CN103222425A - Efficient and rapid propagation technology suitable for southern highbush blueberry - Google Patents
Efficient and rapid propagation technology suitable for southern highbush blueberry Download PDFInfo
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- CN103222425A CN103222425A CN2013100550331A CN201310055033A CN103222425A CN 103222425 A CN103222425 A CN 103222425A CN 2013100550331 A CN2013100550331 A CN 2013100550331A CN 201310055033 A CN201310055033 A CN 201310055033A CN 103222425 A CN103222425 A CN 103222425A
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Abstract
The invention relates to an efficient and rapid propagation technology suitable for southern highbush blueberry. Excellent culture seedlings are obtained by the following steps of a. selecting materials; b, carrying out primary culture to obtain aseptic seedlings; c, optimizing subculture; d, establishing a leaf regeneration system; and e, carrying out rooting culture of tub seedlings. According to the technology, young stems of O'Neil blueberries are used as test materials, a proliferation medium, the leaf regeneration system, a rooting medium and a plantlet medium are optimized, the optimum proliferation culture, the leaf regeneration and the rooting culture are combined, and an efficient seedling survival medium is added, so that excellent tissue culture seedlings can be obtained efficiently, rapidly and continuously.
Description
Technical field
The present invention relates to a kind of blueberry high efficiency quick breeding technology, be specifically related to a kind of suitable south high clump blueberry high efficiency quick breeding technology.
Background technology
Blueberry belongs to Ericaceae (Ericaeae) blueberry subfamily (Vaccinioideae) Vaccinium (Vaccinium).The blueberry fruit is rich in elements such as anthocyan, unsaturated fatty acid, tannic acid and calcium, potassium, zinc, iron, vitamin B group content is particularly outstanding, classified as one of five big healthy food by international food and agricultural organization, the king who can be rated as world's fruit, be developed in recent years integrate the most rapidly nutrition and health care the 3rd generation fruit variety
[1-4]In recent years, developed country markets such as blueberry fresh fruit and the fashionable America and Europe of processed goods, though cost an arm and a leg but still supply falls short of demand, oneself far can not satisfy the demand of local and world market the output in the producing region, North America that it is traditional, for this reason in the world other countries oneself priority introduces a fine variety.There is the wide world that can produce blueberry in China, and southern red yellow earth land area reaches 2179600km
2, account for 22% of national land area, wherein 2040000km
2Soil belongs to acid, and a big chunk pH＜5.0
[5]Because soil acidity is big and the fertility deficiency, level of agricultural production is lower, and living standards of the people are low.Blueberry is particularly suitable for acidity and strongly acidic soil, just the red-yellow soil area desirable crop of shaking off poverty and setting out on the road to prosperity.
(O ' Neal) is one of representative kind of south high clump blueberry to the Ao Ni'er, the big grain of fruit, base of a fruit trace is little, the fruit powder is less, the hard anti-accumulating of pulp matter, high yield, thick flavor, fragrance is had higher rating in the high clump blueberry kind of south, and most of area plantation in the Yangtze river basin suits.Along with the scale of introducing a fine variety enlarges day by day, the good seed demand sharply increases, the seedling underproduce.Tissue culture relies on its vegetative unique advantage, can remove virus and new varieties more rapidly, the original characteristic of retaining variety, variability is little, and can in less space He in the short period, obtain a large amount of clonal tissue culture seedlings apace, the main path that becomes the blueberry detoxification and breed fast.At present, oneself obtains certain achievement the research of blueberry tissue culture technique both at home and abroad, domestic stem with bud induce and propagation aspect the research report more, and accounted for bigger ratio in that blade is carried out direct Study on Regeneration abroad.We from stem with bud induce that propagation, blade are directly regenerated, take root in the bottle, to each link of hardening just the high clump in south blueberry Ao Ni'er set up a kind of industrial breeding technique system that is suitable for the blueberry nursery stock, to satisfy the needs of commodity construction of base.
Summary of the invention
Defective at the prior art existence, the objective of the invention is to propose a kind of suitable south high clump blueberry high efficiency quick breeding technology, tissue culture relies on its vegetative unique advantage, can remove virus and new varieties more rapidly, the original characteristic of retaining variety, variability is little, and can in less space He in the short period, obtain a large amount of clone tissue cultural seedlings of free apace, the main path that becomes the blueberry detoxification and breed fast, the present invention has set up the at a high speed fast numerous effective ways of a kind of detoxification with regard to the lagophthalmos blueberry.
Technical scheme of the present invention is:
A kind of suitable south high clump blueberry high efficiency quick breeding technology is characterized in that,
A, selection: vegetable material is the south high clump blueberry kind Ao Ni'er of the rich biotechnology company of intelligence plantation plantation, and explant material is selected for use and given birth to young spray then;
B, initial culture obtain aseptic seedling: the living then spray with field growing is initial explant, tripping.Behind dirts such as the clean dust of detergent solution, the running water flushing.Forward on the superclean bench, successively explant is carried out the surface sterilizing of 50s and 10min respectively with 75% alcohol and 0.1% mercuric chloride, then use aseptic water washing 4 times, 2 minutes/time, inhale with aseptic filter paper at last and remove the explant surface moisture, spray segment after the sterilization is formed the long stem-segment with single bud of 1-2cm, be placed on then in the initial culture base and cultivate, induce sprouting of lateral bud;
The optimization of c, successive transfer culture: after treating the stem segment with axillary buds elongation of stem-segment with single bud, stem segment with axillary buds downcut to transfer in the proliferated culture medium (pH=5.2) of the improvement WPM medium that contains the variable concentrations hormone combinations+20g/L sucrose+8.8g/L daily output agar powder, carry out illumination cultivation, the illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark; The improvement method of improvement WPM medium is: with Ca (NO
3)
24H
2O, KNO
3Replace the CaCl in the former WPM medium
2, K
2SO
4
The foundation of d, leaf regeneration system: 2 months aseptic seedling plant leaf of successive transfer culture is downcut, downcut blade by the vertical vane master pulse from 1/3 place of blade tip end and be divided into blade tip end and petiole end, in the blake bottle with blade tip end, petiole end and complete this equal back side of three parts of leaf improvement WPS medium+20g/L sucrose+9g/L agar powder (pH=5.2) that is inoculated in different salinity and different ZT concentration placed apart down, every bottle each about 10, each processed group repeats 12 bottles.Carry out the dark cultivation of 20d earlier, dark temperature of cultivating is 25 ± 2 ℃, carry out illumination cultivation subsequently, the illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark, the seedling number of statistics blade bud ratio and the corresponding formation of each blade behind the 30d, thus obtain best leaf regeneration system;
E, rooting of vitro seedling are cultivated: the aseptic seedling plant of successive transfer culture is downcut apart from about the 2.5～3.0cm of top, be inoculated at random in the blake bottle of the improvement WPS medium+20g/L sucrose+9g/L agar powder (pH=5.2) that contains different salinity, different I BA concentration and the combination of different activities concentration of carbon and carry out illumination cultivation, the illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark, the record start rootage duration, the upgrowth situation of statistics rooting rate and take root number and observation seedling behind the 60d, thus filter out optimum root media;
F, hardening: in advance 3d opens bottle cap with rooting tube plantlet in the bottle and carries out transition, clean the root medium, transplant at random, in the booth of growing seedlings that keeps humidity 100%, cultivate 20d in different hardening matrix, transition 3d under the outdoor conditions of humidity 80%, light transmittance 70% then, the canopy film is thrown off in progressively ventilation more fully behind the 7d, statistics survival rate after 2 weeks, the amount of planting of every cover matrix is no less than 50 strains, the number of repetition of each test is no less than 3 times, and the statistics survival rate is to finish hardening.
Preferably, the initial culture base is produced agar powder+1.0mg/L ZT daily, its pH=5.2 for improvement WPM medium+20g/L sucrose+8.8g/L among the step b.
Preferably, among the step c, after treating the stem segment with axillary buds elongation of stem-segment with single bud, stem segment with axillary buds downcut to transfer in the proliferated culture medium (pH=5.2) of the improvement WPM medium that contains variable concentrations ZT, 6-BA or TDZ+20g/L sucrose+8.8g/L daily output agar powder, carry out illumination cultivation.
Preferably, among the step c, the incidence of indefinite bud and the ZT concentration in the medium have confidential relation; When the ZT mass concentration is 0.5mg/L, to cultivate about 10d and produce yellowish green callus at basal part of stem, the bud of growing thickly appears in the joint place, and the rate of increase reaches 5.02 times behind the 28d, and is mainly the major branch bud, and the bud of growing thickly is sturdy, and the growth impetus is good, is fresh and tender shape; When the ZT mass concentration is elevated to 1mg/L, begin to grow and be more or less the same when time of breaking up and 0.5mg/L, its rate of increase increases slightly to some extent; When the ZT mass concentration equaled and is higher than 2mg/L, regrowth began growth and differentiating phenomenon shifts to an earlier date, and occurred 2 branches on the young sprout branch that majority breaks up out, and the rate of increase reaches about 10 times, because the side shoot bud is prone to vitrifying seedling phenomenon a little less than making the seedling growing way more; When using 6-BA, explant is sprout growth not, growth retardation.When using TDZ, form a small amount of callus at the explant base portion behind the 10d, though the energy rudiment, the low and poor growth of germination rate; Take all factors into consideration from aspects such as the rate of increase, the bud growing way of growing thickly, zeatin costs, subculture medium is the best with WPM+0.5mg/L ZT.
Preferably, in steps d, the seedling number of statistics blade bud ratio and the corresponding formation of each blade in the blake bottle of different salinity, high salt concentration is unfavorable for the regeneration of blade, its regeneration induction seedling rate is 0-2.7%; Macroelement concentration is reduced half, can obviously promote the regeneration of blade, blade regeneration induction seedling rate is increased to 23.1-70.9%; In the blade different parts, the regrowth inductivity of blade tip end is minimum, and the petiole end is placed in the middle, and the inductivity of full leaf is the highest.
Preferably, in steps d, the seedling number of statistics blade bud ratio and the corresponding formation of each blade in the blake bottle of variable concentrations ZT or TDZ; TDZ can not form regrowth by inducer blade, and blade forms callus in incision after cultivating 10d, and along with the prolongation of incubation time, callus is brownization slowly, does not see that regrowth forms; ZT induces effect obviously to be better than TDZ to the leaf regeneration seedling, the incidence of indefinite bud and the ZT in the medium have confidential relation, ZT induces the leaf regeneration seedling and has the significant concentration effect, blade tip end inductivity when 0.5mg/L ZT is 0%, along with the rising inductivity of ZT concentration increases gradually, the inductivity when 5mg/L ZT reaches 32.3%; The inductivity of petiole end and full leaf all increases gradually with the rising of ZT mass concentration, and inductivity all reaches the highest when 2.0mg/L ZT, full the regrowth inductivity of petiole end and leaf is respectively 54.3% and 71.4%, the inductivity during 5mg/L ZT but all descends; There is notable difference in the regrowth inductivity of different parts blade, and the inductivity of blade tip end is minimum, petiole end placed in the middle, and the inductivity of full leaf is the highest; Low concentration ZT mainly induces and forms Dan Shengmiao, and high concentration ZT help the clustering formation of seedling; The seedling of living again that blade tip end blade is induced mainly is formed at incision, and the regrowth that petiole end and full leaf are induced mainly is formed at petiole one side; Select for use full leaf to carry out the leaf regeneration seedling and induce, the best ZT mass concentration that its regrowth is induced is 2.0mg/L.
Preferably, in step e, there is material impact in different salinity to the root induction of single bud seedling, and high salt concentration is unfavorable for the bud seedling rooting, and beginning rootage duration length and rooting rate only are 30%; When macroelement reduces by half, begin to take root behind about 14d and rooting rate be 75%; When macroelement reduces to original 1/4 the time, the beginning rootage duration be about 7 days in advance and rooting rate up to 100%, the seedling growing way is strong; Macroelement reduces at original 1/8 o'clock, though beginning rootage duration and rooting rate and macroelement reduce to original 1/4 similar, yet because the shortage of nutriment, a little less than the seedling growing way, suitably reduce the growth that salinity helps taking root and do not influence seedling, the salinity of the root media of taking root in Ao Ni'er's test-tube plantlet bottle selects for use macroelement to reduce to original 1/4.
Preferably, in step e, active carbon has material impact to taking root in the blake bottle, when in the medium during non-activity charcoal, the all long earlier callus of basal part of stem that inserts in the medium is taken root then thereon, because vascular bundle is obstructed between the rhizome, stem promptly stops growing soon, the leaf look rubescent to come off, and the seedling growing way is very poor; After adding active carbon in the medium, basal part of stem seldom or not long callus and directly taking root from stem, thereby help the growth of seedling; There is concentration effect in active carbon to taking root, along with the rising of concentration of activated carbon, rooting rate is higher, take root number the more and root longer, the seedling growing way is better; When active carbon was 0.05%, rooting rate was 58%-75%, every strain several 1-3 bars of taking root, and the long 1-3cm of root, the seedling growth is slower; When active carbon was increased to 0.2%, rooting rate improved about 20%, and every strain is taken root for counting and reached more than 4, the long 4-6cm of root, and the seedling growth is fast; IBA is taken root to the Ao Ni'er and is also had concentration effect, and high concentration IBA more helps taking root, and there are synergistic effect between the two in active carbon and IBA, and when both concentration were all high, rooting rate and seedling growing way were all better.Effective rooting rate at 0.2% active carbon, 0.6mg/L and 0.8mg/L IBA all reaches 100%, but initial time of taking root of the latter and root are long all Zao and long than the former, the latter about 7 days visible basal part of stem have root to form, grow 4-6 bar root about 45 days, root reaches about 6cm, more than the height of seedling 9cm, leaf look dark green blade is big, the seedling robust growth, in Ao Ni'er's bottle in the root media best concentration of activated carbon and IBA concentration be respectively 0.2% and 0.8mg/L, effectively rooting rate is up to 100%.
Preferably, among the step f: hardening matrix is selected the peat composed of rotten mosses: river sand=1: 1; The perhaps peat composed of rotten mosses: garden mould=1: 1; The perhaps peat composed of rotten mosses: quartz sand=2～3: 1; The perhaps peat composed of rotten mosses: perlite=1～2: 1; Perhaps pure water liver moss.
Preferably, among the step f: rooting tube plantlet is grown in five kinds of different hardening matrix, and wherein the effect of pure water liver moss is best, and survival rate is up to 100%, and the amount of growth of nursery stock reaches more than the 3cm, newly-increased blade 6-8 piece; Effect next be 2～3: the combination of 1 the peat composed of rotten mosses and quartz sand, its survival rate is 84%, the amount of growth of nursery stock is higher; The survival rate of 1: 1 the peat composed of rotten mosses and river sand matrix only is 50%, but it is very high to survive the quality of seedling, shows as growth pier reality, robust plant, amount of growth and " peat composed of rotten mosses+quartz sand " quite; The effect of the peat composed of rotten mosses+garden mould matrix and the peat composed of rotten mosses+pearlite interstitial substance is placed in the middle.
Useful technique effect of the present invention is:
For obtaining the technical system of south high clump blueberry Ao Ni'er high efficiency quick breeding, be test material with Ao Ni'er's tender stem segments, proliferated culture medium, leaf regeneration system, root media, hardening matrix are optimized.The result shows that the proliferated culture medium of improvement WPM+0.5mg/L ZT can make the young sprout rate of increase improve more than 4 times, and grow thickly seedling robust growth and fast growth; Reduce salinity and help inducing of leaf regeneration seedling, the regeneration that is better than cutting off blade is induced in the regeneration of full leaf, and the former regeneration rate in the medium of 1/2 improvement WPM+2.0mg/L ZT is up to 71%, and the main seedling that clusters that forms; The root media of 1/4 improvement WPM+0.2% activated carbon+0.8mg/L IBA can make the rooting of vitro seedling rate reach 100%; With the sphagna moss is that hardening matrix can make the survival rate of seedling up to 100%.Therefore, enrichment culture, leaf regeneration and the culture of rootage of the best combined, adds that effective seedling survives matrix, can be efficiently, fast, continue to obtain good Ao Ni'er's group and cultivate seedling.
Description of drawings
Fig. 1 is the histograms of the different hardening matrix of the suitable south of the embodiment of the invention high clump blueberry high efficiency quick breeding technology to the influence of test-tube plantlet survival rate.
The object of the invention realization, functional characteristics and advantage will be in conjunction with the embodiments, are described further with reference to accompanying drawing.
Embodiment
Should be appreciated that specific embodiment described herein only in order to explanation the present invention, and be not used in qualification the present invention.
The concrete a kind of suitable south high clump blueberry high efficiency quick breeding technology of implementing may further comprise the steps:
A, selection: vegetable material is the south high clump blueberry kind Ao Ni'er of the rich biotechnology company of intelligence plantation plantation, and explant material is selected for use and given birth to young spray then;
B, initial culture obtain aseptic seedling: the living then spray with field growing is initial explant, tripping.Behind dirts such as the clean dust of detergent solution, the running water flushing.Forward on the superclean bench, successively explant is carried out the surface sterilizing of 50s and 10min respectively with 75% alcohol and 0.1% mercuric chloride, then use aseptic water washing 4 times, 2 minutes/time, inhale with aseptic filter paper at last and remove the explant surface moisture, spray segment after the sterilization is formed the long stem-segment with single bud of 1-2cm, be placed on then in the initial culture base and cultivate, induce sprouting of lateral bud;
The optimization of c, successive transfer culture: after treating the stem segment with axillary buds elongation of stem-segment with single bud, stem segment with axillary buds downcut to transfer in the proliferated culture medium (pH=5.2) of the improvement WPM medium that contains the variable concentrations hormone combinations+20g/L sucrose+8.8g/L daily output agar powder, carry out illumination cultivation, the illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark; The improvement method of improvement WPM medium is: with Ca (NO
3)
24H
2O, KNO
3Replace the CaCl in the former WPM medium
2, K
2SO
4
The foundation of d, leaf regeneration system: 2 months aseptic seedling plant leaf of successive transfer culture is downcut, downcut blade by the vertical vane master pulse from 1/3 place of blade tip end and be divided into blade tip end and petiole end, in the blake bottle with blade tip end, petiole end and complete this equal back side of three parts of leaf improvement WPS medium+20g/L sucrose+9g/L agar powder (pH=5.2) that is inoculated in different salinity and different ZT concentration placed apart down, every bottle each about 10, each processed group repeats 12 bottles.Carry out the dark cultivation of 20d earlier, dark temperature of cultivating is 25 ± 2 ℃, carry out illumination cultivation subsequently, the illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark, the seedling number of statistics blade bud ratio and the corresponding formation of each blade behind the 30d, thus obtain best leaf regeneration system;
E, rooting of vitro seedling are cultivated: the aseptic seedling plant of successive transfer culture is downcut apart from about the 2.5～3.0cm of top, be inoculated at random in the blake bottle of the improvement WPS medium+20g/L sucrose+9g/L agar powder (pH=5.2) that contains different salinity, different I BA concentration and the combination of different activities concentration of carbon and carry out illumination cultivation, the illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark, the record start rootage duration, the upgrowth situation of statistics rooting rate and take root number and observation seedling behind the 60d, thus filter out optimum root media;
F, hardening: in advance 3d opens bottle cap with rooting tube plantlet in the bottle and carries out transition, clean the root medium, transplant at random in different hardening matrix, under the condition of group training chamber, cultivate 20d in the booth of growing seedlings of maintenance humidity 100%, transition 3d under the outdoor conditions of humidity 80%, light transmittance 70% then, progressively ventilation again, throw off the canopy film behind the 7d fully, statistics survival rate after 2 weeks, the amount of planting of every cover matrix is no less than 50 strains, the number of repetition of each test is no less than 3 times, and the statistics survival rate is to finish hardening.
The invention is characterized in that the initial culture base is produced agar powder+1.0mg/L ZT daily, its pH=5.2 for improvement WPM medium+20g/L sucrose+8.8g/L among the step b.
The invention is characterized in, among the step c, after treating the stem segment with axillary buds elongation of stem-segment with single bud, stem segment with axillary buds downcut to transfer in the proliferated culture medium (pH=5.2) of the improvement WPM medium that contains variable concentrations ZT, 6-BA or TDZ+20g/L sucrose+8.8g/L daily output agar powder, carry out illumination cultivation.
The invention is characterized in that among the step c, the incidence of indefinite bud and the ZT concentration in the medium have confidential relation; When the ZT mass concentration is 0.5mg/L, to cultivate about 10d and produce yellowish green callus at basal part of stem, the bud of growing thickly appears in the joint place, and the rate of increase reaches 5.02 times behind the 28d, and is mainly the major branch bud, and the bud of growing thickly is sturdy, and the growth impetus is good, is fresh and tender shape; When the ZT mass concentration is elevated to 1mg/L, begin to grow and be more or less the same when time of breaking up and 0.5mg/L, its rate of increase increases slightly to some extent; When the ZT mass concentration equaled and is higher than 2mg/L, regrowth began growth and differentiating phenomenon shifts to an earlier date, and occurred 2 branches on the young sprout branch that majority breaks up out, and the rate of increase reaches about 10 times, because the side shoot bud is prone to vitrifying seedling phenomenon a little less than making the seedling growing way more; When using 6-BA, explant is sprout growth not, growth retardation.When using TDZ, form a small amount of callus at the explant base portion behind the 10d, though the energy rudiment, the low and poor growth of germination rate; Take all factors into consideration from aspects such as the rate of increase, the bud growing way of growing thickly, zeatin costs, subculture medium is the best with WPM+0.5mg/L ZT.
The invention is characterized in, in steps d, the seedling number of statistics blade bud ratio and the corresponding formation of each blade in the blake bottle of different salinity, high salt concentration is unfavorable for the regeneration of blade, its regeneration induction seedling rate is 0-2.7%; Macroelement concentration is reduced half, can obviously promote the regeneration of blade, blade regeneration induction seedling rate is increased to 23.1-70.9%; In the blade different parts, the regrowth inductivity of blade tip end is minimum, and the petiole end is placed in the middle, and the inductivity of full leaf is the highest.
The invention is characterized in, in steps d, the seedling number of statistics blade bud ratio and the corresponding formation of each blade in the blake bottle of variable concentrations ZT or TDZ; TDZ can not form regrowth by inducer blade, and blade forms callus in incision after cultivating 10d, and along with the prolongation of incubation time, callus is brownization slowly, does not see that regrowth forms; ZT induces effect obviously to be better than TDZ to the leaf regeneration seedling, the incidence of indefinite bud and the ZT in the medium have confidential relation, ZT induces the leaf regeneration seedling and has the significant concentration effect, blade tip end inductivity when 0.5mg/L ZT is 0%, along with the rising inductivity of ZT concentration increases gradually, the inductivity when 5mg/L ZT reaches 32.3%; The inductivity of petiole end and full leaf all increases gradually with the rising of ZT mass concentration, and inductivity all reaches the highest when 2.0mg/L ZT, full the regrowth inductivity of petiole end and leaf is respectively 54.3% and 71.4%, the inductivity during 5mg/L ZT but all descends; There is notable difference in the regrowth inductivity of different parts blade, and the inductivity of blade tip end is minimum, petiole end placed in the middle, and the inductivity of full leaf is the highest; Low concentration ZT mainly induces and forms Dan Shengmiao, and high concentration ZT help the clustering formation of seedling; The seedling of living again that blade tip end blade is induced mainly is formed at incision, and the regrowth that petiole end and full leaf are induced mainly is formed at petiole one side; Select for use full leaf to carry out the leaf regeneration seedling and induce, the best ZT mass concentration that its regrowth is induced is 2.0mg/L.
The invention is characterized in that in step e, there is material impact in different salinity to the root induction of single bud seedling, high salt concentration is unfavorable for the bud seedling rooting, and beginning rootage duration length and rooting rate only are 30%; When macroelement reduces by half, begin to take root behind about 14d and rooting rate be 75%; When macroelement reduces to original 1/4 the time, the beginning rootage duration be about 7 days in advance and rooting rate up to 100%, the seedling growing way is strong; Macroelement reduces at original 1/8 o'clock, though beginning rootage duration and rooting rate and macroelement reduce to original 1/4 similar, yet because the shortage of nutriment, a little less than the seedling growing way, suitably reduce the growth that salinity helps taking root and do not influence seedling, the salinity of the root media of taking root in Ao Ni'er's test-tube plantlet bottle selects for use macroelement to reduce to original 1/4.
The invention is characterized in, in step e, active carbon has material impact to taking root in the blake bottle, when in the medium during non-activity charcoal, the all long earlier callus of basal part of stem that inserts in the medium is taken root then thereon, because vascular bundle is obstructed between the rhizome, stem promptly stops growing soon, the leaf look rubescent to come off, and the seedling growing way is very poor; After adding active carbon in the medium, basal part of stem seldom or not long callus and directly taking root from stem, thereby help the growth of seedling; There is concentration effect in active carbon to taking root, along with the rising of concentration of activated carbon, rooting rate is higher, take root number the more and root longer, the seedling growing way is better; When active carbon was 0.05%, rooting rate was 58%-75%, every strain several 1-3 bars of taking root, and the long 1-3cm of root, the seedling growth is slower; When active carbon was increased to 0.2%, rooting rate improved about 20%, and every strain is taken root for counting and reached more than 4, the long 4-6cm of root, and the seedling growth is fast; IBA is taken root to the Ao Ni'er and is also had concentration effect, and high concentration IBA more helps taking root, and there are synergistic effect between the two in active carbon and IBA, and when both concentration were all high, rooting rate and seedling growing way were all better.Effective rooting rate at 0.2% active carbon, 0.6mg/L and 0.8mg/L IBA all reaches 100%, but initial time of taking root of the latter and root are long all Zao and long than the former, the latter about 7 days visible basal part of stem have root to form, grow 4-6 bar root about 45 days, root reaches about 6cm, more than the height of seedling 9cm, leaf look dark green blade is big, the seedling robust growth, in Ao Ni'er's bottle in the root media best concentration of activated carbon and IBA concentration be respectively 0.2% and 0.8mg/L, effectively rooting rate is up to 100%.
The invention is characterized in that among the step f: hardening matrix is selected the peat composed of rotten mosses: river sand=1: 1; The perhaps peat composed of rotten mosses: garden mould=1: 1; The perhaps peat composed of rotten mosses: quartz sand=2～3: 1; The perhaps peat composed of rotten mosses: perlite=1～2: 1; Perhaps pure water liver moss.
The invention is characterized in that among the step f: rooting tube plantlet is grown, and wherein the effect of pure water liver moss is best, and survival rate is up to 100%, and the amount of growth of nursery stock reaches more than the 3cm in five kinds of different hardening matrix, newly-increased blade 6-8 piece; Effect next be 2～3: the combination of 1 the peat composed of rotten mosses and quartz sand, its survival rate is 84%, the amount of growth of nursery stock is higher; The survival rate of 1: 1 the peat composed of rotten mosses and river sand matrix only is 50%, but it is very high to survive the quality of seedling, shows as growth pier reality, robust plant, amount of growth and " peat composed of rotten mosses+quartz sand " quite; The effect of the peat composed of rotten mosses+garden mould matrix and the peat composed of rotten mosses+pearlite interstitial substance is placed in the middle.
During concrete enforcement:
1 materials and methods
1.1 material
Vegetable material is the south high clump blueberry kind Ao Ni'er (0 ' Neal) of the rich biotechnology company of intelligence plantation plantation.Explant material is selected for use and is given birth to young spray then.
1.2 method
1.2.1 initial culture obtains aseptic seedling
Living then spray with field growing is initial explant, tripping.Behind dirts such as the clean dust of detergent solution, the running water flushing.Forward on the superclean bench, successively explant is carried out the surface sterilizing of 50s and 10min respectively, then, 2 minutes/time, inhale with aseptic filter paper at last and remove the explant surface moisture with aseptic water washing 4 times with 75% alcohol and 0.1% mercuric chloride.Branch is cut into the long stem-segment with single bud of 1-2cm, under aseptic condition, stem-segment with single bud is inoculated in the initial culture base of improvement WPM medium+20g/L sucrose+8.8g/L daily output agar powder+1.0mg/L ZT, the pH=5.2 of initial culture base, carry out illumination cultivation, induce sprouting of lateral bud, stem segment with axillary buds extends more than the 5cm after cultivating 30d, and gives birth to 4-5 sheet leaf.The illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark.
1.2.2 the optimization of successive transfer culture
The aseptic seedling that initial culture obtains is downcut, being cut into transfers behind the stem-segment with single bud carries out illumination cultivation in the proliferated culture medium (pH=5.2) of the improvement WPM of different hormone concentrations medium (respectively handle the hormone concentration combination and see Table 1)+20g/L sucrose+8.8g/L daily output agar powder, cultivate propagation multiple and the upgrowth situation of observing bud behind the 40d.The illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark.The concrete improvement method of improvement WPM medium: with Ca (NO
3)
24H
2O, KNO
3Replace the CaCl in the former WPM medium
2, K
2SO
4
1.2.2 the foundation of leaf regeneration system
2 months aseptic seedling plant leaf of successive transfer culture is downcut, downcut blade by the vertical vane master pulse from 1/3 place of blade tip end and be divided into blade tip end and petiole end, in the blake bottle with blade tip end, petiole end and complete this equal back side of three parts of leaf improvement WPS medium+20g/L sucrose+9g/L agar powder (pH=5.2) that is inoculated in different salinity (respectively handle salinity and see Table 2) and different ZT concentration (respectively handle ZT concentration and see Table 3) placed apart down, every bottle each about 10, each processed group repeats 12 bottles.Carry out the dark cultivation (25 ± 2 ℃ of temperature) of 20d earlier, carry out illumination cultivation subsequently, the illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark.The seedling number of statistics blade bud ratio and the corresponding formation of each blade behind the 30d, thus best leaf regeneration system obtained.
1.2.3 rooting of vitro seedling is cultivated
The aseptic seedling plant of successive transfer culture is downcut apart from about the 2.5～3.0cm of top, be inoculated at random and contain different salinity (respectively handle the salinity combination and see Table 4), (the illumination cultivation condition is: 25 ± 2 ℃ of temperature to carry out illumination cultivation in the improvement WPS medium+20g/L sucrose+9g/L agar powder (pH=5.2) of different I BA concentration and different activities concentration of carbon combination (each IBA concentration and activated carbon concentration combination see Table 5), light intensity 2500Lx, photoperiod 16h illumination/8h dark), the record start rootage duration, the upgrowth situation of statistics rooting rate and take root number and observation seedling behind the 60d, thus filter out optimum root media.
1.2.4 hardening
In advance 3d opens bottle cap with rooting tube plantlet in the bottle and carries out transition, clean the root medium, transplant at random in different hardening matrix (each substrate combination is seen Fig. 1), in the booth of growing seedlings that keeps humidity 100%, cultivate 20d, transition 3d under the outdoor conditions of humidity 80%, light transmittance 70% then, the canopy film is thrown off in progressively ventilation more fully behind the 7d, statistics survival rate after 2 weeks.The amount of planting of every cover matrix is no less than 50 strains.The number of repetition of each test is no less than 3 times, and the statistics survival rate is to finish hardening.
2 results and analysis
2.1 the optimization of successive transfer culture
The aseptic seedling that obtains is cut on the medium that stem-segment with single bud is transferred to different hormone concentrations carries out subculture medium optimization.The result of table 1 shows that the cultivation effect of ZT obviously is better than 6-BA and TDZ.In ZT, the incidence of indefinite bud and the ZT in the medium have confidential relation.When the ZT mass concentration is 0.5mg/L, to cultivate about 10d and produce yellowish green callus at basal part of stem, the bud of growing thickly appears in the joint place, and the rate of increase reaches 5.02 times behind the 28d, and is mainly the major branch bud, and the bud of growing thickly is sturdy, and the growth impetus is good, is fresh and tender shape.When the ZT mass concentration is elevated to 1mg/L, begin to grow and be more or less the same when time of breaking up and 0.5mg/L, its rate of increase increases slightly to some extent.When the ZT mass concentration equaled and is higher than 2mg/L, regrowth began growth and differentiating phenomenon shifts to an earlier date, and occurred 2 branches on the young sprout branch that majority breaks up out, and the rate of increase reaches about 10 times, because the side shoot bud is prone to vitrifying seedling phenomenon a little less than making the seedling growing way more.WPM and other hormone combinations effect all are worse than the WPM+ZT combination.When using 6-BA, explant is sprout growth not, growth retardation.When using TDZ, form a small amount of callus at the explant base portion behind the 10d, though the energy rudiment, the low and poor growth of germination rate.Therefore, take all factors into consideration from aspects such as the rate of increase, the bud growing way of growing thickly, zeatin costs, subculture medium is the best with WPM+0.5mg/L ZT.
The different hormone concentrations of table 1 are to the influence of blueberry bud propagation
2.2 the foundation of leaf regeneration system
2.2.1 the influence that salinity is induced leaf regeneration
By vertical vane master pulse direction blade is cut blade tip end and petiole end two parts from blade tip 1/3, and full leaf, vacuum side of blade is inoculated in the blake bottle that contains different salinity down, and statistics leaf regeneration rate the results are shown in Table 2 after 30 days.As can be seen from Table 2, high salt concentration is unfavorable for the regeneration of blade, and its regeneration induction seedling rate only is 0-2.7%.Macroelement concentration is reduced half, can obviously promote the regeneration of blade, blade regeneration induction seedling rate is increased to 23.1-70.9%.In the blade different parts, the regrowth inductivity of blade tip end is minimum, and the petiole end is placed in the middle, and the inductivity of full leaf is the highest.
The influence that the different salinity of table 2 are induced blade different parts regrowth
The withered blade of brownization does not count wherein.
2.2.2ZT concentration is to the live again influence of seedling of inducer blade
ZT is a kind of basic element of cell division of high vigor, has the effect of breaking plant apical dominance, promotion axillary bud sprouting, evoking adventive bud formation.Variable concentrations ZT induces the effect difference to the seedling of living again of blade different parts, the results are shown in Table 3.As can be seen from Table 3, ZT induces and has the significant concentration effect the blade seedling of living again, and blade tip end inductivity when 0.5mg/L ZT is 0%, and along with the rising inductivity of ZT concentration increases gradually, the inductivity when 5mg/L ZT reaches 41%; The inductivity of petiole end and full leaf all increases gradually with the rising of ZT mass concentration, and inductivity all reaches the highest when 2.0mg/L ZT, full the seedling inductivity of living again of petiole end and leaf is respectively 54% and 71%, the inductivity during 5mg/LZT but all descends.There is notable difference in the seedling inductivity of living again of different parts blade, and the inductivity of blade tip end is minimum, petiole end placed in the middle, and the inductivity of full leaf is the highest.Low concentration ZT mainly induces and forms Dan Shengmiao, and high concentration ZT help the clustering formation of seedling.The seedling of living again that blade tip end blade is induced mainly is formed at incision, and the seedling of living again that petiole end and full leaf are induced mainly is formed at petiole one side.Based on the above results, select for use full leaf to carry out the blade seedling of living again and induce, the best ZT mass concentration that its seedling of living again is induced is 2.0mg/L.
Table 3 different quality concentration ZT is to the live again influence of seedling of inducer blade
2.3 take root in the bottle of test-tube plantlet
2.3.1 the influence of salinity to taking root
There is material impact in different salinity to the root induction of single bud seedling.Can find out from table 4: high salt concentration is unfavorable for the bud seedling rooting, and beginning rootage duration length and rooting rate only are 30%; When macroelement reduces by half, begin to take root behind about 14d and rooting rate be 75%; When macroelement reduces to original 1/4 the time, the beginning rootage duration be about 7 days in advance and rooting rate up to 100%, the seedling growing way is strong; Macroelement reduces at original 1/8 o'clock, though beginning rootage duration and rooting rate and macroelement reduce to original 1/4 similar, yet owing to the shortage of nutriment, a little less than the seedling growing way.Therefore suitably reduce the growth that salinity helps taking root and do not influence seedling, the salinity of the root media of taking root in Ao Ni'er's test-tube plantlet bottle all selects for use macroelement to reduce to original 1/4.
The influence of the different salinity of table 4 to taking root
2.3.2 active carbon and growth hormone influence to taking root
Active carbon has material impact to taking root in the blueberry bottle.As seen from Table 5, when in the medium during non-activity charcoal, all long earlier callus of basal part of stem that inserts in the medium is taken root then thereon, and owing to vascular bundle between the rhizome is obstructed, stem promptly stops growing soon, and the leaf look rubescent to come off, and the seedling growing way is very poor.After adding active carbon in the medium, basal part of stem seldom or not long callus and directly taking root from stem, thereby help the growth of seedling.There is concentration effect in active carbon to taking root, along with the rising of concentration of activated carbon, rooting rate is higher, take root number the more and root longer, the seedling growing way is better.When active carbon was 0.05%, rooting rate was 58%-75%, every strain several 1-3 bars of taking root, and the long 1-3cm of root, the seedling growth is slower; When active carbon was increased to 0.2%, rooting rate improved about 20%, and every strain is taken root for counting and reached more than 4, the long 4-6cm of root, and the seedling growth is fast.IBA is taken root to the Ao Ni'er and is also had concentration effect, and high concentration IBA more helps taking root.There are synergistic effect between the two in active carbon and IBA, and when both concentration were all high, rooting rate and seedling growing way were all better.Effective rooting rate at 0.2% active carbon, 0.6mg/L and 0.8mg/L IBA all reaches 100%, but initial time of taking root of the latter and root are long all Zao and long than the former, the latter about 7 days visible basal part of stem have root to form, grow 4-6 bar root about 45 days, root reaches about 6cm, more than the height of seedling 9cm, leaf look dark green blade is big, the seedling robust growth.Based on the above results, in Ao Ni'er's bottle in the root media best concentration of activated carbon and IBA concentration be respectively 0.2% and 0.8mg/L, effectively rooting rate is up to 100%.
Table 5 different activities carbon and of the influence of IBA concentration to taking root in the tissue cultivating seedling bottle
2.4 the optimization of hardening matrix
The seedling of taking root in the bottle is shifted to an earlier date 3d open bottle cap, transplant on 5 kinds of different substrates behind the medium of clean root, add up survival rate behind the 20d.As can be seen from Figure 1, the effect of sphagna moss is best, and survival rate is up to 100%, and the amount of growth of nursery stock reaches more than the 3cm, newly-increased blade 6-8 piece.Effect next be 2～3: the combination of 1 the peat composed of rotten mosses and quartz sand, its survival rate is 84%, the amount of growth of nursery stock is higher.The survival rate of 1: 1 the peat composed of rotten mosses and river sand matrix only is 50%, but it is very high to survive the quality of seedling, shows as growth pier reality, robust plant, amount of growth and " peat composed of rotten mosses+quartz sand " quite.The effect of the peat composed of rotten mosses+garden mould matrix and the peat composed of rotten mosses+pearlite interstitial substance is placed in the middle.
Oneself there are some researches show, the fast numerous medium of blueberry is best with the effect of improvement WPM, and bud induce and breed in the effect of the basic element of cell division most important
[Zeatin is a kind of basic element of cell division of high vigor, and therefore, we have mainly adopted improvement WPM medium, and emphasis has been inquired into the effect of zeatin.Originally studies show that, utilize 0.5mg/L ZT to carry out enrichment culture, the propagation multiple reaches more than 4 times, and the blastogenesis of growing thickly is long healthy and strong, fast growth, and by 2-3 subculture, its propagation multiple can reach 50-60 doubly; The a little higher than 0.5mg/L ZT of the propagation multiple of 1mg/L ZT; More than or equal to the propagation multiple of 2mg/L ZT up to more than 12 doubly, degree of growth is slow too much for the seedling of growing thickly, the appearance of vitrification phenomenon simultaneously reduces effective seedling number.Because the ZT price is expensive, take all factors into consideration cultivation effect the best of 0.5mg/L ZT from the upgrowth situation of production cost, propagation multiple and seedling.
Aspect leaf regeneration, relevant both at home and abroad studies show that, adopts the hormones of different concentrations combination can induce different high clump blueberry kind leaf regenerations, and there is breed difference in the blueberry leaf regeneration.Relevant Ao Ni'er's leaf regeneration does not appear in the newspapers as yet.Originally discover that high salt concentration can not induce Ao Ni'er's leaf regeneration, reduce salinity and help Ao Ni'er's leaf regeneration seedling and induce.By leaf regeneration is induced optimization, find that the inductivity of full leaf is better than cutting off leaf simultaneously, full leaf regeneration rate the best under 1/2 improvement WPM+2.0mg/L ZT condition, up to 71%, and the main seedling that clusters that forms.Many and the high leaf regeneration rate of the quantity of tissue cultivating seedling blade, again in conjunction with enrichment culture, its propagation multiple is considerable, is higher than the result of forefathers in high clump blueberry proliferation research far away.
Because blueberry is the difficult plant that takes root, the current main both at home and abroad mode that adopts outside sprout-cultivating-bottle.Comparatively speaking, take root in the tissue culture flasks and have more advantage than outside sprout-cultivating-bottle, the former survival rate height, growth are fast.We have carried out a bottle interior root media optimization to the Ao Ni'er, obtained the best prescription of taking root: 1/4 improvement WPM+0.2% activated carbon+0.8mg/L IBA prescription, can make young sprout about 7d, begin to take root, rooting rate is up to 100%, seedling stalwartness and fast growth, but about 45d is with regard to the bottle outlet hardening.This result obviously is better than the result in high clump blueberry is taken root research in the past.
The soil pH value of blueberry suitable growth is 4.5～5.5.Aspect hardening, how the multiple formulations of domestic report is the blueberry growth with consideration all provides suitable pH value, and they are basic medium with turfy soil or its analog, and the additional support material is regulated the permeability of matrix again.Result from this research, the pH value is the key factor that the test-tube plantlet domestication survives, but not decisive factor, the group of normal growth training domestication seedling key is the gas permeability and the moisture capacity of matrix, and liver moss is an organic substrate, has advantages such as preserve moisture, breathe freely, the pH value is suitable, easy to use, himself has active factors, be beneficial to and bring out root system, can form root mass at short notice, and promote its growth.The root system of growing in the liver moss is energetic, and it is fast to form the root system group, can obtain high quality seedling.This research adopts the little shed of plastics to preserve moisture and the sphagna moss is a hardening matrix, and seedling percent is up to 100%, and growth of seedling is fast and healthy and strong, and the hardening effect is better than obviously that domestic what reported is the hardening matrix of basic medium with turfy soil.Therefore, this research has been carried out a series of optimization from leaf regeneration, enrichment culture, culture of rootage to this several links of hardening, having set up a cover can be efficiently, fast, continue to obtain good south high clump blueberry Ao Ni'er and organize the technical system of cultivating seedling, help blueberry industrialized development with China.
The above only is the preferred embodiments of the present invention; be not so limit claim of the present invention; every equivalent structure transformation that utilizes specification of the present invention and accompanying drawing content to be done; or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.
Claims (10)
1. a suitable south high clump blueberry high efficiency quick breeding technology is characterized in that,
A, selection: vegetable material is the south high clump blueberry kind Ao Ni'er of the rich biotechnology company of intelligence plantation plantation, and explant material is selected for use and given birth to young spray then;
B, initial culture obtain aseptic seedling: the living then spray with field growing is initial explant, tripping.Behind dirts such as the clean dust of detergent solution, the running water flushing.Forward on the superclean bench, successively explant is carried out the surface sterilizing of 50s and 10min respectively with 75% alcohol and 0.1% mercuric chloride, then use aseptic water washing 4 times, 2 minutes/time, inhale with aseptic filter paper at last and remove the explant surface moisture, spray segment after the sterilization is formed the long stem-segment with single bud of 1-2cm, be placed on then in the initial culture base and cultivate, induce sprouting of lateral bud;
The optimization of c, successive transfer culture: after treating the stem segment with axillary buds elongation of stem-segment with single bud, stem segment with axillary buds downcut to transfer in the proliferated culture medium (pH=5.2) of the improvement WPM medium that contains the variable concentrations hormone combinations+20g/L sucrose+8.8g/L daily output agar powder, carry out illumination cultivation, the illumination cultivation condition is: temperature 25+2 ℃, light intensity 2500Lx, photoperiod 16h illumination/8h dark; The improvement method of improvement WPM medium is: with Ca (NO
3)
24H
2O, KNO
3Replace the CaCl in the former WPM medium
2, K
2SO
4
The foundation of d, leaf regeneration system: 2 months aseptic seedling plant leaf of successive transfer culture is downcut, downcut blade by the vertical vane master pulse from 1/3 place of blade tip end and be divided into blade tip end and petiole end, in the blake bottle with blade tip end, petiole end and complete this equal back side of three parts of leaf improvement WPS medium+20g/L sucrose+9g/L agar powder (pH=5.2) that is inoculated in different salinity and different ZT concentration placed apart down, every bottle each about 10, each processed group repeats 12 bottles.Carry out the dark cultivation of 20d earlier, dark temperature of cultivating is 25 ± 2 ℃, carry out illumination cultivation subsequently, the illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark, the seedling number of statistics blade bud ratio and the corresponding formation of each blade behind the 30d, thus obtain best leaf regeneration system;
E, rooting of vitro seedling are cultivated: the aseptic seedling plant of successive transfer culture is downcut apart from about the 2.5～3.0cm of top, be inoculated at random in the blake bottle of the improvement WPS medium+20g/L sucrose+9g/L agar powder (pH=5.2) that contains different salinity, different I BA concentration and the combination of different activities concentration of carbon and carry out illumination cultivation, the illumination cultivation condition is: 25 ± 2 ℃ of temperature, light intensity 2500Lx, photoperiod 16h illumination/8h dark, the record start rootage duration, the upgrowth situation of statistics rooting rate and take root number and observation seedling behind the 60d, thus filter out optimum root media;
F, hardening: in advance 3d opens bottle cap with rooting tube plantlet in the bottle and carries out transition, clean the root medium, transplant at random, in the booth of growing seedlings that keeps humidity 100%, cultivate 20d in different hardening matrix, transition 3d under the outdoor conditions of humidity 80%, light transmittance 70% then, the canopy film is thrown off in progressively ventilation more fully behind the 7d, statistics survival rate after 2 weeks, the amount of planting of every kind of matrix is no less than 50 strains, the number of repetition of each test is no less than 3 times, and the statistics survival rate is to finish hardening.
2. suitable south according to claim 1 high clump blueberry high efficiency quick breeding technology is characterized in that the initial culture base is produced agar powder+1.0mg/L ZT daily, its pH=5.2 for improvement WPM medium+20g/L sucrose+8.8g/L among the step b.
3. suitable south according to claim 1 high clump blueberry high efficiency quick breeding technology, it is characterized in that, among the step c, after treating the stem segment with axillary buds elongation of stem-segment with single bud, stem segment with axillary buds downcut to transfer in the proliferated culture medium (pH=5.2) of the improvement WPM medium that contains variable concentrations ZT, 6-BA or TDZ+20g/L sucrose+8.8g/L daily output agar powder, carry out illumination cultivation.
4. suitable south according to claim 3 high clump blueberry high efficiency quick breeding technology is characterized in that among the step c, the incidence of indefinite bud and the ZT concentration in the medium have confidential relation; When the ZT mass concentration is 0.5mg/L, to cultivate about 10d and produce yellowish green callus at basal part of stem, the bud of growing thickly appears in the joint place, and the rate of increase reaches 5.02 times behind the 28d, and is mainly the major branch bud, and the bud of growing thickly is sturdy, and the growth impetus is good, is fresh and tender shape; When the ZT mass concentration is elevated to 1mg/L, begin to grow and be more or less the same when time of breaking up and 0.5mg/L, its rate of increase increases slightly to some extent; When the ZT mass concentration equaled and is higher than 2mg/L, regrowth began growth and differentiating phenomenon shifts to an earlier date, and occurred 2 branches on the young sprout branch that majority breaks up out, and the rate of increase reaches about 10 times, because the side shoot bud is prone to vitrifying seedling phenomenon a little less than making the seedling growing way more; When using 6-BA, explant is sprout growth not, growth retardation.When using TDZ, form a small amount of callus at the explant base portion behind the 10d, though the energy rudiment, the low and poor growth of germination rate; Take all factors into consideration from aspects such as the rate of increase, the bud growing way of growing thickly, zeatin costs, subculture medium is the best with WPM+0.5mg/L ZT.
5. suitable south according to claim 1 high clump blueberry high efficiency quick breeding technology is characterized in that,
In steps d, the seedling number of statistics blade bud ratio and the corresponding formation of each blade in the blake bottle of different salinity, high salt concentration is unfavorable for the regeneration of blade, its regeneration induction seedling rate is 0-2.7%; Macroelement concentration is reduced half, can obviously promote the regeneration of blade, blade regeneration induction seedling rate is increased to 23.1-70.9%; In the blade different parts, the regrowth inductivity of blade tip end is minimum, and the petiole end is placed in the middle, and the inductivity of full leaf is the highest.
6. suitable south according to claim 1 high clump blueberry high efficiency quick breeding technology is characterized in that, in steps d, and the seedling number of statistics blade bud ratio and the corresponding formation of each blade in the blake bottle of variable concentrations ZT or TDZ; TDZ can not form regrowth by inducer blade, and blade forms callus in incision after cultivating 10d, and along with the prolongation of incubation time, callus is brownization slowly, does not see that regrowth forms; ZT induces effect obviously to be better than TDZ to the leaf regeneration seedling, the incidence of indefinite bud and the ZT in the medium have confidential relation, ZT induces the leaf regeneration seedling and has the significant concentration effect, blade tip end inductivity when 0.5mg/LZT is 0%, along with the rising inductivity of ZT concentration increases gradually, the inductivity when 5mg/L ZT reaches 32.3%; The inductivity of petiole end and full leaf all increases gradually with the rising of ZT mass concentration, and inductivity all reaches the highest when 2.0mg/L ZT, full the regrowth inductivity of petiole end and leaf is respectively 54.3% and 71.4%, the inductivity during 5mg/L ZT but all descends; There is notable difference in the regrowth inductivity of different parts blade, and the inductivity of blade tip end is minimum, petiole end placed in the middle, and the inductivity of full leaf is the highest; Low concentration ZT mainly induces and forms Dan Shengmiao, and high concentration ZT help the clustering formation of seedling; The seedling of living again that blade tip end blade is induced mainly is formed at incision, and the regrowth that petiole end and full leaf are induced mainly is formed at petiole one side; Select for use full leaf to carry out the leaf regeneration seedling and induce, the best ZT mass concentration that its regrowth is induced is 2.0mg/L.
7. suitable south according to claim 1 high clump blueberry high efficiency quick breeding technology, it is characterized in that in step e, there is material impact in different salinity to the root induction of single bud seedling, high salt concentration is unfavorable for the bud seedling rooting, and beginning rootage duration length and rooting rate only are 30%; When macroelement reduces by half, begin to take root behind about 14d and rooting rate be 75%; When macroelement reduces to original 1/4 the time, the beginning rootage duration be about 7 days in advance and rooting rate up to 100%, the seedling growing way is strong; Macroelement reduces at original 1/8 o'clock, though beginning rootage duration and rooting rate and macroelement reduce to original 1/4 similar, yet because the shortage of nutriment, a little less than the seedling growing way, suitably reduce the growth that salinity helps taking root and do not influence seedling, the salinity of the root media of taking root in Ao Ni'er's test-tube plantlet bottle selects for use macroelement to reduce to original 1/4.
8. suitable south according to claim 1 high clump blueberry high efficiency quick breeding technology, it is characterized in that, in step e, active carbon has material impact to taking root in the blake bottle, and when in the medium during non-activity charcoal, all long earlier callus of basal part of stem that inserts in the medium is taken root then thereon, because vascular bundle is obstructed between the rhizome, soon stem promptly stops growing, and the leaf look rubescent to come off, and the seedling growing way is very poor; After adding active carbon in the medium, basal part of stem seldom or not long callus and directly taking root from stem, thereby help the growth of seedling; There is concentration effect in active carbon to taking root, along with the rising of concentration of activated carbon, rooting rate is higher, take root number the more and root longer, the seedling growing way is better; When active carbon was 0.05%, rooting rate was 58%-75%, every strain several 1-3 bars of taking root, and the long 1-3cm of root, the seedling growth is slower; When active carbon was increased to 0.2%, rooting rate improved about 20%, and every strain is taken root for counting and reached more than 4, the long 4-6cm of root, and the seedling growth is fast; IBA is taken root to the Ao Ni'er and is also had concentration effect, and high concentration IBA more helps taking root, and there are synergistic effect between the two in active carbon and IBA, and when both concentration were all high, rooting rate and seedling growing way were all better.Effective rooting rate at 0.2% active carbon, 0.6mg/L and 0.8mg/L IBA all reaches 100%, but initial time of taking root of the latter and root are long all Zao and long than the former, the latter about 7 days visible basal part of stem have root to form, grow 4-6 bar root about 45 days, root reaches about 6cm, more than the height of seedling 9cm, leaf look dark green blade is big, the seedling robust growth, in Ao Ni'er's bottle in the root media best concentration of activated carbon and IBA concentration be respectively 0.2% and 0.8mg/L, effectively rooting rate is up to 100%.
9. suitable south according to claim 1 high clump blueberry high efficiency quick breeding technology is characterized in that among the step f: hardening matrix is selected the peat composed of rotten mosses: river sand=1: 1; The perhaps peat composed of rotten mosses: garden mould=1: 1; The perhaps peat composed of rotten mosses: quartz sand=2～3: 1; The perhaps peat composed of rotten mosses: perlite=1～2: 1; Perhaps pure water liver moss.
10. suitable south according to claim 8 high clump blueberry high efficiency quick breeding technology, it is characterized in that, among the step f: rooting tube plantlet is grown in five kinds of different hardening matrix, wherein the effect of pure water liver moss is best, survival rate is up to 100%, the amount of growth of nursery stock reaches more than the 3cm, newly-increased blade 6-8 piece; Effect next be 2～3: the combination of 1 the peat composed of rotten mosses and quartz sand, its survival rate is 84%, the amount of growth of nursery stock is higher; The survival rate of 1: 1 the peat composed of rotten mosses and river sand matrix only is 50%, but it is very high to survive the quality of seedling, shows as growth pier reality, robust plant, amount of growth and " peat composed of rotten mosses+quartz sand " quite; The effect of the peat composed of rotten mosses+garden mould matrix and the peat composed of rotten mosses+pearlite interstitial substance is placed in the middle.
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| CN105028193A (en) * | 2015-06-19 | 2015-11-11 | 山东省林业科学研究院 | Breeding method for generating micro adventitious buds through induction of legacy leaves |
| CN106106182A (en) * | 2016-08-05 | 2016-11-16 | 四川省农业科学院经济作物育种栽培研究所 | A kind of root media cultivated for blueberry tissue culture Seedling and cultural method thereof |
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| CN109997698A (en) * | 2019-05-15 | 2019-07-12 | 西南林业大学 | It is a kind of using stem section as the camphor tree leaf blueberry method for tissue culture of explant |
| CN111657144A (en) * | 2020-06-24 | 2020-09-15 | 广西壮族自治区中国科学院广西植物研究所 | Tissue culture rapid propagation method of Nangao blueberry variety' Aunier |
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