CN103210845A - Method for obtaining aseptic seedling by using pinus massoniana seed - Google Patents
Method for obtaining aseptic seedling by using pinus massoniana seed Download PDFInfo
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- CN103210845A CN103210845A CN2013101499826A CN201310149982A CN103210845A CN 103210845 A CN103210845 A CN 103210845A CN 2013101499826 A CN2013101499826 A CN 2013101499826A CN 201310149982 A CN201310149982 A CN 201310149982A CN 103210845 A CN103210845 A CN 103210845A
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Abstract
The invention discloses a method for obtaining an aseptic seedling by using a pinus massoniana seed. By virtue of good water absorption and holding performance of sponge, the sterilized pinus massoniana seed is placed in the sponge to culture for 10-20 days to obtain the aseptic seedling which grows strongly. When the method disclosed by the invention is applied to culturing the pinus massoniana aseptic seedling, the time for the emerging of seedling is obviously reduced, and nursery stock growth, especially root growth is superior to that in agar culture; simultaneously, experimental procedure is simplified and culture efficiency is improved; in addition, as the sponge can be repeatedly used without generating wastes, seedling propagation cost is saved and environment is protected; and the aseptic seedling propagation method disclosed by the invention is economical, efficient, simple and convenient to operate, suitable for researche and production of biotechnologies such as the isolated culture of the pinus massoniana and transgenosis, and has important significance on the improved variety propagation of the pinus massoniana and biotechnology researches.
Description
Technical field
The invention belongs to masson pine tissue culture technique field, relate in particular to a kind of method of utilizing masson pine seed to obtain aseptic seedling.
Background technology
Masson pine (Pinus massoniana) is China important dual-purpose seeds of fat material in south, and it is of many uses, economic worth is high, is the primary raw material seeds of industries such as China woods slurry paper (plate), rosin and building.Except that timber and two large purposes of rosin, the full tree of masson pine all can utilize, and pollen, needle and bark are rich in polyphenoils, can be used as good anti-ageing and anti-oxidation health product; Pine nut oil-containing 30% can be made paint, soap and lubricating oil; Cone can refine crude oil; Pine stump can extract pine tar, can also cultivate precious traditional Chinese medicine---Poria cocos.
Through the genetic improvement research in more than 30 years, masson pine fine provenance-superior stand-seed collecting seed production stand-primary seed garden-improvement was built up for multi-level breeding bases such as seed orchards in Guangxi, was promoting to have brought into play important function aspect the masson pine improved variety process.Compare with other commerical tree species, the vegetative propagation difficulty of masson pine is bigger, and wherein tissue culture is difficult more.But, capture the target that the pine tree tissue culture technique is world's breeding scholar always because the tissue culture of plant has advantages such as reproduction coefficient is big, seedling growth is neat.Traditional aseptic seedling is cultivated agar powder (bar) medium that adopt more, needs to operate comparatively loaded down with trivial details through agar powder (bar) heating for dissolving-sterilization-cooling three steps of condensing; In addition, aseptic seedling is cultivated owing to there are various problems such as percentage of seedgermination and pollution, and the planting percent of aseptic seedling generally can only reach 5～20%, makes 80～95% medium become discarded object and is difficult to recycling and causes waste; Simultaneously, the gas permeability of agar is relatively poor, and seedling growth especially root growth is good inadequately.Therefore, seek a kind of simple, economical and efficiently method to cultivate aseptic seedling be to carry out the matter of utmost importance that researchs such as masson pine tissue culture and gene engineering need to be resolved hurrily.
Summary of the invention
The technical problem to be solved in the present invention provides a kind ofly operates the masson pine seed of utilizing simple and easy, economical and efficient and obtains the method for aseptic seedling.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: utilize masson pine seed to obtain the method for aseptic seedling, with water-absorbing sponge as medium.
The above-mentioned method of utilizing masson pine seed to obtain aseptic seedling may further comprise the steps:
＜1〉medium is prepared
The Chang ╳ of clip is wide to be 3-5cm ╳ 1-2cm sponge, and sponge is fully absorbed water, and the sponge that suctions water is put into tissue culture bottle, tightens bottle cap, sterilization 20min under 121 ℃ of autoclaves;
＜2〉seed disinfection
Adopt 0.1% mercuric chloride to the seed 8-10min that carries out disinfection, aseptic water washing 4-5 time;
＜3〉inoculation
Under aseptic condition with step＜2 seed that disinfects is inoculated into step＜1 the sponge medium in;
＜4〉cultivate
With step＜3〉tissue culture bottle place under the 23-25 ℃ of condition dark the cultivation 10-20 days.
Step＜1〉in sponge lie at the bottom of the tissue culture bottle step＜3 in seed be inoculated into the sponge upper surface.
Step＜1〉in sponge be converted into the right angle and paste the bottle wall and vertically be placed on (moisture content of sponge forms graded, and vertical adherent moisture content is lower than horizontal positioned) at the bottom of the tissue culture bottle, step＜3 in seed be inoculated into the parallel bottle sponge upper surface at the end.
The adherent height 0.5-1.5cm of sponge.
The above-mentioned method of utilizing masson pine seed to obtain aseptic seedling, further comprising the steps of:
＜5〉induce
With step＜4〉middle robust growth, the aseptic seedling of high 3-7cm is taken out proliferation-inducing or the callus induction that is used for leaf, stem and bud, and with all the other ablastous seeds cleanings, sponge is cleaned and dries.
Cultivate the problem that adopts agar to exist as medium at present aseptic seedling, the inventor adopts the sponge with good suction and water retention property to set up the method for utilizing masson pine seed to obtain aseptic seedling as medium, the masson pine seed after the sterilization is put into water-absorbing sponge cultivate the aseptic seedling that can obtain robust growth in 10-20 days.Use the present invention and cultivate the masson pine aseptic seedling, seedling-growing time obviously shortens, and seedling growth especially root growth is better than the agar cultivation; Simplify experimental procedure simultaneously, improved culture efficiency; In addition, because sponge can reuse and not produce discarded object, saved the reproductive-cost of growing seedlings and protected environment again.Method for cultivating aseptic seedling economical and efficient of the present invention and easy and simple to handle is applicable to the research and the production of biotechnologys such as the cultured in vitro of masson pine and transgenosis, and is significant to stock breeding and the biotechnology research of masson pine.
Description of drawings
Fig. 1 is that the present invention utilizes masson pine seed to obtain the reference state figure (sponge is vertical) of the method embodiment 1 to 3 of aseptic seedling.
Fig. 2 is that the present invention utilizes masson pine seed to obtain the reference state figure (sponge keeps flat) of the method embodiment 4 to 5 of aseptic seedling.
Among the figure: 1 tissue culture bottle, 2 sponges, 3 seeds.
Embodiment
Further specify the present invention below in conjunction with drawings and Examples.
Embodiment 1
＜1〉medium is prepared
The Chang ╳ of clip is wide to be about 3cm ╳ 2cm sponge, place running water fully to absorb water sponge, the sponge that will suction water with tweezers is put into tissue culture bottle, as shown in Figure 1, sponge is converted into right angle subsides bottle walls (adherent height 1cm) and vertically is placed at the bottom of the tissue culture bottle, tighten bottle cap, sterilization 20min under 121 ℃ of autoclaves;
＜2〉seed disinfection
Flowing water flushing 5h, 0.5% liquor potassic permanganate soaks 20min, and flowing water washes down, and adopts 0.1% mercuric chloride to the seed 8min that carries out disinfection, aseptic water washing 5 times in superclean bench;
＜3〉inoculation
With step＜2〉seed that disinfects is inoculated into step＜1 on superclean bench the sponge medium in the sponge upper surface at the parallel bottle end;
＜4〉cultivate
With step＜3〉tissue culture bottle place under 24 ℃ of conditions dark the cultivation 15 days;
＜5〉induce
With step＜4〉middle robust growth, the aseptic seedling of high about 5.5cm is taken out the proliferation-inducing that is used for bud, all the other is not germinateed or mouldy seed cleaning, and sponge is cleaned and dries and can reuse.
＜1〉medium is prepared
The Chang ╳ of clip is wide to be about 4.5cm ╳ 2cm sponge, place running water fully to absorb water sponge, the sponge that will suction water with tweezers is put into tissue culture bottle, and sponge is converted into right angle subsides bottle walls (adherent height 1.5cm) and vertically is placed at the bottom of the tissue culture bottle, tighten bottle cap, sterilization 20min under 121 ℃ of autoclaves;
＜2〉seed disinfection
Flowing water flushing 6h, 0.3% liquor potassic permanganate soaks 30min, and flowing water washes down, and adopts 0.1% mercuric chloride to the seed 10min that carries out disinfection, aseptic water washing 4 times in superclean bench;
＜3〉inoculation
With step＜2〉seed that disinfects is inoculated into step＜1 on superclean bench the sponge medium in the sponge upper surface at the parallel bottle end;
＜4〉cultivate
With step＜3〉tissue culture bottle place under 25 ℃ of conditions dark the cultivation 18 days;
＜5〉induce
With step＜4〉middle robust growth, the aseptic seedling of high about 7cm is taken out the proliferation-inducing that is used for bud, all the other is not germinateed or mouldy seed cleaning, and sponge is cleaned and dries and can reuse.
Embodiment 3
＜1〉medium is prepared
The Chang ╳ of clip is wide to be about 2.8cm ╳ 1.5cm sponge, place running water fully to absorb water sponge, the sponge that will suction water with tweezers is put into tissue culture bottle, and sponge is converted into right angle subsides bottle walls (adherent height 0.8cm) and vertically is placed at the bottom of the tissue culture bottle, tighten bottle cap, sterilization 20min under 121 ℃ of autoclaves;
＜2〉seed disinfection
Flowing water flushing 5h, 0.5% liquor potassic permanganate soaks 15min, and flowing water washes down, and adopts 0.1% mercuric chloride to the seed 9min that carries out disinfection, aseptic water washing 5 times in superclean bench;
＜3〉inoculation
With step＜2〉seed that disinfects is inoculated into step＜1 on superclean bench the sponge medium in the sponge upper surface at the parallel bottle end;
＜4〉cultivate
With step＜3〉tissue culture bottle place under 23 ℃ of conditions dark the cultivation 17 days;
＜5〉induce
With step＜4〉middle robust growth, the aseptic seedling of high about 6.8cm is taken out the proliferation-inducing that is used for bud, all the other is not germinateed or mouldy seed cleaning, and sponge is cleaned and dries and can reuse.
Embodiment 4
＜1〉medium is prepared
The Chang ╳ of clip is wide to be about 2.3cm ╳ 1.5cm sponge, places running water fully to absorb water sponge, and the sponge that will suction water with tweezers is put into tissue culture bottle, and as shown in Figure 2, sponge lies at the bottom of the tissue culture bottle, tightens bottle cap, in 121 ℃ of autoclaves sterilization 20min down;
＜2〉seed disinfection
Flowing water flushing 5.5h, 0.4% liquor potassic permanganate soaks 18min, and flowing water washes down, and adopts 0.1% mercuric chloride to the seed 11min that carries out disinfection, aseptic water washing 5 times in superclean bench;
＜3〉inoculation
With step＜2〉seed that disinfects is inoculated into step＜1 on superclean bench the sponge upper surface;
＜4〉cultivate
With step＜3〉tissue culture bottle place under 23 ℃ of conditions dark the cultivation 20 days;
＜5〉induce
With step＜4〉middle robust growth, the aseptic seedling of high about 6.0cm is taken out the proliferation-inducing that is used for bud, all the other is not germinateed or mouldy seed cleaning, and sponge is cleaned and dries and can reuse.
Embodiment 5
＜1〉medium is prepared
The Chang ╳ of clip is wide to be about 3.0cm ╳ 1.7cm sponge, places running water fully to absorb water sponge, and the sponge that will suction water with tweezers is put into tissue culture bottle, and sponge lies at the bottom of the tissue culture bottle, tightens bottle cap, in 121 ℃ of autoclaves sterilization 20min down;＜2〉seed disinfection
Flowing water flushing 4h, 0.4% liquor potassic permanganate soaks 20min, and flowing water washes down, and adopts 0.1% mercuric chloride to the seed 9min that carries out disinfection, aseptic water washing 4 times in superclean bench;
＜3〉inoculation
With step＜2〉seed that disinfects is inoculated into step＜1 on superclean bench the sponge upper surface;
＜4〉cultivate
With step＜3〉tissue culture bottle place under 25 ℃ of conditions dark the cultivation 20 days;
＜5〉induce
With step＜4〉middle robust growth, the aseptic seedling of high about 5.8cm is taken out the proliferation-inducing that is used for bud, all the other is not germinateed or mouldy seed cleaning, and sponge is cleaned and dries and can reuse.
Reference examples
＜1〉medium is prepared
Take by weighing 7g/ and rise agar strip, agar strip is shredded or smash with cracker, add suitable quantity of water heating for dissolving while stirring, dissolving is finished with beaker and is settled to 1 liter, fully mixes thoroughly, pours into while hot in the blake bottle, every bottle of 30-40mL, tighten bottle cap, sterilization 20min naturally cools to and condenses under 121 ℃ of autoclaves;
＜2〉seed disinfection
Flowing water flushing 4.5h, 0.2% liquor potassic permanganate soaks 25min, flowing water washes down, in superclean bench with 0.1% mercuric chloride to the seed 9min that carries out disinfection, aseptic water washing 5 times;
＜3〉inoculation
With step＜2〉seed that disinfects is inoculated into step＜1 on superclean bench agar medium in;
＜4〉cultivate
With step＜3〉tissue culture bottle be positioned under 25 ℃ of conditions dark the cultivation 45 days;
＜5〉induce
With step＜4〉in robust growth, the aseptic seedling of high about 5.6cm is taken out and is carried out the bud proliferation-inducing, all the other is not germinateed or the blake bottle of mouldy seed is put into the autoclave heating and made the agar dissolving, pours agar in the waste liquid basin concentrated abandoning.
Table 1 aseptic seedling culture effect comparison sheet
Conclusion: as seen from Table 1, use the embodiment 1 to 5 that this method is handled, germination rate is 20～25%, and height of seedling is 5.5～7.0cm, and incubation time is 15～20 days, robust growth; In the reference examples, germination rate has only 15%, and height of seedling is 5.6cm, and incubation time is 45 days, and a little less than the growth, the germination rate of embodiment and contrast and height of seedling are close substantially, and embodiment is slightly high; The embodiment of the invention all is better than reference examples on incubation time and upgrowth situation.
Wherein the germination rate of embodiment 1～3 and embodiment 4～5, seedling bud height and 3 aspects of upgrowth situation do not have significant difference, but embodiment 1～3 is better than embodiment 4～5 on incubation time.So being converted into the method for pasting the bottle wall in the right angle, the sponge among the embodiment 1～3 has certain advantage.
The incubation time average out to of embodiment 1～5 18 days obviously is better than 45 days of reference examples, and incubation time shortens about 30 days, has clear superiority.The upgrowth situation of embodiment 1～5 is better than reference examples, and reason is the good permeability of sponge in agar, causes the aseptic seedling well developed root system of growing on the sponge medium, seedling growth stalwartness.Because Plant Tissue Breeding needs 23-25 ℃ of 24h temperature control; carry out under the 8h artificial lighting condition; the incubation time shortening can not only effectively save production cost and can also accelerate the production cycle of tissue cultivating seedling in 30 days; simultaneously; sponge among the embodiment can reuse and not produce discarded object, thereby has protected environment.
Claims (6)
1. a method of utilizing masson pine seed to obtain aseptic seedling is characterized in that with water-absorbing sponge as medium.
2. the method for utilizing masson pine seed to obtain aseptic seedling according to claim 1 is characterized in that may further comprise the steps:＜1〉the medium preparation
The Chang ╳ of clip is wide to be 3-5cm ╳ 1-2cm sponge, and sponge is fully absorbed water, and the sponge that suctions water is put into tissue culture bottle, tightens bottle cap, sterilization 20min under 121 ℃ of autoclaves;
＜2〉seed disinfection
Adopt 0.1% mercuric chloride to the seed 8-10min that carries out disinfection, aseptic water washing 4-5 time;
＜3〉inoculation
Under aseptic condition with step＜2 seed that disinfects is inoculated into step＜1 the sponge medium in;
＜4〉cultivate
With step＜3〉tissue culture bottle place under the 23-25 ℃ of condition dark the cultivation 10-20 days.
3. the method for utilizing masson pine seed to obtain aseptic seedling according to claim 2 is characterized in that: step＜1〉in sponge lie at the bottom of the tissue culture bottle step＜3 in seed be inoculated into the sponge upper surface.
4. the method for utilizing masson pine seed to obtain aseptic seedling according to claim 3 is characterized in that: step＜1〉in sponge be converted into the right angle and paste the bottle wall and vertically be placed at the bottom of the tissue culture bottle step＜3 in seed be inoculated into the parallel bottle sponge upper surface at the end.
5. the method for utilizing masson pine seed to obtain aseptic seedling according to claim 4 is characterized in that: the adherent height 0.5-1.5cm of described sponge.
6. the method for utilizing masson pine seed to obtain aseptic seedling according to claim 2 is characterized in that further comprising the steps of:
＜5〉induce
With step＜4〉middle robust growth, the aseptic seedling of high 3-7cm is taken out proliferation-inducing or the callus induction that is used for leaf, stem and bud, and with all the other ablastous seeds cleanings, sponge is cleaned and dries.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103734008A (en) * | 2013-12-26 | 2014-04-23 | 柳州赛特生物科技研发中心 | Liquid culture medium for tissue culture intermediate propagation of phalaenopsis and corresponding culture method |
| CN104186324A (en) * | 2014-09-09 | 2014-12-10 | 齐齐哈尔大学 | Method for inducing mature zygotic embryo somatic cell embryos of pinus sylvestris var mongolica |
| CN106688885A (en) * | 2016-11-15 | 2017-05-24 | 邹少英 | Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation |
| CN110754483A (en) * | 2019-10-29 | 2020-02-07 | 贵州大学 | Pinus massoniana seed coating agent and preparation method and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103734008A (en) * | 2013-12-26 | 2014-04-23 | 柳州赛特生物科技研发中心 | Liquid culture medium for tissue culture intermediate propagation of phalaenopsis and corresponding culture method |
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| CN104186324B (en) * | 2014-09-09 | 2016-08-24 | 齐齐哈尔大学 | The abductive approach of Pinus sylvestnis var. mongolica Litv. mature zygotic embryos somatic embryo |
| CN106688885A (en) * | 2016-11-15 | 2017-05-24 | 邹少英 | Method for quickly obtaining masson pine vegetative propagation seedlings for afforestation |
| CN110754483A (en) * | 2019-10-29 | 2020-02-07 | 贵州大学 | Pinus massoniana seed coating agent and preparation method and application thereof |
| CN110754483B (en) * | 2019-10-29 | 2021-04-09 | 贵州大学 | Pinus massoniana seed coating agent and preparation method and application thereof |
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