CN1994063A - Rice breeding method incorporating high photosynthetic efficiency and presenility resistant property - Google Patents
Rice breeding method incorporating high photosynthetic efficiency and presenility resistant property Download PDFInfo
- Publication number
- CN1994063A CN1994063A CN 200610161649 CN200610161649A CN1994063A CN 1994063 A CN1994063 A CN 1994063A CN 200610161649 CN200610161649 CN 200610161649 CN 200610161649 A CN200610161649 A CN 200610161649A CN 1994063 A CN1994063 A CN 1994063A
- Authority
- CN
- China
- Prior art keywords
- rice
- premature senescence
- gene
- senescence resistance
- high light
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a rice seeding method which comprises that: leading decay gene into rice seed to obtain the rice system; crossing the rice with C4 plant PEPC gene stable representation with the rice system, to obtain F1 system; checking, selecting the rice with high light effect and anti-decay character; cultivating F1, to obtain two flower systems; checking, selecting the rice pure system with high light effect and anti-decay character.
Description
Technical field
The present invention relates to molecule physiology breeding field, especially a kind of the evaluation by gene engineering, conventional breeding and physiology organically combines, and cultivates a kind of rice breeding method that merges high light efficiency and premature senescence resistance proterties.
Background technology
The three series mating of of short stemization of rice varieties and indica hybrid rice is two important milestones on the modern rice breeding history, and they significantly improve rice yield.China's paddy rice average yield has reached 6t/hm
2, Jiangsu has surpassed 8t/hm
2, leaf area index all reaches about 8～10, improves quite difficulty of output by increasing population leaf area again.The utility rate of luminous energy of analyzing existing high-yield rice kind is 1～1.5%, and desirable utility rate of luminous energy can reach 3～5%, and as seen, improving the paddy rice photosynthetic efficiency may be one of effective way that will improve from now on rice yield.
As everyone knows, output, the quality trait of most proterties, the especially crops of plant are subjected to controlled by multiple genes.Improve these quantitative characters, only depend on change certain gene wherein to be difficult to prove effective, and must be simultaneously to controlling a plurality of encoding genes of proterties, even controlling gene carries out genetic transformation, and they stably being expressed and heredity just can reach its intended purposes in transfer-gen plant and offspring thereof, this not only needs number of research projects to confirm, and a plurality of genes of polymerization also can run into the phenomenon of gene silencing, not only influence the effect that transforms, but also costly.Nearest data shows, by gene engineering with corn C
4It is an approach that effectively improves photosynthetic efficiency that photosynthetic gene imports paddy rice.
According to CO
2The difference of assimilation mode, higher plant is divided into C
3, C
4And crassulacean acid (CAM) plant, wherein, C
4Plant has photosynthetic efficiency height, CO
2Compensation point is low, almost do not have advantages such as photorespiration, especially under conditions such as high light, high temperature, arid, and C
4Plant has obvious growth advantage and moisture and nutrient utilization, and biological yield is also higher, and plant physiology and thremmatology man are devoted to for a long time with C
4Photosynthesis characteristics imports C
3Crop is to improve its photosynthetic efficiency.But crop C in the section
4Plant corn and C
3The hybrid of plant paddy rice is sterile owing to the irregular pairing of chromosome or genetic disorder show, and therefore, proves effective by being difficult on the conventional breeding.Over past ten years, C
4A series of key enzymes of photosynthetic pathway are the clone and the existing further investigation of expression characteristic thereof of two kinases (PPDK) genes of phosphoric acid enol pyruvic acid carboxylase (PEPC), NADP-malic acid (NADP-ME) and pyruvate phosphate.C particularly
4Photosynthetic gene successfully imports C
3The important breakthrough of plant research, the PEPC activity of its transfer corn pepc gene paddy rice is C up to 44-81 times of original seed
4The 2-3 of the PEPC activity of plant corn doubly, oxygen presses down and has photosyntheticly also descended 20%, seed production improves the transgenic paddy rice material of 10-40% than original seed.Stable seed selection along with the pepc gene paddy rice of the commentaries on classics corn of high expressed, increasing result of study has confirmed that high expressed changes corn pepc gene paddy rice and has high light efficiency characteristic: under the natural conditions of China's summer high light intensity and high temperature, change the pepc gene rice plant and have high light efficiency characteristic and output raising 10-20%, its fast light inhibition ability under the light rejection condition strengthens, and the genetic research of the rice material by utilize changeing pepc gene also shows: pepc gene is the phase key enzyme, efficiently expressing of the high photosynthetic capacity of paddy rice of its decision, and the high light efficiency characteristic of carrying the pepc gene paddy rice be can be stable heredity, Jiao Demao, Gao Dongying, Li Xia etc. have applied for national inventing patent in December 21 calendar year 2001 at " changing the breeding method of the paddy rice energy stably express of C4 plant pepc gene over to ", and being awarded patent right, the patent No. is ZL011380128.
In recent years, the super hybridization rice breeding of China had obtained huge progress, and a collection of high-quality, high yielding variety are bred in succession, for the food problem that solves the China and even the world has been made significant contribution.But it is withered that most of super hybridization rice late growth stages experience low temperature are prone to cauline leaf green grass or young crops, and early ageing takes place, and seriously affects the performance of super hybridization rice output.By genetic improvement, it will be one of effective way that further improves rice yield that the early ageing of control paddy rice takes place.
Many subspecies super hybridization rices are at late growth stage, the easy yellow of blade, and it is very fast that physiological performance is that chlorophyll and protein content descend, and causes photosynthetic rate obviously to descend, not only influence grouting, and seriously influenced the yield and quality of crop.Studies show that: the photosynthesis related gene, reduce as transcriptional level in rbcs (Rubisco small subunit) and cab (chlorophyll a/b is in conjunction with albumen) the gene aging course.Utilize differential screening and reduction hybridization technique (SSH), people are from arabidopsis, rape, potato, barley, tomato, cucumber, wheat, paddy rice, identify in the seeding corn and other crops and obtained expressing between declining period the gene that increases, these genes are commonly called senescence-associated gene (SAGs) or old and feeble up-regulated gene (senescence-upregulated genes), but the many liquid manure controls of the conventional now relevant method that delays senility from cultivation, the research that imports above-mentioned senescence-associated gene rests in the theoretical research more, because these expression of gene are relevant with specific factor, some is only expressed in the naturally-aged process, some is induced in the aging course in specific adverse circumstance and expresses, as only at moisture, just express under specific the coercing such as nutrition, therefore, it is still very difficult to obtain stable premature senescence resistance material by gene engineering merely, and the adaptability on agricultural breeding is limited.
Nearest studies show that: the active oxygen species level of glutathione in the controlling plant body plays an important role, glutathione is resisted abiotic oxidation environment stresses such as high light, low temperature and soil be barren plant can both cause sharp increase, deletion mutant at the glutathione that only contains wild species 5%, show as the oxidation adverse circumstance more responsive, the glutathione molecule as if having to the Perception Features of the signaling molecule of oxidation adverse circumstance " meet sharp and increase ", glutathione can with the reactive oxygen species chemically reactive, with the reduced glutathione (GSH) of enzymatic coupling to H
2O
2Detoxification play a key role, the oxidation of GSH self sulfydryl forms oxidized form of glutathione (GSSG), prevents the sex change of the albumen of being induced by the oxidation adverse circumstance, thus protected protein.Gene clone and genetically modified studies show that from the enzyme of glutathione metabolism: increased the synthesis capability of the glutathione of transfer-gen plant, can stop the generation of the oxidation adverse circumstance in its chloroplast.With the endoplasmic reticulum of overexpression studies show that in conjunction with the protein-bonded genetically modified plants of endoplasmic reticulum of albumen or antisense: the endoplasmic reticulum of overexpression by the mixing of sulfydryl albumen such as GSH, may help photosynthetic mechanism to stop endogenous oxidation adverse circumstance in conjunction with albumen.How this achievement is used as yet in the rice breeding process is not appeared in the newspapers.
Summary of the invention
The objective of the invention is to: low at the ubiquitous photosynthetic efficiency of present paddy rice, easily old and feeble, especially easily the old and feeble practical problem that directly has influence on the yield and quality of paddy rice, we are by existing ZL01138012.8 patented technology, further investigation provides a kind of rice breeding method that merges high light efficiency and premature senescence resistance proterties.
Our susceptibility that studies show that paddy rice early ageing generation and the variation of glutathione homologue have very high correlation, (γ-ECS) is the rate-limiting enzyme in the route of synthesis committed step of glutathione to gamma-glutamyl amine cysteine synthase gene, as long as can in rice varieties, import γ-ECS gene, just the glutathione in the energy inducing paddy rice is synthetic, the early ageing of control paddy rice.
The object of the present invention is achieved like this: a kind of rice breeding method that merges high light efficiency and premature senescence resistance proterties is characterized in that:
A) the early ageing related gene is imported in the rice germplasm of high-quality, obtain the rice strain of premature senescence resistance;
B) will have C
4The rice germplasm of plant pepc gene stably express and the rice strain's hybridization with premature senescence resistance characteristic obtain the F1 hybrid;
C) physiology that the F1 hybrid is carried out high light efficiency and premature senescence resistance characteristic is identified, filters out the rice material with high light efficiency and premature senescence resistance characteristic;
The F1 with two kinds of good physiological properties that d) will filter out cultivates by pollen, obtains the stable flower training strain system with two kinds of good physiological properties;
E) be to carry out the physiology of high light efficiency, premature senescence resistance to identify to stable flower training strain, the screening back obtains the paddy rice pure lines of described two the good physiological traits of fusion.
In the present invention: the rice breeding method of high light efficiency of fusion according to claim 1 and premature senescence resistance proterties is characterized in that: the rice germplasm of described high-quality is the fragrant round-grained rice in Soviet Union Shanghai; Be meant in the described rice germplasm: change in the fragrant round-grained rice in Soviet Union Shanghai by the γ-ECS gene of agrobacterium mediation method with corn with early ageing related gene importing high-quality; In the described rice germplasm with early ageing related gene importing high-quality, the rice strain that obtains premature senescence resistance is meant: by agrobacterium mediation method with the γ-ECS gene of corn change over to obtain the rice strain of premature senescence resistance in the fragrant round-grained rice in Soviet Union Shanghai after, the chlorophyll content of sword-like leave and photochemistry efficient originally when blooming 7 days and 21 days by mensuration, filter out the Fv/Fm decay be not higher than 10% or the chlorophyll content decay be not higher than 20% premature senescence resistance material H
1, and make H
1In generation, multiplied H
2, the rice plant of γ-ECS gene is carried in acquisition.
In the present invention: described will have C
4The rice germplasm of plant pepc gene stably express is meant with the rice strain's hybridization with premature senescence resistance characteristic: have C
4The rice germplasm of plant pepc gene stably express is a male parent, is female parent with the rice plant that carries γ-ECS gene.
In the present invention: the described physiology that carries out high light efficiency and premature senescence resistance characteristic is identified, filters out the rice material with high light efficiency and premature senescence resistance characteristic and is meant: by measuring C
4Active and the leaf photosynthesis speed of photosynthetic enzyme, and measure when blooming 7 days and 21 days sword-like leave originally photochemistry efficient (Fv/Fm) and chlorophyll content, filter out the PEPC activity between 250～1099 μ mol/mgChl.h, photosynthetic rate numerical value is at 16.2～30.8 μ mol/m
2.s between, Fv/Fm decay simultaneously be not higher than 10% or the chlorophyll content decay be not higher than 20% rice material.
In the present invention: the described F1 with two kinds of good physiological properties that will filter out cultivates by pollen, obtaining the stable flower training strain system with two kinds of good physiological properties is meant: the spike of rice of choosing the F1 monokaryotic stage, putting into 4 ℃ handled 10 days down, be inoculated in then in the medium of H8, induce callus, change seedling differentiation among the differential medium MS6 again over to, after the flower training seed of acquisition is planted, keep dliploid, thereby obtain stable flower training strain system.
In the present invention: described H
8The prescription of medium is: be minimal medium with M8, add 2,4-D 1mg/L+ methyl (NAA) the 3mg/L+ basic element of cell division (KT) 1mg/L+ abscisic acid (ABA) 3mg/L+ proline 100mg/L+ caseinhydrolysate 300mg/L+ sucrose 50g/L+ agar 8g/L; Described MS6 culture medium prescription is: with MS minimal medium, and additional 2mg/LKT+1mg/L NAA+ sucrose 30g/L+ agar 6g/L.
The invention has the advantages that: overcome expense and reticent deficiency that a plurality of genes of polymerization may bring, in conjunction with conventional hybridization, physiology is identified and anther cultural characteristics, forms a kind of fast effective plant breeding method.With the paddy rice is example, and under the existing high-yield culturing condition of China, today that various good super hybridization rices combinations continue to bring out, the performance degree of good physiological trait is to be related to the performance of output of kind and the key factor of promoted extension.But the performance of each physiological trait is again the coefficient result of h and E, even also be difficult to improvement by molecular biology and engineered modern biotechnology, and a lot of senescence-associated gene (SAGs) or the old and feeble up-regulated gene (senescence-upregulated genes) that obtains will be identified in the crop, all importing paddy rice had both spent huge, and multiple gene polymerization also gene silencing can occur, be difficult to reach the result of expectation, and this method is set up corresponding relation by the expression and the good physiology shape of single key gene, two materials are simply merged, overcome the gene multiple gene and imported the gene silencing that brings, the cycle length of conventional breeding method and the unsettled difficult problem of the physiological trait that the plant ecological environmental differences causes, obtain to have the material of a good physiological trait respectively by the importing of single key gene, thereby overcome the genetic disorder between different cultivars, both spent low, being convenient to the breeder again implements, cultivation by flower pesticide, material settling out with screening under nature and the artificial condition, not only reduced workload, and overcome the long deficiency of conventional hybridization breeding cycle, from having improved the accuracy and the breeding efficiency of seed selection.
Embodiment
Embodiment 1 γ-ECS gene changes over to Soviet Union's fragrant round-grained rice in Shanghai
2003, with corn, γ-ECS gene changes over to, concrete grammar is as follows: the paddy rice mature seed of the fragrant round-grained rice in Shanghai of will reviving shells, and soaks 15min with 0.1% mercuric chloride solution after 75% alcohol is handled 1min, uses rinsed with sterile water again 3 times, be inoculated into M8 is minimal medium, (prescription of inducing mature embryo callus is M8+2mg/L2,4-D1mg/LABA+1mg/L6-BA, 3% sucrose+7% agar) 28 ℃ of dark conditions are cultivated 30d down on the evoked callus medium of the kind of hormone and proportioning.Downcut the yellow-white dense callus that induces from seed embryonic shield sheet position, be immersed in the bacterium liquid of the Agrobacterium strain GV3010 that carries γ-ECS gene, contaminated 5 minutes, cultivate 48h then altogether, insert again that (the screening and culturing based formulas is except containing antibiotic in the screening culture medium of the kanamycin contain 500mg/L cephalosporin and 50mg/L, other the formula components with the mature embryo inducing culture is identical), behind 3 week, the callus that successfully changes γ-ECS over to promptly survives and becomes resistant calli, there is not the callus of transgene promptly dead, with resistant calli, be transferred in the differential medium, (the differentiation culture based formulas is MS+2,4-D0.2mg/L+6-BA2.0mg/L+KT2.0mg/L+ sucrose 30g/L, pH5.6), induce into seedling behind 2 week, be the rice plant that carries γ-ECS.These plant are planted big Tanaka, the chlorophyll content of sword-like leave and photochemistry efficient (Fv/Fm) originally when blooming 7 days and 21 days by mensuration, filter out the Fv/Fm decay be not higher than 10% or the chlorophyll content decay be not higher than 20% premature senescence resistance material H
1, receive seed, in order to accelerate to obtain seed, with H
1Arrive the plantation of base, Hainan in the winter time for seed, obtain H
2For seed, realized results two generations material in a year.
Fragrant round-grained rice in Shanghai and the γ-ECS-Soviet Union fragrant round-grained rice in Shanghai of will reviving adopts the method for gene magnification (PCR) at first to carry out the Molecular Identification of gene, γ-ECS expression of gene is that amount with the gene magnification under equal in quality of this gene is a standard determination, the number of landing that obtains γ-ECS according to the NCBI gene library is AJ302783., sequences Design by this gene a right margin PCR primer, the sequence of primer is the primer of 5 '-AGGAGCGCCACTAAGTTCAA and a left margin, primer sequence is 5 '-TGCAAAGTTGCCTTTCCCTTT, method by PCR is identified, brightness with the black and white of the gene expression electrophoresis picture of the fragrant round-grained rice in not genetically modified Soviet Union Shanghai is 1, rice material contrast with changeing γ-ECS obtains genetically modified expression intensity.Result's (referring to table 1) shows: γ-ECS-Soviet Union fragrant round-grained rice in Shanghai is compared with the not genetically modified Soviet Union fragrant round-grained rice in Shanghai, has tangible premature senescence resistance.
The evaluation of table 1 γ-ECs-Soviet Union fragrant round-grained rice premature senescence resistance in Shanghai
| Kind | γ-ECS expression of gene | Chlorophyll content decay (%) | Photochemistry efficient (Fv/Fm) originally |
| The fragrant round-grained rice in Soviet Union Shanghai | 1 | 28 | 25 |
| γ-ECS-Soviet Union fragrant the round-grained rice in Shanghai | 15.2 | 12 | 9 |
Described premature senescence resistance characteristic mainly is to judge by the decay of measuring chlorophyll fluorescence parameters Fv/Fm and chlorophyll content.
The mensuration of chlorophyll fluorescence parameters Fv/Fm: (Hansatech UK) measures the chlorophyll fluorescence kinetics parameter with the FMS2 luminoscope.Do with light 1200 μ molm
-2S
-1, saturation pulse light 4000 μ molm
-2S
-1, flash of light (0.9 μ molm
-2S
-1) 2s, interval 30s, each material repeats for 3 times.The fluorescence kinetics parameter is calculated as follows: F
v/ F
m(F
m-F
0) F
m, F wherein
0Be initial fluorescent intensity, F
mBe maximum fluorescence intensity, F
vBe variable fluorescence intensity.The decay of photochemistry efficient=(F originally
v/ F
M7-F
v/ F
M21)/F
v/ F
M7) * 100%
The mensuration of chlorophyll content: first day that blooms in paddy rice begins to list, at the 7th day and 21 days that blooms, get 10 leaf disks on the sword-like leave respectively, each leaf disk diameter is 0.5cm, the ethanol of putting into 10ml96% is in the volumetric flask extracting, and 30 ℃ of extractings are 7 days darkling, measures 665 and the light absorption of 649nm then on spectrophotometer, thereby measure the chlorophyll content of blade, chlorophyllous decay=(Chl
7-Chl
21)/Chl
7) * 100%
The acquisition of the F1 of the embodiment 2 γ-ECS-Soviet Union fragrant round-grained rice in Shanghai/commentaries on classics pepc gene paddy rice
2004, with the material H with premature senescence resistance of embodiment 1 acquisition
2Germplasm is as female parent, has a C according to what the method for the ZL01138012.8 patent disclosure of applications such as Jiao Demao, Gao Dongying, Li Xia obtained
4The rice germplasm of plant pepc gene stably express is a male parent, by hybridization, obtains 112 hybrid F1 seeds.
The F1 screening of embodiment 3 high light efficiencies and premature senescence resistance characteristic
112 hybrid F1 materials that all obtain were planted in winter in 2004 by embodiment 2 in Hainan, by identify PEPC activity, the photosynthetic rate of blade at heading stage, measure sword-like leave bloom back chlorophyll content and Fv/Fm after the 7th day and 21 days in flowering stage, filter out the rice material F of 55 plant height light efficiencies and premature senescence resistance
1Material, all between 260～1099 μ mol/mgChl.h, photosynthetic rate numerical value is at 17.2～29.8 μ mol/m for the material PEPC activity that filters out
2.s between, Fv/Fm decay 5%～10%, chlorophyll content decay at 12-20%, all has high photosynthesis efficiency and premature senescence resistance characteristic, proving can be by sexual hybridization, the material of two kinds of good physiological properties is made up in acquisition.
The evaluation of high light efficiency characteristic is by measuring C
4Active and the leaf photosynthesis speed of photosynthetic enzyme PEPC.
The mensuration of photosynthetic rate:, measure the photosynthetic CO of 2 leaves of paddy rice disjunctor in booting stage with the photosynthetic mensuration of LI-6400 system
2Gas exchange.Source of the gas is compressed air (CO
2Volume fraction is 350 μ l/L), light source is a Halogen lamp LED, changes the light intensity that arrives leaf surface by the distance of regulating between lamp and the leaf chamber.First-class dynamic water tank is arranged to reduce the temperature (the leaf room temperature remains on 30 ℃, and relative moisture is 60%) that arrives the leaf chamber, light quantum flux density (PFD), system CO between lamp and the leaf chamber
2Volume fraction is directly measured by this system, measures 800 μ mol/m
2.s the photosynthetic rate under the light intensity, every bit is measured 4-6 repetition.
The mensuration of PEPC activity: water intaking rice second from the bottom blade 0.5g at heading stage, clean with distilled water, use the filter paper suck dry moisture, be cut into small pieces, be placed in 4 ℃ of freezing in advance mortars with scissors, add a small amount of liquid nitrogen (196 ℃), freezing blade, grind into powder, [composition that extracts buffer solution is: 50mmol/L Tris-HCl (pH7.8) to add the extraction buffer extraction of 2ml then, contain the 5mmol/L dithiothreitol (DTT), 1mmol/L MgCl
2With 2% polyvinylpyrrolidone], lapping liquid is got 40 μ l and is added in the 960 μ l absolute ethyl alcohols with four layers of filtered through gauze, filtrate, measures absorbance value respectively under 649nm and 665nm, calculates chlorophyll content.Remaining filtrate is at 10000 * g, and 4 ℃ of centrifugal 20min, supernatant are the crude enzyme liquid of measuring enzymic activity.The distilled water that in measuring the enzymic activity reaction system, adds 495 μ l, 50 μ molHepes-KOH (pH8.0), 10mmol NaHCO
3, 5mmol MgCl
2, 0.2 μ mol NADH, 50 μ l crude enzyme liquids, the 1.5unit malate dehydrogenase adds 5 μ mol PEP behind the mixing, and reaction 5min measures the variation of light absorption under the 340nm.
The mensuration of premature senescence resistance characteristic and decision method are identical with the method that relates among the embodiment 1.
The acquisition and the evaluation of the stabilizing material of the embodiment 4 γ-ECS-Soviet Union fragrant round-grained rice in Shanghai/commentaries on classics pepc gene paddy rice
For the above-mentioned two kinds of good physiological properties of rapid polymerization and fast and stable get off, 2005,55 strains that continuation obtains embodiment 3 have rice material positive season plantation in Nanjing of high light efficiency and premature senescence resistance characteristic, by the anther culture fast and stable, the physiological property of simultaneously further high light efficiency of repetitive identified and premature senescence resistance is investigated the stability of two kinds of physiological traits the offspring.Specific practice is rice plant vertically to be broken with knife at a minute evil phase rice material, is divided into two, and divides in two groups of basin alms bowls of being transplanted to 10L, and one group is used for anther culture, and another group is used for the repetitive identified of high light efficiency premature senescence resistance characteristic.
Be used for anther cultural rice material August 15, get the flower pesticide of F1 monokaryotic stage and cultivate, anther culture medium is named and is H
8, its prescription is: be minimal medium with M8, add 2,4-D1mg/L+ methyl (NAA) the 3mg/L+ basic element of cell division (KT) 1mg/L+ abscisic acid (ABA) 3mg/L+ proline 100mg/L+ caseinhydrolysate 300mg/L+ sucrose 50g/L+ agar 8g/L.Spike of rice with the γ-ECS-Soviet Union fragrant round-grained rice in Shanghai/commentaries on classics pepc gene paddy rice F1 monokaryotic stage, putting into 4 ℃ handled 10 days down, be inoculated in then in the medium of H8, anther culture is inoculated 4561 pieces of flower pesticide altogether, obtain 537 callus lines, and γ-the ECS-Soviet Union fragrant round-grained rice in Shanghai in contrast, also at 545 pieces of flower pesticide of F1 monokaryotic stage inoculation, obtain 46 callus lines, the callus lines that two above-mentioned materials are induced all in MS6 seedling differentiation (the MS6 culture medium prescription is: be minimal medium with MS, additional 2mg/L KT+1mg/L NAA+ sucrose 30g/L+ agar 6g/L), wherein γ-ECS-Soviet Union fragrant the round-grained rice in Shanghai/commentaries on classics pepc gene paddy rice F1 differentiates 75 clumps of green seedlings, and contrast γ-ECS-Soviet Union fragrant round-grained rice in Shanghai differentiates 11 clumps of green seedlings.
Table 2 Rice Anther Culture situation
| Kind or combination | The flower pesticide number of inoculation (piece) | Go out more number (healing rate %) | Green seedling number (seedling greening-rate) |
| γ-ECS-Soviet Union fragrant the round-grained rice in Shanghai | 545 | 46(8.44％) | 11(2％) |
| γ-ECS-Soviet Union fragrant the round-grained rice in Shanghai/commentaries on classics pepc gene paddy rice | 4561 | 537(11.77％) | 75(1.64％) |
The rice material that another group is used for the physiology evaluation carries out the evaluation of high light efficiency and premature senescence resistance characteristic according to the method for embodiment 3 in flowering stage, F2 separation occurred for material as a result, from the planting material in 55 strain F1 generations, all between 270～1019 μ mol/mgChl.h, photosynthetic rate numerical value is at 17.2～30.8 μ mol/m for the PEPC activity of the 20 strain rice materials of identifying
2.s between, Fv/Fm decay 7%～10% or the chlorophyll content decay at 13-20%, all have high photosynthesis efficiency and premature senescence resistance characteristic, the physiology authenticate technology that shows high light efficiency and premature senescence resistance is reliable, the progeny material that can stable screening goes out to have two kinds of good photosynthetic physiological characteristics, but separation can appear in the progeny material of the good physiological property that obtains by sexual hybridization merely.
In order to obtain the seed of stable flower training material, further plant the green seedling of 75 strains that the F1 flower pesticide of γ-ECS-Soviet Union fragrant round-grained rice in Shanghai/commentaries on classics pepc gene paddy rice induces November in the same year in Hainan, and (plant is little to determine monoploid according to the external form of plant, the grain husk shell is little, shaky), dliploid (solid normal) and polyploid (plant is bigger, and awns, shaky or solid extremely low is arranged).75 from green seedling, 25 strain dliploids are arranged, press individual plant results seed.The high light efficiency that 25 strain paddy rice liploid plants carry out and the qualification result of premature senescence resistance characteristic show: all between 290～1069 μ mol/mgChl.h, photosynthetic rate numerical value is at 16.2～28.8 μ mol/m for their PEPC activity
2.s between, Fv/Fm decay 6%～10% or the chlorophyll content decay at 14-20%, prove that anther culture has all kept high photosynthesis efficiency and premature senescence resistance characteristic.
Embodiment 5 stablizes the premature senescence resistance that the strain of the γ-ECS-Soviet Union fragrant round-grained rice in Shanghai/commentariess on classics pepc gene paddy rice is and the evaluation of high light efficiency physiological property
The 25 strain diploid rice plant that will obtain in 2006 divide individual plant to tie up to the Nanjing plantation, and each strain system finds in its stability of field observation that with 15 of 3 10L basin alms bowl plantations, 5 of every basin kinds all these strain systems all are stable, do not occur separating.Pass through to repeat the high light efficiency of embodiment 3 and the authentication method of premature senescence resistance in the flowering stage of these strain systems, all between 299～1019 μ mol/mgChl.h, photosynthetic rate numerical value is at 17.2～29.8 μ mol/m for their PEPC activity
2.s between, Fv/Fm decay 5%～10% or the chlorophyll content decay at 12-20%, but prove high photosynthesis efficiency and the premature senescence resistance characteristic of anther culture fast and stable paddy rice.
Further investigate its yield traits, it is well relevant to find that high photosynthesis efficiency and premature senescence resistance characteristic and economic flow rate also have, it is good that screening acquires 6 strain plant types at last, have premature senescence resistance and the good physiological trait of high light efficiency and thousand kernel weight and more be sheerly than changeing pepc gene paddy rice (male parent) and γ-ECS-Soviet Union's fragrant round-grained rice in Shanghai (female parent), name and be EP, [EP gets the first English alphabet abbreviation that is respectively γ-ECS and Photosynthesis (photosynthesis) and forms], 6 strains systems names respectively and is EP-1, EP-2, EP-3, EP-4, EP-5, EP-6 (the results are shown in Table 3), show and to obtain stabilizing material fast by the method for embodiment 1 to 4 with two kinds of good physiological properties.
The evaluation of high light efficiency of table 3 and premature senescence resistance characteristic
| Kind or combination | PEPC activity (μ mol/mg.h) | Photosynthetic rate (μ mol/m 2.s) | Chlorophyll content decay (%) | Photochemistry efficient decay (%) originally | 1000 weights of seed output (g) |
| Change the pepc gene paddy rice | 1244-1321 | 32.5 | 25 | 19 | 25.4 |
| γ-ECS-Soviet Union fragrant the round-grained rice in Shanghai | 55.4 | 15.9 | 12 | 9 | 27.6 |
| EP-1 | 967 | 20.1 | 19 | 8 | 29.3 |
| EP-2 | 1089 | 29.8 | 20 | 7 | 30.2 |
| EP-3 | 238 | 23.9 | 20 | 9 | 29.4 |
| EP-4 | 357 | 19.9 | 18 | 9.8 | 31.9 |
| EP-5 | 275 | 17.4 | 14 | 10 | 32.1 |
| EP-6 | 679 | 16.2 | 17 | 6 | 30.5 |
More than each embodiment be not to concrete restriction of the present invention.
Claims (6)
1, a kind of rice breeding method that merges high light efficiency and premature senescence resistance proterties is characterized in that:
A) the early ageing related gene is imported in the rice germplasm of high-quality, obtain the rice strain of premature senescence resistance;
B) will have C
4The rice germplasm of plant pepc gene stably express and the rice strain's hybridization with premature senescence resistance characteristic obtain the F1 hybrid;
C) physiology that the F1 hybrid is carried out high light efficiency and premature senescence resistance characteristic is identified, filters out the rice material with high light efficiency and premature senescence resistance characteristic;
The F1 with two kinds of good physiological properties that d) will filter out cultivates by pollen, obtains the stable flower training strain system with two kinds of good physiological properties;
E) be to carry out the physiology of high light efficiency, premature senescence resistance to identify to stable flower training strain, the screening back obtains the paddy rice pure lines of described two the good physiological traits of fusion.
2, the rice breeding method of high light efficiency of fusion according to claim 1 and premature senescence resistance proterties is characterized in that: the rice germplasm of described high-quality is the fragrant round-grained rice in Soviet Union Shanghai; Be meant in the described rice germplasm: change in the fragrant round-grained rice in Soviet Union Shanghai by the gamma-glutamyl amine cysteine synthase γ-ECS gene of agrobacterium mediation method with corn with early ageing related gene importing high-quality; In the described rice germplasm with early ageing related gene importing high-quality, the rice strain that obtains premature senescence resistance is meant: by agrobacterium mediation method with the γ-ECS gene of corn change over to obtain the rice strain of premature senescence resistance in the fragrant round-grained rice in Soviet Union Shanghai after, the chlorophyll content of sword-like leave and photochemistry efficient originally when blooming 7 days and 21 days by mensuration, filter out the Fv/Fm decay be not higher than 10% or the chlorophyll content decay be not higher than 20% premature senescence resistance material H
1, and make H
1In generation, multiplied H
2In generation, the rice plant of γ-ECS gene is carried in acquisition.
3, the rice breeding method of high light efficiency of fusion according to claim 1 and premature senescence resistance proterties is characterized in that: described will have C
4The rice germplasm of plant pepc gene stably express is meant with the rice strain's hybridization with premature senescence resistance characteristic: have C
4The rice germplasm of plant pepc gene stably express is a male parent, is female parent with the rice plant that carries γ-ECS gene.
4, the rice breeding method of high light efficiency of fusion according to claim 1 and premature senescence resistance proterties, it is characterized in that: the described physiology that carries out high light efficiency and premature senescence resistance characteristic is identified, filters out the rice material with high light efficiency and premature senescence resistance characteristic and is meant: by measuring C
4Active and the leaf photosynthesis speed of photosynthetic enzyme, and measure when blooming 7 days and 21 days sword-like leave originally photochemistry efficient (Fv/Fm) and chlorophyll content, filter out the PEPC activity between 250～1089 μ mol/mgChl.h, photosynthetic rate numerical value is at 16.2～29.8 μ mol/m
2.s between, Fv/Fm decay simultaneously be not higher than 10% or the chlorophyll content decay be not higher than 20% rice material.
5, the rice breeding method of high light efficiency of fusion according to claim 1 and premature senescence resistance proterties, it is characterized in that: the described F1 with two kinds of good physiological properties that will filter out cultivates by pollen, obtaining the stable flower training strain system with two kinds of good physiological properties is meant: the spike of rice of choosing the F1 monokaryotic stage, putting into 4 ℃ handled 10 days down, be inoculated in then in the medium of H8, induce callus, change seedling differentiation among the differential medium MS6 again over to, after the flower training seed that obtains is planted, keep dliploid, thereby obtain stable flower training strain system.
6, the rice breeding method of high light efficiency of fusion according to claim 5 and premature senescence resistance proterties is characterized in that: described H
8The prescription of medium is: be minimal medium with M8, add 2,4-D1mg/L+ methyl (NAA) the 3mg/L+ basic element of cell division (KT) 1mg/L+ abscisic acid (ABA) 3mg/L+ proline 100mg/L+ caseinhydrolysate 300mg/L+ sucrose 50g/L+ agar 8g/L; Described MS6 culture medium prescription is: with MS minimal medium, and additional 2mg/L KT+1mg/LNAA+ sucrose 30g/L+ agar 6g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006101616497A CN100497637C (en) | 2006-12-30 | 2006-12-30 | Rice breeding method incorporating high photosynthetic efficiency and presenility resistant property |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006101616497A CN100497637C (en) | 2006-12-30 | 2006-12-30 | Rice breeding method incorporating high photosynthetic efficiency and presenility resistant property |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1994063A true CN1994063A (en) | 2007-07-11 |
| CN100497637C CN100497637C (en) | 2009-06-10 |
Family
ID=38249342
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006101616497A Expired - Fee Related CN100497637C (en) | 2006-12-30 | 2006-12-30 | Rice breeding method incorporating high photosynthetic efficiency and presenility resistant property |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100497637C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103141376A (en) * | 2013-03-29 | 2013-06-12 | 江苏省农业科学院 | Cultivation method of rice germplasm with high-resistant starch and low-amylose starch |
| CN105830758A (en) * | 2016-03-30 | 2016-08-10 | 中棉种业科技股份有限公司 | Method for identifying premature senescence resistance of cotton |
| CN109380076A (en) * | 2018-12-12 | 2019-02-26 | 怀化市共生农业系统工程研究所(普通合伙) | A kind of breeding method of high photosynthetic efficiency rice |
| CN114350701A (en) * | 2022-01-06 | 2022-04-15 | 武汉大学 | Method for preparing angiosperm haploid by egg cell specific expression gene ECS and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1149917C (en) * | 2001-12-21 | 2004-05-19 | 焦德茂 | Breeding technique capable of making C4 plant PEPC gene transferred rice stably express |
-
2006
- 2006-12-30 CN CNB2006101616497A patent/CN100497637C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103141376A (en) * | 2013-03-29 | 2013-06-12 | 江苏省农业科学院 | Cultivation method of rice germplasm with high-resistant starch and low-amylose starch |
| CN103141376B (en) * | 2013-03-29 | 2014-08-06 | 江苏省农业科学院 | Cultivation method of rice germplasm with high-resistant starch and low-amylose starch |
| CN105830758A (en) * | 2016-03-30 | 2016-08-10 | 中棉种业科技股份有限公司 | Method for identifying premature senescence resistance of cotton |
| CN109380076A (en) * | 2018-12-12 | 2019-02-26 | 怀化市共生农业系统工程研究所(普通合伙) | A kind of breeding method of high photosynthetic efficiency rice |
| CN114350701A (en) * | 2022-01-06 | 2022-04-15 | 武汉大学 | Method for preparing angiosperm haploid by egg cell specific expression gene ECS and application |
| CN114350701B (en) * | 2022-01-06 | 2024-04-19 | 武汉大学 | Method for preparing angiosperm haploid by egg cell specific expression gene ECS and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100497637C (en) | 2009-06-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shao et al. | Using CRISPR/Cas9 genome editing system to create MaGA20ox2 gene‐modified semi‐dwarf banana | |
| CN102776201B (en) | Application of OsELF 3 gene in controlling heading stage of paddy rice | |
| CN101142894A (en) | Wild-rice distant hybridization high-efficient cultivating superior progeny method | |
| CN100355897C (en) | Method for promoting salt and drought tolerance of maize and wheat by combining betA,NHX1,PPase gene and transgene technology | |
| CN102732526B (en) | Application of OsSRO1c gene in controlling rice drought resistance | |
| CN110117320A (en) | Cotton GhCAL-D07 gene is promoting the application in flowering of plant | |
| CN102719433B (en) | Application of osa-MIR167a gene for regulating and controlling plant type of paddy rice | |
| de Almeida et al. | Somatic embryogenesis from flower tepals of Hippeastrum aiming regeneration of virus-free plants | |
| CN108085334B (en) | Improved method for transforming barley microspore by agrobacterium | |
| CN109112140A (en) | Application of the OsSN28 gene in control rice high temperature resistant | |
| CN100497637C (en) | Rice breeding method incorporating high photosynthetic efficiency and presenility resistant property | |
| CN101705234A (en) | Application of DSM1gene of MAPKKK family genes in controlling rice drought resistance | |
| CN103060285B (en) | Application of OsPP18 gene on control of rice drought resistance | |
| Pan et al. | Aerenchyma formation in the root of leaf‐vegetable sweet potato: Programmed cell death initiated by ethylene‐mediated H2O2 accumulation | |
| CN110819635A (en) | Application of HAN homologous gene of leguminous plant in regulating and controlling number of root nodules of leguminous plant | |
| Bouharmont | Application of somaclonal variation and in vitro selection to plant improvement | |
| CN105028184A (en) | Rice fragrant and soft rice variety selecting and culturing method | |
| CN101948870B (en) | Method for reducing branch quantity of plant and improving chlorophyll and anthocyanin contents of plant | |
| CN107593626A (en) | Utilize the method for live body plant hand-feeding propagation corn leaf aphids | |
| CN1768575A (en) | Method for improving beet salt-resistance, draught-resistance and anti-herbicide chlorsulfuron characteristics by multi-gene transformation and application | |
| Venema et al. | Analysis of low-temperature tolerance of a tomato (Lycopersicon esculentum) cybrid with chloroplasts from a more chilling-tolerant L. hirsutum accession | |
| CN104726488A (en) | Method for culturing stress-resistance herbicide-resistance transgenic aerobic rice | |
| CN114027180A (en) | Culture method and application of shiny-leaved yellowhorn polyploidy | |
| CN103088056A (en) | Application of gene PHYB in controlling paddy rice low temperature stress tolerance | |
| CN113234700A (en) | Rice calcineurin B protein and application thereof in breeding |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090610 Termination date: 20111230 |