CN112369327A - Method for improving seedling rate of hybrid seeds of long-male wild rice and cultivated rice - Google Patents
Method for improving seedling rate of hybrid seeds of long-male wild rice and cultivated rice Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a method for improving the seedling rate of hybrid seeds of oryza longistaminata and oryza sativa, and relates to the field of biotechnology breeding. Adopting a good cultivated rice recovery system as a female parent, hybridizing with the long-male wild rice, taking the young embryo for in-vitro culture 7-9 days after pollination, promoting the young embryo to grow and develop simultaneously on the culture medium by utilizing an improved young embryo in-vitro culture medium, transplanting the test-tube seedling of the young embryo in-vitro development into a seedling raising tray after indoor water culture seedling hardening for one week when the root system is developed and the seedling is 8-10cm after in-vitro culture for two weeks. The invention can effectively prevent the premature senility of distant hybrid embryos of long-male wild rice and cultivated rice, so that a large number of distant hybrid embryos continue to develop into normal seedlings, and the survival rate of hybrid seeds is effectively improved; meanwhile, the cultivation period of the wild hybrid progeny is shortened, and a foundation is laid for effectively utilizing the germplasm resources of the long-male wild rice and quickly breeding the breeding progeny of the long-male wild rice and the cultivated rice.
Description
Technical Field
The invention relates to the field of biotechnology breeding, in particular to a method for improving the seedling rate of hybrid seeds of long-male wild rice and cultivated rice.
Background
Rice is one of the most important food crops in the world, and rice is used as a main food source in more than 50% of the world population. In China, rice plays a very important role in the life of people, rice is the main food of more than 60% of the population in China, and China is the biggest rice producing and consuming country in the world. Therefore, the yield per unit of rice and the resistance of the rice to plant diseases and insect pests are improved, and the method has important significance for ensuring food safety in China and the world.
However, cultivated rice is too narrow in breeding resources of cultivated rice varieties due to long-term artificial selection, and the rice breeding has a bottleneck problem that the yield, the resistance and the quality are difficult to further break through. The best way to break this situation is to increase the genetic diversity of the cultivar. In the process of long evolution of wild rice serving as an original ancestor of cultivated rice, in order to adapt to different geographical regions and complex ecological environments, various excellent characteristics such as disease resistance, insect resistance, stress resistance and the like are gradually formed, wherein the long stamen wild rice is a wild kindred species of the existing cultivated rice and stores a plurality of excellent genes which are not available in the conventional cultivated rice or disappear. Therefore, the long stamen wild rice can be used as an excellent germplasm resource to improve the situation that the genetic base of the current cultivated rice variety is narrow.
The long anther, big stigma and rhizome are the most obvious characteristics of long stamen wild rice, meanwhile, long stamen wild rice has stronger cold resistance and drought resistance, is almost immune to rice bacterial leaf blight and has extremely high resistance to rice blast, and long stamen wild rice also shows extremely high resistance in the aspect of insect resistance, particularly to common pests such as rice planthopper and leafhopper.
In recent years, many developing countries using rice as staple food have more and more people and less cultivated land area, so scientists gradually turn their attention to develop and utilize some favorable characters of long stamen wild rice to broaden the resistance inheritance of cultivated rice varieties to biotic or abiotic stress, thereby improving the existing cultivated varieties and improving the quality and yield of rice.
However, the selfing of the long-male wild rice is almost not fruitful, fertile hybrids are difficult to generate when the long-male wild rice is hybridized with the cultivated rice, and the characteristic of strong reproductive isolation is shown, so that the filial generation of the long-male wild rice and the cultivated rice is effectively obtained, and the improvement of the seedling rate of hybrid seeds of the long-male wild rice and the cultivated rice has important significance for effectively utilizing the long-male wild rice.
Disclosure of Invention
The invention provides a method for improving the seedling rate of hybrid seeds of long-male wild rice and cultivated rice, which can effectively prevent premature senescence of distant hybrid embryos of the cultivated rice and the long-male wild rice, so that a large number of distant hybrid embryos continue to develop into normal seedlings, and the survival rate of the hybrid seeds is effectively improved; meanwhile, the cultivation period of the wild hybrid progeny is shortened, and a foundation is laid for effectively utilizing the germplasm resources of the long-male wild rice and quickly breeding the progeny of the long-male wild rice and the cultivated rice.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for improving the seedling rate of hybrid seeds of oryza longistaminata and oryza sativa comprises the following steps:
(1) obtaining of hybrid embryo
Hybridizing the excellent cultivated rice recovery line serving as a female parent and the long-male wild rice serving as a male parent to obtain hybrid embryos of the cultivated rice and the long-male wild rice;
(2) sterilization inoculation of immature embryos
Collecting hybridized seeds after the cultivated rice and the long-male wild rice are pollinated for 7-9 days, peeling glumes, putting the hybrid seeds with the glumes removed in a sterilized container, and sterilizing the hybrid seeds on a super-clean workbench;
(3) in vitro culture of immature embryos
And (3) picking the complete immature embryos by using tweezers on a superclean bench, and placing the complete immature embryos in a container filled with a culture medium for carrying out isolated culture on the immature embryos, wherein the isolated culture medium of the immature embryos is as follows: 1/2MS + plant gel 3g + NAA0.2mg;
(4) hardening and transplanting tissue culture seedlings
After the young embryo is cultured in vitro for 2 days, the embryo and the radicle develop and grow on the culture medium, and the in vitro culture efficiency of the young embryo is counted after one week (the in vitro culture efficiency of the young embryo is equal to the number of green seedlings/the number of inoculated young embryos multiplied by 100%);
after 20 days, when the tissue culture seedlings cultured in vitro by the young embryos have three leaves and one heart of 8-10cm, taking the seedlings out of the culture medium, carefully washing off the culture medium attached to the root systems of the seedlings under running water, properly cutting off overlong leaves and root systems, and then putting the seedlings into a container filled with clear water to be subjected to seedling hardening culture in a culture chamber;
and 5-7d, transferring the seedlings into a seedling raising tray, placing the seedling raising tray in a greenhouse, and performing conventional water and fertilizer management.
Preferably, in step (1), the hybrid is bagged, and the name of the parent and the name of the female parent and the pollination date are marked on the hybrid bag.
As a preferable aspect of the above, in the step (2), the hybrid sterilization specifically includes: firstly, alcohol with the volume percentage of 70% is used for disinfecting the surface of hybrid seeds for 30s, then sodium hypochlorite solution with the effective chlorine content of 1.5-2% is used for soaking the hybrid seeds for 15min, then sterile water is used for cleaning for 5 times, more than 2min each time, and finally the disinfected hybrid seeds are placed in a 15cm culture dish filled with sterile paper for standby.
Preferably, in steps (3) and (4), the culture conditions are a constant temperature culture chamber with a culture temperature of 28 ℃, an air relative humidity of 70% and an illumination intensity of 4000-.
Preferably, in step (3), 3 to 4 immature embryos are inoculated per container.
As a preferable mode of the above, in the steps (2), (3) and (4), a 50ml triangular flask is used as a container.
Due to the structure, the invention has the advantages that:
1. the invention firstly proposes that the young embryo is taken for in vitro culture 7-9 days after the hybridization of the wild rice and the conventional rice, so as to realize the early embryo taking after pollination, thereby effectively preventing the premature senility of distant hybrid embryos of the long-male wild rice and the cultivated rice, and enabling a large amount of distant hybrid embryos to be cultured under in vitro conditions so as to continue to develop into normal seedlings;
2. the culture medium for in vitro culture of the immature embryos can ensure that the embryos and radicles simultaneously and rapidly develop and grow under the in vitro condition of the immature embryos, the in vitro culture efficiency of the cultured immature embryos for 10d is 97.3 percent, only one culture medium is needed for one-time seedling formation, rooting culture is not needed, the culture time is greatly shortened, the culture efficiency and the seedling formation efficiency are improved, and the experimental expectation can be achieved in a short time;
3. the method has the advantages of simple embryo taking operation, seedling hardening and transplanting, high survival rate, no need of operation under a microscope and no need of scraping endosperm, and is simple and easy to operate by picking the complete embryo on an ultraclean workbench by using ophthalmologic forceps; when hardening off the seedlings, only the culture medium attached to the root system during culture needs to be washed away, the culture medium is changed into clear water, the culture medium is continuously placed in the original culture environment, and the culture bottle sealing film is opened to harden the seedlings for 5-7 days for transplanting; after 10 days, the survival rate is counted, and the seedling hardening survival rate of the method reaches 99.1 percent;
in conclusion, the in vitro culture method of the young embryo is simple and easy to operate, the culture period is short, the efficiency is high, the seedling rate of the young embryo in vitro culture is as high as 96.4 percent, and the seedling rate in the hybridization of the long-male wild rice and the cultivated rice is improved.
Detailed Description
The technical solution of the present invention will be clearly and completely described below. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
(1) Obtaining of hybrid embryo
The long-male wild rice is S1029, S1032 and S1035, and is preserved after the rice stumps of Wuhan and Hainan provinces are bred every year. The excellent cultivated rice restorer lines IP094, IP095 and IP098 are sown in stages at intervals of 10 days from 5 months to 10 days in 2020. And cross pollination is carried out on the long male wild rice and the cultivated rice in 8 months, 10 days to 9 months and 15 days in the current year. And (5) bagging after hybridization, wherein the names of the male parent and the female parent and the pollination date are marked on the hybridization bag.
(2) Sterilization inoculation of immature embryos
Collecting hybridized seeds after the cultivated rice and the long-male wild rice are pollinated for 7-9 days, stripping glumes, putting the hybrid seeds without the glumes in a sterilized 50ml triangular flask, and sterilizing the hybrid seeds on a clean bench. Firstly, disinfecting hybrid seeds on the surface by using alcohol with the volume percentage of 70%, then soaking the hybrid seeds in sodium hypochlorite solution with the effective chlorine content of 1.5-2% for 15min, then washing the hybrid seeds with sterile water for 5 times, wherein the time is more than 2min each time, and finally placing the disinfected hybrid seeds in a 15cm culture dish filled with sterile paper for later use.
(3) In vitro culture of immature embryos
And (3) picking the complete immature embryos by using tweezers on a clean bench, and placing the complete immature embryos in a 50ml triangular flask filled with a culture medium for in vitro culture of the immature embryos. Three restorer lines are combined with 9 long male wild rice lines to inoculate 450 young embryos. The culture conditions are that the temperature is 28 ℃, the air relative humidity is 70%, the illumination intensity is 4000-; the embryo in vitro culture medium comprises: 1/2MS + plant gel 3g + NAA0.2mg.
(4) Hardening and transplanting tissue culture seedlings
After 2 days of embryo in vitro culture, the embryo and the radicle are grown on the culture medium, and the embryo in vitro culture efficiency (green seedling number/inoculated embryo number × 100%) is counted after one week. 438 well-developed green seedlings are obtained, and the in vitro culture efficiency of the immature embryos is 97.3 percent. After 20 days, when tissue culture seedlings cultured in vitro by the young embryos have three leaves and one heart of about 8-10cm, taking the seedlings out of the culture medium, carefully washing the culture medium attached to the root systems of the seedlings under running water, properly cutting off overlong leaves and root systems, putting the seedlings in a 50ml triangular flask containing clear water, carrying out seedling hardening culture in a culture room, carrying out culture temperature of 28 ℃, air relative humidity of 70%, illumination intensity of 4000 and 5000Lx, illuminating for 14h every day, and after 5-7 days, transferring the seedlings into a seedling raising tray, and putting the seedling raising tray in a greenhouse for conventional water and fertilizer management. After 10 days, 438 transplanted seedlings are counted, 434 seedlings coexist, and the survival rate (survival rate is survival seedling number/total transplanted seedling number multiplied by 100%) reaches 99.1%.
The in vitro culture method of the young embryo is simple and easy to operate, the culture period is short, the efficiency is high, and the seedling rate of the in vitro culture of the young embryo is up to 96.4 percent (the seedling rate is the in vitro culture efficiency of the young embryo multiplied by the survival rate).
The invention overcomes the defects that the existing wild rice and cultivated rice hybrid is sterile or semi-sterile, the hybrid embryo is easy to senilism and can not develop into a normal plant, and simultaneously, the in vitro development seedling rate of the immature embryo is greatly improved by stripping off the immature embryo as early as possible and improving the culture medium for the in vitro culture of the immature embryo.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. A method for improving the seedling rate of hybrid seeds of long-male wild rice and cultivated rice is characterized in that: the method comprises the following steps:
(1) obtaining of hybrid embryo
Hybridizing the excellent cultivated rice recovery line serving as a female parent and the long-male wild rice serving as a male parent to obtain hybrid embryos of the cultivated rice and the long-male wild rice;
(2) sterilization inoculation of immature embryos
Collecting hybridized seeds after the cultivated rice and the long-male wild rice are pollinated for 7-9 days, peeling glumes, putting the hybrid seeds with the glumes removed in a sterilized container, and sterilizing the hybrid seeds on a super-clean workbench;
(3) in vitro culture of immature embryos
And (3) picking the complete immature embryos by using tweezers on a superclean bench, and placing the complete immature embryos in a container filled with a culture medium for carrying out isolated culture on the immature embryos, wherein the isolated culture medium of the immature embryos is as follows: 1/2MS + plant gel 3g + NAA0.2mg;
(4) hardening and transplanting tissue culture seedlings
After the embryo is cultured in vitro for 2d, the embryo and the radicle develop and grow on the culture medium, and the embryo in vitro culture efficiency is counted after one week;
after 20 days, when the tissue culture seedlings cultured in vitro by the young embryos have three leaves and one heart of 8-10cm, taking the seedlings out of the culture medium, carefully washing off the culture medium attached to the root systems of the seedlings under running water, properly cutting off overlong leaves and root systems, and then putting the seedlings into a container filled with clear water to be subjected to seedling hardening culture in a culture chamber;
and 5-7d, transferring the seedlings into a seedling raising tray, placing the seedling raising tray in a greenhouse, and performing conventional water and fertilizer management.
2. The method of claim 1, wherein: in the step (1), bags are sleeved after hybridization, and the names of parents and the pollination date are marked on the hybridization bags.
3. The method of claim 1, wherein: in the step (2), the hybrid sterilization specifically comprises: firstly, alcohol with the volume percentage of 70% is used for disinfecting the surface of hybrid seeds for 30s, then sodium hypochlorite solution with the effective chlorine content of 1.5-2% is used for soaking the hybrid seeds for 15min, then sterile water is used for cleaning for 5 times, more than 2min each time, and finally the disinfected hybrid seeds are placed in a 15cm culture dish filled with sterile paper for standby.
4. The method of claim 1, wherein: in the steps (3) and (4), the culture conditions are a constant temperature culture chamber with the culture temperature of 28 ℃, the relative air humidity of 70% and the illumination intensity of 4000-.
5. The method of claim 1, wherein: in step (3), 3-4 immature embryos are inoculated in each container.
6. The method of claim 1, wherein: in the steps (2), (3) and (4), a 50ml triangular flask was used as a container.
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| CN104871964A (en) * | 2015-06-12 | 2015-09-02 | 云南省农业科学院生物技术与种质资源研究所 | Method for increasing breeding efficiency of wild rice and cultivated rice distant hybridization embryo rescue |
| CN106888962A (en) * | 2016-08-24 | 2017-06-27 | 云南大学 | The method for cultivating perennial rice restorer using hero wild rice vegetative propagation characteristic long |
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- 2020-11-13 CN CN202011272330.8A patent/CN112369327A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104542299A (en) * | 2015-01-26 | 2015-04-29 | 云南省农业科学院生物技术与种质资源研究所 | Method for improving culture efficiency of anther of filial generation of Oryza rufipogon |
| CN104871964A (en) * | 2015-06-12 | 2015-09-02 | 云南省农业科学院生物技术与种质资源研究所 | Method for increasing breeding efficiency of wild rice and cultivated rice distant hybridization embryo rescue |
| CN106888962A (en) * | 2016-08-24 | 2017-06-27 | 云南大学 | The method for cultivating perennial rice restorer using hero wild rice vegetative propagation characteristic long |
Non-Patent Citations (1)
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