Breeding method for tetraploid <i>Ricinus communis</i>

Abstract

The present invention provides a breeding method for tetraploid ricinus communis. The method comprises: collecting a ricinus communis germplasm resource, performing morphologic character analysis and chromosome ploidy identification, selecting good diploid varieties, carrying out mutagenesis by using colchicine and oryzalin; performing chromosome ploidy identification on F1 generation seeds, selecting homozygous tetraploids, and performing economic character analysis and identification on the F2 generation, so as to breed good tetraploid ricinus communis.

Claims

1. A breeding method for tetraploid Ricinus communis L., comprising the following steps: providing diploid Ricinus communis L. seeds, subjecting the naked embryos of the Ricinus communis L. seeds obtained after artificial removal of endosperms to a forced absorption method with mixed mutagens to perform a doubling process for tetraploidization, subjecting the newborn F1 young leaves to chromosome analysis and ploidy identification, removing chimeras and aneuploids, and screening for homozygous tetraploid plants, which are then subjected to large-scale planting and cultivation management according to conventional methods for cultivating Ricinus communis L.

2. The breeding method for tetraploid Ricinus communis L. according to claim 1, wherein the mixed mutagens are a mixed solution of colchicine and oryzalin.

3. The breeding method for tetraploid Ricinus communis L. according to claim 1, wherein the basic number of the Ricinus communis L. chromosome is x=10, and the diploid chromosome number of somatic cells is 2n=2x=20.

4. A breeding method for tetraploid Ricinus communis L., comprising the following steps: 1) providing diploid Ricinus communis L. seeds; 2) subjecting the Ricinus communis L. seeds to sterile disinfection using conventional disinfection methods, then removing the endosperms, stripping the seed embryos out, placing the seed embryos into 1/2MS medium, and incubating the seed embryos at 23 C.2 C. without lighting until the seed embryos have germinated; 3) transferring into an aqueous solution containing mixed mutagens of colchicine and oryzalin at a certain mass concentration, to perform a doubling process in darkness at a lower temperature of 15-20 C. for 48-72 hours; 4) transferring into 1/2MS medium, to continue to cultivate at 23 C.2 C. with a lighting of 1000 Lux, 16 hours/day; 5) transplanting the seedlings in bottles into disinfected soil when the seedlings have developed into 4 true leaf stage and their roots have a length of 3-5 cm; at 3 days before transplanting, opening the caps of bottles for acclimatization; the transplanted seedlings must be carefully managed; 6) subjecting the newborn young leaves of F1 seed embryo to chromosome analysis and ploidy identification, removing chimeras and aneuploids, screening for homozygous tetraploid plants; and 7) subjecting the Ricinus communis L. tetraploids to large-scale planting and cultivation management according to conventional methods for cultivating Ricinus communis L., subjecting the F1 seeds of tetraploid Ricinus communis L. to chromosome analysis and ploidy identification again, removing chimeras and aneuploids, screening for a new homozygous tetraploid Ricinus communis L. germplasm.

5. The breeding method for tetraploid Ricinus communis L. according to claim 4, wherein the conventional disinfection method is as follows: disinfecting with 75% alcohol for 60 seconds, rinsing with sterile water, and then disinfecting with 2% sodium hypochlorite for 15 minutes, rinsing with sterile water for 3 times.

6. The breeding method for tetraploid Ricinus communis L. according to claim 4, wherein the mass percent concentrations of the colchicine and oryzalin solutions are 0.02%, respectively; and the final concentration of each of the mixed mutagens is 0.01%.

7. The breeding method for tetraploid Ricinus communis L. according to claim 4, wherein the conditions for the management of transplanted seedlings are as follows: maintaining the temperature at 25 C.2 C., the humidity at 65%5; the soil condition is normal sandy loams.

8. The breeding method for tetraploid Ricinus communis L. according to claim 4, wherein the steps of chromosome analysis and ploidy identification are as follows: harvesting newborn young leaves at 8-9 a.m., pre-processing with 0.002M 8-hydroxyquinoline for 3-4 hours, then preparing chromosome specimens of Ricinus communis L. through a wall degradation hypotonic method utilized in the preparation of chromosome specimens of plants, removing chimeric and aneuploid plants, screening for plants whose somatic cell chromosome is 2n=4x=40 to obtain homozygous tetraploid plants.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the appearance of diploid Ricinus communis L. plants (a) with a chromosome number of 2n=2x=20 (b).

(2) FIG. 2 shows the appearance of tetraploid Ricinus communis L. plants (a) with a chromosome number of 2n=4x=40 (b).

(3) FIG. 3 shows the comparison of appearance between diploid Ricinus communis L. seeds (b) and tetraploid Ricinus communis L. seeds (a).

(4) FIG. 4 shows the picture of Ricinus communis L. germplasm resources nursery.

DETAILED DESCRIPTION

(5) The prominent features and significant advances of the present invention can be presented in the following examples without any limitation of the invention.

(6) A breeding method for tetraploid Ricinus communis L. provided by the present invention comprises the following steps: 1) collecting germplasm resources: collecting 49 cultivars and wild resources of Ricinus communis L. from different regions of China and 5 foreign varieties (see table 1), including Yunnan, Sichuan, Jiangsu, Xinjiang, Shandong, Shanxi, Hebei, Tianjin of China and Israel and France, etc., to establish Ricinus communis L. germplasm resources nursery (see FIG. 4);

(7) TABLE-US-00001 TABLE 1 Types, varieties and sources of 54 Ricinus communis L. germplasm resources NO. type variety source 01 wild Yun-R 2014115 Yunnan 02 cultivated Ya-R No. 1 Sichuan 03 cultivated large grain type of Ricinus communis Jiangsu L. 04 variety Yun-R No. 4 Shanxi 05 variety Yun-R No. 5 Shanxi 06 variety Fen-R No. 10 Shanxi 07 variety economic Ricinus communis L. No. 1 Shanxi 08 variety economic Ricinus communis L. No. 2 Shanxi 09 variety Zhongbei No. 3 Shanxi 10 variety Zhongbei No. 4 Shanxi 11 variety Tong-R No. 9 Neimeng 12 variety Tong-R No. 10 Neimeng 13 variety KB.sub.2 Xinjiang 14 variety Green-R No. 1 Tianjin 15 variety Green-R No. 2 Tianjin 16 variety Zi-R No. 1 Shandong 17 variety Zi-R No. 2 Shandong 18 variety Zi-R No. 3 Shandong 19 variety Zi-R No. 4 Shandong 20 variety Zi-R No. 5 Shandong 21 variety Zi-R No. 7 Shandong 22 variety Zi-R No. 8 Shandong 23 variety Zi-R No. 9 Shandong 24 hybrid 89-3 S207 Shandong 25 hybrid Jiaxiang No. 2 Shandong 26 hybrid C205 1002E Shandong 27 hybrid CS219 E088 Shandong 28 hybrid CS219 9018 Shandong 29 hybrid C208 9018 Shandong 30 hybrid C208 E06 Shandong 31 hybrid Tian 1-8 E06 Shandong 32 hybrid Tian 2-18 S206 Hainan 33 hybrid Tianqi 101 Feng Hainan 34 hybrid Tian 2-18 E07 Shandong 35 hybrid 1002 E06 Shandong 36 hybrid 1002 9018 Shandong 37 hybrid 904 E088 Shandong 38 hybrid 9041C E07 Shandong 39 hybrid 1002 101 Shandong 40 hybrid C205 10 Shandong 41 hybrid C213 E088 Shandong 42 hybrid Tian 1-8 Qiongxi E088 Shandong 43 hybrid Tian 2-18 S206 Shandong 44 hybrid 9041C Cai 07 Shandong 45 hybrid C208 101 Shandong 46 variety Hang-R N0. 8 Shandong 47 variety Hang-R N0. 10 Shandong 48 variety Hang-R N0. 11 Shandong 49 farm variety Qianxi-2015504 Hebei 50 foreign variety C855 Israel 51 foreign variety KA93 Israel 52 foreign variety dwarf-2014309 France 53 foreign variety dwarf-2014411 France 54 foreign variety dwarf-2014412 France 2) subjecting the Ricinus communis L. germplasm resources to cytological analysis and chromosomal ploidy identification: preparing chromosome specimens of Ricinus communis L. through a wall degradation hypotonic method utilized in the preparation of chromosome specimens of plants. The basic number of the Ricinus communis L. chromosome is x=10, and the diploid chromosome number of somatic cells is 2n=2x=20; 3) selecting excellent diploid Ricinus communis L. lines according to the results of morphological analysis, economic characteristic analysis and chromosomal ploidy identification; selecting excellent diploid Ricinus communis L. varieties as materials for inducing tetraploids; 4) subjecting the seeds to sterile disinfection by using conventional disinfection methods, then removing the endosperms, stripping the seed embryos out, placing them into 1/2MS medium, and incubating under conditions of 23 C.2 C. without lighting until the seed embryos have germinated; the conventional disinfection method is as follows: disinfecting with 75% alcohol for 60 seconds, rinsing with sterile water, and then disinfecting with 2% sodium hypochlorite for 15 minutes, rinsing with sterile water for 3 times; 5) performing artificial mutagenesis of tetraploids: transferring the germinated young embryos into a solution containing mixed mutagens of colchicine and oryzalin at a certain mass concentration, to perform a doubling process in darkness at a lower temperature of 15-20 C. for 48-72 hours; the mixed mutagens were prepared as follows: firstly, preparing 0.02% colchicine (mass percentage concentration) and 0.02% oryzalin solutions, respectively, and then mixing them at a volume ratio of 1:1, resulting in that the final concentrations of the colchicine and oryzalin were 0.01% respectively; 6) transferring into 1/2MS medium, to continue to cultivate at 23 C.2 C. with a lighting of 1000 Lux, 16 hours/day; 7) transplanting the seedlings into disinfected soil when their true leaves have achieved more than 4 and their root systems have developed to normal; at 3 days before transplanting, opening the caps of bottles for acclimatization. The doubling treated seedlings have been largely damaged. Thus, the management should be strengthened after transplanting, by maintaining the temperature at 25 C.2 C., and the humidity at about 65%, to reduce the abnormal death of the seedlings. 8) chromosome analysis and ploidy identification: harvesting newborn young leaves at 8-9 a.m., pre-processing with 0.002M 8-hydroxyquinoline for 3-4 hours, then preparing chromosome specimens of Ricinus communis L. through a wall degradation hypotonic method utilized in the preparation of chromosome specimens of plants, removing chimeric and aneuploid plants, screening for plants whose somatic cell chromosome is 2n=4x=40 to obtain homozygous tetraploid plants, continue to cultivate the seedlings; 9) large-scale transplantation: performing large-scale planting and cultivation management according to conventional methods for cultivating Ricinus communis L., subjecting the F1 seeds of tetraploid Ricinus communis L. to chromosome analysis and ploidy identification again, removing chimeras and aneuploids, performing repeated identification as chimeras and aneuploids may exist in the F1 generation of Ricinus communis L. at lower frequency, screening for new homozygous tetraploid Ricinus communis L. germplasms.

SPECIFIC APPLICATION EXAMPLE

(8) 54 cultivars and wild resources of Ricinus communis L. were collected from different regions of China from May to December 2013. Firstly, they were subjected to chromosome analysis and ploidy identification to ensure the basic number of the Ricinus communis L. chromosome is x=10 and the chromosomes of somatic cells are all diploid, i.e. 2n=2x=20. In April 2014, excellent diploid Ricinus communis L. selected from the above germplasm resources were used as raw materials for artificial mutagenesis of tetraploids. Firstly, the seeds were subjected to sterile disinfection by using conventional disinfection methods, i.e. disinfecting with 75% alcohol for 60 seconds under sterile condition, rinsing with sterile water, disinfecting with 2% sodium hypochlorite for 15 minutes, rinsing with sterile water for 3 times. Then the endosperms were removed, and the seed embryos were stripped out and were placed into 1/2MS medium to incubate under conditions of 23 C.2 C. without lighting until the seed embryos had germinated. Then artificial mutagenesis of tetraploids was performed: the germinated young seed embryos were transferred into a solution containing mixed mutagens of colchicine and oryzalin at a certain mass concentration, to perform a doubling process in darkness at a lower temperature of 15-20 C. for 48-72 hours, and subsequently transferred into 1/2MS medium, to continue to cultivate at 23 C.2 C. with a lighting of 1000 Lux, 16 hours/day.

(9) The mixed mutagens were prepared as follows: firstly, preparing aqueous solutions of 0.02% colchicine (mass percentage concentration) and 0.02% oryzalin, respectively, and then mixing them at a volume ratio of 1:1, resulting in that the final concentrations of the colchicine and oryzalin were 0.01% respectively.

(10) In May 2014, the seedlings, as the materials for artificial mutagenesis, were transplanted, and then were individually identified for the ploidy, i.e., pre-process: young leaves were harvested at 8-9 a.m. and placed into 0.002M 8-hydroxyquinoline at 20-22 C. for 3-4 hours.fwdarw.pre-hypotonic treatment: hypotonic treatment with 0.075M KCl at 22 C. for 15 minutes.fwdarw.wall degradation: enzymatic treatment with 2.5% mixed enzymes (pectinase+cellulase) at 25 C. for 60 minutes.fwdarw.post-hypotonic treatment: hypotonic treatment with 0.075M KCl at 25 C. for 15 minutes.fwdarw.fixing: performing fixing treatment with a fixing solution of 3:1 (methanol:glacial acetic acid) for above 30 minutes.fwdarw.preparation of chromosome specimen.fwdarw.staining: staining with a staining solution of 40:1 (phosphate buffer : Giemsa) for 60 minutes.fwdarw.observation with microscope, chromosome analysis and ploidy identification were performed, homozygous tetraploid plants were selected and chimeric and aneuploid plants were eliminated. In June 2014, the homozygous tetraploid plants were subjected to large-scale planting and cultivation management according to conventional methods for cultivating Ricinus communis L. In October 2014, F1 seeds of the tetraploid Ricinus communis L. were harvested. In December 2014, the seeds of tetraploid Ricinus communis L. were subjected to chromosome analysis again to eliminate the chimeras and aneuploids that may exist in the F1 seeds at low frequency. In April 2015, the tetraploid Ricinus communis L. were planted on large-scale (see FIGS. 1 to 3 for a comparison result of diploid Ricinus communis L. and tetraploid Ricinus communis L.).

(11) In the method, mixed mutagens are employed and the endosperms are removed during the germination of the Ricinus communis L. seeds to force the naked embryos to absorb mutagens to interrupt the formation of spindles, which greatly increase the frequency of mutagenesis and result in a new line of homozygous Ricinus communis L. tetraploids in China for the first time. Chromosome analysis and identification performed on F1 young leaves demonstrate the reduced confounding of chimeras and aneuploids and the great improvement in the screening frequency of homozygous tetraploids.