CN107135945A - The tissue culture medium (TCM) and its rapid propagation method of a kind of lime tree - Google Patents
The tissue culture medium (TCM) and its rapid propagation method of a kind of lime tree Download PDFInfo
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- CN107135945A CN107135945A CN201710316354.0A CN201710316354A CN107135945A CN 107135945 A CN107135945 A CN 107135945A CN 201710316354 A CN201710316354 A CN 201710316354A CN 107135945 A CN107135945 A CN 107135945A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a kind of tissue culture medium (TCM) of lime tree and its rapid propagation method, the method for aiming to provide a kind of commercial tissue culture of sprout breeding of lime tree that can quickly breed, its drip irrigation device is, including inducing culture, proliferated culture medium, root media and explant sterilization method, environmental Kuznets Curves and operating procedure, inducing culture includes：MS culture mediums：4630~4640mg；Fill in the grand TDZ of benzene：0.001~0.010mg；Gibberellin：2~3mg；Agar：5~8g；Sucrose：25~40g；Inositol：90~110mg, adds water and complements to 1 liter, proliferated culture medium includes：MS culture mediums：4630~4640mg；Fill in the grand TDZ of benzene：0.001~0.010mg；Gibberellin：2~3mg；Agar：5~8g；Sucrose：25~40g；Inositol：90~110mg, adds water and complements to 1 liter, root media includes：2315~2320mg；Indolebutyric acid IBA：1.0mg~2.0mg；Methyl α-naphthyl acetate NAA：1.0mg~2.0mg；Agar：5~8g；Sucrose：10~20g；Add water and complement to 1 liter.
Description
Technical field
The present invention relates to technical field of plant propagation, the tissue culture medium (TCM) of more particularly to a kind of lime tree and its quickly breed
Method.
Background technology
European leaflet forges (Tilia Greenspire), belongs to Tiliaceae Tilia, lime tree blade can change colour, relatively thicker
Rough, anti-flue dust and toxic gas ability is strong, and timber is excellent, is one of most popular shade tree and shade tree in the world, but mesh
Preceding China lime tree is wild mostly, and lime tree is still at an early stage as shade tree, and artificial propagation cultivation is seldom, with cities and towns
The development of gardens industry, country's lime tree nursery stock demand is big at present, and supply falls short of demand.
Lime tree seed is due to kind of a skin os osseum matter impermeability, embryo dormancy and there is mortifier in seed, causes lime tree seed
Germination percentage is very low, uneven emergence, while Flores tiliae protandry is thus almost sterile from flower, cross-pollination, setting percentage is relatively low,
It is usually no more than 10%.In addition at home and abroad, the Study on tissue culture of lime tree is less, it is impossible to adapt to batch production large-scale production.
Lime tree seedling is bred using tissue culture technique, the breeding cycle is short, and efficiency high, cost is low, can work in a short time
Factory's metaplasia produces a large amount of high quality seedlings, thus by selecting suitable type of culture medium, rational plant growth regulator of arranging in pairs or groups, really
Fixed suitable cultivation temperature, the condition such as illumination, for the foundation of lime tree tissue cultures and fast-propagation system, with important meaning
Justice.
At present, Publication No. CN105746106A Chinese patent discloses a kind of propagation method of tilia miqueliana, and it includes
Five steps complete to breed, step (1)：It can be gathered when seed maturity, seed uses the side of " push away and shine threshing " after adopting go back to
Method is handled；Step (2)：The storage of seed is generally using " ventilation is dry to hide ", three kinds of sides of " cryopreservation " and " storage of profit sand "
Method；Step (3)：Presprouting of seeds is handled, and seed is immersed in 10 to 20 DEG C of water 4 to 6 hours, and 0.5% content is utilized before sowing
Liquor potassic permanganate soak 1 to 2 hours, be then multiplied by 15 centimetres with 30 by way of drilling and sowed for spacing；Step
Suddenly (four)：Utilize prior art cuttage in slotting machine the spray of processing number, slotting machine is arched, plastic foil kettle cover is used, using canopy
Shelter from heat or light and spray cooling outside, light intensity is that relative humidity is maintained at 80% to 90% in 20% to the 40% of full sun, canopy in canopy；Step
Suddenly (five)：The sapling for growing the half a year that expires is transplanted in crop field.
Although the propagation method of this tilia miqueliana solves to carry out ectogenesis to tilia miqueliana by five steps, according to
Seed vigor in growth course can not so be solved low, the problem of germination rate is low, therefore should need suitable culture medium with
And environment regulation so that lime tree can quick flourish, it is difficult to solve Growth Process of Tilia amurensis breeding and seedling, induce
The problem of difficulty, difficulty of taking root, so as to carry out scale, industrial seedling rearing.
The content of the invention
It is an object of the invention to provide a kind of tissue culture medium (TCM) of lime tree, it has is grown thickly using lime tree dormancy axillary bud deriving
The tissue culture technique of bud carries out fast culture breeding to lime tree, and proliferation rate is higher, while rooting rate is preferably, can be with effectively save
Cost, carries out large-scale fast breeding so that lime tree can carry out the advantage of large-scale industrialized production.
The present invention above-mentioned technical purpose technical scheme is that：
A kind of tissue culture medium (TCM) of lime tree, including inducing culture, proliferated culture medium and root media, the Fiber differentiation
Base includes following component：MS culture mediums：4630~4640mg；Fill in the grand TDZ of benzene：0.001~0.010mg；Gibberellin：2~3mg；
Agar：5~8g；Sucrose：25~40g；Inositol：90~110mg, adds water and complements to 1 liter, the proliferated culture medium includes as follows
Component：MS culture mediums：4630~4640mg；Fill in the grand TDZ of benzene：0.001~0.010mg；Gibberellin：2~3mg；Agar：5~8g；
Sucrose：25~40g；Inositol：90~110mg, adds water and complements to 1 liter, the root media includes following component：2315~
2320mg；Indolebutyric acid IBA：1.0mg~2.0mg；Methyl α-naphthyl acetate NAA：1.0mg~2.0mg；Agar：5~8g；Sucrose：10~
20g；Add water and complement to 1 liter.
As preferred：The inducing culture includes following component：MS culture mediums：4636.43mg；Fill in the grand TDZ of benzene：
0.005mg；Gibberellin：2.5mg；Agar：6g；Sucrose：30g；Inositol：100mg, adds water and complements to 1 liter；The Multiplying culture
Base includes following component：MS culture mediums：4636.43mg；Fill in the grand TDZ of benzene：0.005mg；Gibberellin：2.0mg；Agar：6g；Sugarcane
Sugar：30g；Inositol：100mg, adds water and complements to 1 liter；The root media includes following component：2318.22mg；Indoles fourth
Sour IBA：1.5mg；Methyl α-naphthyl acetate NAA：1.5mg；Agar：6g；Sucrose：15g；Add water and complement to 1 liter.
Set by using above-mentioned technical proposal, MS culture mediums are to use most common culture medium at present, and it has higher
Inorganic salt concentration, the mineral nutrition needed for ensure that tissue growth can also accelerate the growth of callus.Due in formula
Ion concentration it is high, preparing, during storage and sterilization etc., even if some compositions are slightly different, do not interfere with interionic yet
Balance, and TDZ is as a plant growth regulators, and with very strong cytokine activity, its basic element of cell division is lived
Tens times higher than the cytokine activity of general plant growth regulator of property can promote plant sprout to hundred times
Regeneration and breeding, break bud stops eye, promotes seed to sprout, and promotes callus growth, delays plant senescence etc..And can be with
The growth and development process of plant is adjusted to the effect of other plant hormones and physiological activator.And gibberellin is mainly used in
Accelerate the growth of cell, improve the content of auxin in plant, and auxin directly adjusts the growth of cell, and cell is divided
Splitting also has facilitation, can promote the expansion of cell, in addition, and gibberellin also has suppression ripe, and lateral bud dormancy declines
Always, the physiological action of tuber character.Fluid nutrient medium is converted into solid medium or semi-solid training by agar as coagulator
Support base.Add the solid medium of the agar advantage compared with fluid nutrient medium and be easy to operate, ventilation problem is easily solved, just
In frequent observational study, and sucrose supplies the nutrient required for cell growth then as the standard carbon source of culture medium.Indolebutyric acid
IBA is primarily to facilitate plant main root and grown, raising germination percentage, survival rate, for promoting explant to take root, and methyl α-naphthyl acetate NAA
Same to promote the growth of plant as a kind of hormone, explant starts the hormone combination that the key grown is mainly culture medium
With concentration, the basic element of cell division of higher concentration and the auxin of low concentration should be typically used, the suppression of apical dominance can be released
Make and use, induction produces Multiple Buds.The too high easy generation callus of auxin concentration；Hormone combination is appropriate, then stem apex is upward
Grow into no offspring.And by inducing culture inducing cell callus regeneration, by proliferated culture medium so that explant is given birth to
Terminal bud is grown, finally by root media so that terminal bud takes root for ensureing to survive.
It is a further object of the present invention to provide a kind of rapid propagation method of lime tree, it has numerous using tissue culture technique
Educate lime tree seedling, the breeding cycle is short, and efficiency high, cost is low, can a large amount of high quality seedlings of factorial praluction in a short time advantage.
The present invention above-mentioned technical purpose technical scheme is that：
A kind of rapid propagation method of lime tree, including following incubation step：
S1：From explant, explant is from the full semi-lignified branch stem apex of healthy lime tree current-year branch, sprout
Part；
S2：Sterilization and Initial culture, lime tree explant blade are cut off, clear water is rinsed well, on the table flushed
Explant is put into the alcohol that concentration is 75% and sterilized 30 seconds, then with aseptic water washing 3~4 times, explant is placed on into sterilization
Soaked 60 minutes in liquid, complete after sterilization, explant is inoculated into inducing culture；Under the conditions of intensity of illumination 1500Lx,
16 DEG C of temperature, half-light culture 1 week, after turn 23 DEG C of temperature, light application time 16h.Culture 6 weeks.
S3：Squamous subculture：In inducing culture in 30~40 days, explant stem apex occurs that explant being transferred during young sprout
Into proliferated culture medium, under certain culture environment, it is transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
S3：Squamous subculture：In inducing culture in 30~40 days, explant stem apex occurs that explant being transferred during young sprout
Into proliferated culture medium, under certain culture environment, it is transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
Set by using above-mentioned technical proposal, stem apex is the conventional explant of Plant Tissue Breeding.Because stem apex
Not only fast growth, breeding potential height, are not likely to produce hereditary variation, and are the effective ways for obtaining virus-free nursery stock, therefore
Explant is from the full semi-lignified branch stem apex part of healthy lime tree annual shoot bud.By the explant chosen and right
Whole experimental situation and appliance carry out effective cleaning sterilization, are brought when reducing the bacterium in air, virus and operation
Bacterium infection explant, so as to influence the success rate of experiment, therefore explant is entered by the immersion of alcohol and thimerosal
The effective sterilization of row, is placed into proliferated culture medium when young sprout occurs in explant, the propagation of cell is carried out, so as to grow
Go out terminal bud, by the healthy and strong lime tree tissue-cultured seedling of squamous subculture, take 2~3cm terminal bud, be put into root media and complete training of taking root
Support, because the body fluid pH value of usual plant is 5.80 or so, therefore during culture medium allotment the control of solution pH value 5.80 or so, and
The agar added under this pH value is preferable into white coagulability, too high if the too low culture medium of pH value can not solidify, and solidifies
It is really up to the mark.
Further set：The thimerosal includes following component：1 part and 3 parts sterilized waters of bleaching water.
Set by using above-mentioned technical proposal, the thimerosal made by using white cat bleaching water can be effectively right
Explant carries out disinfection sterilization.
Further set：Culture environment is that, in intensity of illumination 1500Lx, temperature is 25 DEG C, and light application time 16 is small in the S3
When, in dark condition, temperature is 23 DEG C, 8 hours standing times.
Set by using above-mentioned technical proposal, the photoperiod carries out continuous 16h illumination, 8h dark, is conducive to Shoot Tip Culture
Differentiation and propagation with bud.Enhancing illumination is conducive to rooting of vitro seedling, and play the role of for test tube transplantation of seedlings it is good, but can not
Intensity of illumination is too strong, once long-time intense direct illumination root, can suppress the growth of root.
Further set：The culture medium passes through sterilization treatment, and the culture medium configured is placed in autoclave,
0.105MPa pressure, in the case that temperature is 121 DEG C, sterilize 15~30min
Set by using above-mentioned technical proposal, not only need to carry out explant effective sterilization treatment, with greater need for for training
Support base and carry out sterilization treatment, reduce due to the germ contamination culture medium being infected with operating process.
Further set：Antioxidant is also added with the culture medium, the antioxidant is cysteine or anti-bad
Hematic acid.
Set by using above-mentioned technical proposal, in the medium, use the antioxidant energy such as cysteine, ascorbic acid
Enough browning phenomenons for more efficiently avoiding or mitigating many explants.
Further set：0.1~0.5g activated carbon is also added with the culture medium.
Set by using above-mentioned technical proposal, activated carbon is due to itself being the stronger adsorbent of adsorptivity, Ke Yigeng
The browning phenomenon of good mitigation explant.
In summary, the invention has the advantages that：Sterilized using this method, with preferable sterilization success rate,
Coordinate inducing culture simultaneously, explant rudiment success rate is higher, can effectively set up sterile regenerating system, and use and be somebody's turn to do
The lime tree tissue-cultured seedling propagation success rate that method is finally obtained is higher, well developed root system.
Brief description of the drawings
Fig. 1 is the rapid propagation method schematic flow sheet of lime tree.
Embodiment
The present invention is described in further detail below in conjunction with accompanying drawing.
Embodiment 1：A kind of tissue culture medium (TCM) of lime tree, as shown in figure 1, including inducing culture, proliferated culture medium and
Root media, the inducing culture includes following component：MS culture mediums：4630mg；Fill in the grand TDZ of benzene：0.001mg；It is red mould
Element：2g；Agar：5g；Sucrose：25g；Inositol：90mg, adds water and complements to 1 liter, the proliferated culture medium includes following component：
MS culture mediums：4630mg；Fill in the grand TDZ of benzene：0.001mg；Gibberellin：2mg；Agar：5g；Sucrose：25g；Inositol：90mg, is added
Water complements to 1 liter, and the root media includes following component：2315mg；Indolebutyric acid IBA：1.0mg；Methyl α-naphthyl acetate NAA：
1.0mg；Agar：5g；Sucrose：10g；Add water and complement to 1 liter.
A kind of rapid propagation method of lime tree, as shown in Figure 1：
S1：From explant, explant is from the full semi-lignified branch stem apex part of the raw twig of healthy lime tree；
S2：Sterilization and Initial culture, lime tree explant blade are cut off, clear water is rinsed well, on the table flushed
Explant is put into the alcohol that concentration is 75% and sterilized 30 seconds, then with aseptic water washing 3~4 times, explant is placed on into sterilization
Soaked 60 minutes in liquid, complete after sterilization, explant is inoculated into inducing culture；Under the conditions of intensity of illumination 1500Lx,
16 DEG C of temperature, half-light culture 1 week, after turn 23 DEG C of temperature, light application time 16h is cultivated 6 weeks.
S3：Squamous subculture：In inducing culture in 30~40 days, explant stem apex occurs that explant being transferred during young sprout
Into proliferated culture medium, under certain culture environment, it is transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
Embodiment 2：A kind of tissue culture medium (TCM) of lime tree, including inducing culture, proliferated culture medium and root media,
The inducing culture includes following component：MS culture mediums：4640mg；Fill in the grand TDZ of benzene：0.010mg；Gibberellin：3mg；Agar：
8g；Sucrose：40g；Inositol：110mg, adds water and complements to 1 liter, the proliferated culture medium includes following component：MS culture mediums：
4640mg；Fill in the grand TDZ of benzene：0.010mg；Gibberellin：3mg；Agar：8g；Sucrose：40g；Inositol：110mg, adds water and complements to 1
Rise, the root media includes following component：2320mg；Indolebutyric acid IBA：2.0mg；Methyl α-naphthyl acetate NAA：2.0mg；Agar：
8g；Sucrose：20g；Add water and complement to 1 liter.
A kind of rapid propagation method of lime tree, as shown in Figure 1：
S1：From explant, explant is from the full semi-lignified branch stem apex part of the raw twig of healthy lime tree；
S2：Sterilization and Initial culture, lime tree explant blade are cut off, clear water is rinsed well, on the table flushed
Explant is put into the alcohol that concentration is 75% and sterilized 30 seconds, then with aseptic water washing 3~4 times, explant is placed on into sterilization
Soaked 60 minutes in liquid, complete after sterilization, explant is inoculated into inducing culture；Under the conditions of intensity of illumination 1500Lx,
16 DEG C of temperature, half-light culture 1 week, after turn 23 DEG C of temperature, light application time 16h is cultivated 6 weeks.
S3：Squamous subculture：In inducing culture in 30~40 days, explant stem apex occurs that explant being transferred during young sprout
Into proliferated culture medium, under certain culture environment, it is transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
Embodiment 3：A kind of tissue culture medium (TCM) of lime tree, including inducing culture, proliferated culture medium and root media,
The inducing culture includes following component：MS culture mediums：4636.43mg；Fill in the grand TDZ of benzene：0.005mg；Gibberellin：2.5mg；
Agar：6g；Sucrose：30g；Inositol：100mg, adds water and complements to 1 liter, the proliferated culture medium includes following component：MS is cultivated
Base：4636.43mg；Fill in the grand TDZ of benzene：0.005mg；Gibberellin：2.0mg；Agar：6g；Sucrose：30g；Inositol：100mg, is added
Water complements to 1 liter, and the root media includes following component：2318.22mg；Indolebutyric acid IBA：1.5mg；Methyl α-naphthyl acetate NAA：
1.5mg；Agar：6g；Sucrose：15g；Add water and complement to 1 liter.
A kind of rapid propagation method of lime tree, as shown in Figure 1：
S1：From explant, explant is from the full semi-lignified branch stem apex part of the raw twig of healthy lime tree；
S2：Sterilization and Initial culture, lime tree explant blade are cut off, clear water is rinsed well, on the table flushed
Explant is put into the alcohol that concentration is 75% and sterilized 30 seconds, then with aseptic water washing 3~4 times, explant is placed on into sterilization
Soaked 60 minutes in liquid, complete after sterilization, explant is inoculated into inducing culture；Under the conditions of intensity of illumination 1500Lx,
16 DEG C of temperature, half-light culture 1 week, after turn 23 DEG C of temperature, light application time 16h is cultivated 6 weeks.
S3：Squamous subculture：In inducing culture in 30~40 days, explant stem apex occurs that explant being transferred during young sprout
Into proliferated culture medium, under certain culture environment, it is transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
Embodiment 4：A kind of tissue culture medium (TCM) of lime tree, including inducing culture, proliferated culture medium and root media,
The inducing culture includes following component：MS culture mediums：4636.43mg；Fill in the grand TDZ of benzene：0.008mg；Gibberellin：2.9g；
Agar：6g；Sucrose：30g；Inositol：100mg, adds water and complements to 1 liter, the proliferated culture medium includes following component：MS is cultivated
Base：4636.43mg；Fill in the grand TDZ of benzene：0.008mg；Gibberellin：2.9mg；Agar：6g；Sucrose：30g；Inositol：100mg, is added
Water complements to 1 liter, and the root media includes following component：2318.22mg；Indolebutyric acid IBA：1.8mg；Methyl α-naphthyl acetate NAA：
1.8mg；Agar：6g；Sucrose：15g；Add water and complement to 1 liter.
A kind of rapid propagation method of lime tree, as shown in Figure 1：
S1：From explant, explant is from the full semi-lignified branch stem apex part of the raw twig of healthy lime tree；
S2：Sterilization and Initial culture, lime tree explant blade are cut off, clear water is rinsed well, on the table flushed
Explant is put into the alcohol that concentration is 75% and sterilized 30 seconds, then with aseptic water washing 3~4 times, explant is placed on into sterilization
Soaked 60 minutes in liquid, complete after sterilization, explant is inoculated into inducing culture；Under the conditions of intensity of illumination 1500Lx,
16 DEG C of temperature, half-light culture 1 week, after turn 23 DEG C of temperature, light application time 16h is cultivated 6 weeks.
S3：Squamous subculture：In inducing culture in 30~40 days, explant stem apex occurs that explant being transferred during young sprout
Into proliferated culture medium, under certain culture environment, it is transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
Embodiment 5：A kind of tissue culture medium (TCM) of lime tree, including inducing culture, proliferated culture medium and root media,
The inducing culture includes following component：MS culture mediums：4636.43mg；Fill in the grand TDZ of benzene：0.002mg；Gibberellin：2.1g；
Agar：6g；Sucrose：30g；Inositol：100mg, adds water and complements to 1 liter, the proliferated culture medium includes following component：MS is cultivated
Base：4636.43mg；Fill in the grand TDZ of benzene：0.002mg；Gibberellin：2.1mg；Agar：6g；Sucrose：30g；Inositol：100mg, is added
Water complements to 1 liter, and the root media includes following component：2318.22mg；Indolebutyric acid IBA：1.1mg；Methyl α-naphthyl acetate NAA：
1.1mg；Agar：6g；Sucrose：15g；Add water and complement to 1 liter.
A kind of rapid propagation method of lime tree, as shown in Figure 1：
S1：From explant, explant is from the full semi-lignified branch stem apex part of the raw twig of healthy lime tree；
S2：Sterilization and Initial culture, lime tree explant blade are cut off, clear water is rinsed well, on the table flushed
Explant is put into the alcohol that concentration is 75% and sterilized 30 seconds, then with aseptic water washing 3~4 times, explant is placed on into sterilization
Soaked 60 minutes in liquid, complete after sterilization, explant is inoculated into inducing culture；Under the conditions of intensity of illumination 1500Lx,
16 DEG C of temperature, half-light culture 1 week, after turn 23 DEG C of temperature, light application time 16h is cultivated 6 weeks.
S3：Squamous subculture：In inducing culture in 30~40 days, explant stem apex occurs that explant being transferred during young sprout
Into proliferated culture medium, under certain culture environment, it is transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
Embodiment 6：A kind of tissue culture medium (TCM) of lime tree, including inducing culture, proliferated culture medium and root media,
The inducing culture includes following component：MS culture mediums：4636.43mg；Fill in the grand TDZ of benzene：0.005mg；Gibberellin：2.0g；
Agar：6g；Sucrose：30g；Inositol：100mg, adds water and complements to 1 liter, the proliferated culture medium includes following component：MS is cultivated
Base：4636.43mg；Fill in the grand TDZ of benzene：0.005mg；Gibberellin：2.0mg；Agar：6g；Sucrose：30g；Inositol：100mg, is added
Water complements to 1 liter, and the root media includes following component：2318.22mg；Indolebutyric acid IBA：1.5mg；Methyl α-naphthyl acetate NAA：
2mg；Agar：6g；Sucrose：15g；Add water and complement to 1 liter.
A kind of rapid propagation method of lime tree, as shown in Figure 1：
S1：From explant, explant is from the full semi-lignified branch stem apex part of the raw twig of healthy lime tree；
S2：Sterilization and Initial culture, lime tree explant blade are cut off, clear water is rinsed well, on the table flushed
Explant is put into the alcohol that concentration is 75% and sterilized 30 seconds, then with aseptic water washing 3~4 times, explant is placed on into sterilization
Soaked 60 minutes in liquid, complete after sterilization, explant is inoculated into inducing culture；Under the conditions of intensity of illumination 1500Lx,
16 DEG C of temperature, half-light culture 1 week, after turn 23 DEG C of temperature, light application time 16h is cultivated 6 weeks.
S3：Squamous subculture：In inducing culture in 30~40 days, explant stem apex occurs that explant being transferred during young sprout
Into proliferated culture medium, under certain culture environment, it is transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
Embodiment 7：A kind of tissue culture medium (TCM) of lime tree, including inducing culture, proliferated culture medium and root media,
The inducing culture includes following component：MS culture mediums：4636.43mg；Fill in the grand TDZ of benzene：0.005mg；Gibberellin：
2.5g；Agar：6g；Sucrose：30g；Inositol：100mg, adds water and complements to 1 liter, the proliferated culture medium includes following component：
MS culture mediums：4636.43mg；Fill in the grand TDZ of benzene：0.005mg；Gibberellin：2.0mg；Agar：6g；Sucrose：30g；Inositol：
100mg, adds water and complements to 1 liter, the root media includes following component：2318.22mg；Indolebutyric acid IBA：：2mg；
Methyl α-naphthyl acetate NAA：1.5mg；Agar：6g；Sucrose：15g；Add water and complement to 1 liter.
A kind of rapid propagation method of lime tree, as shown in Figure 1：
S1：From explant, explant is from the full semi-lignified branch stem apex part of the raw twig of healthy lime tree；
S2：Sterilization and Initial culture, lime tree explant blade are cut off, clear water is rinsed well, on the table flushed
Explant is put into the alcohol that concentration is 75% and sterilized 30 seconds, then with aseptic water washing 3~4 times, explant is placed on into sterilization
Soaked 60 minutes in liquid, complete after sterilization, explant is inoculated into inducing culture；Under the conditions of intensity of illumination 1500Lx,
16 DEG C of temperature, half-light culture 1 week, after turn 23 DEG C of temperature, light application time 16h is cultivated 6 weeks.
S3：Squamous subculture：In inducing culture in 30~40 days, explant stem apex occurs that explant being transferred during young sprout
Into proliferated culture medium, under certain culture environment, it is transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
Above-described embodiment 1 to embodiment 7 is subjected to Viral diagnosis after being sterilized to explant, is put into while observing explant
Young sprout growing state after inducing culture, is put into after proliferated culture medium and breeds multiplying power, is put into the life of the terminal bud after root media
Root rate and coefficient of taking root carry out analysis contrast, as a result as shown in the table：
It can be drawn from upper table, can be fast by more suitable proportioning inducing culture, proliferated culture medium and root media
Speed breeding lime tree, the rooting rate of lime tree reaches more than 90%, while coefficient of taking root reaches more than 4, while can be with by observation
It was found that root system is all more flourishing, healthy and strong, while breeding multiplying power at 3~5 times or so, the factorial praluction of entirely appropriate lime tree has
Beneficial to large-scale production high-quality lime tree seedling.
This specific embodiment is only explanation of the invention, and it is not limitation of the present invention, people in the art
Member can make the modification without creative contribution to the present embodiment as needed after this specification is read, but as long as at this
All protected in the right of invention by Patent Law.
Claims (8)
1. a kind of tissue culture medium (TCM) of lime tree, including inducing culture, proliferated culture medium and root media, its feature exist
In：The inducing culture includes following component：MS culture mediums：4630~4640mg；Fill in the grand TDZ of benzene：0.001~0.010mg；
Gibberellin：2~3mg；Agar：5~8g；Sucrose：25~40g；Inositol：90~110mg, adds water and complements to 1 liter, the propagation
Culture medium includes following component：MS culture mediums：4630~4640mg；Fill in the grand TDZ of benzene：0.001~0.010mg；Gibberellin：2~
3mg；Agar：5~8g；Sucrose：25~40g；Inositol：90~110mg, adds water and complements to 1 liter, the root media includes
Following component：2315~2320mg；Indolebutyric acid IBA：1.0mg~2.0mg；Methyl α-naphthyl acetate NAA：1.0mg~2.0mg；Agar：5
~8g；Sucrose：10~20g；Add water and complement to 1 liter.
2. a kind of tissue culture medium (TCM) of lime tree according to claim 1, it is characterised in that：The inducing culture is included such as
Lower component：MS culture mediums：4636.43mg；Fill in the grand TDZ of benzene：0.005mg；Gibberellin：2.5mg；Agar：6g；Sucrose：30g；Flesh
Alcohol：100mg, adds water and complements to 1 liter, the proliferated culture medium includes following component：MS culture mediums：4636.43mg；Fill in benzene grand
TDZ：0.005mg；Gibberellin：2.0mg；Agar：6g；Sucrose：30g；Inositol：100mg, adds water and complements to 1 liter, described to take root
Culture medium includes following component：2318.22mg；Indolebutyric acid IBA：1.5mg；Methyl α-naphthyl acetate NAA：1.5mg；Agar：6g；Sucrose：
15g；Add water and complement to 1 liter.
3. a kind of rapid propagation method of lime tree, it is characterised in that：Including following incubation step：
S1：From explant, explant is from the full semi-lignified branch stem apex portion of healthy lime tree current-year branch, sprout
Point；
S2：Sterilization and Initial culture, lime tree explant blade are cut off, clear water is rinsed well, on the table flushed
Explant is put into the alcohol that concentration is 75% and sterilized 30 seconds, then with aseptic water washing 3~4 times, explant is placed on into thimerosal
Middle immersion 60 minutes, completes after sterilization, explant is inoculated into inducing culture；Under the conditions of intensity of illumination 1500Lx, temperature
16 DEG C of degree, half-light culture 1 week, after turn 23 DEG C of temperature, light application time 16h, culture 6 weeks；
S3：Squamous subculture：In inducing culture in 30~40 days, explant is transferred to increasing when young sprout occurs in explant stem apex
Grow in culture medium, under certain culture environment, be transferred to every surrounding in new proliferated culture medium；
S4：Culture of rootage：By the lime tree tissue-cultured seedling of squamous subculture, when terminal bud occurs in explant, 2~3cm terminal bud is taken, is put into
Root media, until completion culture of taking root.
4. a kind of rapid propagation method of lime tree according to claim 3, it is characterised in that：The thimerosal includes as follows
Component：1 part and 3 parts sterilized waters of bleaching water.
5. a kind of rapid propagation method of lime tree according to claim 3, it is characterised in that：Ring is cultivated in the step S3
Border is that, in intensity of illumination 1500Lx, temperature is 25 DEG C, light application time 16 hours, and in dark condition, temperature is 23 DEG C, standing time
8 hours.
6. a kind of rapid propagation method of lime tree according to claim 3, it is characterised in that：The culture medium is by going out
Bacterium is handled, and the culture medium configured is placed in autoclave, in 0.105MPa pressure, in the case that temperature is 121 DEG C, sterilizing
15~30min.
7. a kind of rapid propagation method of lime tree according to claim 3, it is characterised in that：Also added in the culture medium
There is antioxidant, the antioxidant is cysteine.
8. a kind of rapid propagation method of lime tree according to claim 3, it is characterised in that：Also added in the culture medium
There is 0.1~0.5g activated carbon.
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| CN108040715A (en) * | 2017-12-04 | 2018-05-18 | 大新县科学技术情报研究所 | A kind of mountain planting method of buerretiodendron hsienmu |
| CN109220808A (en) * | 2018-11-09 | 2019-01-18 | 安徽东方金桥农林科技股份有限公司 | A kind of lime tree root tissues cultural method |
| CN110663552A (en) * | 2019-11-08 | 2020-01-10 | 中国科学院昆明植物研究所 | Tissue culture and rapid propagation method of Yunnan tung tree |
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| CN108040715A (en) * | 2017-12-04 | 2018-05-18 | 大新县科学技术情报研究所 | A kind of mountain planting method of buerretiodendron hsienmu |
| CN109220808A (en) * | 2018-11-09 | 2019-01-18 | 安徽东方金桥农林科技股份有限公司 | A kind of lime tree root tissues cultural method |
| CN110663552A (en) * | 2019-11-08 | 2020-01-10 | 中国科学院昆明植物研究所 | Tissue culture and rapid propagation method of Yunnan tung tree |
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