CN101503671B - Method for improving Fraxinus mandshurica somatic embryo development synchronization - Google Patents
Method for improving Fraxinus mandshurica somatic embryo development synchronization Download PDFInfo
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- CN101503671B CN101503671B CN2009100715520A CN200910071552A CN101503671B CN 101503671 B CN101503671 B CN 101503671B CN 2009100715520 A CN2009100715520 A CN 2009100715520A CN 200910071552 A CN200910071552 A CN 200910071552A CN 101503671 B CN101503671 B CN 101503671B
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Abstract
The invention discloses a method for improving the synchronization of the development of the somatic embryos of fraxinus mandshurica, which relates to a method for improving the synchronization of the development of somatic embryos. The method solves the problems of the prior technology for the development of the somatic embryos of the fraxinus mandshurica, such as asynchronous development of the somatic embryos, low survival rate of somatic embryo regeneration plants caused by low somatic embryo development consistency, and high somatic embryo abnormal rate caused by chemical regulation. The method comprises the following steps that: I) explants are inoculated in a somatic embryo inductive culture medium to be cultured to form spherical somatic embryos; II) the spherical somatic embryos are inoculated in a synchronization regulating culture medium to be cultured to form mature somatic embryos; and III) the mature somatic embryos are inoculated in a germination culture medium after being dried to be cultured to form fraxinus mandshurica plantlets, and thus the improvement in the synchronization of the development of the somatic embryos of the fraxinus mandshurica is completed. The method realizes the physical regulation of the synchronous development of the somatic embryos of the fraxinus mandshurica, reduces the somatic embryo abnormal rate to less than 50 percent, and improves the somatic embryo development consistency to more than 80 percent and the survival rate to 64 to 81 percent.
Description
Technical field
The present invention relates to a kind of method that improves somatic embryo development synchronization.
Background technology
Cortex Fraxini mandshuricae (Fraxinus mandshurica Rupr.) belongs to the Oleaceae Fraxinus, is one of important precious hard wealthy seeds in China northeast.Its trunk is perfectly straight, and the age of tree is long, and wood quality is good, and beautiful texture is widely used in aspects such as military project, building, machinery, aviation, vehicle, house fitting, sports equipment manufacturing.The Cortex Fraxini mandshuricae seed has the deep dormancy characteristic, utilizes traditional seminal propagation nursery stock to have long, problem such as the seedling moulding is slow, and the breeding cycle is long of seed treatment time, is difficult to obtain at short notice Cortex Fraxini mandshuricae high quality seedling a large amount of, that have specific good character.Somatic embryo in the plant tissue culture technique is grown the favor that advantages such as technology is big with its breeding quantity, reproduction speed fast, the descendant inheritting proterties is stable are subjected to people, and the Fraxinus mandshurica somatic embryo fetal hair is educated technology provides an important method and means for solving Cortex Fraxini mandshuricae seed problem deficient and that Cortex Fraxini mandshuricae excellent strain germplasm is preserved.
At present, the Fraxinus mandshurica somatic embryo fetal hair is educated and is existed serious somatic embryo to grow asynchronization in the technology, be the somatic embryo development stage inconsistent, the somatic embryo difference in size is very big, the consistence of somatic embryo development poor (less than 7%) causes the survival rate of somatic embryo regeneration plant low (less than 12%); And exist and to utilize chemical regulation, cause the problem that the somatocyte postembryonal development is undesired, somatic embryo is lopsided (up to 75%), can't realize the batch production and the automatic production of Fraxinus mandshurica somatic embryo, can not satisfy the needs of big area afforestation.
Summary of the invention
The objective of the invention is to educate and exist somatic embryo to grow asynchronization in the technology in order to solve existing Fraxinus mandshurica somatic embryo fetal hair, the consistence of somatic embryo development is poor, cause the survival rate of somatic embryo regeneration plant low and utilize chemical regulation, cause the high problem of somatic embryo abnormal rate, and a kind of method that improves Fraxinus mandshurica somatic embryo development synchronization that provides.
The method that improves Fraxinus mandshurica somatic embryo development synchronization realizes according to the following steps: one, the Cortex Fraxini mandshuricae explant is inoculated in the somatic embryo inducement substratum, temperature be 22～30 ℃, humidity be 60%～70% unglazedly obtain the spheroplast somatic embryo according to being cultured under the condition; Two, the spheroplast somatic embryo being inoculated in the synchronously regulating and controlling substratum, is that 22～30 ℃, humidity are 60%～70%, changed the unglazed of fresh synchronously regulating and controlling substratum in per 20～30 days and cultivated 40～60 days according under the condition in temperature, mature somatic embryo; Three, mature somatic embryo is peeled off out from explant be placed on the aseptic empty culture dish, in temperature is that 22～30 ℃, humidity are dry 10～30min under 60%～70% the natural lighting condition, be inoculated in the germination medium then, be in temperature that 22～30 ℃, humidity are 60%～70%, illumination every day 14～18h, intensity of illumination are 1500～2000Lux, cultivated 60～80 days under per condition of changing fresh germination medium in 20～30 days, get the Cortex Fraxini mandshuricae plantlet, promptly finish the raising Fraxinus mandshurica somatic embryo development synchronization; Wherein the somatic embryo inducement substratum is to be minimum medium with the MS1/2 substratum in the step 1, and also comprise the acid hydrolyzed casein of 400～700mg, the sucrose of 40000～70000mg, the naa of 1.0～1.5mg, the 6-benzylaminopurine of 0.1～0.5mg and the agar of 6000～7000mg in every 1L somatic embryo inducement substratum, the pH value is 5.8; The synchronously regulating and controlling substratum is to be minimum medium with the MS1/2 substratum in the step 2, and also comprises the sucrose of 20000～50000mg and the agar of 6000～7000mg in every 1L synchronously regulating and controlling substratum, and the pH value is 5.8; Germination medium is to be minimum medium with the MS1/2 substratum in the step 3, and also comprise the sucrose of 20000～25000mg, the acid hydrolyzed casein of 100～200mg, the naa of 0.01～0.05mg and the agar of 6000～7000mg in every 1L germination medium, the pH value is 5.8.
Cost of the present invention is low, simple to operate, by regulating the sucrose concentration in the synchronously regulating and controlling substratum, can regulate the osmotic pressure of substratum, and then realization is to the physical regulating of Fraxinus mandshurica somatic embryo synchronization growth, the somatocyte postembryonal development of having avoided chemical regulation to bring is undesired, somatic embryo odd-shaped problem, the somatic embryo abnormal rate is reduced to below 50%, and the consistence of somatic embryo development reaches more than 80%; The drying that the present invention adopts can improve the survival rate of Fraxinus mandshurica somatic embryo regeneration plant, survival rate is up to 64%～81%, and its plant resistance obviously strengthens after the sprouting of dried somatic embryo, is transplanted in the soil media, then all can survive more than 90%, and late growing stage is influenced hardly.
Embodiment
Embodiment one: the method that present embodiment improves Fraxinus mandshurica somatic embryo development synchronization realizes according to the following steps: one, the Cortex Fraxini mandshuricae explant is inoculated in the somatic embryo inducement substratum, temperature be 22～30 ℃, humidity be 60%～70% unglazedly obtain the spheroplast somatic embryo according to being cultured under the condition; Two, the spheroplast somatic embryo being inoculated in the synchronously regulating and controlling substratum, is that 22～30 ℃, humidity are 60%～70%, changed the unglazed of fresh synchronously regulating and controlling substratum in per 20～30 days and cultivated 40～60 days according under the condition in temperature, mature somatic embryo; Three, mature somatic embryo is peeled off out from explant be placed on the aseptic empty culture dish, in temperature is that 22～30 ℃, humidity are dry 10～30min under 60%～70% the natural lighting condition, be inoculated in the germination medium then, be in temperature that 22～30 ℃, humidity are 60%～70%, illumination every day 14～18h, intensity of illumination are 1500～2000Lux, cultivated 60～80 days under per condition of changing fresh germination medium in 20～30 days, get the Cortex Fraxini mandshuricae plantlet, promptly finish the raising Fraxinus mandshurica somatic embryo development synchronization; Wherein the somatic embryo inducement substratum is to be minimum medium with the MS1/2 substratum in the step 1, and also comprise the acid hydrolyzed casein of 400～700mg, the sucrose of 40000～70000mg, the naa of 1.0～1.5mg, the 6-benzylaminopurine of 0.1～0.5mg and the agar of 6000～7000mg in every 1L somatic embryo inducement substratum, the pH value is 5.8; The synchronously regulating and controlling substratum is to be minimum medium with the MS1/2 substratum in the step 2, and also comprises the sucrose of 20000～50000mg and the agar of 6000～7000mg in every 1L synchronously regulating and controlling substratum, and the pH value is 5.8; Germination medium is to be minimum medium with the MS1/2 substratum in the step 3, and also comprise the sucrose of 20000～25000mg, the acid hydrolyzed casein of 100～200mg, the naa of 0.01～0.05mg and the agar of 6000～7000mg in every 1L germination medium, the pH value is 5.8.
Be to get the monolithic cotyledon as the Cortex Fraxini mandshuricae explant after the radicle on the Cortex Fraxini mandshuricae embryo and plumular axis are cut in the present embodiment step 1.
The MS1/2 substratum is the MS substratum that all elements content all reduces by half in the present embodiment step 1.
The somatic embryo inducement substratum is through 121 ℃ of sterilization 20min, naturally cooling in the present embodiment step 1.
Pass through to regulate the sucrose concentration in the synchronously regulating and controlling substratum in the present embodiment step 2, can regulate the osmotic pressure of substratum, and then realization is to the physical regulating of Fraxinus mandshurica somatic embryo synchronization growth, the consistence of somatic embryo development reaches more than 80%, and the somatic embryo abnormal rate is reduced to below 50%.
Drying is to carry out on aseptic Bechtop in the present embodiment step 3, and the diameter in the aseptic empty culture dish is 9.5 centimetres, puts 10～20 individual cells embryos in each culture dish.
The dry survival rate that can improve the Fraxinus mandshurica somatic embryo regeneration plant in the present embodiment step 3, survival rate is up to 64%～81%, and dried somatic embryo is sprouted its plant resistance of back and is obviously strengthened, be transplanted in the soil media, then all can survive more than 90%, and late growing stage is influenced hardly.
Embodiment two: present embodiment and embodiment one not to be both in the step 1 in temperature be that 25 ℃, humidity are 65% unglazedly cultivate according under the condition.Other step and parameter are identical with embodiment one.
Embodiment three: present embodiment and embodiment two not to be both in the step 2 in temperature be that 25 ℃, humidity are 65%, changed the unglazed according to cultivating 50 days under the condition of fresh synchronously regulating and controlling substratum in per 25 days.Other step and parameter are identical with embodiment two.
Embodiment four: present embodiment and embodiment three not to be both in the step 3 in temperature be that 25 ℃, humidity are dry 20min under 65% the natural lighting condition.Other step and parameter are identical with embodiment three.
Embodiment five: not being both in the step 3 in temperature of present embodiment and embodiment four is that 25 ℃, humidity are 65%, illumination every day 16h, intensity of illumination are 1800Lux, cultivated 75 days under per condition of changing fresh germination medium in 25 days.Other step and parameter are identical with embodiment four.
Embodiment six: the method that present embodiment improves Fraxinus mandshurica somatic embryo development synchronization realizes according to the following steps: one, the Cortex Fraxini mandshuricae explant is inoculated in the somatic embryo inducement substratum, temperature be 25 ℃, humidity be 70% unglazedly obtain the spheroplast somatic embryo according to being cultured under the condition; Two, the spheroplast somatic embryo is inoculated in the synchronously regulating and controlling substratum, temperature be 25 ℃, humidity be 70%, changed fresh synchronously regulating and controlling substratum in per 30 days unglazed according to cultivating 60 days under the condition, mature somatic embryo; Three, mature somatic embryo is peeled off out from explant be placed on the aseptic empty culture dish, in temperature is that 28 ℃, humidity are dry 10min under 60% the natural lighting condition, be inoculated in the germination medium then, be in temperature that 25 ℃, humidity are 70%, illumination every day 16h, intensity of illumination are 2000Lux, cultivated 70 days under per condition of changing fresh germination medium in 30 days, get the Cortex Fraxini mandshuricae plantlet, promptly finish the raising Fraxinus mandshurica somatic embryo development synchronization; Wherein the somatic embryo inducement substratum is to be minimum medium with the MS1/2 substratum in the step 1, and also comprise the acid hydrolyzed casein of 400～700mg, the sucrose of 40000～70000mg, the naa of 1.0～1.5mg, the 6-benzylaminopurine of 0.1～0.5mg and the agar of 6000～7000mg in every 1L somatic embryo inducement substratum, the pH value is 5.8; The synchronously regulating and controlling substratum is to be minimum medium with the MS1/2 substratum in the step 2, and also comprises the sucrose of 20000～50000mg and the agar of 6000～7000mg in every 1L synchronously regulating and controlling substratum, and the pH value is 5.8; Germination medium is to be minimum medium with the MS1/2 substratum in the step 3, and also comprise the sucrose of 20000～25000mg, the acid hydrolyzed casein of 100～200mg, the naa of 0.01～0.05mg and the agar of 6000～7000mg in every 1L germination medium, the pH value is 5.8.
The consistence of somatic embryo development reaches 81% in the present embodiment, and the somatic embryo abnormal rate is 10%, and the survival rate of Cortex Fraxini mandshuricae plantlet reaches 80.2%, and plantlet is transplanted in the soil media 92% can be survived.
Embodiment seven: the method that present embodiment improves Fraxinus mandshurica somatic embryo development synchronization realizes according to the following steps: one, the Cortex Fraxini mandshuricae explant is inoculated in the somatic embryo inducement substratum, temperature be 25 ℃, humidity be 70% unglazedly obtain the spheroplast somatic embryo according to being cultured under the condition; Two, the spheroplast somatic embryo is inoculated in the synchronously regulating and controlling substratum, temperature be 25 ℃, humidity be 60%, changed fresh synchronously regulating and controlling substratum in per 30 days unglazed according to cultivating 60 days under the condition, mature somatic embryo; Three, mature somatic embryo is peeled off out from explant be placed on the aseptic empty culture dish, in temperature is that 25 ℃, humidity are dry 30min under 70% the natural lighting condition, be inoculated in the germination medium then, be in temperature that 25 ℃, humidity are 60%, illumination every day 16h, intensity of illumination are 2000Lux, cultivated 70 days under per condition of changing fresh germination medium in 20 days, get the Cortex Fraxini mandshuricae plantlet, promptly finish the raising Fraxinus mandshurica somatic embryo development synchronization; Wherein the somatic embryo inducement substratum is to be minimum medium with the MS1/2 substratum in the step 1, and also comprise the acid hydrolyzed casein of 400～700mg, the sucrose of 40000～70000mg, the naa of 1.0～1.5mg, the 6-benzylaminopurine of 0.1～0.5mg and the agar of 6000～7000mg in every 1L somatic embryo inducement substratum, the pH value is 5.8; The synchronously regulating and controlling substratum is to be minimum medium with the MS1/2 substratum in the step 2, and also comprises the sucrose of 20000～50000mg and the agar of 6000～7000mg in every 1L synchronously regulating and controlling substratum, and the pH value is 5.8; Germination medium is to be minimum medium with the MS1/2 substratum in the step 3, and also comprise the sucrose of 20000～25000mg, the acid hydrolyzed casein of 100～200mg, the naa of 0.01～0.05mg and the agar of 6000～7000mg in every 1L germination medium, the pH value is 5.8.
The consistence of somatic embryo development reaches 85% in the present embodiment, and the somatic embryo abnormal rate is 15%, and the survival rate of Cortex Fraxini mandshuricae plantlet reaches 70%, and plantlet is transplanted in the soil media 90% can be survived.
Claims (5)
1. method that improves Fraxinus mandshurica somatic embryo development synchronization, it is characterized in that the method that improves Fraxinus mandshurica somatic embryo development synchronization realizes according to the following steps: one, the Cortex Fraxini mandshuricae explant is inoculated in the somatic embryo inducement substratum, temperature be 22～30 ℃, humidity be 60%～70% unglazedly obtain the spheroplast somatic embryo according to being cultured under the condition; Two, the spheroplast somatic embryo being inoculated in the synchronously regulating and controlling substratum, is that 22～30 ℃, humidity are 60%～70%, changed the unglazed of fresh synchronously regulating and controlling substratum in per 20～30 days and cultivated 40～60 days according under the condition in temperature, mature somatic embryo; Three, mature somatic embryo is peeled off out from explant be placed on the aseptic empty culture dish, in temperature is that 22～30 ℃, humidity are dry 10～30min under 60%～70% the natural lighting condition, be inoculated in the germination medium then, be in temperature that 22～30 ℃, humidity are 60%～70%, illumination every day 14～18h, intensity of illumination are 1500～2000Lux, cultivated 60～80 days under per condition of changing fresh germination medium in 20～30 days, get the Cortex Fraxini mandshuricae plantlet, promptly finish the raising Fraxinus mandshurica somatic embryo development synchronization; Wherein the somatic embryo inducement substratum is to be minimum medium with the MS1/2 substratum in the step 1, and also comprise the acid hydrolyzed casein of 400～700mg, the sucrose of 40000～70000mg, the naa of 1.0～1.5mg, the 6-benzylaminopurine of 0.1～0.5mg and the agar of 6000～7000mg in every 1L somatic embryo inducement substratum, the pH value is 5.8; The synchronously regulating and controlling substratum is to be minimum medium with the MS1/2 substratum in the step 2, and also comprises the sucrose of 20000～50000mg and the agar of 6000～7000mg in every 1L synchronously regulating and controlling substratum, and the pH value is 5.8; Germination medium is to be minimum medium with the MS1/2 substratum in the step 3, and also comprise the sucrose of 20000～25000mg, the acid hydrolyzed casein of 100～200mg, the naa of 0.01～0.05mg and the agar of 6000～7000mg in every 1L germination medium, the pH value is 5.8.
2. a kind of method that improves Fraxinus mandshurica somatic embryo development synchronization according to claim 1 is characterized in that in the step 1 in temperature being that 25 ℃, humidity are 65% unglazedly cultivate according under the condition.
3. a kind of method that improves Fraxinus mandshurica somatic embryo development synchronization according to claim 2 is characterized in that in the step 2 in temperature being that 25 ℃, humidity are 65%, changed the unglazed according to cultivating 50 days under the condition of fresh synchronously regulating and controlling substratum in per 25 days.
4. a kind of method that improves Fraxinus mandshurica somatic embryo development synchronization according to claim 3 is characterized in that in the step 3 in temperature being that 25 ℃, humidity are dry 20min under 65% the natural lighting condition.
5. a kind of method that improves Fraxinus mandshurica somatic embryo development synchronization according to claim 4 is characterized in that in the step 3 being in temperature that 25 ℃, humidity are 65%, illumination every day 16h, intensity of illumination are 1800Lux, cultivated 75 days under per condition of changing fresh germination medium in 25 days.
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| CN102888379A (en) * | 2012-11-06 | 2013-01-23 | 东北林业大学 | Method for establishing fraxinus mandshurica suspension culture system |
| CN102948369A (en) * | 2012-11-28 | 2013-03-06 | 东北林业大学 | Method for improving inductivity of ashtree somatic embryo |
| CN104094840A (en) * | 2013-04-02 | 2014-10-15 | 东北林业大学 | Establishment method for Fraxinus rhynchophylla Hance. suspension culture system |
| CN105941155B (en) * | 2016-05-31 | 2018-04-20 | 东北林业大学 | A kind of method that Manchurian ash is quickly bred using suspension culture techniques |
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