CN110199880A - A kind of cultural method covering mulberry polyploid seedling - Google Patents
A kind of cultural method covering mulberry polyploid seedling Download PDFInfo
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Abstract
The present invention provides a kind of cultural methods for covering mulberry polyploid seedling, this method includes carrying out preculture after choosing stem section to illiteracy mulberry tissue-cultured seedling, then polyploid is obtained using colchicine-induced to the stem section for starting to generate callus, obtains covering the polyploid seedling of mulberry by Multiplying culture and culture of rootage.This method can overcome the problems, such as that illiteracy mulberry tissue cultures browning is serious, multiploid induction rate is low, chimera is more after induction.
Description
Technical field
The present invention relates to forest raising technology fields, and in particular to a kind of cultural method for covering mulberry polyploid seedling.
Background technique
It covers mulberry (Morus mongolica), alias rock mulberry is ideal Eco-economic Type tree species, to the water of northern area
Soil is kept and restoration of the ecosystem can play very positive effect.It is mainly distributed on Daqunshan Mountains mountainous region, In The South of Yinshan Mountains loess hill
Area, Daxing'an Mountainrange south mountainous region and Kezuohou Banner Daqinggou Nature Reserve.
Currently, domestic less to the research for covering mulberry.Liu Ling etc. (2016) studies its geographical distribution and source, recognizes
Exist to cover mulberry before the Quaternary ice age, form specific feature during adapting to environment, has to cold stronger
Adaptability.Tissue cultures and polyploid germ plasm resource innovative research for mulberry are relatively more, but there are nursery stock tissue cultures are easy
Browning is generated, growth coefficient is lower, phenomena such as growing compared with slow, multiploid induction rate is relatively low, be also easy to produce chimera.It is high
Beautiful rosy clouds etc. (2013) lure the seed and seedling of 109 material of osmanthus Sang You 62, Yuesang 11 and husky 2 × human relations using colchicine
It leads, successfully obtains tetraploid, inductivity is up to 33.3%.Li Xiaoshuan etc. (2017) induces long fringe mulberry with colchicine,
Finally only obtain 3 plants of polyploid materials.Wang Qianling (2009) induces the adventitious bud of mulberry tree using different methods for inducing, only
It is applicable in drip method and obtains two parts of tetraploid material, 2 parts of chimera materials are only obtained using infusion process.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The present invention is induced based on Vitro Plant method for tissue culture through callus from stem segment, is soaked using colchicine
The processing of stain method covers mulberry polyploid material to obtain.This method successfully solves serious browning in tissue culture procedures, mentions
The high inductivity of polyploid, reduces the formation of chimera, saves experimentation cost.
To achieve the above object, technical scheme is as follows:
The present invention relates to a kind of cultural methods for covering mulberry polyploid seedling, comprising the following steps:
(1) stem section is intercepted on Cong Mengsang tissue-cultured seedling, is inoculated in precultivation medium and carries out preculture, obtain preculture stem
Section；
(2) the preculture stem section is inoculated in the induced medium containing colchicine and carries out Fiber differentiation, obtained
Stem section after induction；
(3) stem section after the induction is inoculated in proliferated culture medium and carries out Multiplying culture, obtain Multiple Buds；
(4) Multiple Buds are inoculated in root media and carry out culture of rootage, obtain rooted seedling.
Preferably, the step of stem section is intercepted in step (1), on Cong Mengsang tissue-cultured seedling includes: the illiteracy mulberry for choosing robust growth
Tissue-cultured seedling obtains stem section after being cut off blade and petiole, stem section is truncated into the segment of 1~1.5cm long, there are 1 for every segment
Axillary bud, from segment one end, progress is longitudinal sectional, obtains the notch that length is 1mm.
Preferably, in step (1), by from covering on mulberry tissue-cultured seedling in the laterally inserted culture medium of stem section that intercepts, only upper surface
Expose culture medium, then carries out preculture.
Preferably, in step (1), the precultivation medium is basic culture medium with MS, and add KT, 6-BA, NAA,
PVP and active carbon obtain, and the concentration of KT is 2.0mg/L in the precultivation medium, and the concentration of 6-BA is 0.5mg/L, NAA's
Concentration is 0.5mg/L, and the concentration of PVP is 0.5g/L, and the concentration of active carbon is 1.0g/L.
Preferably, in step (1), the preculture condition are as follows: intercept stem section on Cong Mengsang tissue-cultured seedling and be inoculated in preculture
It is 25 ± 2 DEG C in temperature in culture medium, daily light application time is 8h, and intensity of illumination 2500xl, interlunation is the item of 16h
72h or more is cultivated under part.
Preferably, in step (2), the induced medium not add the MS of agar as basic culture medium, and add KT,
6-BA, NAA, PVP, colchicine and active carbon obtain, and the concentration of KT is 2.0mg/L in the induced medium, and 6-BA's is dense
Degree is 0.5mg/L, and the concentration of NAA is 0.5mg/L, and the concentration of PVP is 0.5g/L, and the concentration of colchicine is 30mg/L, activity
The concentration of charcoal is 1.0g/L.
Preferably, in step (2), the inducing culturing condition are as follows: the preculture stem section is inoculated in induced medium
In, temperature be 20 ± 2 DEG C, daily light application time be 8h, intensity of illumination 2500xl, interlunation be 16h under conditions of train
Support 72h or more.
Preferably, after step (2), the stem section after the induction is taken out, with aseptic water washing 3~4 times, use is sterile
After filter paper blots surface moisture, it is transferred to proliferated culture medium and is cultivated.
Preferably, in step (3), the proliferated culture medium is identical as the composition of precultivation medium.
Preferably, in step (3), the Multiplying culture condition are as follows: the stem section after the induction is inoculated in Multiplying culture
It is 25 ± 2 DEG C in temperature in base, daily light application time is 8h, intensity of illumination 2500xl, under conditions of interlunation is 16h
Culture 20~30 days, until induction seedling grows to 30cm or more.
Preferably, after step (3), Chromosome Analysis is carried out to the blade of the Multiple Buds, analysis instrument is
Flow cytometer determines its chromosome number, filters out after tetraploid continues 20~30d of culture, carries out culture of rootage.
Preferably, in step (4), the root media is basic culture medium with MS, and adds IBA and obtain, the life
The concentration of IBA is 0.3mg/L in root culture medium.
Preferably, in step (4), the culture of rootage condition are as follows: the Multiple Buds are inoculated in root media,
Temperature be 25 ± 2 DEG C, daily light application time be 8h, intensity of illumination 2500xl, interlunation be 16h under conditions of culture 15~
25 days.
Preferably, further include step (5) after step (4): after the rooted seedling is cleaned, 40 are cultivated in matrix
~60d obtains transplanted seedling.
Preferably, turf in the matrix, vermiculite, perlite volume ratio be 3:3:2.
Beneficial effects of the present invention:
It covers Sang Yusang and is different kind, there are larger differences during tissue cultures and polyploid germ plasm resource are formulated
It is different.The present invention provides a kind of cultural methods for covering mulberry polyploid seedling, carry out preculture to covering after mulberry tissue-cultured seedling chooses stem section,
Then polyploid is obtained using colchicine-induced to the stem section for starting to generate callus, by Multiplying culture and culture of rootage
Obtain covering the polyploid seedling of mulberry afterwards.This method can overcome cover mulberry tissue cultures browning is serious, multiploid induction rate is low,
The more problem of chimera after induction.
Detailed description of the invention
Fig. 1 is with the DNA content figure in flow cytometry diploid.
Fig. 2 is with the DNA content figure in flow cytometry tetraploid.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical solution of the present invention will be carried out below
Detailed description.Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Base
Embodiment in the present invention, those of ordinary skill in the art are obtained all without making creative work
Other embodiment belongs to the range that the present invention is protected.
The present embodiments relate to a kind of cultural methods for covering mulberry polyploid seedling, method includes the following steps:
(1) stem section is intercepted on Cong Mengsang tissue-cultured seedling, is inoculated in precultivation medium and carries out preculture, obtain preculture stem
Section.
In one embodiment of the invention, cover mulberry tissue-cultured seedling to obtain in the following manner: it is strong that mid or late May chooses growth
Strong illiteracy Sang Zuowei maternal plant, in maternal plant middle and upper part clip current year raw spray.After removing the blade on branch, branch is washed with dish washing liquid
Then 30min is rinsed with tap water in surface.Branch after cleaning is moved into superclean bench, segment is cut to, per small
Contain number >=2 of bud in section.Above-mentioned segment is placed in the alcohol of 75% volume fraction and sterilizes 1min, then rushed with sterile water
It washes 3 times or more, then impregnates 6min with the mercuric chloride solution of 0.1% mass concentration, finally use aseptic water washing 3~4 times, with sterile filter
Paper adsorbs residual moisture, the stem section after being sterilized.
Stem section after sterilizing is inoculated in after cultivating 20d in the first culture medium, which occurs rudiment.By the stem after rudiment
Section is inoculated in the second culture medium, is obtained and is covered mulberry tissue-cultured seedling.Wherein, the first culture medium is MS culture medium.Second culture medium is with MS
For basic culture medium, and add KT, 6-BA, NAA, PVP and active carbon obtains, wherein the concentration of KT is 2.0mg/L, and 6-BA's is dense
Degree is 0.5mg/L, and the concentration of NAA is 0.5mg/L, and the concentration of PVP is 0.5g/L, and the concentration of active carbon is 1.0g/L, the second training
Ingredient and the content for supporting base are identical as precultivation medium in subsequent step.
In one embodiment of the invention, the step of stem section is intercepted on Cong Mengsang tissue-cultured seedling includes: selection robust growth
Tissue-cultured seedling, obtain stem section after being cut off blade and petiole, stem section be truncated into the segment of 1~1.5cm long, there are 1 for every segment
A axillary bud, from segment one end, progress is longitudinal sectional, obtains the notch that length is 1mm.
Wherein, from one end of segment stem section, progress is longitudinal sectional, is the growth area in order to increase callus, is conducive to callus
It organizes the formation of.Specifically, there is the ability to heal automatically after plant is injured.After causing notch, the traumatin of incision is opened
Begin active, it has activated the parenchyma cell of incision, and parenchyma cell just accelerates to divide, and is formed quickly in cut surface by thin-walled
The callus of cell composition.
In one embodiment of the invention, the stem section intercepted on mulberry tissue-cultured seedling will be covered to be inoculated in precultivation medium
Mode are as follows: by the laterally inserted culture medium of stem section, only culture medium is exposed in upper surface, then carries out preculture.It should be noted that
During preculture, stem section can be can also be longitudinally inserted into laterally inserted culture medium.Since the notch in stem section end is vertical
To, therefore just can make notch is as much as possible to contact with culture medium for stem section is laterally inserted, accelerate the differentiation of callus
Journey.
In one embodiment of the invention, precultivation medium is with MS for basic culture medium, and add KT, 6-BA,
NAA, PVP and active carbon obtain.The concentration of KT is 2.0mg/L in precultivation medium, and the concentration of 6-BA is 0.5mg/L, NAA's
Concentration is 0.5mg/L, and the concentration of PVP is 0.5g/L, and the concentration of active carbon is 1.0g/L.
Wherein, MS culture medium is at present using most common culture medium, and formula is well known to tissue culture field.The culture medium
Inorganic salt concentration with higher, mineral nutrition needed for capable of guaranteeing tissue growth can also accelerate the growth of callus.By
Ion concentration in formula is high,, will not shadow even if some ingredients are slightly different durings preparation, storage and disinfection etc.
Ring interionic balance.KT is kinetin, belongs to plant cytokinin, and chemical name is 6-nonylaminopurine (or N6- furan
Furfuryl adenine), it can promote cell differentiation, division, growth；Evoked callus sprouts；Release apical dominance.6-BA is benzyl
Aminoadenine is the artificial synthesized basic element of cell division, has the characteristics that efficient, stable, cheap and easy to use, the master of 6-BA
Acting on is the formation for promoting bud, can also be occurred with evoked callus.NAA is methyl α-naphthyl acetate, belongs to auximone, can be promoted
Cell division and expansion.In tissue culture, the effect of auxins is the formation of evoked callus, the generation and examination of embryoid
Guan Miao's takes root.Usually auxin and the basic element of cell division are used cooperatively, to induce the generation of axillary bud and adventitious bud.IBA is Yin
Diindyl butyric acid, belongs to auximone, branches out to plant or the top bud-end of bud, seedling etc. is formed with facilitation, can also be by it
Replace with heteroauxin.PVP is polyvinylpyrrolidone, can form complex compound with specific polyphenolic substance (such as tannin), improve
In tissue cultures the problem of browning.It is such as added without and will lead to Brown death.The effect of active carbon is absorption in vitro culture
In mortifier (such as phenolic substances), inactivate polyphenol oxidase and peroxidase, to prevent browning.
In one embodiment of the invention, the actual conditions of preculture are as follows: the stem section that will be intercepted from illiteracy mulberry tissue-cultured seedling
It is inoculated in precultivation medium, is 25 ± 2 DEG C in temperature, daily light application time is 8h, intensity of illumination 2500xl, when dark
Between to cultivate 72h or more under conditions of 16h, obtain preculture stem section.
(2) after the completion of preculture, preculture stem section is inoculated in the induced medium containing colchicine and is induced
Culture, the stem section after being induced.
In one embodiment of the invention, induced medium is not to add the MS of agar as basic culture medium, and adds
KT, 6-BA, NAA, PVP, colchicine and active carbon obtain.The concentration of KT is 2.0mg/L, the concentration of 6-BA in induced medium
For 0.5mg/L, the concentration of NAA is 0.5mg/L, and the concentration of PVP is 0.5g/L, and the concentration of colchicine is 30mg/L, active carbon
Concentration be 1.0g/L.
Usual induced medium is fluid nutrient medium, therefore is wherein added without agar.Colchicine is a kind of alkaloid, again
Name colchicin.Colchicine can inhibit mitosis, destroy spindle, chromosome is made to be stuck in metaphase.It is this by the autumn
Abnormal division, referred to as Colchcine mitosis caused by narcissus element.In such mitosis, although chromosome is vertical
It splits, but cell does not divide, two daughter cells cannot be formed, thus make chromosome doubling.From nineteen thirty-seven American scholar cloth Simon Rex
Sharp (A.F.Blakeslee) etc., after being succeeded with the chromosome number that colchicine doubles the plants such as datura, colchicine
Just it is widely used in the research and plant breeding of cytology, science of heredity.In the present invention, it is terrible for colchicine being added to be
To illiteracy mulberry tetraploid.
In one embodiment of the invention, the actual conditions of Fiber differentiation are as follows: the stem section after preculture is inoculated in and is lured
It leads in culture medium, is 20 ± 2 DEG C in temperature, daily light application time is 8h, and intensity of illumination 2500xl, interlunation is 16h's
Under the conditions of cultivate 72h or more.
In one embodiment of the invention, after Fiber differentiation, the stem section after induction is taken out, aseptic water washing is used
It 3~4 times, after blotting surface moisture with aseptic filter paper, is transferred to proliferated culture medium and is cultivated.
(3) after the completion of Fiber differentiation, the stem section after induction is inoculated in proliferated culture medium and carries out Multiplying culture, obtains clump
It sprouts.
In one embodiment of the invention, proliferated culture medium is identical as the ingredient of precultivation medium and content.
In one embodiment of the invention, the actual conditions of Multiplying culture are as follows: the stem section after induction is inoculated in proliferation
It is 25 ± 2 DEG C in temperature in culture medium, daily light application time is 8h, and intensity of illumination 2500xl, interlunation is the item of 16h
It is cultivated 20~30 days under part, until induction seedling grows to 30cm or more, obtains Multiple Buds.
Since Fiber differentiation can generate tetraploid, diploid and chimera, after Multiplying culture, need to growing thickly
The blade of bud carries out Chromosome Analysis, determines its chromosome number.Then it filters out tetraploid and continues 20~30d of culture
Afterwards, culture of rootage is carried out.
The present invention uses the ploidy after flow cytometry induction, method particularly includes: in superclean bench, from every plant
1~2 leaf of clip on Multiple Buds.After label, with flow cytometry ploidy.In one embodiment of the invention, when
When concentration of the colchicine in induced medium is 30~50mg/L, in Multiple Buds the ratio of tetraploid be 12%~
25%.
(4) after the completion of Multiplying culture, obtained Multiple Buds is inoculated in root media and carry out culture of rootage, are given birth to
Offspring.
In one embodiment of the invention, root media is with MS for basic culture medium, and adds IBA and obtain.It is raw
The concentration of IBA is 0.3mg/L in root culture medium.IBA is indolebutyric acid, branches out to plant or the top bud-end of bud, seedling etc. is formed
There is facilitation, heteroauxin can also be replaced with.
In one embodiment of the invention, the actual conditions of culture of rootage are as follows: Multiple Buds are inoculated in root media
In, temperature be 25 ± 2 DEG C, daily light application time be 8h, intensity of illumination 2500xl, interlunation be 16h under conditions of train
It supports 15~25 days, obtains rooted seedling.
It further, further include step (5) after step (4): after rooted seedling is cleaned, in the nursery that matrix is housed
40~60d of culture obtains transplanted seedling in disk, is transplanted to Field planting when growth of seedling to 10~15cm height.
In one embodiment of the invention, the bottle seedling that rooted seedling first carries out 1 week is taken exercise, and specific steps include: first to unscrew
Bottle cap 3d, then twist-off closure temper 3~4d, then take out seedling and wash away root culture medium in warm water, then with containing 1% it is more
Bacterium spirit aqueous solution soaking 30s.Then it is transplanted and carries out hardening in Medium Culture.The volume ratio of turf, vermiculite, perlite in matrix
For 3:3:2.Hardening is between 20~25 DEG C in temperature, and humidity carries out under conditions of being 75%~85%.
Then the present invention is by using the autumn to obtained callus to preculture is carried out after covering mulberry tissue-cultured seedling selection stem section
Narcissus element induces to obtain polyploid, then passes through Multiplying culture and culture of rootage, obtains the polyploid seedling for covering mulberry.Specifically,
The present invention reduces the melting brown rate of breeding in Fiber differentiation by addition PVP and active carbon, and is suitable for by selection
Pre-incubation time, and suitable colchicine is added in the callus idiophase, reduce the generation of chimera, improves more
The inductivity of times body.
Embodiment 1-1
(1) cultivate and cover mulberry tissue-cultured seedling: mid or late May chooses the illiteracy Sang Zuowei maternal plant of robust growth, cuts in maternal plant middle and upper part
Take current year raw spray.After removing the blade on branch, branch surface is washed with dish washing liquid, then rinses 30min with tap water.It will
Branch after cleaning moves into superclean bench, is cut to segment, number >=2 containing bud on every segment.By above-mentioned segment
It is placed in the alcohol of 75% volume fraction and sterilizes 1min, then use aseptic water washing 3 times or more, then with the liter of 1% mass concentration
Mercury solution impregnates 6min, finally uses aseptic water washing 3~4 times, adsorbs residual moisture with aseptic filter paper, the stem after being sterilized
Section.
Stem section after sterilizing is inoculated in after cultivating 20d in the first culture medium, which occurs rudiment.By the stem after rudiment
Section is inoculated in the second culture medium, is obtained and is covered mulberry tissue-cultured seedling.Wherein, the first culture medium is MS culture medium.Second culture medium is with MS
For basic culture medium, and add KT, 6-BA, NAA, PVP and active carbon obtains, wherein the concentration of KT is 2.0mg/L, and 6-BA's is dense
Degree is 0.5mg/L, and the concentration of NAA is 0.5mg/L, and the concentration of PVP is 0.5g/L, and the concentration of active carbon is 1.0g/L.
(2) preculture: choosing the tissue-cultured seedling of robust growth, obtains stem section after being cut off blade and petiole, and stem section is cut
For the segment of 1~1.5cm long, there are 1 axillary buds for every segment, and from segment one end, progress is longitudinal sectional, obtain the notch that length is 1mm.
By in the laterally inserted culture medium of stem section, only culture medium is exposed in upper surface.It is 25 ± 2 DEG C in temperature, daily light application time is 8h, light
It is 2500xl according to intensity, culture 72h or more, obtains preculture stem section under conditions of interlunation is 16h.
Precultivation medium is with MS for basic culture medium, and adds KT, 6-BA, NAA, PVP and active carbon obtains.Pre- training
The concentration for supporting KT in culture medium is 2.0mg/L, and the concentration of 6-BA is 0.5mg/L, and the concentration of NAA is 0.5mg/L, the concentration of PVP
For 0.5g/L, the concentration of active carbon is 1.0g/L.
(3) Fiber differentiation: the stem section after preculture is inoculated in induced medium, is 20 ± 2 DEG C in temperature, every daylight
It is 8h, intensity of illumination 2500xl, culture 72h or more, the stem after being induced under conditions of interlunation is 16h according to the time
Section.After Fiber differentiation, the stem section after induction is taken out, with aseptic water washing 3~4 times, blots surface water with aseptic filter paper
Point.
Induced medium adds KT, 6-BA, NAA, PVP, colchicine not add the MS of agar as basic culture medium
It is obtained with active carbon.The concentration of KT is 2.0mg/L in induced medium, and the concentration of 6-BA is 0.5mg/L, and the concentration of NAA is
The concentration of 0.5mg/L, PVP are 0.5g/L, and the concentration of colchicine is 30mg/L, and the concentration of active carbon is 1.0g/L.
(4) Multiplying culture: the stem section after induction is inoculated in proliferated culture medium, is 25 ± 2 DEG C in temperature, daily illumination
Time is 8h, and intensity of illumination 2500xl, interlunation is cultivates 20~30 days under conditions of 16h, until induction seedling is grown to
30cm or more obtains Multiple Buds.Proliferated culture medium is identical as the ingredient of precultivation medium and content.
(5) Chromosome Analysis: determining the chromosome number of Multiple Buds using flow cytometer, then filters out four times
After body continues 20~30d of culture, culture of rootage is carried out.Fig. 1 and Fig. 2 is the DNA content figure in Diploid and Tetraploid respectively, is said
It is bright that tetraploid can be obtained using colchicine-induced, but there is also the diploids that chromosome number variation does not occur.It is indulged in figure
Coordinate is population, and abscissa is fluorescence intensity.The DNA content in plant can be calculated by peak figure, and judges cell inner dyeing
The ploidy level of body.
(6) culture of rootage: Multiple Buds are inoculated in root media, are 25 ± 2 DEG C in temperature, daily light application time is
8h, intensity of illumination 2500xl, interlunation to cultivate 15~25 days under conditions of 16h, obtain rooted seedling.
Root media is with MS for basic culture medium, and adds IBA and obtain.The concentration of IBA is in root media
0.3mg/L。
(7) acclimatization and transplants: the bottle cap 3d of container where first unscrewing rooted seedling, then twist-off closure temper 3~4d, then take out
Seedling washes away root culture medium in warm water, then with the carbendazim aqueous solution soaking 30s containing 1%.It is transplanted in Medium Culture
Hardening is carried out, 40~60d obtains transplanted seedling, is transplanted to Field planting when growth of seedling to 10~15cm height.
Turf in matrix, vermiculite, perlite volume ratio be 3:3:2, hardening is between 20~25 DEG C in temperature, and humidity is
It is carried out under conditions of 75%~85%.
Embodiment 1-2 to embodiment 1-5
The reagent type and dosage in (2) precultivation medium are changed the step, observation different additive combination is to preculture
The influence of stem section melting brown rate and growing state.The precultivation medium that each embodiment uses is with MS+0.5mg/L 6-BA+
Based on 0.5mg/L NAA+2.0mg/L KT, other cultural methods the results are shown in Table 1 with embodiment 1-1.
Table 1
As can be seen from Table 1, compared with embodiment 1-1, change the additional amount of PVP and active carbon, cover mulberry preculture stem section
Melting brown rate is risen, the decline of ratio shared by normal stem section.Illustrate that PVP and active carbon, which is added, is able to suppress browning.
Embodiment 2-1 to embodiment 2-27
It changes the step the dosage of the colchicine in pre-incubation time and step (3) induced medium in (2) and lures
Lead incubation time.Chromosome Analysis is carried out after Fiber differentiation and Multiplying culture, and Multiple Buds are determined using flow cytometer
Chromosome number, and the tetraploid in Multiple Buds and chimera percentage is calculated.Other cultural methods are the same as embodiment 1-
1, it the results are shown in Table 2.
Table 2
As can be seen from Table 2, pre-incubation time and Fiber differentiation overlong time or too short, are unable to get tetraploid.Meanwhile
When the additional amount of colchicine 20~50mg/L change when, also will affect tetraploid proportion.Therefore by pre-incubation time
It is controlled at 3 days with the Fiber differentiation time, and colchicine concentration is controlled in 30mg/L, can obtained in the stem section after induction
To the higher tetraploid of ratio.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. a kind of cultural method for covering mulberry polyploid seedling, which comprises the following steps:
(1) stem section is intercepted on Cong Mengsang tissue-cultured seedling, is inoculated in precultivation medium and carries out preculture, obtain preculture stem section；
(2) the preculture stem section is inoculated in the induced medium containing colchicine and carries out Fiber differentiation, induced
Stem section afterwards；
(3) stem section after the induction is inoculated in proliferated culture medium and carries out Multiplying culture, obtain Multiple Buds；
(4) Multiple Buds are inoculated in root media and carry out culture of rootage, obtain rooted seedling.
2. cultural method according to claim 1, which is characterized in that in step (1), intercept stem section on Cong Mengsang tissue-cultured seedling
The step of include: choose robust growth illiteracy mulberry tissue-cultured seedling, obtain stem section after being cut off blade and petiole, stem section be truncated into 1
The segment of~1.5cm long, there are 1 axillary buds for every segment, and from segment one end, progress is longitudinal sectional, obtain the notch that length is 1mm.
3. cultural method according to claim 1, which is characterized in that in step (1), by what is intercepted from illiteracy mulberry tissue-cultured seedling
In the laterally inserted culture medium of stem section, only culture medium is exposed in upper surface, then carries out preculture.
4. cultural method according to claim 1, which is characterized in that in step (1), the precultivation medium is with MS
Basal medium, and add KT, 6-BA, NAA, PVP and active carbon obtains, the concentration of KT is in the precultivation medium
The concentration of 2.0mg/L, 6-BA are 0.5mg/L, and the concentration of NAA is 0.5mg/L, and the concentration of PVP is 0.5g/L, the concentration of active carbon
For 1.0g/L；
Preferably, the preculture condition are as follows: intercept stem section on Cong Mengsang tissue-cultured seedling and be inoculated in precultivation medium, in temperature
It is 25 ± 2 DEG C, daily light application time is 8h, intensity of illumination 2500xl, culture 72h or more under conditions of interlunation is 16h.
5. cultural method according to claim 1, which is characterized in that in step (2), the induced medium is not to add
The MS of agar is basic culture medium, and adds KT, 6-BA, NAA, PVP, colchicine and active carbon and obtain, the Fiber differentiation
The concentration of KT is 2.0mg/L in base, and the concentration of 6-BA is 0.5mg/L, and the concentration of NAA is 0.5mg/L, and the concentration of PVP is 0.5g/
L, the concentration of colchicine are 30mg/L, and the concentration of active carbon is 1.0g/L；
Preferably, the inducing culturing condition are as follows: the preculture stem section is inoculated in induced medium, temperature be 20 ±
2 DEG C, daily light application time is 8h, intensity of illumination 2500xl, culture 72h or more under conditions of interlunation is 16h.
6. cultural method according to claim 1, which is characterized in that in step (3), the proliferated culture medium and preculture
The composition of culture medium is identical；
Preferably, the Multiplying culture condition are as follows: the stem section after the induction is inoculated in proliferated culture medium, is 25 in temperature
± 2 DEG C, daily light application time be 8h, intensity of illumination 2500xl, interlunation be 16h under conditions of cultivate 20~30 days, until
Induction seedling grows to 30cm or more.
7. cultural method according to claim 1, which is characterized in that after step (3), to the blade of the Multiple Buds
Chromosome Analysis is carried out, analysis instrument is flow cytometer, determines its chromosome number, filters out tetraploid and continues to cultivate
After 20~30d, culture of rootage is carried out.
8. cultural method according to claim 1, which is characterized in that in step (4), the root media is using MS as base
Basal culture medium, and add IBA and obtain, the concentration of IBA is 0.3mg/L in the root media；
Preferably, the culture of rootage condition are as follows: the Multiple Buds are inoculated in root media, are 25 ± 2 DEG C in temperature,
Daily light application time is 8h, intensity of illumination 2500xl, interlunation be 16h under conditions of cultivate 15~25 days.
9. cultural method according to claim 1, which is characterized in that further include step (5) after step (4): by institute
State rooted seedling clean after, in matrix cultivate 40~60d obtain transplanted seedling.
10. cultural method according to claim 9, which is characterized in that the body of turf, vermiculite, perlite in the matrix
Product is than being 3:3:2.
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