CN103299901A - In-vitro rapid proliferation method of Masui dauphine fig - Google Patents
In-vitro rapid proliferation method of Masui dauphine fig Download PDFInfo
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Abstract
The invention discloses an in-vitro rapid proliferation method of a Masui dauphine ficus carica. The in-vitro rapid proliferation method comprises the steps of A, explant preparation; B, induction; C, proliferation; and D, rooting and transplanting. The in-vitro rapid proliferation method is characterized in that a young stem segment of the Masui dauphine fig with an auxiliary bud is selected as an explant in the step A; the basic culture medium in a culture medium component selected in the steps B, C and D is chloride-free MS culture medium of anhydrous calcium chloride in macroelements replaced by calcium nitrate terahydrate; the induction medium in the step B is the chloride-free MS culture medium, 20-30 mg/L of cane sugar, and 5.8-7.5 mg/l of agar powder; the pH is 5.8-6.2; the proliferation medium in the step C is chloride-free MS culture medium, 0.5-2.0 mg/L of 6-benzyladenine, 0.05-0.1 mg/L of naphthylacetic acid, 0.2-1.0 mg/L of gibberellins, 20-30 mg/L of cane sugar, and 5.8-7.5 mg/L of agar powder; pH is 5.8-6.2; the rooting culture medium in the step D is 1/2 of chloride-free MS culture medium, 0.3-0.6 mg/L of indolebutyric acid, 20-30 mg/L of cane sugar, and 5.8-7.5 mg/L of agar powder; pH is5.8-6.2. Compared with the prior art, the explants is economic and convenient to select; and the increase rate can be up to 28.4fold.
Description
Technical field: the invention belongs to the fig tissue culture technique, particularly a kind of rapid in-vitro propagation method of Mace clothing Tao Fen fig.
Background technology: Mace clothing Tao Fen fig, be commonly called as huge fig greatly with its fruit, originate in California, USA, introduce China from Japan.Show that through planting experimentally to observe Mace clothing Tao Fen has one of fig improved seeds of promoting future at present most, the cultivation of suitable areas to the south, the Changjiang river.
Seedling propagation on Mace clothing Tao Fen fig is produced at present, adopt cottage propagations on the one hand more, also just like prior art document 1 Chinese patent ZL 200910155405.1 disclosed exotic fig grating cultivation methods, but compare with the stripped fast culture seedling propagation of test tube that the many rates of increase of material source are high, because material source is few, reproduction rate is low, time-consuming, be subject to seasonal restrictions seriously, site area takies problems such as big, is difficult to satisfy the needs of current commerial growing and popularization; On the other hand, from the breeding angle, fig is hypanthodium, and Xiao Hua is latent to be born in the cryptomere clinanthium, can not utilize the conventional hybridization breeding technique to carry out breed improvement and renewal.Along with the maturation of biotechnology in fruit breeding used, the application of transgenic technology on the fig prefered method of improving the breed beyond doubt particularly.And transgenic technology is to organize that to cultivate be prerequisite, be a special kind of skill on basis therefore, to want to utilize transgenic technology to carry out the fast breeding system that the fig breeding just must be set up the fig cultured in vitro earlier.The deficiency of Mace clothing Tao Fen fig tree maximum is exactly not resist cold, and meets with-5 ℃ and following low temperature and just subjects to freeze injury, causes the serious underproduction, even the withered heavy losses of full garden tree body.Utilize transgenic technology will carry directed the importing in the Mace clothing Tao Fen fig of genetic fragment of cold-resistant gene, the cold resistance of energy orderly improvement Mace clothing Tao Fen fig, thereby the cold-resistant kind that seed selection makes new advances.
Prior art document 2 " the fig tissue is cultivated and quick propagating technology research " gardening journal the 24th phase in 1997, Li Kang etc. reported the Xinjiang breeding in it dried live in peace you adopt " inducing culture MS+6-BA1.5mg/L+NAA1mg/L+GA0.2mg/L+PG100mg/L; sucrose 3%, agar 0.5%; " " test-tube plantlet propagation appropriate media is MS+6-BA1.0mg/L+NAA0.1mg/L+GA0.2mg/L, and effective propagation can reach 3 times in every month." " rooting rate is up to 96.7% ".
Prior art document 3 research of regenerative system " the fig tissue cultivate " forestry scientific research the 6th phase of calendar year 2001, Duan Xinling etc. have reported that " test material is taken from Xinjiang Tarim University of Agricultural Reclamation and planted 3 years living fig treelets of institute of section." " minimal medium has MS, LS, B5, N64 kind "; in " 3 brief summary ", " found out the suitable proliferated culture medium of fig, be MS+BA1.0mg/L+NAA0.1mg/L+GA0.2mg/L, the quick breeding of sucrose 30g/L, agar 6.5-7.0g/L, pH=5.5～7.0 optimum figs." in " 3 brief summary ", " (4) can make rooting rate up to 98.1% on root induction.”
Prior art document 4 " fig Study on tissue culture " northern fruit tree the 9th phase in 2002, Zhu Jianhuas etc. have been reported " this is tested used fig and takes from Liaoning agricultural occupation technical college greenhouse ", " the additional BA0.5mg/L+NAA0.1mg/L's of MS medium is the optimal medium that axillalry bud is induced concerning axillalry bud ", in listed " table 2 different hormone combinations is to the influence of fig propagation ", all adopted " the MS medium adds hormone ", high proliferation coefficient 5.72 in the table; Also be all to have adopted " the MS medium adds hormone " in " influence that the different medium of table 3 are taken root to fig ".
Prior art document 5 " fig stem section exsomatize quick propagating technology " forestry science and technology the 6th phase in 2002, Song Yinong etc. have reported " the tender tip of clip 5cm on the A212 fig plant of cultivating in the greenhouse ", the minimal medium that all adopts in " test of 1.2 medium " all is MS medium, in " 2 results and analysis " " 2.1 macroelement increase with macroelement the mitogenetic multiple of the influence of mitogenetic multiple; the mitogenetic multiple of 2MS, MS medium reach respectively 7.8 and the average plant height of 7.6. with the MS medium for the highest, reach 3.4cm." " 3.1 carry out fig stem section exsomatizes when breeding fast, and 7g/L is mitogenetic medium with MS+BA1.0mg/L+NAA0.2mg/L+ sugar 30g/L+ agar, and mitogenetic training number reaches more than 9 in " 3 brief summary "." " 3.2 take root with 1/2MS+IBA0.5mg/L+ sugar 20g/L+ agar 7g/L for well, and 6-7 is after week in cultivation, and rooting rate reaches more than 90%.”
The fig tissue of prior art is cultivated and is bred fast and has following deficiency and need improvements in sum:
1, all there is not ' the relevant report of the rapid in-vitro propagation method of Mace clothing Tao Fen ' fig among the prior art document 2-5.Because genotype is often determining the success or failure of cultured in vitro, different genotype requires different to the kind of different hormones and concentration collocation, and therefore necessity works out the method for a cover Mace clothing Tao Fen fig rapid in-vitro propagation fully.
2, the minimal medium that adopts among the prior art document 2-5 all is MS medium, and contains the anhydrous calcium chloride in the macroelement in the MS nutrient media components, that is to say to belong to chloride MS medium.As everyone knows, tobacco is fear-chlorion crop, and chlorion has generally acknowledged ill effect to the growth of tobacco, and chloride MS medium uses can or can not also exist the generation ill effect in the rapid in-vitro propagation of Mace clothing Tao Fen fig, is a problem that is worth research.
3, the explant that adopts among the prior art document 2-5 becomes the seedling effect preferably on the problem, there is no report using the band terminal bud still to make the examination material with the stem section of axillalry bud.
This shows that study and design a kind of explant and choose more economic convenience, the rapid in-vitro propagation method that has increased substantially the Mace clothing Tao Fen fig of the rate of increase again is necessary.
Summary of the invention: the objective of the invention is to overcome the deficiencies in the prior art, study and design a kind of explant and choose more economic convenience, increased substantially the rapid in-vitro propagation method of the Mace clothing Tao Fen fig of the rate of increase again.
The present invention seeks to be achieved through the following technical solutions:
A kind of rapid in-vitro propagation method of Mace clothing Tao Fen fig comprises that steps A, explant prepare, and step B, induces, and step C, propagation, are taken root and transplanted at step D, it is characterized in that
---in the steps A, select to be in the tender stem segments of Mace clothing Tao Fen fig band axillalry bud of vigorous growth as explant;
---the minimal medium during selected inducing culture, proliferated culture medium, root media formed among step B, step C, the step D is the no chlorine MS medium of replacing the anhydrous calcium chloride in the macroelement with four water-calcium nitrate;
---the inducing culture among the step B is on the inducing culture of no chlorine MS medium+sucrose 20～30mg/L+ agar powder 5.8～7.5mg/L, pH=5.8～6.2;
---the proliferated culture medium among the step C is no chlorine MS medium+6-benzyladenine 0.5～2.0mg/L+ methyl 0.05～0.1mg/L+ gibberellin 0.2～1.0mg/L+ sucrose 20～30mg/L+ agar powder 5.8～7.5mg/L, pH=5.8～6.2;
---the root media among the step D is 1/2 no chlorine MS medium+indolebutyric acid 0.3～0.6mg/L+ sucrose 20～30mg/L+ agar powder 5.8～7.5mg/L, pH=5.8～6.2.
Purpose of the present invention can also take following further technical measures to realize:
Described no chlorine MS medium is to replace 1.8～2.4 times of anhydrous calcium chloride weight consumption in the former MS medium macroelement component with four water-calcium nitrate.
Described steps A, explant prepare also to comprise following technical measures of carrying out successively:
1), flushing: clip be in vigorous growth ' the young shoot of Mace clothing Tao Fen ' fig band axillalry bud is cut into the stem section of band simple bud with it, and bud top is cut and stayed 0.5cm long, the stem section is cut and is stayed 1-1.5cm long under the bud, clean repeatedly several times with washing powder, flowing water flushing 2h, standby in the freshness protection package of packing into:
2), anti-browning is handled: the freshness protection package that simple bud will be housed is put into 4 ℃ refrigerator, and precooling was taken out after 4 hours, dried surface moisture, places the ascorbic acid solution of 1000mg/L to soak 30 minutes, and is standby;
3), surface sterilization: will carry out the simple bud that anti-browning is handled, earlier with 75% alcohol-pickled 30 seconds, aseptic clear water flushing 2 times, aseptic paper is dried surface moisture, changes in 0.1% mercuric chloride solution to sterilize 8 minutes again, rock bottle incessantly, explant is fully contacted with solution, wash explant number time repeatedly with aseptic clear water again, until residual mercuric chloride is rinsed well fully, blot the explant surface moisture with aseptic paper at last, standby;
4), cuttage: it is long respectively the clip of each explant to be cut again 0.2cm, according to the polarity of bud, the lower end cuttage is gone in the inducing culture, cultivates 20-30 days, treats can downcut when seedling grows to 0.4-0.5cm, forwards to and breeds cultivation in the proliferated culture medium.
Described inducing culture namely is the no chlorine MS medium with the anhydrous calcium chloride in the four water-calcium nitrate replacement macroelement that does not add hormone.
The rapid in-vitro propagation method of the Mace clothing Tao Fen fig that the invention process obtains has compared with prior art shown following beneficial effect:
1, explant is chosen more economic convenience.
Explant is selected comparative trial: the tender stem segments of choosing band axillalry bud and band terminal bud is studied as explant examination material, wash successively respectively, anti-browning processing and surface sterilization, cuttage is to the inducing culture of no chlorine MS+ sucrose 30mg/L+ agar powder 7mg/L then, and pH is adjusted into 5.8.
Result of the test is as shown in table 1:
Table 1 different explants type is to the influence of cuttage planting percent
Conclusion (of pressure testing): axillalry bud all can be cultivated into healthy seedling with terminal bud, though the terminal bud planting percent is higher by 3% than axillalry bud, terminal bud is through after anti-browning and disinfecting, and very easily albefaction becomes the seedling albefaction rate up to 17%, is nearly 3 times of axillalry bud albefaction rate 6%; And, treat that the maternal plant terminal bud quantity that selection is used is less than axillalry bud greatly, obviously more economical convenient as explant with the tender stem segments of band axillalry bud, namely source of drawing material is wide, becomes the seedling albefaction rate low.
2, increased substantially the rate of increase.
The minimal medium screening test: interpolation 6-BA1mg/L and the seedling of NAA0.05mg/L are cultivated in MS, 3/4MS, 1/2MS and four kinds of minimal mediums of no chlorine MS of the present invention respectively, every bottle of inoculation of medium 1 strain seedling, every processing is done 10 bottles, cultivates the 30d " Invest, Then Investigate ".Above-mentioned 6-BA namely refers to 6-benzyladenine, and NAA namely refers to methyl.
Result of the test is as shown in table 2:
The different minimal mediums of table 2 are to the influence of fig propagation multiple
Conclusion (of pressure testing): seedling growing way in no chlorine MS minimal medium of the present invention is best, the propagation multiple is the highest, reach 26.7 times, the amount of callus differentiation is also more, delicate and glittering and translucent, the plant albefaction rate is minimum, illustrates that no chlorine MS minimal medium is more suitable for ' Mace clothing Tao Fen ' the fig fast culture that exsomatizes.
This shows, the anhydrous calcium chloride in the macroelement among the minimal medium MS is replaced into the employing of the no chlorine MS minimal medium that four water-calcium nitrate obtains, and is first major reason that the present invention has increased substantially the rate of increase.
Screening hormone kind and concentration proportioning test: selecting no chlorine MS medium of the present invention for use is minimal medium additional hormone: 6-BA, IBA, NAA and GA again
3, carry out the combo test respectively by variable concentrations.Above-mentioned 6-BA namely refers to 6-benzyladenine, and IBA namely refers to indolebutyric acid, and NAA namely refers to methyl, GA
3Namely refer to gibberellin.
Result of the test sees Table 3:
The different hormone kinds of table 3 and concentration proportioning are to the influence of fig rapid in-vitro propagation
Conclusion (of pressure testing): add 6-BA1mg/L, NAA0.05mg/L and GA at the same time
3On the no chlorine MS medium of 1mg/L, test-tube plantlet plant growing way is the most healthy and the strongest, and the leaf look green, and blade is big, and the children is tender and plump, and the plant plant height is the highest, on average can reach 6.8cm; Internode is the longest, reaches 1cm, and the multiple of propagation is the highest, reaches 28.4 times.
Those skilled in the art is known: the plant plant height increases, and internode elongates, and also can utilize simple bud to breed.Adopt the prescription of reporting among the prior art document 2-5, the fig plant is all short and small relatively, and internode and cripetura thereof are unfavorable for that the single axillalry bud of clip breeds, and the plant that can only utilize callus to be differentiated to form carries out single-plant propagation, and reproduction rate is low.Plant plant height of the present invention is up to 9cm, on average can reach 6.8cm, is 2 times of 3.4cm of report such as Song Yi farming in the prior art document 5.Therefore the present invention is except the single-plant propagation that can utilize callus and break up out, and plant can also the single axillalry bud of clip be used for breeding because internode has elongated, so has increased breeding rate greatly.
Further show thus, proliferated culture medium does not have chlorine MS medium+6-benzyladenine 0.5～2.0mg/L+ methyl 0.05～0.1mg/L+ gibberellin 0.2～1.0mg/L+ sucrose 20～30mg/L+ agar powder 5.8～7.5mg/L, no chlorine MS medium+6-BA1mg/L+NAA0.05mg/L+GA is wherein especially adopted in the employing of pH=5.8～6.2
31mg/L+ sucrose 30mg/L+ agar powder 7mg/L, the proliferated culture medium of pH=5.8 is second major reason that the present invention has increased substantially the rate of increase.
It is 1/2 no chlorine MS medium+indolebutyric acid 0.3～0.6mg/L+ sucrose 20～30mg/L+ agar powder 5.8～7.5mg/L that the present invention selects root media for use, pH=5.8～6.2, and through test, rooting rate reaches 100%, and transplanting survival rate reaches 98%.
Further show again thus, selecting root media for use is 1/2 no chlorine MS medium+indolebutyric acid 0.3～0.6mg/L+ sucrose 20～30mg/L+ agar powder 5.8～7.5mg/L, pH=5.8～6.2 are compared with prior art document 2～5, have improved rooting rate and transplanting survival rate then.
In sum, can reach 3 times with every month effective propagation in the prior art document 2, rooting rate is up to 96.7% and compares, compare up to 98.1% with the rooting rate in the prior art document 3, compare with the high proliferation coefficient 5.72 in the prior art document 4, reach more than 9 with the mitogenetic training number in the prior art document 5, rooting rate reaches more than 90% to be compared, the propagation multiple of the present invention in implementing is up to 28.4 times, through test, rooting rate reaches 100%, and transplanting survival rate reaches 98%, obviously compared with prior art increased substantially the rate of increase, and rooting rate, transplanting survival rate is all quite high.
Embodiment: in conjunction with the embodiments implementation detail of the present invention is described as follows:
Embodiment 1 used no chlorine MS medium is to replace 1.8 times of anhydrous calcium chloride weight consumption in the former MS medium macroelement component with four water-calcium nitrate;
Steps A, explant are prepared: select to be in the tender stem segments of Mace clothing Tao Fen fig band axillalry bud of vigorous growth as explant; Technical measures of carrying out successively:
1), flushing: clip be in vigorous growth ' the young shoot of Mace clothing Tao Fen ' fig band axillalry bud is cut into the stem section of band simple bud with it, and bud top is cut and stayed 0.5cm long, the stem section is cut and is stayed 1～1.5cm long under the bud, clean repeatedly several times with washing powder, flowing water flushing 2h, standby in the freshness protection package of packing into:
2), anti-browning is handled: the freshness protection package that simple bud will be housed is put into 4 ℃ refrigerator, and precooling was taken out after 4 hours, dried surface moisture, places the ascorbic acid solution of 1000mg/L to soak 30 minutes, and is standby;
3), surface sterilization: will carry out the simple bud that anti-browning is handled, earlier with 75% alcohol-pickled 30 seconds, aseptic clear water flushing 2 times, aseptic paper is dried surface moisture, changes in 0.1% mercuric chloride solution to sterilize 8 minutes again, rock bottle incessantly, explant is fully contacted with solution, wash explant number time repeatedly with aseptic clear water again, until residual mercuric chloride is rinsed well fully, blot the explant surface moisture with aseptic paper at last, standby.
Step B, induce: it is long respectively the clip of each explant to be cut again 0.2cm, polarity according to bud, the lower end cuttage is gone in the inducing culture, cultivated 20～30 days, treat to downcut when seedling grows to 0.4～0.5cm, forward to and breed cultivation in the proliferated culture medium, inducing culture is on the inducing culture of no chlorine MS medium+sucrose 20mg/L+ agar powder 5.8mg/L, pH=5.8;
Step C, propagation: the seedling that explant induction among the step B is grown downcuts when growing to 0.4～0.5cm, be transferred on the proliferated culture medium, proliferated culture medium is no chlorine MS medium+6-benzyladenine 0.5mg/L+ methyl 0.05mg/L+ gibberellin 0.2mg/L+ sucrose 20mg/L+ agar powder 5.8mg/L, pH=5.8; When height of seedling average out to 6～7cm, when adopting single-plant propagation, the advantage plant clip single axillalry bud breeding that internode has been elongated;
Step D, take root and transplant: the bud of growing thickly that propagation produces in step C grows tall to 1.5～2.5cm, when having 2～4 blades, be inoculated on the root media from its base portion cutting-out, root media is 1/2 no chlorine MS medium+indolebutyric acid 0.3mg/L+ sucrose 20mg/L+ agar powder 5.8mg/L, pH=5.8; Cultivate the root that produced 3～25 whites from bastem portion in 15～20 days, can transplant.
Embodiment 2 used no chlorine MS medium are to replace 2.4 times of anhydrous calcium chloride weight consumption in the former MS medium macroelement component with four water-calcium nitrate;
Steps A, explant are prepared: identical with embodiment 1;
Step B, induce: it is long respectively the clip of each explant to be cut again 0.2cm, polarity according to bud, the lower end cuttage is gone in the inducing culture, cultivated 20～30 days, treat to downcut when seedling grows to 0.4～0.5cm, forward to and breed cultivation in the proliferated culture medium, inducing culture is on the inducing culture of no chlorine MS medium+sucrose 30mg/L+ agar powder 7.5mg/L, pH=6.2;
Step C, propagation: the seedling that explant induction among the step B is grown downcuts when growing to 0.4～0.5cm, be transferred on the proliferated culture medium, proliferated culture medium is no chlorine MS medium+6-benzyladenine 2.0mg/L+ methyl 0.1mg/L+ gibberellin 1.0mg/L+ sucrose 30mg/L+ agar powder 7.5mg/L, pH=6.2; When height of seedling average out to 6～7cm, when adopting single-plant propagation, the advantage plant clip single axillalry bud breeding that internode has been elongated;
Step D, take root and transplant: the bud of growing thickly that propagation produces in step C grows tall to 1.5～2.5cm, when having 2～4 blades, be inoculated on the root media from its base portion cutting-out, root media is 1/2 no chlorine MS medium+indolebutyric acid 0.6mg/L+ sucrose 30mg/L+ agar powder 7.5mg/L, pH=6.2; Cultivate the root that produced 3～25 whites from bastem portion in 15～20 days, can transplant.
Embodiment 3 used no chlorine MS medium are to replace 2.0 times of anhydrous calcium chloride weight consumption in the former MS medium macroelement component with four water-calcium nitrate;
Steps A, explant are prepared: identical with embodiment 1;
Step B, induce: it is long respectively the clip of each explant to be cut again 0.2cm, polarity according to bud, the lower end cuttage is gone in the inducing culture, cultivated 20～30 days, treat to downcut when seedling grows to 0.4～0.5cm, forward to and breed cultivation in the proliferated culture medium, inducing culture is on the inducing culture of no chlorine MS medium+sucrose 25mg/L+ agar powder 7mg/L, pH=6.0;
Step C, propagation: the seedling that explant induction among the step B is grown downcuts when growing to 0.4～0.5cm, be transferred on the proliferated culture medium, proliferated culture medium is no chlorine MS medium+6-benzyladenine 1.0mg/L+ methyl 0.08mg/L+ gibberellin 0.6mg/L+ sucrose 25mg/L+ agar powder 7mg/L, pH=6.0; When height of seedling average out to 6～7cm, when adopting single-plant propagation, the advantage plant clip single axillalry bud breeding that internode has been elongated;
Step D, take root and transplant: the bud of growing thickly that propagation produces in step C grows tall to 1.5～2.5cm, when having 2～4 blades, be inoculated on the root media from its base portion cutting-out, root media is 1/2 no chlorine MS medium+indolebutyric acid 0.4mg/L+ sucrose 25mg/L+ agar powder 7mg/L, pH=6.0; Cultivate the root that produced 3～25 whites from bastem portion in 15～20 days, can transplant.
Embodiment 4 used no chlorine MS medium are to replace 2.2 times of anhydrous calcium chloride weight consumption in the former MS medium macroelement component with four water-calcium nitrate;
Steps A, explant are prepared: identical with embodiment 1;
Step B, induce: it is long respectively the clip of each explant to be cut again 0.2cm, polarity according to bud, the lower end cuttage is gone in the inducing culture, cultivated 20～30 days, treat to downcut when seedling grows to 0.4～0.5cm, forward to and breed cultivation in the proliferated culture medium, inducing culture is on the inducing culture of no chlorine MS medium+sucrose 28mg/L+ agar powder 7mg/L, pH=6.1;
Step C, propagation: the seedling that explant induction among the step B is grown downcuts when growing to 0.4～0.5cm, be transferred on the proliferated culture medium, proliferated culture medium is no chlorine MS medium+6-benzyladenine 1.0mg/L+ methyl 0.05mg/L+ gibberellin 1.0mg/L+ sucrose 30mg/L+ agar powder 7mg/L, pH=5.8; When height of seedling average out to 6～7cm, when adopting single-plant propagation, the advantage plant clip single axillalry bud breeding that internode has been elongated;
Step D, take root and transplant: the bud of growing thickly that propagation produces in step C grows tall to 1.5～2.5cm, when having 2～4 blades, be inoculated on the root media from its base portion cutting-out, root media is 1/2 no chlorine MS medium+indolebutyric acid 0.5mg/L+ sucrose 28mg/L+ agar powder 6mg/L, pH=5.9; Cultivate the root that produced 3～25 whites from bastem portion in 15～20 days, can transplant.
More than show and described basic principle of the present invention, principal character and advantage of the present invention.The technical staff of the industry should understand, and the present invention is not comprised the restriction of the foregoing description of embodiment; Without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention; The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (3)
1. the rapid in-vitro propagation method of a Mace clothing Tao Fen fig comprises that steps A, explant prepare, and step B, induces, and step C, propagation, are taken root and transplanted at step D, it is characterized in that
---in the steps A, select to be in the tender stem segments of Mace clothing Tao Fen fig band axillalry bud of vigorous growth as explant;
---the minimal medium during selected inducing culture, proliferated culture medium, root media formed among step B, step C, the step D is the no chlorine MS medium of replacing the anhydrous calcium chloride in the macroelement with four water-calcium nitrate;
---the inducing culture among the step B is on the inducing culture of no chlorine MS medium+sucrose 20～30mg/L+ agar powder 5.8～7.5mg/L, pH=5.8～6.2;
---the proliferated culture medium among the step C is no chlorine MS medium+6-benzyladenine 0.5～2.0mg/L+ methyl 0.05～0.1mg/L+ gibberellin 0.2～1.0mg/L+ sucrose 20～30mg/L+ agar powder 5.8～7.5mg/L, pH=5.8～6.2;
---the root media among the step D is 1/2 no chlorine MS medium+indolebutyric acid 0.3～0.6mg/L+ sucrose 20～30mg/L+ agar powder 5.8～7.5mg/L, pH=5.8～6.2.
2. the rapid in-vitro propagation method of Mace clothing Tao Fen fig according to claim 1 is characterized in that described no chlorine MS medium is to replace 1.8～2.4 times of anhydrous calcium chloride weight consumption in the former MS medium macroelement component with four water-calcium nitrate.
3. the rapid in-vitro propagation method of Mace clothing Tao Fen fig according to claim 1 is characterized in that described steps A, explant also comprise following technical measures of carrying out successively in preparing:
1), flushing: clip is in the young shoot of the Mace clothing Tao Fen fig band axillalry bud of vigorous growth, and it is cut into the stem section of band simple bud, and the bud top is cut and stayed 0.5cm long, the stem section is cut and is stayed 1～1.5cm long under the bud, clean repeatedly several times with washing powder, flowing water flushing 2h, standby in the freshness protection package of packing into:
2), anti-browning is handled: the freshness protection package that simple bud will be housed is put into 4 ℃ refrigerator, and precooling was taken out after 4 hours, dried surface moisture, places the ascorbic acid solution of 1000mg/L to soak 30 minutes, and is standby;
3), surface sterilization: will carry out the simple bud that anti-browning is handled, earlier with 75% alcohol-pickled 30 seconds, aseptic clear water flushing 2 times, aseptic paper is dried surface moisture, changes in 0.1% mercuric chloride solution to sterilize 8 minutes again, rock bottle incessantly, explant is fully contacted with solution, wash explant number time repeatedly with aseptic clear water again, until residual mercuric chloride is rinsed well fully, blot the explant surface moisture with aseptic paper at last, standby.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN107094621A (en) * | 2017-04-13 | 2017-08-29 | 江苏农林职业技术学院 | A kind of fig seedling method for tissue culture |
| CN110720393A (en) * | 2019-11-01 | 2020-01-24 | 中国计量大学 | Method for tissue culture and rapid propagation of ficus microcarpa |
| CN110731268A (en) * | 2019-11-01 | 2020-01-31 | 中国计量大学 | Tissue culture and rapid propagation method for ficus carica fruits |
| CN110896855A (en) * | 2019-11-01 | 2020-03-24 | 中国计量大学 | Tissue culture and rapid propagation method for ficus variabilis |
| CN116649217A (en) * | 2023-06-21 | 2023-08-29 | 杭州拾光欣雅生物技术有限公司 | Proliferation culture method of fig callus |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106417009A (en) * | 2016-08-28 | 2017-02-22 | 李志勇 | Fig tissue culture and rapid propagation technology and method thereof |
| CN107094621A (en) * | 2017-04-13 | 2017-08-29 | 江苏农林职业技术学院 | A kind of fig seedling method for tissue culture |
| CN107094621B (en) * | 2017-04-13 | 2019-03-08 | 江苏农林职业技术学院 | A kind of fig seedling method for tissue culture |
| CN110720393A (en) * | 2019-11-01 | 2020-01-24 | 中国计量大学 | Method for tissue culture and rapid propagation of ficus microcarpa |
| CN110731268A (en) * | 2019-11-01 | 2020-01-31 | 中国计量大学 | Tissue culture and rapid propagation method for ficus carica fruits |
| CN110896855A (en) * | 2019-11-01 | 2020-03-24 | 中国计量大学 | Tissue culture and rapid propagation method for ficus variabilis |
| CN110731268B (en) * | 2019-11-01 | 2021-08-03 | 中国计量大学 | Tissue culture and rapid propagation method for ficus carica |
| CN116649217A (en) * | 2023-06-21 | 2023-08-29 | 杭州拾光欣雅生物技术有限公司 | Proliferation culture method of fig callus |
| CN116649217B (en) * | 2023-06-21 | 2024-06-04 | 杭州拾光欣雅生物技术有限公司 | Proliferation culture method of fig callus |
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