CN104351051A - Intermediate propagation aseptic seedling of tender Taiwan calocedrus formosana leaves - Google Patents
Intermediate propagation aseptic seedling of tender Taiwan calocedrus formosana leaves Download PDFInfo
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- CN104351051A CN104351051A CN201410584582.2A CN201410584582A CN104351051A CN 104351051 A CN104351051 A CN 104351051A CN 201410584582 A CN201410584582 A CN 201410584582A CN 104351051 A CN104351051 A CN 104351051A
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Abstract
The invention discloses an intermediate propagation aseptic seedling of tender Taiwan calocedrus formosana leaves. The tender Taiwan calocedrus formosana leaves are used as explants which are inoculated into a transition culture medium for culture, good explants are selected and inoculated to a callus induction culture medium for culture, multiple shoots are further separated from callus, and the multiple shoots are cultured in a strong plantlet and rootage culture medium. A matching formula of a plant growth adjusting agent and a natural plant additive is selected for Taiwan calocedrus formosana aseptic seedlings according to the matching ratio of the plant growth adjusting agent, so that vegetative intermediate propagation of the tender Taiwan calocedrus formosana leaves is achieved. A great amount of aseptic seedlings are obtained, the excellent hereditary feature is maintained, and great significance is achieved. Compared with seeding and ordinary vegetative propagation, according to the intermediate propagation, the propagation coefficients of the Taiwan calocedrus formosana seedlings are greatly increased. The intermediate propagation aseptic seedling is beneficial for landscaping, returning of the grain plots to forestry, and forest planting.
Description
Technical field
The invention belongs to biological technical field, particularly the Vegetative tissue of plant is cultivated, and particularly relates to Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling.
Background technology
Taiwan Xiao Nan (Calocedrus formosana (Florin) Florin) is also known as Xiao Nan, young, the yellow meat tree of yellow meat, the evergreen megaphanerophyte of Cupressaceae Xiao Phoebe, for the peculiar gymnosperm in Taiwan, branch, in the mountain region of north Taiwan and middle part height above sea level 300 ~ 1900 meters, is the Major Tree Species Planted of Taiwan temperate warm zone (500 ~ 1800m).One of Taiwan coniferous tree five wood.Taiwan Xiao Nan is tree-like is straight dry oval taper, and bark bronzing, cutter can flow out pale red resin after cutting.Material is careful, and be not subject to the erosion of termite moth, the inclined yellowish-brown of color and luster is high-class timber.With the same column Taiwan such as Taiwan Japan cypress, Chamaecyparis formosensis, fragrant China fir, Taiwania cryptomerioides needle five wood.The same pearl of male and female, cone lignification, be oblong, seed has two wings.Leaf is flakey, tiny but more elongated than Japan cypress.Due to the natural forest of felling energetically in past, cause Taiwan Xiao Nan natural group amount extremely rare, and group there is aging to be inclined to.
Show that Taiwan Xiao Nan elite stand is rare through consulting pertinent literature, solid not good, cottage propagation survival rate is not high, and poor growth.(Zhong Zhende, 2000) with different forest cultivation operating types, the influence research that Taiwan Xiao Nan blossoms and bears fruit is shown, to be completely cured, to prune and to be completely cured and the Taiwan Xiao Nan elite stand of the mode process such as pruning life in 24 years, but within continuous 2 years, only promote the plant blossom of 0 ~ 2.4%, poor effect.(loyal emerging Guo, 2002) are with GA
3process Taiwan Xiao Nan animated period of blooming, 93.9% can bloom, but male flower drops in a large number in advance, causes ripening rate not high.(Guo Baozhang, 1989) carry out Taiwan Xiao Nan cottage propagation test, but survival rate is less than 2%, and the growth of cuttage seedlings survived is slow.
Through consulting pertinent literature, yet there are no the report in about Taiwan Xiao Nan tissue cultures, cuttage etc. in continent at present.And Taiwan's scholars does not have comparatively quantum jump in the Xiao Nan Study on tissue culture of Taiwan yet, Lin Zhimou etc. (1997) point out that immature embryo adds in the WPM medium of 600mg/Lcasein enzymatic hydrolysate, 500mg/L yeast extract, 0.5mg/L BA and 1mg/L NAA to induce and sprout, but bud ratio is lower.With seedling stem section and the blade of 4 ~ 6 months, axillalry bud can be induced to occur at WPM+0.05 mg/LNAA+1mg/LBA, but sprout has vitrification phenomenon, be seeded in WPM+0.01mg/LBA+0.05mg/LTDZ medium with identical explant, adventitious bud induction frequency also only can reach 60%.The inducing effect of callus in MS+ 1mg/LNAA+0.1mg/LBA medium is better, but the effect that callus is divided into Multiple Buds is again bad.
Comprehensively think through consulting pertinent literature: though existing aspect, Taiwan related scientific research personnel have carried out the research of tissue cultures aspect to Taiwan Xiao Nan at present, but general effect is bad, have no complete tissue-culturing rapid propagation system report, and the present invention has explored the optimal medium formula and cultural method that utilize Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling, the present invention has had certain originality and novelty.
Summary of the invention
The object of the invention is to set up one and apply mechanically Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling, thus construct and do not rely on simple seminal propagation or cottage propagation to carry out the traditional planting models of cultivating, the seedling utilizing tissue culture technology to breed maintains maternal characteristic to greatest extent, a large amount of proterties can be obtained in a short time consistent simultaneously, the Taiwan Xiao Nan aseptic seedling of detoxification, for the protection of wild Taiwan Xiao Nan resource, the recovery of group and utilize Taiwan Xiao Nan in Continental Area as conceding the land to forestry, seeds and the Appropriate application of afforestation etc. provide a kind of new technical support.
The present invention realizes by following technology
Explant is cultivated
1) introduce from Taiwan Taiwan Xiao Nan being planted in matrix is in basin humous, water depending on soil moisture content, be placed in the ventilation that faces south for subsequent use, when needs explant material, the blade that top newly grows is positioned at stainless steel scissors clip, as explant material source, explant collection is suitable for and is carried out at the 8:00 ~ 10:00 of fine day.
2) aseptic seedling obtains
The explant material of clip, is placed in clean beaker, and instillation 1-3 drips common liquid detergent, and 1 polysorbas20, covers gauze and colligation is good, is placed in running water down-flow water and rinses 5h, explant material after flushing is on superclean bench, first use 75% alcohol-pickled 50s, with aseptic water washing 3 times, then sterilize the mercuric chloride that the tweezers after the explant sterilizing after rinsing put into 0.1% 8min, aseptic water washing 6 times, each 1.5min, then explant is put into preprepared sterile petri dish, with moisture residual on aseptic filter paper blotting material, before inoculation, blade is cut into the segment of 1.5cm with aseptic stainless steel blade, then the explant cut is inoculated in transitional culture medium, (MS (without inositol)+0.01 ~ 3.0mg/LNAA+5 ~ 7g/L agar).
Until cultivate 15 ~ 20d in transitional culture medium time, the free of contamination explant of picking carries out callus induction.
3) callus induction
Explant free of contamination in transitional culture medium is transferred to callus inducing medium training (MS+0.01mg/L ~ 3.0mg/LNAA+0.01 ~ 2.0mg/L2,4-D+0.01 ~ 1.0mg/L6-BA+25g/L sucrose+5 ~ 7g/L agar).Explant traverse during inoculation, and light pressure explant material makes it fully to contact with medium; Culturing room's temperature 25 DEG C, humidity 70%, intensity of illumination 2200lx, every day light application time 12h, after 7d, visible rice white Lax callus grows from incision, until callus grow to about 1.5cm wide time, proceed to inducing clumping bud medium culture.
4) inducing clumping bud and increment
Loose, peak green, the callus that grows fine to be transferred on redifferential medium (inducing clumping bud and proliferated culture medium:
WPM+0.01 ~ 1.0mg/LNAA+0.01mg/L ~ 2.0mg/LTDZ+0.01 ~ 1.5mg/LKT+0.01mg/L ~ 1.5mg/LGA
3+ 30g/L sucrose+5 ~ 7g/L agar).Callus is gently pressed to make it fully to contact with medium with tweezers, culturing room's temperature 25 DEG C, humidity 70%, intensity of illumination 2200lx, every day light application time 12h, cultivate after 30d, visible Multiple Buds grows in a large number, continue to cultivate 35d, can well differentiated be obtained, the tufted seedling that growing way is vigorous.
5) strong seedling culture
Intercept the tufted seedling of high 4 ~ 5cm, be inoculated on strong seedling culture base, (appropriate (5-10mL) bananas juice+30g/L sucrose+5 ~ 7g/L agar of Rooting and hardening-off culture base: WPM+0.01 ~ 3.0mg/LNAA+0.01mg/L ~ 2.0mg/L IBA+).Culturing room's temperature 25 DEG C, humidity 70%, intensity of illumination 2200lx, every day, light application time 12h, can obtain healthy and strong Taiwan Xiao Nan aseptic seedling after cultivating 7d.
The present invention, in inoculation operating process, when carrying out induction of callus inoculation, needs first to be disposed across on medium by Taiwan Xiao Nan explant, and light pressure explant material makes it fully to contact with medium; When carrying out inducing clumping bud, callus is transferred on redifferential medium, needs gently to press callus to make it fully to contact with medium with tweezers.Avoid that inoculation material out-of-shape causes contact insufficient with medium and growth that is that cause is bad even dead.
Technical scheme of the present invention, has following beneficial effect: relative to traditional planting seed or cottage propagation, and reproduction speed is fast, from explant to seedling, only need 4 months; Reproduction coefficient is high, and indefinite bud exponentially increases; Be not affected by the external environment, the four seasons all can carry out seedling breeding; Seedling good stability, is separated or the situation of decay occurs less; Seedling, by group training, is all detoxic seedling, is beneficial to high yield, few damage by disease and insect; Small investment; only need a small amount of primary explant just can obtain a large amount of aseptic seedling; high financial profit; can large-scale planting; solve this bottleneck of source of seedling, the wild environment cultivation of plantlet in vitro is conducive to the recovery of wild population, can contain that the predatoriness of wild resource is excavated; increase conceding the land to forestry, afforestation, Landscape Trees, good economy, society and ecological benefits can be produced.
Embodiment
Embodiment 1
A basin Taiwan Xiao Nan is introduced for subsequent use from Taiwan, be arranged in stainless steel scissors clip the blade that top newly grows and be placed in clean beaker, instillation 1-3 drips common liquid detergent, 1 polysorbas20, cover gauze and colligation is good, be placed in running water down-flow water and rinse 3-5h, material after flushing is on superclean bench, first use 75% alcohol-pickled 50s, with aseptic water washing 3 times, then sterilize the mercuric chloride that the tweezers after the explant sterilizing after rinsing put into 0.1% 8min, aseptic water washing 6 times, each 1.5min, then explant is put into preprepared sterile petri dish, with moisture residual on aseptic filter paper blotting material, the segment of 1.5cm is cut into aseptic stainless steel blade, then the explant cut is inoculated in transitional culture medium, until cultivate 15 ~ 20d in transitional culture medium time, the free of contamination explant of picking carries out callus induction, explant traverse during inoculation, and light pressure explant material makes it fully to contact with medium, culturing room's temperature 25 DEG C, humidity 70%, intensity of illumination 2200lx, every day light application time 12h, after 7d, visible rice white Lax callus grows from incision, until callus grow to about 1.5cm wide time, picking loosens, peak green, the callus grown fine is transferred on redifferential medium, callus is gently pressed to make it fully to contact with medium with tweezers, culturing room's temperature 25 DEG C, humidity 70%, intensity of illumination 2200lx, every day light application time 12h, after cultivating 30d, visible Multiple Buds grows in a large number, continue to cultivate 35d, well differentiated can be obtained, the tufted seedling that growing way is vigorous, intercept the tufted seedling of high 4 ~ 5cm, be inoculated on strong seedling culture base, culturing room's temperature 25 DEG C, humidity 70%, intensity of illumination 2200lx, every day, light application time 12h, can obtain healthy and strong Taiwan Xiao Nan aseptic seedling after cultivating 14d.
Medium that scheme uses is: explant is cultivated: humus soil.
The transitional culture medium of this invention above-mentioned:
MS (without inositol)+0.01 ~ 3.0mg/LNAA+5 ~ 7g/L agar.
The callus induction optimal medium of this invention:
MS+0.01mg/L ~ 3.0mg/LNAA+0.01 ~ 2.0mg/L2,4-D+0.01 ~ 1.0mg/L6-BA+25g/L sucrose+5 ~ 7g/L agar.
Inducing clumping bud and the propagation optimal medium of this invention:
WPM+0.01 ~ 1.0mg/LNAA+0.01mg/L ~ 2.0mg/LTDZ+0.01 ~ 1.5mg/LKT+0.01mg/L ~ 1.5mg/LGA
3+ 30g/L sucrose+5 ~ 7g/L agar.
The strong plantlets and rootage optimal medium of this invention:
WPM+0.01 ~ 3.0mg/LNAA+0.01mg/L ~ appropriate the bananas juice of 2.0mg/L IBA++30g/L sucrose+5 ~ 7g/L agar.
Claims (6)
1. a Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling, is characterized in that: follow these steps to:
1) explant is cultivated: plant Taiwan Xiao Nan in having in humus soil for subsequent use, when needs explant material, is positioned at the blade that top newly grows, originates as explant material with stainless steel scissors clip;
2) aseptic seedling obtains: the explant material of clip, is placed in clean beaker, and instillation 1-3 drips common liquid detergent, and 1 polysorbas20, covers gauze and colligation is good, is placed in running water down-flow water and rinses 3-5h, explant material after flushing is on superclean bench, first use 75% alcohol-pickled 50s, with aseptic water washing 3 times, then sterilize the mercuric chloride that the tweezers after the explant sterilizing after rinsing put into 0.1% 8min, aseptic water washing 6 times, each 1.5min, then explant is put into preprepared sterile petri dish, with moisture residual on aseptic filter paper blotting material, before inoculation, blade is cut into the segment of 1.5cm with aseptic stainless steel blade, then the explant cut is inoculated in transitional culture medium, until cultivate 15 ~ 20d in transitional culture medium time, the free of contamination explant of picking carries out callus induction,
3) callus induction: explant free of contamination in transitional culture medium is transferred to callus inducing medium and cultivates, explant traverse during inoculation, and light pressure explant material makes it fully to contact with medium; Culturing room's temperature 25 DEG C, humidity 70%, intensity of illumination 2200lx, every day light application time 12h, after 7d, visible rice white Lax callus grows from incision, until callus grow to about 1.5cm wide time, proceed to inducing clumping bud medium culture;
4) inducing clumping bud and propagation: loose, peak green, the callus that grows fine are transferred on redifferential medium, callus is gently pressed to make it fully to contact with medium with tweezers, culturing room's temperature 25 DEG C, humidity 70%, intensity of illumination 2200lx, every day light application time 12h, after cultivating 30d, visible Multiple Buds grows in a large number, continues to cultivate 35d, well differentiated can be obtained, the tufted seedling that growing way is vigorous;
5) strong seedling culture: the tufted seedling intercepting high 4 ~ 5cm, is inoculated on strong seedling culture base, and WPM is minimal medium, culturing room's temperature 25 DEG C, humidity 70%, intensity of illumination 2200lx, and every day, light application time 12h, obtained healthy and strong Taiwan Xiao Nan aseptic seedling after cultivating 14d.
2. a kind of Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling according to claims 1, is characterized in that: the Taiwan Xiao Nan kind described in step 1) is introduced from Taiwan.
3. a kind of Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling according to claims 1, it is characterized in that: described step 2) middle transitional culture medium, take MS as minimal medium, do not add inositol and sucrose, add the NAA of 5 ~ 7g/L agar, 0.01 ~ 3.0mg/L, pH value is adjusted to 6.0 at normal temperatures, autoclaving 20min.
4. the one according to claims 1 utilizes Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling, it is characterized in that: callus inducing medium in described step 3), take MS as minimal medium, add NAA, the 0.01 ~ 2.0mg/L2 of 0.01mg/L ~ 3.0mg/L, the sucrose of 6-BA, 25g/L of 4-D, 0.01 ~ 1.0mg/L, pH value are adjusted to 6.0 under normal temperature, 5 ~ 7g/L agar, autoclaving 20min.
5. a kind of Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling according to claims 1, it is characterized in that: redifferential medium in described step 4), take WPM as minimal medium, add KT, 0.01mg/L ~ 1.5mg/LGA of the TDZ of NAA, 0.01mg/L ~ 2.0mg/L of 0.01 ~ 1.0mg/L, 0.01 ~ 1.5mg/L
3, the sucrose of 30g/L, pH value be adjusted to 6.0 under normal temperature, 5 ~ 7g/L agar, autoclaving 20min, gently presses callus to make it fully to contact with medium during inoculation with tweezers.
6. a kind of Taiwan Xiao Nan young leaflet tablet Fast-propagation aseptic seedling according to claims 1, it is characterized in that: strong seedling culture base in described step 5): take WPM as minimal medium, add the bananas juice of IBA, 5-10mL of NAA, 0.01mg/L ~ 2.0mg/L of 0.01 ~ 3.0mg/L, the sucrose of 30g/L, pH value are adjusted to 6.0 under normal temperature, 5 ~ 7g/L agar, autoclaving 20min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107896985A (en) * | 2017-05-27 | 2018-04-13 | 江苏农林职业技术学院 | A kind of Japanese cypress Initial culture method |
| CN109275563A (en) * | 2018-09-30 | 2019-01-29 | 内蒙古农业大学 | A kind of Chinese juniper rapid propagation in vitro method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07274981A (en) * | 1994-04-13 | 1995-10-24 | Takeda Chem Ind Ltd | Production of hinokitiol by tissue culture |
| CN101218894A (en) * | 2007-09-26 | 2008-07-16 | 南京林业大学 | Embryogenesis and plant strain regeneration method for hybrid somatic cell of slash pine and cuban pine |
| TW200840525A (en) * | 2007-04-13 | 2008-10-16 | Tien-Fu Chang | Manufacturing method of natural smokeless incense |
-
2014
- 2014-10-28 CN CN201410584582.2A patent/CN104351051B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07274981A (en) * | 1994-04-13 | 1995-10-24 | Takeda Chem Ind Ltd | Production of hinokitiol by tissue culture |
| TW200840525A (en) * | 2007-04-13 | 2008-10-16 | Tien-Fu Chang | Manufacturing method of natural smokeless incense |
| CN101218894A (en) * | 2007-09-26 | 2008-07-16 | 南京林业大学 | Embryogenesis and plant strain regeneration method for hybrid somatic cell of slash pine and cuban pine |
Non-Patent Citations (2)
| Title |
|---|
| JIM-MIN FANG: "TERPENOIDS FROM LEAVES OF CALOCEDRUS FORMOSANA", 《PHYTOCHEMISTRY》 * |
| 刘洪谔: "台湾珍贵针叶树种引种造林试验结果", 《浙江林学院学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107896985A (en) * | 2017-05-27 | 2018-04-13 | 江苏农林职业技术学院 | A kind of Japanese cypress Initial culture method |
| CN109275563A (en) * | 2018-09-30 | 2019-01-29 | 内蒙古农业大学 | A kind of Chinese juniper rapid propagation in vitro method |
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