CN100496224C - Tissue culture regeneration method for wheatgrass mature embryo - Google Patents
Tissue culture regeneration method for wheatgrass mature embryo Download PDFInfo
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- CN100496224C CN100496224C CNB2006100084977A CN200610008497A CN100496224C CN 100496224 C CN100496224 C CN 100496224C CN B2006100084977 A CNB2006100084977 A CN B2006100084977A CN 200610008497 A CN200610008497 A CN 200610008497A CN 100496224 C CN100496224 C CN 100496224C
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Abstract
The present invention relates to mature embryo tissue cultivating regeneration method of wheatgrass. During seed selection and treatment, plump seed with the lemma and the palea eliminated is first soaked in solution containing 2, 4-D in 1.0-4.0 mg/L for 1.5-3.0 hr and sterilized, and the mature embryo is then inoculated to callus inducing culture medicine with increased AgNO3 solution to culture. The present invention has the advantages of capacity of promoting the generation one callus organ and somatic cell to raise callus generating rate, capacity of promoting the generation of seed bud, and capacity of increasing the number of adventitious bud the explant generates and raising the regeneration frequency of the plant.
Description
Technical field:
The present invention relates to a kind of cultivation regenerating method for wheatgrass mature embryo tissue, especially relate to the cultural method of a kind of wheatgrass mature embryo regeneration induction plant.
Background technology:
The meadow of China is the greenery patches ecosystem of national area maximum, but mainly is in the border area, northwest, belongs to the arid and semi-arid lands mostly, ecological environment frailty.In recent decades, owing to reasons such as overgraze, irrational utilization, estrepement denudations, causing meadow ' three to be changed ' phenomenon is on the rise.And these areas can be very abundant for the land resources of development and use, can utilize the strong plant of resisting drought, salt and alkali to improve these regional ecotopes, improves land utilization rate.Because most of herbage breeding cycles are longer, character inheritance is comparatively complicated, and is bigger with conventional breeding method cultivation new varieties difficulty; Caused few, the poor quality of China's herbage production kind quantity, and the resistance horizontality is all not high.The new varieties that how can cultivate the high yield, high-quality, resistance and the disease and insect resistance excellent that are fit to the different regions of China at short notice just become urgency problem to be solved of herbage breeding.
Summary of the invention:
The object of the present invention is to provide a kind of cultivation regenerating method for wheatgrass mature embryo tissue that can effectively improve wheatgrass somatic embryo and bud occurrence frequency.
Purpose of the present invention is implemented by following technical scheme: it includes following steps, (1): the choosing and handling of seed; (2): mature embryo is inoculated on the callus of induce medium cultivates (3): the mature embryo of callus of induce medium culture changed in the subculture medium cultivate; (4): the good callus of selecting growth conditions changes over to and carries out differentiation culture on the differential medium; (5): wait to differentiate to change in the root media behind the seedling and take root;
The choosing and handling of a, described seed: choose viable full seed, after seed at room temperature is soaked in water, peel off flower glume, be put into and contain 1.0mg/L-4.0mg/L2, soaked 1.5 hours-3.0 hours in the solution of 4-D, disinfect again;
Described callus of induce medium: MS minimal medium+0.1mol/L-0.5mol/L mannitol+1mg/L-10mg/L 2,4-D+ concentration is 0.2%-1.5%AgNO
3+ mass percent is sucrose+mass percent 0.55%-0.85% agar of 2%-10%, the pH value 5.8～6.0 of wherein said callus of induce medium, and the conventional method autoclaving is used more.
The invention has the advantages that: can promote the organ generation of callus and the generation of somatic embryo, improve healing rate; Promote some regeneration difficulties to plant the generation of bud; Increasing explant produces the number of indefinite bud and improves plant regeneration frequency.
Description of drawings:
Fig. 1 is the process chart of mature embryo tissue culture regenerating method.
Embodiment:
Embodiment 1: cultivation regenerating method for wheatgrass mature embryo tissue includes following steps,
(1): the choosing and handling of seed: choose viable full seed, seed at room temperature is soaked in water behind 2～4h, peel off flower glume, be put into and contain 1.0mg/L 2, soaked 1 and a half hours in the solution of 4-D, then on superclean bench with 75% alcohol disinfecting 30s, behind the aseptic water washing at least 3 times with 0.2% mercuric chloride sterilization 5min, again with aseptic water washing at least 3 times; Place the culture dish that is covered with wet aseptic filter paper, choose as far as possible less embryo from scultellum with endosperm with dissecting needle; When stripping embryo, suitably embryo is clipped broken, be seeded on the callus inducing medium, 50 mature embryos of every ware inoculation;
(2): mature embryo is inoculated on the callus of induce medium cultivates, 26 ℃ of dark cultivations 14 days, wherein the callus of induce medium is: MS minimal medium+0.1mol/L mannitol+1mg/L2,4-D+ concentration are 0.2%AgNO
3+ mass percent is sucrose+mass percent 0.55% agar of 2%, the pH value 5.8～6.0 of wherein said callus of induce medium, and the conventional method autoclaving is used more.
(3): the mature embryo of callus of induce medium culture changed in the subculture medium cultivated 20 days, subculture 2～3 times: subculture medium: MS+0.2mol/L mannitol+2.0mg/L 2,4-D+0.3mg/L water quality shape abscisic acid ABA+ mass percent is sucrose+mass percent 0.55% agar of 2%, the pH value 5.8～6.0 of wherein said medium, the conventional method autoclaving;
(4): the good callus of selecting growth conditions changes over to and carries out differentiation culture on the differential medium; Differential medium wherein: MS+3.0mg/L KT+1.0mg/L NAA+ mass percent is sucrose+mass percent 0.55% agar of 2%, the pH value 5.8～6.0 of wherein said medium, conventional method autoclaving; Condition of culture is 26 ℃ of following 16h illumination cultivation, and intensity of illumination is 3000～40001x, every 30 days subcultures once.
(5): wait to differentiate to change in the root media behind the seedling and take root, wherein root media is that 1/2MS+0.5mg/L NAA+ mass percent is sucrose+mass percent 0.55% agar of 2%, the pH value 5.8～6.0 of wherein said medium, the conventional method autoclaving.
Embodiment 2: cultivation regenerating method for wheatgrass mature embryo tissue includes following steps,
(1): the choosing and handling of seed, choose viable full seed, seed at room temperature is soaked in water behind 2～4h, peel off flower glume, be put in the solution that contains 3.0mg/L 2,4-D and soaked 3 hours, then on superclean bench with 75% alcohol disinfecting 30s, behind the aseptic water washing at least 3 times with about 30% clorox sterilization 45min, again with aseptic water washing at least 3 times; Place the culture dish that is covered with wet aseptic filter paper, the part that will contain embryo with blade is downcut, and is seeded on the callus inducing medium, 50 mature embryos of every ware inoculation;
(2): mature embryo is inoculated on the callus of induce medium cultivates, 26 ℃ of dark cultivations 14 days, wherein the callus of induce medium is: MS minimal medium+0.5mol/L mannitol+10mg/L2,4-D+ concentration are 1.5%AgNO
3+ mass percent is sucrose+mass percent 0.85% agar of 10%, the pH value 5.8～6.0 of wherein said callus of induce medium, and the conventional method autoclaving is used more;
(3): the mature embryo of callus of induce medium culture changed in the subculture medium cultivated 20 days, subculture 2～3 times: subculture medium: MS+0.2mol/L mannitol+2.0mg/L2,4-D+0.6mg/L water quality shape abscisic acid ABA+ mass percent is sucrose+mass percent 0.85% agar of 10%, the pH value 5.8～6.0 of wherein said medium, the conventional method autoclaving;
(4): the good callus of selecting growth conditions changes over to and carries out differentiation culture on the differential medium; Differential medium wherein: MS+3.0mg/L KT+1.0mg/L NAA+ mass percent is sucrose+mass percent 0.85% agar of 10%, the pH value 5.8～6.0 of wherein said medium, conventional method autoclaving; Condition of culture is 26 ℃ of following 16h illumination cultivation, and intensity of illumination is 3000～40001x, every 30 days subcultures once.
(5): wait to differentiate to change in the root media behind the seedling and take root, wherein root media is that 1/2MS+0.5mg/L NAA+ mass percent is sucrose+mass percent 0.85% agar of 10%, the pH value 5.8～6.0 of wherein said medium, the conventional method autoclaving.
Embodiment 3: cultivation regenerating method for wheatgrass mature embryo tissue includes following steps,
(1): the choosing and handling of seed, choose viable full seed, seed at room temperature is soaked in water behind 2～4h, peel off flower glume, be put in the solution that contains 2.0mg/L 2,4-D and soaked 2 hours, then on superclean bench with 75% alcohol disinfecting 30s, behind the aseptic water washing at least 3 times with about 50% clorox sterilization 25min, again with aseptic water washing at least 3 times; Place the culture dish that is covered with wet aseptic filter paper, choose as far as possible less embryo from scultellum with endosperm with dissecting needle; When stripping embryo, suitably embryo is clipped broken, be seeded on the callus inducing medium, 50 mature embryos of every ware inoculation;
(2): mature embryo is inoculated on the callus of induce medium cultivates, 26 ℃ of dark cultivations 14 days, wherein the callus of induce medium is: MS minimal medium+0.3mol/L mannitol+5.5mg/L2,4-D+ concentration are 0.85%AgNO
3+ mass percent is sucrose+mass percent 0.7% agar of 3%, the pH value 5.8～6.0 of wherein said callus of induce medium, and the conventional method autoclaving is used more;
(3): the mature embryo of callus of induce medium culture changed in the subculture medium cultivated 20 days, subculture 2～3 times: subculture medium: MS+0.2mol/L mannitol+2.0mg/L2,4-D+0.6mg/L water quality shape abscisic acid ABA+ mass percent is sucrose+mass percent 0.7% agar of 3%, the pH value 5.8～6.0 of wherein said medium, the conventional method autoclaving;
(4): the good callus of selecting growth conditions changes over to and carries out differentiation culture on the differential medium; Differential medium wherein: MS+3.0mg/L KT+1.0mg/L NAA+ mass percent is sucrose+mass percent 0.7% agar of 3%, the pH value 5.8～6.0 of wherein said medium, conventional method autoclaving; Condition of culture is 26 ℃ of following 16h illumination cultivation, and intensity of illumination is 3000～40001x, every 30 days subcultures once.
(5): wait to differentiate to change in the root media behind the seedling and take root, wherein root media is that 1/2MS+0.5mg/L NAA+ mass percent is sucrose+mass percent 0.7% agar of 3%, the pH value 5.8～6.0 of wherein said medium, the conventional method autoclaving.
Embodiment 4: the contrast of the cultivation of the present invention and prior art mature embryo regeneration induction plant
1.1 experiment material
(1), this test selects for use the mature seed of 4 kinds of perennial wheatgrass to be confession examination material.Press embodiment 1, embodiment 2, and the cultivation regenerating method for wheatgrass mature embryo tissue of embodiment 3 is cultivated, and wait to differentiate to change over to behind the seedling and carry out interpretation of result after taking root in the root media.
(2), simultaneously choose viable full seed, handle, under following condition of culture, cultivate again afterwards as the method for embodiment 1.
Mature embryo is inoculated on the callus inducing medium, and 26 ℃ of dark cultivations 14 days change over to then in the subculture medium and cultivated subculture 2～3 times 20 days.The good callus of selecting growth conditions changes on the differential medium and breaks up, and condition of culture is 26 ℃ of following 16h illumination cultivation, and intensity of illumination is 3000～4000lx, every 30 days subcultures once.Wait to differentiate to change in the root media behind the seedling and take root.
Wherein: the callus of induce medium is: MS+ mannitol 0.2mol/L+2, and the 4-D2.0mg/L+ mass percent is sucrose+mass percent 0.7% agar of 3%, the pH value 5.8～6.0 of wherein said callus of induce medium, conventional method autoclaving;
Subculture medium: MS+ mannitol 0.2mol/L+2, the 4-D2.0mg/L+6BA0.3mg/L+ mass percent is sucrose+mass percent 0.7% agar of 3%, the pH value 5.8～6.0 of wherein said medium, conventional method autoclaving;
Differential medium: MS+KT 3.0mg/L+NAA 1.0mg/L+ mass percent is sucrose+mass percent 0.7% agar of 3%, the pH value 5.8～6.0 of wherein said medium, conventional method autoclaving;
Root media: 1/2MS+NAA 0.5mg/L+ mass percent is sucrose+mass percent 0.7% agar of 3%, the pH value 5.8～6.0 of medium wherein, conventional method autoclaving; Press the method for embodiment 1 and cultivate, wait to differentiate to change over to behind the seedling and carry out interpretation of result after taking root in the root media.
2, two kinds of methods are to the influence (as following table) of wheatgrass tissue culture regeneration
As can be seen from the table, 4 kinds of wheatgrasses are seeded on the different callus of induce medium, healing rate exists than big-difference, and healing rate of the present invention on average exceeds 11.6% than the healing rate of prior art, thereby makes differentiation rate also improve 13% accordingly.
3, reason
AgNO
3Effect: can promote the organ of callus that the generation with somatic embryo takes place, improve healing rate; Promote some regeneration difficulties to plant the generation of bud; Increasing explant produces the number of indefinite bud and improves plant regeneration frequency.
Mechanism of action: AgNO
3Can act on the ethylene action position competitively, thereby ethene suppressing activity (ethene synthetic amount and bud differentiation capability are negative correlation, and ethene not only suppresses the differentiation of explant bud, has also suppressed the growth of regrowth) promotes plant organ to take place and the somatic embryo generation.Ag
+Existence make ethene can not disturb the synthetic of polyamines, AgNO
3By the synthetic raising somatic embryo of promotion polyamines and the frequency of bud generation.
Claims (1)
1, cultivation regenerating method for wheatgrass mature embryo tissue, it includes following steps, (1): the choosing and handling, (2) of seed: mature embryo is inoculated on the callus of induce medium cultivates, (3): the mature embryo of callus of induce medium culture changed in the subculture medium cultivate; (4): the good callus of selecting growth conditions changes over to and carries out differentiation culture on the differential medium; (5): wait to differentiate to change in the root media behind the seedling and take root; It is characterized in that:
The choosing and handling of a, described seed: choose viable full seed, after seed at room temperature is soaked in water, peel off flower glume, be put into and contain 1.0mg/L-4.0mg/L2, soaked 1.5 hours-3.0 hours in the solution of 4-D, disinfect again;
B, described callus of induce medium: MS minimal medium+0.1mol/L-0.5mol/L mannitol+1mg/L-10mg/L2,4-D+ concentration is 0.2%-1.5%AgNO
3+ mass percent is sucrose+mass percent 0.55%-0.85% agar of 2%-10%, the pH value 5.8～6.0 of wherein said callus of induce medium, and the conventional method autoclaving is used more.
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| CNB2006100084977A CN100496224C (en) | 2006-01-28 | 2006-01-28 | Tissue culture regeneration method for wheatgrass mature embryo |
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| CN102870671A (en) * | 2011-07-11 | 2013-01-16 | 中国科学院植物研究所 | High-efficiency aseptic seedling acquisition method from grass seed |
| CN107637213A (en) * | 2017-10-16 | 2018-01-30 | 河北科技师范学院 | A kind of method for improving wheatgrass germination percentage and planting percent |
| CN109832193B (en) * | 2017-11-28 | 2021-05-11 | 内蒙古农业大学 | Culture method for inducing and regenerating mature embryo callus of roegneria kamoji |
| CN110384045B (en) * | 2019-09-03 | 2021-11-09 | 南通大学 | Method for preserving and recovering aseptic material from ice grass leaves |
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Non-Patent Citations (4)
| Title |
|---|
| 基因型和脱落酸对冰草成熟胚组织培养再生体系的影响. 徐春波等.中国草学会第六届二次会议暨国际学术研讨会论文集. 2004 |
| 基因型和脱落酸对冰草成熟胚组织培养再生体系的影响. 徐春波等.中国草学会第六届二次会议暨国际学术研讨会论文集. 2004 * |
| 蒙农杂种冰草成熟胚愈伤组织的诱导及植株再生. 张辉等.草地学报，第13卷第1期. 2005 |
| 蒙农杂种冰草成熟胚愈伤组织的诱导及植株再生. 张辉等.草地学报，第13卷第1期. 2005 * |
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