CN104221859B - A kind of fast numerous cultural method of Garcinia mangostana - Google Patents
A kind of fast numerous cultural method of Garcinia mangostana Download PDFInfo
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- CN104221859B CN104221859B CN201410439705.3A CN201410439705A CN104221859B CN 104221859 B CN104221859 B CN 104221859B CN 201410439705 A CN201410439705 A CN 201410439705A CN 104221859 B CN104221859 B CN 104221859B
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Abstract
The present invention relates to plant tissue culture technique, disclose a kind of fast numerous cultural method of Garcinia mangostana, comprise the following steps: (1) Explant surface sterilizing；(2) induction period of adventitious embryo；(3) the outer implant multiplicative stage；(4) regeneration bud root induction.Applying the fast numerous cultural method of Garcinia mangostana of the present invention and breed Garcinia mangostana seedling, growth coefficient is up to about 5.4, and gained test tube Seedling remains the good characteristic of maternal plant, will not produce character segregation phenomenon, and seedling early growth is in order.1 seed was cultivated through 1 year and can be produced 1500 strain left and right Garcinia mangostana seedlings, reaches quick, to breed Garcinia mangostana seedling in a large number purpose, Garcinia mangostana is planted industry and has great practical significance.
Description
Technical field
The present invention relates to field of plant tissue culture technique, particularly relate to a kind of fast numerous cultural method of Garcinia mangostana.
Background technology
The tradition of Garcinia mangostana (GarciniaMangostanaL.) is bred and can be passed through seed growing, but the vigor of Garcinia mangostana seed can not maintain for a long time, and Garcinia mangostana seed growing coefficient is low, seed amount is few, 1 fruit produces at most 2 seeds (a lot of fruits do not have seed), these factor drastic delimitations Garcinia mangostana quick, breed in a large number.Further, since mangosteen self-characteristic, it is impossible to whole year production seed, causes seed supply not enough；Secondly, seedling needs 10-15 just to bloom to bear fruit, and growth cycle is long, industry development cost height, it is difficult to meet the demand in market.In Garcinia mangostana reproductive process, also have to be received by improved variety scion by engrafting method and seedling is improved.As the paper " Hainan Cortex Garciniae seedling breeding technology " being published on " guangdong agricultural science " magazine in June, 2014 such as massed troops, describe the raising technology of Cortex Garciniae seedling and grafting.Although although observing and Garcinia mangostana can being made to shift to an earlier date result phenomenon by grafting, but due to affinity problem between scion and stock, once run into strong wind, occurring, easily caused by grafting part, problems such as rupturing, fracture, therefore the method for grafting there is also significant limitation on Garcinia mangostana seedling breeding.Additionally, also have been reported that the branch with bud point of the seed Seedling selecting life in 1 year, soak root-growing agent, cottage propagation Garcinia mangostana seedling in greenhouse sand bed.Although cuttage branch is easy to take root, but little seedling to have bud to count out less, breed coefficient extremely low, it is also difficult to realize breeding on a large scale of Garcinia mangostana seedling.
Adventitious embryo is to have the embryo that the plant soma of same morphing process is formed with germ cell.The Garcinia mangostana of cultivar only opens female flower, rare male flower.Owing to Garcinia mangostana is parthenogenesis, it does not have be divided into embryo form, and form adventitious embryo, lack qualitative variation.And Garcinia mangostana seed has the structure of uniqueness, seed two ends are not have WD plumule and radicle by what two thin cambium layer were connected to.During seed germination, bud is formed from one end of procambia, and root grows from the seed other end.Due to the special mode of reproduction of the unisexuality of its existence and kernel texture; the adventitious embryo of Garcinia mangostana saves the character of female parent, and can stablize heredity, will not produce the phenomenon of qualitative variation; therefore, the research of Garcinia mangostana adventitious embryo is significant for large-scale cultivation Garcinia mangostana seedling.
Summary of the invention
In order to overcome Garcinia mangostana seed growing coefficient low and other mating system breeds the deficiency existed in industry in Garcinia mangostana, the present invention proposes a kind of fast numerous cultural method of Garcinia mangostana.
The technical scheme is that and be achieved in that:
A kind of fast numerous cultural method of Garcinia mangostana, comprises the following steps:
(1) Explant surface sterilizing
The Garcinia mangostana seed obtained from ripe mangosteen, with added with the solution washing of detergent or liquid detergent or running water 2-5h；Dip 5-30s with ethanol again, dip while shake Garcinia mangostana seed, aseptic water washing；The last 15-20min that soaks in the disinfectant solution containing sodium hypochlorite and Tween 80, immersion limit, limit shake Garcinia mangostana seed, aseptic water washing, standby；
(2) adventitious embryo induction period
Through the Garcinia mangostana seed stripping and slicing that step (1) processes, proceed to and MS culture medium carries out inducing culture；
(3) the outer implant multiplicative stage
Outer implant proceeds to WPM and cultivates evoking adventive bud；Treat that adventitious bud grows blade, after cultivation, take blade crosscut 3-5mm, cultivate 40-60d and produce bud point；Proceed to proliferated culture medium from the excision of outer implant when bud point grows into regeneration bud and carry out enrichment culture；
(4) regeneration bud root induction
When regeneration bud grows to 13-17mm, proceed to root media root induction, move on to nursery after cultivation and heel in.
Further, detergent used by described step (1) or liquid detergent aqueous solution mass concentration are 1-3%, and the volumetric concentration of ethanol is 65-75%, and the volumetric concentration of sodium hypochlorite is 5-8%, and the volumetric concentration of Tween 80 is 0.1-0.3%.
Further, the described Garcinia mangostana seed adventitious embryo of step (2) is separated into 3-6 block.
Further, step (2) used medium is the MS culture medium of the BA adding NAA and the 0-5mg/L that mass concentration is 0.3-0.8mg/L, or interpolation mass concentration is the 2 of 2.0-4.0mg/L, the MS culture medium of the BA of 4-D and 0-5mg/L, light application time 8-12h/d carries out inducing culture.
Further, step (3) is cultivated outer implant and is grown the sucrose of BA, 10-30g/L that the culture medium used by blade is interpolation mass concentration 2-6mg/L and the WPM culture medium of the Phytagel of 2-3g/L, carries out crosscut after leaf culture 30-50d；Regeneration bud proliferated culture medium is the WPM culture medium adding the BA that mass concentration is 0.9-1.3mg/L.
Further, step (3) is cultivated outer implant and is grown the WPM that the culture medium used by blade is the Phytagel adding the sucrose of BA, 15-25g/L of 4-5mg/L, 2.4-2.6g/L；Regeneration bud proliferated culture medium is the WPM culture medium adding the BA that mass concentration is 1.0-1.2mg/L.
Further, the described regeneration bud height of step (3) excises when 5-6mm.
Further, step (4) described root media is the 1/2MS culture medium adding the IBA that mass concentration is 0.1-1.0mg/L, and condition of culture is intensity of illumination 2000Lx, light application time 8-12 hour/day, and incubation time is 40-60d.
Further, step (4) test tube Seedling stem height 5-7cm, root length 3-5cm moves on to nursery and heels in.
Beneficial effects of the present invention:
The present invention utilizes the gynecogenic characteristic of Garcinia mangostana, its adventitious embryo does not have, as outer implant, the phenomenon that characters of progenies separates, by fast numerous cultural method of the present invention, growth coefficient is about 5.4, can be quick, large scale cultivating Garcinia mangostana seedling, compensate for Garcinia mangostana and adopt seminal propagation, grafting, the breeding coefficient that the method breedings such as cuttage exist is low, growth cycle is long, the problems such as seedling wind resistance difference, the seedling produced remains the good characteristic of maternal plant, upgrowth situation is good, character segregation phenomenon will not be produced, 1 Garcinia mangostana seed was cultivated through 1 year and can be produced 1500 strain Garcinia mangostana seedlings, substantially increase the reproduction speed of Garcinia mangostana, it is capable of quickly, stably breed the purpose of Garcinia mangostana seedling, Garcinia mangostana is planted industry there is great practical significance.
Detailed description of the invention
Technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described embodiment is only a part of embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, the every other embodiment that those of ordinary skill in the art obtain under not making creative work premise, broadly fall into the scope of protection of the invention.
Embodiment 1
A kind of fast numerous cultural method of Garcinia mangostana, comprises the following steps:
(1) Explant surface sterilizing
The Garcinia mangostana seed obtained from ripe mangosteen, washes 1-2 time with the aqueous solution added with mass concentration 1% detergent or liquid detergent or running water 1-5h；Dip 5-15s with the ethanol of volume fraction 65% again, dip while shake Garcinia mangostana seed, aseptic water washing 2-4 time；The last 15-20min that soaks in the disinfectant solution of volumetric concentration 5% sodium hypochlorite and volumetric concentration 0.1% Tween 80, immersion limit, limit shake Garcinia mangostana seed, aseptic water washing 3-5 time, the Garcinia mangostana seed after surface sterilization, sterilizing is standby；For avoiding Garcinia mangostana seed activity to reduce, the seed taken out from fruit need to process in 5d；
(2) inducing culture of adventitious embryo
The Garcinia mangostana seed processed through step (1) is cut into 3 pieces, proceeds to inducing culture in MS culture medium, and light application time is 8h/d, adds the naphthalene acetic acid (1-Naphthaleneaceticacid, NAA) of 0.3mg/L in culture medium；(3) propagation of induced tissue
Outer implant proceeds to xylophyta culture medium (WoodyPlantmedium, WPM culture medium) cultivate evoking adventive bud, it is 2mg/LBA, 10g/L sucrose and 2g/LPhytagel that WPM culture medium contains mass concentration, Adventitious bud culture cultivates 30d after growing blade, take blade crosscut 3-5mm to cultivate, after MS culture medium culturing 40d, every leaf explant can produce the bud point of about 50；Each bud point can grow up to a regeneration bud, and regeneration bud height, when 5-6mm, after growing the basic structure sprouted, excises from outer implant, carries out enrichment culture in the WPM of 0.9mg/LBA cultivates；
(4) regeneration bud root induction
When regeneration bud grows to 13mm, 1/2MS culture medium is added lower 8 hours/day of IBA, 2000Lx illumination condition of 0.1mg/L, carries out root induction, after cultivating 45 days, after test tube Seedling stem height 5-7cm, root length 3-5cm, move on to nursery heel in.
Embodiment 2
A kind of fast numerous cultural method of Garcinia mangostana, comprises the following steps:
(1) Explant surface sterilizing
The Garcinia mangostana seed obtained from ripe mangosteen, washes 1-2 time with the aqueous solution added with mass concentration 2% detergent or liquid detergent or running water 1-5h；Dip 10-20s with the ethanol of volumetric concentration 70% again, dip while shake Garcinia mangostana seed, aseptic water washing 2-4 time；The last 15-20min that soaks in the disinfectant solution of volumetric concentration 7% sodium hypochlorite and 0.2% Tween 80, immersion limit, limit shake Garcinia mangostana seed, aseptic water washing 3-5 time, the Garcinia mangostana seed after surface sterilization, sterilizing is standby；For avoiding Garcinia mangostana seed activity to reduce, the seed taken out from fruit need to process in 5d；
(2) inducing culture of adventitious embryo
The Garcinia mangostana seed processed through step (1) is cut into 4 pieces and is seeded in inducing culture in MS culture medium, light application time is 10h/d, culture medium is added the NAA and and the BA (6-benzyladenine, 6-Benzylaminopurine) of 2.0mg/L of 0.5mg/L；
(3) propagation of induced tissue
Outer implant proceeds to WPM culture medium culturing evoking adventive bud, WPM culture medium contains 4.5mg/LBA, 20g/L sucrose and 2.5g/LPhytagel, after adventitious bud grows blade, takes blade crosscut 3-5mm and cultivate after cultivating 40d, after cultivating 50d, outside every leaf, implant can produce the bud point of about 50；Each bud point can grow up to a regeneration bud, and regeneration bud height, after 5-6mm grows the basic structure sprouted, excises from outer implant, carries out enrichment culture in the WPM of 1.1mg/LBA cultivates；
(4) regeneration bud is taken root
When regeneration bud grows to 15mm, adding 10h/d under IBA, the 2000Lx illumination condition of 0.5mg/L in 1/2MS culture medium, carry out root induction, cultivation 50d moves on to nursery after test tube Seedling stem height 5-7cm, root length 3-5cm and heels in.
Embodiment 3
The Garcinia mangostana seed obtained from ripe mangosteen, comprises the following steps:
(1) Explant surface sterilizing
The Garcinia mangostana seed obtained from ripe mangosteen, washes 1-2 time with the aqueous solution added with mass concentration 3% detergent or liquid detergent or running water 1-5h；Dip 20-30s with the ethanol of volume fraction 75% again, dip while shake Garcinia mangostana seed, aseptic water washing 2-4 time；The last 15-20min that soaks in the disinfectant solution of volumetric concentration 5-8% sodium hypochlorite and 0.3% Tween 80, immersion limit, limit shake Garcinia mangostana seed, aseptic water washing 3-5 time, the Garcinia mangostana seed after surface sterilization, sterilizing is standby；For avoiding Garcinia mangostana seed activity to reduce, the seed taken out from fruit need to process in 5d；
(2) inducing culture of adventitious embryo
The Garcinia mangostana seed processed through step (1) is cut into 6 pieces, is seeded in inducing culture in MS culture medium, and light application time is 12h/d, adds the BA of NAA and the 5mg/L of 0.8mg/L in culture medium；
(3) propagation of induced tissue
Outer implant proceeds to WPM culture medium culturing evoking adventive bud, and WPM culture medium contains 6mg/LBA, 30g/L sucrose and 3.0g/LPhytagel, grows after blade until adventitious bud, after cultivating 50d, taking leaf crosscut 3-5mm to cultivate, after cultivating 60d, the every outer implant of leaf can produce about 50 bud points；Each bud point can grow up to a regeneration bud, and regeneration bud height, after 5-6mm grows the basic structure sprouted, excises from outer implant, carries out enrichment culture in the WPM of 1.3mg/LBA cultivates；
(4) regeneration bud root induction
When regeneration bud grows to 17mm, adding 12h/d under IBA, the 2000Lx illumination condition of 1.0mg/L, root induction in 1/2MS culture medium, cultivation 60d moves on to nursery after test tube Seedling stem height 5-7cm, root length 3-5cm and heels in.
Embodiment 4
A kind of fast numerous cultural method of Garcinia mangostana, wherein adds the 2,4-D of 2mg/L in MS culture medium in step (2), and all the other operations are with embodiment 1.
Embodiment 5
A kind of fast numerous cultural method of Garcinia mangostana, wherein adds the BA of 3mg/L2,4-D and 2.0mg/L in MS culture medium in step (2), all the other operations are with embodiment 2.
Embodiment 6
A kind of fast numerous cultural method of Garcinia mangostana, wherein adds the BA of 4mg/L2,4-D and 5mg/L in MS culture medium in step (2), all the other operations are with embodiment 3.
The fast numerous cultural method breeding effect of Garcinia mangostana of the present invention is observed
Taking 30 well-developed Garcinia mangostana seeds, be allocated as 6 groups, often group 5, the cultural method being respectively adopted in above 6 embodiments carries out numerous cultivation 1 year soon, calculates the Garcinia mangostana test tube Seedling number of gained.Experimental result is in Table 1.
The fast numerous cultural method breeding effect of watch 1 Garcinia mangostana of the present invention
In above example, comprehensive whole group of training process, more relative relative to embodiment 4-6 of the tissue cultured seedling number of embodiment 1-3 gained, the production as Garcinia mangostana tissue cultured seedling is described, NAA has more obvious advantage.Therefore, actual production preferentially adopts the tissue culture method described in embodiment 1-3.
Adopting the fast numerous cultural method of Garcinia mangostana of the present invention, breed coefficient up to about 5.4, a seed cultivated, through 1 year, the test tube Seedling that can produce about 1200-1500 strain.Above 6 embodiment gained test tube Seedlings all save the character of its female parent, do not occur the problem such as trait segregation and qualitative variation in cultivation, it is achieved that quickly, stably breed on a large scale, to realizing, Garcinia mangostana implantation in large scale is significant for Garcinia mangostana seedling.
The adventitious embryo cutting number impact on Garcinia mangostana induced bud number
In the inducing culture stage, Garcinia mangostana adventitious embryo being cut into 1-6 block, other operations, with embodiment 1, investigate the cutting number impact on Garcinia mangostana induced bud number that adventitious embryo is different.
Under the same terms, adventitious embryo is cut into 3-6 block than not cutting or be cut into 2 pieces of many about 20-40% of induced bud number produced, and is cut into 6 pieces of ratios and is cut into 3 pieces of induced bud numbers many 30.4%.Therefore adventitious embryo is cut into 3-6 block by the fast numerous cultural method of Garcinia mangostana of the present invention, and with 6 pieces for best.
The impact on Garcinia mangostana induced bud number of the induction period light application time
In the inducing culture stage, light application time is respectively provided with 0,4,8,12 hours/day, and other operations, with embodiment 3, investigate the light application time impact on inducing effect.
It is shown that under same culture conditions, 8-12 hour/day about 15-17% more than the induced bud number that other condition of culture produce of illumination, therefore in the fast numerous cultural method step (2) of Garcinia mangostana of the present invention, light application time is 8-12 hour/day.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included in protection scope of the present invention.
Claims (7)
1. the fast numerous cultural method of Garcinia mangostana, it is characterised in that comprise the following steps:
(1) Explant surface sterilizing
The Garcinia mangostana seed obtained from ripe mangosteen, with added with the solution washing of detergent or liquid detergent or running water 2-5h；Dip 5-30s with ethanol again, dip while shake Garcinia mangostana seed, aseptic water washing；The last 15-20min that soaks in the disinfectant solution containing sodium hypochlorite and Tween 80, immersion limit, limit shake Garcinia mangostana seed, aseptic water washing, standby；
(2) adventitious embryo induction period
Through the Garcinia mangostana seed stripping and slicing that step (1) processed, the MS culture medium or the interpolation mass concentration that proceed to the BA adding NAA and the 0-5mg/L that mass concentration is 0.3-0.8mg/L are the 2 of 2.0-4.0mg/L, in the MS culture medium of the BA of 4-D and 0-5mg/L, light application time 8-12h/d induces adventitious embryo；
(3) multiplicative stage
The adventitious embryo that step (2) obtains is proceeded to the sucrose of BA, 10-30g/L of adding mass concentration 2-6mg/L and the WPM culture medium evoking adventive bud of the Phytagel of 2-3g/L；Treat that adventitious bud grows blade, after cultivation, take blade crosscut 3-5mm, produce bud point at MS culture medium culturing 40-60d；Proceed to proliferated culture medium from blade excision when bud point grows into regeneration bud and carry out enrichment culture；Proliferated culture medium is the WPM culture medium adding the BA that mass concentration is 0.9-1.3mg/L；
(4) regeneration bud root induction
When regeneration bud grows to 13-17mm, proceed to root media root induction, move on to nursery after cultivation and heel in；Described root media is the 1/2MS culture medium adding the IBA that mass concentration is 0.1-1.0mg/L.
2. a kind of fast numerous cultural method of Garcinia mangostana according to claim 1, it is characterized in that: detergent used by step (1) or liquid detergent aqueous solution mass concentration are 1-3%, the volumetric concentration of ethanol is 65-75%, the volumetric concentration of sodium hypochlorite is 5-8%, and the volumetric concentration of Tween 80 is 0.1-0.3%.
3. a kind of fast numerous cultural method of Garcinia mangostana according to claim 1, it is characterised in that: step (2) gained adventitious embryo is separated into 3-6 block.
4. a kind of fast numerous cultural method of Garcinia mangostana according to claim 1, it is characterised in that: the culture medium of step (3) evoking adventive bud is the WPM culture medium of the Phytagel adding the sucrose of BA, 15-25g/L of 4-5mg/L, 2.4-2.6g/L；Crosscut is carried out after leaf culture 30-50d；Proliferated culture medium is the WPM culture medium adding the BA that mass concentration is 1.0-1.2mg/L.
5. a kind of fast numerous cultural method of Garcinia mangostana according to claim 1, it is characterised in that: the described regeneration bud height of step (3) excises when 5-6mm.
6. a kind of fast numerous cultural method of Garcinia mangostana according to claim 1, it is characterised in that: step (4) condition of culture is intensity of illumination 2000Lx, light application time 8-12h/d, incubation time 40-60d.
7. a kind of fast numerous cultural method of Garcinia mangostana according to claim 1, it is characterised in that: step (4) test tube Seedling stem height 5-7cm, root length 3-5cm moves on to nursery and heels in.
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| CN106497860A (en) * | 2016-11-04 | 2017-03-15 | 广州赛莱拉干细胞科技股份有限公司 | Culture medium and the preparation method of Garcinia mangostana cell secondary metabolitess |
| CN107396726A (en) * | 2017-07-19 | 2017-11-28 | 福建师范大学 | A kind of method for culturing seedlings for improving Common zenia cuttage root-taking quantity |
| CN115413540B (en) * | 2022-09-30 | 2023-05-16 | 中国热带农业科学院海口实验站 | Breeding method for improving survival rate of mangosteen seedlings |
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| CN108323369A (en) * | 2018-03-14 | 2018-07-27 | 佛山市三水区嘉信农业技术研究院（普通合伙） | A kind of mangosteen implantation methods rich in various trace elements |
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