CN103210845B - 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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- CN103210845B CN103210845B CN201310149982.6A CN201310149982A CN103210845B CN 103210845 B CN103210845 B CN 103210845B CN 201310149982 A CN201310149982 A CN 201310149982A CN 103210845 B CN103210845 B CN 103210845B
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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 technical field of tissue culture, particularly relate to a kind of method utilizing masson pine seed to obtain aseptic seedling.
Background technology
Masson pine (Pinus massoniana) is the important dual-purpose seeds of fat material of south China, and it is of many uses, economic worth is high, is the primary raw material seeds of the industries such as China woods slurry paper (plate), rosin and building.Except timber and rosin two large purposes, masson pine is entirely set and all can utilize, and pollen, needle and bark are rich in polyphenoils, can be used as excellent anti-ageing and antioxidant health-care product; Pine nut oil-containing 30%, can manufacture 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 improvements research of more than 30 years, masson pine fine provenance-superior stand-seed collecting seed production stand-Primary seed orchard-improve was built up for the multi-level Seed multiplication base such as seed orchard in Guangxi, in promotion masson pine improved variety process, played important function.Compared with other commerical tree species, the vegetative propagation difficulty of masson pine is comparatively large, and wherein tissue cultures is more difficult.But the tissue cultures due to plant has the advantages such as reproduction coefficient is large, seedling growth is neat, captures the target that pine tissues culture technique is world's breeding scholar always.Traditional Aseptic seedling culture many employings agar powder (bar) medium, needs, through agar powder (bar) heating for dissolving-sterilizing-cooling condensation three step, to operate comparatively loaded down with trivial details; In addition, owing to there is the various problems such as percentage of seedgermination and pollution in Aseptic seedling culture, the planting percent of aseptic seedling generally can only reach 5 ~ 20%, makes the medium of 80 ~ 95% become discarded object and be difficult to recycling and cause waste; Meanwhile, the gas permeability of agar is poor, and seedling growth especially root growth is good not.Therefore, find a kind of simple, economical and method cultivates aseptic seedling is efficiently carry out the researchs such as masson pine tissue cultures and gene engineering matter of utmost importance urgently to be resolved hurrily.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of masson pine seed that utilizes operating 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, using water-absorbing sponge as medium.
The above-mentioned method utilizing masson pine seed to obtain aseptic seedling, comprises the following steps:
<1> medium prepares
The Chang ╳ of clip is wide is 3-5cm ╳ 1-2cm sponge, is fully absorbed water by sponge, the sponge suctioning water is put into tissue culture bottle, tightens bottle cap, sterilizing 20min under 121 DEG C of autoclaves;
<2> seed disinfection
0.1% mercuric chloride is adopted to carry out disinfection 8-10min to seed, aseptic water washing 4-5 time;
<3> inoculates
Aseptically the seed that step <2> disinfects is inoculated in the sponge medium of step <1>;
<4> cultivates
The tissue culture bottle of step <3> is placed in light culture 10-20 days under 23-25 DEG C of condition.
In step <1>, sponge lies at the bottom of tissue culture bottle, and in step <3>, seed is inoculated into sponge upper surface.
In step <1>, sponge is converted into right angle subsides bottle wall and is vertically placed on (the moisture content formation graded of sponge at the bottom of tissue culture bottle, vertically adherent moisture content is lower than horizontal positioned), in step <3>, seed is inoculated into the sponge upper surface at the bottom of parallel bottle.
The adherent height 0.5-1.5cm of sponge.
The above-mentioned method utilizing masson pine seed to obtain aseptic seedling, further comprising the steps of:
<5> induces
By robust growth in step <4>, the aseptic seedling of high 3-7cm takes out the proliferation-inducing or callus induction that are used for leaf, stem and bud, and by all the other ablastous Seed cleaning ups, sponge is cleaned and dries.
Adopt agar as medium Problems existing for current Aseptic seedling culture, inventor adopts the sponge with good water suction and water retention property to establish the method utilizing masson pine seed to obtain aseptic seedling as medium, the masson pine seed after sterilization is put into water-absorbing sponge and cultivates the aseptic seedling that 10-20 days can obtain robust growth.Application the present invention cultivates masson pine aseptic seedling, and seedling-growing time obviously shortens, and seedling growth especially root growth is better than agar cultivation; Simplify experimental procedure simultaneously, improve culture efficiency; In addition, can reuse due to sponge and not produce discarded object, having saved nursery reproductive-cost 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 research and the production of the biotechnology such as cultured in vitro and transgenosis of masson pine, to the stock breeding of masson pine and biotechnology research significant.
Accompanying drawing explanation
Fig. 1 is that the present invention utilizes masson pine seed to obtain the reference state figure (sponge is vertical) of the embodiment of the method 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 embodiment of the method 4 to 5 of aseptic seedling.
In figure: 1 tissue culture bottle, 2 sponges, 3 seeds.
Embodiment
The present invention is further illustrated below in conjunction with drawings and Examples.
Embodiment 1
<1> medium prepares
The Chang ╳ of clip is wide is about 3cm ╳ 2cm sponge, sponge is placed in running water fully absorb water, with tweezers, the sponge suctioning water is put into tissue culture bottle, as shown in Figure 1, sponge is converted into right angle subsides bottle wall (adherent height 1cm) and is vertically placed at the bottom of tissue culture bottle, tighten bottle cap, sterilizing 20min under 121 DEG C of autoclaves;
<2> seed disinfection
Running water 5h, 0.5% liquor potassic permanganate soaks 20min, and flowing water washes down, and adopts 0.1% mercuric chloride to carry out disinfection 8min to seed, aseptic water washing 5 times in superclean bench;
<3> inoculates
The seed disinfected by step <2> is inoculated into the sponge upper surface in the sponge medium of step <1> at the bottom of parallel bottle on superclean bench;
<4> cultivates
The tissue culture bottle of step <3> to be placed under 24 DEG C of conditions light culture 15 days;
<5> induces
By robust growth in step <4>, the aseptic seedling of high about 5.5cm takes out the proliferation-inducing being used for bud, all the other is not germinateed or mouldy Seed cleaning up, and sponge is cleaned and dries and can reuse.
Embodiment 2
<1> medium prepares
The Chang ╳ of clip is wide is about 4.5cm ╳ 2cm sponge, sponge is placed in running water fully absorb water, with tweezers, the sponge suctioning water is put into tissue culture bottle, sponge is converted into right angle subsides bottle wall (adherent height 1.5cm) and is vertically placed at the bottom of tissue culture bottle, tighten bottle cap, sterilizing 20min under 121 DEG C of autoclaves;
<2> seed disinfection
Running water 6h, 0.3% liquor potassic permanganate soaks 30min, and flowing water washes down, and adopts 0.1% mercuric chloride to carry out disinfection 10min to seed, aseptic water washing 4 times in superclean bench;
<3> inoculates
The seed disinfected by step <2> is inoculated into the sponge upper surface in the sponge medium of step <1> at the bottom of parallel bottle on superclean bench;
<4> cultivates
The tissue culture bottle of step <3> to be placed under 25 DEG C of conditions light culture 18 days;
<5> induces
By robust growth in step <4>, the aseptic seedling of high about 7cm takes out the proliferation-inducing being used for bud, all the other is not germinateed or mouldy Seed cleaning up, and sponge is cleaned and dries and can reuse.
Embodiment 3
<1> medium prepares
The Chang ╳ of clip is wide is about 2.8cm ╳ 1.5cm sponge, sponge is placed in running water fully absorb water, with tweezers, the sponge suctioning water is put into tissue culture bottle, sponge is converted into right angle subsides bottle wall (adherent height 0.8cm) and is vertically placed at the bottom of tissue culture bottle, tighten bottle cap, sterilizing 20min under 121 DEG C of autoclaves;
<2> seed disinfection
Running water 5h, 0.5% liquor potassic permanganate soaks 15min, and flowing water washes down, and adopts 0.1% mercuric chloride to carry out disinfection 9min to seed, aseptic water washing 5 times in superclean bench;
<3> inoculates
The seed disinfected by step <2> is inoculated into the sponge upper surface in the sponge medium of step <1> at the bottom of parallel bottle on superclean bench;
<4> cultivates
The tissue culture bottle of step <3> to be placed under 23 DEG C of conditions light culture 17 days;
<5> induces
By robust growth in step <4>, the aseptic seedling of high about 6.8cm takes out the proliferation-inducing being used for bud, all the other is not germinateed or mouldy Seed cleaning up, and sponge is cleaned and dries and can reuse.
Embodiment 4
<1> medium prepares
The Chang ╳ of clip is wide is about 2.3cm ╳ 1.5cm sponge, and sponge is placed in running water and fully absorbs water, with tweezers, the sponge suctioning water is put into tissue culture bottle, as shown in Figure 2, sponge lies at the bottom of tissue culture bottle, tightens bottle cap, sterilizing 20min under 121 DEG C of autoclaves;
<2> seed disinfection
Running water 5.5h, 0.4% liquor potassic permanganate soaks 18min, and flowing water washes down, and adopts 0.1% mercuric chloride to carry out disinfection 11min to seed, aseptic water washing 5 times in superclean bench;
<3> inoculates
The seed disinfected by step <2> is inoculated into the sponge upper surface of step <1> on superclean bench;
<4> cultivates
The tissue culture bottle of step <3> to be placed under 23 DEG C of conditions light culture 20 days;
<5> induces
By robust growth in step <4>, the aseptic seedling of high about 6.0cm takes out the proliferation-inducing being used for bud, all the other is not germinateed or mouldy Seed cleaning up, and sponge is cleaned and dries and can reuse.
Embodiment 5
<1> medium prepares
The Chang ╳ of clip is wide is about 3.0cm ╳ 1.7cm sponge, and sponge is placed in running water and fully absorbs water, with tweezers, the sponge suctioning water is put into tissue culture bottle, sponge lies at the bottom of tissue culture bottle, tightens bottle cap, sterilizing 20min under 121 DEG C of autoclaves; <2> seed disinfection
Running water 4h, 0.4% liquor potassic permanganate soaks 20min, and flowing water washes down, and adopts 0.1% mercuric chloride to carry out disinfection 9min to seed, aseptic water washing 4 times in superclean bench;
<3> inoculates
The seed disinfected by step <2> is inoculated into the sponge upper surface of step <1> on superclean bench;
<4> cultivates
The tissue culture bottle of step <3> to be placed under 25 DEG C of conditions light culture 20 days;
<5> induces
By robust growth in step <4>, the aseptic seedling of high about 5.8cm takes out the proliferation-inducing being used for bud, all the other is not germinateed or mouldy Seed cleaning up, and sponge is cleaned and dries and can reuse.
Reference examples
<1> medium prepares
Take 7g/ and rise agar strip, agar strip is shredded or smashes with cracker, add suitable quantity of water heating for dissolving while stirring, dissolving completes and is settled to 1 liter with beaker, fully mixes thoroughly, pours in blake bottle while hot, every bottle of 30-40mL, tighten bottle cap, under 121 DEG C of autoclaves, sterilizing 20min, naturally cools to condensation;
<2> seed disinfection
Running water 4.5h, 0.2% liquor potassic permanganate soaks 25min, and flowing water washes down, and to carry out disinfection 9min in superclean bench with 0.1% mercuric chloride to seed, aseptic water washing 5 times;
<3> inoculates
The seed disinfected by step <2> is inoculated in the agar medium of step <1> on superclean bench;
<4> cultivates
The tissue culture bottle of step <3> to be positioned under 25 DEG C of conditions light culture 45 days;
<5> induces
By robust growth in step <4>, the aseptic seedling of high about 5.6cm is taken out and is carried out Shoot propagation induction, all the other not to be germinateed or the blake bottle of mouldy seed is put into autoclave heating agar is dissolved, agar is poured into concentrated in waste liquid basin abandoning.
Table 1 Aseptic seedling culture effectiveness comparison table
Conclusion: as seen from Table 1, the embodiment 1 to 5 of application this method process, germination rate is 20 ~ 25%, and height of seedling is 5.5 ~ 7.0cm, and incubation time is 15 ~ 20 days, robust growth; In reference examples, germination rate only has 15%, and height of seedling is 5.6cm, and incubation time is 45 days, grows more weak, and embodiment is substantially close with height of seedling with the germination rate contrasted, and embodiment is slightly high; On incubation time and upgrowth situation, the embodiment of the present invention is all better than reference examples.
Wherein high and upgrowth situation 3 aspects of the germination rate of embodiment 1 ~ 3 and embodiment 4 ~ 5, seedling bud do not have significant difference, but embodiment 1 ~ 3 is better than embodiment 4 ~ 5 on incubation time.So the method that the sponge in embodiment 1 ~ 3 is converted into right angle subsides bottle wall has certain advantage.
The incubation time average out to of embodiment 1 ~ 5 18 days, be obviously better than 45 days of reference examples, incubation time shortens about 30 days, has clear superiority.The upgrowth situation of embodiment 1 ~ 5 is better than reference examples, and reason is that the good permeability of sponge is in agar, causes the aseptic seedling well developed root system grown on sponge medium, and seedling growth is healthy and strong.Because Plant Tissue Breeding need at 24h temperature control 23-25 DEG C; carry out under 8h artificial lighting condition; incubation time shortens 30 days and can not only effectively save production cost can also accelerate the production cycle of plantlet in vitro; simultaneously; sponge in embodiment can reuse and not produce discarded object, thus protects environment.
Claims (4)
1. utilize masson pine seed to obtain a method for aseptic seedling, it is characterized in that comprising the following steps:
<1> medium prepares
The Chang ╳ of clip is wide is 3-5cm ╳ 1-2cm sponge, is fully absorbed water by sponge, the sponge suctioning water is put into tissue culture bottle, tightens bottle cap, sterilizing 20min under 121 DEG C of autoclaves;
<2> seed disinfection
0.1% mercuric chloride is adopted to carry out disinfection 8-10min to seed, aseptic water washing 4-5 time;
<3> inoculates
Aseptically the seed that step <2> disinfects is inoculated in the sponge medium of step <1>;
<4> cultivates
The tissue culture bottle of step <3> is placed in light culture 10-20 days under 23-25 DEG C of condition.
2. the method utilizing masson pine seed to obtain aseptic seedling according to claim 1, it is characterized in that: in step <1>, sponge is converted into right angle and pastes bottle wall and be vertically placed at the bottom of tissue culture bottle, and in step <3>, seed is inoculated into the sponge upper surface at the bottom of parallel bottle.
3. the method utilizing masson pine seed to obtain aseptic seedling according to claim 2, is characterized in that: the adherent height 0.5-1.5cm of described sponge.
4. the method utilizing masson pine seed to obtain aseptic seedling according to claim 3, characterized by further comprising following steps:
<5> induces
By robust growth in step <4>, the aseptic seedling of high 3-7cm takes out the proliferation-inducing or callus induction that are used for leaf, stem and bud, and by all the other ablastous Seed cleaning ups, sponge is cleaned and dries.
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| CN201310149982.6A CN103210845B (en) | 2013-04-26 | 2013-04-26 | Method for obtaining aseptic seedling by using pinus massoniana seed |
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| CN201310149982.6A CN103210845B (en) | 2013-04-26 | 2013-04-26 | Method for obtaining aseptic seedling by using pinus massoniana seed |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103734008B (en) * | 2013-12-26 | 2016-04-13 | 广西森仙源生物科技有限公司 | A kind of butterfly orchid tissue cultivating Fast-propagation liquid nutrient medium and corresponding cultural method |
| 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 |
| 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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| CN2322221Y (en) * | 1998-03-24 | 1999-06-02 | 徐万茂 | Experiment appliance for student to observe seed sprouting |
| CN201601959U (en) * | 2010-02-05 | 2010-10-13 | 浙江大学 | Watering-free seed germination box with scales |
| CN201957411U (en) * | 2011-01-30 | 2011-09-07 | 柳永丁 | Germinator |
| CN202587804U (en) * | 2012-06-03 | 2012-12-12 | 左颖 | Astragalus smicus seed germination box |
| CN102884974A (en) * | 2012-07-02 | 2013-01-23 | 杨建伟 | Sponge seedling raising bed |
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2013
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN2322221Y (en) * | 1998-03-24 | 1999-06-02 | 徐万茂 | Experiment appliance for student to observe seed sprouting |
| CN201601959U (en) * | 2010-02-05 | 2010-10-13 | 浙江大学 | Watering-free seed germination box with scales |
| CN201957411U (en) * | 2011-01-30 | 2011-09-07 | 柳永丁 | Germinator |
| CN202587804U (en) * | 2012-06-03 | 2012-12-12 | 左颖 | Astragalus smicus seed germination box |
| CN102884974A (en) * | 2012-07-02 | 2013-01-23 | 杨建伟 | Sponge seedling raising bed |
Non-Patent Citations (1)
| Title |
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| 海绵作发芽床衬垫物的研究探讨;彭云芳;《种子》;19940625(第3期);第46页左栏最后一段，第47页右栏第2-10段 * |
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