CN106386477A - Tissue culture and rapid propagation method for Ziziphus jujube Mill.cv.Jinsi 4 - Google Patents
Tissue culture and rapid propagation method for Ziziphus jujube Mill.cv.Jinsi 4 Download PDFInfo
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- CN106386477A CN106386477A CN201610348279.1A CN201610348279A CN106386477A CN 106386477 A CN106386477 A CN 106386477A CN 201610348279 A CN201610348279 A CN 201610348279A CN 106386477 A CN106386477 A CN 106386477A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
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Abstract
The invention specifically relates to a tissue culture and rapid propagation method for Ziziphus jujube Mill.cv.Jinsi 4, belonging to the technical field of tissue culture of plants. The method comprises the following steps: construction of explants; initiation culture; subculture; rooting culture; seedling hardening; transplanting; etc. According to the invention, research is carried out on the species Ziziphus jujube Mill.cv.Jinsi 4, optimal mediums are used for different stages of the species, and a high propagation coefficient and a high rooting rate are obtained; the method is simple to operate and low in cost and can efficiently and conveniently acquire a great number of tissue culture seedlings of Ziziphus jujube Mill.cv.Jinsi 4; and the obtained tissue culture seedlings are high in survival rate and nursery stock can retain excellent hereditary features of selected trees. The method provided by the invention can realize mass propagation and rapid promotion of Ziziphus jujube Mill.cv.Jinsi 4; and Ziziphus jujube Mill.cv.Jinsi 4 can be planted in northern mountain areas of Guangxi Province, China in large scale, especially in mountain areas with the Karst landform.
Description
Technical field
The invention belongs to plant tissue culture technical field is and in particular to a kind of tissue culture and rapid propagation method of Jujube 4.
Background technology
" spun gold 4 " Zao Shi Shandong Fruit-tree Inst. nineteen ninety selection-breeding from the jujube tree of growing directly from seeds of No. 2 natural hybridizations of spun gold
The fine individual plant going out.Have fruit big, neat, sugary high, quality is excellent, disease-resistant, cracking resistance fruit, result are early, pole high yield, degeneration-resistant, adapt to
The advantages of property is strong.The closely long tubular of Jujube 4 fruit, two hold level with both hands, and middle part is slightly thick.10～12 grams of Mean Fruit Weight, regularity is high.Really
Shoulder is put down and slightly tiltedly, is no substantially obstructed low-lying area, and ring low-lying area is medium deep wide, fruit point nick, fruit surface smooth, and light is gorgeous, peel carefully thin rich toughness,
Ripe phase light green white in vain, is in light brown red after coloring.Pulp white, quality densification is tender and crisp, and juice is more, the extremely sweet micro- acid of taste, no
Miscellaneous taste, the excellent taste such as bitter peppery grass is pungent.Thin skin flesh is rich, and the little meat of core is thick, and delicate fragrance is sweet, both can eat raw, and can make dry, with it again
Excellent quality and abundant nutrition enjoy consumers welcomed." spun gold 4 " Fructus Jujubae has the characteristics that early real, high yield, strong stress resistance,
It is the seeds that south China hillocky and mountainous region develops great development prospect, have become as the main cultivation in south China area Fructus Jujubae kind.
At present, in actual cultivation, " spun gold 4 " Fructus Jujubae is carried out using methods such as root turion Seedling plant division, cuttage and graftings mostly
Breed, using the breeding of root evil seedling separation, need to combine with propagation by grafiting, not only time-consuming, planting percent is relatively low, and easily infect various
Plant viruses, and carry out breeding that breeding potential is very low using methods such as root turion Seedling plant division, cuttage and graftings, the speed of growth is slow, weak
Sick seedling ratio height, it is impossible to carry out whole year production, far can not meet the wilderness demand in market.Thus have impact on it and large-area push away
Extensively.Under this situation, the effective way of quickly breeding breeding nursery stock has then been become using jujube tree tissue culture technique.Tissue culture
（Tissue Culture）It is aseptically, the technology of separation in the medium culture of ex vivo plant tissue, with tissue
Cultural method Fast-propagation, has the genetic identity of whole strain, robust growth, well developed root system, transplanting survival rate are high, nutrient quality
Well, the advantages of yield is high.
The Study on tissue culture of jujube tree starts from the 70's Mos of 2O century, and so far, existing more than 20 kind be not with
Same type explant establishes aseptic in vitro breeding system.Tissue culture method for jujube tree also has pertinent literature to report, such as：1、
【Autograph】Rabdosia japonica group culturation rapid propagating technology is studied【Author】Continuous nine such as, Li Chunli, Sun Jianshe【Source】Beijing Forestry University is learned
Report, 2003,25（3）【Summary】Using tissue culture technique, the group culturation rapid propagating technology of Rabdosia japonica is studied.Prove for 3, April
It is the best period gathering explant in 1 year, when cultivating to explant in this stage, effective germination rate highest.With MS it is
Minimal medium filters out optimum hormone concentration and the proportioning in suitable Rabdosia japonica tissue culture each stage, respectively：Primary culture medium
MS+6-BA 0.8 mg/L+IBA 0.4mg/L；Proliferated culture medium MS+ 6-BA 1.2 mg/L+IBA 0.5mg/L；Root culture
Base 1/2MS+IBA 3.0mg/L simultaneously illustrates the relation of subculture number and growth coefficient and rooting rate, proposes Rabdosia japonica subculture
Number is advisable with about 8 generations, and 8 instead of should carry out root culture afterwards.2、【Autograph】Ziziphi Spinosae tissue-culturing rapid propagation is studied【Author】Dai Li, Liu
Meng Jun, Wang Jiurui, Zhou Junyi【Source】Jouranl of Agricultural University of Hebei, 2005,28（2）【Summary】:With resting bud for trying material, build
Found the tissue culture rapid propagation system of Ziziphi Spinosae.The most suitable primary culture medium is MS+ BA1.0+ IBA0.1Or MS+BA2.0+NAA0.1, propagation training
Foster base is MS+BA2.0-3.0, germination rate and strong bud rate have all exceeded 70%.During enrichment culture, the bud ratio of bottom stem section is higher than upper
Portion's stem section, but the ratio sprouting strong bud is then of a relatively high with top stem section.1/2 MS+IBA0.5-1.0It is Ziziphi Spinosae numerous training of taking root soon
Foster appropriate media, rooting rate reaches as high as 92%.So far, people in terms of the group training research of jujube tree achieved with one
A little achievements, the technology with tissue culture propagation jujube tree improved seeds is progressively applied in production, but, the tissue culture of Fructus Jujubae is more tired
Difficulty, relatively costly, the Reproduction Conditions between different cultivars differ greatly, and the tissue culture method of the jujube tree of different cultivars is also different.Mesh
Before, in breeding of " spun gold 4 " Fructus Jujubae, due to being bred using methods such as traditional root turion Seedling plant division, cuttage and graftings, deposit
Breeding, breeding potential is very low, and the speed of growth is slow, far can not meet market wilderness demand the problems such as；Therefore, develop one kind
Tissue culture and rapid propagation method for " spun gold 4 " Fructus Jujubae carries out culture breeding, can promote the Fast-propagation of excellent genetic stockss, realize
The amount reproduction of " spun gold 4 " jujube tree and Rapid Popularization, are the effective ways of the problems such as solve the market demand.
Content of the invention
It is an object of the invention to provide a kind of easy and simple to handle, low production cost, rooting rate height, breeding potential is high, can be applicable to
The tissue culture and rapid propagation method of the Jujube 4 of large-scale commercial nursery, can effectively, easily be obtained at short notice using the method
Obtain a large amount of Jujube 4 tissue cultured seedlinies, and the tissue culture shoot survival percent being obtained is high, suitable Guangxi karst landform mountain area is planted.
For achieving the above object, the present invention adopts the following technical scheme that：
A kind of tissue culture and rapid propagation method of Jujube 4, comprises the following steps：
(1) foundation of explant：In at the beginning of 3 months, clip 16-18cm length, give birth to the sturdy root turion bar of high-quality then, rinsed with tap water
After 10 minutes, it is to soak 10-13 minute in 0.4% washing powder solution in mass concentration, then is rinsed well with tap water, then will
Root turion bar after cleaning is placed in water planting culture in culturing room, and every 22-26 hour changes a water, and cultivation temperature is 27-28 DEG C, illumination
Time 17-19h/d, intensity of illumination 2000～3500lx, cultivate to growing after the water planting bud of 2～3cm, take out root turion bar, cleaning,
After sterilization, clip water planting bud is as explant；
（2）Primary culture：Water planting bud is proceeded to through in the primary culture medium of high temperature sterilize, the sprouting of evoking adventive bud, cultivate bar
Part is 25～28 DEG C of temperature, and intensity of illumination is 2000-2500lx, light application time 16-17h/d, aseptic training in culturing room
Support, after sprouting sprouts, be transferred to culture in mitogenetic culture medium, be 2000-2200lx in intensity of illumination, light application time
14-16h/d, under conditions of temperature is 27 DEG C, cultivates 40-45 days；
（3）Successive transfer culture：By plant stem section transfer sterilized after subculture medium in cultivate, be 25-26 in cultivation temperature
DEG C, light intensity 2000～25O0lx, light application time is culture 32-34d under conditions of 15h/d；
（4）Root culture：Aseptically, the band base of leaf section that clip long 2-2.5cm successive transfer culture obtains, inserts root culture
Carry out root culture in base；26～28 DEG C of cultivation temperature, light intensity 1800～20001x, light application time 14-16h/d；
（5）Seedling exercising：As rooting tube plantlet root length 1.0～2.0 cm after root culture, tissue cultured seedling is carried out indoor refining
Seedling, seedling exercising temperature is：27-28 DEG C, intensity of illumination is 4000-6000lx, after indoor bottle refines 4-6 days, uncovered seedling exercising 5-7 days；
（6）Transplant：Take out test tube seedling, the culture medium on clean root, being transplanted to mass concentration is 0.2%KMnO4Solution disinfection mistake
Coconut palm chaff+Vermiculitum+thin river sand according to 2-3:5:1 ratio is done in the nutrient cup of substrate, pours water, and holding air humidity is
85%-90%, 25～26 DEG C of temperature, it is to avoid direct sunlight, after 35-38 days, removal greenhouse carries out full exposure seedling exercising 5-6d, and
When water, then transplant land for growing field crops.
The tissue culture and rapid propagation method of described Jujube 4, step（1）Middle cultivate to growing after the water planting bud of 2～3cm to root
The concretely comprising the following steps of tiller bar cleaning and sterilizing：Take out root turion bar, with 75% alcohol-pickled 35s, aseptic water washing 3-4 time, drain the water
Point, being put into mass concentration is 0.1%HgCL2Soaking disinfection 7 minutes in solution, then use aseptic water washing 7-8 time, are pressed from both sides with tweezers
Take cleaned stem section, be placed on the inoculation dish of sterilizing, blot surface moisture, clip water planting bud conduct with the filter paper sterilizing
Explant.
The tissue culture and rapid propagation method of described Jujube 4, step（2）In the composition of primary culture medium be：MS+ N6 benzyl
Base adenine（6-BA) 1.2mg/L+naphthalene acetic acid（NAA）0.15～0.25mg/L+3.5% molasses+agar 6g/L, medium pH
It is worth for 6.0-6.3.
The tissue culture and rapid propagation method of described Jujube 4, step（2）In the composition of mitogenetic culture medium be：MS+ N6Benzyl
Base adenine（6-BA) 2.2mg/L+ naphthalene acetic acid（NAA）0.5mg/L+vitamin C 1.6-1.8mg/L+ glutamic acid 1.7～
2.0mg/L+3.5% molasses+agar 7g/L, culture medium pH value is 6.0.
The tissue culture and rapid propagation method of described Jujube 4, step（3）The composition of middle subculture medium is：MS+ N6Benzyl
Adenine（6-BA) 2.2mg/L+ naphthalene acetic acid（NAA）0.5-0.6mg/L+vitamin C 0.7-0.9mg/L+ vitamin B2
1.5-1.8mg/L+ glutamic acid 1.7～2.0mg/L+3.5% molasses+agar 8g/L, culture medium pH value is 6.1.
The tissue culture and rapid propagation method of described Jujube 4, step（4）The composition of middle root media is：1/2MS+ NAA
0.05mg/L+ IAA0.15 mg/L+IBA 0.7～1.0 mg/L+5% molasses+agar 8g/L, pH=6.0-6.2.
Beneficial effects of the present invention are：
1st, the present invention is a kind of tissue culture and rapid propagation method for " spun gold 4 " Fructus Jujubae, for grinding that " spun gold 4 " Fructus Jujubae kind is carried out
Study carefully, test through continuous, obtained being best suitable for the various culture medium used by this jujube tree tissue-culturing rapid propagation, the culture medium of the present invention is adopted
Carbon source is molasses（The non crystallized containing sugar liquors that sugar refinery sugar industry is separated）, instead of conventional sucrose, not only
Reduce the cost of culture medium, and molasses be rich in nutritional labeling, containing various monosaccharide, thick protein and mineral it is easier to
Utilized by plant.Glutamic acid is all added, glutamic acid can not only provide organic in the mitogenetic culture medium of the present invention and subculture medium
Nitrogen source, the also growth to plant and adventitious bud simultaneously, the differentiation of adventitious embryo plays a driving role, and can by plant cell very
Fast absorption；The vitamin C adding in the medium and vitamin B2, can strengthen the nutrition of culture medium, beneficial to the growth of explant
Develop.
2nd, the tissue culture and rapid propagation method of the Jujube 4 of the present invention can solve the breeding potential existing using traditional mating system
Very low, the problems such as the speed of growth is slow, the tissue culture and rapid propagation method using the Jujube 4 of the present invention can be greatly enhanced spun gold 4
The reproduction speed of number Fructus Jujubae, reduces weak seedling, the proportion of sick seedling, accelerates Rapid Popularization and the exploitation of new varieties；Using
The method carries out culture breeding to Jujube 4, can promote the Fast-propagation of excellent genetic stockss, realizes " spun gold 4
Number " amount reproduction of jujube tree and Rapid Popularization, solve the demand in market.
3rd, the tissue culture and rapid propagation method operation easier of the Jujube 4 of the present invention is low, low production cost, and rooting rate is up to 88%
More than, the tissue culture shoot survival percent of production high it is easy to interior on a large scale promote the use of, using the tissue culture of the Jujube 4 of the present invention
Quick-breeding method can obtain a large amount of neat and consistent, the regeneration plant of stabilization characteristics of genetics, and result of the test is stable, and repeatability is good,
Can be applicable to large-scale commercial nursery, nursery stock can keep the excellent hereditary character of select tree, the big face in suitable In Northern Guangxi mountain area
Long-pending ecological region planting, particularly karst landform mountain area are planted.
Specific embodiment
Embodiment 1
A kind of tissue culture and rapid propagation method of Jujube 4, comprises the following steps：
(1) foundation of explant：In at the beginning of 3 months, clip 16cm length, the then sturdy root turion bar of raw high-quality, with tap water flushing 10
After minute, it is to soak 10 minutes in 0.4% washing powder solution in mass concentration, then is rinsed well with tap water, after then cleaning
Root turion bar be placed in culturing room water planting culture, change one within every 22 hours
Secondary water, cultivation temperature is 27-28 DEG C, light application time 17h/d, and intensity of illumination 3500lx is cultivated to the water growing 2～3cm
After training bud, take out root turion bar, the alcohol-pickled 35s being 75% with mass concentration, aseptic water washing 3 times, drain away the water, be put into matter
Amount concentration is 0.1%HgCL2Soaking disinfection 7 minutes in solution, then use aseptic water washing 7 times, with the stem that tweezers gripping is cleaned
Section, is placed on the inoculation dish of sterilizing, blots surface moisture with the filter paper sterilizing, clip water planting bud is as explant；
（2）Primary culture：Water planting bud is proceeded to through in the primary culture medium of high temperature sterilize（The composition of primary culture medium is：MS+
N6 benzyladenine（6-BA) 1.2mg/L+naphthalene acetic acid（NAA）0.15mg/L+3.5% molasses+agar 6g/L, medium pH
It is worth for 6.0-6.3）The sprouting of evoking adventive bud, condition of culture is 25 DEG C of temperature, and intensity of illumination is 2500lx, light application time
16h/d, aseptic culture in culturing room, after sprouting sprouts, it is transferred to culture in mitogenetic culture medium, mitogenetic culture medium
Composition is：MS+ N6Benzyladenine（6-BA) 2.2mg/L+ naphthalene acetic acid（NAA）0.5mg/L+vitamin C 1.6mg/L+ paddy
Propylhomoserin 2.0mg/L+3.5% molasses+agar 7g/L, culture medium pH value is 6.0；It is 2000lx in intensity of illumination, light application time
16h/d, under conditions of temperature is 27 DEG C, cultivates 40 days；
（3）Successive transfer culture：By plant stem section transfer sterilized after subculture medium in cultivate in, the composition of subculture medium
For：MS+ N6Benzyladenine（6-BA) 2.2mg/L+ naphthalene acetic acid（NAA）0.5mg/L+vitamin C 0.7mg/L+ vitamin
B2 1.8mg/L+ glutamic acid 1.8mg/L+3.5% molasses+agar 8g/L, culture medium pH value is 6.1；It is 25- in cultivation temperature
26 DEG C, light intensity 2000lx, light application time is culture 34d under conditions of 15h/d；
（4）Root culture：Aseptically, the band base of leaf section that clip long 2-2.5cm successive transfer culture obtains, inserts root culture
Carry out root culture in base；The composition of root media is：1/2MS+ NAA 0.05mg/L+ IAA0.15 mg/L+IBA
0.7mg/L+5% molasses+agar 8g/L, pH=6.0-6.2；26～28 DEG C of cultivation temperature, light intensity 18001x, light application time 16h/
d；
（5）Seedling exercising：As rooting tube plantlet root length 1.0～2.0 cm after root culture, tissue cultured seedling is carried out indoor refining
Seedling, seedling exercising temperature is：27-28 DEG C, intensity of illumination is 4000lx, after indoor bottle refines 6 days, uncovered seedling exercising 5 days；
（6）Transplant：Take out test tube seedling, the culture medium on clean root, being transplanted to mass concentration is 0.2%KMnO4Solution disinfection mistake
Coconut palm chaff+Vermiculitum+thin river sand according to 2:5:1 ratio is done in the nutrient cup of substrate, pours water, and holding air humidity is 85%-
90%, 25～26 DEG C of temperature, it is to avoid direct sunlight, after 35 days, removal greenhouse carries out full exposure seedling exercising 5d, waters in time, then
Transplant land for growing field crops.
Embodiment 2
A kind of tissue culture and rapid propagation method of Jujube 4, comprises the following steps：
(1) foundation of explant：In at the beginning of 3 months, clip 17cm length, the then sturdy root turion bar of raw high-quality, with tap water flushing 10
After minute, it is to soak 12 minutes in 0.4% washing powder solution in mass concentration, then is rinsed well with tap water, after then cleaning
Root turion bar be placed in culturing room water planting culture, change one within every 24 hours
Secondary water, cultivation temperature is 27-28 DEG C, light application time 18h/d, and intensity of illumination 3000lx is cultivated to the water growing 2～3cm
After training bud, take out root turion bar, with 75% alcohol-pickled 35s, aseptic water washing 4 times, drain away the water, being put into mass concentration is
0.1%HgCL2Soaking disinfection 7 minutes in solution, then use aseptic water washing 8 times, with the stem section that tweezers gripping is cleaned, place
On the inoculation dish of sterilizing, blot surface moisture with the filter paper sterilizing, clip water planting bud is as explant；
（2）Primary culture：Water planting bud is proceeded to through in the primary culture medium of high temperature sterilize（The composition of primary culture medium is：MS+
N6 benzyladenine（6-BA) 1.2mg/L+naphthalene acetic acid（NAA）0.20mg/L+3.5% molasses+agar 6g/L, medium pH
It is worth for 6.0-6.3）The sprouting of evoking adventive bud, condition of culture is 25～28 DEG C of temperature, and intensity of illumination is 2300lx, during illumination
Between 16h/d, aseptic culture in culturing room, after sprouting sprouts, be transferred in mitogenetic culture medium culture, mitogenetic culture medium
Composition be：MS+ N6Benzyladenine（6-BA) 2.2mg/L+ naphthalene acetic acid（NAA）0.5mg/L+vitamin C 1.7mg/L+
Glutamic acid 1.8mg/L+3.5% molasses+agar 7g/L, culture medium pH value is 6.0；It is 2100lx in intensity of illumination, light application time
15h/d, under conditions of temperature is 27 DEG C, cultivates 43 days；
（3）Successive transfer culture：By plant stem section transfer sterilized after subculture medium in cultivate in, the composition of subculture medium
For：MS+ N6Benzyladenine（6-BA) 2.2mg/L+ naphthalene acetic acid（NAA）0.6mg/L+vitamin C 0.8mg/L+ vitamin
B2 1.7mg/L+ glutamic acid 1.9mg/L+3.5% molasses+agar 8g/L, culture medium pH value is 6.1；It is 25- in cultivation temperature
26 DEG C, light intensity 23O0lx, light application time is culture 33d under conditions of 15h/d；
（4）Root culture：Aseptically, the band base of leaf section that clip long 2-2.5cm successive transfer culture obtains, inserts root culture
Carry out root culture in base；The composition of root media is：1/2MS+ NAA 0.05mg/L+ IAA0.15 mg/L+IBA
0.8mg/L+5% molasses+agar 8g/L, pH=6.0-6.2；27 DEG C of cultivation temperature, light intensity 19001x, light application time 15h/d；
（5）Seedling exercising：As the rooting tube plantlet root length 1.0～2.0cm after root culture, tissue cultured seedling is carried out indoor refining
Seedling, seedling exercising temperature is：27-28 DEG C, intensity of illumination is 5000lx, after indoor bottle refines 5 days, uncovered seedling exercising 6 days；
（6）Transplant：Take out test tube seedling, the culture medium on clean root, being transplanted to mass concentration is 0.2%KMnO4Solution disinfection mistake
Coconut palm chaff+Vermiculitum+thin river sand according to 3:5:1 ratio is done in the nutrient cup of substrate, pours water, and holding air humidity is 85%-
90%, 25～26 DEG C of temperature, it is to avoid direct sunlight, after 37 days, removal greenhouse carries out full exposure seedling exercising 6d, waters in time, then
Transplant land for growing field crops.
Embodiment 3
A kind of tissue culture and rapid propagation method of Jujube 4, comprises the following steps：
(1) foundation of explant：In at the beginning of 3 months, clip 18cm length, the then sturdy root turion bar of raw high-quality, with tap water flushing 10
After minute, it is to soak 13 minutes in 0.4% washing powder solution in mass concentration, then is rinsed well with tap water, after then cleaning
Root turion bar be placed in culturing room water planting culture, change one within every 26 hours
Secondary water, cultivation temperature is 27-28 DEG C, light application time 19h/d, and intensity of illumination 2000lx is cultivated to the water growing 2～3cm
After training bud, take out root turion bar, the alcohol-pickled 35s being 75% with mass concentration, aseptic water washing 4 times, drain away the water, be put into matter
Amount concentration is 0.1%HgCL2Soaking disinfection 7 minutes in solution, then use aseptic water washing 7 times, with the stem that tweezers gripping is cleaned
Section, is placed on the inoculation dish of sterilizing, blots surface moisture with the filter paper sterilizing, clip water planting bud is as explant；
（2）Primary culture：Water planting bud is proceeded to through in the primary culture medium of high temperature sterilize（The composition of primary culture medium is：MS+
N6 benzyladenine（6-BA) 1.2mg/L+naphthalene acetic acid（NAA）0.25mg/L+3.5% molasses+agar 6g/L, medium pH
It is worth for 6.0-6.3）The sprouting of evoking adventive bud, condition of culture is 25～28 DEG C of temperature, and intensity of illumination is 2500lx, during illumination
Between 16h/d, aseptic culture in culturing room, after sprouting sprouts, be transferred in mitogenetic culture medium culture, mitogenetic culture medium
Composition be：MS+ N6Benzyladenine（6-BA) 2.2mg/L+ naphthalene acetic acid（NAA）0.5mg/L+vitamin C 1.8mg/L+
Glutamic acid 1.7mg/L+3.5% molasses+agar 7g/L, culture medium pH value is 6.0；It is 2200lx in intensity of illumination, light application time
14h/d, under conditions of temperature is 27 DEG C, cultivates 45 days；
（3）Successive transfer culture：By plant stem section transfer sterilized after subculture medium in cultivate in, the composition of subculture medium
For：MS+ N6Benzyladenine（6-BA) 2.2mg/L+ naphthalene acetic acid（NAA）0.5mg/L+vitamin C 0.9mg/L+ vitamin
B2 1.5mg/L+ glutamic acid 2.0mg/L+3.5% molasses+agar 8g/L, culture medium pH value is 6.1；It is 25- in cultivation temperature
26 DEG C, light intensity 25O0lx, light application time is culture 32d under conditions of 15h/d；
（4）Root culture：Aseptically, the band base of leaf section that clip long 2-2.5cm successive transfer culture obtains, inserts root culture
Carry out root culture in base；The composition of root media is：1/2MS+ NAA 0.05mg/L+ IAA0.15 mg/L+IBA1.0
Mg/L+5% molasses+agar 8g/L, pH=6.0-6.2；26～28 DEG C of cultivation temperature, light intensity 20001x, light application time 14h/d；
（5）Seedling exercising：As rooting tube plantlet root length 1.0～2.0 cm after root culture, tissue cultured seedling is carried out indoor refining
Seedling, seedling exercising temperature is：27-28 DEG C, intensity of illumination is 6000lx, after indoor bottle refines 4 days, uncovered seedling exercising 7 days；
（6）Transplant：Take out test tube seedling, the culture medium on clean root, being transplanted to mass concentration is 0.2%KMnO4Solution disinfection mistake
Coconut palm chaff+Vermiculitum+thin river sand according to 3:5:1 ratio is done in the nutrient cup of substrate, pours water, and holding air humidity is 85%-
90%, 25～26 DEG C of temperature, it is to avoid direct sunlight, after 38 days, removal greenhouse carries out full exposure seedling exercising 6d, waters in time, then
Transplant land for growing field crops.
The breeding results of the Jujube 4 tissue cultured seedling below obtaining for the tissue culture and rapid propagation method of Jujube 4 of the present invention：
Plantation place：Laibin city of Guangxi Xiangzhou County（Xiangzhou County is located at In Northern Guangxi, minimum subzero 2 degree of weather winter, summer 32
Degree, hills hillside is more, suitable jujube growth）.
As can be seen from the above table, the tissue culture and rapid propagation method of Jujube 4 of the present invention, breeding coefficient is high, and rooting rate reaches more than 88%,
More than 86%, field-transplanting survival rate, more than 89%, can adapt to rapidly outdoor nutrient cup transplanting survival rate after tissue culture transplantation of seedlings
Environment, growth is rapid, and anti-external world poor environment ability is strong.Using the method for the present invention, Jujube 4 is bred, can have
Imitate, easily obtain a large amount of Jujube 4 tissue cultured seedlinies at short notice, and nursery stock can keep the excellent heritability of select tree
Shape, suitable large area region plantation, drive the production of fruit trees of locality.
Claims (6)
1. a kind of tissue culture and rapid propagation method of Jujube 4 is it is characterised in that comprise the following steps：
(1) foundation of explant：In at the beginning of 3 months, clip 16-18cm length, give birth to the sturdy root turion bar of high-quality then, rinsed with tap water
After 10 minutes, it is to soak 10-13 minute in 0.4% washing powder solution in mass concentration, then is rinsed well with tap water, then will
Root turion bar after cleaning is placed in water planting culture in culturing room, and every 22-26 hour changes a water, and cultivation temperature is 27-28 DEG C, illumination
Time 17-19h/d, intensity of illumination 2000～3500lx, cultivate to growing after the water planting bud of 2～3cm, take out root turion bar, cleaning,
After sterilization, clip water planting bud is as explant；
（2）Primary culture：Water planting bud is proceeded to through in the primary culture medium of high temperature sterilize, the sprouting of evoking adventive bud, cultivate bar
Part is 25～28 DEG C of temperature, and intensity of illumination is 2000-2500lx, light application time 16-17h/d, aseptic training in culturing room
Support, after sprouting sprouts, be transferred to culture in mitogenetic culture medium, be 2000-2200lx in intensity of illumination, light application time
14-16h/d, under conditions of temperature is 27 DEG C, cultivates 40-45 days；
（3）Successive transfer culture：By plant stem section transfer sterilized after subculture medium in cultivate, be 25-26 in cultivation temperature
DEG C, light intensity 2000～25O0lx, light application time is culture 32-34d under conditions of 15h/d；
（4）Root culture：Aseptically, the band base of leaf section that clip long 2-2.5cm successive transfer culture obtains, inserts root culture
Carry out root culture in base；26～28 DEG C of cultivation temperature, light intensity 1800～20001x, light application time 14-16h/d；
（5）Seedling exercising：As rooting tube plantlet root length 1.0～2.0 cm after root culture, tissue cultured seedling is carried out indoor refining
Seedling, seedling exercising temperature is：27-28 DEG C, intensity of illumination is 4000-6000lx, after indoor bottle refines 4-6 days, uncovered seedling exercising 5-7 days；
（6）Transplant：Take out test tube seedling, the culture medium on clean root, being transplanted to mass concentration is 0.2%KMnO4Solution disinfection mistake
Coconut palm chaff+Vermiculitum+thin river sand according to 2-3:5:1 ratio is done in the nutrient cup of substrate, pours water, and holding air humidity is
85%-90%, 25～26 DEG C of temperature, it is to avoid direct sunlight, after 35-38 days, removal greenhouse carries out full exposure seedling exercising 5-6d, and
When water, then transplant land for growing field crops.
2. the tissue culture and rapid propagation method of Jujube 4 according to claim 1 is it is characterised in that described step（1）Middle culture
Root turion bar cleaning and sterilizing is concretely comprised the following steps after the water planting bud of 2～3cm to growing：Take out root turion bar, with 75% alcohol-pickled
35s, aseptic water washing 3-4 time, drain away the water, being put into mass concentration is 0.1%HgCL2Soaking disinfection 7 minutes in solution, then
With aseptic water washing 7-8 time, with the stem section that tweezers gripping is cleaned, it is placed on the inoculation dish of sterilizing, with the filter paper sterilizing
Blot surface moisture, clip water planting bud is as explant.
3. the tissue culture and rapid propagation method of Jujube 4 according to claim 1 is it is characterised in that described step（2）In open
The composition of dynamic culture medium is：MS+ N6 benzyladenine 1.2mg/L+naphthalene acetic acid 0.15～0.25mg/L+3.5% molasses+agar
6g/L, culture medium pH value is 6.0-6.3.
4. the tissue culture and rapid propagation method of Jujube 4 according to claim 1 is it is characterised in that described step（2）In point
The composition of raw culture medium is：MS+ N6Benzyladenine 2.2mg/L+ naphthalene acetic acid 0.5mg/L+vitamin C 1.6-1.8mg/L+
Glutamic acid 1.7～2.0mg/L+3.5% molasses+agar 7g/L, culture medium pH value is 6.0.
5. the tissue culture and rapid propagation method of Jujube 4 according to claim 1 is it is characterised in that described step（3）Middle subculture
The composition of culture medium is：MS+ N6Benzyladenine 2.2mg/L+ naphthalene acetic acid 0.5-0.6mg/L+vitamin C 0.7-0.9mg/L
+ vitamin B2 1.5-1.8mg/L+ glutamic acid 1.7～2.0mg/L+3.5% molasses+agar 8g/L, culture medium pH value is
6.1.
6. the tissue culture and rapid propagation method of Jujube 4 according to claim 1 is it is characterised in that described step（4）In take root
The composition of culture medium is：1/2MS+ NAA 0.05mg/L+ IAA0.15 mg/L+IBA 0.7～1.0 mg/L+5% molasses+fine jade
Fat 8g/L, pH=6.0-6.2.
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| CN103190347A (en) * | 2013-04-26 | 2013-07-10 | 北京林业大学 | Teapot dates tissue culturing method |
| CN103461124A (en) * | 2013-09-15 | 2013-12-25 | 德州市农业科学研究院 | Tissue culture and rapid propagation process for seedless golden silk jujube |
| CN104381126A (en) * | 2014-10-06 | 2015-03-04 | 新疆农垦科学院 | Jun date virus-free tissue culture rapid-propagation technology |
| CN104996300A (en) * | 2015-07-27 | 2015-10-28 | 合肥一诺生物科技有限公司 | Tissue cultivating method of choerospondias axillaries |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103190347A (en) * | 2013-04-26 | 2013-07-10 | 北京林业大学 | Teapot dates tissue culturing method |
| CN103461124A (en) * | 2013-09-15 | 2013-12-25 | 德州市农业科学研究院 | Tissue culture and rapid propagation process for seedless golden silk jujube |
| CN104381126A (en) * | 2014-10-06 | 2015-03-04 | 新疆农垦科学院 | Jun date virus-free tissue culture rapid-propagation technology |
| CN104996300A (en) * | 2015-07-27 | 2015-10-28 | 合肥一诺生物科技有限公司 | Tissue cultivating method of choerospondias axillaries |
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