CN104756870B - Hold up the tissue culture propagation method of sky tree chitting piece - Google Patents
Hold up the tissue culture propagation method of sky tree chitting piece Download PDFInfo
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Abstract
The present invention relates to a kind of tissue culture propagation method holding up sky tree chitting piece, technical scheme includes holding up a day pretreatment for seeds, sterilization process, the steps such as initial culture, become to have the aseptic seedling of two pairs of true leaves by holding up sky tree cultivating seeds, described pretreatment is put into 30-35 DEG C of sterilized water embathe 2-5 min for being held up sky tree chitting piece, described sterilization process embathes 2-5 min for being put into by seed in 0.8-0.9 ‰ mercuric chloride solution, by 30-35 DEG C of sterile water wash, described initial culture for being inoculated into MS+100 mg/L inositol+2.5 mg/L 6-BA+1.0 mg/L NAA+10 mg/L Vc+30 g/L sucrose by seed, the culture medium of pH6.6 is cultivated, cultivate and control intensity of illumination 1500-3000 lux, light application time 12 h/d, temperature 25 ± 3 DEG C。Outer implant sterilizing is sterilizable material by the present invention so that sterilizable material can carry out expanding propagation further, takes root, and cultivates aseptic seedling, relative to tradition method for culturing seedlings, has rate of increase height, the annual advantage producing nursery stock free of discontinuities。
Description
Technical field
The present invention relates to plant Fast Asexual Propagation Technique field, specifically a kind of tissue culture propagation method holding up sky tree chitting piece。
Background technology
Holding up sky tree (ParashoreachinensisWangHsie.), Dipterocarpaceae willow Eucalyptus, evergreen high megaphanerophyte, for seeds the most in great numbers, most representational in Tropical Asian rainforest。Trunk may be up to five or six ten meters, ranks on crown canopy, and timber is hard, rotproofness is strong, and the grain of wood is straight, even structure, handling ease, slicing face is smooth, and decorative pattern is attractive in appearance, for manufacturing the senior material of various furniture, there is Important Economic and be worth and scientific research value, be the rarity of state。Hold up the distribution of sky tree narrow, be only distributed in Xishuangbanna mend freshwater mussel and Guang Nalixin stockaded village to scape wafts within the scope of 20 square kilometres of a band；Growing environment major part is original Tropical ravine rainforest and mountane rain forest, many growths in flakes, forms independent group, forms peculiar natural landscape, is the first class of protection plant of China。
Hold up sky tree and blossom and bear fruit later, be typically in the diameter of a cross-section of a tree trunk 1.3 meters above the ground and reach more than 40 centimetres and just blossom and bear fruit, solid rareness, and just blossom and bear fruit 1 time every 2-5, shedding is serious, not easily collects seed。Holding up the germination of sky tree quickly, on forest land, l-4d just germinates, and at high temperature and rainy environment, some mellow fruit is still on elite stand, and seed just germinates, drastically influence the success rate of cultivating seedlings。Holding up sky tree according to epicormic branch cuttage or cuttage vegetative propagation technique, seasonal strong, the time of cuttage survival rate more than 70% is in annual March to July。Including aldehydes matter owing to holding up a day branch bar, epicormic branch cuttage brownization is serious, and cuttage adventitious root time of origin is long, as met arid or high temperature plum rains adverse weather condition, holds up day branch bar easily withered or rotten lethal。Special because holding up sky tree biological nature, seriously limit the exploitation of this resource。Employing group training vegetative propagation technique is the effective way promoting to hold up sky tree development。Owing to holding up the densely covered fine hair of sky tree tender stem segments, sterilizing difficulty is big。Hold up sky tree tissue-culturing rapid propagation brood body to obtain, explore the method holding up sky tree chitting piece sterilizing particularly important。
Summary of the invention
The invention aims to keep Seed Vitality, improve the rate of increase, annual production nursery stock free of discontinuities, it is provided that a kind of tissue culture propagation method holding up sky tree chitting piece。
The solution of the present invention is by being achieved in that:
A kind of tissue culture propagation method holding up sky tree chitting piece, it is characterised in that: including pretreatment, sterilizing, initial culture operation, main operational steps is:
(1) pretreatment: removed by the seed wing holding up sky tree chitting piece, after rinsing seed with clear water, then divests kind of a shell, is placed in 30-35 DEG C of sterilized water and embathes 2-5min, obtain embryo standby；
(2) sterilizing: be placed in by standby embryo in mass concentration 0.8-0.9 ‰ mercuric chloride solution and soak, by 30-35 DEG C of sterile water wash 5-8 time, obtains sterilizing embryo and waits to cultivate；
(3) initial culture: sterilizing embryo is inoculated in initial culture base and makes an initial culture, after inoculation 3d, embryo starts plant division, Embryo stem extension, when inoculation 20-30h has brown compound matter to produce, embryo is transferred in new initial culture base and makes secondary initial culture, transferring 2-3 time, obtaining length has two pairs of true leaves, terminal bud the tissue cultured seedling with a pair of stipules。
The time of above-described immersion is 2-5min。
The volumetric concentration of above-described initial culture based formulas is: MS+80-120mg/L inositol+8-10g/L carrageenan+2-3mg/L6-BA+0.5-1.5mg/LNAA+5-15mg/LVC+15-45g/L sucrose, pH6.0-7.0。
It is 1500-3000lux that above-described initial culture controls intensity of illumination, and light application time is 10-15h/d, and temperature is 25 ± 3 DEG C。
The volumetric concentration of above-described initial culture based formulas is: MS+100mg/L inositol+9g/L carrageenan+2.5mg/L6-BA+1.0mg/LNAA+15mg/LVC+30g/L sucrose, PH6.6。
The condition of above-described initial culture is: intensity of illumination 2000lux, light application time 12h/d, temperature 25 DEG C。
The present invention possesses advantages below and good effect:
(1) present invention adopts 0.8-0.9 ‰ mercuric chloride solution to carry out sterilizing to holding up a day seeds embryo, both can effectively kill the antibacterial being attached to outer planting surface and fungal spores, and embryo can be prevented again poisoning, keeps the vitality of outer implant, obtains the aseptic explant of high-quality。
(2) present invention is in the initial culture stage, when inoculating 20-30h and having brown compound matter to produce, is transferred to by embryo in new culture medium and cultivates, transfer the cultivation of 2-3 time, can effectively prevent embryo brownization, improve and cultivate breeding success rate, Senescence rate 3-10%, aseptic rate 70-94%。
(3) present invention is by the optimization to initial culture base and condition of culture, and inositol promotes the formation of embryoid and bud, shortens incubation time, improves culture efficiency, adds VC simultaneously in the medium and can effectively prevent the browning in incubation。
(4) the inventive method operating process is easy, mild condition, and cultivation cycle is short, and the rate of increase is high, can meet the annual requirement producing nursery stock free of discontinuities, have good economic benefit and social benefit。
Accompanying drawing explanation
Fig. 1 holds up day seeds embryo to be seeded in the condition diagram on initial culture base with after 0.9 ‰ mercuric chloride sterilizings。
Fig. 2 is kind of an embryo elongate condition figure。
Fig. 3 is the condition diagram that embryo grows a pair true leaf。
Fig. 4 is the condition diagram that embryo grows two pairs of true leaves。
Detailed description of the invention
Describing a kind of tissue culture propagation method holding up sky tree chitting piece of the present invention below in conjunction with embodiment, these descriptions are not the restriction to present invention。
Embodiment 1
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo；Embryo is placed in 35 DEG C of sterilized water and embathes 3min, again embryo is placed in mass concentration 0.9 ‰ mercuric chloride solution and soaks 3min, by 35 DEG C of sterile water wash 7 times, it is then seeded into containing volumetric concentration MS+100mg/L inositol+9g/L carrageenan+2.5mg/L6-BA+1.0mg/LNAA+10mg/LVC+30g/L sucrose, the initial culture base of pH=6.6 is cultivated, initial culture controls intensity of illumination 2000lux, light application time 12h/d, temperature 25 DEG C, when inoculation 20-25h has brown compound matter to produce, embryo is transferred in new initial culture base and cultivates, transfer 3 times, obtain length and have two pairs of true leaves, terminal bud the tissue cultured seedling with a pair of stipules。
It is higher that this example cultivates breeding success rate, Senescence rate 3%, aseptic rate 94%。
Embodiment 2
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo；Embryo is placed in 30 DEG C of sterilized water and embathes 2min, again embryo is placed in mass concentration 0.8 ‰ mercuric chloride solution and soaks 2min, by 30 DEG C of sterile water wash 5 times, it is then seeded into containing volumetric concentration MS+80mg/L inositol+8g/L carrageenan+2mg/L6-BA+0.5mg/LNAA+5mg/LVC+15g/L sucrose, the initial culture base of pH=6.0 is cultivated, initial culture controls intensity of illumination 1500lux, light application time 10h/d, temperature 22 DEG C, when inoculation 25-30h has brown compound matter to produce, embryo is transferred in new initial culture base and cultivates, transfer 3 times, obtain length and have two pairs of true leaves, terminal bud the tissue cultured seedling with a pair of stipules。
It is higher that this example cultivates breeding success rate, Senescence rate 1%, aseptic rate 88%。
Embodiment 3
The tissue culture propagation method holding up sky tree chitting piece in the present embodiment is as follows:
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo；Embryo is placed in 32 DEG C of sterilized water and embathes 4min, again embryo is placed in mass concentration 0.8 ‰ mercuric chloride solution and soaks 4min, by 32 DEG C of sterile water wash 6 times, it is then seeded into containing volumetric concentration MS+90mg/L inositol+10g/L carrageenan+3mg/L6-BA+1.5mg/LNAA+7mg/LVC+24g/L sucrose, the initial culture base of pH=6.3 is cultivated, initial culture controls intensity of illumination 1800lux, light application time 13h/d, temperature 23 DEG C, when inoculation 20-23h has brown compound matter to produce, embryo is transferred in new initial culture base and cultivates, transfer 2 times, obtain length and have two pairs of true leaves, terminal bud the tissue cultured seedling with a pair of stipules。
It is higher that this example cultivates breeding success rate, Senescence rate 4%, aseptic rate 80%。
Embodiment 4
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo；Embryo is placed in 33 DEG C of sterilized water and embathes 5min, again embryo is placed in mass concentration 0.8 ‰ mercuric chloride solution and soaks 5min, by 33 DEG C of sterile water wash 8 times, it is then seeded into containing volumetric concentration MS+110mg/L inositol+8.5g/L carrageenan+2.8mg/L6-BA+1.2mg/LNAA+12mg/LVC+38g/L sucrose, the initial culture base of pH=6.8 is cultivated, initial culture controls intensity of illumination 2500lux, light application time 14h/d, temperature 27 DEG C, when inoculation 24-28h has brown compound matter to produce, embryo is transferred in new initial culture base and cultivates, transfer 3 times, obtain length and have two pairs of true leaves, terminal bud the tissue cultured seedling with a pair of stipules。
It is higher that this example cultivates breeding success rate, Senescence rate 7%, aseptic rate 75%。
Embodiment 5
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo；Embryo is placed in 34 DEG C of sterilized water and embathes 3min, again embryo is placed in mass concentration 0.9 ‰ mercuric chloride solution and soaks 5min, by 34 DEG C of sterile water wash 7 times, it is then seeded into containing volumetric concentration MS+120mg/L inositol+9.5g/L carrageenan+2.3mg/L6-BA+0.8mg/LNAA+15mg/LVC+45g/L sucrose, the initial culture base of pH=7.0 is cultivated, initial culture controls intensity of illumination 3000lux, light application time 15h/d, temperature 28 DEG C, when inoculation 26-30h has brown compound matter to produce, embryo is transferred in new initial culture base and cultivates, transfer 2 times, obtain length and have two pairs of true leaves, terminal bud the tissue cultured seedling with a pair of stipules。
It is higher that this example cultivates breeding success rate, Senescence rate 10%, aseptic rate 70%。
Comparative example 1
The seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo；Embryo is placed in sterilized water and embathes 5min, again embryo is placed in mass concentration 1 ‰ mercuric chloride solution and soaks 8min, by sterile water wash 6 times, it is then seeded in the initial culture base containing volumetric concentration 1/2MS+IBA0.3mL/L+6-BA0.4mL/L+ sugar 30g+ agar 5g and cultivates, initial culture controls intensity of illumination 3000lux, light application time 12h/d, temperature 26 DEG C, when there being brown compound matter to produce, embryo is transferred to 1/2MS+6-BA0.4mg/L+IBA0.3mg/L+VC0.2mg/L+AC0.5g/L, 1/2MS+6-BA0.4mg/L+IBA0.3mg/L+VC0.2mg/L, the initial culture base of 1/2MS+VC0.2mg/L+PVP2g/L+AC0.5g/L is cultivated, obtain length and have a pair true leaf, terminal bud the tissue cultured seedling with a pair of stipules。
It is higher that this comparative example cultivates breeding success rate, Senescence rate 18%, aseptic rate 65%。
Claims (4)
1. hold up the tissue culture propagation method of sky tree chitting piece, it is characterised in that: including pretreatment, sterilizing, initial culture operation, main operational steps is:
(1) pretreatment: removed by the seed wing holding up sky tree chitting piece, after rinsing seed with clear water, then divests kind of a shell, is placed in 30-35 DEG C of sterilized water and embathes 2-5min, obtain embryo standby；
(2) sterilizing: be placed in by standby embryo in mass concentration 0.8-0.9 ‰ mercuric chloride solution and soak, by 30-35 DEG C of sterile water wash 5-8 time, obtains sterilizing embryo and waits to cultivate；
(3) initial culture: sterilizing embryo is inoculated in initial culture base and makes an initial culture, after inoculation 3d, embryo starts plant division, Embryo stem extension, when inoculation 20-30h has brown compound matter to produce, embryo is transferred in new initial culture base and makes secondary initial culture, transferring 2-3 time, obtaining length has two pairs of true leaves, terminal bud the tissue cultured seedling with a pair of stipules；
The time of described immersion is 2-5min；
The formula of described initial culture base is: MS+80-120mg/L inositol+8-10g/L carrageenan+2-3mg/L6-BA+0.5-1.5mg/LNAA+5-15mg/LVC+15-45g/L sucrose, pH6.0-7.0；
It is 1500-3000lux that described initial culture controls intensity of illumination, and light application time is 10-15h/d, and temperature is 25 ± 3 DEG C。
2. the tissue culture propagation method holding up sky tree chitting piece according to claim 1, it is characterized in that: the formula of described initial culture base is: MS+100mg/L inositol+9g/L carrageenan+2.5mg/L6-BA+1.0mg/LNAA+15mg/LVC+30g/L sucrose, pH6.6。
3. the tissue culture propagation method holding up sky tree chitting piece according to claim 1, it is characterised in that: the condition of described initial culture is: intensity of illumination 2000lux, light application time 12h/d, temperature 25 DEG C。
4. the tissue culture propagation method holding up sky tree chitting piece, it is characterised in that: the seed wing holding up sky tree chitting piece is removed, after rinsing with clear water, then divests kind of a shell and obtain complete embryo；Embryo is placed in 35 DEG C of sterilized water and embathes 3min, again embryo is placed in mass concentration 0.9 ‰ mercuric chloride solution and soaks 3min, by 35 DEG C of sterile water wash 7 times, it is then seeded into being made up of component MS+100mg/L inositol+9g/L carrageenan+2.5mg/L6-BA+1.0mg/LNAA+10mg/LVC+30g/L sucrose, the initial culture base of pH=6.6 is cultivated, initial culture controls intensity of illumination 2000lux, light application time 12h/d, temperature 25 DEG C, when inoculation 20-25h has brown compound matter to produce, embryo is transferred in new initial culture base and cultivates, transfer 3 times, obtain length and have two pairs of true leaves, terminal bud the tissue cultured seedling with a pair of stipules。
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