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Agrobacterium-mediated Transformation of Mature Ginseng Embryos
农杆菌介导的成熟人参胚胎的遗传转化   

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Abstract

Ginseng refers to species within the genus Panax and is a slow-growing perennial herb from the Araliaceae family. The most widely used Panax species is Panax ginseng Meyer (Korean ginseng). Panax japonicus (Japanese ginseng), Panax notoginseng (Chinese ginseng), and Panax quinquefolium (American ginseng). Due to the various pharmaceutical importance of ginsenosides, ginseng plant has been cultivated for its highly valued root over 2,000 years as a medicinal plant in East Asian countries particularly in China, Korea, and Japan and North America. Korean ginseng (Panax ginseng C. A. Meyer) consists of nine cultivars from three Jakyung, Chungkyung, and Hwangsook lines. Cultivar “Yunpoong” has characteristics to have more axillary shoots and lateral roots compared to other cultivars. Thus “Yunpoong”: Ginseng seeds are relatively more feasible for regeneration of adventitious roots during gene transformation. Here, we describe how to prepare and treat ginseng seeds after harvest till the ginseng immature embryos are ready for the germination, and to be used in Agrobacterium tumefaciens-mediated gene transformation.

Keywords: Panax ginseng(人参), Adventitious root(不定根), Agrobacterium tumefaciens(农杆菌介导法), Binary vector(二进制向量), Regeneration(再生)

Materials and Reagents

  1. Ginseng seeds, in which zygotic embryos were in a mature state (4 mm in length)
  2. Agrobacterium tumefaciens C58C1 (pMP90) strain
  3. Binary (pCAMBIA1390) vector
  4. MS powder (Duchefa Biochemie, catalog number: M0222 )
  5. Sucrose (Duchefa Biochemie, catalog number: S0809 )
  6. 2-(N-morpholino) ethanesulfonic acid (MES) monohydrate (MB cell, catalog number: MB-M4837 )
  7. Phytoagar (Duchefa Biochemie, catalog number: P1003) or Gelrite (Duchefa Biochemie, catalog number: G1101)
  8. Yeast extract (Duchefa Biochemie, catalog number: Y1333 )
  9. Peptone (Duchefa Biochemie, catalog number: P1328 )
  10. NaCl (Affymetrix, catalog number: 4171746 )
  11. Bactoagar (BD, catalog number: 214010 )
  12. Hygromycin (Duchefa Biochemie, catalog number: H0192 )
  13. Kanamycin (Duchefa Biochemie, catalog number: K0126 )
  14. Cefotaxime (Duchefa Biochemie, catalog number: C0111 )
  15. 6-benzylaminopurine (BAP) (Duchefa Biochemie, catalog number: B0904 )
  16. 2,4-dichlorophenoxyacetic acid (2,4-D) (Duchefa Biochemie, catalog number: D0911 )
  17. Indole-3-butyric acid (IBA) (Duchefa Biochemie, catalog number: I0902 )
  18. Sucrose
  19. Phyto agar
  20. 70% ethyl alcohol (EtOH)
  21. 2% (v/v) sodium hypochlorite (NaOCl) (Sigma-Aldrich, catalog number: 425044-1L )
  22. B5 medium (Duchefa Biochemie, catalog number: G0209 )
  23. MS (Murashige & Skoog) medium (see Recipes)
  24. YEP medium (see Recipes)
  25. Antibiotics stock solution (in ddH2O) (see Recipes)
  26. Plant growth regulators stock solution (see Recipes)

Equipment

  1. Laminar flow cabinet (HEPA filter 99.99% efficient on particle of 0.3 μ and vertical type)
  2. Shaking incubator (100~200 rpm on a gyratory shaker)
  3. Centrifuge (3,000 x g) (Labnet International, catalog number: C2500-R-230V )
  4. Micropipette
  5. Forceps (Phoenix Technologies, catalog number: ESD-16 )
  6. Erlenmeyer flask (50/100 ml) or autoclavable plastic tube for seed sterilization
  7. Autoclave machine
  8. Growth chamber (23 °C ± 2 °C)
  9. Sterile filter paper
  10. Petri dish

Procedure

  1. Korean ginseng (Panax ginseng Meyer cv. “Yunpoong”) (Lee et al., 2011) seeds were stratified in humidified sand at 15 °C for 4 months (Figure 1) since the zygotic embryo right after the harvest were in an immature globular stage (about 200 μm in length).


    Figure 1. Diagram of ginseng seeds stratification. Ginseng seeds were stacked as depicted in the diagram inside of square cement pad or wooden square box. The seeds were mixed with river sand 1:3 (seed:sand) or often sandwiched in net bag with sand on top and down and kept at 15 ~20 °C, 10~15% moisture by watering once or twice a day. Sand within 2 mm size is good and coarse sand within 4 mm.

  2. Then the seeds were further stratified for 100 days at 2-4 °C to break seed dormancy. After stratification for about 6.5 -7 months, the zygotic embryos reach in mature stage (Figure 2C-D, about 4.0-4.5 mm in length), which means the seeds are ready to germinate.
  3. Once uncover the hard seed coat using fingers, the seeds were immersed in 70% (v/v) EtOH for 1 min, surface sterilized in 2% (v/v) NaOCl for 15 min, and washed three times with sterilized distilled water (Figure 2A-B) in the clean bench (Figure 2A). The zygotic embryos from the seed were carefully dissected using fingers and forceps, placed on MS (Murashige and Skoog, 1962) basal medium vertically containing 3% (w/v) sucrose and 0.8% (w/v) phytoagar, and grown for 5-10 days (Figure 3A).
  4. An Agrobacterium tumefaciens C58C1 (pMP90) (Bechtold and Pelletier, 1998) strain containing a plant binary vector (pCAMBIA1390) harboring gene of interest was cultured in 5 ml of YEP liquid medium overnight (170 rpm for 20 h) at 28 °C. The next day morning, 5 ml of A. tumefaciens cells was subcultured in 100 ml of erlenmeyer flask till the OD600 nm reach 0.6. Bacteria cells were washed with liquid MS medium containing 3% (w/v) sucrose twice and resuspended in the same media for the ginseng explants infection.
    The cotyledon explants (Figures 2E and 3A, 4 mm) were used for A. tumefaciens transformation. A pCAMBIA1390 vector contains hptII and nptII genes under the cauliflower mosaic virus (CaMV) 35S promoter was used (Lee et al., 2010; Kim et al., 2014).


    Figure 2. Ready-to-go ginseng seeds for transformation and sterilization. A. Clean Bench. B. Ginseng seeds for sterilization. C. Ginseng seed with and without hard seed coat, then ginseng embryo covered with endosperm. D. Ginseng embryo. E. Ginseng cotyledons ready for the Agrobacterium co-cultivation.

  5. The explants were dipped in the bacterial solution for 15 min, subsequently blotted with sterile filter paper, and co-cultivated on MS medium containing 3% (w/v) sucrose and 1 µg/ml 2, 4-dichlorophenoxyacetic acid (2, 4-D) for 2 days (Figure 3B).
  6. Thereafter, the explants were cultured on MS selection medium (called callus induction medium) with 3% (w/v) sucrose, 1 µg/ml 2, 4-D, 0.5 µg/ml 6-benzylaminopurine (BAP), 250 µg/ml cefotaxime, and 50 µg/ml hygromycin (Figure 3C). After subculturing three to four times every two weeks on selection medium, the surviving cotyledons producing calli were cultured on the same medium without antibiotics (Figure 3D), and adventitious roots were induced from calli on B5 medium with 3% (w/v) sucrose and 3 mg/L indole-3-butyric acid (IBA) (Figure 3E).
  7. Adventitious roots were excised from the maternal explants prior to sub-culturing in liquid B5 medium with 3% sucrose and 2 mg/L IBA, which was replaced every 5 weeks (Figure 3F).


    Figure 3. Ginseng transformation by Agrobacterium tumefaciens for the transgenic adventitious root induction. A. Five days further grown cotyledons. B. Cotyledon explants dipped in the bacterial solution for 15 min, subsequently blotted with sterile filter paper, and co-cultivated on MS medium containing 3% (w/v) sucrose and 1 µg/ml 2, 4-dichlorophenoxyacetic acid (2, 4-D) for 2 days. C. Transgenic calli selection on MS selection medium, 1 µg/ml 2,4-D, 0.5 µg/ml BAP, 250 µg/ml cefotaxime, and 50 µg/ml hygromycin containing 3% (w/v) sucrose. D. Survived calli were cultured on the same medium without antibiotics. E. Adventitious roots on B5 medium containing 3% (w/v) sucrose and 3 mg/L IBA. F. Subculture in liquid B5 medium containing 3% sucrose and 2 mg/L IBA.

Recipes

  1. MS (Murashige & Skoog) medium
    2.2 g of MS powder
    30 g of sucrose
    0.5 g of 2-(N-morpholino) ethanesulfonic acid (MES) monohydrate
    8 g of phytoagar or 0.25% of Gelrite
    Adjust pH to 5.6 with 1 N KOH
    Autoclave at 121 °C for 15 min
  2. YEP medium
    10 g of yeast extract
    10 g of peptone
    5 g of NaCl and 15 g of Bactoagar
    Adjust pH to 7.0 with 1 N HCl
    Add to dH2O to 1,000 ml
    Autoclave at 121 °C for 15 min
  3. Antibiotics stock solution (in ddH2O)
    Hygromycin 50 mg/ml
    Kanamycin 50 mg/ml
    Cefotaxime 200 mg/ml
    Filter sterilize (0.2 μm)
  4. Plant growth regulators stock solution
    1. 6-benzylaminopurine (BAP): 0.5 mg/ml in 1 N NaOH
      Dissolve 50 mg of BAP in small amount of 1 N NaOH
      Bring to volume 100 ml with ddH2O
      Autoclave or filter sterilize (0.2 μm)
      Freeze at -20 °C
    2. 2,4-dichlorophenoxyacetic acid (2,4-D): 1 mg/ml in 1 N NaOH
      Dissolve 100 mg of 2, 4-D in small amount of 1 N NaOH
      Bring to volume 100 ml with ddH2O
      Autoclave or filter sterilize (0.2 μm)
      Freeze at -20 °C
    3. Indole-3-butyric acid (IBA): 10 mg/ml in 1 N NaOH
      Dissolve 1 g of IBA in small amount of 1 N NaOH
      Bring to volume 100 ml with ddH2O
      Autoclave or filter sterilize (0.2 μm)
      Freeze at -20 °C

Acknowledgments

This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ00952902), Rural Development Administration, Republic of Korea to ORL.

References

  1. Bechtold, N. and Pelletier, G. (1998). In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82: 259-266.
  2. Kim, Y. J., Lee, O. R., Oh, J. Y., Jang, M. G. and Yang, D. C. (2014). Functional analysis of 3-hydroxy-3-methylglutaryl coenzyme a reductase encoding genes in triterpene saponin-producing ginseng. Plant Physiol 165(1): 373-387.
  3. Lee, O. R., Kim, S. J., Kim, H. J., Hong, J. K., Ryu, S. B., Lee, S. H., Ganguly, A. and Cho, H. T. (2010). Phospholipase A(2) is required for PIN-FORMED protein trafficking to the plasma membrane in the Arabidopsis root. Plant Cell 22(6): 1812-1825.
  4. Lee, O. R., Sathiyaraj, G., Kim, Y. J., In, J. G., Kwon, W. S., Kim, J. H. and Yang, D. C. (2011). Defense genes induced by pathogens and abiotic stresses in Panax ginseng CA Meyer. J Gins Res 35(1): 1-11.
  5. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15(3): 473-497.

简介

人参是指属于Panax的物种,并且是来自五加科(Araliaceae)的缓慢生长的多年生草本植物。最广泛使用的人参是人参 Meyer(高丽人参)。 (日本人参),三七(中国人参)和中国人参[美国人参]。 由于人参皂苷的各种药物重要性,人参植物作为一种药用植物在东亚国家,特别是在中国,韩国,日本和北美地区作为药用植物已经培育了其高价值的根。高丽人参(人人参C. A. Meyer )由来自Jakyung,Chungkyung和Hwangsook三个品系的9个品种组成。栽培品种"云蓬"与其他栽培品种相比具有腋芽和侧根更多的特点。因此"Yunpoong":人参种子在基因转化过程中对不定根的再生相对更可行。在这里,我们描述如何在收获后准备和处理人参种子,直到人参未成熟胚准备好发芽,并用于根癌土壤杆菌介导的基因转化。

关键字:人参, 不定根, 农杆菌介导法, 二进制向量, 再生

材料和试剂

  1. 人参种子,其中合子胚处于成熟状态(长度为4mm)
  2. 根癌农杆菌 C58C1(pMP90)菌株
  3. 二进制(pCAMBIA1390)向量
  4. MS粉末(Duchefa Biochemie,目录号:M0222)
  5. 蔗糖(Duchefa Biochemie,目录号:S0809)
  6. 2-(N-吗啉代)乙磺酸(MES)一水合物(MB电池,目录号:MB-M4837)
  7. Phytoagar(Duchefa Biochemie,目录号:P1003)或Gelrite(Duchefa Biochemie,目录号:G1101)
  8. 酵母提取物(Duchefa Biochemie,目录号:Y1333)
  9. 蛋白胨(Duchefa Biochemie,目录号:P1328)
  10. NaCl(Affymetrix,目录号:4171746)
  11. Bactoagar(BD,目录号:214010)
  12. 潮霉素(Duchefa Biochemie,目录号:H0192)
  13. 卡那霉素(Duchefa Biochemie,目录号:K0126)
  14. 头孢噻肟(Duchefa Biochemie,目录号:C0111)
  15. 6-苄氨基嘌呤(BAP)(Duchefa Biochemie,目录号:B0904)
  16. 2,4-二氯苯氧基乙酸(2,4-D)(Duchefa Biochemie,目录号:D0911)
  17. 吲哚-3-丁酸(IBA)(Duchefa Biochemie,目录号:I0902)
  18. 蔗糖
  19. 植物琼脂
  20. 70%乙醇(EtOH)
  21. 2%(v/v)次氯酸钠(NaOCl)(Sigma-Aldrich,目录号:425044-1L)
  22. B5培养基(Duchefa Biochemie,目录号:G0209)
  23. MS(Murashige& Skoog)培养基(参见Recipes)
  24. YEP介质(参见配方)
  25. 抗生素储备溶液(在ddH 2 O中)(参见配方)
  26. 植物生长调节剂储备溶液(见配方)

设备

  1. 层流柜(HEPA过滤器,粒径0.3μ和垂直型99.99%效率)
  2. 摇匀培养箱(在旋转振荡器上100〜200rpm)
  3. 离心机(3000×g)(Labnet International,目录号:C2500-R-230V)
  4. 微量移液器
  5. 镊子(Phoenix Technologies,目录号:ESD-16)
  6. 锥形烧瓶(50/100ml)或可高压灭菌的塑料管用于种子灭菌
  7. 高压釜机
  8. 生长室(23℃±2℃)
  9. 无菌滤纸
  10. 培养皿

程序

  1. 将种子在加湿砂中在15℃下分层4个月(图1)(参见图1),其中所述的人参(E参) ),因为收获后即刻的合子胚处于未成熟的球状阶段(长度约200μm)。


    图1.人参种子分层图。人参种子如图所示在方形水泥垫或木制方形盒内堆叠。将种子与河沙1:3(种子:沙)混合,或经常夹在具有沙子的网袋中上下,并通过每天浇水一次或两次保持在15〜20℃,10〜15%水分。 2毫米以内的砂是好的,粗砂在4毫米以内
  2. 然后将种子在2-4℃下进一步分层100天以破坏种子休眠。分层约6.5-7个月后,合子胚达到成熟阶段(图2C-D,长度约4.0-4.5mm), 这意味着种子准备发芽。
  3. 一旦用手指暴露硬种子包衣,将种子浸入70%(v/v)EtOH中1分钟,在2%(v/v)NaOCl中表面灭菌15分钟,并用无菌蒸馏水图2A-B)在清洁台(图2A)中。使用手指和镊子小心地切割来自种子的合子胚,置于垂直含有3%(w/v)蔗糖和0.8%(w/v)植物琼脂的MS(Murashige和Skoog,1962)基础培养基上, -10天(图3A)。
  4. 将含有目的基因的植物二元载体(pCAMBIA1390)的根癌土壤杆菌C58C1(pMP90)(Bechtold和Pelletier,1998)菌株在5ml YEP液体培养基中培养过夜(170rpm,20小时)在28℃。第二天早上,5ml的A。根癌土细胞在100ml锥形瓶中传代培养直至OD 600nm达到0.6。用含有3%(w/v)蔗糖的液体MS培养基洗涤细菌细胞两次,并重悬于用于人参外植体感染的相同培养基中。
    将子叶外植体(图2E和3A,4mm)用于根瘤土壤杆菌转化。 pCAMBIA1390载体在花椰菜花叶病毒(CaMV)35S启动子下包含 hptII 和 nptII 基因(Lee等人,/em>,2010; Kim ,2014)。


    图2.用于转化和灭菌的即食型人参种子。 A. Clean Bench。 B.人参种子灭菌。 C.有和没有硬种子外壳的人参种子,然后用胚乳覆盖的人参胚。 D.人参胚。 E.人参子叶准备用于土壤杆菌共培养
  5. 将外植体浸在细菌溶液中15分钟,随后用无菌滤纸吸干,并在含有3%(w/v)蔗糖和1μg/ml 2,4-二氯苯氧基乙酸(2,4)的MS培养基-D)2天(图3B)。
  6. 然后,将外植体在含有3%(w/v)蔗糖,1μg/ml 2,4-D,0.5μg/ml 6-苄基氨基嘌呤(BAP),250μg/ml 6-苄基氨基嘌呤(BAP)的MS选择培养基(称为愈伤组织诱导培养基) ml头孢噻肟和50μg/ml潮霉素(图3C)。在选择培养基上每两周传代培养三至四次后,将产生愈伤组织的存活子叶在不含抗生素的相同培养基上培养(图3D),并在含有3%(w/v)蔗糖的B5培养基上从愈伤组织诱导不定根和3mg/L吲哚-3-丁酸(IBA)(图3E)。
  7. 在含有3%蔗糖和2mg/L IBA的液体B5培养基中继代培养之前,从母体外植体切除不定根,每5周更换一次(图3F)。


    图3.通过根癌土壤杆菌进行的人参转化用于转基因不定根诱导。 A.五天进一步生长子叶。 B.子叶外植体浸在细菌溶液中15分钟,随后用无菌滤纸吸干,并在含有3%(w/v)蔗糖和1μg/ml 2,4-二氯苯氧基乙酸(2, 4-D)2天。 C.在MS选择培养基,含有3%(w/v)蔗糖的1μg/ml 2,4-D,0.5μg/ml BAP,250μg/ml头孢噻肟和50μg/ml潮霉素的转基因愈伤组织选择。 D.在没有抗生素的相同培养基上培养存活的愈伤组织。 E.不定根在含有3%(w/v)蔗糖和3mg/L IBA的B5培养基上。 F.在含有3%蔗糖和2mg/L IBA的液体B5培养基中的继代培养

食谱

  1. MS(Murashige& Skoog)培养基
    得到2.2g的MS粉末
    30克蔗糖 0.5克2-(N-吗啉代)乙磺酸(MES)一水合物 8g植物琼脂或0.25%Gelrite
    用1N KOH将pH调节至5.6 121℃高压灭菌15分钟
  2. YEP介质
    10g酵母提取物 10g蛋白胨
    5g NaCl和15g Bactoagar
    用1N HCl调节pH至7.0 添加到dH 2 O到1,000ml
    121℃高压灭菌15分钟
  3. 抗生素储备溶液(在ddH 2 O中)
    潮霉素50mg/ml
    卡那霉素50mg/ml
    头孢噻肟200mg/ml
    过滤灭菌(0.2μm)
  4. 植物生长调节剂储备液
    1. 6-苄氨基嘌呤(BAP):0.5mg/ml在1N NaOH中 将50mg BAP溶解在少量1N NaOH中 用ddH 2 O 2将体积调至100ml 高压灭菌或过滤灭菌(0.2μm)
      在-20°C下冻结
    2. 2,4-二氯苯氧基乙酸(2,4-D):在1N NaOH中1mg/ml 将100mg的2,4-D溶于少量1N NaOH中 用ddH 2 O 2将体积调至100ml 高压灭菌或过滤灭菌(0.2μm)
      在-20°C下冻结
    3. 吲哚-3-丁酸(IBA):在1N NaOH中10mg/ml 将1g IBA溶解在少量1N NaOH中 用ddH 2 O 2将体积调至100ml 高压灭菌或过滤灭菌(0.2μm)
      在-20°C下冷冻

致谢

这项工作得到了下一代生物绿色21计划(SSAC,拨款#:PJ00952902),大韩民国农村发展局给ORL的资助。

参考文献

  1. Bechtold,N。和Pelletier,G。(1998)。 农杆菌介导的转化成虫拟南芥植物通过真空浸润。 Methods Mol Biol 82:259-266。
  2. Kim,Y.J.,Lee,O.R.,Oh,J.Y.,Jang,M.G.and Yang,D.C。(2014)。 3-羟基-3-甲基戊二酰辅酶a编码三萜皂草苷生产人参中基因的还原酶功能分析。 Plant Physiol 165(1):373-387。
  3. Lee,O.R.,Kim,S.J.,Kim,H.J.,Hong,J.K.,Ryu,S.B.,Lee,S.H.,Ganguly,A.and Cho,H.T.(2010)。 磷脂酶A(2)是PIN- FORMED蛋白质运输到拟南芥根中的质膜。 植物细胞22(6):1812-1825。
  4. Lee,O.R.,Sathiyaraj,G.,Kim,Y.J.,In,J.G.,Kwon,W.S.,Kim,J.H。和Yang,D.C。 由人参中的病原体和非生物胁迫诱导的防御基因CA Meyer。 J Gins Res 35(1):1-11。
  5. Murashige,T。和Skoog,F。(1962)。 用于快速增长的修订媒介 用烟草组织培养物进行生物测定。 生理药理学 15(3):473-497。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Lee, O. R., Han, J. H. and Kim, Y. (2014). Agrobacterium-mediated Transformation of Mature Ginseng Embryos. Bio-protocol 4(24): e1362. DOI: 10.21769/BioProtoc.1362.
  2. Kim, Y. J., Lee, O. R., Oh, J. Y., Jang, M. G. and Yang, D. C. (2014). Functional analysis of 3-hydroxy-3-methylglutaryl coenzyme a reductase encoding genes in triterpene saponin-producing ginseng. Plant Physiol 165(1): 373-387.
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