CN104365584A - Method for improving survival rate of plant seeds by N-acylethanolamines after ultralow-temperature preservation and application of N-acylethanolamines - Google Patents
Method for improving survival rate of plant seeds by N-acylethanolamines after ultralow-temperature preservation and application of N-acylethanolamines Download PDFInfo
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Abstract
The invention provides a method for improving survival rate of plant seeds by N-acylethanolamines (NAE) after ultralow-temperature preservation and application of N-acylethanolamines. The method comprises the following steps: taking plumular axes of the plant seeds and applying the N-acylethanolamines to the plumular axes of the plant seeds; carrying out pretreatment and an ultralow-temperature preservation experiment; and carrying out a germination force experiment to detect the vitality. Meanwhile, the invention provides application of the N-acylethanolamines to the ultralow-temperature preservation of the plant seeds. Before the seeds with the plumular axes which are sensitive to dehydration are subjected to the ultralow-temperature preservation, the survival rate of the seeds can be remarkably improved if an inhibitor NAE (100 micromoles) of PLDalpha is exogenously applied;lipidomics analysis is carried out in a whole process and shows that in an ultralow-temperature preservation process of the plumular axis seeds treated with the NAE, substrate phospholipids (PG, PE and PC) for hydrolysis of PLD alpha are remarkably improved and the formation of membrane lipid is changed, so that the survival rate of the plumular axes after the ultralow-temperature preservation process is improved; and the method has a very good application prospect in the preservation of germplasm resources of the sensitive seeds.
Description
Technical field:
The invention belongs to seed physiology and seed storage technical field, particularly, relate to the method by survival rate after the preservation of N-acyl ethanol amine raising plant seed ultralow temperature and application thereof.
Background technology:
Seed Deposit is the important means that plant germplasm resource is preserved for a long time, and it can provide important material in kind and genetic resources for new crop varieties seed selection and breed improvement, ecological system degradation or the field such as reconstruction, Plant diversity conservation.According to the statistical report display that the World Food Programme of the United Nations (FAO) is up-to-date, between 1996 to 2009, the germ plasm resource of the wild plant of global all kinds of seed bank Collection and conservation is increased to 133.2 ten thousand parts (FAO, 2010) from 900,000 parts.But the seed having the plant of 7-25% (having the plant up to 47% in tropical rain forest) to produce, due to not dehydration tolerance, cannot be preserved by conventional seed bank Techniques of preserving.According to the Desiccation-tolerance of seed, seed storage behavior can be divided into three kinds: orthodox seed (Orthodoxseed), osculant seed (Intermediate seed) and recalcitrant seeds (Recalcitrant seed).Orthodox seed can be dried to low water content (2-5%) and seed vitality is unaffected, and seed longevity with seed moisture content and storage temperature reduction and increase.Recalcitrant seeds does not tolerate excessive dehydration, usually when seed moisture content is lower than will death during 12-31%.The dehydration tolerance of osculant seed is between above two types, but seed will be subject to physiological loss when low temperature.The species great majority producing " recalcitrant " seed and " osculant " seed belong to Main Constructive Species in the torrid zone, subtropical forest or Important Economic plant, as Dipterocarpaceae (Dipterocarpaceae), Fagaceae (Fagaceae), Lauraceae (Lauraceae) etc.Therefore, to grope and to set up a set of new germ plasm resource preservation system extremely urgent.
Ultralow temperature preservation (cryopreservation) refers to that (-196 DEG C) preserve the method for biomaterial under liquid nitrogen temperature.Lot of experimental data proves, ultralow temperature preservation technology realizes the recalcitrant unique approach (Li and Pritchard2009) preserved for a long time with osculant seed at present, scientists has used ultralow temperature preservation technology successfully cannot preserve at conventional seed bank coffee, Chinese chestnut etc., but the recalcitrant and osculant seed with Important Economic value establishes a series of successful preservation system.Although ultralow temperature preservation technology has had an example of successful Application in the preservation of germ plasm resource, but due to the difference of plant species, the process in each species ultralow temperature preservation steps is caused to be had any different, and after ultralow temperature preservation the survival rate of species and regeneration rate lower, therefore, this technical bottleneck constrains greatly to recalcitrant and osculant seed and To body material extensive preservation.The survival rate how effectively improving species is the major issue that ultralow temperature preservation faces.
The permeability barrier around cell or organelle that cell membrane is made up of phospholipid bilayer and associated protein and cholesterol and glycolipid.Cell membrane is the organ being the most easily subject to environmental factor stresses injury, and its damage directly can cause cell death.Low temperature is one of Stress Factors producing Cell Membrane Injury.Plant membrane lipid mainly contains 8 kinds of fundamental types about 120 kinds of molecules (molecular species), comprise 6 kinds of phosphatide: phosphatidyl glycerol (phosphatidylglycerol, PG), phosphatidylinositols (phosphatidylinositol, PI), phosphatidylserine (phosphatidylserine, PS), phosphatidyl-ethanolamine (phosphatidylethanolamine, PE), phosphatid ylcholine (phosphatidylcholine, PC), phosphatidic acid (phosphatidic acid, PA) and 2 kinds of glycolipids: single galactose two glyceride (monogalactosyldiacylglycerol, MGDG), two galactose two glyceride (digalactosyldiacylglycerol, DGDG).Dissimilar film fat molecular structure is different, and its biophysics is different with biochemical property.Plant to conform, comprises the change of temperature by adjustment film fat composition.Low temperature result in each constituent of cell membrane lipid and changes, thus make originally orderly bimolecular hierarchal arrangement structure (lamellar phase), become (non-laminar phase) structure (Steponkus et al. of the starlike arrangement of disorderly radiation, 1993), the permeability of film is caused to change, cell sap and a large amount of inorganic ions and the Small molecular such as amino acid, soluble sugar are by trend extracellular seepage, destroy normal metabolic pathway, finally cause cell death.The conical molecular structure of the uniqueness of PA and MGDG, is considered to the formation (" non-laminar phase " namely in film phase transformation, Hexagonal II phase) being conducive to membrane damage, and finally causes film seepage, thus cell death.PA can be produced by many metabolic pathways, and wherein an important channel is produced by phospholipase D (phospholipase D, PLD) hydrolytic phosphatide (PC, PE, PG, PS).There is increasing evidence to show in recent years, if external source applies specific inhibitor N-acyl ethanol amine (N-acylethanolamines, NAE) of PLD α, the activity of PLD α can be suppressed, thus affect the alpha mediated physiology course of PLD.NAE is a class trace lipid composition in animal and plant cell, research finds, the activity of PLD α can by different NAE molecules in inhibiting (molecule from NAE12:0 to NAE18:3 all can suppress), and this suppression is that PLD alpha specific suppresses, such inhibition (Austin-Brown and Chapman, 2002) is not but had for other members (PLD β and PLD γ) in PLD family.
At present, apply there are no external source the report that N-acyl ethanol amine improves survival rate after the preservation of seed ultralow temperature in prior art.
Summary of the invention:
The object of the present invention is to provide and a kind of apply NAE by external source, change the composition of film fat, thus the survival rate improved after the preservation of osculant seed ultralow temperature and the method for regeneration rate and the application of the method.
In order to realize above-mentioned purpose of the present invention, present invention employs following technical scheme:
After the preservation of raising plant seed ultralow temperature, the method for survival rate, gets embryo of a plant seed axle, N-acyl ethanol amine is put on plant seed plumular axis, carries out pretreatment and ultralow temperature preservation experiment, and carries out germination experiment detection vigor.
As described in the preservation of raising seed ultralow temperature after the method for survival rate; that plant seed plumular axis is stripped out; aseptically add N-acyl ethanol amine; 25 DEG C of dark treatment 3 hours; drying process 4 hours is carried out afterwards with the silica gel of high-temperature sterilization; plumular axis is put into Cryopreservation pipe; insert after preserving 1 week in liquid nitrogen; cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating; the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, and then sprouts under being transferred to light.
As as described in the preservation of raising seed ultralow temperature after the method for survival rate, that plant seed plumular axis is stripped out, aseptically every 10 plumular axis add the PLD alpha specific inhibitor N-acyl ethanol amine of 1ml100 μM, 25 DEG C of dark treatment 3 hours, the silica gel of high-temperature sterilization is used to carry out drying process 4 hours afterwards, every 10 plumular axis are put into 1ml Cryopreservation pipe, preserve in direct insertion liquid nitrogen, after 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, sprout under transferring to light afterwards.
As described in the preservation of raising seed ultralow temperature after the method for survival rate, after getting osculant peeling plant seeds, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards, under aseptic technique, the plumular axis of seed is taken out, every 10 plumular axis are a repetition, each process 5 repetition, puts into the treatment fluid of 1ml water, 100 μMs of DMSO and 100 μM NAE respectively, is placed in 25 DEG C of dark treatment 3 hours; Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours, every 10 plumular axis are put into 1ml Cryopreservation pipe, put into liquid nitrogen fast and carry out preservation 1 week, after 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, sprout under transferring to light afterwards, significantly increase through NAE pretreated seed plumular axis survival rate and regeneration rate.
As described in the preservation of raising seed ultralow temperature after the method for survival rate, after getting the peeling of plant osculant seed, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards, under aseptic technique, the plumular axis of seed is taken out, every 10 plumular axis are a repetition, each process 5 repetition, puts into the treatment fluid of 1ml water, 100 μMs of DMSO and 100 μM NAE respectively, is placed in 25 DEG C of dark treatment 3 hours; Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours, every 10 plumular axis are put into 1ml Cryopreservation pipe, put into liquid nitrogen fast and carry out preservation after 1 week, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, carry out lipid extraction afterwards and detect and analyze; Every 10 plumular axis are added in the isopropyl alcohol of 3ml preheating, 15min is fixed in 75 DEG C of water-baths, after room temperature cooling, add in the extract of chloroform and methyl alcohol and extract 3 days, merge supernatant, dry up with nitrogen, at 105 DEG C of drying in oven seeds, and weighing obtains dry weight, the fat proposed carries out lipid and detects analysis.
Invention also provides the application of N-acyl ethanol amine in the preservation of plant seed ultralow temperature.
As as described in application, that plant seed plumular axis is stripped out, aseptically every 10 plumular axis add the PLD alpha specific inhibitor N-acyl ethanol amine of 1ml100 μM, 25 DEG C of dark treatment 3 hours, the silica gel of high-temperature sterilization is used to carry out drying process 4 hours afterwards, every 10 plumular axis are put into 1ml Cryopreservation pipe, preserve in direct insertion liquid nitrogen, after 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, sprout under transferring to light afterwards.
As described in application, being N-acyl ethanol amine by reducing PLD alpha active, changing film fat composition, improve the survival rate of plumular axis after ultralow temperature preservation and regeneration rate.
As described in application, after getting osculant peeling plant seeds, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards, under aseptic technique, the plumular axis of seed is taken out, every 10 plumular axis are a repetition, each process 5 repetition, puts into the treatment fluid of 1ml water, 100 μMs of DMSO and 100 μM NAE respectively, is placed in 25 DEG C of dark treatment 3 hours; Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours, every 10 plumular axis are put into 1ml Cryopreservation pipe, put into liquid nitrogen fast and carry out preservation 1 week, after 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, sprouts under transferring to light afterwards.
As described in application, after getting the peeling of plant osculant seed, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards, under aseptic technique, the plumular axis of seed is taken out, every 10 plumular axis are a repetition, each process 5 repetition, puts into the treatment fluid of 1ml water, 100 μMs of DMSO and 100 μM NAE respectively, is placed in 25 DEG C of dark treatment 3 hours; Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours, every 10 plumular axis are put into 1ml Cryopreservation pipe, put into liquid nitrogen fast and carry out preservation after 1 week, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, carry out lipid extraction afterwards and detect and analyze; Every 10 plumular axis are added in the isopropyl alcohol of 3ml preheating, 15min is fixed in 75 DEG C of water-baths, after room temperature cooling, add in the extract of chloroform and methyl alcohol and extract 3 days, merge supernatant, dry up with nitrogen, at 105 DEG C of drying in oven seeds, and weighing obtains dry weight, the fat proposed carries out lipid and detects analysis.
The application of N-acyl ethanol amine of the present invention in the preservation of osculant seed ultralow temperature; that N-acyl ethanol amine is by regulation and control PLD α; and then change film fat composition, the osculant seed after the process of N-acyl ethanol amine, the application that survival rate and regeneration rate significantly improve after ultralow temperature preservation.
The present invention is in osculant seed Camellia reticulata (Camillia reticulate) and Pu'er tea (CamilliaSinensis) ultralow temperature preserving process; find the pretreatment applying N-acyl ethanol amine by carrying out external source to the plumular axis of these two species, the survival rate after its ultralow temperature preservation significantly raises.As compared to the sample processed through water and dimethyl sulfoxide (DMSO) (DMSO), through the sample of N-acyl ethanol amine process, its survival rate has significant difference.By carrying out lipidomics analysis to the sample of different disposal; find the sample of N-acyl ethanol amine process; wherein significantly can raise as the substrate phosphatide (PG, PE, PC) of the PLD α enzyme effect of being hydrolyzed; show thus; the sample of N-acyl ethanol amine process is the activity by reducing PLD α; change the composition of film fat, thus improve the survival rate of plumular axis after ultralow temperature preservation.
Therefore; the present invention's N-acyl ethanol amine passes through regulation and control PLD α; change the composition of film fat; improve the survival rate of osculant seed in ultralow temperature preservation; not only to the mechanism disclosing membrane damage in seed ultralow temperature process, there is substantial value, and in the Germ-plasma resources protection to osculant seed, have important realistic meaning and good application prospect.
Accompanying drawing illustrates:
Fig. 1 external source applies water, after 100 μMs of DMSO and 100 μM NAE on the impact of plumular axis in ultralow temperature preserving process of Camellia reticulata and tea;
On the impact that film fat composition changes in ultralow temperature preserving process of Camellia reticulata plumular axis after Fig. 2 external source applies water, 100 μMs of DMSO and 100 μM NAE.
Embodiment:
Below in conjunction with accompanying drawing, further illustrate essentiality content of the present invention with embodiments of the invention, but do not limit the present invention with this.
Embodiment 1:
After external source applies 100 μMs of N-acyl ethanol amines (N-acylethanolamines, NAE), the survival rate of the plumular axis significantly improving Camellia reticulata and Pu'er tea in ultralow temperature preserving process and regeneration rate.
The plumular axis that osculant seed Camellia reticulata (Camillia reticulate) and tea (Camilliasinensis) seed are got in this experiment respectively carries out pretreatment and ultralow temperature preservation experiment, and carries out germination experiment detection vigor.After the peeling of the seed of Camellia reticulata and Pu'er tea, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards.Under aseptic technique, taken out by the plumular axis of seed, every 10 plumular axis are a repetition, and each process 5 repetition is put into the treatment fluid of 1ml water, 100 μMs of DMSO and 100 μM NAE respectively, is placed in 25 DEG C of dark treatment 3 hours.Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours.Every 10 plumular axis are put into 1ml Cryopreservation pipe, puts into liquid nitrogen fast and carry out preservation 1 week.After 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, sprouts under transferring to light afterwards.Significantly increase (Fig. 1) through NAE pretreated seed plumular axis survival rate and regeneration rate.
Embodiment 2:
After external source applies 100 μMs of N-acyl ethanol amines (N-acylethanolamines, NAE), change the film fat composition of Camellia reticulata plumular axis in ultralow temperature preserving process, thus the survival rate that improve after the preservation of plumular axis ultralow temperature and regeneration rate.
The plumular axis that osculant seed Camellia reticulata (Camillia reticulate) seed is chosen in this experiment carries out pretreatment and ultralow temperature preservation experiment, and carries out lipid attempt detection analysis.After the seed of Camellia reticulata is removed the peel, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards.Under aseptic technique, taken out by the plumular axis of seed, every 10 plumular axis are a repetition, and each process 5 repetition is put into the treatment fluid of 1ml water, 100 μMs of DMSO and 100 μM NAE respectively, is placed in 25 DEG C of dark treatment 3 hours.Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours.Every 10 plumular axis are put into 1ml Cryopreservation pipe, puts into liquid nitrogen fast and carry out preservation 1 week.After 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, carry out lipid extraction afterwards and detect and analyze.Every 10 plumular axis are added in the isopropyl alcohol of 3ml preheating, in 75 DEG C of water-baths, fixes 15min.After room temperature cooling, add in the extract of chloroform and methyl alcohol and extract 3 days, merge supernatant, dry up with nitrogen.At 105 DEG C of drying in oven seeds, and weighing obtains dry weight.The fat proposed is addressed to lipid inspection center of the U.S. and carries out detection analysis.Result shows; at the sample of N-acyl ethanol amine process; wherein significantly can raise as the substrate phosphatide (PG, PE, PC) of the PLD α enzyme effect of being hydrolyzed; show thus; the sample of N-acyl ethanol amine process is the activity by reducing PLD α; change the composition of film fat, thus improve the survival rate of plumular axis after ultralow temperature preservation (Fig. 2).
N-acyl ethanol amine (N-acylethanolamines, NAE) by suppressing the activity of PLD α enzyme, the substrate phosphatide (PG, PE, PC) of its effect of being hydrolyzed significantly is raised, change the composition of film fat, thus the relevant physiological biochemical test that improve the survival rate of plumular axis after ultralow temperature preservation and regeneration rate proves, this regulatory pathway is in ultralow temperature preserving process, play an important role, the present invention understands the membrane damage of this approach in ultralow temperature preservation have substantial value to deep; In addition the present invention also provides new thinking and technology for ultralow temperature preservation desiccation sensitive type seed, has important economic implications and application prospect.Finally, it is also to be noted that what enumerate above is only wherein several specific embodiment of the present invention.Obviously, the invention is not restricted to above embodiment, many improvement can also be had.The improvement that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (10)
1. by the method for survival rate after the preservation of N-acyl ethanol amine raising plant seed ultralow temperature; it is characterized in that getting embryo of a plant seed axle; N-acyl ethanol amine is put on plant seed plumular axis, carries out pretreatment and ultralow temperature preservation experiment, and carry out germination experiment detection vigor.
2. the method by survival rate after the preservation of N-acyl ethanol amine raising seed ultralow temperature as claimed in claim 1, plant seed plumular axis is it is characterized in that to strip out, aseptically add N-acyl ethanol amine, 25 DEG C of dark treatment 3 hours, drying process 4 hours is carried out afterwards with the silica gel of high-temperature sterilization, plumular axis is put into Cryopreservation pipe, insert after preserving 1 week in liquid nitrogen, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, and then sprout under being transferred to light.
3. the method by survival rate after the preservation of N-acyl ethanol amine raising seed ultralow temperature as claimed in claim 1, plant seed plumular axis is it is characterized in that to strip out, aseptically every 10 plumular axis add the PLD alpha specific inhibitor N-acyl ethanol amine of 1ml100 μM, 25 DEG C of dark treatment 3 hours, the silica gel of high-temperature sterilization is used to carry out drying process 4 hours afterwards, every 10 plumular axis are put into 1ml Cryopreservation pipe, preserve in direct insertion liquid nitrogen, after 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, sprout under transferring to light afterwards.
4. the method by survival rate after the preservation of N-acyl ethanol amine raising seed ultralow temperature as claimed in claim 1, after it is characterized in that getting osculant peeling plant seeds, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards, under aseptic technique, the plumular axis of seed is taken out, every 10 plumular axis are a repetition, each process 5 repetition, puts into the treatment fluid of 1ml water, 100 μMs of DMSO and 100 μM NAE respectively, is placed in 25 DEG C of dark treatment 3 hours; Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours, every 10 plumular axis are put into 1ml Cryopreservation pipe, put into liquid nitrogen fast and carry out preservation 1 week, after 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, sprout under transferring to light afterwards, significantly increase through NAE pretreated seed plumular axis survival rate and regeneration rate.
5. the method by survival rate after the preservation of N-acyl ethanol amine raising seed ultralow temperature as claimed in claim 1, after it is characterized in that getting the peeling of plant osculant seed, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards, under aseptic technique, the plumular axis of seed is taken out, every 10 plumular axis are a repetition, each process 5 repetition, puts into the treatment fluid of 1ml water, 100 μMs of DMSO and 100 μM NAE respectively, is placed in 25 DEG C of dark treatment 3 hours; Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours, every 10 plumular axis are put into 1ml Cryopreservation pipe, put into liquid nitrogen fast and carry out preservation after 1 week, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, carry out lipid extraction afterwards and detect and analyze; Every 10 plumular axis are added in the isopropyl alcohol of 3ml preheating, 15min is fixed in 75 DEG C of water-baths, after room temperature cooling, add in the extract of chloroform and methyl alcohol and extract 3 days, merge supernatant, dry up with nitrogen, at 105 DEG C of drying in oven seeds, and weighing obtains dry weight, the fat proposed carries out lipid and detects analysis.
The application of 6.N-acyl ethanol amine in the preservation of plant seed ultralow temperature.
7. apply as claimed in claim 6, plant seed plumular axis is it is characterized in that to strip out, aseptically every 10 plumular axis add the PLD alpha specific inhibitor N-acyl ethanol amine of 1ml100 μM, 25 DEG C of dark treatment 3 hours, the silica gel of high-temperature sterilization is used to carry out drying process 4 hours afterwards, every 10 plumular axis are put into 1ml Cryopreservation pipe, preserve in direct insertion liquid nitrogen, after 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, sprout under transferring to light afterwards.
8. applying as claimed in claim 6, it is characterized in that N-acyl ethanol amine is by reducing PLD alpha active, changes film fat composition, improves the survival rate of plumular axis after ultralow temperature preservation.
9. apply as claimed in claim 6, after it is characterized in that getting osculant peeling plant seeds, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards, under aseptic technique, the plumular axis of seed is taken out, every 10 plumular axis are a repetition, each process 5 repetition, put into respectively 1ml water, the treatment fluid of 100 μMs of DMSO and 100 μM NAE, be placed in 25 DEG C of dark treatment 3 hours; Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours, every 10 plumular axis are put into 1ml Cryopreservation pipe, put into liquid nitrogen fast and carry out preservation 1 week, after 1 week Liquid nitrogen storage, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, the recovery media 25 DEG C afterwards plumular axis being placed in WPM and 6-BA is secretly sprouted 7 days, sprouts under transferring to light afterwards.
10. apply as claimed in claim 6, after it is characterized in that getting the peeling of plant osculant seed, with 75% within alcohol-pickled 1 minute, carry out surface sterilization, clean with clear water afterwards, under aseptic technique, the plumular axis of seed is taken out, every 10 plumular axis are a repetition, each process 5 repetition, put into respectively 1ml water, the treatment fluid of 100 μMs of DMSO and 100 μM NAE, be placed in 25 DEG C of dark treatment 3 hours; Plumular axis is taken out, the silica gel being placed on high-temperature sterilization carries out drying 4 hours, every 10 plumular axis are put into 1ml Cryopreservation pipe, put into liquid nitrogen fast and carry out preservation after 1 week, cryopreservation tube is taken out rapidly from liquid nitrogen the water-bath 2 minutes putting into 40 DEG C of preheating, carry out lipid extraction afterwards and detect and analyze; Every 10 plumular axis are added in the isopropyl alcohol of 3ml preheating, 15min is fixed in 75 DEG C of water-baths, after room temperature cooling, add in the extract of chloroform and methyl alcohol and extract 3 days, merge supernatant, dry up with nitrogen, at 105 DEG C of drying in oven seeds, and weighing obtains dry weight, the fat proposed carries out lipid and detects analysis.
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| CN107484461A (en) * | 2017-09-12 | 2017-12-19 | 中国科学院华南植物园 | A kind of method that the pocket orchid seed germination rate for improving cryopreservation is handled using sodium hypochlorite |
| CN112514751A (en) * | 2020-11-30 | 2021-03-19 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Long-term storage and recovery method of cassava crossbreeding capsule |
| CN112514751B (en) * | 2020-11-30 | 2022-06-17 | 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） | Long-term storage and recovery method of cassava crossbreeding capsule |
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