Method for Producing Rice Haploid by Rice X Maize Hybridization

Abstract

The present invention provides a novel method for producing the rice haploid, i.e., producing the rice haploid by ricemaize hybridization. In this method, rice is used as the female parent, and the rice panicle is emasculated and then pollinated with fresh maize pollens; the emasculated panicle is sprayed with 2, 4-D solution at 50-200 mg/L 24 h after pollination, and after 15-20 days the rice panicle is cut off to collect caryopses; and the haploid embryos are obtained by dissectting caryopses asepticlly, then inoculated and cultured with MS medium, then the haploid embryos directly germinate into rice haploid seedlings. Compared with the existing main methods for producing the rice haploid, such as anther culture and isolated microspore culture, the rice haploid production method of the present invention reduces the dependence on rice genotypes, does not produce mixture of haploid and diploid plants, contains no albino seedling, and is simpler in technical operation.

Claims

1. A method for producing the rice haploid by ricemaize hybridization, wherein the rice haploid is produced by the elimination of maize genome in wide cross between rice and maize.

2. The method for producing the rice haploid by ricemaize hybridization of claim 1, comprising the following steps: A. emasculation: after heading and before flowering of the rice, for each panicle, cut off the young base spikelets and keep the upper and middle spikelets, and then the panicle is emasculated and bagged after emasculation according to routine methods; B. pollination: the emasculated rice panicle is pollinated with fresh maize pollens when the stigmas of rice florets develop to mature; C. production of haploid embryo: 24 h after the pollination, the pollinated rice panicle is sprayed with 2, 4-dichlorophenoxyacetic acid (2,4-D) solution, and continually grows on the maternal plant; D. cutting of panicle and peeling of caryopsis: the pollinated rice panicle is cut off after growing on the maternal plant for 15-20 days, to collect the caryopses produced through ricemaize hybridization; and E. embryo rescue: dissect the sterilized caryopses on the aseptic bench by stereomicroscope to obtain haploid embryos, and inoculate the haploid embryo into the MS medium, to obtain the rice haploid plant after germination of the haploid embryo.

3. The method for producing the rice haploid by ricemaize hybridization of claim 2, wherein in step C, 24 h after the pollinating, the pollinated rice panicle is sprayed with 2,4-D solution at concentration of 50-200 mg/L.

4. The method for producing the rice haploid by ricemaize hybridization of claim 3, wherein in step C the pollinated rice plant is grown in an artificial climate room or an artificial climate chamber under artificial light (about 2 000 lux) 14 hours 271 C. or 10 hours 201 C. alternately, and keeping 85% humidity.

5. The method for producing the rice haploid by ricemaize hybridization of claim 2, wherein in step D, the pollinated panicle is cut off from the maternal plant when the length of the haploid embryos reach 0.5-1 mm.

6. The method for producing the rice haploid by ricemaize hybridization of claim 2, wherein in step E, the culture medium is MS medium, and the haploid embryos are first cultured under dark condition (231 C.), then moved to light condition (about 2 000 lux, 231 C.) after the haploid embryos germinate into buds, so as to obtain the rice haploid plant.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] To describe the technical solutions in the embodiments of the present invention or in the prior art more clearly, the following briefly describes the accompanying drawings required for describing the embodiments or the prior art. Apparently, the accompanying drawings in the following description show some embodiments of the present invention, and a person of ordinary skill in the art may still derive other drawings from these accompanying drawings without creative efforts.

[0026] FIG. 1 is a photograph showing the difference of caryopses between a maize pollen pollinated panicle (left) and a self-crossing panicle (right) in Example 1;

[0027] FIG. 2 is a comparative photograph of a normal diploid rice plant of Example 2 (left) and a haploid plant obtained in Example 2 (right);

[0028] FIG. 3 is a photograph showing a normal diploid rice plant of Example 3 (right) and a haploid plant obtained in Example 2 (left) in an artificial climate cabinet;

[0029] FIG. 4 is a photograph of the self-crossed caryopsis and the out-crossed caryopsis obtained through cultivation in Example 3, where in this figure, the left picture (having no embryo and endosperm) and the middle picture (having embryo but having no endosperm) are the out-crossed caryopsis, the right picture is the self-crossed caryopsis (having embryo and endosperm);

[0030] FIG. 5 is a photograph of an out-crossed caryopsis obtained in Example 3 and a haploid embryo thereof;

[0031] FIG. 6 is a diagram showing the results of chromosome ploidy detection on a normal diploid plant; and

[0032] FIG. 7 is a diagram showing the results of chromosome ploidy detection on a haploid plant obtained in Example 3.

[0033] FIG. 8 is a photograph showing differences in plant size and seed-set just before harvest between a normal rice plant (left) and a haploid rice plant (right) obtained in Example 3.

DESCRIPTION OF THE EMBODIMENTS

[0034] In order to make the objectives, technical solutions and advantages of the present invention more apparent, the technical solution of present invention will be described in detail below. Apparently, the described embodiments are merely a part rather than all of the embodiments of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] In the present invention, all parts and percentages are units of weight, and all equipment and raw materials are commercially available or commonly used in the industry, unless otherwise specified. The methods in the following Examples are all routine methods in the art, unless otherwise specified.

Example 1

[0036] A. material planting: a japonica rice variety Yunjing 37 (bred by Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Yunnan, China) was subjected to sowed and seedlings raising in March, 2017, and transplanted into a pot in early May; and a maize (a maize inbred line SW6, (developed by Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Yunnan, China) was sowed every 10 days from May to June, so that the flowering periods of the rice and the maize would meet from July to September, and thus hybridization pollination could be conducted.

[0037] B. emasculation: after heading and before flowering of the rice, for each panicle, cut off the young base spikelets and keep the upper and middle spikelets, and then the panicle was emasculated and bagged according to routine methods;

[0038] C. pollination: pollinating was conducted with fresh maize pollens when the stigma of rice florets developed to mature;

[0039] D. production of haploid embryos: 24 h after pollination, the maize-pollen-pollinated rice panicles were sprayed with 2,4-D solution at 50 mg/L, and the pollinated rice panicles were continually grown on their maternal plants;

[0040] E. cutting of panicle and peeling of caryopses: the rice panicle was cut off after the pollinated rice panicle had grown for 20 days, to collect caryopses produced from ricemaize hybridization; and

[0041] F. embryo rescue: the sterilized caryopses were peeled on an aseptic bench by stereomicroscope to obtain haploid embryos, and the haploid embryos were inoculated into the MS medium, in which the haploid embryos were first darkly cultured, and then were subjected to light culture after the haploid embryos germinated into buds, to obtain green haploid plantlets.

Example 2

[0042] A. material planting: an indica rice variety Liangyou 2186 (developed by Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Yunnan, China) was sowed and seedlings raising in March, 2017, and transplanted into pots in early May; and a maize inbred line SW6 (developed by Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Yunnan, China) was sowed every 10 days from May to June, so that the flowering periods of the rice and the maize would meet from July to September, and thus hybridization pollination could be conducted.

[0043] B. emasculation: after heading and before flowering of the rice, for each panicle, cut off the young base spikelets and keep the upper and middle spikelets, and then the panicle was emasculated and bagged according to routine methods;

[0044] C. pollination: pollinating was conducted with fresh maize pollens when the rice stigmas developed to mature;

[0045] D. production of haploid embryos: 24 h after pollination, the maize-pollen-pollinated rice panicles were sprayed with 2,4-D solution at 200 mg/L, and the pollinated rice panicles were continually grown on their maternal plants;

[0046] E. cutting of panicle and peeling of caryopses: the rice panicle was cut off after the pollinated rice panicle had grown for 18 days, to collect caryopses produced through ricemaize hybridization; and

[0047] F. embryo rescue: the sterilized caryopses were peeled on an aseptic bench by stereomicroscope to obtain haploid embryos, and the haploid embryos were inoculated into the MS medium, in which the haploid embryos were first darkly cultured, and then were moved to light culture after the haploid embryos germinated into buds, to obtain green haploid plantlets.

Example 3

[0048] A. material planting: a japonica rice variety Yunjing 37 (developed by Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Yunnan, China) was sowed and seedlings raising in March, 2016, and transplanted into a pot in early May; and a maize inbred line SW6, developed by Institute of Food Crops, Yunnan Academy of Agricultural Sciences, Yunnan, China, was sowed every 10 days from May to June, so that the flowering periods of the rice and the maize would meet from July to September, and thus hybridization pollination could be conducted.

[0049] B. emasculation: after heading and before flowering of the rice, for each panicle, cut off the young base spikelets and keep the upper and middle spikelets, and then the panicle was emasculated and bagged according to routine methods;

[0050] C. pollination: pollinating was conducted with fresh maize pollens when the rice stigmas developed to mature;

[0051] D. production of haploid embryo: 24 h after pollination, the maize-pollen-pollinated rice panicles were sprayed with 2,4-D solution at 200 mg/L, and the pollinated rice panicles were continually grown on their maternal plants;

[0052] E. cutting of panicle and peeling of caryopses: the rice panicle was cut off after the pollinated rice panicle had grown for 15 days, to collect caryopses produced through ricemaize hybridization; and

[0053] F. embryo rescue: the sterilized caryopses were peeled on an aseptic bench by stereomicroscope to obtain haploid embryos, and the haploid embryos were inoculated into the MS medium, in which the haploid embryos were first darkly cultured, and then were moved to light culture after the haploid embryos germinated into buds, to obtain green haploid plantlets.

[0054] To demonstrate the rice plants obtained by the method of the present invention was haploid plants, the genome ploidy of the rice plants were detected by flow cytometry. The detection method was: first a cell nucleus was isolated, and then AT bases on the chromosomes were stained with DAPI dyeing solution, and then the intensity of fluorescence emitted by the stained AT bases were detected with a flow cytometer. The used instrument was CyFlow Space under the brand of Sysmex Partec, the kit was CyStain UV Precise P kit available from Sysmex Partec, and the ploidy of the samples could be determined based on the position of the DNA peak value detected by the flow cytometer. The DNA detection results of the normal diploid rice plant was shown in FIG. 6, and the DNA detection results of the rice plant obtained by the method of the present invention was shown in FIG. 7; it could be seen from FIG. 6 and FIG. 7 that, the ordinate (the number of isolated cell nucleuses) showed that the number of isolated cell nucleuses of the normal diploid rice plant to be detected was smaller than that of the plant produced by the present invention, and the abscissa (the DNA content) showed relatively higher main peaks in both the channel 100 and the channel 50 (the peak on the left side was an interference peak), and the abscissas in FIGS. 6 and 7 showed that the DNA content of the normal diploid rice plant was twice the DNA content of the rice plant produced by the method of the present invention, which proved that the rice plant produced by the method of the present invention was a rice haploid. In FIG. 8, the size of haploid rice plant obtained from the present invention was obviously smaller than that of normal diploid rice plant.

[0055] The aforementioned description is only specific embodiments of the present invention, and the claimed scope of the present invention is not limited thereto. Changes or substitutions can come into the mind of those of skills in the art readily, without departing from the technical scope disclosed by the present invention. These changes or substitutions all should fall within the claimed scope of the present invention. Therefore, the claimed scope of the present invention shall be determined by the claimed scope of the appended claims.