CN101341854B - Cultivation method for vegetative organ in vitro cluster bud of adult tree of mandarin orange - Google Patents
Cultivation method for vegetative organ in vitro cluster bud of adult tree of mandarin orange Download PDFInfo
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- CN101341854B CN101341854B CN2008101070523A CN200810107052A CN101341854B CN 101341854 B CN101341854 B CN 101341854B CN 2008101070523 A CN2008101070523 A CN 2008101070523A CN 200810107052 A CN200810107052 A CN 200810107052A CN 101341854 B CN101341854 B CN 101341854B
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Abstract
The invention discloses a method which comprises the following steps: strong mature tangerine plants which have no disease and insect damage and have fruit are selected; young branch within 20 days and randomly selected to grow in the year are taken as explants; the explants are cleaned and disinfected, and then inoculated and cultured. Axillary bud primary culture medium: MS or improved MS+6-BA0.1 to 0.6mg/L+Kt0.1 to 0.6mg/L+NAA0.01 to 0.1mg/L. Improved MS+6-BA0.2 to 0.5mg/L+Kt0.04 to 0.4mg/L+Zt0.2 to 0.5mg/L+NAA0.01 to 0.05mg/L. The culture method and the substrate prescription of the invention provide required environment conditions for the mature tangerine stem axillary bud vitro regeneration. Plenty of regeneration buds can be obtained if the method of the invention is used for the vitro regeneration of Nanfeng tangerine. The tangerine vitro regeneration buds can be used for germplasm preservation and batch high-grade germchit grafting and culture in test tubes.
Description
Technical field
The present invention relates to a Plant Biotechnology, relate in particular to a kind of cultivation method for vegetative organ exsomatized treeing sprout of bearing tree of mandarin orange.
Background technology
Oranges and tangerines occupy first of world's all kinds of fruits production, and its fruits nutrition is abundant, and color, smell and taste are held concurrently excellent, both can eat raw, can process juice product again.Nanfeng County's tangerine is famous improved seeds in the wide skin tangerine of citrus.Tangerine source, Nanfeng County goes out newborn tangerine, is again the elaboration in the newborn tangerine, outstanding feature is that skin is thin golden yellow, sour and sweet palatability, give off a strong fragrance, the juice multinuclear is few, being described as " king in the tangerine " has won fame both at home and abroad, it is the distinctive good citriculture kind of China.
The stripped axillalry bud of tangerine bearing tree stem organ is grown thickly to cultivate quick breeding high quality seedling and germplasm {in vitro} conservation is had important function, and it can keep the genetic stability of kind preferably.It is more extensive that China utilizes tissue culture to carry out the quick research work of breeding of oranges and tangerines, behind the big unit report of the seventies king in last century so far, many reported literatures have been arranged the exsomatize generation of indefinite bud or clump bud of the various plants that belongs to of oranges and tangerines section, but the explant of selecting for use almost mostly is a tissue of not getting over the virgin phase, the report of rarely found adult attitude trophosome tissue culture regeneration bud.Reason has 2 points: the one, become the branch stem explant surface sterilizing difficulty of annual bearing mandarin tree; The 2nd, the attitude oranges and tangerines nutrition organs tissue of growing up exsomatizes and breaks up difficulty again.And the adult attitude stem section cultured in vitro of oranges and tangerines is the important technological platform of oranges and tangerines excellent strain factorial seedling growth and germplasm {in vitro} conservation thereof etc., must at first set up effective cultured in vitro regenerating system.
Summary of the invention
The objective of the invention is to: a kind of cultivation method for vegetative organ exsomatized treeing sprout of bearing tree of mandarin orange is provided, corresponding culture matrix is provided simultaneously.
The inventive method may further comprise the steps:
(1.1) the adult excellent strain of selecting healthy and strong no damage by disease and insect tangerine to tie fruit, getting and taking out living 20 days then is explant with interior shoot;
(1.2) to the explant cleaning and sterilizing;
(1.3) explant is seeded in axillalry bud and starts and to be cultured to the axillary bud growth point on the medium and to begin to sprout, and the yellow green small salient point occurs around the base portion of bud, continues to start on the medium at axillalry bud and grows 12～15 days, and main axillalry bud elongation this moment rapidly;
When (1.4) cultivating 30～40 days, the axillalry bud that grows divided from explant (female branch) cut off, be inoculated into induced bundle bud differentiation on the bud proliferated culture medium, cultivated 30～40 days, on average each culture differentiates 4～6 of the buds of growing thickly, bud length 2～3cm;
(1.5) cut apart subculture, budlet becomes the piece successive transfer culture, and big bud cuts into terminal bud and stipes, is inoculated into successive transfer culture on the bud proliferated culture medium, changes generation once, reproduction coefficient 4～6 in later per 30～40 days; Build up clump bud vegetative propagation system, the above bud of the 2cm of breeding can carry out graft seedling growth, and seedling is made stripped quality saving or subculture expands numerous.
The used medium of the inventive method is as follows:
(1) modified MS medium, improvement MS contains the organic principle of following improvement: glycine 2.0mg/L, VB
11.0～2.0mg/L, VB
61.0～2.0mg/L, nicotinic acid 0.5～1.0mg/L, inositol 100mg/L, folic acid 0.1～1.0mg/L, sucrose 40～55g/L, other components unchanged.
(2) axillalry bud starts medium: MS+6-BA0.1～0.6mg/L+Kt0.1～0.6mg/L+NAA0.01～0.1mg/L.
(3) bud proliferated culture medium: improvement MS+6-BA0.2～0.5mg/L+Kt0.04～0.4mg/L+Zt0.2～0.5mg/L+NAA0.01～0.05mg/L.
Condition of culture of the present invention is as follows:
(1) culture parameters: 24 ± 2 ℃ of constant temperature, relative moisture 60%～70%, intensity of illumination are 1000～16000lx, sunshine, duration was 11～14 hours/day.
(2) technical parameter: the explant pollution rate is lower than 5%～10%; The axillalry bud inductivity 75%～87% of sprouting; The bud growth coefficient 4～6 of growing thickly; Subculture cycle 30～40 days.
To in the explant cleaning and sterilizing process sterilization being embathed with slant acidity toothpaste solution by elder generation in the explant cleaning and sterilizing process, embathe sterilization with alkaline detergent liquid again in the inventive method; Use 75% ethanolic solution and 0.1%～0.5%HgCl at last
2Solution disinfection.
Cultural method of the present invention and matrix formulations provide essential environmental condition for the adult attitude stipes axillalry bud Regeneration in Vitro of tangerine.Carry out the cultured in vitro of Nanfeng County's tangerine with method of the present invention, can obtain a large amount of regeneration buds.Utilize such oranges and tangerines Regeneration in Vitro bud both can make the quality saving material, the stem end that can cut 1cm again carries out test tube engrafting and cultivating high quality seedling in enormous quantities.This in-vitro propagate or germplasm {in vitro} conservation to Nanfeng County's tangerine and citrus elite germplasm all has wide practical value.
Description of drawings
Fig. 1 is Nanfeng County of the present invention tangerine attitude stipes axillalry bud induced map of sprouting of growing up;
Fig. 2 is the bud of the present invention enrichment culture figure of growing thickly.
Embodiment
The present invention is described in detail below in conjunction with embodiment.
Embodiment 1:
(1), select the adult excellent strain fruit tree of healthy and strong no damage by disease and insect Nanfeng County tangerine, get take out then living 20d (my god) take out living shoot with interior adult attitude stem;
(2), with running water flush away dust, use slant acidity commodity toothpaste water-soluble in right amount again, soak shoot 6～10min, scrub branches and leaves with banister bruss again after, the running water rinsing is clean; Embathe 6～10min with alkaline detergent liquid again, in the running water thread water rinsing clean, repair behind the old leaf branch with distilled water flushing 1～3 time, filter paper blots and carries out surface sterilizing in the sterile chamber of packing into behind the water droplet and handle; Go on the superclean bench, earlier with alcohol solution dipping 20～30s of 75%, aseptic water washing 2～4 times, 0.1%～0.5%HgCl again
2Soak oranges and tangerines shoot 6～10min, behind the soup that inclines, use aseptic water washing 5～6 times.Then under aseptic condition, it is segment (band stipes) about 0.5～1cm that the oranges and tangerines shoot is cut into length, is inoculated into ready axillalry bud and starts on the medium and cultivate.
(3), be cultured to the axillary bud growth point and begin to sprout, and around the base portion of bud the yellow green small salient point appears, continue to start on the medium and grew 12～15 days at axillalry bud, main axillalry bud elongation this moment rapidly,
When inoculation back is cultivated 30～40 days, the axillalry bud that grows divided from explant (female branch) cut off, be inoculated into induced bundle bud differentiation on the bud proliferated culture medium again, cultivated 30～40 days, on average each culture differentiates 4～6 of the buds of growing thickly, bud length 2～3cm;
(4), cut apart subculture, budlet becomes the piece successive transfer culture, big bud cuts into terminal bud and stipes, is inoculated into successive transfer culture on the bud proliferated culture medium, changes generation once, reproduction coefficient 4～6 in later per 30～40 days; Build up clump bud vegetative propagation system, the above bud of the 2cm of breeding can carry out graft seedling growth, and seedling is made stripped quality saving or subculture expands numerous.
(5) condition of culture
1. culture medium prescription
Each constituents such as the macroelement in the medium, trace element, organic principle, plant hormone, carbohydrate are carried out repeatedly obtaining on orthogonal design and the gradient experimental basis.Minimal medium of the present invention is improvement MS: identical in macroelement, molysite, micro-three and the MS medium; Organic principle (improvement) consumption is: glycine 2.0/L, VB
11.0～2.0mg/L, VB
61.0～2.0mg/L, nicotinic acid 0.5～1.0mg/L, inositol 100mg/L, folic acid 0.1～1.0mg/L; Sucrose changes 40～55g/L into.(1) axillalry bud starts medium: MS (or improvement MS)+6-BA0.1～0.6mg/L (unit down together)+Kt0.1～0.6+NAA0.01～0.1; (2) bud proliferated culture medium: improvement MS+6-BA0.2～0.5+Kt0.04～0.4+Zt0.2～0.5+NAA0.01～0.1; The pH value 5.8～6.0 of above medium, agar powder 0.6%～0.62%, and the 25min that under 118～120 ℃ of HTHPs, sterilizes.
2. culture environment
Culture environment is provided: constant temperature (24 ± 2) ℃, relative moisture 60%～70%, intensity of illumination are 1000～1600lx, sunshine duration 11～14h (hour)/d (my god)
Embodiment 2:
Select the adult excellent strain fruit tree of healthy and strong no damage by disease and insect tangerine, get take out then living 20d (my god) take out living shoot with interior adult attitude stem, during to the explant cleaning and sterilizing earlier with alcohol solution dipping 20～30s of 75%, aseptic water washing 2～4 times, 0.1% soaks oranges and tangerines shoot 10min again, under aseptic condition, it is segment (band stipes) about 0.5～1cm that the oranges and tangerines shoot is cut into length, is inoculated on the ready medium and cultivates.(1) axillalry bud starts medium: MS+6-BA0.1mg/L (unit down together)+Kt0.1+NAA0.01; (2) bud proliferated culture medium: improvement MS+6-BA0.2+Kt0.04+Zt0.2+NAA0.01, other is identical with embodiment 1.
Embodiment 3:
(1) axillalry bud starts medium: improvement MS+6-BA0.2mg/L (unit down together)+Kt0.6+NAA0.1; (2) bud proliferated culture medium: improvement MS+6-BA0.3+Kt0.4+Zt0.5+NAA0.1, other is identical with embodiment 1 or 2.
Embodiment 4:
(1) axillalry bud starts medium: MS+6-BA0.3mg/L (unit down together)+Kt0.5+NAA0.08; (2) bud proliferated culture medium: improvement MS+6-BA0.5+Kt0.08+Zt0.3+NAA0.08, other is identical with embodiment 1.
Embodiment 5:
(1) axillalry bud starts medium: MS+6-BA0.4mg/L (unit down together)+Kt0.3+NAA0.06; (2) bud proliferated culture medium: improvement MS+6-BA0.4+Kt0.1+Zt0.4+NAA0.06, other is identical with embodiment 1.
Embodiment 6:
(1) axillalry bud starts medium: improvement MS+6-BA0.5mg/L (unit down together)+Kt0.1+NAA0.05; (2) bud proliferated culture medium: improvement MS+6-BA0.3+Kt0.2+Zt0.0.5+NAA0.05, other is identical with embodiment 1.
Embodiment 7:
(1) axillalry bud starts medium: improvement MS+6-BA0.5mg/L (unit down together)+Kt0.1～0.4+NAA0.06; (2) bud proliferated culture medium: improvement MS+6-BA0.2+Kt0.28+Zt0.3+NAA0.04, other is identical with embodiment 1.
Embodiment 8:
(1) axillalry bud starts medium: MS+6-BA0.6mg/L (unit down together)+Kt0.1+NAA0.01; (2) bud proliferated culture medium: improvement MS+6-BA0.2+Kt0.3+Zt0.2+NAA0.03, other is identical with embodiment 1.
Embodiment 9:
(1) axillalry bud starts medium: improvement MS+6-BA0.4mg/L (unit down together)+Kt0.2+NAA0.07; (2) bud proliferated culture medium: improvement MS+6-BA0.3+Kt0.36+Zt0.4+NAA0.07.
Embodiment 10:
(1) axillalry bud starts medium: MS+6-BA0.2mg/L (unit down together)+Kt0.6+NAA0.1; (2) bud proliferated culture medium: improvement MS+6-BA0.4+Kt0.4+Zt0.3+NAA0.1, other is identical with embodiment 1.
Embodiment 11:
(1) axillalry bud starts medium: improvement MS+6-BA0.3mg/L (unit down together)+Kt0.3+NAA0.09; (2) bud proliferated culture medium: improvement MS+6-BA0.4+Kt0.32+Zt0.4+NAA0.09, other is identical with embodiment 1.
Experimental result such as table 1, table 2:
Table 1: the influence that hormone prescription starts and breeds the oranges and tangerines bud
*
| The medium numbering | 6-BAmg/L | Ktmg/L | NAAmg/L | Inoculation explant number (individual) | Just on average sprout and count (individual) for axillalry bud | Inductivity (%) | Subculture is grown or is on average bred number (strain *) | In generation, bred mean (doubly) * |
| 1 | 1.0 | 1.0 | 1.0 | 40 | 0 | — | — | — |
| 2 | 1.0 | 0.4 | 0.1 | 40 | 0 | — | — | — |
| 3 | 1.0 | 0.04 | 0.01 | 40 | 0 | — | — | — |
| 4 | 0.5 | 1.0 | 0.1 | 40 | 0 | — | — | — |
| 5 | 0.5 | 0.4 | 0.01 | 40 | 34 | 85 | 90 | 2.65 |
| 6 | 0.5 | 0.04 | 1.0 | 40 | 2 | 5 | 0 | 0 |
| 7 | 0.1 | 1.0 | 0.01 | 40 | 3 | 7.5 | 4 | 1.33 |
| 8 | 0.1 | 0.4 | 1.0 | 40 | 1 | 2.5 | 0 | 0 |
| 9 | 0.1 | 0.04 | 0.1 | 40 | 30 | 75 | 48 | 1.6 |
*: minimal medium is MS
*: minimal medium is improvement MS
On the basis of table 1 The selection result, continue to have furtherd investigate hormone list factor, multifactor effect, found out the suitable proportion scope that several hormones are sprouted to the oranges and tangerines bud and the bud of growing thickly takes place.
The successful cultivation of the adult attitude regeneration bud of oranges and tangerines also is just for later successive transfer culture.Usually experiment cuts first generation that the female branch of explant sprouts when exsomatizing the axillalry bud successive transfer culture, substantially cultivate at subculture medium MS and to add on the exogenous hormone (preferred hormone cooperate with scope), irreversible dedifferentiation nearly all took place in 10 days in the stripped oranges and tangerines bud of culture, produced a moisture high callus indefiniteness, loose.By a series of researchs of improvement MS organic principle consumption, found that the differentiation again that can effectively keep bud with hormone combinations is joined in the organic principle consumption combination of improvement, obtained the success of regeneration bud successive transfer culture.Table 2 is wherein 1 examples of research improvement organic principle.
Table 2 improvement MS prescription is to the influence of oranges and tangerines bud propagation
*
| The medium numbering | VB 1mg/L | VB 6mg/L | Nicotinic acid mg/L | Sucrose g/L | Folic acid mg/L | Regeneration bud is on average bred number | The long situation of subculture blastogenesis |
| ? | ? | ? | ? | ? | ? | (doubly) | ? |
| 1 * | 0.4 | 0.5 | 0.5 | 30 | 0.0 | 0.00 | The bud callusization |
| 2 | 0.4 | 1.0 | 1.0 | 40 | 0.1 | 1.23 | The long propagation of blastogenesis is slow |
| 3 | 0.4 | 2.0 | 2.0 | 50 | 0.5 | 1.06 | The long propagation of blastogenesis is slow |
| 4 | 0.4 | 4.0 | 4.0 | 60 | 1.0 | 0.68 | Most of bud callusization |
| 5 | 0 | 0.5 | 1.0 | 50 | 1.0 | 0.56 | Most of bud callusization |
| 6 | 0 | 1.0 | 0.5 | 60 | 0.5 | 0.78 | Most of bud callusization |
| 7 | 0 | 2.0 | 4.0 | 30 | 0.1 | 0.00 | The bud callusization |
| 8 | 0 | 4.0 | 2.0 | 40 | 0.0 | 0.65 | Most of bud callusization |
| 9 | 1.0 | 0.5 | 2.0 | 60 | 0.1 | 1.78 | The long propagation of blastogenesis is slow |
| 10 | 1.0 | 1.0 | 4.0 | 50 | 0.0 | 1.85 | The long propagation of blastogenesis is slow |
| 11 | 1.0 | 2.0 | 0.5 | 40 | 1.0 | 3.93 | Bud is bred |
| 12 | 1.0 | 4.0 | 1.0 | 30 | 0.5 | 1.35 | The long propagation of blastogenesis is slow |
| 13 | 2.0 | 0.5 | 4.0 | 40 | 0.5 | 2.32 | Bud propagation better has lopsided bud |
| 14 | 2.0 | 1.0 | 2.0 | 30 | 1.0 | 1.24 | The long propagation of blastogenesis is slow |
| 15 | 2.0 | 2.0 | 1.0 | 60 | 0.0 | 4.21 | Bud propagation has lopsided bud more |
| 16 | 2.0 | 4.0 | 0.5 | 50 | 0.1 | 2.54 | Bud propagation better elongation is slow |
*: No. 1 medium component is identical with MS
*: other composition that uses in 1～No. 16 medium is all identical with hormone.
Claims (3)
1. cultivation method for vegetative organ exsomatized treeing sprout of bearing tree of mandarin orange is characterized in that: may further comprise the steps:
(1.1) the adult excellent strain of selecting healthy and strong no damage by disease and insect tangerine to tie fruit, getting and taking out living 20 days then is explant with interior shoot;
(1.2) to the explant cleaning and sterilizing;
(1.3) explant is seeded in axillalry bud and starts and to be cultured to the axillary bud growth point on the medium and to begin to sprout, and the yellow green small salient point occurs around the base portion of bud, continues to start on the medium at axillalry bud and grows 12～15 days, and main axillalry bud elongation this moment rapidly; Axillalry bud starts medium: MS or improvement MS+6-BA0.1～0.6mg/L+Kt0.1～0.6mg/L+NAA0.01～0.1mg/L; Identical in the modified MS medium, macroelement, molysite, micro-three and MS medium, improvement MS contains the organic principle of following improvement: glycine 2.0mg/L, VB
11.0～2.0mg/L, VB
61.0～2.0mg/L, nicotinic acid 0.5～1.0mg/L, inositol 100mg/L, folic acid 0.1～1.0mg/L, sucrose 40～55g/L;
When (1.4) cultivating 30～40 days, the axillalry bud that grows is cut off from the explant branch, be inoculated into induced bundle bud differentiation on the bud proliferated culture medium, cultivated 30～40 days, average each culture differentiates 4～6 of the buds of growing thickly, long 2～the 3cm of bud, bud proliferated culture medium: improvement MS+6-BA0.2～0.5mg/L+Kt0.04～0.4mg/L+NAA0.01～0.05mg/L;
(1.5) cut apart subculture, budlet becomes the piece successive transfer culture, and big bud cuts into terminal bud and stipes, is inoculated into successive transfer culture on the bud proliferated culture medium, changes generation once, reproduction coefficient 4～6 in later per 30～40 days; Build up clump bud vegetative propagation system, the above bud of the 2cm of breeding can carry out graft seedling growth, and seedling is made stripped quality saving or subculture expands numerous.
2. cultivation method for vegetative organ exsomatized treeing sprout of bearing tree of mandarin orange according to claim 1, it is characterized in that: condition of culture is as follows:
(2.1) culture parameters: 24 ± 2 ℃ of constant temperature, relative moisture 60%～70%, intensity of illumination are 1000～1600lx, sunshine, duration was 11～14 hours/day;
(2.2) technical parameter: the explant pollution rate is lower than 5%～10%; The axillalry bud inductivity 75%～87% of sprouting; The bud growth coefficient 4～6 of growing thickly; Subculture cycle 30～40 days.
3. cultivation method for vegetative organ exsomatized treeing sprout of bearing tree of mandarin orange according to claim 1 is characterized in that: to embathing sterilization with slant acidity toothpaste solution earlier in the explant cleaning and sterilizing process, embathe sterilization with alkaline detergent liquid again; Use 75% ethanolic solution and 0.1%～0.5%HgCl at last
2Solution disinfection.
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| CN104126499A (en) * | 2014-07-22 | 2014-11-05 | 湖南农业大学 | Method for obtaining orange filial generation through embryo rescuing |
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| CN106035087B (en) * | 2016-06-16 | 2017-11-28 | 杭州师范大学 | A kind of method of Changshan grapefruit fast asexual propagation |
| CN108739373B (en) * | 2018-04-23 | 2022-03-04 | 湖南科技学院 | Method for rapid propagation and detoxification of grapefruit stem tips |
| CN117814034A (en) * | 2024-01-31 | 2024-04-05 | 中国热带农业科学院香料饮料研究所 | Seedling raising method for bread fruits |
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| CN104126499A (en) * | 2014-07-22 | 2014-11-05 | 湖南农业大学 | Method for obtaining orange filial generation through embryo rescuing |
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