CN103004610A - Method for high-salt screening of saline-alkali resistant rosa chinensis plant - Google Patents
Method for high-salt screening of saline-alkali resistant rosa chinensis plant Download PDFInfo
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Abstract
The invention relates to a method for high-salt screening of a saline-alkali resistant rosa chinensis plant. In particular, rosa chinensis which is planted in Tibet plateau is obtained by the method for high-salt screening of saline-alkali resistant rosa chinensis. The method comprises the steps of inducing obtained rosa chinensis explant culture medium suitable for rosa chinensis explants out of a sprout texture and then irrigating and screening by a high-salt culture medium to obtain the saline-alkali resistant rosa chinensis; cutting a fibroid branch with thick vascular bundle into 10cm stem sections, sterilizing the culture mediums by 0.5% potassium permanganate according to the ratio of vermiculite to perlite to nutrient soil being 10:2:1, and then spraying the medium by 0.3% rooting liquid, wherein high-salt screening is diluting and irrigating by sodium chloride liquid culture mediums B with different concentrations, and selecting the plant which can normally grow. By adopting the method, the growth boundaries of fresh flowers are broken through; five saline-alkali resistant rosa chinensises can be obtained from genetic modification and individual gene screening, and a blank no fresh flower is available in golmud is filled.
Description
Technical field
The present invention relates to the method for a kind of high salt Selection of Salt-Tolerant alkali Chinese rose plant, be specially the method for utilizing high salt Selection of Salt-Tolerant alkali Chinese rose and obtain the Chinese rose that survives in Qinghai-Tibet Platean plantation.
Background technology
Chinese rose (Rosa chinensis) belongs to the rose family, is evergreen or machaka, is described as and spends middle queen, claims again " Chinese rose ", and the nature florescence is from 5 to November.Tianjin have the title in " township of Chinese rose ", and the Chinese rose cultivation history is long, and Tianjin in 1984 is decided to be Chinese rose after the city flower, and Chinese rose produces and cultivation has a great development, and spreads all over the street greens, city, meaning residing " peace, friendship, prosperity, development ".The fresh flower and do not grow in the high Golmud City that is positioned at 2900 meters of Qinghai-Tibet height above sea level, not only there is not fresh flower in the people life here, even in the gala occasion meeting, do not have fresh flower yet, several rows of decoration artificial flower racks down, seriously affected solemn scene, this is that radiation activity is large because the sunshine time is long, high and cold anoxic, short of rain windy, day and night temperature is large, and flower growth is difficulty very.
The geographical position, area, Golmud City that is positioned at the Qinghai-Tibet Platean is extremely important, she is the last leg that leads to Tibet, it is the important place of Tibet material stock, it also is maximum city, the whole nation, area is 139535.85 square kilometres, also larger than Zhejiang Province, Golmud is called again the salt lake city, strides the Kun Lun Mountain that contains towering high mystery.The Golmud City is because of high and cold anoxic, and short of rain windy, day and night temperature is large, and the sunshine time is long, and radiation activity is large, does not have fresh flower.Although Tianjin is located in bank of the Bohai Sea, soil also has certain salinization of soil, and the soil salinity and alkalinity of Ge-ermu is larger, is not suitable for the Chinese rose growth.
Break the growth boundary of fresh flower, must start with from the screening of improvement of genes, idiotype, because the reason that plant can not fine growth in the saline land is that plant can't absorb enough moisture and finishes process of growth.The saline-resisting and alkaline-resisting gene that plant has can make plant cell in certain environment stress scope, initiatively accumulates the osmotic pressure that some osmotic adjustments such as proline strengthen cell, stops the cell dehydration, guarantees the normal g and D of plant.But only have the individual osmotic adjustment that could produce than strongly hydrophilic of only a few resistant gene of salt type, plant cell is escaped injury or alleviate injury.The a plurality of genes of Salt And Alkali Tolerance physiological reaction process need, a plurality of metabolic pathway concur, even if the plant of same kind, because of growth course, growing environment and the trickle genotypic difference of individuality, all can cause plant to the existing geology in long-day, saline land and plateau, weather, reaction that environmental condition is different.Utilize the cell engineering method, a series of degeneration-resistant metabolic pathways in stress cell, excite the biological function of related gene, make competent genotype produce the osmotic pressure that osmotic adjustment strengthens cell, stop the cell dehydration, form the Salt And Alkali Tolerance function, this need to do a large amount of salt tolerance experiments, could will choose than the arge gene type of high resistance to cold and diseases.
Summary of the invention
The method that the purpose of this invention is to provide a kind of high salt Selection of Salt-Tolerant alkali Chinese rose plant, be specially the method for utilizing high salt Selection of Salt-Tolerant alkali Chinese rose, utilize high salt culture medium to induce the adversity gene of Chinese rose self to express, need not cultivate through the tissue of gene engineering and plant, but obtain the plant of Salt And Alkali Tolerance within a short period of time, thereby the Chinese rose plant that acquisition survives in the Qinghai-Tibet Platean plantation, make Ge-ermu grow Chinese rose, greening to Golmud fresh flower production and saline land has very important theory and practice meaning, and it is numerous to be applicable to a large amount of expansions of the large regional Chinese rose of other saline and alkaline.The present invention has broken the growth boundary of fresh flower, starts with from improvement of genes, idiotype screening, obtains 5 kinds of Salt And Alkali Tolerance Chinese roses.
The step that the method for high salt Selection of Salt-Tolerant alkali Chinese rose plant provided by the invention comprises:
1) preparation of Chinese rose explant cultivation matrix
Vermiculite, perlite and Nutrition Soil be with the mass ratio of 5-10:2:1 preparation matrix, and on matrix lamination 0.5-1 centimetre river sand, after the sterilization of 0.2-0.5% potassium permanganate, the taking root liquid with 0.1-0.3% sprays matrix again.Vermiculite and perlite mainly have been ventilative, and the effect that moisture retention is strong provides a better growing conditions to plant; Pressing river sand mainly is fixation (Fig. 1).
2) preparation of Chinese rose explant
Choose the thicker wood fibre Chinese rose branch of vascular bundle, be cut into the stem section of 8-15cm, each stem section is stayed 2-3 sheet leaf.The morphology lower end of Stem Sections in Rosa Chinensis Jacq is immersed in the taking root liquid of 1-3% and soaked 0.5-1 hour, cuttage enters in the matrix of preparation, and waters (culture medium A is MS+0.4mg/L 6-BA+0.8 mg/L NAA) with the culture medium A of diluting 10 times.18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2000～4000Lx.
3) Chinese rose bud tissue induces and high salt screening
After two weeks of cuttage, the Chinese rose explant begins to have new bud to grow, and inducing with root media B(medium B with the bud tissue that contains NaCl successively is MS+1mg/L 6-BA+NaCl) screen:
In the first～two week, NaCl concentration is 6.0 ‰, 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2000～4000Lx.
The the 3rd～all around, NaCl concentration is 7.0 ‰, 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2000～4000Lx.
In the five～six week, NaCl concentration is 8.0 ‰, 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2000～4000Lx.
4) survive the transplanting of Chinese rose after the screening
The Chinese rose plant (choosing the plant of energy normal growth) that survives is planted in saline and alkaline flowerpot, and normal management is transplanted to the saline land after surviving, saline land pH value 8.7-9.0; Total salt amount is 0.5%-0.85%; Water (culture medium C is MS+0.4mg/L6-BA+0.8 mg/L NAA+1mg/L IBA) behind 100 times of the culture medium C dilute with waters, implement normal extensive management.
5) detection of resistance molecule
Draw the calibration curve of proline, curvilinear equation y=27.015x+1.7929, R
2=0.9936.
Get respectively the Chinese rose of planting in the Salt And Alkali Tolerance Chinese rose of screening and the alkali prevention groove, every kind of Chinese rose is got five plant, every plant is got each 0.5g of blade, put respectively in the Boiling tube, then the sulfosalicylic acid solution that adds respectively 5ml 3% to each pipe, in boiling water bath, extract in the 10min(leaching process and will often shake), cooled and filtered is in clean test tube, and filtrate is the extract of proline.
Extract 2.5ml before drawing, ice acetic acid 2.5ml, 2.5% acid ninhydrine solution 4ml puts 100 ℃ of water-bath 1h, adds 4ml toluene mixing after the cooling, leaves standstill 12-16 hour.Second day in cuvette, take toluene solution as blank, is measured absorbance (A) value with the suction pipe gentle aspiration upper strata red toluene solution of proline at 520mm wavelength place.
Find the content of proline the sample determination liquid from calibration curve, press proline content in the formula calculation sample:
Proline content (μ gg
-1Fw)=[X * extract total amount (ml)]/when mensuration [sample fresh weight (g) * extract consumption (ml)]
In the formula: the proline content (μ g) of X-from calibration curve, check in
Generally free proline content is very low in the plant corpus, under adverse environmental factor proline content can increase sharply ten several times to tens times, so proline content has become a biochemical indicator of resistance in the plant corpus.Under salt stress, the free proline content in the degeneration-resistant Chinese rose plant can obviously raise.
6) transportation, cultivation and transplanting:
Salt And Alkali Tolerance Chinese rose seedling (the Salt And Alkali Tolerance Chinese rose seedling tissue that screening obtains is cultivated numerous 1000 strains of expansion) packages by normal condition insulation, moisturizing, lucifuge in batches, directly transport the indoor greenhouse cultivation of Germo urban district, to survive Chinese rose be transplanted to outdoor, normal management, survival rate is up to 85%.
The invention provides the Chinese rose that survives in the Qinghai-Tibet Platean plantation of a kind of method of utilizing high salt Selection of Salt-Tolerant alkali Chinese rose and generation thereof.Utilize high salt culture medium to induce the adversity gene of Chinese rose self to express, need not cultivate through the tissue of gene engineering and plant, but obtain the plant of Salt And Alkali Tolerance within a short period of time, the present invention has broken the growth boundary of fresh flower, start with from improvement of genes, idiotype screening, obtain 5 kinds of Salt And Alkali Tolerance Chinese roses, filled up the blank that Golmud does not have fresh flower.Thereby obtain the Chinese rose plant that survives in the Qinghai-Tibet Platean plantation, make Ge-ermu grow Chinese rose, the greening in Golmud fresh flower production and saline land is had very important theory and practice meaning, and it is numerous to be applicable to a large amount of expansions of the large regional Chinese rose of other saline and alkaline.
Description of drawings
The Chinese rose explant of Fig. 1 for obtaining.
Fig. 2 is that the bud of Chinese rose is induced with saline and alkaline screening: a as NaCl screening is arranged, and b screens without NaCl.
Fig. 3 plants in saline and alkaline flowerpot for the Chinese rose of screening.
Fig. 4 Salt And Alkali Tolerance seedling arrives Golmud and carries out that booth is planted experimentally and success.
The Salt And Alkali Tolerance rose flower plant that grows first in plateau, Fig. 5 Golmud.
Embodiment
The invention will be further described below in conjunction with specific embodiment.
Materials and methods
(1) Chinese rose material: from genetic engineering research institute of University Of Tianjin Experimental Base.
(2) experiment equipment: nutritive cube, ultraviolet specrophotometer, wide-mouth bottle.
(3) matrix agent material: vermiculite, perlite, river sand, potassium permanganate, MS minimal medium, sodium chloride, sucrose, 6-BA(6-benayl aminopurine), NAA (methyl α-naphthyl acetate), IBA(heteroauxin).Root-inducing powder is the serial root-inducing powder of green energy that Shaanxi Pu Sidun agricultural Co., Ltd produces, the 30g dress.
(4) utilize the cell engineering method, by saline and alkaline stimulation stress cell in a series of degeneration-resistant metabolic pathways, excite the biological function of related gene, will choose than the arge gene type of high resistance to cold and diseases.
Embodiment
Take Hybrid Tea as material, filter out the Chinese rose that is suitable at the saline land normal growth through high salt, screening process is as follows:
(1) preparation of Chinese rose explant cultivation matrix
Vermiculite, perlite and Nutrition Soil be with the mass ratio of 10:2:1 preparation matrix, and on matrix the river sand of 1 centimetre of lamination, after the potassium permanganate solution with 0.5% is sterilized, with 0.3% taking root liquid matrix is sprayed again.Vermiculite and perlite mainly have been ventilative, and the effect that moisture retention is strong provides a better growing conditions to plant; Pressing approximately 1 centimetre river sand, mainly is fixation.
(2) preparation of Chinese rose explant
Choose the thicker wood fibre branch of vascular bundle, be cut into the stem section about 10cm, each stem section is stayed 2-3 sheet leaf.The morphology lower end of Stem Sections in Rosa Chinensis Jacq immersed in 3% the taking root liquid and soaked 1 hour, cuttage enters in the matrix of preparation, and waters (liquid nutrient medium A is MS+ 0.4mg/L 6-BA+ 0.8 mg/L NAA) with the liquid nutrient medium A that dilutes 10 times.18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2 000～4 000Lx.。
(3) Chinese rose bud tissue induces and high salt screening
After two weeks of cuttage, the Chinese rose explant begins to have new bud to grow, and induce with root media B(medium B with dilution 5-10 bud tissue doubly this moment is MS+1 mg/L 6-BA+NaCl) screen screening conditions:
In the first～two week, NaCl concentration is 6.0 ‰,, survival rate 13.5%; 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2 000～4 000Lx;
The the 3rd～all around, NaCl concentration is 7.0 ‰, survival rate 9.2%; 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2 000～4 000Lx;
In the five～six week, NaCl concentration is 8.0 ‰, survival rate 2.8%, 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2 000～4 000Lx;
(4) survive the transplanting of Chinese rose after the screening
With the plantlet of transplant that survives to the saline land, pH value 8.7-9.0, total salt amount is 7.5 ‰-8.5 ‰; Water (culture medium C is MS+0.4mg/L6-BA+0.8 mg/L NAA+1mg/L IBA) behind 100 times of the culture medium C dilute with waters, implement normal extensive management.
(5) detection of resistance molecule
Draw the calibration curve of proline, curvilinear equation y=27.015x+1.7929, R
2=0.9936
Get respectively the Chinese rose of planting in the Salt And Alkali Tolerance Chinese rose of screening and the alkali prevention groove, every kind of Chinese rose is got five plant, every plant is got each 0.5g of blade, put respectively in the Boiling tube, then the sulfosalicylic acid solution that adds respectively 5ml 3% to each pipe, in boiling water bath, extract in the 10min(leaching process and will often shake), cooled and filtered is in clean test tube, and filtrate is the extract of proline.
Draw proline extract 2.5ml, ice acetic acid 2.5ml, 2.5% acid ninhydrine solution 4ml puts 100 ℃ of water-bath 1h, adds 4ml toluene mixing after the cooling, standing over night.Second day in cuvette, take toluene solution as blank, is measured absorbance (A) value with the suction pipe gentle aspiration upper strata red toluene solution of proline at 520mm wavelength place.
Find the content of proline the sample determination liquid from calibration curve, press proline content in the formula calculation sample:
Proline content (μ gg
-1Fw)=[X * extract total amount (ml)]/when mensuration [sample fresh weight (g) * extract consumption (ml)]
In the formula: the proline content (μ g) of X-from calibration curve, check in.
The measurement result of the degeneration-resistant Chinese rose of table 1 leaf texture free proline content
| The Salt And Alkali Tolerance Chinese rose | Alkali prevention groove plantation Chinese rose | |
| OD 520 | 0.879 | 0.095 |
| Pro（ug/g·fw） | 55.8509 | 11.5716 |
Upper table shows, under salt stress, the free proline content in the whole strain of degeneration-resistant Chinese rose raises 5 times.Proved that under normal conditions free proline content is very low in the plant corpus, but under adverse environmental factor can increase sharply decades of times to hundred times of proline content, so proline content has become a biochemical indicator of resistance in the plant corpus.
(6) transportation and cultivation:
In batches Salt And Alkali Tolerance Chinese rose seedling (tissue is cultivated the Salt And Alkali Tolerance seedling) packages by normal condition insulation, moisturizing, lucifuge, directly transports the regional indoor greenhouse cultivation in Qinghai-Tibet Golmud City, will survive Chinese rose be transplanted to outdoor, normal management, survival rate is up to 85%.
The way far away transportation of Salt And Alkali Tolerance seedling is the key of success or failure, need to be incubated, moisturizing, root can not dry see light and guarantee the metabolic fundamental prerequisite of plant, carrying 1000 strain cell engineering Salt And Alkali Tolerance seedlings, successfully arrive at Golmud through Beijing used during taking train, plant experimentally and success in Salt And Alkali Tolerance seedling arrival Golmud, Fig. 4 is that the Salt And Alkali Tolerance seedling arrives Golmud and carries out that booth is planted experimentally and success, plateau, Golmud fresh flower of having grown first, and Fig. 5 is the Salt And Alkali Tolerance rose flower plant that grows first in plateau, Golmud.Make the Tianjin Chinese rose climb up the Qinghai-Tibet Platean, filled up the blank that Golmud does not have fresh flower.
Obtain 5 kinds of Salt And Alkali Tolerance Chinese roses according to method of the present invention, plantation survives in the Qinghai-Tibet Platean:
(1) plants:
Morphological feature: the pink colour corymb, petal is more, and look fickle in love is lighter, and late April at florescence is to October, the fructescence 9-11 month.Evergreen or the half evergreen upright shrub because of environment, First Year can be up to 0.5-1 rice, and Second Year can be up to 2 meters of 1.5-.
The gardens purposes: the Chinese rose pattern is gorgeous, and monthly open, the florescence is longer, can plant in flower garden, flower bed, also can make the gaily decorated basket, garland, potted landscape and be used as cut-flower etc.
(2) plants:
Morphological feature: red single giving birth to or corymb, petal is more, and late April at florescence is to October, the fructescence 9-11 month.Evergreen or the half evergreen upright shrub because of environment, First Year can be up to 0.5-1 rice, and Second Year can be up to 2 meters of 1.5-.
The gardens purposes: the Chinese rose pattern is gorgeous, and monthly open, the florescence is longer, can plant in flower garden, flower bed, also can make the gaily decorated basket, garland, potted landscape and be used as cut-flower etc.
(3) plants:
Morphological feature: red solitary flower order, petal is many, and late April at florescence is to October, the fructescence 9-11 month.Evergreen or the half evergreen upright shrub because of environment, First Year can be up to 0.5-1 rice, and Second Year can be up to 2 meters of 1.5-.
The gardens purposes: the Chinese rose pattern is gorgeous, and monthly open, the florescence is longer, can plant in flower garden, flower bed, also can make the gaily decorated basket, garland, potted landscape and be used as cut-flower etc.
(4) plants:
Morphological feature: pink colour solitary flower order, petal is many, and late April at florescence is to October, the fructescence 9-11 month.Evergreen or the half evergreen upright shrub because of environment, First Year can be up to 0.5-1 rice, and Second Year can be up to 2 meters of 1.5-.
The gardens purposes: the Chinese rose pattern is gorgeous, and monthly open, the florescence is longer, can plant in flower garden, flower bed, also can make the gaily decorated basket, garland, potted landscape and be used as cut-flower etc.
(5) plants:
Morphological feature: pigment-redness-lilac corymb, petal is more, and look fickle in love is lighter, and late April at florescence is to October, 9 ~ November of fructescence.Evergreen or the half evergreen upright shrub because of environment, First Year can be up to 0.5-1 rice, and Second Year can be up to 2 meters of 1.5-.
The gardens purposes: the Chinese rose pattern is gorgeous, and monthly open, the florescence is longer, can plant in flower garden, flower bed, also can make the gaily decorated basket, garland, potted landscape and be used as cut-flower etc.
The invention provides the Chinese rose that survives in the Qinghai-Tibet Platean plantation of a kind of method of utilizing high salt Selection of Salt-Tolerant alkali Chinese rose and generation thereof, need not cultivate through the tissue of gene engineering and plant, but obtain the plant of Salt And Alkali Tolerance within a short period of time, the a large amount of expansions that are used for the Salt And Alkali Tolerance Chinese rose are numerous, and the greening in Golmud fresh flower production and saline land is had very important theory and practice meaning.
Claims (5)
1. the method for high salt Selection of Salt-Tolerant alkali Chinese rose plant is characterized in that the step that it comprises:
1) preparation of Chinese rose explant cultivation matrix
Vermiculite, perlite and Nutrition Soil be with the mass ratio of 5-10:2:1 preparation matrix, and on matrix lamination 0.5-1 centimetre river sand, after the sterilization of 0.2-0.5% potassium permanganate, the taking root liquid with 0.1-0.3% sprays matrix again;
2) preparation of Chinese rose explant
Choose the thicker wood fibre Chinese rose branch of vascular bundle, be cut into the stem section of 8-15cm, each stem section is stayed 2-3 sheet leaf, the morphology lower end of Stem Sections in Rosa Chinensis Jacq is immersed in the taking root liquid of 1-3% and soaked 0.5-1 hour, cuttage enters in the matrix of preparation, and water with 10 times liquid nutrient medium A of dilution, cultivated for two weeks;
3) Chinese rose bud tissue induces and high salt screening
After two weeks of cuttage, the Chinese rose explant begins to have new bud to grow, and induces with root media B with the bud tissue that contains variable concentrations NaCl successively and screens;
In the first～two week, NaCl concentration is 6.0 ‰, 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2000～4000Lx;
The the 3rd～all around, NaCl concentration is 7.0 ‰, 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2000～4000Lx;
In the five～six week, NaCl concentration is 8.0 ‰, 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2000～4000Lx;
4) survive the transplanting of Chinese rose after the screening
Choose the Chinese rose plant of normal growth and plant in saline and alkaline flowerpot, normal management is transplanted to the saline land after surviving, saline land pH value 8.7-9.0; Total salt amount is 0.5%-0.85%, and waters the culture medium C of 100 times of dilutions, implements normally management;
5) by the detection method of general proline content, to the detection of the resistance molecule of Salt And Alkali Tolerance Chinese rose
Draw the calibration curve of proline, curvilinear equation y=27.015x+1.7929, R
2=0.9936;
From the Salt And Alkali Tolerance Chinese rose of screening, extract the extract obtain proline, measure the content of proline wherein, find the content of proline the sample determination liquid from calibration curve, press proline content in the formula calculation sample:
Proline content (μ gg
-1Fw)=[X * extract total amount (ml)]/when mensuration [sample fresh weight (g) * extract consumption (ml)]
In the formula: the proline content (μ g) of X-from calibration curve, check in;
6) transportation, cultivation and transplanting:
In batches Salt And Alkali Tolerance Chinese rose seedling packages by normal condition insulation, moisturizing, lucifuge, directly transports the indoor greenhouse cultivation in Golmud, will survive Chinese rose be transplanted to outdoor, normal management.
2. method according to claim 1 is characterized in that the prescription of described liquid nutrient medium A is: MS+ 0.4mg/L 6-BA+ 0.8 mg/L NAA.
3. method according to claim 1 is characterized in that step 2) described condition of culture: 18～26 ℃ of cultivation temperature, illumination 12h/d, illuminance 2000～4000Lx.
4. method according to claim 1 is characterized in that the prescription of described medium B is: MS+1 mg/L 6-BA+NaCl.
5. method according to claim 1 is characterized in that the prescription of described culture medium C is: MS+ 0.4mg/L 6-BA+0.8 mg/L NAA+1mg/L IBA.
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