CN104488717A - Method for obtaining multiple-ploidy regeneration plant by culturing flower bud of green Chinese onion - Google Patents

Method for obtaining multiple-ploidy regeneration plant by culturing flower bud of green Chinese onion Download PDF

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CN104488717A
CN104488717A CN201410806368.7A CN201410806368A CN104488717A CN 104488717 A CN104488717 A CN 104488717A CN 201410806368 A CN201410806368 A CN 201410806368A CN 104488717 A CN104488717 A CN 104488717A
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bud
culture
explant
shallot
ploidy
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赵泓
王永勤
王贤
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Beijing Academy of Agriculture and Forestry Sciences
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

The invention belongs to the field of obtaining regeneration plants by using a tissue culture technology and provides a method for obtaining a multiple-ploidy regeneration plant by culturing a flower bud of green Chinese onion. The method comprises the following steps: carrying out adventitious-bud induced culture on the flower bud of the green Chinese onion, which is taken as an explant, and carrying out seedling culture on an adventitious bud to obtain the multiple-ploidy regeneration plant. The adventitious-bud induced culture also comprises the three steps of heat shock, dark culture and light culture. The method provided by the invention has the advantages that the flower bud of the green Chinese onion is adopted as the explant, and the multiple-ploidy regeneration plant can be obtained from the adventitious bud; with a proper adventitious bud induced culture medium, the induction efficiency of the adventitious bud is improved; and the operation is simple, the price of the components of the culture medium is low and the application value is high.

Description

A kind of shallot flower bud culture obtains the method for multiple ploidy regeneration plant
Technical field
The invention belongs to and obtain regeneration plant field with tissue culture technique, particularly a kind of by the bud of shallot is carried out adventitious bud induction culture as explant, then indefinite bud is carried out the method that seedling cultivation obtains seedling plants.
Background technology
Shallot (Allium fistulosum L.) is Liliaceae (Liliaceae) allium, and originating from Siberia, Chinese the north and northeast and the Central Asia, is important vegetables and the flavouring of the East Asian countries such as Chinese Japanese Korea S.Shallot is typical cross pollinated plant, and inbreeding of more generation decline is serious.Usually can only carry out 2-3 for selfing, be difficult to obtain genotype and the on all four inbred line of phenotype.Large allium biennial plant, is bred as an Elite inbred and at least needs 8-10, and the excellent proterties transformation of some complexity may need the longer time.Utilize Doubled haploid breeding technology can acquired character is consistent in the short period of time inbred line, significantly improve efficiency of selection.In addition, the Double-haploid population of acquisition, can be used as the ideal material of the genetic analysis of important character, molecular labeling and quantitative character research.
Tetraploid shallot growing way is obviously better than dliploid shallot, and plant height, green onion shank diameter and green onion leaf all significantly increase, thus directly causes output to increase, increasing peasant income.Although utilize the chemical reagent such as colchicine can obtain tetraploid plant, the toxicity of bypassing colchicine is not said, doubles effect usually unstable during agent treated, and plant is in follow-up Natural growth process, and its ploidy also likely changes.
Report is had no about the cultivation of shallot double haploid and tetraploid induction.The onion belonged to together obtains haplobiont by ovule, Unpollinated ovules, flower bud culture.Keller and Korzun (1996) reviews the trial of not cultivating Haploid production plant by andro gamete.Campion and Alloni, Keller1990 report and utilize Unpollinated ovules to obtain haplobiont.Afterwards, numerous researcher improves condition of culture, and is applied to germ plasm resource improvement.
In current research practice, not yet occur one with shallot bud for explant, cultivation obtains the method for multiple ploidy regeneration plant, cannot set up shallot Doubled haploid breeding technology and the tetraploid shallot inductive technology of stability and high efficiency.So, a kind of maturation of current needs, stable method, shallot bud is carried out tissue cultures and obtains shallot double haploid and tetraploid plant, for setting up stable shallot Doubled haploid breeding technical system and tetraploid induction technical system provides technical support, for shallot is breeding hybridized, molecule marking research, genetic map construction etc. lay the foundation.
Summary of the invention
The invention provides a kind of method that shallot flower bud culture obtains multiple ploidy regeneration plant, the bud of shallot is carried out adventitious bud induction culture as explant by the present invention, then indefinite bud is carried out the regeneration plant that seedling cultivation obtains multiple ploidy.The present invention can set up stable shallot Doubled haploid breeding and tetraploid induction technical system, for shallot is breeding hybridized, molecule marking research, genetic map construction etc. lay the foundation.
The object of the invention is to be achieved through the following technical solutions:
Shallot flower bud culture obtains a method for multiple ploidy regeneration plant, comprises the following steps:
Explant treatment step: the explant choosing shallot material, disinfection, the explant after being sterilized; Adventitious bud inducing step: be placed on inducing culture by the explant after described sterilization, carries out adventitious bud inducing, obtains indefinite bud; Regeneration bud incubation step: described indefinite bud is placed on seedling medium, carries out regeneration bud cultivation, obtain seedling plants.
Further, described adventitious bud inducing step comprises: shock step: be placed in by the explant after described sterilization on inducing culture and carry out Heat thermostability, obtain the explant after heat shock; Light culture step: the explant after described heat shock is placed on inducing culture and carries out light culture process, obtain the explant after light culture; Illumination cultivation step: the explant after described light culture is carried out illumination cultivation process, obtains indefinite bud.
Further, in described shock step, described Heat thermostability is: be that 33 DEG C of lucifuges are cultivated as 3-9 days in temperature; In described light culture step, described light culture is treated to: be that 23-27 DEG C of lucifuge is cultivated as 2-4 week in temperature; In described illumination cultivation step, described illumination cultivation is treated to: illumination condition is 16 hours/day, and temperature is 23-27 DEG C, and the time is more than 2 weeks.
Further, described inducing culture is, based on minimal medium, adds 6-benzyladenine (6-BA) 1.0-5.0mg/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 1.0-5.0mg/L.
Further, described seedling medium is MS minimal medium.
Further, in described inducing culture, the minimal medium of employing is MS minimal medium, and/or B5 minimal medium.
Further, described shallot material is flat bar green onion, Tianjin five leaf is neat, five leaves are neat.
Further, described explant is the bud of the excision anthocaulus before blooming before 1-3 days, and/or the bud of reservation 1-2mm anthocaulus before blooming before 1-3 days.
The present invention compared with the prior art tool has the following advantages:
1, the present invention adopts the bud of shallot as explant, can obtain multiple ploidy regeneration plant by indefinite bud.
2, the present invention adopts suitable adventitious bud induction culture base, and in the process of adventitious bud inducing, carry out Heat thermostability, light culture process, illumination cultivation process, and various factors acts synergistically, and improves the induced efficiency of indefinite bud.
3, the present invention is easy and simple to handle, and medium component is cheap, and using value is high.
4, the tetraploid plant output that the present invention obtains obviously increases, thus increases farmers' income.
Accompanying drawing explanation
Fig. 1: after light culture terminates in embodiment 2, the photo of the explant after the light culture on inducing culture A.
Fig. 2: after light culture terminates in embodiment 4, the photo of the explant after the light culture on inducing culture B.
Fig. 3: flow cytomery result in embodiment 4.Left-half: A is monoploid regeneration plant, and B is contrast; Right half part: A is contrast, and B, C are mixoplod regeneration plant.
Embodiment
Shallot flower bud culture obtains a method for multiple ploidy regeneration plant, comprises the following steps:
Step one, explant process:
Choose the explant of shallot material (farm variety flat bar green onion, or the common kind such as Tianjin five leaf is neat, five Ye Qi), disinfection on superclean bench, the shallot explant after being sterilized.
What above-mentioned explant was chosen the is shallot bud of before blooming 1 ~ 3 day, can be the bud of excision anthocaulus, or retain the bud of 1 ~ 2mm anthocaulus;
Above-mentioned disinfect be operating as: on superclean bench, carry out surface sterilization 30 ~ 60 seconds with 75% alcoholic solution, then sterilize 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis, aseptic water washing 3 ~ 4 times.
Adopt the bud of shallot as explant, multiple ploidy regeneration plant can be obtained by indefinite bud.
Step 2, adventitious bud inducing:
Shallot explant after sterilization is placed on inducing culture, through adventitious bud inducing: shock step, light culture step, illumination cultivation step, obtain indefinite bud;
(1), heat shock: the shallot explant after sterilization is placed on inducing culture (A, B two kinds) and carries out Heat thermostability, obtain the explant after heat shock; This Heat thermostability is: be that 33 DEG C of lucifuges cultivate in temperature be 3 ~ 9 days, is preferably 3 ~ 5 days;
(2), light culture: the explant after heat shock is placed on inducing culture (A, B two kinds) and carries out light culture process, obtain the explant after light culture; This light culture is treated to: be 23 ~ 27 DEG C in temperature, is preferably 25 DEG C, and it is 2 ~ 4 weeks that lucifuge is cultivated;
(3), illumination cultivation: the explant after light culture is carried out illumination cultivation process, obtains indefinite bud; This illumination cultivation is treated to: be 23 ~ 27 DEG C in temperature, is preferably 25 DEG C, carries out illumination cultivation more than 2 weeks under 16hr illumination condition.
Above-mentioned inducing culture A is, based on B5 minimal medium, adds 6-benzyladenine (6-BA) 1.0 ~ 5.0mg/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 1.0 ~ 5.0mg/L;
Above-mentioned inducing culture B is, based on MS minimal medium, adds 6-benzyladenine (6-BA) 1.0 ~ 5.0mg/L, 2,4-dichlorphenoxyacetic acid (2,4-D) 1.0 ~ 5.0mg/L;
Exemplarily, inducing culture A is based on B5 minimal medium, add any or any scope between the two in 6-BA 1mg/L, 1.5mg/L, 2mg/L, 2.5mg/L, 3mg/L, 3.5mg/L, 4mg/L, 4.5mg/L, 5mg/L etc., be preferably 2mg/L, add 2, in 4-D 1mg/L, 1.5mg/L, 2mg/L, 2.5mg/L, 3mg/L, 3.5mg/L, 4mg/L, 4.5mg/L, 5mg/L etc., any or any scope between the two, is preferably 2mg/L; Inducing culture B is based on MS minimal medium, add any or any scope between the two in 6-BA 1mg/L, 1.5mg/L, 2mg/L, 2.5mg/L, 3mg/L, 3.5mg/L, 4mg/L, 4.5mg/L, 5mg/L etc., be preferably 2mg/L, add 2, in 4-D1mg/L, 1.5mg/L, 2mg/L, 2.5mg/L, 3mg/L, 3.5mg/L, 4mg/L, 4.5mg/L, 5mg/L etc., any or any scope between the two, is preferably 2mg/L;
Above-mentioned B5 minimal medium consists of:
Macroelement:
Potassium nitrate KNO 32500mg/L, magnesium sulfate MgSO 47H 2o 250mg/L, calcium chloride CaCl 22H 2o 750mg/L, ammonium sulfate (NH 4) 2sO 4134mg/L, sodium dihydrogen phosphate NaH 2pO 42H 2o 150mg/L;
Trace element:
Potassium iodide KI 0.75mg/L, boric acid H 3bO 33mg/L, manganese sulphate MnSO 4h 2o 5mg/L, zinc sulphate ZnSO 47H 2o 2mg/L, sodium molybdate Na 2moO 42H 2o 0.25mg/L, cobalt chloride CoCl 26H 2o 0.025mg/L, copper sulphate CuSO 45H 2o 0.025mg/L;
Molysite:
Two ethylenediamine hydrate tetraacethyl disodium Na 2-EDTA 37.3mg/L, ferrous sulfate FeSO 44H 2o27.8mg/L;
Organic:
Inositol 100mg/L, nicotinic acid 1mg/L, puridoxine hydrochloride 1mg/L, thiamine 10mg/L;
Carbohydrate: sucrose 100g/L;
Agar: 7g/L;
PH＝5.8。
Above-mentioned MS minimal medium consists of:
Macroelement:
Potassium nitrate KNO 31900mg/L, ammonium nitrate NH 4nO 31650mg/L, magnesium sulfate MgSO 47H 2o 370mg/L, potassium dihydrogen phosphate KH 2pO 4170mg/L, calcium chloride CaCl 22H 2o440mg/L;
Trace element:
Manganese sulphate MnSO 4h 2o 16.9mg/L, zinc sulphate ZnSO 47H 2o 8.6mg/L, boric acid H 3bO 36.2mg/L, potassium iodide KI 0.83mg/L, sodium molybdate Na 2moO 42H 2o 0.25mg/L, copper sulphate CuSO 45H 2o 0.025mg/L, cobalt chloride CoCl 26H 2o 0.025mg/L;
Molysite:
Two ethylenediamine hydrate tetraacethyl disodium Na 2-EDTA 37.3mg/L, ferrous sulfate FeSO 44H 2o27.8mg/L;
Organic: glycine 2.0mg/L, puridoxine hydrochloride 0.5mg/L, thiamine 0.1mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L;
Carbohydrate: sucrose 100g/L;
Agar: 7g/L;
PH＝5.8。
Adopt suitable adventitious bud induction culture base, and in the process of adventitious bud inducing, carry out Heat thermostability, light culture process, illumination cultivation process, improve the induced efficiency of indefinite bud.
Step 3, regeneration bud are cultivated:
Indefinite bud is placed on MS minimal medium (i.e. seedling medium), carries out regeneration bud cultivation, obtain seedling plants.
After at least 2 weeks, when the blade of seedling plants grows to more than 6cm, cut 100 ~ 200mg blade, carry out ploidy analysis with flow cytometer (BD FACSCount); Contrast with the seedling of donor plant, according to the ploidy of the position judgment regeneration plant of peak value, peak allows ± variation of 5%.
Embodiment 1:
The shallot flower bud culture of the present embodiment obtains the method for multiple ploidy regeneration plant, comprises the following steps:
(1), explant process:
Choose the common kinds such as farm variety flat bar green onion or Tianjin five leaf are neat, five Ye Qi, bloom 1-3 days before, the shallot bud of excision anthocaulus is as explant, on superclean bench, surface sterilization is carried out 30 ~ 60 seconds with 75% alcoholic solution, sterilize 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis again, aseptic water washing 3 ~ 4 times, the shallot explant after being sterilized.
(2), adventitious bud inducing:
(2.1), heat shock: the shallot explant after sterilization is placed on inducing culture A (B5+6-BA2mg/L+2,4-D 2mg/L) and carries out Heat thermostability: is that 33 DEG C of lucifuges cultivate 5 days in temperature, obtain the explant after heat shock;
(2.2), light culture: the explant after heat shock is carried out light culture process: be 25 DEG C in temperature, lucifuge is cultivated 4 weeks, obtains the explant after light culture;
(2.3), illumination cultivation: the explant after light culture is carried out illumination cultivation process: be 25 DEG C in temperature, cultivate more than 2 weeks under the illumination condition of 16 hours/day, obtain indefinite bud.
(3), regeneration bud is cultivated:
Indefinite bud is placed on MS minimal medium (i.e. seedling medium), carries out regeneration bud cultivation, obtain seedling plants.
After at least 2 weeks, when the blade of seedling plants grows to more than 6cm, cut 100-200mg blade, carry out ploidy analysis with flow cytometer (BD FACSCount); Contrast with the seedling of donor plant, according to the ploidy of the position judgment regeneration plant of peak value, peak allows ± variation of 5%.
The Ploidy Identification of the vegetative seedling plant of the present embodiment is as follows:
The present embodiment shares about 500, bud, through above-mentioned cultivation, and 6 regeneration plants obtaining with flow cytomery.In these 6 regeneration plants, monoploid 0, dliploid 4, tetraploid 1, mixoplod 1, heteroploid 0.Contrast flow cytomery result, find tetraploid regeneration plant dark green leaf, during plant height 6-10cm, blade is sturdy, and width is about 5mm, without tillering; Dliploid regeneration plant blade yellow green is to peak green; Dliploid and mixoplod regeneration plant very alike, the most 3-5 of tiller number, width of blade is about 1mm, and a small amount of tiller number is extremely many, and blade is very thin.
Embodiment 2:
The shallot flower bud culture of the present embodiment obtains the method for multiple ploidy regeneration plant, comprises the following steps:
(1), explant process:
Choose the common kinds such as farm variety flat bar green onion or Tianjin five leaf are neat, five Ye Qi, bloom 1-3 days before, the shallot bud that retains 1-2mm anthocaulus is as explant, on superclean bench, surface sterilization is carried out 30 ~ 60 seconds with 75% alcoholic solution, sterilize 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis again, aseptic water washing 3 ~ 4 times, the shallot explant after being sterilized.
(2), adventitious bud inducing:
(2.1), heat shock: the shallot explant after sterilization is placed on inducing culture A (B5+6-BA2mg/L+2,4-D 2mg/L) and carries out Heat thermostability: is that 33 DEG C of lucifuges cultivate 5 days in temperature, obtain the explant after heat shock;
(2.2), light culture: the explant after heat shock is carried out light culture process: be 25 DEG C in temperature, lucifuge is cultivated 4 weeks, obtains the explant after light culture;
(2.3), illumination cultivation: the explant after light culture is carried out illumination cultivation process: be 25 DEG C in temperature, cultivate more than 2 weeks under the illumination condition of 16 hours/day, obtain indefinite bud.
(3), regeneration bud is cultivated:
Indefinite bud is placed on MS minimal medium (i.e. seedling medium), carries out regeneration bud cultivation, obtain seedling plants.
After at least 2 weeks, when the blade of seedling plants grows to more than 6cm, cut 100-200mg blade, carry out ploidy analysis with flow cytometer (BD FACSCount); Contrast with the seedling of donor plant, according to the ploidy of the position judgment regeneration plant of peak value, peak allows ± variation of 5%.
The Ploidy Identification of the vegetative seedling plant of the present embodiment is as follows:
The present embodiment shares about 500, bud, through above-mentioned cultivation, and 17 regeneration plants obtaining with flow cytomery.In these 17 regeneration plants, monoploid 1, dliploid 12, tetraploid 1, mixoplod 3, heteroploid 0.Contrast flow cytomery result, find tetraploid regeneration plant dark green leaf, during plant height 6-10cm, blade is sturdy, and width is about 5mm, without tillering; Monoploid, dliploid regeneration plant blade yellow green are to peak green; Monoploid blade is thin and delicate, and during plant height 6-10cm, width of blade, less than 1mm, is tillered extremely many; Dliploid and mixoplod regeneration plant very alike, the most 3-5 of tiller number, width of blade is about 1mm, and a small amount of tiller number is extremely many, and blade is very thin.
Embodiment 3:
The shallot flower bud culture of the present embodiment obtains the method for multiple ploidy regeneration plant, comprises the following steps:
(1), explant process:
Choose the common kinds such as farm variety flat bar green onion or Tianjin five leaf are neat, five Ye Qi, bloom 1-3 days before, the shallot bud of excision anthocaulus is as explant, on superclean bench, surface sterilization is carried out 30 ~ 60 seconds with 75% alcoholic solution, sterilize 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis again, aseptic water washing 3 ~ 4 times, the shallot explant after being sterilized.
(2), adventitious bud inducing:
(2.1), heat shock: the shallot explant after sterilization is placed on inducing culture B (MS+6-BA2mg/L+2,4-D 2mg/L) and carries out Heat thermostability: is that 33 DEG C of lucifuges cultivate 3 days in temperature, obtain the explant after heat shock;
(2.2), light culture: the explant after heat shock is carried out light culture process: be 25 DEG C in temperature, lucifuge is cultivated 4 weeks, obtains the explant after light culture;
(2.3), illumination cultivation: the explant after light culture is carried out illumination cultivation process: be 25 DEG C in temperature, cultivate more than 2 weeks under the illumination condition of 16 hours/day, obtain indefinite bud.
(3), regeneration bud is cultivated:
Indefinite bud is placed on MS minimal medium (i.e. seedling medium), carries out regeneration bud cultivation, obtain seedling plants.
After at least 2 weeks, when the blade of seedling plants grows to more than 6cm, cut 100-200mg blade, carry out ploidy analysis with flow cytometer (BD FACSCount); Contrast with the seedling of donor plant, according to the ploidy of the position judgment regeneration plant of peak value, peak allows ± variation of 5%.
The Ploidy Identification of the vegetative seedling plant of the present embodiment is as follows:
The present embodiment shares about 500, bud, through above-mentioned cultivation, and 11 regeneration plants obtaining with flow cytomery.In these 11 regeneration plants, monoploid 0, dliploid 4, tetraploid 2, mixoplod 3, heteroploid 2.Contrast flow cytomery result, find tetraploid regeneration plant dark green leaf, during plant height 6-10cm, blade is sturdy, and width is about 5mm, without tillering; Dliploid regeneration plant blade yellow green is to peak green; Dliploid and mixoplod regeneration plant very alike, the most 3-5 of tiller number, width of blade is about 1mm, and a small amount of tiller number is extremely many, and blade is very thin.
Embodiment 4:
The shallot flower bud culture of the present embodiment obtains the method for multiple ploidy regeneration plant, comprises the following steps:
(1), explant process:
Choose the common kinds such as farm variety flat bar green onion or Tianjin five leaf are neat, five Ye Qi, bloom 1-3 days before, the shallot bud that retains 1-2mm anthocaulus is as explant, on superclean bench, surface sterilization is carried out 30 ~ 60 seconds with 75% alcoholic solution, sterilize 12 ~ 15 minutes with 2.5% liquor natrii hypochloritis again, aseptic water washing 3 ~ 4 times, the shallot explant after being sterilized.
(2), adventitious bud inducing:
(2.1), heat shock: the shallot explant after sterilization is placed on inducing culture B (MS+6-BA2mg/L+2,4-D 2mg/L) and carries out Heat thermostability: is that 33 DEG C of lucifuges cultivate 7 days in temperature, obtain the explant after heat shock;
(2.2), light culture: the explant after heat shock is carried out light culture process: be 25 DEG C in temperature, lucifuge is cultivated 4 weeks, obtains the explant after light culture;
(2.3), illumination cultivation: the explant after light culture is carried out illumination cultivation process: be 25 DEG C in temperature, cultivate more than 2 weeks under the illumination condition of 16 hours/day, obtain indefinite bud.
(3), regeneration bud is cultivated:
Indefinite bud is placed on MS minimal medium (i.e. seedling medium), carries out regeneration bud cultivation, obtain seedling plants.
After at least 2 weeks, when the blade of seedling plants grows to more than 6cm, cut 100-200mg blade, carry out ploidy analysis with flow cytometer (BD FACSCount); Contrast with the seedling of donor plant, according to the ploidy of the position judgment regeneration plant of peak value, peak allows ± variation of 5%.
The Ploidy Identification of the vegetative seedling plant of the present embodiment is as follows:
The present embodiment shares about 500, bud, through above-mentioned cultivation, and 42 regeneration plants obtaining with flow cytomery.In these 42 regeneration plants, monoploid 2, dliploid 23, tetraploid 5, mixoplod 9, heteroploid 3.
Fig. 3 is flow cytomery result in the present embodiment.In the left-half of Fig. 3, A is monoploid regeneration plant, and B is contrast; In the left-half of Fig. 3, A is contrast, and B, C are mixoplod regeneration plant.
Contrast flow cytomery result, find tetraploid regeneration plant dark green leaf, during plant height 6-10cm, blade is sturdy, and width is about 5mm, without tillering; Monoploid, dliploid regeneration plant blade yellow green are to peak green; Monoploid blade is thin and delicate, and during plant height 6-10cm, width of blade, less than 1mm, is tillered extremely many; Dliploid and mixoplod regeneration plant very alike, the most 3-5 of tiller number, width of blade is about 1mm, and a small amount of tiller number is extremely many, and blade is very thin.
Embodiment 1-4 is discussed below:
1, in haploid breeding, genotype is one of important restraining factors, no matter is what plant, and what is originated (egagametophyte or male gametophyte) (Dunwell 2010, Chen 2011).No matter which kind of genotype, shallot must just can the dedifferentiation of active cell and differentiation function again through the Heat thermostability of certain hour and light culture.
2, derive from petal, generation that anther wall, the dliploid somatic cell of joining the tissue such as capsule also have callus, embryoid or indefinite bud on the inducing culture be applicable to.In order to avoid these to disturb as far as possible, should the tissue of removing body cell derived or organ to greatest extent.
3, in embodiment 1-4, the bud be placed on A, B two kinds of medium has differentiation adventitious buds.A, B two kinds of medium have different effects to cell induction.In embodiment 2, in 2 culture dishes, on the inducing culture of B5+ hormone, callus is only had to produce less (Fig. 1); And in embodiment 4, in 2 culture dishes, on the inducing culture of MS+ hormone, explant is easy to grow callus (Fig. 2), unstable by Calli Differentiation plant ploidy out, type is more various.
4, no matter use the explant of which kind of form, no matter adopt any medium, the temperature that the megaspore breeding of onion is cultivated is all similar, namely 25 ± 2 DEG C (Puddephat 1999, Liu Bing river 2012).At this temperature, no matter adopt which kind of medium, shallot bud does not all have callus, embryoid or indefinite bud to occur.With reference to other research (Liu Gongshe 1995, Liu's bolt peach 2008) in this field, we have carried out the high-temperature cultivation of certain hour to the bud after sterilization, and result obtains regeneration plant.This shows that the Heat thermostability of certain hour is cultivated the megaspore of shallot is required, and series of experiments also shows simultaneously, two weeks (more than) light culture also most important.Point out in the research that Han (1997) preserves for a long time at Study In Asian lily haploidy callus, 25 DEG C of dark culturing are optimal conditions.
5, flow cytometer is utilized can to carry out ploidy analysis (Chen Bin 2007) fast and accurately to plant.Compared with the microspores culture of other plant, it is larger that shallot megaspore cultivates the regeneration plant varied in ploidy obtained.Analyzing this is because the regeneration plant of about 2/3rds comes from callus, therefore chromosome number instability (Xu Qi river 2001).The regeneration plant ploidy come from the inducing culture of B5+ hormone is stablized, but is derived from petal inside base and middle and lower part due to it, is therefore necessary to carry out molecular markers for identification to liploid plant, to judge its chromosomal homogeneity.Also to the concrete position that indefinite bud occurs be determined simultaneously, with its source clear and definite, the double haploid culture technique of Improvement and perfection shallot.
Bibliography of the present invention is as follows:
1、J.Keller1990Culture of unpollinated ovules,ovaries,and flower buds insome species of the genus Allium and haploid induction via gynogenesis in onion(Allium cepa L.)Euphytica 47:241-247
2、B.Campionand C.Alloni.1990Induction of haploid plants in onion(Allium cepa L.)by in vitro culture of unpollinated ovules.Plant Cell,Tissue andOrgan Culture 20:1-6
3、I.J.Puddephat,H.T.Robinson,B.M.Smith and J.Lynn 1999Influence ofstock plant pretreatment on gynogenic embryo induction from flower buds ofonion.Plant Cell,Tissue and Organ Culture 57:145-148
4, Liu Shuantao, Zhao Zhizhong, Sun little Lei, Lu Jin east 2008 custard squash Unfertilized Ovules in-vitro inducings plant the key factor of regeneration.North China agronomy report 23 (2): 96-100,
5, Liu Gongshe, Li Yan, Liu Fan, Cao Ming celebrate 1995 high temperature affects Chinese cabbage microspores culture.Botany Gazette 37:140-146
6, Chen Bin, Zhao Hong, Geng San economize, Zhang Baoxi, Zhang Yueyun, Liu Fan 2007 diversity of forming of pepper anther culture regeneration strain colony ploidy.North China agronomy report, 22 (1): 123-158
7, Xu Qijiang, Chen Dian, Li Guiying 2001 tillered-onion tissue cultures chromosome number changes.Northeast Agricultural University's journal, 32 (4): 313-319,
8、J.M.Dunwell,2010,Haploids in flowering plants:origins andexploitation.Plant Biotechnology Journal,8:377–424.
9、J.F.Chen,L.C.,A.A.Malik and K.G.Mbira.2011In vitro haploid anddihaploid production via unfertilized ovule culture.Plant Cell,Tissue and OrganCulture.104:311-319
10、D.Sh.Han,Y.Niimi,M.Nakano.Long term maintenance of anther-drivedhaploid callus line of the Asiatic hybrid“Connecticut King’2000.Plant Cell,Tissue and Organ Culture,06-2000,61(3):215-219
11、E.R.J.Keller,L.Korzun.Ovary and ovule culture for haploid production.IN:S.M.Jain,S.K.Sopory,R.E.Veilleux(eds)In vitro Haploid Production inHigher Plants,Vol 1.Kluwer,Dordrecht,1996,pp145-176.

Claims (9)

1. shallot flower bud culture obtains a method for multiple ploidy regeneration plant, it is characterized in that: comprise the following steps:
Explant treatment step:
Choose the explant of shallot material, disinfection, the explant after being sterilized;
Adventitious bud inducing step:
Explant after described sterilization is placed on inducing culture, carries out adventitious bud inducing, obtain indefinite bud;
Regeneration bud incubation step:
Described indefinite bud is placed on seedling medium, carries out regeneration bud cultivation, obtain seedling plants.
2. shallot flower bud culture obtains the method for multiple ploidy regeneration plant according to claim 1, it is characterized in that:
Described adventitious bud inducing step comprises:
Shock step: the explant after described sterilization is placed on inducing culture and carries out Heat thermostability, obtain the explant after heat shock;
Light culture step: the explant after described heat shock is placed on inducing culture and carries out light culture process, obtain the explant after light culture;
Illumination cultivation step: the explant after described light culture is carried out illumination cultivation process, obtains indefinite bud.
3. shallot flower bud culture obtains the method for multiple ploidy regeneration plant according to claim 2, it is characterized in that:
In described shock step, described Heat thermostability is: be that 33 DEG C of lucifuges cultivate in temperature be 3 ~ 9 days;
In described light culture step, described light culture is treated to: be that 23 ~ 27 DEG C of lucifuges cultivate in temperature be 2 ~ 4 weeks;
In described illumination cultivation step, described illumination cultivation is treated to: illumination condition is illumination 16 hours/day, and temperature is 23 ~ 27 DEG C, and the time is more than 2 weeks.
4. according to claim 1 or 2, shallot flower bud culture obtains the method for multiple ploidy regeneration plant, it is characterized in that:
Described inducing culture is, based on minimal medium, adds 6-benzyladenine 1.0 ~ 5.0mg/L, 2,4-dichlorphenoxyacetic acid, 1.0 ~ 5.0mg/L; Preferably, 6-benzyladenine 2.0mg/L is added, 2,4-dichlorphenoxyacetic acid 2.0mg/L.
5. according to claim 1 or 2, shallot flower bud culture obtains the method for multiple ploidy regeneration plant, it is characterized in that: described seedling medium is MS minimal medium.
6. shallot flower bud culture obtains the method for multiple ploidy regeneration plant according to claim 4, it is characterized in that:
In described inducing culture, described minimal medium is MS minimal medium, and/or B5 minimal medium.
7. shallot flower bud culture obtains the method for multiple ploidy regeneration plant according to claim 1, it is characterized in that: described shallot material is flat bar green onion, Tianjin five leaf is neat, five leaves are neat.
8. shallot flower bud culture obtains the method for multiple ploidy regeneration plant according to claim 1, it is characterized in that: described explant is 1 ~ 3 day bud before blooming.
9. according to claim 1 or 8, shallot flower bud culture obtains the method for multiple ploidy regeneration plant, it is characterized in that: described explant is the bud of excision anthocaulus, and/or retains the bud of 1 ~ 2mm anthocaulus.
CN201410806368.7A 2014-12-22 2014-12-22 Method for obtaining multiple-ploidy regeneration plant by culturing flower bud of green Chinese onion Pending CN104488717A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105660109A (en) * 2016-01-15 2016-06-15 沃华农业科技（江苏）股份有限公司 Factory-like sowing method for green Chinese onions
CN111657147A (en) * 2020-06-30 2020-09-15 富阿丽 Differentiation culture medium and differentiation culture method for culture breeding of green Chinese onion flowers
CN111657146A (en) * 2020-06-30 2020-09-15 富阿丽 Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101960991A (en) * 2010-11-15 2011-02-02 南京农业大学 Onion callus induction method and special culture medium thereof
CN102939900A (en) * 2012-11-22 2013-02-27 北京市农林科学院 Method for obtaining (diploid) haploid plant through induction of OT type lily anther and culture medium thereof
CN103782910A (en) * 2014-02-13 2014-05-14 中国农业科学院蔬菜花卉研究所 Isolated culture method of unfertilized ovary of lilium

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101960991A (en) * 2010-11-15 2011-02-02 南京农业大学 Onion callus induction method and special culture medium thereof
CN102939900A (en) * 2012-11-22 2013-02-27 北京市农林科学院 Method for obtaining (diploid) haploid plant through induction of OT type lily anther and culture medium thereof
CN103782910A (en) * 2014-02-13 2014-05-14 中国农业科学院蔬菜花卉研究所 Isolated culture method of unfertilized ovary of lilium

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
刘颖颖等: "不同因素对大蒜未受精子房愈伤组织诱导的影响", 《山东农业科学》 *
张松等: "大葱（Allium fistulosum L.）花蕾培养再生植株的研究", 《山东农业大学学报》 *
张松等: "葱属植物组织培养研究进展", 《山东农业大学学报》 *
李江等: "大通鸡腿葱未受精子房愈伤组织诱导研究", 《青海大学学报（自然科学版）》 *
陈佩度: "《作物育种生物技术》", 31 May 2001, 中国农业出版社 *
黄晓梅等: "组织培养中大葱染色体倍性变异研究", 《中国农学通报》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105660109A (en) * 2016-01-15 2016-06-15 沃华农业科技（江苏）股份有限公司 Factory-like sowing method for green Chinese onions
CN105660109B (en) * 2016-01-15 2018-06-19 沃华农业科技（江苏）股份有限公司 A kind of type of seeding of shallot batch production
CN111657147A (en) * 2020-06-30 2020-09-15 富阿丽 Differentiation culture medium and differentiation culture method for culture breeding of green Chinese onion flowers
CN111657146A (en) * 2020-06-30 2020-09-15 富阿丽 Induction culture medium and induction culture method for culture breeding of green Chinese onion flowers

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