CN112400693A - Pretreatment method for improving induction of calluses of mature embryos of indica rice - Google Patents
Pretreatment method for improving induction of calluses of mature embryos of indica rice Download PDFInfo
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- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract description 46
- 240000002582 Oryza sativa Indica Group Species 0.000 title claims abstract description 39
- 230000006698 induction Effects 0.000 title claims abstract description 39
- 238000002203 pretreatment Methods 0.000 title claims abstract description 18
- 210000002257 embryonic structure Anatomy 0.000 title claims description 20
- 229950009390 symclosene Drugs 0.000 claims abstract description 37
- YRIZYWQGELRKNT-UHFFFAOYSA-N 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione Chemical compound ClN1C(=O)N(Cl)C(=O)N(Cl)C1=O YRIZYWQGELRKNT-UHFFFAOYSA-N 0.000 claims abstract description 34
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 14
- 235000007164 Oryza sativa Nutrition 0.000 claims description 28
- 235000009566 rice Nutrition 0.000 claims description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 18
- 239000007864 aqueous solution Substances 0.000 claims description 15
- 239000008223 sterile water Substances 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 229920002148 Gellan gum Polymers 0.000 claims description 3
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- -1 trichloroisocyanuric acid compound Chemical class 0.000 claims description 3
- 235000021186 dishes Nutrition 0.000 claims description 2
- 241000209094 Oryza Species 0.000 claims 3
- 238000007654 immersion Methods 0.000 claims 2
- 238000005406 washing Methods 0.000 claims 1
- 230000002068 genetic effect Effects 0.000 abstract description 12
- 230000009466 transformation Effects 0.000 abstract description 8
- 239000005556 hormone Substances 0.000 abstract description 5
- 229940088597 hormone Drugs 0.000 abstract description 5
- 230000009261 transgenic effect Effects 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 3
- 240000007594 Oryza sativa Species 0.000 description 25
- 239000001963 growth medium Substances 0.000 description 15
- 230000035876 healing Effects 0.000 description 9
- 238000002791 soaking Methods 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 5
- 230000035784 germination Effects 0.000 description 5
- 230000006872 improvement Effects 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 240000008467 Oryza sativa Japonica Group Species 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 1
- 229960001669 kinetin Drugs 0.000 description 1
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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
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明属于转基因技术领域，具体提供了一种提高籼稻成熟胚愈伤组织诱导率的前处理方法。去壳的籼稻种子，利用质量浓度为0.5％‑1％的30％三氯异氰尿酸溶液浸泡8‑12h，而后按照传统方法进行种子消毒和接种于诱导培养基，可以在不调整传统诱导培养基配方、激素类型及浓度等条件下，显著提高籼稻种子的出愈率和显著增加愈伤组织的质量，为解决籼稻诱导率比较低提供新的途径。本发明简便易行，不仅节省大量时间和精力，而且也促进遗传转化进程。
The invention belongs to the technical field of transgenic, and specifically provides a pretreatment method for improving the induction rate of mature embryo callus of indica rice. The hulled indica rice seeds are soaked in a 30% trichloroisocyanuric acid solution with a mass concentration of 0.5%-1% for 8-12 hours, and then the seeds are sterilized and inoculated in the induction medium according to the traditional method. Under the conditions of basic formula, hormone type and concentration, the callus rate of indica rice seeds and the quality of callus were significantly increased, which provided a new way to solve the low induction rate of indica rice. The invention is simple and easy to implement, not only saves a lot of time and energy, but also promotes the process of genetic transformation.
Description
Technical Field
The invention belongs to the technical field of transgenosis, and particularly provides a pretreatment method for improving the induction of calluses of mature embryos of indica rice.
Background
Rice is one of the most important food crops in the world, and the survival of more than half of the world population is maintained. In recent years, with the increase of the population of the world and the reduction of the culturable area, the improvement of the yield and quality of rice has an important meaning for solving the worldwide food crisis. The further development of molecular biology and molecular genetics makes the transgenic technology an important tool for improving the yield and quality of rice. The tissue culture technology is an important component of the transgenic technology and is widely applied to rice genetic transformation and variety improvement. The most used system for rice genetic transformation is a callus regeneration system. The callus induction rate of japonica rice varieties is high, a high-efficiency genetic transformation system is established, and the transgenic breeding has obtained favorable results in japonica rice; however, indica rice which accounts for more than 80% of rice varieties is difficult to genetically transform due to low callus induction rate, and the application of a transgenic technology in indica rice is severely restricted.
The rice somatic tissue culture is a basic technology of rice genetic transformation and plays an important role in rice genetic improvement, so that the establishment of a high-efficiency regeneration system is a prerequisite for realizing genetic transformation of genes. Because the mature embryo of the rice has the advantages of wide source, no limitation of the material selection by seasons and geographical environments, convenient operation, difficult pollution and the like, the mature embryo of the rice is an ideal material for genetic operation, but the callus induction rates of different varieties of rice are very different; even under the theoretically optimal conditions, the callus induction rate of mature embryos of many indica rice varieties with excellent traits is still low. In the traditional method, the callus induction of the mature embryo of the rice is improved by adopting measures on the improvement of a culture medium and other aspects of tissue culture to induce the callus, for example, different culture media, different hormone types and concentrations are tried out, and a small amount of kinetin is added in the induction culture medium to improve the differentiation quality of the callus; organic additives (e.g. hydrolysed casein) are added to improve the callus quality of certain varieties. However, the results of previous studies show that the genetic background of rice determines the induction process in tissue culture, and the main factor influencing the induction of rice callus is the genotype of rice varieties. The rice variety has abundant resources and high-level genetic diversity, and the induction of the calluses of different mature embryos of the indica rice is improved by trying different culture media, different hormone types and concentrations, culture medium improvement and other aspects of tissue culture by using a traditional method, so that the method is time-consuming and labor-consuming, and ideal results are not necessarily obtained.
Based on this, a method widely applicable to pretreatment for callus induction of indica rice materials is highly demanded in the art.
Disclosure of Invention
The invention aims to solve the technical problem that the invention provides a callus induction pretreatment method which is widely suitable for indica rice materials. After the pretreatment, the callus induction rate of the indica rice can be improved without adjusting the formula, the hormone type and the concentration of an induction culture medium used in the traditional method. The pretreatment method of the invention not only saves time and labor, but also accelerates the genetic transformation progress of indica rice.
The invention is realized by the following means:
a pretreatment method for improving callus induction of mature embryos of indica rice comprises the following steps:
(1) hulling mature embryo of long-shaped rice
Selecting full, non-mildew and mature indica rice seeds. Hulling with manual or miniature huller, taking care to keep embryo intact.
(2) Preparation of pretreatment reagent
30 percent of trichloroisocyanuric acid compound and sterilized water, and preparing trichloroisocyanuric acid solution with the mass concentration of 0.5 to 1 percent according to the mass ratio.
(3) Pretreatment of mature embryo
The rice seeds after shelling are washed clean with clear water and rinsed 3-5 times with sterile water. Soaking the hulled rice seeds for 8-12h at 25-37 ℃ by using trichloroisocyanuric acid solution with the mass concentration of 0.5-1%; rinsing with sterile water for 3-5 times, and sucking dry the seeds with sterile filter paper.
(4) Sterilization of mature embryos
Treating the rice seeds soaked in the trichloroisocyanuric acid solution with 75% alcohol for 2min, and rinsing with sterile water for 2-3 times; sterilizing with 5-10% sodium hypochlorite for 15-20min, or sterilizing with 0.15% mercuric chloride for 12min, and rinsing with sterile water for 2-3 times; sucking dry the seeds with sterile filter paper; then inoculated into callus induction medium.
The callus induction culture medium comprises the following components in percentage by weight: NB minimal medium +2,4-D (4mg/L) + sucrose (30g/L) + plant gel phytagel (2.7g/L), pH 5.8.
The invention has the beneficial effects that:
according to the method, husked indica rice seeds are soaked in 30% trichloroisocyanuric acid solution with the mass concentration of 0.5% -1% for 8-12h, then the seeds are disinfected and inoculated in a traditional induction culture medium according to a traditional method, the callus growth rate of the indica rice seeds can be obviously improved, the quality of callus can be obviously improved, the tedious work of trying to adjust the formula of the induction culture medium, the types of hormones, the concentration conditions and the like in order to improve the callus induction rate in the traditional method is avoided, and a new way is provided for solving the problem of low indica rice induction rate; the method is simple and easy to implement, not only saves a large amount of time and energy, but also promotes the genetic transformation process.
Drawings
FIG. 1 shows callus induction conditions of 93-11 indica rice seeds soaked in 1% 30% trichloroisocyanuric acid aqueous solution for 4-72 h; a. the treatment times of b, c, d, e, f and g were 4h, 8h, 12h, 24h, 36h, 48h and 72h, respectively.
FIG. 2 shows callus induction of indica rice seeds treated with 30% trichloroisocyanuric acid aqueous solution with a mass concentration of 1% and water for 12 hours; a. b, treating indica rice seeds by water and 1 percent of 30 percent trichloroisocyanuric acid aqueous solution for 12 hours respectively.
Detailed Description
Example 1
A pretreatment method for improving callus induction of mature embryos of indica rice comprises the following steps:
(1) hulling mature embryo of long-shaped rice
Selecting full, non-mildew and mature indica rice seeds. Hulling with manual or miniature huller, taking care to keep embryo intact.
(2) Preparation of pretreatment reagent
30 percent of trichloroisocyanuric acid compound and sterilized water, and preparing trichloroisocyanuric acid solution with the mass concentration of 0.5 to 1 percent according to the mass ratio.
(3) Pretreatment of mature embryo
The rice seeds after shelling are washed clean with clear water and rinsed 3-5 times with sterile water. Soaking the hulled rice seeds for 8-12h at 25-37 ℃ by using trichloroisocyanuric acid solution with the mass concentration of 0.5-1%; rinsing with sterile water for 3-5 times. The seeds were blotted dry on sterile filter paper.
(4) Sterilization of mature embryos
Treating the rice seeds soaked in the trichloroisocyanuric acid solution with 75% alcohol for 2min, and rinsing with sterile water for 2-3 times; sterilizing with 5-10% sodium hypochlorite for 15-20min, or sterilizing with 0.15% mercuric chloride for 12min, and rinsing with sterile water for 2-3 times; the seeds were blotted dry on sterile filter paper. Inoculating to callus induction culture medium.
The callus induction culture medium comprises the following components in percentage by weight: NB minimal medium +2,4-D (4mg/L) + sucrose (30g/L) + plant gel phytagel (2.7g/L), pH 5.8.
The key steps (2) and (3) are implemented in the following specific steps:
the 30% trichloroisocyanuric acid aqueous solution with the concentration of 1% can effectively promote the germination of seeds. Soaking indica rice seeds such as 93-11 and Mudgo for 0-72h by using 1% 30% trichloroisocyanuric acid aqueous solution, sterilizing mature embryos after pretreatment, and inoculating 10-12 seeds in each induction culture medium plate and 3 dishes in each treatment. Taking water soaking or not as a reference, and counting the healing rate of the mature embryos soaked in different concentrations for different times after 21 days of inoculation. The results show that the cure rate is higher for treatment 12h, and the specific results are shown in table 1 and fig. 1.
The test result of the output rate calculation method is counted according to the following formula:
the survival rate (%) is the number of seeds growing out callus/the number of inoculated seeds multiplied by 100%
TABLE 1 cure rate of trichloroisocyanuric acid solution with 1% mass concentration after pretreatment for 12h
From the results in table 1, it is understood that the germination rate and the healing rate increase with time in the first 4 hours after the pretreatment with the trichloroisocyanuric acid solution having a mass concentration of 1%, but the germination rate and the healing rate decrease with time at the 8 th hour, and the germination rate and the healing rate are the highest and then gradually decrease after 12 hours. And the germination rate and the recovery rate when reaching 12h are obviously higher than the time points of 1h, 4h, 8h, 24h, 36h, 48h and 72h, and the difference has statistical significance (P is less than 0.05).
Taking 1% as a base point, preparing 30% trichloroisocyanuric acid aqueous solutions with mass concentrations of 0.5% and 1.5%, and respectively soaking indica rice seeds of 93-11, Mudgo and the like for 8h, 12h and 24 h. After the pretreatment is finished, the mature embryo is disinfected, 10-12 seeds are inoculated to each induction culture medium plate, and 3 plates are inoculated to each treatment. After 21 days of inoculation, the callus rate of the mature embryos soaked in different concentrations for different times is counted. The result shows that no callus is obtained when 30% trichloroisocyanuric acid aqueous solution with the mass concentration of 1.5% is treated for 8 hours, 12 hours and 24 hours; callus is obtained when 30% trichloroisocyanuric acid water solution with the mass concentration of 0.5% is processed for 8 hours, 12 hours and 24 hours, so that the healing rate is higher when the callus is processed for 12 hours, and specific results are shown in table 2.
TABLE 2 cure rate of trichloroisocyanuric acid solution pre-treatment at 0.5% and 1.5% by mass for 8, 12 and 24h
0.1% is used as a variation to prepare 30% trichloroisocyanuric acid aqueous solution with mass concentration of 0.6%, 0.7%, 0.8%, 0.9%, 1.1%, 1.2%, 1.3% and 1.4%. Soaking indica rice seeds of 93-11, Mudgo, etc. for 8, 12 and 24h respectively. After the pretreatment is finished, the mature embryo is disinfected, 10-12 seeds are inoculated to each induction culture medium plate, and 3 plates are inoculated to each treatment. After 21 days of inoculation, the callus rate of the mature embryos soaked in different concentrations for different times is counted. The results show that the higher healing rate can be obtained by treating 0.6-0.9% of 30% trichloroisocyanuric acid aqueous solution for 8-12h, and the specific results are shown in Table 3.
TABLE 3 cure rate at 8, 12 and 24h pre-treatment with trichloroisocyanuric acid solution with mass concentration of 0.5% -1.5%
According to the results of comparison of the healing rate of 0-72h of indica rice seeds treated by 30% trichloroisocyanuric acid aqueous solution with the concentration gradient, the indica rice seeds treated by 0.5-1% of 30% trichloroisocyanuric acid aqueous solution for 8-12h can obtain higher healing rate.
Indica rice seeds are treated by 30 percent trichloroisocyanuric acid aqueous solution with the mass concentration of 1 percent for 12 hours. After the pretreatment is finished, the mature embryo is disinfected, 10-12 seeds are inoculated to each induction culture medium plate, and 3 plates are inoculated to each treatment. The callus was weighed 21 days after inoculation with or without water soaking as a control, and it was analyzed whether there was a significant difference between 12h treatment of indica rice seeds with 1% aqueous 30% trichloroisocyanuric acid and water soaking. The results show that there is a significant difference between 12h of indica rice seeds treated with 30% trichloroisocyanuric acid aqueous solution with mass concentration of 1% and the quality of callus soaked with water, as shown in table 4 and fig. 2.
TABLE 4 callus weight pretreated with and without trichloroisocyanuric acid solution for 12h
| Categories | Using trichloroisocyanuric acid | Without using trichloroisocyanuric acid |
| Weight (g) | 0.4667±0.1413** | 0.1191±0.0505 |
Note: p < 0.01.
The results show that for most indica rice seeds, 30 percent trichloroisocyanuric acid solution with the mass concentration of 0.5 to 1 percent is used for soaking for 8 to 12 hours, the healing rate of the indica rice seeds is obviously improved compared with that of the indica rice seeds soaked by water, and the quality of callus is also obviously improved compared with that of the indica rice seeds soaked by water.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
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| CN107129993A (en) * | 2016-02-26 | 2017-09-05 | 华中农业大学 | A kind of modified glyphosate-resistant gene and breeding method of glyphosate-resistant rice |
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| CN107129993A (en) * | 2016-02-26 | 2017-09-05 | 华中农业大学 | A kind of modified glyphosate-resistant gene and breeding method of glyphosate-resistant rice |
Non-Patent Citations (3)
| Title |
|---|
| 曾晓珊等: "籼粳稻对农杆菌遗传转化反应的差异及提高转化率的研究", 《湖南农业大学学报(自然科学版)》 * |
| 钟华鑫等: "DMSO浸种对水稻种子愈伤组织诱导、再分化的影响", 《武汉植物学研究》 * |
| 高立宏等: "水稻转座子Pong特异性RNAi载体TPAS的构建和农杆菌介导的遗传转化条件的优化", 《东北师大学报（自然科学版）》 * |
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