CN106234212A - A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification - Google Patents
A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification Download PDFInfo
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- CN106234212A CN106234212A CN201510887004.0A CN201510887004A CN106234212A CN 106234212 A CN106234212 A CN 106234212A CN 201510887004 A CN201510887004 A CN 201510887004A CN 106234212 A CN106234212 A CN 106234212A
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- vitrification
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- fragariae ananssae
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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 present invention relates to a kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification, comprise the steps: 1) stem apex sterilization inoculation, inducing culture carries out induction cultivation, it is thus achieved that Regenerated plantlet；2) on the subculture medium adding variable concentrations 6-BA, carry out 3 times subculture multiplication to cultivate；3) on root media, the plant forming successive transfer culture carries out root induction.The method have the advantages that: in first, second and third subculture multiplication incubation of Fructus Fragariae Ananssae test tube Seedling, reduce the 6-BA concentration in culture matrix by generation, on the premise of ensureing the growth coefficient that Fructus Fragariae Ananssae test tube Seedling is higher, controlled strawberries Vitrification, during first three time subculture multiplication is cultivated, Fructus Fragariae Ananssae test tube seedling proliferation Coefficient Mean reaches 6.01, and glass rate mean control is 26.25%.
Description
Technical field
The present invention relates to a kind of Strawberry tissue culture method, refer in particular to a kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification.
Background technology
At present, plant tissue culture technique is acknowledged as solving Strawberry Virus, Fast-propagation Fructus Fragariae Ananssae detoxic seedling most efficient method.Strawberry tissue culture is divided into the processes such as the process of outer implant, initial culture, subculture multiplication cultivation, root culture, rooting culture, and the outer implant type of employing has the Fructus Fragariae Ananssae tissues such as stem apex, flower pesticide, root, leaf and petiole.
But, in Fructus Fragariae Ananssae subculture multiplication is cultivated, Vitrification Occurred is a great problem being badly in need of solving.Vitrified test tube Seedling occurs, and its organizational structure and physiological function are abnormal, and differentiation and proliferation ability is low and difficulty of taking root, and has a strong impact on the large-scale production of Fructus Fragariae Ananssae test tube Seedling.The generation of vitrification phenomenon all has relation with hormone concentration, agar concentration, additive and the ion concentration proportioning etc. in culture medium.
Li Sheng is at " plant Vitrification phenomenal research progress " (Gansu Agriculture University's journal 2003, think in 3(1): 1-16) that high concentration 6-BA has certain facilitation to vitrified, reach to reduce vitrification generation by reducing 6-BA concentration in culture medium.But the culture medium using low concentration 6-BA carries out Fructus Fragariae Ananssae subculture multiplication cultivation, and the growth coefficient of Fructus Fragariae Ananssae adventitious bud is relatively low.Simultaneously along with the increase of subculture number, continuing many generations uses the culture medium of same concentrations 6-BA, in actual production process or can there is higher vitrification phenomenon.
Summary of the invention
It is an object of the invention to provide a kind of tissue training controlling Fructus Fragariae Ananssae Vitrification
Breeding method, this is, during one can improve Fructus Fragariae Ananssae test tube seedling proliferation coefficient, effectively control Fructus Fragariae Ananssae enrichment culture, vitrified method for tissue culture occurs.
The present invention through the following steps that technical scheme realize:
1) stem apex sterilization inoculation, carries out induction cultivation, it is thus achieved that Regenerated plantlet on inducing culture；
2) on the subculture medium adding variable concentrations 6-BA, carry out three times subculture multiplication to cultivate；
3) on root media, subculture multiplication is cultivated the plant formed and carries out root induction.
Present invention additionally comprises techniques below scheme: inoculating the stem apex semicircle spherical apical meristem size chosen in described step 1) is 0.3 mm ~ 0.4 mm.In described step 1), inducing culture based formulas is: MS minimal medium+1.5 mg L-1 6-BA+0.1 mg·L-1 NAA+10 g·L-1Agar+30 g L-1Sucrose, culture medium pH value 5.5 ~ 6.0.In described step 1), the inducing culture time is 35～40 d.Described step 2) in carry out the test tube Seedling of subculture multiplication cultivation be differentiated to form vitrified plantlet does not occurs.Described step 2) in first, second and third subculture multiplication to cultivate the 6-BA concentration added in MS minimal medium used different, other compositions are the same, and other compositions include agar 10 g L-1+ sucrose 30 g L-1+0.1 mg·L-1NAA, culture medium pH value 5.5 ~ 6.0, the 6-BA concentration added in first, second and third subculture multiplication is cultivated is respectively 1.0mg L-1、0.8mg·L-1、0.6 mg·L-1.Described step 2) in every time subculture multiplication incubation time be 25～30 d.Described step 3) prescription of rooting medium is: 1/2 MS minimal medium+10 g L-1Agar+20 g L-1Sucrose.Described step 1), described step 2), conditions of tissue culture is in described step 3): cultivating indoor cultivation, cultivation temperature 16 DEG C ~ 22 DEG C, intensity of illumination is 1500 lx ~ 2000 lx, every day light application time 12 h.
The present invention specifically comprises the following steps that (1), by successively peelling off spire through strawberry stem tip ecto-entad under anatomical lens of sterilization, until exposing the spherical apical meristem of semicircle, cutting 0.3 mm ~ 0.4 about mm size stem apex and being inoculated in containing 1.5 mg L-1 6-BA、0.1 mg·L-1 NAA、10 g·L-1Agar, 30 g L-1Induction cultivation, culture medium pH value 5.5 ~ 6.0 is carried out in the MS culture medium of sucrose.Cultivate indoor cultivation, cultivation temperature 16 DEG C ~ 22 DEG C, intensity of illumination is 1500 lx ~ 2000 lx, every day light application time 12 h.
Do not occur that vitrified plantlet proceeds by subculture multiplication and cultivates by be differentiated to form after (2) 35～40 d, cultivate once every 25 d～30 d subculture multiplication, culture medium is MS minimal medium, subculture multiplication is cultivated in addition to the 6-BA concentration added is different every time, other uniform component samples, other compositions include 0.1 mg L-1 NAA、10 g·L-1Agar, 30 g L-1Sucrose, culture medium pH value 5.5 ~ 6.0.The 6-BA concentration added in first, second and third subculture multiplication is cultivated is respectively 1.0mg L-1、0.8mg·L-1、0.6 mg·L-1。
(3) will subculture multiplication cultivate in formed whole plant, and plant height reaches the test tube Seedling of 2 more than cm and is inoculated into root induction in 1/2 MS culture medium.Other compositions of culture medium include 10 g L-1Agar, 20 g L-1Sucrose.
The invention has the beneficial effects as follows: in first, second and third subculture multiplication incubation of Fructus Fragariae Ananssae test tube Seedling, reduce the 6-BA concentration in culture matrix by generation, on the premise of ensureing the growth coefficient that Fructus Fragariae Ananssae test tube Seedling is higher, controlled strawberries Vitrification, during first three time subculture multiplication is cultivated, Fructus Fragariae Ananssae test tube seedling proliferation Coefficient Mean reaches 6.01, and glass rate mean control is 26.25%.
Accompanying drawing explanation
Fig. 1 is the process chart of the present invention.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment:
The terminal bud of stolon the top 3～4 cm length newly grown on " beauty " the Fructus Fragariae Ananssae maternal plant choose the 3-5 month robust growth, 30 min are soaked with the tap water containing a small amount of liquid detergent, rinse 30 min with tap water again, finally use aseptic water washing 3 times, then go to superclean bench and carry out sterilization treatment.Concrete Biocidal treatment method is: first with 75% Ethanol Treatment 30 s, uses 0.1% HgCl after aseptic water washing 3 times2Process 7 min, finally with aseptic water washing 5～6 times.
Inoculate on superclean bench after sterilizing.Under anatomical lens, ecto-entad successively peels off spire, until exposing the spherical apical meristem of semicircle, cutting 0.3 mm ~ 0.4 about mm size stem apex and being inoculated in the triangular flask (50 ml) equipped with inducing culture and cultivate, 4 stem apexs of every bottle graft kind.The composition of inducing culture is MS+1.5 mg L-1 6-BA+0.1 mg·L-1 NAA+10 g·L-1Agar+30 g L-1Sucrose, culture medium pH value 5.5 ~ 6.0, change an inducing culture after 15～20 d.Condition of culture is illumination 1500 lx ~ 2000 lx, 12 h/d, temperature 16 DEG C ~ 22 DEG C.
The plantlet being differentiated to form proceeding by after inoculation 35～40 d subculture multiplication cultivate, the 6-BA concentration added in first, second and third subculture multiplication cultivation MS minimal medium used is different, and other compositions are the same, and other compositions include 10 g L-1Agar+30 g L-1Sucrose+0.1 mg L-1NAA, culture medium pH value 5.5 ~ 6.0.The inoculation material that subculture multiplication is cultivated all selects plantlet vitrification phenomenon do not occur, each successive transfer culture time to be 25 d～30 d.Condition of culture is illumination 1500 lx ~ 2000 lx, 12 h/d, temperature 16 DEG C ~ 22 DEG C.
Particular make-up and the successive transfer culture effect of adding the subculture medium of variable concentrations 6-BA in MS culture medium are as shown in table 1.As seen from the experiment, from keeping the Fructus Fragariae Ananssae high-caliber growth coefficient of test tube Seedling, from the point of view of controlling again the incidence rate angle of Vitrification, the test effect processing 1 is best simultaneously.Although the glass rate of process 2 and process 3 test tube Seedlings is lower, but the growth coefficient of test tube Seedling is the highest, is unfavorable for the expanding propagation of test tube Seedling.
Fructus Fragariae Ananssae test tube seedling proliferation and the vitrification of each successive transfer culture are affected by table 1 variable concentrations 6-BA
| Subculture number | Process numbering | 6-BA concentration | Growth coefficient | Glass rate/% |
| Subculture for the first time | CK | 1.5 mg·L-1 | 6.95 a | 7.06 a |
| 1 | 1.0 mg·L-1 | 6.21 b | 0 b | |
| 2 | 0.8 mg·L-1 | 5.10 c | 0 b | |
| 3 | 0.6 mg·L-1 | 3.77 d | 0 b | |
| Subculture for the second time | CK | 1.5 mg·L-1 | 6.24 ab | 56.84 a |
| 1 | 0.8 mg·L-1 | 6.75 a | 48.08 b | |
| 2 | 0.6 mg·L-1 | 5.10 bc | 40.79 c | |
| 3 | 0.4 mg·L-1 | 4.39 c | 27.98 d | |
| Subculture for the third time | CK | 1.5 mg·L-1 | 5.72 a | 41.32 a |
| 1 | 0.6 mg·L-1 | 5.06 b | 30.66 b | |
| 2 | 0.4 mg·L-1 | 4.52 b | 29.27 b | |
| 3 | 0.2 mg·L-1 | 3.29 c | 14.43 c | |
| The average ratio of first three successive transfer culture is relatively | CK | 6.30 a | 35.07 a | |
| 1 | 6.01 a | 26.25 b | ||
| 2 | 4.91 b | 23.35 c | ||
| 3 | 3.82 c | 14.14 d |
Note: small English alphabet represents different disposal difference, represents difference during Duncan analyzes and reaches 0.05 significant level.
Will subculture multiplication cultivate in formed whole plant, and plant height reaches the test tube Seedling of 2 more than cm and is inoculated into root induction in 1/2 MS culture medium.After 15 d～20 d, when test tube shoot root length is to 2 more than cm, number of taking root reaches more than 2, carries out rooting culture during height of seedling 5 more than cm.
Claims (9)
1. the method for tissue culture controlling Fructus Fragariae Ananssae Vitrification, it is characterised in that comprise the steps:
1) stem apex sterilization inoculation, carries out induction cultivation, it is thus achieved that Regenerated plantlet on inducing culture；
2) on the subculture medium adding variable concentrations 6-BA, carry out three times subculture multiplication to cultivate；
3) on root media, subculture multiplication is cultivated the plant formed and carries out root induction.
A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification the most as claimed in claim 1, it is characterised in that: inoculating the stem apex semicircle spherical apical meristem size chosen in described step 1) is 0.3 mm ~ 0.4 mm.
A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification the most as claimed in claim 1, it is characterised in that: in described step 1), inducing culture based formulas is: MS minimal medium+1.5 mg L-1 6-BA+0.1 mg·L-1 NAA+10 g·L-1Agar+30 g L-1Sucrose, culture medium pH value 5.5 ~ 6.0.
A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification the most as claimed in claim 1, it is characterised in that: in described step 1), the inducing culture time is 35～40 d.
A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification the most as claimed in claim 1, it is characterised in that: described step 2) in carry out the test tube Seedling of subculture multiplication cultivation be differentiated to form vitrified plantlet does not occurs.
A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification the most as claimed in claim 1, it is characterized in that: described step 2) in first, second and third subculture multiplication to cultivate the 6-BA concentration added in MS minimal medium used different, other compositions are the same, and other compositions include agar 10 g L-1+ sucrose 30 g L-1+0.1 mg·L-1NAA, culture medium pH value 5.5 ~ 6.0, the 6-BA concentration added in first, second and third subculture multiplication is cultivated is respectively 1.0mg L-1、0.8mg·L-1、0.6 mg·L-1。
A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification the most as claimed in claim 1, it is characterised in that: described step 2) in every time subculture multiplication incubation time be 25～30 d.
A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification the most as claimed in claim 1, it is characterised in that: described step 3) prescription of rooting medium is: 1/2
MS minimal medium+10 g L-1Agar+20 g L-1Sucrose.
A kind of method for tissue culture controlling Fructus Fragariae Ananssae Vitrification the most as claimed in claim 1, it is characterized in that: described step 1), described step 2), conditions of tissue culture is in described step 3): cultivating indoor cultivation, cultivation temperature 16 DEG C ~ 22 DEG C, intensity of illumination is 1500 lx ~ 2000 lx, every day light application time 12 h.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109197592A (en) * | 2018-10-24 | 2019-01-15 | 河南云帮农业科技有限公司 | A method of reducing albumen mulberry tissue culture Vitrification |
| CN112772414A (en) * | 2021-01-08 | 2021-05-11 | 山东农业大学 | Method for improving rapid propagation efficiency of apple rootstock tissue culture seedlings |
| CN114027194A (en) * | 2021-11-17 | 2022-02-11 | 上海农林职业技术学院 | Culture medium and method for devitrification of 'Huanghua' evening primrose tissue culture seedlings |
| CN115191354A (en) * | 2022-07-18 | 2022-10-18 | 华亭市正元生态农业发展有限公司 | Method for producing virus-free strawberry tissue culture seedlings by using descending type gradient culture medium |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109197592A (en) * | 2018-10-24 | 2019-01-15 | 河南云帮农业科技有限公司 | A method of reducing albumen mulberry tissue culture Vitrification |
| CN112772414A (en) * | 2021-01-08 | 2021-05-11 | 山东农业大学 | Method for improving rapid propagation efficiency of apple rootstock tissue culture seedlings |
| CN114027194A (en) * | 2021-11-17 | 2022-02-11 | 上海农林职业技术学院 | Culture medium and method for devitrification of 'Huanghua' evening primrose tissue culture seedlings |
| CN115191354A (en) * | 2022-07-18 | 2022-10-18 | 华亭市正元生态农业发展有限公司 | Method for producing virus-free strawberry tissue culture seedlings by using descending type gradient culture medium |
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