used is: adopt 4 cm for the nucleotide fragments amplification * 6 cm PCR plates, the neat tubule that cuts away PCR plate bottom, the tubule retained on four angles is made support, is placed in the culture dish of lid.
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