WO2019061621A1 - 一种等离子体高通量定向诱变甘蔗抗草甘膦的方法 - Google Patents

一种等离子体高通量定向诱变甘蔗抗草甘膦的方法 Download PDF

Info

Publication number
WO2019061621A1
WO2019061621A1 PCT/CN2017/107910 CN2017107910W WO2019061621A1 WO 2019061621 A1 WO2019061621 A1 WO 2019061621A1 CN 2017107910 W CN2017107910 W CN 2017107910W WO 2019061621 A1 WO2019061621 A1 WO 2019061621A1
Authority
WO
WIPO (PCT)
Prior art keywords
medium
sugarcane
glyphosate
μmol
embryogenic callus
Prior art date
Application number
PCT/CN2017/107910
Other languages
English (en)
French (fr)
Inventor
黄忠兴
孙东磊
徐苑娴
何慧怡
樊丽娜
劳方业
凌秋平
安玉兴
Original Assignee
广东省生物工程研究所（广州甘蔗糖业研究所）
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广东省生物工程研究所（广州甘蔗糖业研究所） filed Critical 广东省生物工程研究所（广州甘蔗糖业研究所）
Priority to US16/315,016 priority Critical patent/US11516979B2/en
Publication of WO2019061621A1 publication Critical patent/WO2019061621A1/zh

Links

Images

Classifications

    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • 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
    • A01H4/008—Methods for regeneration to complete plants
    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • 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
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/123—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • A01H1/1235—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance to glyphosate
    • 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
    • A01H4/002—Culture media for tissue culture

Definitions

  • the invention relates to a method for plasma high-throughput directed mutagenesis of sugarcane glyphosate resistant.
  • Sugarcane is an important sugar crop in China.
  • Sugar cane accounts for more than 90% of China's sugar production.
  • the development of the sugar industry is inseparable from the improvement of sugarcane varieties.
  • the contribution rate of fine varieties to the technological progress of sugar production is 60%. It is the main core technology for the sustainable development of China's sugar industry and the international competitiveness of the sugar industry.
  • Excellent varieties have more or less disadvantages in the production process, such as drought resistance or glyphosate resistance.
  • Sugarcane mutagenesis breeding is a rapid way to improve the cultivation of sugarcane varieties, and it is a situation to alleviate the simplification of the structure of sugarcane varieties in China. Reducing callus pollution during sugarcane mutagenesis is an important key link.
  • the invention of sugarcane callus irradiated by low-energy plasma beam provides good environmental conditions for sugarcane callus and ensures the smooth progress of sugarcane mutagenic breeding. .
  • the present invention provides for the first time a method for high-throughput mutagenesis of sugarcane embryogenic callus, which eliminates the problem that chemical mutagens cause carcinogenicity to humans, and allows technicians to work with peace of mind, not only to solve the traditional problem.
  • the problem of easy contamination of sugarcane callus can also meet the demand of large-scale mutagenesis of sugarcane callus, and solve the problem of inconvenient operation and small amount of processing of test tube slant medium.
  • the mutagenic effect is obviously different and can be based on embryogenicity.
  • the survival rate of callus was quickly determined to determine the time of mutagenesis treatment.
  • the efficiency of mutagenesis was improved by targeted stress screening, and the mutagenesis rate was 2.50%-2.77%.
  • a method for plasma high-throughput directed mutagenesis of sugarcane glyphosate comprises the following steps:
  • the sugarcane embryogenic callus was irradiated and mutagenized under sterile conditions with a plasma instrument.
  • the mutagenic power was 140-200 W
  • the discharge spacing was 35-45 mm
  • the mutagenic time was 110-140 s.
  • the medium CM2 contains: MS medium, 2,4-D 1.2-1.7 ⁇ mol ⁇ L -1 , sucrose 28-32 g ⁇ L -1 , agar 7.5-8.5 g ⁇ L -1 , pH 6.0-6.4 ;
  • the medium CM3 comprises: MS medium, sucrose 28-32 g ⁇ L -1 , agar 7.5-8.5 g ⁇ L -1 , pH 6.0-6.4;
  • the medium CM4 contains: MS medium, NAA 0.4-0.6 ⁇ mol ⁇ L -1 , Kt 0.8-1.2 ⁇ mol ⁇ L -1 , sucrose 48-52 g ⁇ L -1 , agar 7.5-8.5 g ⁇ L -1 , pH value 6.0 to 6.4;
  • the medium CM5 comprises: MS medium, 4.8-5.2 ⁇ mol ⁇ L -1 glyphosate, sucrose 28-32 g ⁇ L -1 , agar 8 g ⁇ L -1 , pH 6.0-6.4;
  • the seedlings that have survived in the previous step are connected to the CM4 for expansion and cultivation, and then transferred to the CM6 for root promotion, refining, and then transplanted on the seedbed.
  • the seedling grows to 100 mm to 150 mm, use 4.8. ⁇ 5.2 ⁇ mol ⁇ L -1 glyphosate spray screening, the survival of the glyphosate resistant strain;
  • the medium CM6 contains: 1/2 MS medium, NAA 0.4-0.6 ⁇ mol ⁇ L -1 , 6-BA 1.8-2.2 ⁇ mol ⁇ L -1 , sucrose 28-32 g ⁇ L -1 , pH 6.0-6.4 .
  • step (1) the mutagenic power is 140 W, the irradiation interval is 40 mm, and the mutagenic time is 120 s.
  • the gas flow rate of nitrogen in the step (1) is 1.0 to 1.4 L/min.
  • the plasma instrument adopts the HPD-280 plasma instrument produced by Nanjing Suman Electronics Co., Ltd.
  • the plasma instrument is sterilized before use, and a container containing 28 to 32% of the H 2 O 2 solution is placed in the treatment chamber, and the vacuum is used for diffuse disinfection.
  • the sugarcane embryogenic callus in the step (1) is a sugarcane embryogenic callus after subculture 2-3 times.
  • the preparation method of the sugarcane embryogenic callus in the step (1) is: after the sugarcane stem grows, the tail of the robust sugarcane plant is cut, the outer blade is stripped, and the alcohol is disinfected under sterile conditions.
  • the CM1 medium is an MS medium containing 1.8-2.2 ⁇ mol ⁇ L -1 2,4-D, 28-32 g ⁇ L -1 sucrose and 7.5-8.5 g ⁇ L -1 agar, and the pH value is 6.0 to 6.4.
  • the number of days of screening culture in the step (5) is 26 to 30 days.
  • the number of days of expansion and cultivation in the step (6) is 28 to 32 days.
  • Figure 1 is the effect of buffer culture days after plasma mutagenesis on the survival rate of sugarcane embryogenic callus
  • FIG. 2 is a graph showing an increase in plant height after spraying 0-5.0 ⁇ mol ⁇ L -1 glyphosate in the glyphosate-resistant strain of the present invention and the control group (CK);
  • Figure 3 shows the damage of anti- and glyphosate-resistant strains after 20 days of spraying 5.0 ⁇ mol ⁇ L -1 glyphosate
  • Figure 4 shows the growth of anti-and glyphosate-resistant strains after 30 days of spraying 5.0 ⁇ mol ⁇ L -1 glyphosate.
  • the left side of the potted plants in the pots were non-glyphosate resistant lines, and the three strains on the right side were side by side. It is a mutant strain.
  • a method for plasma high-throughput directed mutagenesis of sugarcane glyphosate comprises the following steps:
  • the sugarcane embryogenic callus was irradiated and mutagenized under sterile conditions with a plasma instrument.
  • the mutagenic power was 140-200 W
  • the discharge spacing was 35-45 mm
  • the mutagenic time was 110-140 s.
  • the medium CM2 contains: MS medium, 2,4-D 1.2-1.7 ⁇ mol ⁇ L -1 , sucrose 28-32 g ⁇ L -1 , agar 7.5-8.5 g ⁇ L -1 , pH 6.0-6.4 ;
  • the medium CM3 comprises: MS medium, sucrose 28-32 g ⁇ L -1 , agar 7.5-8.5 g ⁇ L -1 , pH 6.0-6.4;
  • the medium CM4 contains: MS medium, NAA 0.4-0.6 ⁇ mol ⁇ L -1 , Kt 0.8-1.2 ⁇ mol ⁇ L -1 , sucrose 48-52 g ⁇ L -1 , agar 7.5-8.5 g ⁇ L -1 , pH value 6.0 to 6.4;
  • the medium CM5 comprises: MS medium, 4.8-5.2 ⁇ mol ⁇ L -1 glyphosate, sucrose 28-32 g ⁇ L -1 , agar 8 g ⁇ L -1 , pH 6.0-6.4;
  • the seedlings that have survived in the previous step are connected to the CM4 for expansion and cultivation, and then transferred to the CM6 for root promotion, refining, and then transplanted on the seedbed.
  • the seedling grows to 100 mm to 150 mm, use 4.8. ⁇ 5.2 ⁇ mol ⁇ L -1 glyphosate spray screening, the survival of the glyphosate resistant strain;
  • the medium CM6 contains: 1/2 MS medium, NAA 0.4-0.6 ⁇ mol ⁇ L -1 , 6-BA 1.8-2.2 ⁇ mol ⁇ L -1 , sucrose 28-32 g ⁇ L -1 , pH 6.0-6.4 .
  • the mutagenic power in step (1) is 140 W
  • the irradiation interval is 40 mm
  • the mutagenic time is 120 s.
  • the gas flow rate of nitrogen in the step (1) is 1.0 to 1.4 L/min.
  • the gas flow rate of nitrogen in the step (1) is 1.2 L/min.
  • the applied voltage is 220V.
  • the plasma instrument adopts a HPD-280 type plasma instrument produced by Nanjing Suman Electronics Co., Ltd.
  • the plasma instrument is sterilized prior to use, and a container containing 28 to 32% of the H 2 O 2 solution is placed in the treatment chamber, and the vacuum is used for diffuse disinfection.
  • the sugarcane embryogenic callus in step (1) is a sugarcane embryogenic callus after subculture 2-3 times.
  • the preparation method of the sugarcane embryogenic callus in the step (1) is: after the sugarcane stem grows, the tail of the robust sugarcane plant is cut, the outer leaf is stripped, and the alcohol is disinfected under sterile conditions.
  • the sugarcane embryogenic callus in step (1) has a size of 0.8-1.2 mm ⁇ 0.8-1.2 mm ⁇ 0.8-1.2 mm, and is discharged into the culture dish, so that each embryogenic callus can be spoke. Upon receipt, the embryogenic callus at the bottom of the dish can also receive discharge radiation.
  • the CM1 medium is an MS medium containing 1.8-2.2 ⁇ mol ⁇ L -1 2,4-D, 28-32 g ⁇ L -1 sucrose and 7.5-8.5 g ⁇ L -1 agar, and the pH value is 6.0 to 6.4.
  • the number of days of screening culture in the step (5) is 26 to 30 days.
  • the number of days of expansion and propagation in the step (6) is 28 to 32 days.
  • the prepared flakes were inoculated into the medium CM1, cultured at 26 ° C to 28 ° C in the dark, 6-8 pieces per dish, induced callus production, and then in the medium CM1 (MS + 2, 4-D 2.0 ⁇ mol ⁇ L -1 + sucrose 30g ⁇ L -1 + agar 8g ⁇ L -1 , pH 6.2) Relay generation 1-2 times, 6-8 pieces per dish, to produce embryogenic callus.
  • the plasma machine adopts the HPD-280 type produced by Nanjing Suman Electronics Co., Ltd. (the powers indicated in the instrument are 40%, 50%, 60% respectively corresponding to specific powers of 140W, 170W, 200W).
  • the injured tissue was mutagenized, the mutagenic power was 140 W, and the irradiation distance was 40 mm.
  • the shielding gas is nitrogen, the gas flow rate is 1.2 L/min, the irradiation mutagenic time is set at 120 s, and the applied voltage is 220V.
  • 30% H 2 O 2 20 mI was placed in a petri dish, placed in a treatment chamber, and vacuum-dissipated for diffuse disinfection.
  • the upper lid of the petri dish covered with the embryogenic callus is then opened, placed in the middle of the processing chamber, and subjected to irradiation mutagenesis according to the plasma operation instructions.
  • the upper lid of the petri dish was covered and sealed with a polyethylene film.
  • the medium CM3 comprises: MS medium, sucrose 28-32 g ⁇ L -1 , agar 7.5-8.5 g ⁇ L -1 , pH 6.2;
  • the viable seedlings were transferred to CM4 for expansion and culture for 30 days, and then transferred to CM6 (1/2MS+NAA 0.5 ⁇ mol ⁇ L -1 +6-BA 2.0 ⁇ mol ⁇ L -1 + sucrose 30g ⁇ L -1 , pH Value 6.2) on the root promotion, refining, and then transplanted on the seedbed, after about 1 month, when the seedling grows to a height of 100mm ⁇ 150mm, spray the leaf surface with 5.0 ⁇ mol ⁇ L -1 glyphosate once, The final survivor is a glyphosate resistant strain.
  • the mutagenic rate % 100% x the number of glyphosate resistant plants / the number of treated embryogenic callus * (i.e., the mutagenicity rate described in Table 1 below).
  • Example 2 On July 15, 2015, the HPD-280 plasma machine was used to induce the embryogenic callus of Yuesu 93-159 sugarcane.
  • the mutagenic power was set to 140W, and the irradiation mutagenesis time was 80s, 100s and 120s respectively. , 140s, 160s, 180s.
  • the other operations in Example 2 were the same as in Example 1. The survival rate and mutagenic rate of each group of embryogenic callus were detected.
  • the test results are shown in Table 1.
  • the survival rate of the embryogenic callus treated with 120s was the highest, 18.3%, which was significantly higher than other treatments, and it could be differentiated into two strains after screening by two rounds of glyphosate.
  • the mutagenic rate was 2.50%. .
  • the survival rate of embryogenic callus% 100% ⁇ the number of embryogenic callus survival/the number of treated embryogenic callus
  • Mutagenesis rate 100% x glyphosate resistant plants / number of treated embryogenic calli.
  • the HPD-280 plasma machine was used to induce the embryogenic callus of Yuesu 93-159 sugarcane, and the irradiation mutagenesis time was 120s.
  • the mutagenic power was set to 140W, 170W, and 200W, respectively.
  • the other operations in the implementation 3 are the same as the implementation 1.
  • the survival rate and mutagenic rate of each group of embryogenic callus were detected.
  • test results are shown in Table 2. Among them, the survival rate of embryogenic callus treated with power of 140W was the highest, which was 62.4%. After two rounds of glyphosate screening, each group could reduce the most differentiated plants, and the highest mutagenesis rate. It is 140W, reaching 2.77%.
  • the HPD-280 plasma machine was used to induce the embryogenic callus of Yuesu 93-159 sugarcane, the power was set to 140W, the callus was mutagenized for 120s, and inoculated on glyphosate solid medium CM2.
  • the buffer culture was set to 8d, 10d, and 13d, respectively, and the other operations in Example 4 were the same as in Example 1. The survival rate of each group of embryogenic callus was examined.
  • the leaves are sprayed with 0, 1.0, 2.5, 4.0, 5.0 ⁇ mol ⁇ L -1 glyphosate respectively.
  • the glyphosate resistant strain is damaged, after 20 days. After still survived, the plant height increased the most, compared with the control, the maximum increase was 9.5mm (Fig. 2), the leaf color remained green, while the control showed the phenomenon of early leaf rolling and late heart (Fig. 3). After 30 days, the control had been browned, and some of the mutants remained greenish (Fig. 4).
  • the mutant strain has strong glyphosate resistance and is a glyphosate resistant strain.
  • the HPD-280 plasma machine was used to induce the embryogenic callus of Yuetang 00-236 sugarcane.
  • the mutagenic power was set to 140W and the irradiation mutagenesis time was 120s.
  • the gas was set to air when mutagenized. Nitrogen flow rate 1.2L/min, nitrogen Gas 0.2L/min.
  • the other operations in Example 6 were the same as in Example 1.
  • the test results are shown in Table 3.
  • the survival rate of the embryogenic callus treated with the protective gas of 1.2 L/min was the highest, which was 22.2%, which was significantly higher than other treatments.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Botany (AREA)
  • Environmental Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Cell Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Physiology (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明提供了一种等离子体高通量定向诱变甘蔗抗草甘膦的方法。该方法为将甘蔗胚性愈伤组织在无菌条件下用等离子体仪器进行辐照诱变处理，诱变功率为140～200W，放电间距35～45mm，诱变时间为110～140s，保护气体为氮气；处理后的愈伤组织进行缓冲培养，中高浓度草甘膦胁迫筛选，分化成苗，瓶苗草甘膦胁迫筛选，盆栽叶面喷草甘膦胁迫筛选。

Description

一种等离子体高通量定向诱变甘蔗抗草甘膦的方法 技术领域
本发明涉及一种等离子体高通量定向诱变甘蔗抗草甘膦的方法。
背景技术
甘蔗(Sugarcane)是我国重要的糖料作物，甘蔗糖占我国食糖产量的90％以上。蔗糖业的发展离不开甘蔗品种改良，优良品种对蔗糖生产的技术进步的贡献率达60％，是我国蔗糖业持续发展和提高蔗糖业国际竞争力的主要核心技术。优良品种在生产过程存在或多或少的缺点，如抗旱性不强或不抗草甘膦等。甘蔗诱变育种是进行栽培甘蔗品种改良的快速途径，是缓解我国甘蔗品种结构单一化的局面。甘蔗诱变育种过程中减少愈伤组织污染是一个重要的关键环节，利用低能等离子束辐照甘蔗愈伤组织的发明给甘蔗愈伤组织提供良好的环境条件，确保甘蔗诱变育种工作的顺利进行。
早期比较传统的甘蔗愈伤组织诱变主要是利用化学诱变剂和Co60-r射线来诱变。化学诱变愈伤组织一方面，由于化学诱变剂对人存在致癌风险；另一方面，操作过程中容易引起污染；再方面处理量有限，不能满足试验需求量；Co60-r射线诱变效果虽好，但需要特殊场地和特殊保护，运行成本高；前人在微生物培养过程中采用试管斜面培养基管口塞棉花置于等离子体机里面诱变，以防污染，防菌效果虽然好，但此种方法存在空间小、操作不方便、处理量偏小等缺点。
发明内容
为了解决上述存在的问题，本发明首次提供了一种高通量诱变甘蔗胚性愈伤组织的方法，杜绝了化学诱变剂对人引起致癌的问题，让技术人员安心工作，不但解决传统甘蔗愈伤组织容易污染的问题，还能适应大批量诱变甘蔗愈伤组织的需求，解决了试管斜面培养基操作不方便、处理量小的问题，其诱变效果差异明显，能根据胚性愈伤组织成活率快速确定诱变处理时间，通过定向胁迫筛选，提高诱变育种效率，诱变率达2.50％-2.77％。
本发明的目的在于提供一种等离子体高通量定向诱变甘蔗抗草甘膦的方法。
本发明所采取的技术方案是：
一种等离子体高通量定向诱变甘蔗抗草甘膦的方法，包括如下步骤：
(1)将甘蔗胚性愈伤组织在无菌条件下用等离子体仪器进行辐照诱变处理，诱变功率为140～200W，放电间距35～45mm，诱变时间为110～140s，保护气体为氮气；
(2)将上步诱变过的甘蔗胚性愈伤组织接种在固体培养基CM2上，用薄膜密封，缓冲 培养8～10d；
所述培养基CM2中含有：MS培养基、2,4-D 1.2～1.7μmol·L-1、蔗糖28～32g·L-1、琼脂7.5～8.5g·L-1，pH值6.0～6.4；
(3)将缓冲培养过的甘蔗愈伤组织依次接种在含1.2～1.7μmol·L-1、2.2～2.7μmol·L-1草甘膦的固体培养基CM3上，用薄膜密封，分别筛选培养8～10d；
所述培养基CM3中含有：MS培养基、蔗糖28～32g·L-1、琼脂7.5～8.5g·L-1，pH值6.0～6.4；
(4)将上步成活的胚性愈伤组织转接到固体分化培养基CM4上，培养至出苗后再继代培养一次；
所述培养基CM4中含有：MS培养基、NAA 0.4～0.6μmol·L-1、Kt 0.8～1.2μmol·L-1、蔗糖48～52g·L-1、琼脂7.5～8.5g·L-1，pH值6.0～6.4；
(5)待上步中的小苗长至20mm-30mm时，转接至CM5上进行筛选培养；
所述培养基CM5中含有：MS培养基、4.8～5.2μmol·L-1草甘膦、蔗糖28～32g·L-1、琼脂8g·L-1，pH值6.0～6.4；
(6)将上步中成活的小苗接到CM4上进行扩繁培养，再转接到CM6上进行促根培养、炼苗，然后移植在苗床上，待苗长至100mm～150mm时，用4.8～5.2μmol·L-1草甘膦喷雾筛选，成活的为抗草甘膦株系；
所述培养基CM6中含有：1/2MS培养基、NAA 0.4～0.6μmol·L-1、6-BA 1.8～2.2μmol·L-1、蔗糖28～32g·L-1，pH值6.0～6.4。
进一步的，步骤(1)中诱变功率为140W，照射间距40mm，诱变时间为120s。
进一步的，步骤(1)中氮气的气流量1.0～1.4L/min。
进一步的，等离子体仪器采用南京苏曼电子有限公司生产的HPD-280型等离子体仪器。
进一步的，等离子体仪器使用前先经过消毒，将盛有28～32％H2O2溶液的容器，放在处理室中，采用抽真空方式进行弥漫消毒。
进一步的，步骤(1)中所述甘蔗胚性愈伤组织为继代2-3次后的甘蔗胚性愈伤组织。
进一步的，步骤(1)中所述甘蔗胚性愈伤组织的制备方法为：待甘蔗茎长出后，割取健壮蔗株尾梢，剥去外部叶片，无菌条件下用酒精消毒，切除外层和两端叶片，留下生长点以上10mm-50mm嫩叶并切成薄片，将薄片接种于CM1培养基中，在26℃～28℃黑暗条件下培养诱导产生愈伤组织，然后在CM1培养基中继代1-2次，使其产生甘蔗胚性愈伤组织。
进一步的，所述CM1培养基为含有1.8～2.2μmol·L-1 2,4-D、28～32g·L-1蔗糖和7.5～8.5g·L-1琼脂的MS培养基，pH值为6.0～6.4。
进一步的，步骤(5)中筛选培养的天数为26～30天。
进一步的，步骤(6)中扩繁培养的天数为28～32天。
本发明的有益效果是：
(1)操作简单、少污染，为甘蔗愈伤组织诱变带来便利。
(2)解决了试管斜面培养基操作不方便、处理量小的问题，可实现大批量诱变，其诱变效果差异明显，根据胚性愈伤组织成活率快速确定诱变处理时间。
(3)诱变效率高，诱变率为2.50％-2.77％
(4)实行定向胁迫筛选，减少了后续筛选的工作量。
(5)突变株抗性判断直观。
附图说明
图1为等离子体诱变后缓冲培养天数对甘蔗胚性愈伤组织成活率的影响；
图2为本发明抗草甘膦株系和对照组(CK)喷施0～5.0μmol·L-1草甘膦后的株高增幅比对图；
图3为喷施5.0μmol·L-1草甘膦后20d抗与非抗草甘膦株系的受害情况；
图4为喷施5.0μmol·L-1草甘膦后30d抗与非抗草甘膦株系的生长情况，盆中的左边3株并排种是非抗草甘膦株系，右边3株并排种是突变株。
具体实施方式
一种等离子体高通量定向诱变甘蔗抗草甘膦的方法，包括如下步骤：
(1)将甘蔗胚性愈伤组织在无菌条件下用等离子体仪器进行辐照诱变处理，诱变功率为140～200W，放电间距35～45mm，诱变时间为110～140s，保护气体为氮气；
(2)将上步诱变过的甘蔗胚性愈伤组织接种在固体培养基CM2上，用薄膜密封，缓冲培养8～10d；
所述培养基CM2中含有：MS培养基、2,4-D 1.2～1.7μmol·L-1、蔗糖28～32g·L-1、琼脂7.5～8.5g·L-1，pH值6.0～6.4；
(3)将缓冲培养过的甘蔗愈伤组织依次接种在含1.2～1.7μmol·L-1、2.2～2.7μmol·L-1草甘膦的固体培养基CM3上，用薄膜密封，分别筛选培养8～10d；
所述培养基CM3中含有：MS培养基、蔗糖28～32g·L-1、琼脂7.5～8.5g·L-1，pH值6.0～6.4；
(4)将上步成活的胚性愈伤组织转接到固体分化培养基CM4上，培养至出苗后再继代培养一次；
所述培养基CM4中含有：MS培养基、NAA 0.4～0.6μmol·L-1、Kt 0.8～1.2μmol·L-1、 蔗糖48～52g·L-1、琼脂7.5～8.5g·L-1，pH值6.0～6.4；
(5)待上步中的小苗长至20mm-30mm时，转接至CM5上进行筛选培养；
所述培养基CM5中含有：MS培养基、4.8～5.2μmol·L-1草甘膦、蔗糖28～32g·L-1、琼脂8g·L-1，pH值6.0～6.4；
(6)将上步中成活的小苗接到CM4上进行扩繁培养，再转接到CM6上进行促根培养、炼苗，然后移植在苗床上，待苗长至100mm～150mm时，用4.8～5.2μmol·L-1草甘膦喷雾筛选，成活的为抗草甘膦株系；
所述培养基CM6中含有：1/2MS培养基、NAA 0.4～0.6μmol·L-1、6-BA 1.8～2.2μmol·L-1、蔗糖28～32g·L-1，pH值6.0～6.4。
优选的，步骤(1)中诱变功率为140W，照射间距40mm，诱变时间为120s。
优选的，步骤(1)中氮气的气流量1.0～1.4L/min。
更优选的，步骤(1)中氮气的气流量1.2L/min。
优选的，步骤(1)中辐照诱变处理时，外加电压为220V。
优选的，等离子体仪器采用南京苏曼电子有限公司生产的HPD-280型等离子体仪器。
优选的，等离子体仪器使用前先经过消毒，将盛有28～32％H2O2溶液的容器，放在处理室中，采用抽真空方式进行弥漫消毒。
优选的，步骤(1)中所述甘蔗胚性愈伤组织为继代2-3次后的甘蔗胚性愈伤组织。
优选的，步骤(1)中所述甘蔗胚性愈伤组织的制备方法为：待甘蔗茎长出后，割取健壮蔗株尾梢，剥去外部叶片，无菌条件下用酒精消毒，切除外层和两端叶片，留下生长点以上10mm-50mm嫩叶并切成薄片，将薄片接种于CM1培养基中，在26℃～28℃黑暗条件下培养诱导产生愈伤组织，然后在CM1培养基中继代1-2次，使其产生甘蔗胚性愈伤组织。
优选的，步骤(1)中甘蔗胚性愈伤组织有大小为0.8～1.2mm×0.8～1.2mm×0.8～1.2mm，排放于培养皿中，让每一粒胚性愈伤组织都能辐照到，培养皿底部的胚性愈伤组织也能接受放电辐照。
优选的，所述CM1培养基为含有1.8～2.2μmol·L-1 2,4-D、28～32g·L-1蔗糖和7.5～8.5g·L-1琼脂的MS培养基，pH值为6.0～6.4。
优选的，步骤(5)中筛选培养的天数为26～30天。
优选的，步骤(6)中扩繁培养的天数为28～32天。
下面结合具体实施例对本发明作进一步的说明。
实施例1
A优良甘蔗栽培种愈伤组织启动
1)接种材料准备
待蔗茎长出后，割取健壮蔗株尾梢，剥去外部叶片，无菌条件下用75％酒精消毒30S，切除外层和两端叶片(叶鞘)，留下生长点以上10mm-50mm嫩叶(嫩鞘)并切成约2mm的薄片，1/2环切后备接种用。
2)甘蔗胚性愈伤组织的获得
将准备好的薄片接种于培养基CM1中，在26℃～28℃黑暗条件下培养，每皿6-8块，诱导产生愈伤组织，然后在培养基CM1(MS+2,4-D 2.0μmol·L-1+蔗糖30g·L-1+琼脂8g·L-1，pH值6.2)中继代1-2次，每皿6-8块，使其产生胚性愈伤组织。
B高通量排布
将继代培养2-3次甘蔗胚性愈伤组织夹成约1mm×1mm×1mm小块，平铺在培养皿(底面宽90mm×高20mm)的底部，让每一粒胚性愈伤组织都能辐照到。盖好培养皿上盖，采用聚乙烯薄膜密封待用。
C等离子体参数设置
等离子体机采用南京苏曼电子有限公司生产的HPD-280型(该仪器中标注的功率40％、50％、60％分别对应具体的功率140W、170W、200W)等离子体机对甘蔗胚性愈伤组织进行诱变，诱变功率为140W，照射间距40mm。保护气体为氮气，气流量1.2L/min，辐照诱变时间设定在120s，外加电压220V。
D等离子体防菌和诱变操作处理
处理前先盛30％H2O2 20mI在培养皿中，放在处理室中，采用抽真空方式进行弥漫消毒。然后将铺满胚性愈伤组织的培养皿上盖打开，置于处理室正中位置，按照等离子体操作说明进行辐照诱变。处理完后盖好培养皿上盖，采用聚乙烯薄膜密封。
E处理后的愈伤组织进行缓冲培养
将诱变过的甘蔗愈伤组织接种在固体培养基CM2(MS+2,4-D 1.5μmol·L-1+蔗糖30·g L-1+琼脂8g·L-1，pH值6.2)上，采用聚乙烯薄膜密封缓冲培养8-10d。同时统计愈伤组织成活数，胚性愈伤组织成活率％＝100％×胚性愈伤组织成活数/处理的胚性愈伤组织数(即为下表1中所述的胚性愈伤组织成活率)。
F胁迫筛选
将缓冲培养过的甘蔗愈伤组织依次接种在含1.5μmol·L-1、2.5μmol·L-1草甘膦的固体培养基CM3上，用薄膜密封，分别筛选培养8～10d；
所述培养基CM3中含有：MS培养基、蔗糖28～32g·L-1、琼脂7.5～8.5g·L-1，pH值6.2；
G抗性胚性愈伤组织分化成苗
选择上步成活的胚性愈伤组织转接到固体分化培养基CM4(MS+NAA 0.5μmol·L-1+激动素Kt 1.0μmol·L-1+蔗糖50g·L-1+琼脂8g·L-1，pH值6.2)上，养至出苗后再继代培养一次。
H瓶苗草甘膦胁迫筛选
待上步小苗长至20mm-30mm时，接到CM5(MS+5.0μmol·L-1草甘膦+蔗糖30g·L-1+琼脂8g·L-1，pH值6.2)上进行筛选培养28d，同时接对照。
I盆栽叶面喷草甘膦胁迫筛选
将成活的小苗接到CM4上进行扩繁培养30d，然后转接到CM6(1/2MS+NAA 0.5μmol·L-1+6-BA 2.0μmol·L-1+蔗糖30g·L-1，pH值6.2)上进行促根、炼苗，然后移植在苗床上，经过1个月左右，待苗长至100mm～150mm高时，用5.0μmol·L-1草甘膦对叶面进行喷雾一次，最终成活者为抗草甘膦株系。统计抗草甘膦株数，诱变率％＝100％×抗草甘膦株数/处理的胚性愈伤组织数*(即为下表1中所述的诱变率)。
实施例2
2015年7月15日利用HPD-280型等离子体机对粤糖93-159甘蔗胚性愈伤组织进行诱变，诱变功率设定为140W，辐照诱变时间分别为80s、100s、120s、140s、160s、180s。实施例2中的其他操作均同实施例1。检测各组胚性愈伤组织成活率及诱变率。
检测结果如表1所示，其中120s处理的胚性愈伤组织成活率最高，为18.3％，显著高于其它处理，而且经过两轮草甘膦筛选后能分化成株，诱变率2.50％。
表1粤糖93-159甘蔗胚性愈伤组织在功率140W，处理时间80-180s的条件下的等离子体诱变情况
Figure PCTCN2017107910-appb-000001
注：同列数据后小写英文字母不同者表示差异显著，大写英文字母不同者表示差异极显著，下同；
胚性愈伤组织成活率％＝100％×胚性愈伤组织成活数/处理的胚性愈伤组织数；
诱变率％＝100％×抗草甘膦株数/处理的胚性愈伤组织数。
实施例3
2016年5月4日利用HPD-280型等离子体机对粤糖93-159甘蔗胚性愈伤组织进行诱变，辐照诱变时间为120s。诱变功率分别设定为140W、170W、200W。实施3中的其他操作均同实施1。检测各组胚性愈伤组织成活率及诱变率。
检测结果如表2所示，其中功率140W处理的胚性愈伤组织成活率最高，为62.4％，经过两轮草甘膦筛选后各组均能少最分化成株，诱变率最高的功率是140W，达到2.77％。
表2粤糖93-159甘蔗胚性愈伤组织在功率140～200W，处理时间为120s的条件下的等离子体诱变情况
Figure PCTCN2017107910-appb-000002
实施例4
利用HPD-280型等离子体机对粤糖93-159甘蔗胚性愈伤组织进行诱变，功率设为140W，诱变处理愈伤组织的时间为120s，接种在草甘膦固体培养基CM2上缓冲培养，时间分别设为8d、10d、13d，实施例4中的其他操作均同实施例1。检测各组胚性愈伤组织成活率。
检测如图1所示，从下图1可知，随着缓冲培养天数的增加，甘蔗胚性愈伤组织的成活率越来越低。缓冲培养的时间应为8d-10d。
实施例5
当诱变后的盆栽苗长至100-150mm高时，叶面分别喷施0、1.0、2.5、4.0、5.0μmol·L-1草甘膦，抗草甘膦株系虽然受害，但经过20d后仍能成活，与对照相比，其株高增幅最大，为9.5mm(图2)，叶色仍保持绿色，而对照则出现前期卷叶、后期枯心的现象(图3)。经过30d后，对照已枯黄，突变株则有些叶片保持青绿(图4)。说明突变株抗草甘膦能力强，为抗草甘膦株系。
实施例6
利用HPD-280型等离子体机对粤糖00-236甘蔗胚性愈伤组织进行诱变，诱变功率设定为140W，辐照诱变时间为120s，诱变时保护气体分别设置为空气、氮气流量1.2L/min、氮 气0.2L/min。实施例6中的其他操作均同实施例1。
检测结果如表3所示，其中保护气体为氮气1.2L/min时处理的胚性愈伤组织成活率最高，为22.2％，明显高于其它处理。
表3诱变时不同保护气体条件下对等离子体诱变情况的影响
Figure PCTCN2017107910-appb-000003
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种等离子体高通量定向诱变甘蔗抗草甘膦的方法，其特征在于，包括如下步骤：
    (1)将甘蔗胚性愈伤组织在无菌条件下用等离子体仪器进行辐照诱变处理，诱变功率为140～200W，放电间距35～45mm，诱变时间为110～140s，保护气体为氮气；
    (2)将上步诱变过的甘蔗胚性愈伤组织接种在固体培养基CM2上，用薄膜密封，缓冲培养8～10d；
    所述培养基CM2中含有：MS培养基、2,4-D 1.2～1.7μmol·L-1、蔗糖28～32g·L-1、琼脂7.5～8.5g·L-1，pH值6.0～6.4；
    (3)将缓冲培养过的甘蔗愈伤组织依次接种在含1.2～1.7μmol·L-1、2.2～2.7μmol·L-1草甘膦的固体培养基CM3上，用薄膜密封，分别筛选培养8～10d；
    所述培养基CM3中含有：MS培养基、蔗糖28～32g·L-1、琼脂7.5～8.5g·L-1，pH值6.0～6.4；
    (4)将上步成活的胚性愈伤组织转接到固体分化培养基CM4上，培养至出苗后再继代培养一次；
    所述培养基CM4中含有：MS培养基、NAA 0.4～0.6μmol·L-1、Kt 0.8～1.2μmol·L-1、蔗糖48～52g·L-1、琼脂7.5～8.5g·L-1，pH值6.0～6.4；
    (5)待上步中的小苗长至20mm-30mm时，转接至CM5上进行筛选培养；
    所述培养基CM5中含有：MS培养基、4.8～5.2μmol·L-1草甘膦、蔗糖28～32g·L-1、琼脂8g·L-1，pH值6.0～6.4；
    (6)将上步中成活的小苗接到CM4上进行扩繁培养，再转接到CM6上进行促根培养、炼苗，然后移植在苗床上，待苗长至100mm～150mm时，用4.8～5.2μmol·L-1草甘膦喷雾筛选，成活的为抗草甘膦株系；
    所述培养基CM6中含有：1/2MS培养基、NAA 0.4～0.6μmol·L-1、6-BA 1.8～2.2μmol·L-1、蔗糖28～32g·L-1，pH值6.0～6.4。
  2. 根据权利要求1所述的方法，其特征在于，步骤(1)中诱变功率为140W，照射间距40mm，诱变时间为120s。
  3. 根据权利要求1所述的方法，其特征在于，步骤(1)中氮气的气流量1.0～1.4L/min。
  4. 根据权利要求1所述的方法，其特征在于，等离子体仪器采用南京苏曼电子有限公司生产的HPD-280型等离子体仪器。
  5. 根据权利要求1所述的方法，其特征在于，等离子体仪器使用前先经过消毒，将盛有28～32％H2O2溶液的容器，放在处理室中，采用抽真空方式进行弥漫消毒。
  6. 根据权利要求1所述的方法，其特征在于，步骤(1)中所述甘蔗胚性愈伤组织为继代2-3 次后的甘蔗胚性愈伤组织。
  7. 根据权利要求1所述的方法，其特征在于，步骤(1)中所述甘蔗胚性愈伤组织的制备方法为：待甘蔗茎长出后，割取健壮蔗株尾梢，剥去外部叶片，无菌条件下用酒精消毒，切除外层和两端叶片，留下生长点以上10mm-50mm嫩叶并切成薄片，将薄片接种于CM1培养基中，在26℃～28℃黑暗条件下培养诱导产生愈伤组织，然后在CM1培养基中继代1-2次，使其产生甘蔗胚性愈伤组织。
  8. 根据权利要求7所述的方法，其特征在于，所述CM1培养基为含有1.8～2.2μmol·L-12,4-D、28～32g·L-1蔗糖和7.5～8.5g·L-1琼脂的MS培养基，pH值为6.0～6.4。
  9. 根据权利要求1所述的方法，其特征在于，步骤(5)中筛选培养的天数为26～30天。
  10. 根据权利要求1所述的方法，其特征在于，步骤(6)中扩繁培养的天数为28～32天。
PCT/CN2017/107910 2017-09-26 2017-10-27 一种等离子体高通量定向诱变甘蔗抗草甘膦的方法 WO2019061621A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/315,016 US11516979B2 (en) 2017-09-26 2017-10-27 Method for conducting high-throughput and directed mutagenesis for sugarcane resistance to glyphosate by plasma

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710880009.XA CN107646676A (zh) 2017-09-26 2017-09-26 一种等离子体高通量定向诱变甘蔗抗草甘膦的方法
CN201710880009.X 2017-09-26

Publications (1)

Publication Number Publication Date
WO2019061621A1 true WO2019061621A1 (zh) 2019-04-04

Family

ID=61131279

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/107910 WO2019061621A1 (zh) 2017-09-26 2017-10-27 一种等离子体高通量定向诱变甘蔗抗草甘膦的方法

Country Status (3)

Country Link
US (1) US11516979B2 (zh)
CN (1) CN107646676A (zh)
WO (1) WO2019061621A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112386085A (zh) * 2020-12-23 2021-02-23 孟政政 一种水果去皮切段装置

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109496839A (zh) * 2018-12-27 2019-03-22 河南师范大学 一种对两色金鸡菊进行物理诱变的育种方法
CN112825767B (zh) * 2021-02-26 2023-01-31 江苏省中国科学院植物研究所 一种利用氧气冷等离子体诱变创制结缕草突变体的方法
CN115918525A (zh) * 2022-09-01 2023-04-07 南京苏曼等离子工程研究院有限公司 一种高压电场低温等离子体激活农作物种子的方法及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102911965A (zh) * 2012-11-16 2013-02-06 南宁奥扬高科农业科技有限公司 一种抗螟虫(BT)和抗除草剂(Bar)共价外源基因导入甘蔗的方法
CN104403974A (zh) * 2014-12-09 2015-03-11 江南大学 一株高产雄甾-4-烯-3,17-二酮的植物甾醇转化菌株的选育
CN105039389A (zh) * 2015-06-29 2015-11-11 广东省农业科学院作物研究所 一种甘蔗无载体框架转基因方法
CN204874543U (zh) * 2015-07-17 2015-12-16 无锡源清天木生物科技有限公司 一种小型等离子体诱变育种仪

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102911965A (zh) * 2012-11-16 2013-02-06 南宁奥扬高科农业科技有限公司 一种抗螟虫(BT)和抗除草剂(Bar)共价外源基因导入甘蔗的方法
CN104403974A (zh) * 2014-12-09 2015-03-11 江南大学 一株高产雄甾-4-烯-3,17-二酮的植物甾醇转化菌株的选育
CN105039389A (zh) * 2015-06-29 2015-11-11 广东省农业科学院作物研究所 一种甘蔗无载体框架转基因方法
CN204874543U (zh) * 2015-07-17 2015-12-16 无锡源清天木生物科技有限公司 一种小型等离子体诱变育种仪

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG XUE: "Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool", APPL MICROBIOL BIOTECHNOL, vol. 98, 27 April 2014 (2014-04-27), XP055587850 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112386085A (zh) * 2020-12-23 2021-02-23 孟政政 一种水果去皮切段装置
CN112386085B (zh) * 2020-12-23 2021-11-23 山东公社联盟食品有限公司 一种水果去皮切段装置

Also Published As

Publication number Publication date
US11516979B2 (en) 2022-12-06
CN107646676A (zh) 2018-02-02
US20210345571A1 (en) 2021-11-11

Similar Documents

Publication Publication Date Title
WO2019061621A1 (zh) 一种等离子体高通量定向诱变甘蔗抗草甘膦的方法
Kacar et al. Gelling agents and culture vessels affect in vitro multiplication of banana plantlets
CN111226792B (zh) 一种斑兰叶种苗的高通量繁育方法
CN112704011B (zh) 一种生姜组织培养的方法
Kaur et al. Optimization of potting mixture for hardening of in vitro raised plants of Tylophora indica to ensure high survival percentage
CN108770692B (zh) 椰子胚诱导培养基和基于椰子合子胚细胞薄层培养获得离体再生植株的方法
CN102487829A (zh) 一种粉用型荸荠综合脱毒快繁的方法
CN112602595A (zh) 一种提高大蒜生长点分化不定芽数量的组培方法
CN106258976B (zh) 一种芥菜型油菜的组织培养快速育苗方法
CN114698549B (zh) 一种葡萄砧木茎段快繁的组培培养基和组培方法
CN105104201B (zh) 一种撒瓦那柏初代组培方法
CN111771730A (zh) 一种百香果脱毒苗的生产方法
CN104488715B (zh) 一种通过花蕾进行宽叶弹簧草鳞茎诱导和植株再生的方法
CN114391474B (zh) 一种获得四倍体艾纳香不定芽的方法
CN105052743B (zh) 一种有效保存黄菖蒲胚性愈伤组织的方法
CN102630566B (zh) 一种南方红豆杉腋芽离体诱导培养生产紫杉醇的方法
KR20100026019A (ko) 양초의 캘러스로부터 재분화를 유도하는 방법 및 배지
CN115250912A (zh) 一种半夏开放式培养的培养基和方法
Subotić et al. Effect of Urea—Type Citokinins on Axillary Shoots Regeneration of Impatiens Walleriana L.
CN109496842B (zh) 一种千里香愈伤组织的诱导方法
CN110521603A (zh) 番茄遗传转化体系的构建方法
CN108651283A (zh) 一种互叶白千层组培苗生根培养方法
KR101664031B1 (ko) 참취의 조직 배양 방법
CN112825767B (zh) 一种利用氧气冷等离子体诱变创制结缕草突变体的方法
CN115735772B (zh) 5-AzaC在苹果砧木组织生根培养或无糖生根培养中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17926895

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17926895

Country of ref document: EP

Kind code of ref document: A1