CN102972293A - Rapid propagation method for cultivating tissue culture seedling by utilizing Mokara axillary bud tissue - Google Patents
Rapid propagation method for cultivating tissue culture seedling by utilizing Mokara axillary bud tissue Download PDFInfo
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- CN102972293A CN102972293A CN201210508576XA CN201210508576A CN102972293A CN 102972293 A CN102972293 A CN 102972293A CN 201210508576X A CN201210508576X A CN 201210508576XA CN 201210508576 A CN201210508576 A CN 201210508576A CN 102972293 A CN102972293 A CN 102972293A
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Abstract
The invention belongs to the technical field of plant cultivation, and relates to a rapid propagation method for cultivating a tissue culture seedling by utilizing a Mokara axillary bud tissue. The method comprises an axillary bud inducing process, a protocorm propagation process, a process of inducing cluster bud with protocorm, a strong seedling cultivation process, and a rooting cultivation process. The Mokara axillary bud tissue is used as an explant; and the Mokara seedling is rapidly bred by protocorm differentiation. Therefore, the propagation speed and the propagation quantity of the Mokara seedling are improved; a rapid propagation system of Mokara tissue culture is built; an effective path is provided for large-scale industrialized production of the Mokara seedling, and the rapid propagation method has great economic benefits.
Description
Technical field
The invention belongs to field of plant growing technology, relate to group training seedling (test-tube plantlet) propagation method in the Plant Biotechnology, be specifically related to a kind of method for quickly breeding that utilizes the blue axillary bud tissue cultivation of Mohs group training seedling.
Background technology
Mohs blue (Mokara) is to be encouraged with blue, the bird tongue is blue, three genus of Hymenocallis americana hybridize out novel orchid all ages by the senior orchid breeding Hou Wei of man of Singapore.The Mohs orchid is with the colored appearance of its uniqueness in recent ten years, and all over the world fashionable, cultivated area and the market demand go up year by year, have risen to one of main orchid cutting flower variety.At present, the propagation method of Mohs orchid mainly adopts traditional single stem to cut off propagation method, be to get the blue plant of the Mohs of growth more than 3～4 years, and robust plant, health of root are flourishing, then carry out single stem and cut off, a strain is once only to cut off 1～2 section, the segment plant that cuts off will grow and just can bloom in 1～2 year, and productive rate is low, length consuming time, cost is high, and efficient is low.
Development along with the industry of flowers and plants, improving constantly of living standards of the people, flowers demand in daily life constantly increases, and the blue traditional single stem of Mohs cuts off propagation method and can not meet the needs of production, and Plant Tissue Breeding becomes the new way of the blue seedling Fast-propagation of Mohs.Plant Tissue Breeding is the plant Sterile Culture Methods Used, claim again cultured in vitro, organ (such as root, stem, leaf, stem apex, flower, fruit etc.) tissue (as forming layer, epidermis, cortex, marrow cell, endosperm etc.) or cell (such as megaspore, microspore, somatic cell etc.) and the protoplast that utilizes plant corpus to exsomatize, under the artificial conditions such as aseptic and suitable synthetic medium and illumination, temperature, can induce callus, indefinite bud, adventive root, form at last the subject of complete plant.At present the domestic research report that the blue tissue of Mohs is cultivated seldom, abroad for the research many places of Mohs orchid in the research to its flower, also relatively less to the report that plant tissue cultivates.
Summary of the invention
The objective of the invention is to provide for the deficiencies in the prior art a kind of method for quickly breeding that utilizes the blue axillary bud tissue cultivation of Mohs group training seedling, take the blue axillalry bud of Mohs as explant, by inducing axillalry bud to produce protocorm, become Multiple Buds by protocorm differentiation again, form at last the group training seedling with root system, effectively improved the reproductive efficiency of the blue factorial seedling growth of Mohs, fill up the technological gap that the blue tissue of Mohs is cultivated, no matter in quantity or on the time, this propagation method will obviously be better than the propagation method that traditional single stem cuts off, and has higher commercial value.
The present invention is the technical scheme that adopts:
A kind of method for quickly breeding that utilizes the blue axillary bud tissue cultivation of Mohs group training seedling comprises axillalry bud Induction Process, Protocorm Multiplication process, protocorm induced bundle sprout process, strong seedling culture process, process of rooting culture, and its concrete steps are as follows:
1, axillalry bud Induction Process
To be inoculated on the inducing culture through the axillalry bud of sterilization and cultivate condition of culture: dark cultivation, 23～27 ℃ of temperature; Be cultured to the axillalry bud Surface Differentiation and go out protocorm.Described inducing culture is to add coconut milk 1～200ml/L, a-methyl α-naphthyl acetate 0.02～0.5mg/L, 6-benzyl aminopurine 1～5mg/L, adenine 0.5～3mg/L on the basis of basal medium.
Composition and the content of described basal medium are respectively: 1. macroelement: KNO
3450～1000mg/L, KH
2PO
4150～250mg/L, (Ca)
3PO
4150～250mg/L, MgSO
47H
2O200～370mg/L, (NH)
2SO
4350～800 mg/L; 2. micro-: FeSO
47H
2O18.5～27.8mg/L, Na
2-EDTA24.86～37.3mg/L, MnSO
4H
2O7.5～14.8mg/L, ZnSO
47H
2O1～3.2mg/L, H
3BO
31～2.5mg/L; 3. inositol 1～250mg/L; 4. sugar 1～20g/L; 5. agar 4.5～5.5g/L; PH is 5.1～5.4.
2, Protocorm Multiplication process
Protocorm is inoculated in breeds cultivation on the proliferated culture medium, condition of culture: secretly cultivate 23～27 ℃ of temperature; 40～50d shifts once.Described proliferated culture medium is to add coconut milk 1～200ml/L, peptone 1～2.00g/L, a-methyl α-naphthyl acetate 0.02～0.5mg/L, 6-benzyl aminopurine 1～5mg/L, adenine 0.5～3mg/L on the basis of basal medium.
3, the protocorm induced bundle process of sprouting
Protocorm after the propagation is inoculated in carries out inducing clumping bud on the inducing clumping bud medium and cultivate, to being differentiated to form budlet, condition of culture: light is cultivated, and intensity of illumination is 1000～2000Lx, 23～27 ℃ of temperature.Described inducing clumping bud medium is to add coconut milk 1～200ml/L, peptone 1～2.00g/L, banana 10～100g/L, potato 10～100g/L on the basis of basal medium.
4, strong seedling culture process
Multiple Buds is inoculated in carries out strong seedling culture on the strong seedling culture base, condition of culture: light is cultivated, and intensity of illumination is 2000～3000Lx, 26～29 ℃ of temperature; After cultivating 35～45d, budlet grows up to the high seedling of 3～5 cm, robust plant.Described strong seedling culture base is to add banana 10～100g/L, potato 10～100g/L, active carbon 1.0～2.0g/L on the basis of basal medium.
5, process of rooting culture
Seedling is transferred to carries out culture of rootage in the root media, condition of culture: light is cultivated, and intensity of illumination is 2000～3000Lx, 26～29 ℃ of temperature; Behind the culture of rootage 60d, the seedling base portion produces a large amount of root systems, forms complete plant.Described root media is to add banana 10～100g/L, potato 10～100g/L, a-methyl α-naphthyl acetate 0.02～0.5mg/L, active carbon 1.0～2.0g/L on the basis of basal medium.
To take root according to routine group training method for transplanting, seedling carries out hardening, the seedling that dries in the air, and then is transplanted to the land for growing field crops and plants.
The present invention is take the blue axillary bud tissue of Mohs as explant, connect the blue seedling of differentiation Fast-propagation Mohs by protocorm, improve reproduction speed and the breeding quantity of the blue seedling of Mohs, set up the rapid propagation system of the blue group training of Mohs, for the large-scale industrialized production of the blue seedling of Mohs provides an effective way, has larger economic benefit.
Description of drawings
Fig. 1 is that axillalry bud is induced design sketch.
Fig. 2 is that protocorm is induced design sketch.
Fig. 3 is the inducing clumping bud design sketch.
Fig. 4 is the strong seedling culture design sketch.
Fig. 5 is the culture of rootage design sketch.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples are used for explanation the present invention, but are not used for limiting the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
1, macroelement composition, Trace Elements, molysite composition and organic components in the basal medium are mixed with the deposit mother liquor by 20 times, 200 times, 200 times and 100 times of concentration respectively, growth regulator 6-benzyl aminopurine, a-methyl α-naphthyl acetate, adenine are mixed with respectively the deposit mother liquor of 1mg/mL, 0.5mg/mL, 1mg/mL, are stored in 4 ℃ the refrigerator.1mol/L watery hydrochloric acid, 1mol/L sodium hydroxide are mainly used in the adjusting of basal medium pH value.
2, utilize conventional medium compound method, prepare corresponding medium of each stage, wherein basal medium is prepared according to the following formulation:
1. macroelement: KNO
3525mg/L; KH
2PO
4250mg/L; (Ca)
3PO
4200 mg/L; MgSO
47H
2O 250 mg/L; (NH)
2SO
4500mg/L; 2. micro-: FeSO
47H
2O 27.0mg/L; Na
2-EDTA 37.0 mg/L; MnSO
4H
2O 10.0mg/L; ZnSO
47H
2O1.0mg/L; H
3BO
31.0mg/L; 3. inositol 100mg/L; 4. sugared 10g/L; 5. agar powder 5.0g/L; PH is adjusted to 5.2.
Embodiment one
1, axillalry bud is induced: blue stem top 30 cm of intercepting Mohs, divest all blades and expose stem, get each axillalry bud that saves as explant, alcohol disinfecting 30 s with 75%, water flushing 1～2 time, at 15 min that sterilize with 2% liquor natrii hypochloritis, water flushing 3～4 times is at last with 2% liquor natrii hypochloritis, 3 min that sterilize, water flushing 3～4 times, whole disinfecting process discontinuous is shaken triangular flask, can make so the abundant contact sterilizing liquid of explant, thereby reach better Disinfection Effect.The stem section that disinfects is cut into the stem section that 4cm and middle part have 2 axillalry buds at least, then the axillalry bud on the stem section is carefully extracted, do not want damaged growing point, be inoculated on the inducing culture and cultivate, 2 axillalry buds of every bottle of culture medium inoculated, through the cultivation of inducing about 15 d, axillalry bud begins to expand, cultivating 30 d left and right sides axillalry buds expands obviously, have bud point to form gradually projection, the axillalry bud that expands behind 45 d is sprouted and is grown up to budlet, and dedifferentiation occurs around the axillalry bud, its Surface Differentiation goes out to have a small amount of protocorm, and growing way is stronger.The whole incubation of inducing is dark cultivation, 25 ± 1 ℃ of cultivation temperature.Induce the differentiation ratio to be about 1:3.The results are shown in Table 1.
Described inducing culture is to add coconut milk 100ml/L, a-methyl α-naphthyl acetate 0.1mg/L, 6-benzyl aminopurine 2.5mg/L, adenine 1mg/L on the basis of basal medium.
2, Protocorm Multiplication: protocorm transferred on the proliferated culture medium cultivate, behind the 45d, protocorm can be bred 5 times, only has the fraction protocorm differentiation to become budlet, and budlet is relatively more healthy and stronger, and this class budlet can be used for strong seedling culture.Can be according to the actual conditions propagation of repeatedly transferring, general 45d switching once, propagation is cultivated as dark and is cultivated 25 ± 1 ℃ of cultivation temperature.The results are shown in Table 1.
Described proliferated culture medium is to add coconut milk 100ml/L, peptone 1.0g/L, a-methyl α-naphthyl acetate 0.05mg/L, 6-benzyl aminopurine 1.0mg/L, adenine 1.0mg/L on the basis of basal medium.
3, the protocorm induced bundle is sprouted: the protocorm after will breeding is transferred on the inducing clumping bud medium and is divided into budlet, cultivates the again differentiation that occurs leaf sheath about 35d, and 50d left and right sides leaf stretches gradually, can obviously observe budlet about 60d and form.The cultivation intensity of illumination in this stage is 1000～2000Lx, 25 ± 1 ℃ of cultivation temperature.Induce survival rate more than 90%.The results are shown in Table 1.
Described inducing clumping bud medium is to add coconut milk 100ml/L, peptone 1.0g/L, banana 20g/L, potato 10g/L on the basis of basal medium.
4, strong seedling culture: will induce the Multiple Buds of generation to be inoculated in and carry out strong seedling culture on the strong seedling culture base, cultivate 35～45d after, budlet grows up to the high seedling of 3～5 cm, robust plant, intensity of illumination 2000～3000 Lx in this stage, 27 ± 1 ℃ of cultivation temperature.
Described strong seedling culture base is to add banana 30g/L, potato 20g/L, activated carbon 1.5g/L on the basis of basal medium.
5, culture of rootage: the seedling that 3～5 cm are high is transferred to and carries out culture of rootage in the root media, intensity of illumination 2000～3000Lx, 27 ± 1 ℃ of cultivation temperature.Cultivate the 30d rooting rate and can reach 100 %, behind cultivation 35～45d, 2～3 roots more than 3 centimetres are arranged, through the cultivation of 60d, the seedling base portion can produce a large amount of root systems, forms complete plant.The results are shown in Table 2.
Described root media is to add banana 50g/L, potato 30g/L, a-methyl α-naphthyl acetate 0.3mg/L, activated carbon 1.5g/L on the basis of basal medium.
Tissue-culture container seedling moved on to carry out by a definite date 15 days hardening in the greenhouse.After the hardening, the careful seedling that takes out, with the medium on the clear water flushing base portion root system, be mixed with again about 2 minutes seedlings that can dry in the air of liquid immersion of 1500 times with 70% Cercobinm, then with sphagna root system is wrapped and be transplanted in the dish of cave, intensity of illumination 4000～5000Lx, 25～30 ℃ of temperature.Transplanting survival rate reaches more than 95%.The results are shown in Table 2.
Embodiment two
Implementation step and tissue-culture container seedling transplanting method be with embodiment one, the results are shown in Table 1, table 2.The medium in basal medium and each stage is respectively:
Basal medium: 1. macroelement: KNO
3685mg/L; KH
2PO
4200mg/L; (Ca)
3PO
4180 mg/L; MgSO
47H
2O 300 mg/L; (NH)
2SO
4400mg/L; 2. micro-: FeSO
47H
2O 22.0mg/L; Na
2-EDTA 31.0 mg/L; MnSO
4H
2O 11.0mg/L; ZnSO
47H
2O2.0mg/L; H
3BO
31.5mg/L; 3. inositol 150mg/L; 4. sugared 15g/L; 5. agar powder 5.0g/L; PH is adjusted to 5.4.
Inducing culture is to add coconut milk 150ml/L, a-methyl α-naphthyl acetate 0.3mg/L, 6-benzyl aminopurine 3.5mg/L, adenine 2mg/L on the basis of basal medium.
Proliferated culture medium is to add coconut milk 130ml/L, peptone 1.5g/L, a-methyl α-naphthyl acetate 0.2mg/L, 6-benzyl aminopurine 3.0mg/L, adenine 2.5mg/L on the basis of basal medium.
The inducing clumping bud medium is to add coconut milk 180ml/L, peptone 1.0g/L, banana 50g/L, potato 40g/L on the basis of basal medium.
The strong seedling culture base is to add banana 60g/L, potato 40g/L, activated carbon 1.0g/L on the basis of basal medium.
Root media is to add banana 20g/L, potato 40g/L, a-methyl α-naphthyl acetate 0.1mg/L, activated carbon 2.0g/L on the basis of basal medium.
Embodiment three
Implementation step and tissue-culture container seedling transplanting method be with embodiment one, the results are shown in Table 1, table 2.The medium in basal medium and each stage is respectively:
Basal medium: 1. macroelement: KNO
3890mg/L; KH
2PO
4170mg/L; (Ca)
3PO
4250 mg/L; MgSO
47H
2O 350 mg/L; (NH)
2SO
4700mg/L; 2. micro-: FeSO
47H
2O 20.0mg/L; Na
2-EDTA 28.0 mg/L; MnSO
4H
2O 8.0mg/L; ZnSO
47H
2O2.5mg/L; H
3BO
32.0mg/L; 3. inositol 200mg/L; 4. sugared 16g/L; 5. agar powder 4.8g/L; PH is adjusted to 5.3.
Inducing culture is to add coconut milk 180ml/L, a-methyl α-naphthyl acetate 0.2mg/L, 6-benzyl aminopurine 4.0mg/L, adenine 1.5mg/L on the basis of basal medium.
Proliferated culture medium is to add coconut milk 160ml/L, peptone 1.2g/L, a-methyl α-naphthyl acetate 0.3mg/L, 6-benzyl aminopurine 2.0mg/L, adenine 2.0mg/L on the basis of basal medium.
The inducing clumping bud medium is to add coconut milk 150ml/L, peptone 2.0g/L, banana 70g/L, potato 60g/L on the basis of basal medium.
The strong seedling culture base is to add banana 50g/L, potato 70g/L, activated carbon 1.5g/L on the basis of basal medium.
Root media is to add banana 70g/L, potato 50g/L, a-methyl α-naphthyl acetate 0.3mg/L, activated carbon, 1.0g/L on the basis of basal medium.
The blue axillary bud tissue of table 1 Mohs is cultivated inductivity
The blue axillary bud tissue of table 2 Mohs is cultivated inductivity and transplanting survival rate
The above only is preferred embodiment of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (1)
1. method for quickly breeding that utilizes the blue axillary bud tissue cultivation of Mohs group training seedling comprises axillalry bud Induction Process, Protocorm Multiplication process, protocorm induced bundle sprout process, strong seedling culture process, process of rooting culture, and its concrete steps are as follows:
1), axillalry bud Induction Process
To be inoculated on the inducing culture through the axillalry bud of sterilization and cultivate condition of culture: dark cultivation, 23～27 ℃ of temperature; Be cultured to the axillalry bud Surface Differentiation and go out protocorm; Described inducing culture is to add coconut milk 1～200ml/L, a-methyl α-naphthyl acetate 0.02～0.5mg/L, 6-benzyl aminopurine 1～5mg/L, adenine 0.5～3mg/L on the basis of basal medium;
Composition and the content of described basal medium are respectively: 1. macroelement: KNO
3450～1000mg/L, KH
2PO
4150～250mg/L, (Ca)
3PO
4150～250mg/L, MgSO
47H
2O200～370mg/L, (NH)
2SO
4350～800 mg/L; 2. micro-: FeSO
47H
2O18.5～27.8mg/L, Na
2-EDTA24.86～37.3mg/L, MnSO
4H
2O7.5～14.8mg/L, ZnSO
47H
2O1～3.2mg/L, H
3BO
31～2.5mg/L; 3. inositol 1～250mg/L; 4. sugar 1～20g/L; 5. agar 4.5～5.5g/L; PH is 5.1～5.4;
2), Protocorm Multiplication process
Protocorm is inoculated in breeds cultivation on the proliferated culture medium, condition of culture: secretly cultivate 23～27 ℃ of temperature; 40～50d shifts once; Described proliferated culture medium is to add coconut milk 1～200ml/L, peptone 1～2.00g/L, a-methyl α-naphthyl acetate 0.02～0.5mg/L, 6-benzyl aminopurine 1～5mg/L, adenine 0.5～3mg/L on the basis of basal medium;
3), the protocorm induced bundle process of sprouting
Protocorm after the propagation is inoculated in carries out inducing clumping bud on the inducing clumping bud medium and cultivate, to being differentiated to form budlet, condition of culture: light is cultivated, and intensity of illumination is 1000～2000Lx, 23～27 ℃ of temperature; Described inducing clumping bud medium is to add coconut milk 1～200ml/L, peptone 1～2.00g/L, banana 10～100g/L, potato 10～100g/L on the basis of basal medium;
4), strong seedling culture process
Multiple Buds is inoculated in carries out strong seedling culture on the strong seedling culture base, condition of culture: light is cultivated, and intensity of illumination is 2000～3000Lx, 26～29 ℃ of temperature; After cultivating 35～45d, budlet grows up to the high seedling of 3～5 cm; Described strong seedling culture base is to add banana 10～100g/L, potato 10～100g/L, active carbon 1.0～2.0g/L on the basis of basal medium;
5), process of rooting culture
Seedling is transferred to carries out culture of rootage in the root media, condition of culture: light is cultivated, and intensity of illumination is 2000～3000Lx, 26～29 ℃ of temperature; Behind the culture of rootage 60d, the seedling base portion produces a large amount of root systems, forms complete plant; Described root media is to add banana 10～100g/L, potato 10～100g/L, a-methyl α-naphthyl acetate 0.02～0.5mg/L, active carbon 1.0～2.0g/L on the basis of basal medium.
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