CN103430841B - Method for promoting jatropha curcas explant to regenerate adventitious buds - Google Patents
Method for promoting jatropha curcas explant to regenerate adventitious buds Download PDFInfo
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Abstract
The invention belongs to the technical field of plant biology, and particularly discloses a method for promoting a jatropha curcas explant to regenerate adventitious buds. The traditional jatropha curcas explant adventitious bud regeneration method is changed by the method, that is hormone is not added to a culture medium for inducing the adventitious buds to regenerate any longer, while a high-concentration thidiazuron solution is used for performing short-term soaking treatment on the jatropha curcas explants of various sources, and short-term and high-strength hormone stimulation is applied on explant cells, so that partial cells re-differentiate directly with higher efficiency to form more adventitious buds. The regeneration efficiency and the quality of the adventitious buds of the jatropha curcas explant can be improved significantly by using the method. In addition, the explant in the method is not required to be subjected to conventional caluus formation or an adventitious bud multiplication stage, so that a regeneration culture cycle can be shortened significantly, and the working efficiency of jatropha curcas related biotechnology breeding is improved correspondingly and greatly.
Description
Technical field
The present invention relates to plant biotechnology field, particularly, relate to a kind of method that promotes Jatropha curcas explant regeneration indefinite bud.
Background technology
Be accompanied by the whole world growing to the demand of fossil fuel, the fossil fuel of storage is about to exhaust, and people more and more pay close attention to reproducible biodiesel.In can producing the candidate crops of biodiesel, belong to Euphorbiaceae Jatropha curcas (
jatroha curcasl.) have obvious advantage, its seed oil content is high, plants benevolence oil content and can reach 40% ~ 60%, meanwhile, contains active component many in jatropha curcas oil, as toxalbumin, leprosy ketone etc. has important agricultural chemicals and pharmaceutical value.
Yet the plantation of wideling popularize Jatropha curcas faces series of problems, although oil content is high in jatropha curcas seed, but seed production is not high; In kind oil, active component is many, but still need to change directly substitute fossil fuels of oily composition; Jatropha curcas is higher to environmental requirement, and a little less than tolerance to cold, distributed areas are narrower.Jatropha comprises 175 kinds, can introduce merit by interspecific cross, but the required cycle is long, can not obtain special foreign gene, therefore mainly change genetic background by genetic transformation.High-frequency plant regeneration system is the basis of genetic transformation.
Jatropha curcas explant can be divided into different types according to source is different.From the Jatropha curcas plant that grows up, directly acquisition blade, petiole can obtain adult plant leaf explant, adult plant petiole explant after treatment.Jatropha curcas seed is carried out to the aseptic seedling that aseptic culture obtains, from aseptic seedling, obtain aseptic hypocotyl, aseptic cotyledon blade and aseptic cotyledon petiole, can obtain after treatment aseptic Hypocotyl Explants, aseptic cotyledon leaf explant and aseptic cotyledon petiole explant.Jatropha curcas seed is cultivated, sprouts through routine the sprouting seedling obtaining, from sprouting seedling, can obtain hypocotyl and true leaf petiole, can obtain after treatment seed seedling Hypocotyl Explants and true leaf petiole explant.
While inducing Jatropha curcas explant regeneration indefinite bud in prior art, normally in Jatropha curcas explant adventitious shoot regeneration medium, add the basic element of cell division, and the most frequently used basic element of cell division is 6-benzyl aminoadenine (6-BA), concentration is 1 ~ 2 mg/l; 6-Furfurylaminopurine (6-KT), concentration is 0.1 ~ 1.0 mg/l or phenyl thiadiazolyl group urea (TDZ), concentration is 0.05 ~ 0.5 mg/l.Under this condition, the adventitious shoot regeneration efficiency of petiole explant is very low, and the quality of sprout is also poor.Meanwhile, all there is cycle long (more than 80 d), problem that regeneration rate is not high in these regenerating systems, has seriously restricted carrying out of Jatropha curcas Study on Genetic Transformation.
Summary of the invention
The defect that the present invention's regeneration rate when overcoming prior art leprosy explant regeneration indefinite bud is low, regeneration bud weight is poor, the regeneration period is long, provides a kind of method that promotes Jatropha curcas explant regeneration indefinite bud.Described method is easy, can significantly shorten whole cultivation cycle, and can obtain indefinite bud more, better quality by described method.
The present invention's above-mentioned purpose that is achieved by the following technical programs:
A method that promotes Jatropha curcas explant regeneration indefinite bud, is characterized in that, comprises the steps:
S1. obtain Jatropha curcas explant;
S2. by 10 ~ 60 mg/l phenyl thiadiazolyl group urea (TDZ) solution immersion treatment 5 ~ 40 min for the Jatropha curcas explant in step S1;
S3. the Jatropha curcas explant after step S2 being processed is seeded on the medium without hormone and cultivates in traverse mode;
Described traverse mode is that the horizontal surface of the cross section of explant and medium is perpendicular;
Described Jatropha curcas explant is plant leaf explant, aseptic Hypocotyl Explants, aseptic cotyledon leaf explant, aseptic cotyledon petiole explant, seed seedling Hypocotyl Explants or the true leaf petiole explant of growing up.
Jatropha curcas explant can be divided into different types according to source is different.From the Jatropha curcas plant that grows up, directly acquisition blade, petiole can obtain adult plant leaf explant, adult plant petiole explant after treatment.Jatropha curcas seed is carried out to the aseptic seedling that aseptic culture obtains, from aseptic seedling, obtain aseptic hypocotyl, aseptic cotyledon blade and aseptic cotyledon petiole, can obtain after treatment aseptic Hypocotyl Explants, aseptic cotyledon leaf explant and aseptic cotyledon petiole explant.Jatropha curcas seed is cultivated, sprouts through routine the sprouting seedling obtaining, from sprouting seedling, can obtain hypocotyl and true leaf petiole, can obtain after treatment seed seedling Hypocotyl Explants and true leaf petiole explant.
Preferably, 20 mg/l phenyl thiadiazolyl group urea solution immersion treatment 20 min for described aseptic Hypocotyl Explants.
Preferably, 20 mg/l phenyl thiadiazolyl group urea solution immersion treatment 40 min for described adult plant leaf explant or aseptic cotyledon leaf explant.
Preferably, 20 mg/l phenyl thiadiazolyl group urea solution immersion treatment 20 min for described aseptic cotyledon petiole explant or true leaf petiole explant.
Meanwhile, inventor finds in previous research work: the optimal processing mode of adult plant petiole explant obtaining from the Jatropha curcas plant that grows up is that the regeneration effect of indefinite bud is best with 3 mg/l phenyl thiadiazolyl group urea solution immersion treatment 5 min.The present invention also finds simultaneously: and seed seedling Hypocotyl Explants is not suitable for adopting the method for phenyl thiadiazolyl group urea solution immersion treatment to carry out evoking adventive bud regeneration.This shows: the splitting ability of the cell of dissimilar Jatropha curcas explant is different, dissimilar Jatropha curcas explant is also different to the susceptibility of TDZ, the complexity that has determined its regenerated adventitious bud is different, so the Jatropha curcas explant of not all type can be adopted and carry out in this way evoking adventive bud regeneration.
Preferably, described adult plant leaf explant or aseptic cotyledon leaf explant, being seeded in traverse mode while cultivating on the medium without hormone, adopt the supine inoculation modes of emplacement of blade following table, and the regeneration effect of its indefinite bud is relatively good.The supine inoculation modes of emplacement of so-called blade following table allows the upper surface of blade be close to media surface in other words, and blade lower surface upward.
The TDZ solution of step S2 adopts conventional method to be mixed with required, particularly, can adopt preparation with the following method: take a certain amount of TDZ powder, with 1mol/l NaOH, fully dissolve, use again deionized water constant volume, adopt 1 mol/l HCl solution to adjust pH to 5.8 ~ 6.0 of TDZ Treatment Solution; Before using, with the water system filter membrane of 0.22 micron, the TDZ Treatment Solution having prepared is carried out to filtration sterilization.
The preparation method of described dissimilar Jatropha curcas explant can be with reference to the art conventional method.
Preferably, the condition of cultivating described in step S3 is: intensity of illumination is 2000 ~ 2500 lx, and light application time is 12 ~ 16 hours/day, and cultivation temperature is 25 ± 1 ℃.
Creation point of the present invention is first to use the of short duration processing of hormone of high concentration, use again the medium culture regenerated adventitious bud without hormone, kind without hormone culture-medium is not emphasis point for purposes of the invention, so long as not containing hormone, and can provide for the regeneration of indefinite bud the medium of sufficient nutrient component can realize the present invention.The MS medium of preferably, take without hormone without the medium of hormone described in step S3 of the present invention is main component.In addition, the described medium without hormone also contains 25 ~ 35 g/l sucrose, 80 ~ 120 mg/l inositols and 6 ~ 8 g/l agar; Medium pH is 5.8 ~ 6.0.Described medium does not limit protection scope of the present invention.
Why tradition Jatropha curcas explant adventitious shoot regeneration cultural method exists adventitious shoot regeneration efficiency very low, the long defect of of poor quality, cycle of sprout, first be because long-time cultivation explant can not be used the basic element of cell division of high concentration, 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 that the basic element of cell division remaining in after adventitious bud formation in medium and explantation tissue has very adverse influence to the further growth of indefinite bud.
The present invention innovates common Jatropha curcas explant adventitious shoot regeneration cultural method, in evoking adventive bud regeneration culture medium, do not add TDZ, the substitute is and utilize the TDZ solution of high concentration Jatropha curcas explant to be carried out to the immersion treatment of short-term, give the stronger stimulation of medium with respect to common interpolation TDZ of cell that explant has differentiation capability, induction forms more indefinite bud.Simultaneously, owing to being only local immersion treatment in short-term, the concentration of TDZ in cell reduces subsequently very soon, has reduced the negative effect to the growth of formed indefinite bud in late stage of culture, can reach and once cultivate the efficient object that operation just can obtain whole plant.
The present inventor has only studied the effect of the adult plant petiole explant adventitious shoot regeneration of high concentration phenyl thiadiazolyl group urea short-term processing method promotion in previous work.But, through a large amount of creativeness tests, found afterwards: the splitting ability of the cell of dissimilar Jatropha curcas explant is different, dissimilar Jatropha curcas explant is also different to the susceptibility of TDZ, the complexity that has determined its regenerated adventitious bud is different, so the Jatropha curcas explant of not all type can be adopted and carry out in this way evoking adventive bud regeneration.
Beneficial effect of the present invention:
The method of Jatropha curcas explant Direct Regeneration indefinite bud of the present invention is easy, can significantly shorten whole cultivation cycle, and the regeneration period of conventional method is more than 80 days, and this method only needs 70 days; Method by Jatropha curcas explant Direct Regeneration indefinite bud of the present invention can obtain indefinite bud more, better quality, existing methodical adventitious shoot regeneration rate is generally 16.08 % ~ 55.11 %, and adopt the adventitious shoot regeneration rate of method of the present invention, is 75.67 % ~ 92.98 %.
figure of description
Fig. 1. the aseptic Hypocotyl Explants of TDZ solution-treated of variable concentrations is the adventitious shoot regeneration cultivation effect of 35 days after 20 minutes; A:10 mg/l; B:20 mg/l; C:30 mg/l; D:60 mg/l; Bar=1 cm.
Fig. 2. aseptic Hypocotyl Explants regenerated adventitious bud is cultivated the effect after 15 days in elongation medium; A: the growth result figure of regenerated adventitious bud in elongation medium; B: the design sketch that regenerated adventitious bud is taken out from elongation medium; Bar=1 cm.
Fig. 3. adopt to be seeded in two ways after 2 types of Hypocotyl Explants 20 min of 20 mg/l TDZ solution immersion treatment and on adventitious shoot regeneration medium, cultivate the effect after 35 days; A: aseptic Hypocotyl Explants traverse mode; B: the perpendicular mode of inserting of aseptic Hypocotyl Explants; C: seed seedling Hypocotyl Explants traverse mode; D: the perpendicular mode of inserting of seed seedling Hypocotyl Explants; Bar=1 cm.
Fig. 4. adopt to be seeded in two ways after 2 types of petiole explant 20 min of TDZ solution immersion treatment of 20 mg/l and on adventitious shoot regeneration medium, cultivate the effect after 35 days; A: true leaf petiole explant traverse mode; B: the perpendicular mode of inserting of true leaf petiole explant; C: aseptic cotyledon petiole explant traverse mode; D: the perpendicular mode of inserting of aseptic cotyledon petiole explant; Bar=1 cm.
Fig. 5. 2 types of petiole explant regenerated adventitious buds are cultivated the effect after 15 days in elongation medium; A, B: true leaf petiole explant regenerated adventitious bud extends effect; C, D: aseptic cotyledon petiole explant regenerated adventitious bud extends effect; Bar=1 cm.
Fig. 6. adopt to be seeded in two ways after 2 types of leaf explant 40 min of 20 mg/l TDZ solution immersion treatment and on adventitious shoot regeneration medium, cultivate the effect after 35 days; A: grow up plant leaf explant upper surface upward, B: grow up plant leaf explant lower surface upward; C: aseptic cotyledon leaf explant upper surface upward; D: aseptic cotyledon leaf explant lower surface upward; Bar=1 cm.
Fig. 7. aseptic cotyledon leaf explant regenerated adventitious bud is cultivated the design sketch after 15 days in elongation medium; Bar=1 cm.
Embodiment
Below in conjunction with the drawings and specific embodiments, further describe the present invention.Unless stated otherwise, the reagent adopting in embodiment and method are conventional reagent and the method for using in this area.
Embodiment 1:
S1. explant is carried out the preparation of the container of short-term basic element of cell division solution-treated
Choose the Petri dish that diameter is 9 cm, be placed in high-pressure steam sterilizing pan, at 121 ℃, sterilizing 20 min under the condition of 0.1 MPa.
S2. the preparation of phenyl thiadiazolyl group urea (TDZ) Treatment Solution.
Accurately take a certain amount of TDZ powder, with 1 mol/l NaOH solution, fully dissolve, then use deionized water constant volume, be mixed with the TDZ Treatment Solution (control group 0 mg/l TDZ solution is aseptic deionized water) of 0,10,20,30,40,50,60 mg/l.Adopt 1 mol/l HCl solution to adjust pH to 5.8 ~ 6.0 of TDZ Treatment Solution; Before using, with the water system filter membrane of 0.22 micron, the TDZ Treatment Solution having prepared is carried out to filtration sterilization.
S3. the aseptic Hypocotyl Explants of Jatropha curcas obtains
The jatropha curcas seed having soaked 2 days is shelled, after 0.1% mercuric chloride solution sterilizing, in superclean bench, with aseptic operation cutter, peel off embryo, the embryo who peels off is seeded to MS medium (mg/l inositol+6, g/l sucrose+100, the MS formula components+30 g/l agar through high-temp steam sterilizing; PH5.8-6.0) upper, cultivate after 12 days, obtain the hypocotyl on seed seedling, with aseptic operation cutter, hypocotyl is cut into the hypocotyl segment that length is about 0.5 cm, can be used as aseptic Hypocotyl Explants.
S4. the basic element of cell division (TDZ) solution-treated jatropha curcas seed seedling Hypocotyl Explants
In superclean bench, the aseptic Hypocotyl Explants cutting is placed in respectively to the ready sterilized culture dish of above-mentioned steps S1, to the TDZ Treatment Solution of each concentration of pouring respectively above-mentioned steps S2 configuration in each culture dish into till submergence petiole explant, cover culture dish lid, after standing 20 min, outwell TDZ solution, retain aseptic hypocotyl, with sterilized tweezers, from culture dish, take out all aseptic Hypocotyl Explants, be placed on aseptic blotting paper, suck the unnecessary moisture in aseptic Hypocotyl Explants surface, the TDZ solution of each concentration has respectively 3 parallel processing.
S5. the aseptic Hypocotyl Explants cutting is placed in to the container of the basic element of cell division (TDZ) solution-treated, to the TDZ solution (0 of pouring variable concentrations in the wide-mouth vial of this container into, 10, 20, 30, 60 mg/l) to (control group 0 mg/l TDZ solution is aseptic deionized water) till all aseptic Hypocotyl Explants of submergence, cover bottle cap, after standing 20 min, outwell TDZ solution, retain plumular axis, with sterilized tweezers, from wide-mouth vial, take out all aseptic Hypocotyl Explants, be placed on aseptic blotting paper, suck the unnecessary liquid in Hypocotyl Explants surface, finally the aseptic Hypocotyl Explants after processing (is placed on Hypocotyl Explants on medium in traverse mode, Hypocotyl Explants is contacted gently with media surface, and make the cross section of Hypocotyl Explants and the horizontal surface of medium perpendicular) be seeded in without hormone MS medium (mg/l inositol+6, g/l sucrose+100, MS formula components+30 g/l agar, pH5.8 ~ 6.0) upper cultivation 35 days, the experimental result that obtains as shown in table 1 and Fig. 1.
The effect of the aseptic Hypocotyl Explants evoking adventive bud of the TDZ solution immersion treatment Jatropha curcas Direct Regeneration of table 1 variable concentrations
Note: data acquisition carries out variance analysis and Duncan multiple ratio (P≤0.05) with SPSS Statistics 17.0 statistical analysis softwares, after data, letter is different represent to process between significant differences.Regeneration rate (%)=(the explant number of the indefinite bud of regenerating/total explant number) * 100%; The bud number of average each explant () the explant number of=regenerated adventitious bud sum/indefinite bud of regenerating.
As can be known from Table 1, with the aseptic Hypocotyl Explants of TDZ solution-treated, do not have indefinite bud to produce, and with the aseptic Hypocotyl Explants of the variable concentrations TDZ solution-treated indefinite bud of all regenerating, but variable concentrations TDZ solution-treated induces the frequency of aseptic Hypocotyl Explants regenerated adventitious bud and the average regeneration bud of each explant to count the existing significant difference of multilist.In addition,, along with the increase of TDZ concentration, the regeneration bud number of adventitious shoot regeneration rate and average each aseptic Hypocotyl Explants shows and first increases the trend reducing afterwards; When wherein TDZ solution concentration is 20 mg/l, the bud number of the regeneration rate of indefinite bud and average each explant is the highest, is respectively 81.91% and 10.16.
Simultaneously, adopt the aseptic Hypocotyl Explants adventitious shoot regeneration of conventional method induction Jatropha curcas: the Hypocotyl Explants cutting directly (is placed on Hypocotyl Explants on medium in traverse mode, Hypocotyl Explants is contacted gently with media surface, and make the cross section of Hypocotyl Explants and the horizontal surface of medium perpendicular) be inoculated in and added variable concentrations TDZ(0,0.1,0.3,0.6,1.2 mg/l) MS medium on cultivate 35 days (control group 0 mg/l TDZ solution is aseptic deionized water), to obtain experimental result as shown in table 2.
Table 2 adopts the aseptic Hypocotyl Explants adventitious shoot regeneration of conventional method induction Jatropha curcas
Note: data acquisition carries out variance analysis and Duncan multiple ratio (P≤0.05) with SPSS Statistics 17.0 statistical analysis softwares, after data, letter is different represent to process between significant differences.Regeneration rate (%)=(the explant number of the indefinite bud of regenerating/total explant number) * 100%; The bud number of average each explant () the explant number of=regenerated adventitious bud sum/indefinite bud of regenerating.
As known from Table 2, when the TDZ concentration of adding is 1.2 mg/l, the bud number of adventitious shoot regeneration rate and average each explant is the highest, is respectively 30.97% and 3.03.
In sum, the data in two tables are known, and the adventitious shoot regeneration effect of the aseptic Hypocotyl Explants of employing short-term high concentration TDZ immersion treatment Jatropha curcas is significantly better than adopting the effect of conventional method.
According to high concentration TDZ described in embodiment, process the aseptic Hypocotyl Explants regenerated adventitious bud obtain at Elongation of adventitious bud medium (mg/l inositol+0.5, g/l sucrose+100, MS formula components+30 mg/l BA(benzyl aminoadenine)+0.2 mg/l KT(kinetin)+0.4 mg/l GA3(gibberellin)+0.2 mg/l IAA(heteroauxin)+10 mg/l arginine+5 g/l agar; PH5.8 ~ 6.0) upper cultivation after 15 days, can obtain the regenerated adventitious bud budling that more growth conditions is good, the results are shown in Figure 2.
The impact of the time of the aseptic Hypocotyl Explants of embodiment 2 TDZ solution immersion treatment Jatropha curcas on Direct Regeneration indefinite bud effect
S1. explant is carried out the preparation of the container of short-term basic element of cell division solution-treated: with embodiment 1.
S2. configure phenyl thiadiazolyl group urea (TDZ) solution of 20 mg/l: with embodiment 1.
S3. obtaining of the aseptic Hypocotyl Explants of Jatropha curcas: with embodiment 1
S4. the aseptic Hypocotyl Explants of the basic element of cell division (TDZ) solution-treated Jatropha curcas: the container that the aseptic Hypocotyl Explants cutting is placed in to the basic element of cell division (TDZ) solution-treated, to pouring concentration in the wide-mouth vial of this container into, be that the TDZ solution of 20 mg/l is to (control group 0 mg/l TDZ solution is aseptic deionized water) till all Hypocotyl Explants of submergence, cover bottle cap, Hypocotyl Explants is carried out to different time (0, 5, 20, 40 min) after TDZ solution immersion treatment, outwell TDZ solution, retain Hypocotyl Explants, with sterilized tweezers, from wide-mouth vial, take out all Hypocotyl Explants, be placed on aseptic blotting paper, suck the unnecessary liquid in Hypocotyl Explants surface, finally the Hypocotyl Explants after processing (is placed on Hypocotyl Explants on medium in traverse mode, Hypocotyl Explants is contacted gently with media surface, and make the cross section of Hypocotyl Explants and the horizontal surface of medium perpendicular) be seeded in without hormone MS medium (mg/l inositol+6, g/l sucrose+100, MS formula components+30 g/l agar, pH5.8 ~ 6.0) upper cultivation 35 days, to obtain experimental result as shown in table 3.
The impact of table 3 TDZ solution immersion treatment time on the aseptic Hypocotyl Explants Direct Regeneration of Jatropha curcas indefinite bud
Note: data acquisition carries out variance analysis and Duncan multiple ratio (P≤0.05) with SPSS Statistics 17.0 statistical analysis softwares, after data, letter is different represent to process between significant differences.Regeneration rate (%)=(the explant number of the indefinite bud of regenerating/total explant number) * 100%; The bud number of average each explant () the explant number of=regenerated adventitious bud sum/indefinite bud of regenerating.
As can be known from Table 3, along with time of the aseptic Hypocotyl Explants of immersion treatment increases, the regeneration bud number of adventitious shoot regeneration rate and average each explant shows and first increases the trend reducing afterwards, take the adventitious shoot regeneration best results of soak time during as 20 min.
Embodiment 3
S1. explant is carried out the preparation of the container of short-term basic element of cell division solution-treated: with embodiment 1.
S2. phenyl thiadiazolyl group urea (TDZ) solution that configures 20 mg/l, concrete configuration method is with embodiment 1.
S3. obtaining of the aseptic Hypocotyl Explants of Jatropha curcas and seed seedling Hypocotyl Explants: concrete grammar is with embodiment 1.
S4. the basic element of cell division (TDZ) solution is processed respectively the aseptic Hypocotyl Explants of Jatropha curcas and seed seedling Hypocotyl Explants: the container that the Hypocotyl Explants of 2 types cutting is placed in to the basic element of cell division (TDZ) solution-treated, to pouring concentration in the wide-mouth vial of this container into, be that the TDZ solution of 20 mg/l is to till all Hypocotyl Explants of submergence, cover bottle cap, Hypocotyl Explants is carried out after the immersion treatment of 20 min, outwell TDZ solution, retain Hypocotyl Explants, with sterilized tweezers, from wide-mouth vial, take out all Hypocotyl Explants, be placed on aseptic blotting paper, suck the unnecessary liquid in Hypocotyl Explants surface, finally the Hypocotyl Explants after processing in two kinds of modes of emplacements (traverse mode: Hypocotyl Explants is placed on medium, Hypocotyl Explants is contacted gently with media surface, and make the cross section of Hypocotyl Explants and the horizontal surface of medium perpendicular.The perpendicular mode of inserting: Hypocotyl Explants is placed on medium, makes the axis of Hypocotyl Explants and media surface perpendicular, the degree of depth that Hypocotyl Explants inserts in medium is 0.1 ~ 0.2 cm.) be seeded in without hormone MS medium (mg/l inositol+6, g/l sucrose+100, MS formula components+30 g/l agar; PH5.8 ~ 6.0) upper cultivation 35 days, experimental results is as shown in table 4.
The impact of table 4 Hypocotyl Explants inoculation modes of emplacement on adventitious shoot regeneration effect
Note: data acquisition carries out variance analysis and Duncan multiple ratio (P≤0.05) with SPSS Statistics 17.0 statistical analysis softwares, after data, letter is different represent to process between significant differences.Regeneration rate (%)=(the explant number of the indefinite bud of regenerating/total explant number) * 100%; The bud number of average each explant () the explant number of=regenerated adventitious bud sum/indefinite bud of regenerating.The aseptic Hypocotyl Explants shelling and obtain after the embryo of seed cultivates 12 days on aseptic MS medium for deriving from Jatropha curcas in explant source 1; Explant source 2 is for deriving from crazy seeds at culture matrix (soil: grit=1:1) the upper seed seedling Hypocotyl Explants obtaining after 12 days with natural conditions cultivation.
Data from table 4 are known, no matter are the Hypocotyl Explants in which kind of source, and the explant adventitious shoot regeneration effect that adopts traverse mode to inoculate is all significantly better than the perpendicular regeneration effect of inserting mode.And seed seedling Hypocotyl Explants adventitious shoot regeneration rate is only 25.70%, be therefore not suitable for adopting the method for phenyl thiadiazolyl group urea solution immersion treatment to induce seed seedling hypocotyl outer planting adventitious shoot regeneration.
Embodiment 4
Distinguish the step of reference example 1,2 and 3, the situation of the adventitious shoot regeneration of the adult plant leaf explant of research variable concentrations TDZ solution-treated, aseptic cotyledon leaf explant, seed seedling Hypocotyl Explants, aseptic cotyledon petiole explant and true leaf petiole explant.
By result, learnt: the concentration of grow up plant leaf explant, the optimal TDZ solution of aseptic cotyledon leaf explant is 20 mg/l, and the processing time is 40 min; The concentration of aseptic cotyledon petiole explant and the optimal TDZ solution of true leaf petiole explant is 20 mg/l, and the processing time is 20 min.And seed seedling Hypocotyl Explants is not suitable for adopting the method for high concentration TDZ solution short-term immersion treatment to carry out evoking adventive bud regeneration.
Embodiment 5: the impact of different vaccination ways on two types of petiole explant regenerated adventitious bud efficiency of Jatropha curcas
Two types of petiole explants described in the present embodiment are respectively aseptic cotyledon petiole explant and true leaf petiole explant.Aseptic cotyledon petiole explant derives from nature germination seed seedling true leaf petiole; True leaf petiole explant derives from axenic germination seed young plant leaf petiole.
The petiole explant of 2 types cutting is placed in to the container of the basic element of cell division (TDZ) solution-treated, to pouring concentration in the wide-mouth vial of this container into, be that the TDZ solution of 20 mg/l is to till all petiole explants of submergence, cover bottle cap, petiole explant is carried out after the immersion treatment of 20 minutes, outwell TDZ solution, retain petiole explant, with sterilized tweezers, from wide-mouth vial, take out all petiole explants, be placed on aseptic blotting paper, suck the unnecessary liquid in petiole explant surface, finally the petiole explant after processing in two kinds of modes of emplacements (traverse mode: petiole explant is placed on medium, petiole explant is contacted gently with media surface, and make the cross section of petiole explant and the horizontal surface of medium perpendicular.The perpendicular mode of inserting: petiole explant is placed on medium, makes the axis of petiole explant and media surface perpendicular, the degree of depth that insert in medium the morphology lower end of petiole explant is 0.1 ~ 0.2 cm.) be seeded in without hormone MS medium (mg/l inositol+6, g/l sucrose+100, MS formula components+30 g/l agar; PH5.8 ~ 6.0) upper cultivation 35 days, experimental results is as shown in table 5 and Fig. 4.
The impact of table 5 petiole explant inoculation modes of emplacement on adventitious shoot regeneration effect
Note: data acquisition carries out variance analysis and Duncan multiple ratio (P≤0.05) with SPSS Statistics 17.0 statistical analysis softwares, after data, letter is different represent to process between significant differences.Regeneration rate (%)=(the explant number of the indefinite bud of regenerating/total explant number) * 100%; The bud number of average each explant () the explant number of=regenerated adventitious bud sum/indefinite bud of regenerating.
Data from table 5 are known, no matter are the petiole explants of which kind of type, adopt the adventitious shoot regeneration effect of traverse vaccination ways to be all significantly better than perpendicular effect of inserting mode.
The petiole explant regenerated adventitious bud of 2 types is at Elongation of adventitious bud medium (mg/l inositol+0.5, g/l sucrose+100, MS formula components+30 mg/l BA(benzyl aminoadenine)+0.2 mg/l KT(kinetin)+0.4 mg/l GA3(gibberellin)+0.2 mg/l IAA(heteroauxin)+10 mg/l arginine+5 g/l agar; PH5.8 ~ 6.0) upper cultivation after 15 days, can obtain the regenerated adventitious bud budling that more growth conditions is good, the results are shown in Figure 5.
Embodiment 6: the impact of different vaccination ways on 2 types of Jatropha curcas blade explant regeneration indefinite bud efficiency
This experiment, selecting described in specification c the cotyledon blade on the seed seedling of cultivating 12 days after surface sterilizing under the stem of Jatropha curcas top blade of the age of tree of growing up and aseptic condition is experiment material, in superclean bench, with aseptic operation cutter, blade is cut into blade fritter that size is about 0.5 * 0.5 cm as explant, the leaf explant cutting is placed in to the container of basic element of cell division solution-treated, to pouring concentration in the wide-mouth vial of this container into, be that the TDZ solution of 20 mg/l is to till all leaf explants of submergence, cover bottle cap, leaf explant is carried out after the TDZ solution immersion treatment of 40 min, outwell TDZ solution, retain blade, with sterilized tweezers, from wide-mouth vial, take out all leaf explants, be placed on aseptic blotting paper, suck the unnecessary liquid in leaf explant surface, finally the leaf explant after processing is inoculated and (made the lower surface of leaf explant upward with two kinds of modes of emplacements, its upper surface contacts with media surface, make the upper surface of leaf explant upward, its lower surface contacts with media surface) on without hormone MS medium, cultivate 35 days, the experimental result that obtains as shown in table 6 and Fig. 6.
The impact of the vaccination ways of table 6 leaf explant on adventitious shoot regeneration effect
Note: data acquisition carries out variance analysis and Duncan multiple ratio (P≤0.05) with SPSS Statistics 17.0 statistical analysis softwares, the different significant differences that represent of letter after data.
Regeneration rate (%)=(the explant number of the indefinite bud of regenerating/total explant number) * 100%; Average bud number () total explant number of=regenerated adventitious bud sum/sprout.
From the data of table 6, when Jatropha curcas blade explant is carried out to regenerated adventitious bud cultivation, the successful of the regenerated adventitious bud of the supine inoculation modes of emplacement of employing blade following table is better than adopting the effect of upper surface inoculation modes of emplacement upward.
Leaf explant regenerated adventitious bud is at Elongation of adventitious bud medium (mg/l inositol+0.5, g/l sucrose+100, MS formula components+30 mg/l BA(benzyl aminoadenine)+0.2 mg/l KT(kinetin)+0.4 mg/l GA3(gibberellin)+0.2 mg/l IAA(heteroauxin)+10 mg/l arginine+5 g/l agar; PH5.8 ~ 6.0) upper cultivation after 15 days, can obtain the regenerated adventitious bud budling that more growth conditions is good.Aseptic seedling cotyledon blade the results are shown in Figure 7 in Elongation of adventitious bud medium culture after 15 days.
Claims (4)
1. a method that promotes Jatropha curcas explant regeneration indefinite bud, is characterized in that, comprises the steps:
S1. obtain Jatropha curcas explant;
S2. by 10 ~ 60 mg/l phenyl thiadiazolyl group urea solution immersion treatment 5 ~ 40 min for the Jatropha curcas explant in step S1;
S3. the Jatropha curcas explant after step S2 being processed is seeded on the medium without hormone and cultivates in traverse mode;
Described traverse mode is that the horizontal surface of the cross section of explant and medium is perpendicular;
Described Jatropha curcas explant is plant leaf explant, aseptic Hypocotyl Explants, aseptic cotyledon leaf explant, aseptic cotyledon petiole explant or the true leaf petiole explant of growing up;
Described adult plant leaf explant or aseptic cotyledon leaf explant, being seeded in traverse mode while cultivating on the medium without hormone, adopt the supine inoculation modes of emplacement of blade following table;
The MS medium that the medium without hormone described in S3 be take without hormone is main component, also comprises 25 ~ 35g/L sucrose, 80 ~ 120mg/L inositol and 6 ~ 8g/L agar; Medium pH is 5.8 ~ 6.0.
2. a kind of method that promotes Jatropha curcas explant regeneration indefinite bud according to claim 1, is characterized in that 20 mg/l phenyl thiadiazolyl group urea solution immersion treatment 20 min for described aseptic Hypocotyl Explants.
3. a kind of method that promotes Jatropha curcas explant regeneration indefinite bud according to claim 1, is characterized in that 20 mg/l phenyl thiadiazolyl group urea solution immersion treatment 40 min for described adult plant leaf explant or aseptic cotyledon leaf explant.
4. a kind of method that promotes Jatropha curcas explant regeneration indefinite bud according to claim 1, is characterized in that 20 mg/l phenyl thiadiazolyl group urea solution immersion treatment 20 min for described aseptic cotyledon petiole explant or true leaf petiole explant.
5.according to claim 1, a method that promotes Jatropha curcas explant regeneration indefinite bud, is characterized in that, the condition of cultivating described in S3 is: intensity of illumination is 2000 ~ 2500 lx, and light application time is 12 ~ 16 hours/day, and cultivation temperature is 25 ± 1 ℃.
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