CN1887065A - Tissue culture propagation process of late spring michelia - Google Patents
Tissue culture propagation process of late spring michelia Download PDFInfo
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Abstract
The present invention is fast propagation tissue culture method of interspecific crossed F1 late spring michelia with M. sphaerantha as female parent and M. yunnanensis as male parent. The method includes taking the stem section of annual or biannual young late spring michelia tree as explant to establish sterile line with high growth coefficient, and two-step process for rooting to obtain high rooting effect and high transplanting survival rate. The late spring michelia is one ideal garden ornamenting tree.
Description
Technical field: the invention belongs to biological technical field, particularly, relate to a kind of tissue culture propagation of Magnoliacea plant process of late spring michelia.
Background technology: Magnoliaceae (Magonliaceae) is ancient angiosperm monoid, and this population plant reduces in imminent danger day by day.Wherein Michelia (Michelia) plant to have many are important ornamental plants in garden, and have higher economic worth, from this genus, isolate multiple sesquiterpene lactone, some has active anticancer; Process of late spring michelia is to be female parent with M. sphaerantha (M.sphaerantha), and Yunnan (M.yunnanensis) with a smile be male parent, belongs to interior interspecific cross F
1The kind that screening cultivates.Tree-like grace, evergreen, white flowers is savory, shrub, is desirable flower garden ornamental tree species; Carry out the research of tissue culture vegetative propagation technique to keeping its heterosis, hybrid vigor, new varieties are promoted has application value realistic.At present, Magnoliacea plant is generally bred by sowing and grafting, and the cottage propagation rooting rate is low big to the elite stand injury, and the tissue culture technique difficulty is bigger, and the report of the method for process of late spring michelia tissue-culturing quick-propagation is not arranged in the prior art.
Summary of the invention: the method that the object of the present invention is to provide a kind of process of late spring michelia tissue-culturing quick-propagation, this method is got the living process of late spring michelia earsh of 1-2 stem section and is made explant, set up aseptic strain with the earsh explant, the growth coefficient height, adopt two step method to take root and make the seedling stalwartness, reproductive process is unimpeded, and rooting efficiency is good, and transplanting survival rate can reach 90%.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
The tissue culture propagation of process of late spring michelia, comprise that mainly inducing differentiation culture, subculture to increase plants cultivation, culture of rootage, transplant step, with MS+BA1mg/l+NAA0.2mg/l is inductive differentiation medium, MS+BA0.8mg/l+NAA0.2mg/l is the shoot proliferation medium, and MS+NAA2mg/l is that root media carries out two secondary roots.
In the said method, inducing the living earsh stem of the preferred process of late spring michelia 1-2 of differentiation culture section is explant, cleans, and spills smart the immersion, changes 0.1% HgCl over to
2Sterilization is 8-10 minute in the solution, use aseptic water washing 4-5 time again, be cut to the segment of 0.5cm band axillalry bud, be inoculated in and add the curing of 0.8% agar, also sterilization 18 minutes in 120 ℃ of conditions of sucrose 3%, PH cultivates 24 ± 2 ℃ of cultivation temperature in 5.6 the MS+BA1mg/l+NAA0.2mg/l medium, intensity of illumination 2000lx, light application time 12h.d
-1
Two secondary roots are that the seedling that will take root takes out, and the root brachymemma is kept sub-fraction concentrate access medium 3-4 week, when waiting to grow 5-7 bar lateral root, the seedling that does not induce root are cut away the bottom callus, insert root media again and carry out the secondary culture of rootage.
Transplant laterite and 2: 1 mixed-matrix of detritus soil that used matrix is preferably sieved, cultivation preferred glass steel greenhouse, place.Transplant the further preferred strawberry soil of used matrix and add 1: 1 mixed matrix of laterite, the white bacterium cleaning/disinfecting of back 500 times of liquid that sieves, the initial stage is put into the greenhouse, keeps the humidity more than 80%, and 25 ℃ of day temperatures are not less than 5 ℃ night.
More specifically, the living earsh stem of the preferred process of late spring michelia 1-2 of technical scheme of the present invention section is an explant, cleans, and spills smart the immersion, changes 0.1% HgCl over to
2Sterilization is 8-10 minute in the solution, use aseptic water washing 4-5 time again, be cut to the segment of 0.5cm band axillalry bud, be inoculated in and add the curing of 0.8% agar, also sterilization 18 minutes in 120 ℃ of conditions of sucrose 3%, PH cultivates 24 ± 2 ℃ of cultivation temperature in 5.6 the MS+BA1mg/l+NAA0.2mg/l medium, intensity of illumination 2000lx, light application time 12h.d
-1Cultivate 4-5 after week, change subculture enrichment culture in the MS+BA0.8mg/l+NAA0.2mg/l medium over to, 8-10 week, wait to differentiate clump bud after, stay 2-3 sheet leaf to downcut the bud more than the 1cm, insert among the root media MS+NAA2mg/l 50-60 days, the seedling of having taken root is taken out, the root brachymemma is kept sub-fraction concentrate access medium 3-4 week, when waiting to grow 5-7 bar lateral root, the seedling that does not induce root is cut away the bottom callus, insert root media again and carry out the secondary culture of rootage; The transplantation of seedlings that will have 3 roots is then gone into strawberry soil and is added 1: 1 mixed transplanting medium of laterite, the white bacterium cleaning/disinfecting of back 500 times of liquid that sieves, and the transplanting initial stage is put into the greenhouse, keeps the humidity more than 80%, and 25 ℃ of day temperatures are not less than 5 ℃ night.
Embodiment: followingly further specify the present invention, but content of the present invention is not exceeded with embodiment with embodiments of the invention.
Embodiment 1:
Getting with M. sphaerantha (M.sphaerantha) is female parent, and Yunnan (M.yunnanensis) with a smile is male parent, interspecific cross F in belonging to
1The living earsh stem of the process of late spring michelia 1-2 section that screening cultivates is an explant, cleans, and spills smart the immersion, changes 0.1% HgCl over to
2Sterilization is 8-10 minute in the solution, use aseptic water washing 4-5 time again, be cut to the segment of 0.5cm band axillalry bud, be inoculated in and add the curing of 0.8% agar, also sterilization 18 minutes in 120 ℃ of conditions of sucrose 3%, PH cultivates 24 ± 2 ℃ of cultivation temperature in 5.6 the MS+BA1mg/l+NAA0.2mg/l medium, intensity of illumination 2000lx, light application time 12h.d
-1Cultivate 4-5 after week, change subculture enrichment culture in the MS+BA0.8mg/l+NAA0.2mg/l medium over to, 8-10 week, wait to differentiate clump bud after, stay 2-3 sheet leaf to downcut the bud more than the 1cm, insert among the root media MS+NAA2mg/l 50-60 days, the seedling of having taken root is taken out, the root brachymemma is kept sub-fraction concentrate access medium 3-4 week, when waiting to grow 5-7 bar lateral root, the seedling that does not induce root is cut away the bottom callus, insert root media again and carry out the secondary culture of rootage; The transplantation of seedlings that will have 3 roots is then gone into strawberry soil and is added 1: 1 mixed transplanting medium of laterite, the white bacterium cleaning/disinfecting of back 500 times of liquid that sieves, and the transplanting initial stage is put into the greenhouse, keeps the humidity more than 80%, and 25 ℃ of day temperatures are not less than 5 ℃ night.
Further elaborate the present invention with detailed process of the test of the present invention and result below.
One. material and method
Getting with M. sphaerantha (M.sphaerantha) is female parent, and Yunnan (M.yunnanensis) with a smile is male parent, interspecific cross F in belonging to
1The current-year branch of the process of late spring michelia bearing tree that can blossom and have seeds that screening cultivates, blade; Get process of late spring michelia 1-2 that cuttage test survives and give birth to and plant viviparous branch, blade, make the explant induction object, the time of drawing materials is divided different times, bearing tree draw materials above two kinds of branch base portion and middle part.
After material is fetched Lao Ye cut off and expose axillalry bud, remove lignification part stem section, stay tender stem, spire, wash a period of time with running water, after writing brush cleaned down gently, 75% alcohol-pickled 30s changed 0.1% HgCl over to
2Solution disinfection, blade sterilization 5-6min, stem section sterilization 8-10min uses aseptic water washing 4-5 time standby then.Medium (1) MS+KT2mg/l (unit down together)+NAA0.2, (2) MS+KT1+NAA0.2, (3) MS+BA1+NAA0.2, (4) MS+BA0.8+NAA0.2, (5) MS+2,4-D1, (6) MS+BA1+NAA0.2+AC0.3g/l, (7) MS+BA0.8+NAA0.2+AC0.3g/l, above medium all adds 0.8% agar and solidifies, sucrose 3%, PH5.6, the 50ml triangular flask is made the primary vaccination culture vessel, 120 ℃ of pressure cooker steam sterilizings, 18min, 24 ± 2 ℃ of culturing room's temperature, intensity of illumination 2000lx, light application time 12h.d
-1Culture of rootage (8) MS+IBA3, (9) MS+NAA3 is cut to the segment that 0.5cm is with axillalry bud with the stem section that disinfects, and blade cuts is 0.5cm
2Sheet, be inoculated into respectively in the medium of above (1)-(7), put into culturing room and cultivate.Transplanting medium is laterite and the detritus soil that sieves, and mixture ratio is 2: 1, and the cultivation place is the fiberglass greenhouse.
Two. result and analysis
1. leaf is seeded in (1) (2) (3) (4) (5) medium culture 2-3 week, around material cut, on the vein of leaf back, grow many callus, they are the green particles shape, the powdery that is white in color that has, wherein (3) (4) (5) are faster than (1) (2) growth, and 4-5 changes them in identical fresh culture cultivation observation after week, and callus can continue to increase, but do not see differentiation and seedling emergence, if rolling bottle does not continue to cultivate 6-8 week at former medium, brownization of medium is serious, also brownization is dead gradually for material, bearing tree, the treelet blade is cultivated performance basically identical indifference.
2. treelet stem section is seeded in the medium of (1) (2) (3) (4), four kinds of hormone combinations can both make the stem segment with axillary buds differentiation, but induce the appearance difference that sprouts bigger, (1) (2) careful length of bud-leaf of inducing, hard, the clump bud is sprouted and is sent out less or not, (3) (4) form of inducing is more sturdy normal, and clump bud number is more.Think (3) MS+BA1+NAA0.2, the most suitable explant induction medium of doing through the test of many times summary.Shoot proliferation is cultivated and is adopted (4) MS+BA0.8+NAA0.2 effect better.In the incubation, medium has browning, but compare with the contrast of bearing tree, brownization do not influence Growth and Differentiation substantially, under the existing condition of culture of setting, can enlarge 4-6 doubly in 75 days, come successive transfer culture with (6) (7) medium that adds activated carbon, can reduce brownization, but can suppress the Growth and Differentiation of clump bud simultaneously yet, the consumption of active carbon is high but suppress phenomenon clearly.
3. bearing tree different parts stem section cultivation results differs greatly, this experiment will divide base portion into below the 50cm, divide top more than the 50cm into, brief summary shows through the different times test of many times, it is extremely low that the top stem explants is received on the medium survival rate, almost whole brownization are dead, base portion stem section cultivation situation is better than top, 8-10% inoculation survival is arranged, this phenomenon and Magnoliaceae branch cutting are taken root for testing and are reported that similarity is arranged, brownization was serious during the material of taking over a job was cultivated in the later stage, through repeatedly changeing still very poor for cultivation effect.
4. take from the stem explants of different times, different plants, induce and break up show widely different.The treelet stem section contrast experiment of bearing tree and cutting survival shows: the treelet material survives than the easy inoculation of bearing tree material, and browning is lighter, induces weak point between differentiation phase, and on the contrary, brownization of bearing tree explant is serious, and induce and grow (seeing Table 1) between differentiation phase,
Table 1 different times different plants stem explants inoculation growing state
| Inoculation time | Explant | Inoculation number (bottle) | Induce fate | Pollute number | Brownization number | Survival rate |
| March | Treelet | 30 | 45 | 12 | 1 | 57％ |
| Grow up and count | 37 | 60 | 16 | 17 | 10％ | |
| August | Treelet | 30 | 45 | 13 | 2 | 50％ |
| Bearing tree | 32 | 60 | 18 | 12 | 6％ | |
| December | Treelet | 44 | 50 | 12 | 1 | 70％ |
| Bearing tree | 30 | 58 | 11 | 12 | 23％ |
The numeral of statistics can be found out from table, and the differentiation of treelet explant induction is easier to, and the dead reason of inoculation mainly is that brownization of material death toll is few, inoculates minimum survival rate more than 50%, is subjected to seasonal effect little owing to pollute; On the contrary, brownization of bearing tree material is serious, and inoculation survival rate is low, and induction time is long, is subjected to seasonal effect big, and induction time is long, and it is good slightly to inoculate situation December, and August, performance was the poorest, and the aseptic strain of inoculation survival is in the successive transfer culture in later stage, and brownization is serious, difficult differentiation.And the treelet material induces clump bud growing way is significantly better than bearing tree, and the later stage enrichment culture also obviously has
5. the two step method rooting method stays 2-3 sheet leaf to downcut the bud more than the 1cm, inserts root media (8) MS+IBA3, (9) two kinds of cultivations of MS+NAA3, begin long root about 30 days, the statistics of taking root after 60 days, rooting rate is at 82%-88%, two kinds of medium situation difference of taking root, (8) root that induces has the 1-2 bar approximately, and thin color is darker, and the root that (9) induce has the 2-3 bar approximately, thick look white, by contrast (9) seedling stalwartness of inducing than (8); Below experiment all uses (9) to make root media, take root for the first time and finish, the growing way of seedling and the length of root are uneven, be not easy to plantation, therefore adopt two secondary roots, with unified taking-up of seedling of having taken root, the root brachymemma is kept sub-fraction concentrate the access medium, can inspire 5-7 bar lateral root 3-4 week, the seedling that does not induce root is cut away the bottom callus, concentrate and insert medium culture of rootage once more; Can improve rooting rate greatly like this, can form healthy and strong neat test-tube plantlet, be convenient to unified transplanting and management, unimpeded with the take root whole group of training method reproductive process that can make process of late spring michelia of two step method.
6. transplant, generally get the field run plant that has more than 3 roots and transplant, transplanting medium adopts strawberry soil to add 1: 1 mixed matrix of laterite, and the white bacterium cleaning/disinfecting of back 500 times of liquid that sieves can be used; The transplanting initial stage is put into the greenhouse, and humidity, the day temperature protected more than 80% are not less than 5 ℃ 25 ℃ of nights, and transplanting survival rate can reach more than 90%.
Describe the crossover process of process of late spring michelia below again in detail:
Process of late spring michelia: M. sphaerantha (Michelia sphaerantha) (♀) X Yunnan is had a smile on one's face (M.yunnanensis) (♂), when maternal flower does not also open, pull open tepal gently, remove stamen, pollen with male parent is pollinated to it, then tepal is restored and the parcel gynoecium, and bagging, remove bag after about 1 week.Hybridization time is 8:00-9:00 in the morning.Not Cheng Gong shedding in about month.Seed is collected in the collecting fruit and the back of drying in the shade behind the seed maturity, flush away oil exosper, the husky Tibetan of at room temperature preserving moisture.1 year early spring, sowing divided transplantation of seedlings in good time.Observe the adaptability and the form shape of seedling.
Maternal: M. sphaerantha (Michelia sphaerantha): evergreen megaphanerophyte, sprout is by brown fine hair.The leaf keratin, the shape of falling ovum Long Circle, the rapid tip of tip tool does not have hair above the leaf, and below by pubescence, middle arteries and veins is recessed on the blade face, the densification of lateral vein network; Petiole does not have the stipule trace.Floral white, sagging, do not open, tepal 12, long 6-7.5cm, wide 1-2.5cm, narrow obovate, stamen is about 2cm, and gynoecium is by pubescence, the florescence 4-5 month.
Male parent: Yunnan is (M.yunnanensis) with a smile: evergreen shrubs, be thick with leaves, 4 meters buds of Gao Keda, spray, above the tender leaf and petiole, bennet close by the calm hair of peony.Leaf hard leather matter, obovate, narrow obovate or the narrow shape of falling ovum ellipse, long 4-10cm, wide 1.5-3.5cm, the blunt or short anxious point of tip circle, the base portion wedge shape, above bottle green, below residual calm hair; The stipule trace be petiole long 2/3 or reach canescence below the acrocont.Bennet is sturdy, floral white, plane or cup-shaped of flare up, fragrance, tepal 6-12 (17), obovate or the shape of falling ovum ellipse, long 3-3.5cm, interior wheel is narrower and small, the long 0.5-1cm of stamen, androecium and gynoecium handle are all by the calm fine, soft fur of bronzing, the florescence 3-4 month.
Process of late spring michelia: evergreen shrubs begins branch to dungarunga from basal part of stem, and branch is many and close, and be turriform: bud, petiole, spire two sides are by brown down.The leaf keratin, long avette or Long Circle, long 6.5-16cm, wide 2.5-5cm, the rapid tip of tip tool or round blunt slightly, the base portion wedge shape, the blade face bottle green, the blade face does not have hair, the residual brown down in the back side, middle arteries and veins is recessed on the blade face, and the every limit 9-11 of lateral vein, petiole pop-up leaf scar are 1/3 of petiole length.The flower milky, tepal (7) 11-12, narrow obovate or cochlear, long 3.5-6cm, wide 1-2cm is 3 and takes turns arrangement, opens to be bell.The florescence 4-5 month.Process of late spring michelia was bred first in 1998, had after this repeatedly cultivated this crossbreed, and the spring in 2003 bloomed first.
Process of late spring michelia proposes the application of plant variety power on June 21st, 2005, accepts.Accept department: new variety of plant protection office of the State Administration of Forestry, application number: 20050039, the applying date: on June 21st, 2005.
Three. conclusion
The suitable living earsh stem of the 1-2 section of selecting of process of late spring michelia tissue-culturing quick-propagation explant.Inductive differentiation medium: MS+BA1+NAA0.2, shoot proliferation medium: MS+BA0.8+NAA0.2, root media is: MS+NAA3.The employing two step method is taken root, and can make whole reproductive process unimpeded.
Hybridization F
1The tissue culture propagating of new varieties " process of late spring michelia " studies show that: blade is easier to induce callus, sprouts but callus is difficult to differentiation, and the stem section is cultivated can induce clump bud.Bearing tree stem section induction time is long, brownization is serious, later stage shoot proliferation performance is poor, uncomfortable cooperation explant, get the living earsh stem of 1-2 section and be easier to induce differentiation, with the aseptic strain growth coefficient height that the earsh explant is set up, the suitable explant source of making tissue-culturing quick-propagation.MS+BA1mg/l+NAA0.2mg/l; MS+BA0.8mg/l+NAA0.2mg/l; more than two kinds of medium better to inducing with the enrichment culture effect; stay 2-3 sheet leaf to cut the bud more than the 1cm; it is better than rooting efficiency on the MS+IBA2mg/l root media to be placed on MS+NAA2mg/l, adopts two step method to take root and can make the seedling stalwartness, and reproductive process is unimpeded; suit to have under the protection facility condition in condition, transplanting survival rate reaches more than 90%.
Claims (6)
1, the tissue culture propagation of process of late spring michelia, comprise that mainly inducing differentiation culture, subculture to increase plants cultivation, culture of rootage, transplant step, it is characterized in that with MS+BA1mg/l+NAA0.2mg/l being inductive differentiation medium, MS+BA0.8mg/l+NAA0.2mg/l is the shoot proliferation medium, and MS+NAA2mg/l is that root media carries out two secondary roots.
2, method according to claim 1, it is characterized in that inducing the living earsh stem of the preferred process of late spring michelia 1-2 of differentiation culture section is explant, cleans, and spills smart the immersion, changes 0.1% HgCl over to
2Sterilization is 8-10 minute in the solution, use aseptic water washing 4-5 time again, be cut to the segment of 0.5cm band axillalry bud, be inoculated in and add the curing of 0.8% agar, also sterilization 18 minutes in 120 ℃ of conditions of sucrose 3%, PH cultivates 24 ± 2 ℃ of cultivation temperature in 5.6 the MS+BA1mg/l+NAA0.2mg/l medium, intensity of illumination 2000lx, light application time 12h.d
-1
3, method according to claim 1, it is characterized in that said two secondary roots are that the seedling of will take root takes out, the root brachymemma is kept sub-fraction concentrate access medium 3-4 week, when waiting to grow 5-7 bar lateral root, the seedling that does not induce root is cut away the bottom callus, insert root media again and carry out the secondary culture of rootage.
4, method according to claim 1 is characterized in that transplanting laterite and 2: 1 the mixed-matrix of detritus soil that used matrix is preferably sieved, cultivation preferred glass steel greenhouse, place.
5, method according to claim 1 is characterized in that transplanting the preferred strawberry soil of used matrix and adds 1: 1 mixed matrix of laterite, and the white bacterium cleaning/disinfecting of back 500 times of liquid sieves, initial stage is put into the greenhouse, keep the humidity more than 80%, 25 ℃ of day temperatures are not less than 5 ℃ night.
6, according to claim 1 or 2 or 3 or 4 or 5 described methods, it is characterized in that the living earsh stem of preferred process of late spring michelia 1-2 section is an explant, clean, spill smart the immersion, change 0.1% HgCl over to
2Sterilization is 8-10 minute in the solution, use aseptic water washing 4-5 time again, be cut to the segment of 0.5cm band axillalry bud, be inoculated in and add the curing of 0.8% agar, also sterilization 18 minutes in 120 ℃ of conditions of sucrose 3%, PH cultivates 24 ± 2 ℃ of cultivation temperature in 5.6 the MS+BA1mg/l+NAA0.2mg/l medium, intensity of illumination 2000lx, light application time 12h.d
-1Cultivate 4-5 after week, change subculture enrichment culture in the MS+BA0.8mg/l+NAA0.2mg/l medium over to, 8-10 week, wait to differentiate clump bud after, stay 2-3 sheet leaf to downcut the bud more than the 1cm, insert among the root media MS+NAA2mg/l 50-60 days, the seedling of having taken root is taken out, the root brachymemma is kept sub-fraction concentrate access medium 3-4 week, when waiting to grow 5-7 bar lateral root, the seedling that does not induce root is cut away the bottom callus, insert root media again and carry out the secondary culture of rootage; The transplantation of seedlings that will have 3 roots is then gone into strawberry soil and is added 1: 1 mixed transplanting medium of laterite, the white bacterium cleaning/disinfecting of back 500 times of liquid that sieves, and the transplanting initial stage is put into the greenhouse, keeps the humidity more than 80%, and 25 ℃ of day temperatures are not less than 5 ℃ night.
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| CN114600772B (en) * | 2022-03-23 | 2023-02-14 | 中国科学院昆明植物研究所 | Tissue culture method and rapid propagation method of michelia figo in remote mountains |
| CN117598204A (en) * | 2024-01-24 | 2024-02-27 | 西南林业大学 | Tissue culture and rapid propagation method and application of michelia yunnanensis |
| CN117598204B (en) * | 2024-01-24 | 2024-03-22 | 西南林业大学 | Tissue culture and rapid propagation method and application of michelia yunnanensis |
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