CN103430850A - Tissue culture method for polyploid hemerocallis middendorfii and culture medium - Google Patents
Tissue culture method for polyploid hemerocallis middendorfii and culture medium Download PDFInfo
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Abstract
The invention provides a tissue culture method for polyploid hemerocallis middendorfii and also discloses a culture medium applicable to the tissue culture method. The tissue culture method provided by the invention can be used for increasing the breeding coefficient of the polyploid hemerocallis middendorfii, and making up for the deficiencies of a conventional tissue culture technology, such as reducing the pollution rate of explants and increasing the callus differentiation rate and transplanting survival rate. By collecting the explants, a disinfection effect, a rooting effect and the transplanted seedling survival rate of the explants are all superior to previous tissue culture rapid propagation technologies for day lily varieties. An established large-scale commodity production system is stable, and the production cycle is only about two months, so that the breeding speed of the polyploid hemerocallis middendorfii is increased, the season limit on breeding is broken, and the industrialized production of seedling culture is realized. The tissue culture formula provided by the invention is the best combination of the mass propagation of the polyploid hemerocallis middendorfii, and can be used for quickly breeding a large number of tissue culture seedlings so as to adapt to the demand of the landscaping market.
Description
Technical field
The invention provides a kind of polyploid hemerocailis middendorffi tissue culture method, also disclose the medium that is applicable to tissue culture method of the present invention simultaneously, belong to the flower cultivation technical field.
Background technology
Tawny daylily is the perennial root flowers, is widely used in recent years in urban afforestation and landscape architecture.In numerous tawny daylily kinds, polyploid hemerocailis middendorffi pattern is abundant, and flower appearance grace, and leaf color jade green, possess cultivation simple and easy, and green sprouting early waits the cultivation advantage.Market is increasing to the demand of polyploid hemerocailis middendorffi in recent years, and simple division propagation speed far can not meet the needs that commercialization is produced, thereby limit the extensive application of polyploid hemerocailis middendorffi in gardens.Therefore, explore technology and the method for polyploid hemerocailis middendorffi Fast-propagation, carry out the research that tissue cultivates and accelerate it and apply and just seem most important.
Summary of the invention
The invention provides a kind of polyploid hemerocailis middendorffi tissue culture method, solve the technological difficulties of the aspects such as the selection of polyploid hemerocailis middendorffi explant, explant acquisition time, explant sterilization method, culture medium prescription, hardening off method.
The invention also discloses the multiple medium that is applicable to tissue culture method of the present invention, can rapid, high volume the polyploid hemerocailis middendorffi of breeding, the problem such as the explant pollution rate that makes up conventional art is high, differentiation rate is low, transplanting survival rate is low, transplanting survival rate can reach 95%.
polyploid hemerocailis middendorffi tissue culture method disclosed by the invention comprises the following steps:
1) take out the flower bud phase plant, gather the healthy and strong tender leaf without damage by disease and insect, growing point, axillalry bud, bud, petal, bennet, the explant of cultivating as tissue, the explant collected is cleaned up with washing powder water, be placed on again under flowing water and rinse 30min, with 75% alcohol disinfecting 5-30s, then use aseptic water washing 3-5 time on superclean bench, use afterwards the 0.1%(mass percent) mercuric chloride sterilization 5-20min;
2) explant after sterilization is inoculated on inducing culture, sends into culturing room and carry out aseptic culture, medium is MS 1L+NAA 0.2mg/L+6-BA 0.5mg/L+2,4-D 0.7mg/L+ sucrose 3 mg/L, agar 9 mg/L; Explant after differentiation is transferred on the shoot proliferation medium and carried out the subculture cultivation, obtain breaking up seedling, subculture medium is MS+NAA 0.2mg/L+6-BA 2.0mg/L+ sucrose 3 mg/L, agar 9 mg/L;
3) will break up seedling and be inoculated on the strong seedling culture base and carry out strong seedling culture, the strong seedling culture base is MS 1L+6-BA 0.2 mg/L+IBA 0.04mg/L+ potato 20g/L+ sucrose 3 mg/L, agar 9 mg/L; Then, healthy and strong bottle seedling is transferred on root media and carried out culture of rootage, root media is MS 1L+NAA 0.02mg/L+ sucrose 3 mg/L, agar 9 mg/L;
4) training of the group after taking root seedling is shifted out to blake bottle, clean the medium of root by hairbrush, root is placed in to the container of filled with water, be placed in seeding room and temper 3-5 days, then be transplanted in the matrix of humus soil: perlite=1:1, obtain finished product.
The working solution concentration unit of described MS medium is mg/L, contains:
Macroelement: NH
4nO
31650, KNO
31900, CaCl
22H
2o 440, MgSO
47H
2o 370, KH
2pO
4170;
Trace element: KI 0.83, H
3bO
36.2, MnSO
44H
2o 22.3, ZnSO
47H
2o 8.6, Na
2mnO
42H
2o 0.25, CuSO
45H
2o 0.0025, CoCl
26H
2o 0.0025; Molysite: FeSO
47H
2o 27.8, Na
2-EDTA2H
2o 37.3;
Inositol 100, hydrochloric acid 0.5, puridoxine hydrochloride 0.5, thiamine hydrochloride 0.1, glycine 2.0.
polyploid hemerocailis middendorffi group training disclosed by the invention is usedinducing culture
, it is characterized in that being made by following raw material:
MS 1L, methyl α-naphthyl acetate 0.2mg/L, 6-benzyl purine 0.5mg/L, 2,4-dichlorphenoxyacetic acid 0.7mg/L, sucrose 3 mg/L, agar 9 mg/L; PH value 5.8-6.0.
the shoot proliferation medium is used in polyploid hemerocailis middendorffi group disclosed by the invention training: it is characterized in that being made by following raw material:
MS 1L, methyl α-naphthyl acetate 0.2mg/L, 6-benzyl purine 2.0mg/L, sucrose 3 mg/L, agar 9 mg/L; PH value 5.8-6.0.
polyploid hemerocailis middendorffi group training strong seedling culture base disclosed by the invention; It is characterized in that being made by following raw material:
MS 1L, 6-benzyl purine 0.2 mg/L, indole-3-butyric acid 0.04mg/L, potato 20g/L, sucrose 3 mg/L, agar 9 mg/L; PH value 5.8-6.0.
polyploid hemerocailis middendorffi group training root media disclosed by the invention; It is characterized in that being made by following raw material:
MS 1L, methyl α-naphthyl acetate 0.02mg/L, sucrose 3 mg/L, agar 9 mg/L; PH value 5.8-6.0.
the working solution concentration unit of described MS is mg/L, contains:
Macroelement: NH
4nO
31650, KNO
31900, CaCl
22H
2o 440, MgSO
47H
2o 370, KH
2pO
4170;
Trace element: KI 0.83, H
3bO
36.2, MnSO
44H
2o 22.3, ZnSO
47H
2o 8.6, Na
2mnO
42H
2o 0.25, CuSO
45H
2o 0.0025, CoCl
26H
2o 0.0025; Molysite: FeSO
47H
2o 27.8, Na
2-EDTA2H
2o 37.3;
Inositol 100, hydrochloric acid 0.5, puridoxine hydrochloride 0.5, thiamine hydrochloride 0.1, glycine 2.0.
the advantage that the present invention has with respect to prior art and progressive being:
The inventive method has improved the reproduction coefficient of polyploid hemerocailis middendorffi, has made up the some shortcomings of traditional tissue culture technique, as reduced explant pollution rate, improved phenylacetic acid and transplanting survival rate etc.All be better than the group culturation rapid propagating technology of tawny daylily kind in the past by aspects such as explant collection, explant Disinfection Effect, rooting efficiency, transplanted seedling survival rates, the scale commodity production stable system of setting up, the production cycle only needs about 2 months.Accelerate polyploid hemerocailis middendorffi reproduction speed, break the season limit of breeding, realize growing seedlings batch production production.To be that the polyploid hemerocailis middendorffi is a large amount of expand numerous best of breeds for of the present invention group of training formula, and breeding group training seedling that can rapid, high volume, adapt to the afforestation market demand.
Embodiment
By following examples, the present invention is further described for example, and do not limit the present invention in any way, under the prerequisite that does not deviate from technical solution of the present invention, within any change that those of ordinary skills made for the present invention easily realize or change all will fall into claim scope of the present invention.
the preparation of embodiment 1 MS:
The working solution concentration unit is mg/L, wherein:
Macroelement: NH
4nO
31650, KNO
31900, CaCl
22H
2o 440, MgSO
47H
2o 370, KH
2pO
4170; Trace element: KI 0.83, H
3bO
36.2, MnSO
44H
2o 22.3, ZnSO
47H
2o 8.6, Na
2mnO
42H
2o 0.25, CuSO
45H
2o 0.0025, CoCl26H2O 0.0025; Molysite: FeSO47H2O 27.8, Na2-EDTA2H2O 37.3; Inositol 100, hydrochloric acid 0.5, puridoxine hydrochloride 0.5, thiamine hydrochloride 0.1, glycine 2.0.Take in proportion above-mentioned raw materials, mix and obtain MS.
embodiment 2 polyploid hemerocailis middendorffi group training inducing cultures
The MS 1L that takes embodiment 1 preparation adds methyl α-naphthyl acetate 0.2mg/L, 6-benzyl purine 0.5mg/L, 2,4-dichlorphenoxyacetic acid 0.7mg/L, sucrose 30 g/L, agar 9 g/L; PH value 5.8-6.0; By above-mentioned raw materials, mix and get final product.
embodiment 3 shoot proliferation medium
The MS 1L that takes embodiment 1 preparation adds methyl α-naphthyl acetate 0.2mg/L, 6-benzyl purine 2.0mg/L, sucrose 30 g/L, agar 9 g/L; PH value 5.8-6.0; By above-mentioned raw materials, mix and get final product.
embodiment 4 strong seedling culture bases
The MS 1L that takes embodiment 1 preparation adds 6-benzyl purine 0.2 mg/L, indole-3-butyric acid 0.04mg/L, potato 20g/L, sucrose 30g/L, agar 9 g/L; PH value 5.8-6.0; By above-mentioned raw materials, mix and get final product.
embodiment 5 root medias
The MS 1L that takes embodiment 1 preparation adds methyl α-naphthyl acetate 0.02mg/L, sucrose 30 g/L, agar 9 g/L; PH value 5.8-6.0; By above-mentioned raw materials, mix and get final product.
embodiment 6
1, be chosen in mid-July (plant is taken out the flower bud phase) for three days on end without after rain, explant being gathered, can effectively reduce the pollution rate of explant.By the outer planting collected
(the flower tawny daylily
bud)body cleans up with washing powder water, then is placed under flowing water and rinses 30min, on superclean bench, with 75% alcohol disinfecting 5-30s, then uses aseptic water washing 3-5 time, uses afterwards the 0.1%(mass percent) mercuric chloride sterilization 5-20min; Wherein, best explant sterilization method: 75% alcohol 10s+0.1%(mass percent) mercuric chloride sterilization 12min.
2, the explant after sterilization is inoculated into to inducing culture (embodiment 2) upper, sends into culturing room and carry out aseptic culture, the condition of culturing room is temperature: 25 ± 1 ℃, and intensity of illumination: 1500lx-2000lx, light application time: 12h/d.Select optimum explant kind and culture medium prescription according to explant induction and differentiation situation general in this process.The bud differentiation is significantly inoculated to piece and transfer to shoot proliferation medium (embodiment 3) above, through 30d, cultivate and can breed 1-2 doubly, obtain breaking up seedling.
3, in order to have improved the quality of breaking up seedling, improve the survival rate of transplanting, by growth, weak differentiation seedling is inoculated on strong seedling culture base (embodiment 4) and carries out strong seedling culture, and the cultivation of process 10-20d can make bottle seedling chap strong.Then, healthy and strong bottle seedling is transferred on root media (embodiment 5) and carried out culture of rootage.
4, to then transplant first through the process of hardening after culture of rootage, could adapt to the requirement of outdoor cropping.Group after taking root training seedling is shifted out to blake bottle, clean the medium of root by hairbrush, be placed in the capsule of filled with water, by the fixing seedling of rubber band, water surface elevation is advisable to be no more than root, is placed in the exercise 3-5 days of seeding room, the upper cover transparent plastic cloth.Then be transplanted in the matrix of humus soil: perlite=1:1.Transplanting survival rate reaches 96% left and right.
To note some in the transplanting process, 1. by tweezers, the soil of seedling root be pinched to reality, allow root fully contact with matrix; 2., in transplanting process, preferably 2-3 seedling is planted in hole, a cave; 3. the seedling after transplanting will put transparent plastic bag with holes, after one week, removes; 4. after transplanting, every two weeks waters the MS nutrient solution (the MS medium that does not contain sucrose and agar) that once dilutes 500 times.Above some all can improve the transplanted seedling survival rate.
embodiment 7
1, be chosen in mid-July (plant is taken out the flower bud phase) for three days on end without after rain, explant being gathered, can effectively reduce the pollution rate of explant.Gather the healthy and strong tender leaf without damage by disease and insect,, axillalry bud, bud, petal, bennet, the explant of cultivating as tissue, by the outer planting collected
(flower tawny daylily growing point
)body cleans up with washing powder water, then is placed under flowing water and rinses 30min, on superclean bench, with 75% alcohol disinfecting 5-30s, then uses aseptic water washing 3-5 time, uses afterwards the 0.1%(mass percent) mercuric chloride sterilization 5-20min; Wherein, best explant sterilization method: 75% alcohol 10s+0.1%(mass percent) mercuric chloride sterilization 12min.
2, the explant after sterilization is inoculated into to inducing culture (embodiment 2) upper, sends into culturing room and carry out aseptic culture, the condition of culturing room is temperature: 25 ± 1 ℃, and intensity of illumination: 1500lx-2000lx, light application time: 12h/d.Select optimum explant kind and culture medium prescription according to explant induction and differentiation situation general in this process.The bud differentiation is significantly inoculated to piece and transfer to shoot proliferation medium (embodiment 3) above, through 30d, cultivate and can breed 1-2 doubly, obtain breaking up seedling.
3, in order to have improved the quality of breaking up seedling, improve the survival rate of transplanting, by growth, weak differentiation seedling is inoculated on strong seedling culture base (embodiment 4) and carries out strong seedling culture, and the cultivation of process 10-20d can make bottle seedling chap strong.Then, healthy and strong bottle seedling is transferred on root media (embodiment 5) and carried out culture of rootage.
4, to then transplant first through the process of hardening after culture of rootage, could adapt to the requirement of outdoor cropping.Group after taking root training seedling is shifted out to blake bottle, clean the medium of root by hairbrush, be placed in the capsule of filled with water, by the fixing seedling of rubber band, water surface elevation is advisable to be no more than root, is placed in the exercise 3-5 days of seeding room, the upper cover transparent plastic cloth.Then be transplanted in the matrix of humus soil: perlite=1:1.Transplanting survival rate reaches 96% left and right.
To note some in the transplanting process, 1. by tweezers, the soil of seedling root be pinched to reality, allow root fully contact with matrix; 2., in transplanting process, preferably 2-3 seedling is planted in hole, a cave; 3. the seedling after transplanting will put transparent plastic bag with holes, after one week, removes; 4. after transplanting, every two weeks waters the MS nutrient solution (the MS medium that does not contain sucrose and agar) that once dilutes 500 times.Above some all can improve the transplanted seedling survival rate.
embodiment 8
1, be chosen in mid-July (plant is taken out the flower bud phase) for three days on end without after rain, explant being gathered, can effectively reduce the pollution rate of explant.By the outer planting collected
(flower tawny daylily axillalry bud
)body cleans up with washing powder water, then is placed under flowing water and rinses 30min, on superclean bench, with 75% alcohol disinfecting 5-30s, then uses aseptic water washing 3-5 time, uses afterwards the 0.1%(mass percent) mercuric chloride sterilization 5-20min; Wherein, best explant sterilization method: 75% alcohol 10s+0.1%(mass percent) mercuric chloride sterilization 12min.
2, the explant after sterilization is inoculated into to inducing culture (embodiment 2) upper, sends into culturing room and carry out aseptic culture, the condition of culturing room is temperature: 25 ± 1 ℃, and intensity of illumination: 1500lx-2000lx, light application time: 12h/d.Select optimum explant kind and culture medium prescription according to explant induction and differentiation situation general in this process.The bud differentiation is significantly inoculated to piece and transfer to shoot proliferation medium (embodiment 3) above, through 30d, cultivate and can breed 1-2 doubly, obtain breaking up seedling.
3, in order to have improved the quality of breaking up seedling, improve the survival rate of transplanting, by growth, weak differentiation seedling is inoculated on strong seedling culture base (embodiment 4) and carries out strong seedling culture, and the cultivation of process 10-20d can make bottle seedling chap strong.Then, healthy and strong bottle seedling is transferred on root media (embodiment 5) and carried out culture of rootage.
4, to then transplant first through the process of hardening after culture of rootage, could adapt to the requirement of outdoor cropping.Group after taking root training seedling is shifted out to blake bottle, clean the medium of root by hairbrush, be placed in the capsule of filled with water, by the fixing seedling of rubber band, water surface elevation is advisable to be no more than root, is placed in the exercise 3-5 days of seeding room, the upper cover transparent plastic cloth.Then be transplanted in the matrix of humus soil: perlite=1:1.Transplanting survival rate reaches 96% left and right.
To note some in the transplanting process, 1. by tweezers, the soil of seedling root be pinched to reality, allow root fully contact with matrix; 2., in transplanting process, preferably 2-3 seedling is planted in hole, a cave; 3. the seedling after transplanting will put transparent plastic bag with holes, after one week, removes; 4. after transplanting, every two weeks waters the MS nutrient solution (the MS medium that does not contain sucrose and agar) that once dilutes 500 times.Above some all can improve the transplanted seedling survival rate.
Claims (5)
1. a polyploid hemerocailis middendorffi tissue culture method is characterized in that comprising the following steps:
1) take out the flower bud phase plant, gather the healthy and strong tender leaf without damage by disease and insect, growing point, axillalry bud, bud, petal, bennet, the explant of cultivating as tissue, the explant collected is cleaned up with washing powder water, be placed on again under flowing water and rinse 30min, with 75% alcohol disinfecting 5-30s, then use aseptic water washing 3-5 time on superclean bench, use afterwards the 0.1%(mass percent) mercuric chloride sterilization 5-20min;
2) explant after sterilization is inoculated on inducing culture, sends into culturing room and carry out aseptic culture, medium is MS 1L+NAA 0.2mg/L+6-BA 0.5mg/L+2,4-D 0.7mg/L+ sucrose 3 mg/L, agar 9 mg/L; Explant after differentiation is transferred on the shoot proliferation medium and carried out the subculture cultivation, obtain breaking up seedling, subculture medium is MS+NAA 0.2mg/L+6-BA 2.0mg/L+ sucrose 3 mg/L, agar 9 mg/L;
3) will break up seedling and be inoculated on the strong seedling culture base and carry out strong seedling culture, the strong seedling culture base is MS 1L+6-BA 0.2 mg/L+IBA 0.04mg/L+ potato 20g/L+ sucrose 3 mg/L, agar 9 mg/L; Then, healthy and strong bottle seedling is transferred on root media and carried out culture of rootage, root media is MS 1L+NAA 0.02mg/L+ sucrose 3 mg/L, agar 9 mg/L;
4) training of the group after taking root seedling is shifted out to blake bottle, clean the medium of root by hairbrush, root is placed in to the container of filled with water, be placed in seeding room and temper 3-5 days, then be transplanted in the matrix of humus soil: perlite=1:1, obtain finished product;
The working solution concentration unit of described MS medium is mg/L, contains:
Macroelement: NH
4nO
31650, KNO
31900, CaCl
22H
2o 440, MgSO
47H
2o 370, KH
2pO
4170;
Trace element: KI 0.83, H
3bO
36.2, MnSO
44H
2o 22.3, ZnSO
47H
2o 8.6, Na
2mnO
42H
2o 0.25, CuSO
45H
2o 0.0025, CoCl
26H
2o 0.0025; Molysite: FeSO
47H
2o 27.8, Na
2-EDTA2H
2o 37.3;
Inositol 100, hydrochloric acid 0.5, puridoxine hydrochloride 0.5, thiamine hydrochloride 0.1, glycine 2.0.
2. inducing culture is used in polyploid hemerocailis middendorffi group training, it is characterized in that being made by following raw material:
MS 1L, methyl α-naphthyl acetate 0.2mg/L, 6-benzyl purine 0.5mg/L, 2,4-dichlorphenoxyacetic acid 0.7mg/L, sucrose 3 mg/L, agar 9 mg/L; PH value 5.8-6.0.
3. the shoot proliferation medium is used in polyploid hemerocailis middendorffi group training: it is characterized in that being made by following raw material:
MS 1L, methyl α-naphthyl acetate 0.2mg/L, 6-benzyl purine 2.0mg/L, sucrose 3 mg/L, agar 9 mg/L; PH value 5.8-6.0.
4. a polyploid hemerocailis middendorffi group is trained and is used the strong seedling culture base; It is characterized in that being made by following raw material:
MS 1L, 6-benzyl purine 0.2 mg/L, indole-3-butyric acid 0.04mg/L, potato 20g/L, sucrose 3 mg/L, agar 9 mg/L; PH value 5.8-6.0.
5. a polyploid hemerocailis middendorffi group is trained and is used root media; It is characterized in that being made by following raw material:
MS 1L, methyl α-naphthyl acetate 0.02mg/L, sucrose 3 mg/L, agar 9 mg/L; PH value 5.8-6.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105340754A (en) * | 2015-12-04 | 2016-02-24 | 定州市绿谷农业科技发展有限公司 | Method suitable for industrialized propagation of hemerocallis middendorfii |
| CN109717075A (en) * | 2017-10-27 | 2019-05-07 | 上海市农业科学院 | A method of hemerocailis middendorffi regeneration plant is obtained by evoking adventive bud |
| CN113491239A (en) * | 2021-08-27 | 2021-10-12 | 黑龙江卉研农业发展有限公司 | Polyploid hemerocallis middendorfii tissue culture and culture medium |
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| JPH104811A (en) * | 1996-06-19 | 1998-01-13 | Gunze Ltd | Proliferation of large amount of plant of genus hemerocallis |
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| CN102265785A (en) * | 2010-06-02 | 2011-12-07 | 上海上房园艺有限公司 | Tissue culturing method of hemerocallis middendorfii poinsettia |
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| JPH104811A (en) * | 1996-06-19 | 1998-01-13 | Gunze Ltd | Proliferation of large amount of plant of genus hemerocallis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105340754A (en) * | 2015-12-04 | 2016-02-24 | 定州市绿谷农业科技发展有限公司 | Method suitable for industrialized propagation of hemerocallis middendorfii |
| CN109717075A (en) * | 2017-10-27 | 2019-05-07 | 上海市农业科学院 | A method of hemerocailis middendorffi regeneration plant is obtained by evoking adventive bud |
| CN113491239A (en) * | 2021-08-27 | 2021-10-12 | 黑龙江卉研农业发展有限公司 | Polyploid hemerocallis middendorfii tissue culture and culture medium |
| CN113491239B (en) * | 2021-08-27 | 2022-01-11 | 黑龙江卉研农业发展有限公司 | Polyploid hemerocallis middendorfii tissue culture and culture medium |
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| CN103430850B (en) | 2015-04-01 |
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