CN1341350A - Method for high-efficiency breeding rice polyploid by combination of tissue culture and chemical induction - Google Patents
Method for high-efficiency breeding rice polyploid by combination of tissue culture and chemical induction Download PDFInfo
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Abstract
The method for cultivating rice polyploid includes the folloiwng steps: using rice testing material, optimized wound tissue culture method, combining tissue culture and colchicin treatment to induce and form polyploid, obtain polyploid plant, determine polyploid plant and cultivate polyploid plant. The application of said invention can successfully induce the Asiatic cultivated rices, including hsien rice, round-grained nonglutinous rice. Java rice and hybrid between them, to reduplicatively form polyloid. It can make reduplicate rice line, variety, hybrid, wild rice strain and hybrid up to 152 kinds, so that said invention can lay the material foundation for making theoretical research of rice genome evolution and heredity and making super rice and super hybrid rice breeding.
Description
Technical field
The present invention relates to the technical method that a kind of high efficiency forms the paddy rice polyploid, basic point is to utilize the explant of seed, young fringe and three different levels of stem section, the advantage of performance tissue culture and chemical induction combination, and high efficiency is cultivated the paddy rice polyploid in high quality.
Background information
Constantly worsen at environment; the population sustainable growth; the soil sharply reduces; under the urgent situation of food shortage potential collision hazard; world's rice breeding circle is carrying out super hybridization rice and super hybridized rice breeding; wherein " the utilizing distant hybridization and double dominancy of polyploid seed selection super hybridization rice " that proposes in disclosed No. 00114471.5 patent application of Chinese patent is a new breeding strategy, causes the attention of Chinese scholars just day by day.Realize the polyploid rice breeding, at first will solve the problem that how to obtain polyploid.For a long time, the method that obtains the paddy rice polyploid mainly contains two kinds, and a kind of is to handle the seed or the seeds germinated of soaking with colchicine; Another kind is to utilize colchicine to handle developmental rice shoot.These two kinds of methods exist two subject matters: the one, and the frequency that doubles to form polyploid rice is low, generally about 5～10%; The 2nd, produce chimera, on the promptly same rice plant, knot dliploid seed on the tassel that has, knot polyploid seed had both been tied the dliploid seed on the tassel that has on the tassel that also has, and also tied the polyploid seed.Influenced the efficient of polyploid rice breeding so greatly, especially be difficult to double to handle with it for several hybrid seeds that are difficult to obtain.Although Fan Kunhua etc. (1991), Huang Huijun etc. (1995) also application organizes cultivation carry out the research of paddy rice polyploid, still there are some problems in aspects such as used explant, condition of culture and induction frequency.Therefore, since nineteen fifty-one Bao Wenkui begins the research of paddy rice polyploid, Chinese scholars mainly is confined to Asia cultivated rice kind, between kind or the research of the autopolyploid of subspecies indica and japonica hybrid, and to African cultivated rice and numerous wild rice, especially right and wrong A genome wild rice do not carry out polyploid double research.
Summary of the invention
The Material Used that the objective of the invention is to avoid above-mentioned multiploid induction to exist in forming is single, range of application is narrow, induce efficient not high, easily produce deficiencies such as chimera, the examination material that a kind of whole-process application paddy rice developmental stage is provided is tissue culture, optimize tissue culture technique, high efficiency, the method for inducing paddy rice polyploid in high quality.
Purpose of the present invention can reach by following measure:
A kind of tissue culture and the chemical induction method that the high efficiency high-quality is cultivated the paddy rice polyploid that combines comprises whole-process application paddy rice examination material, optimizes callus culture method, conjunctive tissue is cultivated and colchicine is handled to induce and formed polyploid, the acquisition of polyploid plant, the evaluation and the cultivation of polyploid plant.
Be detailed incubation step below:
When adopting rice paddy seed and young fringe to make explant, need through following seven cultivation stage: a, callus induction, b, colchicine liquid culture double, c, successive transfer culture, d, the differentiation cultivation of sprouting, e, culture of rootage, f, strong seedling culture, g, the maturation of growing seedlings and polyploid plant are identified.
When adopting rice stem section (the band joint contains resting bud) for explant, need are induced through following seven cultivation stage: a, clump bud and are bred, b, colchicine liquid culture double, c, successive transfer culture, d, the growth of clump bud, e, culture of rootage, f, strong seedling culture, g, the maturation of growing seedlings and polyploid plant are identified.
Specific operation process is:
One, when adopting rice paddy seed and young fringe to make explant.
A, callus induction process are, cultivated rice, wild rice, cultivated rice x wild rice hybrid seed or young fringe are inoculated in N6 (or MS)+2 after sterile-processed, in the inducing culture of 4-D 1～2.5mg/l+NAA 1～2mg/l+6BA (or KT) 0.1～0.5mg/l+ sucrose 5～6%+ caseinhydrolysate 5～150mg/l+ agar 0.65～0.75%, pH5.8～6.2, under 25～30 ℃, dark or illumination 800～10001x, cultivated 25～45 days and form callus;
B, colchicine liquid culture double process and are, the callus of the cultivated rice that will form in inducing culture, wild rice, cultivated rice x wild rice hybrid is transferred to N6 (or MS)+2, doubling in the medium of 4-D1～2.5mg/l+NAA 1～2mg/l+6BA (or KT) 0.1-0.5mg/l+ colchicine 500-700mg/l+ sucrose 3～5%+ caseinhydrolysate 100mg/l, pH5.8～6.2, shaken cultivation is 40～48 hours under 20～22 ℃, 95～105rpm rotating speed;
C, successive transfer culture process are, the callus of the cultivated rice that doubles to handle, wild rice, cultivated rice x wild rice hybrid after fully washing, sterile water is changed in the medium identical with above-mentioned inducing culture under 25～30 ℃ of temperature, dark or illumination 600～1000 1x successive transfer culture 7-10 days;
D, the differentiation incubation of sprouting are, the callus of cultivated rice behind the subculture, wild rice, cultivated rice x wild rice hybrid is transferred in the differential medium of MS+6BA 1～2mg/l+KT 2～3mg/l+NAA 0.2～0.5mg/l+ caseinhydrolysate 100～200mg/l+ sucrose 3%+ agar 0.65～0.75%, pH5.8～6.2, differentiation culture is 20～45 days under 25～30 ℃, illumination 1500～2000 1x, differentiates paddy rice bud seedling;
E, process of rooting culture are, to change over to through the paddy rice bud seedling that the callus differentiation culture goes out in the root media of 1/2MS+6BA 0.1～0.5mg/l+KT 0.1～0.2mg/l+NAA 0.2～0.3mg/l+IAA 0.5～1mg/l+ caseinhydrolysate 100mg/l+ activated carbon 0.03%+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 and take root, grow complete plantlet;
F, strong seedling culture process be, grows into healthy and strong plantlet with deriving from the strong seedling culture base that the complete plantlet of paddy rice that callus is differentiated to form changes 1/2 MS+6BA 0.5mg/l+KT, 0.1～0.2mg/l+NAA, 0.2～0.3mg/l+ paclobutrazol, 2～3mg/l+ caseinhydrolysate, 100～200mg/l+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 over to;
G, the maturation of growing seedlings and polyploid plant qualification process are, healthy and strong plantlet with the strong seedling culture growth, be transplanted to by 2/3 sand and 1/3 fine earth that becomes thoroughly decomposed and form in the cultivating container of compost, transplanting survival is also cultivated plant to ripe, in process of growth, differentiate the polyploid rice plant according to morphological feature (having awns, big grain, leaf to increase thick, cane chap) and cytogenetics evidence (the blade pore increases, root tip chromosomes is multiplied).
Two, when adopting rice stem section (the band joint contains resting bud) for explant.
A, clump bud are induced with breeding and are, the stem section of cultivated rice, wild rice, cultivated rice x wild rice hybrid plant is inoculated into the inducing in the many clumps of bud medium of 2/3MS+6BA 1～3mg/l+KT 2～3mg/l+NAA 0.2～0.5mg/l+ paclobutrazol 3～5mg/l+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 after sterile-processed, under 25～30 ℃, illumination 1500～2000 1x, cultivated 10～30 days and form many clumps of buds;
B, colchicine liquid culture double process and are, the many clumps of buds of the cultivated rice that forms, wild rice, cultivated rice x wild rice hybrid are transferred in the medium of 1/2MS+6BA 1～2mg/l+KT 2～3mg/l+NAA 0.2～0.5mg/l+ paclobutrazol 3～5mg/l+ colchicine 500-700mg/l+ sucrose 2～3%, pH5.8-6.2, and shaken cultivation is 18～24 hours on the shaking table of illumination 500～800 1x, 20～22 ℃ of temperature, rotating speed 95～105rpm;
C, successive transfer culture process are, the many clumps of buds of the cultivated rice that the clump bud is doubled to handle, wild rice, cultivated rice x wild rice hybrid change over to after sterile water fully washs with above-mentioned and induce in the identical medium of the many clumps of bud medium, and successive transfer culture is 7～10 days under 25～30 ℃ of temperature, illumination 1500～2000 1x;
D, clump bud process of growth are, the many clumps of buds of the cultivated rice of successive transfer culture, wild rice, cultivated rice x wild rice hybrid are transferred to after sterile water fully washs in the medium of 1/2MS+6BA 1-2mg/l+KT 2-3mg/l+NAA 0.2-0.5mg/l+ paclobutrazol 3-5mg/L+ caseinhydrolysate 100-200mg/l+ sucrose 2%+ agar 0.65-0.75%, pH5.8-6.2, under illumination 1500-2000 1x, temperature 25-30 ℃, turn out many clumps of bud seedlings;
E, process of rooting culture are, the many clumps of bud seedlings of cultivated rice, wild rice, cultivated rice x wild rice hybrid are changed in the root media of 1/2MS+6BA 0.1～0.5mg/l+KT 0.1～0.2mg/l+NAA 0.2～0.3mg/l+IAA 0.5～1mg/l+ caseinhydrolysate 100mg/l+ activated carbon 0.03%+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 and take root, grow complete plantlet;
F, strong seedling culture process be, grows into healthy and strong plantlet with deriving from the strong seedling culture base that paddy rice plantlet that many clumps of buds propagation form changes 1/2MS+6BA 0.5mg/l+KT 0.1～0.2mg/l+NAA 0.2～0.3mg/l+ paclobutrazol 2～3mg/l+ caseinhydrolysate 100～200mg/l+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 over to;
G, the maturation of growing seedlings and polyploid plant qualification process are, healthy and strong plantlet with the strong seedling culture growth, be transplanted to by 2/3 sand and 1/3 fine earth that becomes thoroughly decomposed and form in the cultivating container of compost, transplanting survival is also cultivated plant to ripe, differentiates the polyploid rice plant according to morphological feature and cytogenetics evidence in process of growth.
Find out that from above application test material of the present invention has obviously different with the research of in the past polyploid rice:
(1) uses prematurity or ripe seed, the stem section of young fringe in growing and heading back band joint is a material, break through common paddy rice multiploid induction and mainly used the limitation of seedling in tillering stage, widened the type of Material Used, in paddy rice from seed development, seedling grows to can be as the examination material in the full growing process of blooming of earing, handle and the formation polyploid plant through tissue culture and colchicine, promptly do not double under the case of successful in the phase I (seed), can handle at second stage (young fringe), first, second stage does not use the phase III (stem section) to handle under the case of successful, guarantees that the minority provenance can double to form polyploid; (2) enlarge the scope of using.Changed the state that original multiploid induction is only used in the cultivated rice of Asia, the multiploid induction that this method is applied to A genome wild rice such as long-grained nonglutinous rice, japonica rice and java rice kind, indica and japonica subspecies hybrid, African cultivated rice kind, Asia cultivated rice and the African cultivated rice species hybrid of Asia cultivated rice, African wild rice and the genomic wild rice species of other non-A and cultivated rice and wild rice hybrid forms.(3) simultaneously also as can be seen, the present invention has optimized method for tissue culture.
The tissue culture reaction of Oryza different plant species exists evident difference.Generally say, cultivated rice is than the easier tissue culture of wild rice, A genome wild rice is more easily cultivated than non-A genome wild rice, thereby shows a basic trend on the easy degree of cultivation, i.e. japonica rice＞java rice＞long-grained nonglutinous rice＞A genome wild rice＞non-A genome wild rice.Because former research only forms at the training method of the tissue culture of long-grained nonglutinous rice, japonica rice and tissue culture and the multiploid induction that medium can not be adapted to more seed rice.So when making explant with seed and young fringe, adopt callus induction, the colchicine liquid culture doubles, successive transfer culture breaks up the cultivation of sprouting, six cultivation stages such as culture of rootage and strong seedling culture; When being explant with the stem section, employing clump bud is induced and is bred, and the colchicine liquid culture doubles, successive transfer culture, the growth of clump bud, culture of rootage and six cultivation stages of strong seedling culture.Inducing the seed that forms callus can be immature seed, also can be mature seed, and young fringe is good with branch stalk for the second time to female stamen formation period, and the stem section must be with joint, promptly contains the bottom sections of resting bud.
The medium of inducing seed or young fringe to form callus is N6 (MS)+2,4-D 1～2.5mg/l+NAA 1～2mg/l+6BA (KT) 0.1～0.5mg/l+ sucrose 5～6%+ caseinhydrolysate 50～150mg/l+ agar 0.65-0.75%, pH5.8-6.2.Easily the japonica rice of cultivating, rice variety and hybrid are adopted the lower content of concentration, and the difficult wild rice of cultivating adopts the combination of higher concentration, cultivate under 25-30 ℃, dark or 800-10001x and form callus in 25-45 days.Two types auxins combination more helps the formation of callus, and the interpolation of the basic element of cell division of low concentration makes callus growth structure and physiological status more help follow-up differentiation culture.
The inducing culture of many clumps of buds is 2/3 MS+6BA 1-3mg/l+KT 2-3mg/l+NAA0.2-0.5mg/l+ paclobutrazol 3-5mg/l+ sucrose 2%+ agar 0.65-0.75%, pH5.8-6.2.
Induce in the formation polyploid in the colchicine processing, the present invention adopts liquid oscilaltion continue to cultivate to be the callus behind inducing culture and many clumps of buds are changed over to and carries out somatic double in the liquid nutrient medium, increased colchicine and entered chance in the cell, improved polyploid and form frequency.Both improved and doubled effect, reduced the colchicine consumption again.
The present invention compared with former live body (being the whole plant under the natural conditions) polyploid rice inductive technology has following advantage:
1. widened test material, vitals----seed, young fringe and stem section (including resting bud) in the overall process that paddy rice grows can be guaranteed just can induce polyploid with a few materials from three levels as the material of induction polyploid.
2. according to the characteristics of different rice materials, optimize culture technique at aspects such as medium component, training methods, not only make cultivated rice kind and hybrid can be used in multiploid induction, and wild rice, cultivated rice x wild rice hybrid also can be used in multiploid induction, enlarged range of application.
3. the technology that adopts tissue culture and chemical induction to combine, brought into play the advantage of a large amount of proliferative cell propagation of tissue culture bud, and the interpolation colchicine doubles to handle in continuing cultivation in the liquid medium within, colchicine can be contacted with cell fully plays effectiveness, improved the frequency that forms polyploid, the 5-10% that is handled by live body adds overtones band, brings up to 46.7%, is up to 75.0%; And reduced the concentration of colchicine.The present invention only uses the colchicine of 0.05-0.07%, and former most of polyploid plant to induce working concentration be 0.2-0.3%.
4. the paddy rice kind shoot survival percent after the colchicine under normal condition is handled is low to be one of subject matter of live plant induction polyploid.Serial training measures such as the present invention adopts inducing under the aseptic condition, breaks up, takes root, strong seedling culture, the efficient that the paddy rice seedling is doubled improves, and the plantlet that has doubled can stalwartness be grown up in test tube, and can be transplanted in the culture vessel, until growing up to ripe rice plant. high-servival rate
5. overcome the shortcoming that live body plant multiploid induction mainly produces polyploid and the chimeric rice plant of dliploid.Because live body plant multiploid induction is used mainly is seedling in germinateing, the rice shoot of perhaps tillering, there are cell that is dividing and the cell that does not divide in its growing point position, the cell of division is suppressed by colchicine and forms polyploid cell, it is grown with the diploid cell that does not change and forms the chimera plant, and dliploid is preponderated often.The present invention utilizes colchicine to handle vigorous developmental callus to be had obviously different with many clumps of bud original hases and whole plant.Can break up independently in the mode of individual cells origin after many cells in the callus double and sprout, grow into complete polyploid rice, the chimera phenomenon is seldom arranged; Growing point cell in the many clumps of bud original hases is under lasting liquid culture is handled, and most cells are doubled and have quadrupled body, thereby forms complete polyploid plant in proliferate subsequently, and the chimera phenomenon is also seldom arranged.
The specific embodiment
Use the inventive method, we comprise long-grained nonglutinous rice, japonica rice, Java successfully with Asian Cultivated Rice Rice varieties and the hybrid between them comprise that especially two is sterile line and restorer, band wide compatibility gene product Kind, band is the sterile line strain to two of herbicide sensitive gene, African cultivated rice, and African wild rice, and Asian Cultivated Rice and African wild rice hybrid, Asian Cultivated Rice and oryza officinalis hybrid (AACC), Asia Continent cultivated rice and granule wild rice hybrid (AABBCC), Asian Cultivated Rice and oryza meyeriana hybrid ( AAGG) etc. induce and double to form polyploid. Make the rice strain, kind, hybrid, the wild rice thing that double Kind and hybrid etc. reached more than 152, realized between the cultivated rice kind, between subspecies, kind, cultivated rice is wild Between seed rice, the filling in the gaps to complete a chain of cultivated rice wild rice genome species hybrid polyploid, advance for carrying out rice genome Change with the theoretical research of heredity and carry out super hybridization rice and Breeding on Super Hybrid Rice has been established solid material foundation.
Specific embodiment and effect are seen attached list.
Subordinate list: use " tissue cultivation and chemical induction combine high-efficiency breeding rice polyploid " embodiment And technique effect.
| Sequence number | Species and kind (being) title | Species and variety type | Explant type | Process material number (individual) | The regeneration plant number | The tetraploid plant number | Add overtones band (%) |
| 1 | W9874s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 10 | 16 | 7 | 43.75 |
| 2 | W9740s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 10 | 17 | 7 | 41.18 |
| 3 | N108s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 15 | 21 | 6 | 28.57 |
| 4 | N9643s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 10 | 13 | 2 | 15.38 |
| 5 | N9899s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 10 | 18 | 4 | 22.22 |
| 6 | N95076s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 15 | 27 | 8 | 29.63 |
| 7 | 810s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 10 | 12 | 3 | 25.0 |
| 8 | PA64s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 15 | 20 | 6 | 30.0 |
| 9 | 8902s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 15 | 17 | 7 | 41.18 |
| 10 | 8077s① | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 15 | 20 | 7 | 35.0 |
| 11 | 3186s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 10 | 12 | 5 | 41.67 |
| 12 | 5088s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 10 | 24 | 12 | 50.0 |
| 13 | 7001s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 10 | 13 | 6 | 46.15 |
| 14 | 29130s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 15 | 54 | 31 | 57.41 |
| 15 | 2301s | Long-grained nonglutinous rice photoperiod-temperature sensitive male sterility system | Seed | 10 | 12 | 5 | 41.67 |
| 16 | A33 | Long-grained nonglutinous rice | Seed | 15 | 17 | 7 | 41.18 |
| Sequence number | Species and kind (being) title | Species and variety type | Explant type | Process material number (individual) | The regeneration plant number | The tetraploid plant number | Add overtones band (%) |
| 17 | Osmanthus 99 | Long-grained nonglutinous rice | Seed | 15 | 33 | 12 | 36.36 |
| 18 | In spend No. 14 | Japonica rice | Seed | 15 | 21 | 7 | 33.33 |
| 19 | New 14 | Long-grained nonglutinous rice | Children's fringe | 15 | 13 | 5 | 38.46 |
| 20 | Become extensive 448 | Long-grained nonglutinous rice, restorer | Children's fringe | 10 | 7 | 3 | 42.86 |
| 21 | A22 | Long-grained nonglutinous rice | Children's fringe | 10 | 4 | 1 | 25.0 |
| 22 | 96-13 | Long-grained nonglutinous rice | Children's fringe | 10 | 7 | 4 | 57.14 |
| 23 | Zhenshan 97B | Long-grained nonglutinous rice | Children's fringe | 15 | 21 | 12 | 57.14 |
| 24 | Nanjing 11 2. | Long-grained nonglutinous rice | Children's fringe | 10 | 21 | 7 | 33.33 |
| 25 | IR36② | Long-grained nonglutinous rice | Children's fringe | 10 | 12 | 5 | 41.67 |
| 26 | 2. Bali draws | Japonica rice | Children's fringe | 10 | 20 | 13 | 65.0 |
| 27 | Qiu Guang 2. | Japonica rice | Children's fringe | 10 | 19 | 9 | 47.37 |
| 28 | Wujin round-grained rice 99-82 | Japonica rice | Children's fringe | 20 | 47 | 32 | 68.09 |
| 29 | Wujin round-grained rice 99-90 | Japonica rice | Children's fringe | 13 | 24 | 14 | 58.33 |
| 30 | Samsara 422 3. | Long-grained nonglutinous rice | Children's fringe | 10 | 11 | 4 | 36.36 |
| 31 | Peiai 64 3. | Long-grained nonglutinous rice | Children's fringe | 10 | 13 | 4 | 30.77 |
| 32 | Training C311 3. | Long-grained nonglutinous rice | Children's fringe | 10 | 14 | 8 | 57.14 |
| 33 | 02428③ | Japonica rice | Children's fringe | 15 | 43 | 26 | 60.47 |
| 34 | Priscilla | Java rice | Children's fringe | 15 | 24 | 9 | 37.50 |
| 35 | Orion | Java rice | Children's fringe | 15 | 20 | 7 | 35.0 |
| 36 | 99041/99004 | The rice variety species hybrid | Children's fringe | 12 | 23 | 17 | 73.91 |
| 37 | H98089/H98082 | Subspecies indica and japonica hybrid | Children's fringe | 10 | 11 | 5 | 45.45 |
| 38 | The IR36/ Bali draws | Subspecies indica and japonica hybrid | Children's fringe | 32 | 38 | 18 | 47.37 |
| 39 | Bali draws/IR36 | Subspecies indica and japonica hybrid | Children's fringe | 30 | 42 | 20 | 47.62 |
| 40 | IR36/ autumn light | Subspecies indica and japonica hybrid | Children's fringe | 25 | 51 | 25 | 49.02 |
| 41 | Qiu Guang/IR36 | Subspecies indica and japonica hybrid | Children's fringe | 28 | 37 | 18 | 48.65 |
| 42 | 11/ autumn of Nanjing light | Subspecies indica and japonica hybrid | Children's fringe | 25 | 38 | 18 | 47.37 |
| 43 | Qiu Guang/Nanjing 11 | Subspecies indica and japonica hybrid | Children's fringe | 30 | 47 | 27 | 57.45 |
| 44 | Nanjing 11/ Bali draws | Subspecies indica and japonica hybrid | Children's fringe | 30 | 42 | 23 | 54.76 |
| 45 | Bali draws/Nanjing 11 | Subspecies indica and japonica hybrid | Children's fringe | 27 | 35 | 22 | 62.86 |
| 46 | Bali draws/93-11 | Subspecies indica and japonica hybrid | Children's fringe | 24 | 27 | 15 | 55.56 |
| 47 | CpsLol 7/C92 | Pawl round-grained rice inter subspecific hybrid | Children's fringe | 30 | 52 | 37 | 51.92 |
| 48 | 962136-2/ autumn light | The japonica rice variety species hybrid | Children's fringe | 27 | 29 | 20 | 68.96 |
| 49 | 98164 | Africa cultivated rice Ag A g | Children's fringe | 12 | 17 | 7 | 41.18 |
| 50 | The Africa wild rice | A genome wild rice | Children's fringe | 7 | 12 | 6 | 50.0 |
| 51 | HDAR001/ Africa wild rice | Cultivation japonica rice/A genome wild rice hybrid | Children's fringe | 10 | 12 | 7 | 58.33 |
| 52 | No. 7/African wild rice of dragon round-grained rice | Cultivation japonica rice/A genome wild rice hybrid | Children's fringe | 10 | 14 | 8 | 57.14 |
| Sequence number | Species and kind (being) title | Species and variety type | Explant type | Process material number (individual) | The regeneration plant number | The tetraploid plant number | Add overtones band (%) |
| 53 | 98168-2/ Africa wild rice | Cultivation japonica rice/A genome wild rice hybrid | Children's fringe | 7 | 11 | 4 | 36.36 |
| 54 | Left I round-grained rice/African wild rice | Cultivation japonica rice/A genome wild rice hybrid | Children's fringe | 15 | 22 | 10 | 45.45 |
| 55 | 98168/ African wild rice | Cultivation japonica rice/A genome wild rice hybrid | Children's fringe | 14 | 24 | 13 | 54.17 |
| 56 | 98186/ African wild rice | Cultivation japonica rice/A genome wild rice hybrid | Children's fringe | 25 | 47 | 21 | 44.68 |
| 57 | The rich short No. 1/African wild rice that accounts for | Cultivation long-grained nonglutinous rice/A genome wild rice hybrid | Children's fringe | 10 | 21 | 15 | 71.42 |
| 58 | Orion/ Africa wild rice | Java rice/A genome wild rice hybrid | Children's fringe | 25 | 47 | 21 | 44.68 |
| 59 | WR001 Oryza me yeriana | Oryza meyeriana, the GG genome | Children's fringe | 5 | 78 | 20 | 25.64 |
| 60 | WR003 O.punctata | Spot wild rice/BB genome | Children's fringe | 15 | 14 | 5 | 35.71 |
| 61 | WR004 O.officinalis | Oryza officinalis, the CC genome | Children's fringe | 15 | 22 | 6 | 27.27 |
| 62 | WR005 O.latifolia | The broad-leaved wild rice, the CCDD genome | Children's fringe | 15 | 19 | 8 | 42.11 |
| 63 | WR006 O.minuta | The granule wild rice, the BBCC genome | Children's fringe | 15 | 23 | 9 | 39.13 |
| 64 | Red awns wild rice/African wild rice | Wild rice hybrid in the A genome | Children's fringe | 6 | 4 | 3 | 75.0 |
| 65 | Zhenshan 97B/oryza officinalis | AC genome species hybrid | Children's fringe | 3 | 7 | 3 | 42.68 |
| 66 | Zhenshan 97B/granule wild rice | ABC genome species hybrid | Children's fringe | 5 | 10 | 7 | 70.00 |
| 67 | The IR36/ oryza meyeriana | AG genome species hybrid | Children's fringe | 3 | 7 | 4 | 57.14 |
| 68 | DXT3s | Japonica rice photoperiod-temperature sensitive male sterility system | Stipes | 12 | 45 | 18 | 40.0 |
| 69 | 98168-2/ Africa wild rice | Cultivation japonica rice/A genome wild rice hybrid | Stipes | 10 | 37 | 19 | 51.35 |
| 70 | Bali draws/IR36 | Subspecies indica and japonica hybrid | Stipes | 10 | 53 | 21 | 39.62 |
| 71 | The IR36/ Bali draws | Subspecies indica and japonica hybrid | Stipes | 10 | 47 | 17 | 36.17 |
| 72 | Bali draws/Nanjing 11 | Subspecies indica and japonica hybrid | Stipes | 10 | 35 | 13 | 37.14 |
| 73 | Nanjing 11/ Bali draws | Subspecies indica and japonica hybrid | Stipes | 10 | 32 | 15 | 46.88 |
| Add up to | 73 kinds, species or hybrids | Minimum induction frequency 15.38% the highest induction frequency 75% | 1042 | 1819 | 850 | Average out to: 46.73 |
* utilize this technology to obtain 152 polyploid varieties, species and hybrids, only have 1-2 again at some The not statistics of living plant is in this table. This table is listed 73 kinds, species and hybrids altogether, and wherein two is hybridization 16 of the sterile lines of rice, long-grained nonglutinous rice 12,6 in japonica rice, 2 of java rices, 1 of African rice, African wild rice 1 Individual, 1 of rice variety species hybrid, 1 of japonica rice variety species hybrid, 14 of subspecies indica and japonica hybrids, pawl round-grained rice Asia Plant 1 of hybrid, cultivated rice/9 of A genome wild rice species hybrids, A genome wild rice species hybrid 1 3 of 5 of individual, non-A genome wild rices, cultivated rice/non-A genome species hybrid; Explant is 18 of seed 49 of individual, young fringes, 6 of stipes.
Annotate:
1. to herbicide Bentazon responsive type;
2. typical long-grained nonglutinous rice and japonica rice are tested kind;
3. wide affine kind.
Claims (6)
1, a kind ofly combines by tissue culture and chemical induction, the method of high-efficiency breeding polyploid rice, adopt rice paddy seed and young fringe to make explant, needing through following seven cultivation stage: a, callus induction it is characterized in that, b, colchicine liquid nutrient medium double, c, successive transfer culture, d, the differentiation cultivation of sprouting, e, culture of rootage, f, strong seedling culture, g, the maturation of growing seedlings and polyploid plant are identified.
2, a kind ofly combine by tissue culture and chemical induction, the method of high-efficiency breeding polyploid rice, adopt rice stem section (the band joint contains resting bud) to be explant, it is characterized in that needing inducing and breeding through following seven cultivation stage: a, clump bud, b, colchicine liquid culture double, c, successive transfer culture, d, the growth of clump bud, e, culture of rootage, f, strong seedling culture, g, the maturation of growing seedlings and polyploid plant are identified.
3, the method for high-efficiency breeding polyploid rice according to claim 1 is characterized in that:
A, callus induction process are, cultivated rice, wild rice, cultivated rice x wild rice hybrid seed or young fringe are inoculated in N6 (or MS)+2 after sterile-processed, in the inducing culture of 4-D 1～2.5mg/l+NAA 1～2mg/l+6BA (or KT) 0.1～0.5mg/l+ sucrose 5～6%+ caseinhydrolysate 50～150mg/l+ agar 0.65～0.75%, pH5.8～6.2, under 25～30 ℃, dark or illumination 800～10001x, cultivated 25～45 days and form callus;
B, colchicine liquid nutrient medium double process and are, the callus of the cultivated rice that will form in inducing culture, wild rice, cultivated rice x wild rice hybrid is transferred to N6 (or MS)+2, doubling in the medium of 4-D 1～2.5mg/l+NAA 1～2mg/l+6BA (or KT) 0.1-0.5mg/l+ colchicine 500-700mg/l+ sucrose 3～5%+ caseinhydrolysate 100mg/l, pH5.8～6.2, shaken cultivation is 40～48 hours under 20～22 ℃, 95～105rpm rotating speed;
C, successive transfer culture process are, the callus of the cultivated rice that doubles to handle, wild rice, cultivated rice x wild rice hybrid after fully washing, sterile water is changed in the medium identical with above-mentioned inducing culture under 25～30 ℃ of temperature, dark or illumination 600～1000 1x successive transfer culture 7-10 days;
D, the differentiation incubation of sprouting are, the callus of cultivated rice behind the subculture, wild rice, cultivated rice x wild rice hybrid is transferred in the differential medium of MS+6BA1～2mg/l+KT 2～3mg/l+NAA 0.2～0.5mg/l+ caseinhydrolysate 100～200mg/l+ sucrose 3%+ agar 0.65～0.75%, pH5.8～6.2, differentiation culture is 20～45 days under 25～30 ℃, illumination 1500～2000 1x, differentiates paddy rice bud seedling;
E, process of rooting culture are, to change over to through the paddy rice bud seedling that the callus differentiation culture goes out in the root media of 1/2MS+6BA 0.1～0.5mg/l+KT 0.1～0.2mg/l+NAA 0.2～03mg/l+IAA 0.5～1mg/l+ caseinhydrolysate 100mg/l+ activated carbon 0.03%+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 and take root, grow complete plantlet;
F, strong seedling culture process be, the complete plantlet of paddy rice changed in the strong seedling culture base of 1/2 MS+6BA 0.5mg/l+KT0.1～0.2mg/l+NAA, 0.2～0.3mg/l+ paclobutrazol, 3～5mg/l+ caseinhydrolysate, 100～200mg/l+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 and grow into healthy and strong plantlet;
G, the maturation of growing seedlings and polyploid plant qualification process are, healthy and strong plantlet with the strong seedling culture growth, be transplanted to by 2/3 sand and 1/3 fine earth that becomes thoroughly decomposed and form in the compost cultivating container, transplanting survival is also cultivated plant to ripe, in process of growth, differentiate the polyploid rice plant according to morphological feature (having awns, big grain, leaf to increase thick, cane chap) and cytogenetics evidence (the blade pore increases, root tip chromosomes is multiplied).
4, the method for high-efficiency breeding polyploid rice according to claim 2 is characterized in that:
A, clump bud are induced with breeding and are, the stem section of cultivated rice, wild rice, cultivated rice x wild rice hybrid plant is inoculated into the inducing in the many clumps of bud medium of 2/3MS+6BA 1～3mg/l+KT 2～3mg/l+NAA 0.2～0.5mg/l+ paclobutrazol 3～5mg/l+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 after sterile-processed, under 25～30 ℃, illumination 1500～2000 1x, cultivated 10～30 days and form many clumps of buds;
B, colchicine liquid culture double process and are, the many clumps of buds of the cultivated rice that forms, wild rice, cultivated rice x wild rice hybrid are transferred to doubling in the medium of 1/2MS+6BA 1～2mg/l+KT 2～3mg/l+NAA 0.2～0.5mg/l+ paclobutrazol 3～5mg/l+ colchicine 500-700mg/l+ sucrose 2～3%, pH5.8-6.2, and shaken cultivation is 18～24 hours on the shaking table of illumination 500～800 1x, 20～22 ℃ of temperature, rotating speed 95～105rpm;
C, successive transfer culture process are, the many clumps of buds of the cultivated rice that doubles to handle, wild rice, cultivated rice x wild rice hybrid are changed over to after sterile water fully washs with above-mentioned induce in the identical medium of the medium that sprouts, successive transfer culture is 7～10 days under 25～30 ℃ of temperature, illumination 1500～2000 1x;
D, clump bud process of growth are, the many clumps of buds of the cultivated rice of successive transfer culture, wild rice, cultivated rice * wild rice hybrid are transferred to after sterile water fully washs in the medium of 1/2MS+6BA 1-2mg/l+KT 2-3mg/l+NAA0.2-0.5mg/l+ paclobutrazol 3-5mg/l+ caseinhydrolysate 100-200mg/l+ sucrose 2%+ agar 0.65-0.75%, pH5.8-6.2, under illumination 1500-2000 1x, temperature 25-30 ℃, turn out many clumps of bud seedlings;
E, process of rooting culture are, many clumps of bud seedlings are changed in the root media of 1/2MS+6BA 0.1～0.5mg/l+KT 0.1～0.2mg/l+NAA 0.2～0.3mg/l+IAA 0.5～1mg/l+ caseinhydrolysate 100mg/l+ activated carbon 0.03%+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 and take root, grow complete plantlet;
F, strong seedling culture process be, grows into healthy and strong plantlet with deriving from the strong seedling culture base that the complete plantlet of paddy rice that many clumps of buds propagation form changes 1/2MS+6BA 0.5mg/l+KT 0.1～0.2mg/l+NAA 0.2～0.3mg/l+ paclobutrazol 2～3mg/l+ caseinhydrolysate 100～200mg/l+ sucrose 2%+ agar 0.65～0.75%, pH5.8～6.2 over to;
G, the maturation of growing seedlings and polyploid plant qualification process are, the healthy and strong plantlet of strong seedling culture growth is transplanted to by 2/3 sand and 1/3 fine earth that becomes thoroughly decomposed forms in the compost cultivating container, transplanting survival is also cultivated plant to ripe, in process of growth, differentiate the polyploid rice plant according to morphological feature and cytogenetics evidence.
5, according to the method for claim 1 or 3 described high-efficiency breeding polyploid rices, it is characterized in that with the prematurity of cultivated rice, wild rice, cultivated rice x wild rice hybrid or ripe seed as explant, perhaps make explant to the young fringe that female stamen forms this developmental stage in period with the branch stalk second time of cultivated rice, wild rice, cultivated rice x wild rice hybrid plant.
6,, it is characterized in that the stem section of the bottom band joint of the stem of having eared with cultivated rice, wild rice, cultivated rice x wild rice hybrid plant is an explant according to the method for claim 2 or 4 described high-efficiency breeding polyploid rices.
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