CN102106259A - Method for improving salt endurance of cereal crops - Google Patents
Method for improving salt endurance of cereal crops Download PDFInfo
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- CN102106259A CN102106259A CN2009102006105A CN200910200610A CN102106259A CN 102106259 A CN102106259 A CN 102106259A CN 2009102006105 A CN2009102006105 A CN 2009102006105A CN 200910200610 A CN200910200610 A CN 200910200610A CN 102106259 A CN102106259 A CN 102106259A
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Abstract
The invention provides a method for improving salt endurance of cereal crops. The method comprises the steps as follows: NaCl is respectively added during the process of differentiation culture of dissociated sporule, embryoid induction and regeneration plants for stress screening; in vitro treatment is performed to sporules of a crop with NaCl; NaCl is respectively added during the induction culture period and the plant differentiation period for stress screening; through layer by layer selection, rooted regeneration plants are obtained, then transplanted into a greenhouse, and are reaped plant by plant after ripening; and reaped seeds are placed on a stress stock culture containing NaCl to germinate, and doubled haplont strains with strong salt endurance are screened out. The implementation method provided by the invention is simple and convenient and is easy to master; and the whole screening process is carried out under complete, uniform and controllable conditions in laboratories, so that the result is real and reliable.
Description
Technical field
The invention belongs to the plant genetics and breeding technical field, relate to the improvement method of seed as the salt-tolerance character of the cereal crop of modes of reproduction.
Background technology
In the cereal crop of seed as modes of reproduction, wheat and paddy rice are the staple food crops of China, and barley is that China makes the primary raw material of beer and the Tibetan area people's staple food crop.Along with developing rapidly of national economy and society, the contradiction that reduce in population growth and arable land becomes increasingly conspicuous.China has the soil that large stretch of salt content does not wait, and the cereal crop of cultivating the salt resistance raising can increase the arable area of China, improves total grain output.
With the cereal crop of seed as modes of reproduction, for example the salt resistance of wheat and paddy rice is relatively poor, and the salt resistance of barley is general, is badly in need of the method by genetic improvement, cultivates the new varieties that salt resistance improves.Usually adopt the mutagenesis means mostly, the mutative material that the screening salt resistance improves in its offspring; Adopt cross breeding method, the proterties reorganization elite plant strain that the screening salt resistance improves in its offspring.The report that obtains the strain system of salt resistance raising by anther culture method is also arranged.
Microspores culture is haploid cell cultivation truly, more more responsive to the reaction that NaCl coerces than flower pesticide, and each carry the cell of salt resistant gene suffered coerce more quite consistently, microspore coerces through NaCl that to cultivate the regeneration plant of surviving the back all be monoploid or doubled haploid plant.Easier acquisition salt resistance improves after giving NaCl continuously to coerce processing in microspore inducing culture, differentiation culture and three processes of culture of rootage variant or recombinant material.
This area still needs to obtain can plant in a large number on China's beach salty soil with the germplasm materials of seed as the cereal crop salt-tolerance character of modes of reproduction, effectively utilizes limited resources.
Summary of the invention
The present invention is directed to the deficiency that lacks the salt tolerant kind in the existing germplasm materials of cereal class, a kind of acquisition of new cereal class salt-resistance strain and the method for vitro Screening are provided, and screening conditions are easy to unified control, be not subjected to the restriction of extraneous natural conditions.
Therefore, first aspect present invention relates to a kind of method that improves the cereal crop salt-tolerance character, and this method comprises:
1) gets this crop flower pesticide Isolated microspore in booting stage, place the pretreatment fluid that is added with NaCl 50～500mg/L, obtain the microspore of survival, salt tolerant;
2) microspore of collection step 1) acquisition places and adds NaCl 50～500mg/L, 2,4,5-T 0.5～2.0mg/L, KT 0.3～1.0mg/L, maltose 60～120g/L, the N of pH 5.6～6.0
6Cultivate on the inducing culture, obtain embryoid;
3) with step 2) the gained embryoid places and adds NaCl 50～500mg/L, 6-BA 0.5～1.5mg/L, KT 0.5～2.0mg/L, NAA 0.01～0.1mg/L and maltose 10～50g/L, pH cultivates on 5.6～6.0 the 2/3MS differential medium, obtains regeneration plant;
4) regeneration plant that step 3) is obtained places and adds NAA 0.02～0.08mg/L, paclobutrazol 2.0～10.0mg/L and sucrose 10～40g/L, cultivates on the 1/2MS strong plantlets and rootage medium of pH5.6～6.0, obtains root, stem, regeneration plant that leaf is complete;
Thereby obtain the cereal crop that salt resistance improves.
In a specific embodiment, described method also comprises:
5) regeneration plant with the step 4) gained moves into soil, treats ripe back results seed; With
6) seed is placed on the medium that adds NaCl 50～500mg/L and germinate, select the strong material of NaCl patience.
In a preferred embodiment, described cereal crop is barley, wheat and paddy rice.
In a preferred embodiment, described N
6Inducing culture contains the NaCl of 200～400mg/L, 2,4 of 0.8～1.5mg/L, and 5-T, the KT of 0.4～0.8mg/L and the maltose of 80～100g/L, pH are 5.6～5.9.
In a preferred embodiment, described 2/3MS differential medium contains the NaCl of 200～400mg/L, the 6-BA of 0.4～1.0mg/L, and the KT of 1.0～1.8mg/L, the NAA of 0.02～0.08mg/L, the maltose of 20～40g/L, pH are 5.7～5.8.
In a preferred embodiment, described 1/2MS strong plantlets and rootage medium contains the NAA of 0.03～0.06mg/L, the MET of 3.0～8.0mg/L, and the sucrose of 20～30g/L, pH are 5.8～5.9.
In a preferred embodiment, this method comprises:
1) gets described crop flower pesticide in booting stage Isolated microspore, place the pretreatment fluid that adds NaCl 50～500mg/L, obtain the microspore of survival, salt tolerant;
2) microspore after the collection and treatment places and adds NaCl 50～500mg/L, 2,4,5-T 0.5～2.0mg/L, KT 0.5～1.0mg/L, maltose 90g/L, the N of pH 5.8
6Cultivate on the inducing culture, obtain embryoid;
3) embryoid places and adds NaCl 50～500mg/L, 6-BA 0.5～1.5mg/L, KT 0.5～2.0mg/L, NAA 0.01～0.1mg/L and maltose 30g/L, and PH cultivates on 5.8 the 2/3MS differential medium, obtains regeneration plant;
4) regeneration plant places and adds NAA 0.05mg/L, paclobutrazol (MET) 5.0mg/L and sucrose 20g/L, cultivates on the 1/2MS strong plantlets and rootage medium of pH5.8, obtains root, stem, regeneration plant that leaf is complete;
5) regeneration plant moves into soil, treats ripe back results seed; With
6) seed is placed on the medium that adds NaCl 50～500mg/L and germinate, select the strong material of NaCl patience.
In a preferred embodiment, described pretreatment fluid contains NaCl 50～500mg/L, mannitol 40～80g/L, CaCl
20.8～1.5g/L and MES 0.6～1.5g/L.
The present invention relates to a kind of cover box that is used to implement method of the present invention on the other hand, and described cover box contains pretreatment fluid as herein described, N
6Inducing culture, 2/3MS differential medium and/or 1/2MS strong plantlets and rootage medium.
In a preferred embodiment, in described cover box:
Described pretreatment fluid contains NaCl 50～500mg/L, mannitol 40～80g/L, CaCl
20.8～1.5g/L and MES 0.6～1.5g/L;
Described N
6Inducing culture contains the NaCl of 200～400mg/L, 2,4 of 0.8～1.5mg/L, and 5-T, the KT of 0.4～0.8mg/L and the maltose of 80～100g/L, pH are 5.6～5.9;
Described 2/3MS differential medium contains the NaCl of 200～400mg/L, the 6-BA of 0.4～1.0mg/L, and the KT of 1.0～1.8mg/L, the NAA of 0.02～0.08mg/L, the maltose of 20～40g/L, pH are 5.7～5.8;
Described 1/2MS strong plantlets and rootage medium contains the NAA of 0.03～0.06mg/L, the MET of 3.0～8.0mg/L, and the sucrose of 20～30g/L, pH are 5.8～5.9.
On the one hand, the application also comprises pretreatment fluid as herein described, N
6Inducing culture, 2/3MS differential medium and 1/2MS strong plantlets and rootage medium.
Embodiment
The application relates to a kind of method that improves the cereal crop salt-tolerance character, and this method comprises:
1) gets this crop flower pesticide Isolated microspore in booting stage, place the pretreatment fluid that adds NaCl 50～500mg/L, obtain the microspore of survival, salt tolerant;
2) microspore after the collection and treatment places and adds NaCl 50～500mg/L, 2,4,5-T 0.5～2.0mg/L, KT 0.3～1.0mg/L, maltose 60～120g/L, the N of pH 5.6～6.0
6Cultivate on the inducing culture, obtain embryoid;
3) embryoid places and adds NaCl 50～500mg/L, 6-BA 0.5～1.5mg/L, KT 0.5～2.0mg/L, NAA 0.01～0.1mg/L and maltose 10～50g/L, and pH cultivates on 5.6～6.0 the 2/3MS differential medium, obtains regeneration plant;
4) regeneration plant places and adds NAA 0.02～0.08mg/L, paclobutrazol 2.0～10.0mg/L and sucrose 10～40g/L, cultivates on the 1/2MS strong plantlets and rootage medium of pH5.6～6.0, obtains root, stem, regeneration plant that leaf is complete;
Can obtain the cereal crop that salt resistance improves thus.
When implementing the application, can choose middle part Xiao Hua microspore development from the land for growing field crops and be in that monokaryon is early stage, the tassel in mid-term, put into refrigerator cold-storage about 12～20 days.During inoculation, with for example saturated about 15～20min of Eusol sterilization tassel, use aseptic water washing then 2～4 times earlier.Each test tube connects about 10～15 tassels, pours NaCl 50～500mg/L, mannitol 40～80g/L, the CaCl of about 15～20ml into
20.8 the extract of～1.5g/L, MES 0.6～1.5g/L, with (for example 3000-4000rpm) rotary-cut of high speed dispersion device hypervelocity, 150 eye mesh screens filter, and (for example 800～100rpm) centrifugal filtrate repeat 2～3 times low speed, collect microspore.
Use NaCl 50～500mg/L, mannitol 40～80g/L, CaCl then
20.8 the extract (pretreatment fluid) of～1.5g/L, MES (2-morpholino b acid) 0.6～1.5g/L in about 25 ℃, dark preliminary treatment microspore 1～3 day.
The preferred concentration range for of NaCl is 200～400mg/L in the extract, more preferably can be about 300mg/L.
The preferred concentration range for of mannitol is 50～70g/L, more preferably about 60g/L.
CaCl
2Preferred concentration range for be 1.0～1.3g/L, more preferably about 1.1g/L.
The preferred concentration range for of MES is 0.8～1.2g/L, more preferably about 1.0～1.2g/L.In a specific embodiment, the concentration of MES is 0.976g/L.
Available inducing culture inducing embryoid body.Inducing culture is with N
6Medium is a minimal medium, is added with NaCl 50～500mg/L, 2,4,5-T 0.5～2.0mg/L, KT 0.3～1.0mg/L, and maltose 60～120g/L, pH 5.6～6.0.
In the inducing culture, the preferred concentration range for of NaCl is 200～400mg/L.In one embodiment, the concentration of NaCl is 300mg/L.
2,4, the preferred concentration range for of 5-T (2,4, the 5-trichlorophenoxyacetic acid) is 0.8～1.5mg/L, more preferably 1.0～1.2mg/L.
The preferred concentration range for of KT (6-chaff aminopurine) is 0.4～0.8mg/L, more preferably 0.5～0.6mg/L.
The preferred concentration range for of maltose is 80～100g/L.In one embodiment, the concentration of maltose is 90g/L.
PH is preferably 5.6～5.9, and more preferably 5.8.
Can will have now before the inducing culture inducing culture washing microspore 1 to 2 time, then with inducing culture with microspore Auto-regulating System of Density of Heavy Medium to 1.0～1.2 * 10
5/ ml gets about 1.5～2ml microspore suspension and is inoculated in culture dish (30 * 15mm), Parafilm seals, and (about 25～28 ℃) were secretly cultivated about 15～25 days under the room temperature.
N
6The prescription of medium as shown in table 1 below (unit: mg/L):
Table 1
N
6Macroelement:
(NH
4)
2SO
4 463
KNO
3 2830
CaCl
2·2H
2O 166
MgSO
4·7H
2O 185
KH
2PO
4 400
N
6Trace element:
H
3BO
3 1.6
KI 0.83
MnSO
4·4H
2O 5.0
ZnSO
4·7H
2O 1.5
N
6Molysite:
Na
2EDTA 37.3
FeSO
4·7H
2O 27.8
N
6Organic element:
Thiamine hydrochloride 1.0
Pyridoxine Hydrochloride 0.5
Glycine 2.0
Nicotinic acid 0.5
Inositol 100.0
N
6Medium can be pressed above-mentioned formulated voluntarily, also can buy finished product to chemical reagents corporations such as Sigma.Then according to the N of formulated this paper described herein
6Inducing culture.It will be appreciated by those skilled in the art that, can make suitable change to the concentration shown in the above-mentioned table 1, for example have about 5% (for example about 3%, about 2%, about 1%, or lower mobility scale) or lower change, prepare the medium that obtains thus and still have required biological function, still can be used to implement the present invention.For example, be example with the inositol, every liter of N
6The inositol (5% amplitude of fluctuation) that can contain about 95～105mg in the medium.The concentration of other composition is so change also.In addition, N
6But the composition in the medium also function of use and the same or analogous composition of character is replaced.
The gained embryoid can be transferred on the differential medium behind the inducing culture.Differential medium is minimal medium with 2/3MS, wherein is added with NaCl 50～500mg/L, 6-BA 0.5～1.5mg/L, KT 0.5～2.0mg/L, NAA 0.01～0.1mg/L and maltose 10～50g/L, and pH is 5.6～6.0.
The NaCl that is added in the differential medium is preferably 200～400mg/L, more preferably 300～400mg/L.
The preferred concentration of 6-BA (6-benzyl aminoadenine) is 0.4～1.0mg/L, more preferably 0.5～0.8mg/L.
The preferred concentration of KT is 1.0～1.8mg/L, more preferably 1.2～1.5mg/L.
Preferred concentration 0.02～0.08mg/L of NAA (methyl), more preferably 0.03～0.06mg/L.In an embodiment, the concentration of NAA is 0.05mg/L.
The concentration of maltose is 20～40g/L, more preferably 20～30g/L.
The pH of differential medium is preferably 5.7～5.8.
The prescription of MS medium as shown in table 2 below (unit: mg/L):
Table 2
The MS macroelement:
NH
4NO
4 1650
KNO
3 1900
CaCl
2·2H
2O 440
MgSO
4·7H
2O 370
KH
2PO
4 170
The MS trace element:
H
3BO
3 36.2
KI 0.83
MnSO
4·4H
2O 22.3
ZnSO
4·7H
2O 8.6
Na
2MoO
4·2H
2O 0.25
CuSO
4·5H
2O 0.025
CoCl
2·6H
2O 0.025
The MS molysite:
Na
2EDTA 37.3
FeSO
4·7H
2O 27.8
The MS organic element:
Thiamine hydrochloride 0.1
Pyridoxine Hydrochloride 0.5
Nicotinic acid 0.5
Glycine 2.0
Inositol 100.0
MS can press above-mentioned formulated voluntarily, also can buy finished product to chemical reagents corporations such as Sigma.The 2/3MS medium then is the macroelement minimizing 1/3 with the MS medium, and trace element, molysite and organic element are constant.Then according to the 2/3MS inducing culture of formulated this paper described herein.It will be appreciated by those skilled in the art that, can make suitable change to the concentration shown in the above-mentioned table 1, for example have about 5% (for example about 3%, about 2%, about 1%, or lower mobility scale) or lower change, prepare the medium that obtains thus and still have required biological function, still can be used to implement the present invention.For example, be example with the inositol, can contain the inositol (5% amplitude of fluctuation) of about 95～105mg in every liter of 2/3MS medium.The concentration of other composition is so change also.In addition, but the composition in the 2/3MS inducing culture also replaced by function of use and the same or analogous composition of character.
In one embodiment, use and to have added NaCl 300mg/L, 6-BA 0.5mg/L, KT 1.5mg/L, NAA 0.05mg/L and maltose 30g/L, PH is that 5.8 2/3MS differential medium is cultivated embryoid.
Can be at 25 ± 1 ℃, differentiation is coerced and was cultivated 25～30 days under the condition of illumination in 10～12 hours every day, until differentiating the green regenerating plant.
The regeneration plant that differentiates can change NAA 0.02～0.08mg/L, paclobutrazol (MET) 2.0～10.0mg/L and sucrose 10～40g/L over to, the 1/2MS strong plantlets and rootage medium of pH5.6～6.0.At 25 ± 1 ℃, the strong plantlets and rootage that carried out under the condition of illumination in 10～12 hours every day 25～30 days is cultivated.
In the strong plantlets and rootage medium, the preferred concentration range for of NAA is 0.03～0.06mg/L, more preferably 0.04～0.05mg/L.
The preferred concentration range for of MET is 3.0～8.0mg/L, more preferably 4.0～6.0mg/L.
In one embodiment, the concentration of MET is 5.0mg/L.
The preferred concentration range for of sucrose is 20～30g/L.In one embodiment, concentration of sucrose is 20g/L.
The pH of strong plantlets and rootage medium is preferably 5.8～5.9.
1/2MS strong plantlets and rootage medium is that the macroelement with the MS medium shown in the table 2 reduces by half, and adds behind the described adding ingredient of the application and preparation obtains.
In one embodiment, the strong plantlets and rootage medium is to add NAA 0.05mg/L, paclobutrazol (MET) 5.0mg/L and sucrose 20g/L, the 1/2MS strong plantlets and rootage medium of pH5.8.
The regeneration plant that obtains through the strong plantlets and rootage medium culture is salt-tolerant plants.
In the application's embodiment, the application's method also can comprise:
5) will move into soil through the regeneration plant that strong plantlets and rootage is cultivated, treat ripe back results seed; With
6) seed is placed on the medium that adds NaCl 50～500mg/L and germinate, select the strong material of NaCl patience.
The salt-tolerant plants of cultivating through strong plantlets and rootage becomes the complete whole plant of root, stem and leaf, transplants and blake bottle is opened hardening in the laboratory earlier in preceding 1 day.Hardening shifts out plant carefully with tweezers after finishing from blake bottle, keep the complete of root system as far as possible, and the medium that flush away adheres to moves into seedling in the pot for growing seedlings, and the back of watering covers with plastic film, preserves moisture 2～3 days.Remove behind the plastic film at 20 ± 1 ℃, grow to maturation under the condition of illumination in 10～12 hours every day.Seed after the maturation is by the strain results.
Can select the culture dish of diameter 9cm for use, clean up back sterilization and oven dry, put into two filter paper.Each strain system chooses about 30～40 healthy seeds, with for example 3% hydrogen peroxide (H
2O
2) sterilization 6～10min, distilled water flushing 2～3 times was soaked in distilled water 6～8 hours at about about 30 ℃ with seed then, preserved moisture after the end and spent the night.Evenly be thrown into after showing money or valuables one carries unintentionally in the middle of the culture dish in seed, add the NaCl solution of 5～15g/L, preferable 8～12g/L, compare with distilled water, and place the illumination box of about 27 ℃ of constant temperature illumination in 10～12 hours every day to germinate in all culture dishes.Add up germination rate, main root length, coleoptile length after 5 days.
Can select germination rate height, main root length, the coleoptile length strain that difference is little compared with the control system to plant in the land for growing field crops.
Therefore, the application also comprises the plant of adopting the screening of the application's method to obtain, and other propagating materials etc.
The application also comprises above-mentioned various extracts and the medium that is used to implement the application's method.
The application provides a kind of extract on the other hand, and described extract contains NaCl 50～500mg/L, mannitol 40～80g/L, CaCl
20.8～1.5g/L, MES 0.6～1.5g/L.
The application also comprises a kind of inducing culture, is used to implement the described screening technique of the application.This inducing culture is with N
6Medium is a minimal medium, is added with NaCl 50～500mg/L, 2,4,5-T 0.5～2.0mg/L, KT 0.3～1.0mg/L, and maltose 60～120g/L, pH 5.6～6.0.
In the inducing culture, the preferred concentration range for of NaCl is 200～400mg/L.In one embodiment, the concentration of NaCl is 300mg/L.
2,4, the preferred concentration range for of 5-T is 0.8～1.5mg/L, more preferably 1.0～1.2mg/L.
The preferred concentration range for of KT is 0.4～0.8mg/L, more preferably 0.5～0.6mg/L.
The preferred concentration range for of maltose is 80～100g/L.In one embodiment, the concentration of maltose is 90g/L.
PH is preferably 5.6～5.9, and more preferably 5.8.
The application also comprises the cover box that contains above-mentioned mannitol extract, inducing culture, differential medium and strong plantlets and rootage medium.Each extract can be divided in the different containers with medium.Randomly, this cover box can comprise that also the reagent enforcement of instructing use wherein to be comprised obtains the specification of the method for the stable material of salt-tolerance character.
Among the application, described cereal crop comprises barley, wheat and paddy rice.
Compare with traditional breeding way, the inventive method is easy, is easy to grasp, implement; The acquisition of salt-resistance strain and the screening of salt-tolerance character are all carried out under the on all four condition in laboratory, are not subjected to the influence of external condition; Utilize microspore as starting material, can obtain a large amount of embryoids, in the microspores culture process, carry out the salt-tolerance character vitro Screening, can strict screen, courageously eliminate; Whole process is all carried out in culturing room, has reduced the use in labour and farmland simultaneously again, but and the anniversary repeated test, screening material constantly is provided, both shortened breeding time, can obtain a large amount of germplasm materials again; Adopt the salt tolerant screening of exsomatizing respectively of microspore, embryoid induction and regeneration plant differentiation three phases, and screen in conjunction with the answer in the salt-resistance strain seed germination process, The selection result is genuine and believable.
Applicant's experiment shows, utilizes the present invention can finish cereal class strain system in the time less than half a year and screens, and obtains to obtain seed after the stable material of salt-tolerance character is planted and is used for the detection of further field.The present invention can shorten breeding time greatly.The gained material is through the screening of three steps, isozygotied, fixed through salt-tolerance character after the chromosome doubling, and the offspring that same strain is, performance is unified.Utilize the present invention, in one-year age, vitro Screening obtains 5 parts of salt-resistance strains.
Therefore, the present invention also comprises the embryoid that each step produced that adopts the inventive method to obtain, the last salt-resistance strain that obtains etc. on the other hand.
Hereinafter will be that present invention is described for example with the barley.Should be understood that under the situation that does not depart from spirit and scope of the invention and can make various modifications and changes the present invention.And also can adopt method of the present invention to be used for other cereal crop, and can produce identical effect.Following examples only are illustrative, and scope of the present invention is limited by the application's claim.If no special instructions, the used percentage of the present invention is percent weight in volume.It in addition, should be understood that the preferable range of above-mentioned each reagent can make up arbitrarily, as long as can realize goal of the invention of the present invention.
Embodiment
Embodiment 1: the salt tolerant screening of barley microspore
Choose middle part Xiao Hua microspore development from the land for growing field crops and be in that monokaryon is early stage, the tassel in mid-term, put into refrigerator cold-storage 15 days.During inoculation, tassel is with saturated Eusol sterilization 15min, aseptic water washing 3～4 times.Each test tube connects 10 tassels, pours 15ml NaCl 300mg/L, mannitol 60g/L, CaCl into
21.1g/L, the extract of MES 0.976g/L, with the high speed dispersion device hypervelocity rotary-cut of 3000rpm, 150 eye mesh screens filter, filtrate is repeated 3 times with the 800rpm low-speed centrifugal, the collection microspore.Microspore is used NaCl 300mg/L, mannitol 60g/L, CaCl
21.1g/L, the extract of MES 0.976g/L is in 25 ℃, dark preliminary treatment 2d.
Embodiment 2: the obtaining of salt tolerant embryoid
Inducing culture is with N
6Medium is a minimal medium, wherein is added with NaCl 300mg/L, 2,4,5-T1.0mg/L, KT 0.5mg/L, and maltose 90g/L, pH 5.8.Before cultivating with microspore with medium washing 1 time, then with medium with microspore Auto-regulating System of Density of Heavy Medium to 1.0～1.2 * 10
5/ ml gets the 1.5ml microspore suspension and is inoculated in culture dish (30 * 15mm), Parafilm seals, 25 ℃ of dark 21d that cultivate.
Embodiment 3: the obtaining of salt tolerant regeneration plant
Behind the inducing culture 21d embryoid is transferred on the differential medium.Differential medium is minimal medium with 2/3MS, wherein is added with NaCl 300mg/L, 6-BA 0.5mg/L, KT 1.5mg/L, NAA 0.05mg/L and maltose 30g/L, and PH is 5.8.At 25 ± 1 ℃, differentiation is coerced and was cultivated 25～30 days under the condition of illumination in 10～12 hours every day, until differentiating the green regenerating plant.
Embodiment 4: the culture of rootage of salt tolerant regeneration plant
Choose the regeneration plant that differentiates, change over to and add NAA 0.05mg/L, paclobutrazol (MET) 5.0mg/L and sucrose 20g/L, the 1/2MS strong plantlets and rootage medium of pH5.8.At 25 ± 1 ℃, the strong plantlets and rootage that carried out under the condition of illumination in 10～12 hours every day 25～30 days is cultivated.
Embodiment 5: the transplanting of salt-tolerant plants and seed results
The salt-tolerant plants of cultivating through strong plantlets and rootage becomes the complete whole plant of root, stem and leaf, transplants and blake bottle is opened hardening in the laboratory earlier in preceding 1 day.Hardening shifts out plant carefully with tweezers after finishing from blake bottle, keep the complete of root system as far as possible, and the medium that flush away adheres to moves into seedling in the pot for growing seedlings, and the back of watering covers with plastic film, and 3d preserves moisture.Remove behind the plastic film at 20 ± 1 ℃, grow to maturation under the condition of illumination in 10～12 hours every day.Seed after the maturation is by the strain results.
Embodiment 6: the screening once more of salt-resistance strain
Select the culture dish of diameter 9cm for use, clean up back sterilization and oven dry, put into two filter paper.Each strain system chooses 30 healthy seeds, with 3% hydrogen peroxide (H
2O
2) sterilization 8min, distilled water flushing 3 times, then seed is soaked in distilled water, 30 ℃, 8h, preserve moisture after the end and spend the night, evenly be thrown in the middle of the culture dish after waiting seed to show money or valuables one carries unintentionally, add NaCl 10g/L solution, compare with distilled water, and place the illumination box of 27 ℃ of constant temperature illumination in 10～12 hours every day to germinate in all culture dishes.Add up germination rate, main root length, coleoptile length behind the 5d.Select germination rate height, main root length, the coleoptile length strain that difference is little compared with the control system to plant in the land for growing field crops.
Embodiment 7
Collect microspore and the processing that barley variety " spends 30 " by embodiment 1, carry out the microspores culture inducing embryoid body by embodiment 2, obtain the regeneration plant of salt tolerant by embodiment 3, carrying out strong plantlets and rootage by embodiment 4 cultivates, press embodiment 5 and transplant and gather in the crops seed, seed after the results carries out further salt-resistance strain screening by embodiment 6, in containing NaCl 10g/L germination fluid, the germination rate of a salt-resistance strain selfing generation is 50%～70%, main root length 2.10～3.60cm, coleoptile length 1.79～2.96cm, select 5 strong strain systems of salt resistance to plant, solid back results seed in the land for growing field crops.
30 in the selfing two generations seed of results is carried out the salt tolerant sprouting test as embodiment 6, and with the seed of starting material in contrast, carry out the salt tolerant sprouting simultaneously.
Experimental results show that, in containing NaCl 10g/L germination fluid, at 27 ± 1 ℃, cultivate under the condition of illumination in 10～12 hours every day, after 5 days, 5 parts of salt-resistance strain selfing two generations seed germination rates 60%～70%, main root length 2.59～3.59cm, coleoptile length be at 1.95～3.02cm, 30 germination rate has only 33.3%, main root length 1.70cm, coleoptile length 1.47cm and starting material is spent.This shows that the salt-tolerance character of salt-resistance strain is stable.Concrete outcome sees the following form 3.
Table 3
Should be understood that the foregoing description only is illustrative.Can on the basis of the foregoing description, under the prerequisite that does not depart from the application's scope and spirit, make suitable change and change to the present invention.
Claims (10)
1. method that improves the cereal crop salt-tolerance character, this method comprises:
1) gets this crop flower pesticide Isolated microspore in booting stage, place the pretreatment fluid that is added with NaCl 50～500mg/L, obtain the microspore of survival, salt tolerant;
2) microspore of collection step 1) acquisition places and adds NaCl 50～500mg/L, 2,4,5-T 0.5～2.0mg/L, KT 0.3～1.0mg/L, maltose 60～120g/L, the N of pH 5.6～6.0
6Cultivate on the inducing culture, obtain embryoid;
3) with step 2) the gained embryoid places and adds NaCl 50～500mg/L, 6-BA 0.5～1.5mg/L, KT 0.5～2.0mg/L, NAA 0.01～0.1mg/L and maltose 10～50g/L, pH cultivates on 5.6～6.0 the 2/3MS differential medium, obtains regeneration plant;
4) regeneration plant that step 3) is obtained places and adds NAA 0.02～0.08mg/L, paclobutrazol 2.0～10.0mg/L and sucrose 10～40g/L, cultivates on the 1/2MS strong plantlets and rootage medium of pH5.6～6.0, obtains root, stem, regeneration plant that leaf is complete;
Thereby obtain the cereal crop of salt tolerant.
2. the method for claim 1 is characterized in that, described method also comprises:
5) regeneration plant with the step 4) gained moves into soil, treats ripe back results seed; With
6) seed is placed on the medium that adds NaCl 50～500mg/L and germinate, select the strong material of NaCl patience.
3. as each described method among the claim 1-2, it is characterized in that described cereal crop is barley, wheat and paddy rice.
4. the method for claim 1 is characterized in that, described N
6Inducing culture contains the NaCl of 200～400mg/L, 2,4 of 0.8～1.5mg/L, and 5-T, the KT of 0.4～0.8mg/L and the maltose of 80～100g/L, pH are 5.6～5.9.
5. the method for claim 1 is characterized in that, described 2/3MS differential medium contains the NaCl of 200～400mg/L, 0.4 the 6-BA of～1.0mg/L, the KT of 1.0～1.8mg/L, the NAA of 0.02～0.08mg/L, the maltose of 20～40g/L, pH are 5.7～5.8.
6. the method for claim 1 is characterized in that, described 1/2MS strong plantlets and rootage medium contains the NAA of 0.03～0.06mg/L, the MET of 3.0～8.0mg/L, and the sucrose of 20～30g/L, pH are 5.8～5.9.
7. the method for claim 1 is characterized in that, this method comprises:
1) gets described crop flower pesticide in booting stage Isolated microspore, place the pretreatment fluid that adds NaCl 50～500mg/L, obtain the microspore of survival, salt tolerant;
2) microspore after the collection and treatment places and adds NaCl 50～500mg/L, 2,4,5-T 0.5～2.0mg/L, KT 0.5～1.0mg/L, maltose 90g/L, the N of pH 5.8
6Cultivate on the inducing culture, obtain embryoid;
3) embryoid places and adds NaCl 50～500mg/L, 6-BA 0.5～1.5mg/L, KT 0.5～2.0mg/L, NAA 0.01～0.1mg/L and maltose 30g/L, and PH cultivates on 5.8 the 2/3MS differential medium, obtains regeneration plant;
4) regeneration plant places and adds NAA 0.05mg/L, paclobutrazol (MET) 5.0mg/L and sucrose 20g/L, cultivates on the 1/2MS strong plantlets and rootage medium of pH5.8, obtains root, stem, regeneration plant that leaf is complete;
5) regeneration plant moves into soil, treats ripe back results seed; With
6) seed is placed on the medium that adds NaCl 50～500mg/L and germinate, select the strong material of NaCl patience.
8. the method for claim 1 is characterized in that, described pretreatment fluid contains NaCl 50～500mg/L, mannitol 40～80g/L, CaCl
20.8～1.5g/L and MES 0.6～1.5g/L.
9. a cover box that is used for implementing each described method of claim 1-8 is characterized in that, described cover box contains the described pretreatment fluid of claim 1, N
6Inducing culture, 2/3MS differential medium and/or 1/2MS strong plantlets and rootage medium.
10. cover box as claimed in claim 9 is characterized in that:
Described pretreatment fluid contains NaCl 50～500mg/L, mannitol 40～80g/L, CaCl
20.8～1.5g/L and MES 0.6～1.5g/L;
Described N
6Inducing culture contains the NaCl of 200～400mg/L, 2,4 of 0.8～1.5mg/L, and 5-T, the KT of 0.4～0.8mg/L and the maltose of 80～100g/L, pH are 5.6～5.9;
Described 2/3MS differential medium contains the NaCl of 200～400mg/L, the 6-BA of 0.4～1.0mg/L, and the KT of 1.0～1.8mg/L, the NAA of 0.02～0.08mg/L, the maltose of 20～40g/L, pH are 5.7～5.8;
Described 1/2MS strong plantlets and rootage medium contains the NAA of 0.03～0.06mg/L, the MET of 3.0～8.0mg/L, and the sucrose of 20～30g/L, pH are 5.8～5.9.
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| CN1230593A (en) * | 1998-12-01 | 1999-10-06 | 海南大学 | Breeding method of halophytes |
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