CN103497928B - Drying method for treating before the sprouting of PIECA ASPERATA somatic embryo - Google Patents
Drying method for treating before the sprouting of PIECA ASPERATA somatic embryo Download PDFInfo
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Abstract
The present invention relates to the drying method for treating before the sprouting of a kind of PIECA ASPERATA somatic embryo.Described method comprises: after the somatic embryo obtaining PIECA ASPERATA, carries out drying and other treatment 0 ~ 21 day, and then sprout the mode of somatic embryo employing " filter paper bridge method ", " empty culture vessel method " or " filter paper method ".The invention provides a kind of PIECA ASPERATA somatic embryo sprout before drying method for treating, include the mummification labelling strategies based on somatic embryo colour-change, the germination rate of somatic embryo can be improved significantly in pole; Meanwhile, the quality that somatic embryo is sprouted also is improved significantly; And the impact of genotype and clone can not be subject to, there is general suitability; For the industrial seedling rearing realizing PIECA ASPERATA somatic embryo has established theoretical basis, and provide practicable technical scheme.
Description
Technical field
The present invention relates to cell engineering sapling multiplication field in forestry, particularly, relate to the drying method for treating before the sprouting of a kind of PIECA ASPERATA somatic embryo.
Background technology
Plant somatocyte embryo generation technique, refers in vitro, and the somatocyte broken up under certain environmental conditions, develops into the process of the embryoid similar with zygotic embryo.Plant somatocyte embryo utilizes one of cell engineering means important channel realizing plant regeneration, utilize artificial means can realize plant to greatest extent to expand numerous measure as a kind of, especially for being difficult to cottage propagation and breeding cycle longer softwood tree seeds, there is huge using value.Meanwhile, because somatic embryo and zygotic embryo have similar morphological structure and growth course, Somatic Embryogenesis has again relative genetic stability; Carry out the research that plant somatocyte embryo occurs, for disclosing the genesis mechanism research of plant embryos and carrying out Study on Genetic Transformation and provide a kind of model study system.
Complete plant somatocyte embryo generation technique comprises the four-stage such as sprouting of the induction of embryo callus, the propagation of embryo callus, the differentiation of somatic embryo and mature somatic embryo; Wherein, the height of somatic embryo germination rate and sprout quality, directly decides the success or failure that the harvest yield of final body embryo seedling and whole body embryo technical system build as the final step of whole technical system sprouting stage of mature somatic embryo.At present, sprout the stage at softwood tree seeds somatic embryo, be germination rate and the sprouting quality of raising somatic embryo, external conventional method carries out mummification (Desiccation) pre-treatment by before somatic embryo sprouting.Usually, phenomenon of losing water is somatic embryo by the stage of maturity to sprouting the most directly responding of transition stage, and somatic embryo can be impelled to be changed to the physiological maturity needed for sprouting by morphological maturity; Therefore, for somatic embryo, give suitable mummification dehydration process, can not only the sprouting ahead of time of somatic embryo in Inhibited differentiation substratum, the sprouting quality and quantity of somatic embryo in the sprouting stage can also be improved.
Generally according to the change of somatic embryo rate-of-loss of coolant, the drying method for treating of somatic embryo can be divided into complete mummification (FullDesiccation) and part mummification (PartialDesiccation) two kinds; The former realizes primarily of tonicity-adjusting substances, and the latter regulates mainly through water vapour change.Although drying and other treatment can improve sprouting frequency, but affect a lot of because have of drying and other treatment effect: between the time that the difference of drying method, drying and other treatment continue, the difference of seeds, even identical seeds different genotype, very big-difference is all existed to the response of identical drying method for treating.If do not consider the factor affecting drying and other treatment effect, adopt single drying and other treatment condition, certainly can not meet the sprouting needs of all genotype or clone somatic embryo.Therefore, have scholar even to propose abroad, the corresponding unique drying and other treatment scheme of each clone possibility, sets up suitable drying and other treatment scheme, needs to explore one by one the somatic embryo mummification condition of each clone if want.This method explored one by one wastes time and energy, and in actual production, feasibility is poor.
PIECA ASPERATA (PiceaasperataMast) is Pinaceae, Picea seeds, is domesticly commonly called as dragon spruce, is China endemic species, the Picea seeds that the distribution of Ye Shi China is the widest.Aiphyllium, up to 45m, the diameter of a cross-section of a tree trunk 1.3 meters above the ground reaches 1m.Mainly be distributed in the Subalpine region area in west Sichuan plateau, the southeast of Gansu and the west and south, Shaanxi, mean sea level is distributed in the high mountain hilly country of 2400 ~ 3600m.Timber yellow-white, more light and soft, texture is straight, and structure is thin, flexible.The starting material of important papermaking, building, furniture and xylon industry.Material is excellent, and growth is fast, and strong adaptability is the Major Tree Species Planted in range of distribution.At present, artificial afforestration seedling is mainly through Seedling propagation, but PIECA ASPERATA solid evening, seed maturity rate is low, and with serious " biennial bearing " phenomenon, supply falls short of demand to cause PIECA ASPERATA good seed.
In the past few years, present inventor attempts building PIECA ASPERATA the Development of Somatic Embryogenesis system first.In the process, the technical system in the stages such as the differentiation of the induction of embryo callus, the propagation of embryo callus, somatic embryo is very ripe, and meets need of production.But, in the germination process of somatic embryo, although through a large amount of exploration work, trial as much as possible has been done on the factor affecting somatic embryo sprouting, though the sprouting condition filtered out can improve the germination rate of PIECA ASPERATA somatic embryo significantly, but germination rate or not high enough (average out to about 20%), still as far as possible highly can not meet the needs of actual production.
Therefore, if before PIECA ASPERATA somatic embryo is sprouted, its suitable drying method for treating can be determined, the germination rate of somatic embryo can be improved significantly and significantly improves the quality of somatic embryo sprouting in pole; And if this treatment process can not be subject to the impact of genotype and clone, there is general suitability, by the industrial seedling rearing for realizing PIECA ASPERATA somatic embryo, provide huge application prospect.
Summary of the invention
In order to solve the problems of the prior art, the object of this invention is to provide the drying method for treating before the sprouting of a kind of PIECA ASPERATA somatic embryo.
To achieve these goals, drying method for treating before PIECA ASPERATA somatic embryo sprouting provided by the invention, comprise: after the somatic embryo obtaining PIECA ASPERATA, drying and other treatment is carried out 0 ~ 21 day to the mode of somatic embryo employing " filter paper bridge method ", " empty culture vessel method " or " filter paper method ", and then sprouts.
Method of the present invention, preferably adopts " empty culture vessel method " or the mode of " filter paper method " to carry out drying and other treatment to somatic embryo.
Method of the present invention, more preferably adopts the mode of " filter paper method " to carry out drying and other treatment to somatic embryo.
Wherein, described " filter paper bridge method " is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel, then this culture vessel (being placed with the culture vessel of PIECA ASPERATA somatic embryo) is placed in another larger culture vessel, water is placed in larger culture vessel, cover with filter paper on the culture vessel being placed with PIECA ASPERATA somatic embryo and make " filter paper bridge ", finally larger culture vessel is sealed.
Wherein, described " empty culture vessel method " is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel, then this culture vessel (being placed with the culture vessel of PIECA ASPERATA somatic embryo) is placed in another larger culture vessel, in larger culture vessel, place water, finally larger culture vessel is sealed.
Wherein, described " filter paper method " is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel being lined with the above filter paper of one deck, then this culture vessel (being placed with the culture vessel of PIECA ASPERATA somatic embryo) is placed in another larger culture vessel, in larger culture vessel, place water, finally larger culture vessel is sealed.
Wherein, in described " filter paper method ", be lined with the number of plies preferably 1 ~ 4 layer of filter paper in the culture vessel of placement PIECA ASPERATA somatic embryo, more preferably 3 layers.
In the present invention, the filter paper placed in culture vessel can size identical, also can be different, but preferably identical.
In the present invention, culture vessel used can be plastic culture dish or glass culture dish, preferred glass culture dish.
Method of the present invention, preferred drying and other treatment 1 ~ 14 day, more preferably 7 ~ 14 days, most preferably 14 days.
Wherein, the condition that somatic embryo carries out drying and other treatment is also comprised: temperature is 23 ~ 25 DEG C; Intensity of illumination is 15 ~ 18 μm of olm
-2s
-1, preferably 15 μm of olm
-2s
-1.
Wherein, adopt the embryo callus of liquid suspension propagation or semi-solid propagation to carry out the differentiation culture of somatic embryo respectively, obtain the somatic embryo of PIECA ASPERATA.
Drying method for treating before PIECA ASPERATA somatic embryo sprouting of the present invention, also comprises: after carrying out drying and other treatment to the somatic embryo of PIECA ASPERATA, carry out mummification mark, and then sprout somatic embryo.
Wherein, the mode of somatic embryo being carried out to mummification mark is: according to the change of somatic embryo color after drying and other treatment, be marked as three types: " unchanged type ", " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type ".
Further, the somatic embryo being labeled as " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type " is sprouted.
Further, the somatic embryo of " green cotyledon and plumular axis and red radicle type " is sprouted to being labeled as.
Drying method for treating before PIECA ASPERATA somatic embryo sprouting provided by the invention, comprise: after the somatic embryo obtaining PIECA ASPERATA, drying and other treatment is carried out 0 ~ 21 day to the mode of somatic embryo employing " filter paper bridge method ", " empty culture vessel method " or " filter paper method ", mummification mark is carried out to somatic embryo, and then sprouts.
Wherein, the mode of somatic embryo being carried out to mummification mark is: according to the change of somatic embryo color after drying and other treatment, be marked as three types: " unchanged type ", " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type ".
Further, the somatic embryo being labeled as " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type " is sprouted.
Further, the somatic embryo of " green cotyledon and plumular axis and red radicle type " is sprouted to being labeled as.
Drying method for treating before PIECA ASPERATA somatic embryo sprouting provided by the invention, comprise: after the somatic embryo obtaining PIECA ASPERATA, the mode of " empty culture vessel method " or " filter paper method " is adopted to carry out drying and other treatment 0 ~ 21 day to somatic embryo, mummification mark is carried out to somatic embryo, and then sprouts.
Wherein, the mode of somatic embryo being carried out to mummification mark is: according to the change of somatic embryo color after drying and other treatment, be marked as three types: " unchanged type ", " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type ".
Further, the somatic embryo being labeled as " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type " is sprouted.
Further, the somatic embryo of " green cotyledon and plumular axis and red radicle type " is sprouted to being labeled as.
Wherein, described " filter paper bridge method " is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel, then this culture vessel (being placed with the culture vessel of PIECA ASPERATA somatic embryo) is placed in another larger culture vessel, water is placed in larger culture vessel, cover with filter paper on the culture vessel being placed with PIECA ASPERATA somatic embryo and make " filter paper bridge ", finally larger culture vessel is sealed.
Wherein, described " empty culture vessel method " is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel, then this culture vessel (being placed with the culture vessel of PIECA ASPERATA somatic embryo) is placed in another larger culture vessel, in larger culture vessel, place water, finally larger culture vessel is sealed.
Wherein, described " filter paper method " is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel being lined with the above filter paper of one deck, then this culture vessel (being placed with the culture vessel of PIECA ASPERATA somatic embryo) is placed in another larger culture vessel, in larger culture vessel, place water, finally larger culture vessel is sealed.
Drying method for treating before PIECA ASPERATA somatic embryo sprouting provided by the invention, comprising: after carrying out drying and other treatment to the somatic embryo of PIECA ASPERATA, then carry out mummification mark, then sprout.
Wherein, the mode of described mummification mark is: according to the changing conditions of somatic embryo color, be marked as three types: " unchanged type ", " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type ".
Wherein, be labeled as the somatic embryo of " green cotyledon and plumular axis and red radicle type ", the drying time needed for it is 0 ~ 21 day, preferably 1 ~ 14 day, more preferably 7 ~ 14 days, most preferably 14 days.
Further, the somatic embryo being labeled as " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type " is sprouted.
Further, the somatic embryo of " green cotyledon and plumular axis and red radicle type " is sprouted to being labeled as.
In the method for the invention, sprout somatic embryo, substratum used is: 1/4LM substratum, adds sucrose 20gL
-1, gel 6gL
-1with gac 2gL
-1, more additional glutamine 500mgL
-1with caseinhydrolysate 1gL
-1.
Up to now, there is not been reported for the domestic drying and other treatment about softwood tree seeds somatic embryo.Present inventor carries out drying and other treatment to the somatic embryo of domestic coniferous trees seeds PIECA ASPERATA first, and research shows that drying and other treatment generally can promote the sprouting of PIECA ASPERATA somatic embryo.Based on the change of somatic embryo color after drying and other treatment, the somatic embryo of " green cotyledon and plumular axis and red radicle type " is marked as the mummification of the most applicable sprouting, this mark drastically increases somatic drying and other treatment efficiency, avoids the blindness of drying and other treatment.Meanwhile, this mark does not rely on genotype and clone, avoids the somatic embryo germination rate of different sources under identical drying method process and improves inconsistent problem.This marker research thinking, not only belongs to pioneering at home, and external a lot of scholar carries out attempting also having no reported success.
Method provided by the invention is that the sprouting state of somatic embryo cannot meet the needs utilizing the Development of Somatic Embryogenesis to realize the large-scale production of plant specially for the PIECA ASPERATA somatic embryo germination rate low problem of low quality with sprouting.Belong to together in the world with existing or similar seeds research report compare, the mummification mode of PIECA ASPERATA somatic embryo provided by the invention, operate more simple, cost is cheaper; The method of the mark of mummification is simultaneously more efficient and novel, has not yet to see the application report of mummification mark, studies the level that is in a leading position.At present, the somatic embryo germination rate of softwood tree seeds is always very low, sprouts quality also not high, greatly constrains the production application of the Development of Somatic Embryogenesis.The mummification mark of the somatic embryo carried out based on somatic embryo colour-change after drying and other treatment, this Research Thinking drastically increases the germination rate of PIECA ASPERATA somatic embryo and sprouts quality, meets the needs of actual production.
In a word, the invention provides the drying method for treating before the sprouting of a kind of PIECA ASPERATA somatic embryo, include the mummification labelling strategies based on somatic embryo colour-change; Method of the present invention can improve the germination rate (reach more than 30%, reach as high as more than 80%) of somatic embryo in pole significantly; Meanwhile, the quality that somatic embryo is sprouted also is improved significantly; And the impact of genotype and clone can not be subject to, there is general suitability; For the industrial seedling rearing realizing PIECA ASPERATA somatic embryo has established theoretical basis, and provide practicable technical scheme.
Accompanying drawing explanation
Fig. 1 be the differentiation generation of liquid suspension propagation institute in a large number and the higher somatic embryo of synchronization degree.
Fig. 2 be the differentiation generation of semi-solid propagation institute in a large number and the higher somatic embryo of synchronization degree.
Fig. 3 is in embodiment 2, with the somatic embryo in " filter paper method " drying and other treatment.
Fig. 4 is in embodiment 5, and drying and other treatment is after 14 days, the somatic embryo of " unchanged type ".
Fig. 5 is in embodiment 5, and drying and other treatment is after 14 days, the somatic embryo of " the light red colour pattern of radicle "; Wherein a is flaxen Cotyledon and embryo shaft portion, and b is pink radicle part.
Fig. 6 is in embodiment 5, and drying and other treatment is after 14 days, the somatic embryo of " green cotyledon and plumular axis and red radicle type "; Wherein a is green Cotyledon and embryo shaft portion, and b is red radicle part.
Fig. 7 is in embodiment 6, and the somatic embryo of " green cotyledon and plumular axis and red radicle type " sprouts the high synchronization of generation and the seedling of robust growth after 3 weeks.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Drying method for treating before PIECA ASPERATA somatic embryo sprouting provided by the invention, research process comprises:
(1) acquisition of high synchronization PIECA ASPERATA somatic embryo;
(2) the mummification mode of applicable PIECA ASPERATA somatic embryo is filtered out;
(3) suitable drying time is screened;
(4) the general applicability demonstration of drying and other treatment mode;
(5) the contrast screening of mummification mark;
(6) the further optimal screening of mummification mode.
In the acquisition of above-mentioned high synchronization PIECA ASPERATA somatic embryo: embryo callus adopts the mode of liquid suspension propagation or semi-solid propagation to carry out, the embryo callus that these two kinds of modes are bred is carried out respectively the differentiation culture of somatic embryo, because division culture medium passes through optimization (conditional filtering namely in PIECA ASPERATA somatic embryo generation system) early stage, therefore all can the large and high synchronized somatic embryo of the amount of acquisition, the drying and other treatment for follow-up somatic embryo provides the material of homogenization to lay a good foundation.
In the screening of above-mentioned applicable PIECA ASPERATA somatic embryo mummification mode: adopt " filter paper bridge method ", " empty culture vessel method " and " filter paper method " three kinds of mummification modes to contrast, preferably " empty culture vessel method " or " filter paper method ", further preferably " filter paper method ".Adopt the mummification mode of " filter paper method ", somatic embryo can not only be made to be in the environment of relatively dry, promote that somatic embryo experiences a sufficient drying process; Meanwhile, adopting the mummification mode of " filter paper method ", can there is colour-change in somatic embryo, for the mummification mark carried out based on colour-change situation provides possibility.
In the screening of above-mentioned suitable drying time: drying time is 0 ~ 21 day, preferably 1 ~ 14 day, more preferably 7 ~ 14 days, most preferably 14 days.Mummification, after 14 days, can improve the germination rate of somatic embryo to greatest extent.
In the general applicability opinion of above-mentioned drying and other treatment mode: the clone adopting liquid suspension propagation and semi-solid propagation two kinds of modes to breed the somatic embryo that breaks up to obtain be material, drying and other treatment is carried out according to method of the present invention, then sprout, all obtain higher germination rate, illustrate that the sprouting promoter action of drying and other treatment of the present invention to the somatic embryo of the different clone of PIECA ASPERATA has general suitability.
In the contrast screening of above-mentioned mummification mark: after drying and other treatment is carried out to the somatic embryo of PIECA ASPERATA, the changing conditions of somatic embryo color, somatic embryo is divided into three types (namely carrying out mummification mark): " unchanged type ", " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type ".The somatic embryo of this three types after drying and other treatment also mark is sprouted respectively, germination rate through statistics " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type " is higher, and the germination rate of " green cotyledon and plumular axis and red radicle type " is the highest.This mummification marker research carried out somatic embryo, belongs to reported first.
In the further optimal screening of above-mentioned mummification mode: in the mummification mode adopting " filter paper method ", in the culture vessel placing PIECA ASPERATA somatic embryo, be lined with the dry aseptic filter paper of more than 1 layer, preferably 1 ~ 4 layer, more preferably 3 layers.This kind of mummification mode not only makes drying and other treatment efficiency improve further, significantly reduces drying and other treatment cost simultaneously.
That is, drying method for treating before PIECA ASPERATA somatic embryo sprouting provided by the invention, comprise: after the somatic embryo obtaining PIECA ASPERATA, drying and other treatment is carried out 0 ~ 21 day to the mode of somatic embryo employing " filter paper bridge method ", " empty culture vessel method " or " filter paper method ", and then sprouts.
As preferred embodiment, adopt the embryo callus of liquid suspension propagation or semi-solid propagation to carry out the differentiation culture of somatic embryo respectively, obtain the somatic embryo of PIECA ASPERATA.
The differentiation culture of described somatic embryo, used medium is: 1/2LM substratum+dormin 16mgL
-1+ PEG(polyoxyethylene glycol) 400050gL
-1+ sucrose 30gL
-1+ gac 1gL
-1+ gel 4gL
-1, additional glutamine 500mgL
-1with caseinhydrolysate 1gL
-1.Use this kind of substratum, and add above-mentioned Related Component and can obtain a large amount of and that synchronization degree is higher somatic embryo.Also can adopt other substratum, but effect of the present invention is more excellent.
Carry out in the differentiation culture of somatic embryo at the embryo callus of liquid suspension propagation, the cell volume of embryo callus and the volume ratio of division culture medium are 1:3.
As preferred embodiment, " empty culture vessel method " or the mode of " filter paper method " is adopted to carry out drying and other treatment to somatic embryo.
As further preferred embodiment, the mode of " filter paper method " is adopted to carry out drying and other treatment to somatic embryo.
" filter paper bridge method " of the present invention is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel, then this culture vessel (being placed with the culture vessel of PIECA ASPERATA somatic embryo) is placed in another larger culture vessel, sterilized water is placed in larger culture vessel, cover with two-layer wetting filter paper on the culture vessel being placed with PIECA ASPERATA somatic embryo and make " filter paper bridge ", finally larger culture vessel is sealed.
" empty culture vessel method " of the present invention is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel, then this culture vessel (being placed with the culture vessel of PIECA ASPERATA somatic embryo) is placed in another larger culture vessel, in larger culture vessel, place sterilized water, finally larger culture vessel is sealed.
" filter paper method " of the present invention is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel being lined with the above aseptic filter paper of one deck, then this culture vessel (being placed with the culture vessel of PIECA ASPERATA somatic embryo) is placed in another larger culture vessel, in larger culture vessel, place water, finally larger culture vessel is sealed.
As preferred embodiment, in described " filter paper method ", the number of plies being lined with filter paper in the culture vessel of placement PIECA ASPERATA somatic embryo is 1 ~ 4 layer, more preferably 3 layers.
In the present invention, the filter paper placed in culture vessel can size identical, also can be different, but preferably identical.Culture vessel used can be plastic culture dish or glass culture dish, preferred glass culture dish.
As further preferred embodiment, drying and other treatment 1 ~ 14 day, more preferably 7 ~ 14 days, most preferably 14 days.
And the condition that somatic embryo carries out drying and other treatment is also comprised: culturing room's temperature (24 ± 1) DEG C, 15 μm of olm
-2s
-1drying and other treatment is carried out under illumination.All operations aseptically carries out, the culture dish adopted, filter paper and provide the deionized water of gnotobasis, uses after all needing autoclaving.
Drying method for treating before PIECA ASPERATA somatic embryo sprouting of the present invention, also comprises: after carrying out drying and other treatment to the somatic embryo of PIECA ASPERATA, carry out mummification mark, and then sprout somatic embryo.
Describedly to the mode that somatic embryo carries out mummification mark be: according to the changing conditions of somatic embryo color after drying and other treatment, be marked as three types: " unchanged type ", " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type ".
As preferred embodiment, the somatic embryo being labeled as " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type " is sprouted.
As further preferred embodiment, the somatic embryo of " green cotyledon and plumular axis and red radicle type " is sprouted to being labeled as.
Drying method for treating before PIECA ASPERATA somatic embryo sprouting provided by the invention, comprising: after carrying out drying and other treatment to the somatic embryo of PIECA ASPERATA, then carry out mummification mark, then sprout; The mode of somatic embryo being carried out to mummification mark is: according to the change of somatic embryo color after drying and other treatment, be marked as three types: " unchanged type ", " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type ".
As preferred embodiment, the somatic embryo being labeled as " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type " is sprouted.
As further preferred embodiment, the somatic embryo of " green cotyledon and plumular axis and red radicle type " is sprouted to being labeled as.
Further, the somatic embryo of " green cotyledon and plumular axis and red radicle type " in the drying method for treating before PIECA ASPERATA somatic embryo provided by the invention is sprouted, is labeled as, drying time needed for it is 0 ~ 21 day, preferably 1 ~ 14 day, more preferably 7 ~ 14 days, most preferably 14 days.
In the method for the invention, sprout somatic embryo, substratum used is: 1/4LM substratum+sucrose 20gL
-1+ gel 6gL
-1+ gac 2gL
-1, additional glutamine 500mgL
-1with caseinhydrolysate 1gL
-1.Also can adopt other substratum, but effect of the present invention is more excellent.
In the method for the invention, break up or sprout in used medium: due to dormin and the glutamine non-refractory of use, can adopt the mode sterilizing of filtration, other all the components all adopt the sterilizing of High Temperature High Pressure mode.
In the method for the invention, the substratum in differential period of somatic embryo and the stage of sprouting: before adding gel, pH is adjusted to 5.8, subsequently 121 DEG C, pressure 101kp high temperature, under condition of high voltage sterilizing use after 20 minutes.Also can adopt other modes, but effect of the present invention is more excellent.
In the method for the invention, the condition that somatic embryo is sprouted is comprised: culture temperature is (24 ± 1) DEG C; First week (with 7 days for the cycle) dark culturing; Second week (with 7 days for the cycle), at 20 μm of olm
-2s
-1under illumination, the photoperiod of every day is that 16 h light are cultivated and 8 h dark are cultivated; Subsequently at 50 μm of olm
-2s
-1under illumination, the photoperiod of every day is that 16 h light are cultivated and 8 h dark are cultivated.Also can adopt other modes, but effect of the present invention is more excellent.
In the method for the invention, sprout somatic embryo, in germination process, somatic embryo is placed horizontally at Semi-solid cell culture primary surface, culture dish inclination 45° angle is placed, and is conducive to cotyledon stretching, extension, plumular axis and radicle and extends; Meanwhile, the radicle of growth is attached to media surface, and when avoiding transplanting, the tender shoot root of children is impaired, is conducive to transplant survival.
The present invention's gel used is plant gel gellangum, adopts Sigma brand; It is a kind of epoxy resin, and the effect with agar is identical, but effect is well a lot, and the rate of set and the transparency that are embodied in substratum all significantly improve.Gac activatedcarbon used in the present invention, same employing Sigma brand, compare with the gac that open market is sold, its activated adsorption ability is stronger, effectively can remove the toxic substances such as the phenols in substratum.Water used in the present invention is deionized water, and purity is 18.2 megaohms.
The present invention's other raw materials used and reagent are the conventional reagent buied from the market, and NM operating method and detection mode are also the operating method of this area routine.
The composition of LM substratum is:
Macroelement: 1.65gL
-1nH
4nO
3, 1.9gL
-1kNO
3, 1.85gL
-1mgSO
47H
2o, 0.34gL
-1kH
2pO
4, 0.022gL
-1caCl
22H
2o;
Trace element: 0.0304gL
-1h
3bO
3, 0.043gL
-1znSO
47H
2o, 0.021gL
-1mnSO
4h
2o, 0.00126gL
-1na
2moO
42H
2o, 0.00415gL
-1kI, 0.0005gL
-1cuSO
45H
2o, 0.000125gL
-1coCl
26H
2o;
Molysite: 0.0278gL
-1feSO
47H
2o, 0.0373gL
-1eDTA2H
2o(ethylenediamine tetraacetic acid (EDTA));
Inositol: 0.1gL
-1myo-Inositol;
Organic salt: 0.0001gL
-1thiamine-HCl(VitaminB
1), 0.0005gL
-1niacinacid(VitaminB
3), 0.0001gL
-1pyroxidone-HCl(VitaminB
6).
The 1/2LM substratum that the present invention occurs, be exactly the composition to above-mentioned LM substratum, macroelement, trace element and molysite are reduced by half, other are constant.The 1/4LM substratum that the present invention occurs is exactly that macroelement, trace element and molysite are reduced to 1/4, and other are constant to above-mentioned LM substratum.The semisolid medium that the present invention occurs, adds gel exactly in above-mentioned 1/2LM substratum.The 1/2LM liquid nutrient medium that the present invention occurs is exactly that macroelement, trace element and molysite are reduced by half, other are constant to above-mentioned LM minimum medium, does not add gel in substratum simultaneously.
First prepare mother liquor during the actual use of each nutritive ingredient in substratum, then dilute use.Macroelement is become 100 times with the mother liquor of molysite, and trace element is mixed with 1000 times, and inositol and organic salt are mixed with 500 times.Wherein, in order to avoid the CaCl in macroelement
22H
2o and other compositions combine and form precipitation, it are prepared separately.
Content of the present invention was carried out on the basis of PIECA ASPERATA somatic embryo generation system construction between 2011 ~ 2013 years.In PIECA ASPERATA somatic embryo generation system building process, by a large amount of simultaneous test, the optimal division culture medium of somatic embryo and germination medium are obtained.The experiment material adopted, preserves one-year age in laboratory, and different clone can represent the ubiquity of PIECA ASPERATA somatic embryo generation plant process, also can represent the general applicability of drying and other treatment mode.
Germination rate of the present invention all refers to added up average germination rate.
Embodiment 1
The acquisition of high synchronization PIECA ASPERATA somatic embryo: be divided into two kinds of differentiation modes to obtain according to the mode of embryo callus propagation: one is the clone 1921 and 1931 for setting up the system of liquid suspension propagation (be 1/2LM liquid nutrient medium), mixed according to cell volume time standing with without the ratio that liquid proliferated culture medium (1/2LM liquid nutrient medium) both volume ratios that hormone adds are 1:3 by the embryo callus of the propagation that suspends, the substratum 5mL getting the interpolation embryo callus shaken up is placed on a filter paper (Whatman
tM2), on, after falling liquid nutrient medium with Büchner funnel suction filtration, the filter paper with embryo callus is transferred on division culture medium and cultivates; Another kind is for the clone 1113,1911,1921,2322,2614 and 2711 of semisolid propagation, and directly tile the embryo callus of switching propagation after 2 weeks filter paper (Whatman
tM2), on, cultivate on division culture medium subsequently.Two kinds of modes adopt identical division culture medium and culture condition: 1/2LM substratum+dormin 16mgL
-1+ PEG400050gL
-1+ sucrose 30gL
-1+ gac 1gL
-1+ gel 4gL
-1, additional glutamine 500mgL
-1with caseinhydrolysate 1gL
-1; Add before gel and medium pH be adjusted to 5.8, subsequently 121 DEG C, pressure 101kp high temperature, under condition of high voltage sterilizing use after 20 minutes; (24 ± 1) DEG C dark culturing, within 7 weeks, (using 7 days for the cycle) break up the mature somatic embryo of generation afterwards as experiment material.
It should be noted that: the clone 1921 of this experiment adopts two kinds of Reproduction methods to study simultaneously, and result can be made more reliable.
These two kinds of Reproduction methods are modes the most frequently used in current embryo callus breeding.Adopt the embryo callus of liquid suspension propagation or semi-solid propagation, carry out the differentiation of somatic embryo respectively, and drying and other treatment is carried out to somatic embryo, the broad applicability of drying and other treatment effect more can be described.Simultaneously, owing to there being the medium component of the most applicable identical embryo differentiate in division culture medium, therefore dark differentiation is after 7 weeks, the embryo callus kept under two kinds of Reproduction methods all can obtain quantity is comparatively large and synchronization degree is very high somatic embryo (as depicted in figs. 1 and 2, wherein Fig. 1 is produce a large amount of of liquid suspension propagation institute differentiation and the somatic embryo that synchronization degree is higher, Fig. 2 be semi-solid propagation differentiation generation in a large number and the higher somatic embryo of synchronization degree); Ensure that the homogeneity of somatic embryo in drying and other treatment process, make drying and other treatment result more reliable.
Embodiment 2
The screening of PIECA ASPERATA somatic embryo mummification mode: with the somatic embryo of 1921 liquid suspension propagation systems differentiation in embodiment 1 for material, adopt external conventional " filter paper bridge method " at present, " filter paper method " that " empty culture vessel method " and this laboratory are set up carries out the comparison of three kinds of drying methods.Particularly: " filter paper bridge method " is placed in plastic culture dish (35 × 12mm) by somatic embryo, subsequently plastic culture dish is placed in larger glass culture dish (87 × 15mm), 10mL sterilized water is placed in glass dish, cover on plastic ware with two-layer moistening filter paper and make " filter paper bridge ", finally glass dish sealed membrane is sealed." empty culture vessel method " operation, with " filter paper bridge method ", is not just done " filter paper bridge "." filter paper method " (as shown in Figure 3, it reflects the integrality in drying and other treatment process) be placed in the plastic culture dish (35 × 12mm) being lined with two-layer aseptic dry filter paper (formed objects) by full grown somatic embryo, subsequently plastic ware is placed in larger sterile glass culture dish (87 × 15mm), 10mL sterilized water is placed in glass dish, make somatic embryo be in the environment of a relatively dry like this, glass dish sealed membrane seals.Subsequently, the somatic embryo of these three kinds of method drying and other treatment is placed on 15 μm of olm
-2s
-1the low light level and at the temperature of (24 ± 1) DEG C mummification cultivate 2 weeks (with 7 days for the cycle).
Observe form and the colour-change of somatic embryo in plastic culture dish, mummification result is as shown in the table:
Note: " the highest percentage of water loss " in upper table refers in drying process, the ratio of somatic embryo fresh weight when the fluid loss that somatic embryo is the highest accounts for drying and other treatment 0 day.This index reflects in different mummification processing mode, the ability of somatic embryo dehydration; The very few drying and other treatment that may cause of dehydration is insufficient, and then can not effectively promote somatic sprouting.
The result of upper table illustrates: " filter paper bridge method ", owing to making the humidity around somatic embryo excessive, the embryo majority in plastic ware is immersed in water, and this processing mode repeatability is poor, and operation is comparatively loaded down with trivial details, empirical comparatively strong, filter paper bridge is slightly dealt with improperly, just easily causes mummification failure." filter paper bridge method " is compared, although there is not the risk making somatic embryo be immersed in water in " empty culture vessel method ", but drying and other treatment found after 2 weeks, somatic embryo is without obvious colour-change, moisture determination is found, when drying and other treatment 1 day, the percentage of water loss of embryo is the highest only had 15.75%, and water ratio raises gradually subsequently; The mark sprouted in conjunction with later stage somatic embryo needs, and for the mummification of PIECA ASPERATA somatic embryo, " empty culture vessel method " is inapplicable equally.Somatic embryo colour-change after " filter paper method " process is obvious; The somatic embryo be just separated from division culture medium is faint yellow, and cotyledon is obvious, and plumular axis is sturdy; When by " filter paper method " drying and other treatment 1 day, the percentage of water loss of embryo accounts for 23.72% of fresh weight, accounts for 41.83% of total water content; Along with the prolongation of drying time, the water content of embryo raises gradually, and along with the change of moisture, plumular axis constantly extends; Drying and other treatment is after 1 week, and radicle portion reddens gradually, and Cotyledon and embryo axle starts to turn green subsequently; Drying and other treatment is after 2 weeks, the embryo showed increased of green cotyledon and plumular axis, color no longer considerable change when continuing drying and other treatment; And the method repeatability very well.Therefore, think more afterwards " filter paper method " be best suited for PIECA ASPERATA somatic embryo sprout before mummification mode, other two kinds of methods are taken second place.
Illustrate: why the highest percentage of water loss of " filter paper bridge method " mummification " cannot be measured ", be because: the somatic embryo majority of " filter paper bridge method " mummification is immersed in water, and measured water ratio has the interference of the water beyond embryo itself; In addition, measure also meaningless, illustrate that this mode can not provide good mummification environment because be immersed in water.
Embodiment 3
The screening of somatic embryo drying time: with the somatic embryo of 1931 liquid suspension propagation systems differentiation in embodiment 1 for material, adopt " filter paper method " mummification mode mentioned in embodiment 2, somatic embryo difference drying and other treatment, after 0 day, 1 day, 4 days, 7 days, 14 days and 21 days (condition of other drying and other treatment is with embodiment 2), is then carried out sprouting contrast experiment respectively.Germination medium is 1/4LM substratum+sucrose 20gL
-1+ gel 6gL
-1+ gac 2gL
-1, additional glutamine 500mgL
-1with caseinhydrolysate 1gL
-1; PH is adjusted to 5.8 by substratum before adding gel, subsequently 121 DEG C, pressure 101kp high temperature, under condition of high voltage sterilizing use after 20 minutes.Sprouting condition is: under the room temperature of (24 ± 1) DEG C, dark culturing one week (with 7 days for the cycle); After forward 20 μm of olm to
-2s
-1cultivate under the light of light intensity one week (with 7 days for the cycle), the photoperiod of every day is 16h illumination cultivation and 8h dark culturing; Subsequently, 50 μm of olm are forwarded to
-2s
-1cultivate under illumination, the photoperiod of every day is 16h illumination and 8h dark culturing.Three weeks (with 7 days for the cycle) add up the normal somatic embryo quantity sprouted afterwards.
Under different mummification time-triggered protocol, the sprouting result of somatic embryo is as shown in the table:
| Drying time (my god) | 0 | 1 | 4 | 7 | 14 | 21 |
| Germination rate (%) | 4.67±1.83 | 7.34±2.80 | 14.00±2.82 | 24.68±3.93 | 56.83±8.25 | 33.50±12.09 |
As seen from the above table: PIECA ASPERATA somatic embryo is extremely low without germination rate during drying and other treatment, the sprouting of time on PIECA ASPERATA somatic embryo of different drying and other treatment has affects very significantly.
Along with the prolongation of drying time, the germination rate of somatic embryo improves gradually.During drying and other treatment 14 days, the germination rate of somatic embryo is the highest can improve 52.16%.Proceed drying and other treatment, when reaching 21 days, somatic embryo germination rate significantly reduces.Illustrate that drying and other treatment can improve the sprouting of PIECA ASPERATA somatic embryo for 2 weeks effectively, other times take second place.
Embodiment 4
The general applicability demonstration of drying and other treatment: the somatic embryo broken up with the clone 1113,1911,1921,2322,2614 and 2711 of propagation semi-solid in embodiment 1 is for material, by " filter paper method " mummification mode mentioned in embodiment 2, drying and other treatment is after 0 day and 14 days (condition of other drying and other treatment is with embodiment 2) respectively, carry out sprouting contrast experiment, germination medium and sprouting condition are with content corresponding in embodiment 3.
Drying and other treatment sprouting result of somatic embryo between different clone is as shown in the table:
| Clone | 1113 | 1911 | 1921 | 2322 | 2614 | 2711 |
| Mummification 0 day germination rate (%) | 0 | 0 | 0.67±0.58 | 0 | 0 | 0 |
| Mummification 1 day germination rate (%) | 6.90±2.73 | 5.61±2.52 | 3.08±2.07 | 2.13±2.99 | 9.24±1.39 | 7.37±2.78 |
| Mummification 14 days germination rates (%) | 53.37±8.04 | 42.05±7.81 | 31.46±9.92 | 47.89±8.82 | 62.96±4.17 | 47.90±8.47 |
As seen from the above table: when the somatic embryo of PIECA ASPERATA is directly sprouted without drying and other treatment, germination rate is extremely low or can not normally sprout; And after the mummification process of 14 days, between different clone, the germination rate of somatic embryo all obtains improving very significantly, namely between different clone, the germination rate of somatic embryo improves from 31.46% to 62.96% not etc.
And according in embodiment 3, after liquid suspension propagation, the somatic embryo of differentiation can improve germination rate by corresponding drying and other treatment mode equally.Embodiment 3 and embodiment 4 combine explanation: the somatic embryo (genotype that different clone & is different) that the embryo callus institute differentiation culture no matter with which kind of Reproduction methods obtained produces, utilize the drying and other treatment mode of the application, the sprouting of somatic embryo can both be promoted; In other words, the specific drying and other treatment mode of the application has ubiquity to the promoter action that PIECA ASPERATA somatic embryo is sprouted.
Embodiment 5
In the mummification mark of somatic embryo: the somatic embryo 1921 and 1931 liquid suspension propagation systems in embodiment 1 broken up is material, adopt " filter paper method " mummification mode mentioned in embodiment 2, respectively the somatic embryo of these two clones is carried out drying and other treatment 14 days (condition of other drying and other treatment is with embodiment 2).After drying and other treatment, according to the changing conditions of somatic embryo color, somatic embryo can be divided into three types, respectively: " unchanged type " (as shown in Figure 4, somatic embryo entirety is faint yellow), " incarnadine radicle type " (as shown in Figure 5, wherein part a is flaxen Cotyledon and embryo axle, and part b is pink radicle), " green cotyledon and plumular axis and red radicle type " (as shown in Figure 6, wherein part a is green Cotyledon and embryo axle, and part b is red radicle).According to morphological observations, although drying and other treatment was expanded before the general comparatively mummification of somatic embryo after 14 days, Embryo stem extension; But how small and weak the somatic embryo of " unchanged type " is, the embryo of the somatic embryo of " incarnadine radicle type " comparatively " unchanged type " is slightly healthy and strong, and the general robust growth of somatic embryo of " green cotyledon and plumular axis and red radicle type ", tool possesses exuberant vitality.
Embodiment 6
In the mummification mark of somatic embryo: according to the mummification labeling pattern of somatic embryo in embodiment 5, the somatic embryo of three types is carried out sprouting simultaneous test respectively, germination medium and sprouting condition are with content corresponding in embodiment 3.
Sprouting result between the different clone of somatic embryo of three types is as shown in the table:
| Somatic embryo type | " unchanged type " | " incarnadine radicle type " | " green cotyledon and plumular axis and red radicle type " |
| Clone 1921 germination rate (%) | 19.17±1.70 | 25.84±3.31 | 81.26±11.66 |
| Clone 1931 germination rate (%) | 22.71±3.38 | 24.24±8.67 | 92.47±33.17 |
As seen from the above table, in two clones of participating in the experiment, after drying and other treatment, the germination rate of dissimilar somatic embryo has very significant difference; Wherein, the germination rate of the somatic embryo of " green cotyledon and plumular axis and red radicle type " comparatively improves other types pole significantly, and reach more than 81.26%, the germination rate of incarnadine radicle type takes second place.Meanwhile, the synchronism that somatic embryo is sprouted also greatly improves, body embryo seedling robust growth, and cotyledon stretches, plumular axis is upright, radicle is sturdy.Sprouted the seedling (as shown in Figure 7, same Fig. 6 of color of somatic embryo) of 3 weeks as can be seen from " green cotyledon and plumular axis and red radicle type " somatic embryo, its young root universal synchronization extends, and the equal contemporaneity of plumular axis is upright, and cotyledon also stretches mostly.
This just illustrates: somatic embryo, after drying and other treatment, divides the type of somatic embryo, then selects suitable type to sprout according to the changing conditions of color, effectively can improve the germination rate of somatic embryo and sprout quality.
In this application, the mark that the somatic embryo of " green cotyledon and plumular axis and red radicle type " after drying and other treatment can be sprouted as a kind of high frequency and high quality.Although the somatic embryo of different clone needs the time of mummification there are differences, because the somatic embryo of different clone all can variable color.Therefore, the frequency occurred according to the somatic embryo of " green cotyledon and plumular axis and red radicle type " and determine that the time of final drying and other treatment whether suitablely to seem more rationally and efficient.
Embodiment 7
The further screening of mummification mode: although " filter paper method " mummification mode that laboratory is set up has very large advantage compared with other mummification modes, but be not difficult to find out that this drying and other treatment mode is still efficient not, be embodied in somatic embryo mummification and adopt aseptic plastic culture dish (35 × 12mm) can only single use; Due to limited space, the somatic embryo quantity that each plastic ware can be placed only has about 30 usually, for production, seem convenient not.Not enough in order to overcome these, with the somatic embryo of 1921 liquid suspension propagation systems differentiation in embodiment 1 for material, 100 full grown somatic embryos are selected from division culture medium (as described in Example 1) and is directly placed in glass dish (87 × 15mm) afterwards, 0 is placed respectively in glass dish, 1, 2, (diameter is 85mm to 3 and 4 layers of aseptic filter paper, place 0 metafiltration paper, be equivalent to " empty culture vessel method "), to form dry environment in various degree around somatic embryo, subsequently glass dish is placed on and fills in the larger culture dish (120 × 20mm) of 10mL sterilized water, after larger culture dish being sealed with sealed membrane, be placed on 15 μm of olm
-2s
-1the low light level and at the temperature of (24 ± 1) DEG C mummification cultivate 2 weeks (with 7 days for the cycle), the frequency of the appearance of statistics " green cotyledon and plumular axis and red radicle type " somatic embryo.
The different filter paper number of plies, the frequency of occurrences of " green cotyledon and plumular axis and red radicle type " somatic embryo under different dry environment (in following table " green cotyledon embryo) is as shown in the table:
| The filter paper number of plies (layer) | 0 | 1 | 2 | 3 | 4 |
| Green cotyledon embryo frequency (%) | 56.41±13.35 | 64.31±10.91 | 64.80±17.35 | 85.42±11.09 | 84.80±15.13 |
As seen from the above table, the different dry environments caused by the different filter paper number of plies have the frequency that " green cotyledon and plumular axis and red radicle type " somatic embryo occurs to be affected extremely significantly.
The frequency that drying and other treatment occurred with " green cotyledon and plumular axis and red radicle type " somatic embryo under three metafiltration paper process after 2 weeks is the highest, reaches 85.42%.Frequency that somatic embryo produces that this kind of mummification mode can not only improve effectively " green cotyledon and plumular axis and red radicle type ", and mummification step can be simplified further, reduce costs (glass dish can reuse), improve drying and other treatment efficiency.
The aim of this research is to provide the somatic embryo drying method for treating of simple to operate, with low cost, a realistic need of production.Change capsule into large ware, although operation is very simple, very practical.In addition, for the Development of Somatic Embryogenesis, an inherently plant production multiplication technique, practical and be efficiently its maximum prerequisite, need to meet economic needs simultaneously.Therefore, unattended operation method how, even very simple, as long as can achieve the goal, just can carry out production application.
Embodiment 8
Liquid suspension propagation and the semi-solid embryo callus of breeding two kinds of modes and breeding is obtained, the high synchronization somatic embryo that differentiation culture obtains according to embodiment 1.
Subsequently, by the high synchronization somatic embryo obtained, adopt " filter paper method " drying and other treatment mode described in embodiment 2, carry out drying and other treatment and sprout (germination medium and sprouting condition are with content corresponding in embodiment 3) afterwards in 14 days.Make the germination rate of somatic embryo bring up to 31.46% to 62.96% and do not wait (concrete data are shown in embodiment 3 and 4), illustrate that the sprouting promoter action of drying and other treatment to PIECA ASPERATA somatic embryo has general suitability.Wherein the drying and other treatment of somatic embryo is aseptically carried out, and comprises aseptic dry filter paper, sterilized water and sterile glass culture dish etc.This step is very crucial, because the sprouting of somatic embryo needs a sterile environment after drying and other treatment; Even if otherwise have a large amount of mummification somatic embryos, can not germinated plantlet be obtained.
Somatic embryo, after 14 days, according to the change of somatic embryo color, can be divided into three types by drying and other treatment, respectively: " unchanged type ", and " incarnadine radicle type ", " green cotyledon and plumular axis and red radicle type ".Sprout contrast experiment to show (germination medium and sprouting condition are with content corresponding in embodiment 3): the somatic embryo of " green cotyledon and plumular axis and red radicle type " obtains very high germination rate (more than 81.26%) and sprout quality preferably that (sprouting seedling synchronism greatly improves, the body embryo seedling robust growth sprouted, cotyledon stretches, plumular axis is upright, radicle is sturdy), particular content is shown in embodiment 5 and 6.
Somatic embryo mummification mode is further optimized: be placed on after 100 full grown somatic embryos are selected from division culture medium in glass dish (87 × 15mm), 3 layers of aseptic filter paper (diameter is 85mm) are placed in glass dish, to form the environment of a relatively dry around somatic embryo, subsequently glass dish is placed on and fills in the larger glass dish (120 × 20mm) of 10ml sterilized water, with sealed membrane by larger glass dish sealing (other conditions are with content corresponding in embodiment 7).Obtain the frequency of occurrences (85.42%) of the highest " green cotyledon and plumular axis and red radicle type " somatic embryo.This kind of mummification mode is not only simple to operate, and with low cost, can meet the needs of large-scale production.
At present, in the domestic patent reported or document, in research strategy, there is not been reported for the drying and other treatment before sprouting about softwood tree seeds somatic embryo, the domestic somatic embryo germination rate about softwood tree seeds is also general very low at present, can not meet the needs of actual production.Drying method for treating of the present invention, especially includes mummification mark, effectively can improve the germination rate of PIECA ASPERATA somatic embryo and sprout quality.Meanwhile, the strategy of mummification marker research, external also not yet have reported success (as adopted osmotic adjustment, but there are no the report of variable color) at present, and correlative study achievement is in a leading position in this field level.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (5)
1. the drying method for treating before the sprouting of PIECA ASPERATA somatic embryo, comprising: after the somatic embryo obtaining PIECA ASPERATA, adopts the mode of " filter paper method " to carry out drying and other treatment 1-14 days, and then sprout somatic embryo;
The condition of somatic embryo being carried out to drying and other treatment also comprises: temperature is 23 ~ 25 DEG C; Intensity of illumination is 15 ~ 18 μm of olm
-2s
-1;
Wherein, adopt the embryo callus of liquid suspension propagation to carry out the differentiation culture of somatic embryo respectively, obtain the somatic embryo of PIECA ASPERATA;
The method also comprises: after carrying out drying and other treatment to the somatic embryo of PIECA ASPERATA, carry out mummification mark, and then sprout somatic embryo;
The mode of somatic embryo being carried out to mummification mark is: according to the change of somatic embryo color after drying and other treatment, be marked as three types: " unchanged type ", " incarnadine radicle type " and " green cotyledon and plumular axis and red radicle type ";
Described " filter paper method " is: be placed on by PIECA ASPERATA somatic embryo in a culture vessel being lined with the above filter paper of one deck, then this culture vessel is placed in another larger culture vessel, in larger culture vessel, place water, finally larger culture vessel is sealed.
2. the drying method for treating before PIECA ASPERATA somatic embryo sprouting according to claim 1, is characterized in that, drying and other treatment 7 ~ 14 days.
3. the drying method for treating before PIECA ASPERATA somatic embryo sprouting according to claim 2, is characterized in that, drying and other treatment 14 days.
4. the drying method for treating before sprouting according to the PIECA ASPERATA somatic embryo in claim 1-3 described in any one, is characterized in that, in described " filter paper method ", the number of plies being lined with filter paper in the culture vessel of placement PIECA ASPERATA somatic embryo is 1 ~ 4 layer.
5. the drying method for treating before PIECA ASPERATA somatic embryo sprouting according to claim 4, is characterized in that, in described " filter paper method ", the number of plies being lined with filter paper in the culture vessel of placement PIECA ASPERATA somatic embryo is 3 layers.
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| CN113575418A (en) * | 2021-08-17 | 2021-11-02 | 青岛农业大学 | Device and culture method for drying treatment of Pinus sylvestris in vitro embryos |
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