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Shoot Regenerative Capacity Assays in Arabidopsis and Tobacco
拟南芥和烟草中嫩芽再生能力的测试   

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Abstract

Plant regeneration refers to a process through which an explant is differentiated to a whole plant under certain conditions. It has been shown that two plant hormones, auxin and cytokinin, play critical roles within this process (Duclercq et al., 2011). Cytokinin induces shoot regeneration, whereas auxin promotes root production. In addition to hormones, recent study has revealed that age cue serves as a common element behind plant cell totipotency (Toledano et al., 2012). Here we present an easy protocol to assess the shoot regenerative capacity of Arabidopsis and tobacco leaves of different ages.

Keywords: Shoot regeneration(再生), Cytokinin(细胞分裂素), Auxin(生长素), Plant age(植物的年龄)

Materials and Reagents

  1. Surgical tape (3M, catalog number: 15300 )
  2. Sterile petri dishes
  3. Seeds: Arabidopsis thaliana (Col-0 ecotype) and tobacco (Nicotiana tabacum cv SR1)
  4. Bacterial strain: Agrobacterium tumefaciens EHA105
  5. Sodium hypochlorite (Sigma-Aldrich, catalog number: L099100 )
    Note: The Pricing and availability are not currently available.
  6. Kanamycin sulfate Streptomyces kanamyceticus (Sigma-Aldrich, catalog number: K0879 )
  7. Timentin (Yeasen, catalog number: 60230ES07 )
  8. Agar (Sangon Biotech, catalog number: A100637 )
  9. Methylester sulfonate (Sangon Biotech, catalog number: MB0341 )
  10. Sucrose (Sigma-Aldrich, catalog number: E001888 )
  11. Glucose (Sigma-Aldrich, catalog number: G8270 )
  12. Ethyl alcohol, Pure (Sigma-Aldrich, catalog number: 459844 )
  13. Deionized water
  14. Soil
  15. Murashige and Skoog (MS) basal medium with vitamin powder (PhytoTechnology, Laboratories®, catalog number: 15B0519117B ) (1 L) (see Recipes)
  16. 1/2 MS medium (1 L) (see Recipes)
  17. MS1 medium (1 L) (see Recipes)
  18. MS2 medium (1 L) (see Recipes)
  19. MS3 medium (1 L) (see Recipes)
  20. Callus-inducing medium (1 L) (see Recipes)
  21. Shoot-inducing medium (1 L) (see Recipes)
  22. 22 mM 2, 4-dichlorophenoxy (2, 4-D) stock solution (Sigma-Aldrich, catalog number: 31518 ) (see Recipes)
  23. 9 mM 3-indoleacetic acid (IAA) (Sigma-Aldrich, catalog number: I2886 ) stock solution (see Recipes)
  24. 20 mM Kinetin (Sigma-Aldrich, catalog number: K0753 ) stock solution (see Recipes)
  25. 50 mM 6-(γ, γ-Dimethylallylamino)purine (2-IP) (Sigma-Aldrich, catalog number: D5912 ) stock solution (see Recipes)
  26. 2 mg/ml 6-benzylaminopurine (6-BA) (Sigma-Aldrich, catalog number: B3408 ) stock solution (see Recipes)
  27. 1 M Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 ) (see Recipes)
  28. 15% bleach (see Recipes)
  29. 10% bleach (see Recipes)
  30. Infection buffer (see Recipes)

Equipment

  1. Clean bench (horizontal laminar flow)
  2. Autoclave (ZEALWAY)
  3. Incubator (25 °C/80 UML) (Percival-scientific, model: CU-41L4 )
  4. Growth chamber
  5. Glass culture bottles (130 mm x 100 mm x 62 mm)
  6. Sterile 5 mm-hole punch
  7. Scalpel (10 cm in length)
  8. Forceps (20 cm in length)
  9. Scissor (15 cm in length)

Procedure

  1. Generation of transgenic tobacco plants
    To study the molecular basis which age regulates plant regeneration capacity, transgenic tobacco plants with gain of function or loss of function candidate genes were generated by using this method (such as miR156 and its targeted gene SPL9).
    1. To get aseptic seedlings, tobacco seeds were sterilized with 15% bleach to shake for 15 min in 2.0 ml tubes. Then wash three times with sterile water. Every seed was germinated on each ½ MS glass bottle. The numbers of aseptic seedlings were determined by the numbers of transgenes. About five aseptic seedlings were needed in each transgene. Seal the glass bottles using surgical tape. Do these in a clean bench. The glass bottles were incubated for 4-5 weeks at 25 °C under long-day conditions (16 h light/8 h dark) (Figure 1A).
    2. Cut the aseptic leaves into pieces (about 1 cm in diameter) using scalpel and forceps. Transfer leaf pieces to MS plates and pre-culture them for 2 days at 25 °C under long-day conditions. Do these in a clean bench.
    3. The candidate genes were cloned into binary vector (such as pCAMBIA2301) and then were delivered into Agrobacterium tumefaciens EHA105 by the freeze-thaw method (Weigel and Glazebrook, 2006) and cultures were incubated for 3 days at 28 °C.
    4. The single positive colony was incubated into 6 ml liquid LB with relevant antibiotics and shook overnight at 28 °C.
    5. Transfer 2 ml cultured Agrobacterium to 100 ml fresh LB with relevant antibiotics and grown with shaking overnight at 28 °C.
    6. The overnight culture of Agrobacterium was centrifuged for 15 min at 4,000 rpm and resuspended in infection buffer (30 g/L glucose, OD600 = 0.8-1.0).
    7. The pre-cultured leaf pieces were transferred into Agrobacterium suspension and infected for 20~30 min. After that, the infected leaf pieces were transferred to MS1 plates and co-cultured for 2 days at 25 °C under long-day conditions. Do these in a clean bench.
    8. The infected leaf pieces were transferred to MS2 plate which contained 6-benzylaminopurine (6-BA), a synthetic cytokinin, to induce the formation of shoots. Then explants were cultured for 3-4 weeks at 25 °C under long-day conditions.
    9. The regenerated adventitious buds (about 1 cm size) on each leaf pieces were isolated by scalpel and forceps (Figure 1B), transferred to a glass culture bottle with MS3 media and cultured for 3-4 weeks.


      Figure 1. Construct the transgenic plants in tobacco by the tissues cultures. A. A tobacco plant grown in glass bottle for 4-5 weeks, bar = 1 cm. B. The regenerated adventitious buds on an explant, bar = 100 μm. C. A regenerated plant which has been cultured 3-4 weeks on MS3 plate, bar = 1 cm.

    10. Subculture once with MS3 media, when the adventitious buds grew up into whole plants.
    11. Finally, transfer regenerated plants from MS3 plates to soil and grow them in growth chamber at 25 °C under long-day conditions.

  2. Regeneration assays using the leaves from tobacco plants of different ages
    To study the relationship between the shoot regenerative capacity and plant age, regeneration assays were performed using the first/second (early), fifth (mid), and ninth/tenth (late) tobacco leaves as explants.
    1. Tobacco seeds were grow on soil in a growth chamber under long-day conditions.
    2. The first/second (early), fifth (mid), and ninth/tenth (late) tobacco leaves were collected and placed on deionized water. To avoid the impact of leaf development on regenerative capacity, the leaves of the same size (1 cm in length) were used.
    3. Detached leaves were sterilized with 10% bleach for 15 min in sterile Petri dishes. Wash three times with sterile water.
    4. Punch on leaves. The leaf dices without vascular tissue (5-mm in diameter) were used as explant.
    5. Transfer explants to MS media with 6-BA of different concentrations (such as: 0.05 mg/L, 0.2 mg/L, and 0.5 mg/L). Explants were cultured at 25 °C in an incubator under long-day conditions.
    6. After about 3-4 weeks, the regenerated shoots appeared. The numbers of explants and regenerated shoots were scored (Figure 2). The regenerative capacity was represented by the number of regenerated shoots in the number of explants.


      Figure 2. Count the regenerated shoots and evaluate the shoot regeneration capacity of explants in tobacco. A. Explants were cultured on MS plates for 3-4 weeks, n = 3 x 9, bar = 0.5 cm. B. The regenerated shoots from explants which were cultured on the MS media with 6-BA, n = 3 x 9, bar = 0.5 cm. C. Shoots that was scored like this, bar = 100 μm. D. One example of representative data on tobacco.

  3. Arabidopsis shoot regeneration assays
    In Arabidopsis, shoot regeneration requires two steps. First, explants were cultured on an auxin-rich callus-inducing medium so that they acquired competence to form shoots. Second, the callus were induced to produce shoots on a shoot-induction medium (Valvekens et al., 1988).
    1. Arabidopsis seeds were sterilized with 15% bleach for 15 min and kept at 4 °C for 2 days under darkness.
    2. The seeds were sowed on 1/2 MS plate and incubated at 22 °C for 7 days under darkness.
    3. The hypocotyls (1 cm in length) were cut out and used as explants for regeneration assays.
    4. The hypocotyl segments were transferred to auxin-rich callus-inducing medium (CIM) and cultured for 7 days 22 °C in an incubator under long-day conditions (16 h light/8 h dark) (Figure 3A).
    5. The calli were then transferred to shoot-inducing medium (SIM) with 2-IP of different concentrations. The shoots will be regenerated by culturing calli at 22 °C under long-day conditions (Figure 3B).
    6. The number of regenerated shoots was scored. The regenerative capacity was represented by the number of regenerated shoots in number of explants.


      Figure 3. The regeneration assays to measure the shoot regeneration capacity of explants in Arabidopsis. A. Arabidopsis hypocotyls were cultured on an auxin-rich callus-inducing medium for 7 days, n = 3 x 10, bar = 1.0 cm. B. The regenerated shoots on shoot-induction medium, n = 3 x 8, bar = 1.0 cm. C. Shoots that was scored like this, bar = 1.0 cm. D. One example of representative data on Arabidopsis.

Recipes

  1. MS medium (1 L)
    4.46 g of MS basal medium with vitamin powder
    0.5 g methylester sulfonate
    20 g sucrose
    8 g agar
    Adjust pH 5.7 with 1 M KOH and autoclave at 121 °C for 15 min
  2. 1/2 MS medium (1 L)
    2.23 g of MS basal medium with vitamin powder
    0.5 g methylester sulfonate
    8 g agar
    Adjust pH 5.7 with 1 M KOH and autoclave at 121 °C for 15 min
  3. MS1 medium (1 L)
    MS medium with 6-BA 2 mg/L
  4. MS2 medium (1 L)
    MS medium with 6-BA 2 mg/L
    100 mg/L kanamycin
    250 mg/L timentin
  5. MS3 medium (1 L)
    MS medium with 100 mg/L kanamycin
    250 mg/L timentin
  6. Callus-inducing medium (1 L)
    MS medium with 2.2 µM 2,4-D and 0.2 µM kinetin
  7. Shoot-inducing medium (1 L)
    MS medium with 0.9 µM IAA and different concentrations of 2-IP
  8. 22 mM 2,4-D stock solution
    Dissolve 0.049 g 2,4-D powder in 10 ml ethanol
    Filter sterilization and stored at -20 °C
  9. 9 mM IAA stock solution
    Dissolve 0.016 g IAA powder in 10 ml ethanol
    Filter sterilization and stored at -20 °C
  10. 20 mM kinetin stock solution
    Add 0.043 g kinetin powder to <10 ml deionized water and dissolve completely by 1 M
    KOH
    Adjust volume to 10 ml
    Filter sterilization and stored at -20 °C
  11. 50 mM 2-IP stock solution
    Add 0.10 g 2-IP powder to <10 ml deionized water and dissolve completely by 1 M KOH
    Adjust volume to 10 ml
    Filter sterilization and stored at -20 °C
  12. 2 mg/ml 6-BA stock solution
    Dissolve 20 mg 6-BA powder in 10 ml DMSO
    Filter sterilization and stored at -20 °C
  13. 1 M KOH
    Dissolve 5.61 g KOH powder in 100 ml deionized water
    Stored at room temperature
  14. 15% bleach
    Add 7.5 ml sodium hypochlorite solution into 42.5 ml deionized water
  15. 10% bleach
    Add 5 ml sodium hypochlorite solution into 45 ml deionized water
  16. Infection buffer
    Dissolve 30 g glucose powder in 1 L deionized water

Acknowledgments

The work in the Wang laboratory is supported by grants from the National Natural Science Foundation of China (31430013; 31222029; 912173023), the State Key Basic Research Program of China (2013CB127000), the Shanghai Outstanding Academic Leader Program (15XD1504100) and the NKLPMG Key Research Program. This protocol was adapted from our recent publications (Zhang et al., 2015a; Zhang et al., 2015b).

References

  1. Duclercq, J., Sangwan-Norreel, B., Catterou, M. and Sangwan, R. S. (2011). De novo shoot organogenesis: from art to science. Trends Plant Sci 16(11): 597-606.
  2. Toledano, H., D'Alterio, C., Czech, B., Levine, E. and Jones, D. L. (2012). The let-7-Imp axis regulates ageing of the Drosophila testis stem-cell niche. Nature 485(7400): 605-610.
  3. Valvekens, D., Van Montagu, M. and Van Lijsebettens, M. (1988). Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci U S A 85(15): 5536-5540.
  4. Weigel, D. and Glazebrook, J. (2006). Transformation of agrobacterium using the freeze-thaw method. CSH Protoc 2006(7).
  5. Zhang, T., Wang, J. and Zhou, C. (2015a). The role of miR156 in developmental transitions in Nicotiana tabacum. Sci China Life Sci 58(3): 253-260.
  6. Zhang, T. Q., Lian, H., Tang, H., Dolezal, K., Zhou, C. M., Yu, S., Chen, J. H., Chen, Q., Liu, H., Ljung, K. and Wang, J. W. (2015b). An intrinsic microRNA timer regulates progressive decline in shoot regenerative capacity in plants. Plant Cell 27(2): 349-360.

简介

植物再生是指在某些条件下外植体分化成整株植物的过程。 已经表明,两种植物激素,植物生长素和细胞分裂素在该过程中起关键作用(Duclercq等人,2011)。 细胞分裂素诱导芽再生,而生长素促进根生产。 除了激素,最近的研究已经揭示年龄提示作为植物细胞全能性背后的共同元素(Toledano等人,2012)。 在这里我们提出了一个简单的协议,以评估不同年龄的拟南芥和烟草叶的射线再生能力。

关键字:再生, 细胞分裂素, 生长素, 植物的年龄

材料和试剂

  1. 手术胶带(3M,目录号:15300)
  2. 无菌培养皿
  3. 种子:拟南芥(Col-0生态型)和烟草( Nicotiana tabacum cv SR1)
  4. 菌株:根癌土壤杆菌 EHA105
  5. 次氯酸钠(Sigma-Aldrich,目录号:L099100)
    注意:价格和可用性目前不可用。
  6. 硫酸卡那霉素链霉菌(Sigma-Aldrich,目录号:K0879)
  7. Timentin(Yeasen,目录号:60230ES07)
  8. 琼脂(Sangon Biotech,目录号:A100637)
  9. 甲基酯磺酸盐(Sangon Biotech,目录号:MB0341)
  10. 蔗糖(Sigma-Aldrich,目录号:E001888)
  11. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  12. 乙醇,Pure(Sigma-Aldrich,目录号:459844)
  13. 去离子水
  14. 土壤
  15. 具有维生素粉的Murashige和Skoog(MS)基础培养基(PhytoTechnology,Laboratories ,目录号:15B0519117B)(1L)
  16. 1/2 MS培养基(1 L)(参见配方)
  17. MS1培养基(1 L)(参见配方)
  18. MS2培养基(1 L)(参见配方)
  19. MS3培养基(1 L)(参见配方)
  20. 愈伤组织诱导培养基(1L)(参见配方)
  21. 拍摄诱导介质(1升)(见配方)
  22. 22mM 2,4-二氯苯氧基(2,4-D)储备溶液(Sigma-Aldrich,目录号:31518)(参见配方)
  23. 9mM 3-吲哚乙酸(IAA)(Sigma-Aldrich,目录号:I2886)储备溶液(参见配方)
  24. 20mM激动素(Sigma-Aldrich,目录号:K0753)储备溶液(参见Recipes)
  25. 50mM 6-(γ,γ-二甲基烯丙基氨基)嘌呤(2-IP)(Sigma-Aldrich,目录号:D5912)储备溶液
  26. 2mg/ml 6-苄基氨基嘌呤(6-BA)(Sigma-Aldrich,目录号:B3408)储备溶液(参见配方)
  27. 1M氢氧化钾(KOH)(Sigma-Aldrich,目录号:P5958)(参见Recipes)
  28. 15%漂白剂(见配方)
  29. 10%漂白剂(见配方)
  30. 感染缓冲区(参见配方)

设备

  1. 清洁工作台(水平层流)
  2. 高压灭菌器(ZEALWAY)
  3. 孵育器(25℃/80UML)(Percival-scientific,型号:CU-41L4)
  4. 生长室
  5. 玻璃培养瓶(130mm×100mm×62mm)
  6. 无菌5mm孔冲头
  7. 手术刀(长度10厘米)
  8. 镊子(长度20厘米)
  9. 剪刀(长度15厘米)

程序

  1. 转基因烟草的生成
    为了研究年龄调节植物再生能力的分子基础,通过使用该方法(例如miR156及其靶向基因SPL9)产生具有功能获得或功能候选基因丧失的转基因烟草植物。
    1. 为了获得无菌幼苗,将烟草种子用15%漂白剂灭菌 在2.0ml管中振荡15分钟。然后用无菌洗涤三次 水。每个种子在每个1/2MS玻璃瓶上发芽。号码 的无菌幼苗通过转基因的数量来确定。关于 在每个转基因中需要五个无菌幼苗。密封玻璃 瓶使用外科胶带。在清洁工作台上做这些。玻璃瓶 在长日照条件下在25℃下孵育4-5周(16小时 光/8小时黑暗)(图1A)。
    2. 将无菌叶子切成片 (直径约1cm)使用解剖刀和镊子。转移叶片 ?并在25℃下长时间预培养2天 条件。在清洁工作台上做这些。
    3. 候选基因 克隆到二元载体(例如pCAMBIA2301 )中,然后递送 通过冻融方法(Weigel)转化到根癌土壤杆菌EHA105中 和Glazebrook,2006),并将培养物在28℃孵育3天
    4. 将单阳性菌落与具有相关抗生素的6ml液体LB培养,并在28℃下摇动过夜
    5. 转移2毫升培养土壤杆菌到100毫升与相关抗生素的新鲜LB,并在28℃摇动过夜生长
    6. 农杆菌的过夜培养物在37℃离心15分钟 并用感染缓冲液(30g/L葡萄糖,OD 600 = 1)重悬浮 ?0.8-1.0)。
    7. 将预培养的叶片转移至农杆菌悬浮液中,感染20?30分钟。之后, 将感染的叶片转移至MS1平板并共培养2 ?天在25℃在长日条件下。在清洁工作台上做这些。
    8. 将感染的叶片转移到MS2平板上 含有6-苄基氨基嘌呤(6-BA),合成细胞分裂素,诱导 形成芽。然后将外植体在25℃培养3-4周 ?℃。长时间条件下
    9. 再生不定芽 (约1cm大小)通过手术刀分离 镊子(图1B),转移到带有MS3的玻璃培养瓶 培养3-4周。


      图1。 A.烟草植物生长 ?在玻璃瓶中4-5周,条= 1cm。 B.再生 外植体上的不定芽,条=100μm。 C.再生植物 其在MS3平板上培养3-4周,条= 1cm
    10. 亚细菌培养一次与MS3媒体,当不定芽长成成整个植物
    11. 最后将再生植物从MS3板转移到土壤和 在长期条件下在25℃的生长室中生长它们。

  2. 使用不同年龄的烟草植物的叶子的再生测定
    为了研究苗再生能力和植物年龄之间的关系,使用第一/第二(早),第五(中)和第九/第十(晚)烟叶作为外植体进行再生测定。
    1. 烟草种子在长日照条件下在生长室中的土壤上生长。
    2. 第一/第二(早),第五(中)和第九/第十(晚)烟草 收集叶并置于去离子水上。避免影响 ?的叶片发育对再生能力,叶片的相同 尺寸(长度为1cm)
    3. 在无菌培养皿中用10%漂白剂将分离的叶子灭菌15分钟。用无菌水洗三次。
    4. 在叶子的拳打。将没有血管组织的叶片(直径5mm)用作外植体
    5. 转移外植体到具有不同浓度的6-BA的MS培养基 (例如:0.05mg/L,0.2mg/L和0.5mg/L)。培养外植体 25℃在孵化器中长时间条件下
    6. 后约3-4 周,再生芽出现。外植体和 对再生芽进行评分(图2)。再生能力为 ?由再生枝条的数量表示 外植体

      图2。 A.外植体在MS平板上培养3-4天 周, n = 3 x 9,bar = 0.5厘米。 B.从外植体再生的芽 其在具有6-BA,n/e = 3×9,bar = 0.5cm的MS培养基上培养。 C.如此刻痕的枝条,bar =100μm。 D.烟草代表性数据的一个例子。

  3. 拟南芥苗再生测定
    在拟南芥中,苗再生需要两个步骤。首先,将外植体在富含生长素的愈伤组织诱导培养基上培养,使得它们获得形成芽的能力。第二,诱导愈伤组织在芽诱导培养基上产生芽(Valvekens等人,1988)。
    1. 将拟南芥种子用15%漂白剂灭菌15分钟,并在黑暗下在4℃下保持2天。
    2. 将种子播种在1/2MS板上,在22℃下在黑暗下孵育7天
    3. 切下下胚轴(长度为1cm)并用作再生测定的外植体
    4. 下胚轴段被转移到富含生长素 愈伤组织诱导培养基(CIM),并在培养箱中在22℃下培养7天 在长日照条件下(16小时光照/8小时黑暗)(图3A)。
    5. 的 ?然后将愈伤组织转移至具有2-IP的芽诱导培养基(SIM) ?不同浓度。芽将通过培养再生 愈伤组织在22℃在长期条件下(图3B)
    6. 号码 的再生枝条。再生能力为 由外植体数目中的再生芽数表示

      图3。 A. 拟南芥下胚轴在富含生长素的 愈伤组织诱导培养基中培养7天,n = 3×10,bar = 1.0cm。 B. The 在芽诱导培养基上再生芽,n = 3×8,bar = 1.0cm。 C.如此刻划的枝条,条= 1.0cm。 D.一个例子 拟南芥的代表性数据。

食谱

  1. MS培养基(1L)
    4.46g含维生素粉末的MS基础培养基
    0.5克甲基磺酸盐 20克蔗糖 8克琼脂
    用1M KOH调节pH 5.7,在121℃高压灭菌15分钟
  2. 1/2 MS培养基(1L)
    2.23g含维生素粉的MS基础培养基 0.5克甲基磺酸盐 8克琼脂
    用1M KOH调节pH 5.7,在121℃高压灭菌15分钟
  3. MS1培养基(1L)
    MS培养基,6-BA 2mg/L
  4. MS2培养基(1L) MS培养基,6-BA 2mg/L 100mg/L卡那霉素
    250mg/L特美汀
  5. MS3培养基(1L)
    MS培养基,具有100mg/L卡那霉素 250mg/L特美汀
  6. 愈伤组织诱导培养基(1L)
    MS培养基,具有2.2μM2,4-D和0.2μM激动素
  7. 拍摄诱导培养基(1 L)
    MS培养基与0.9μMIAA和不同浓度的2-IP
  8. 22mM 2,4-D储液
    将0.049g 2,4-D粉末溶解在10ml乙醇中 过滤灭菌,贮存于-20°C
  9. 9 mM IAA储备液
    将0.016g IAA粉末溶解在10ml乙醇中 过滤灭菌,贮存于-20°C
  10. 20 mM激动素储液
    将0.043g激动素粉末加入到<10ml去离子水中,并通过1M
    完全溶解 KOH
    将体积调整为10 ml
    过滤灭菌,贮存于-20°C
  11. 50 mM 2-IP储液
    将0.10g 2-IP粉末加入到<10ml去离子水中,并用1M KOH完全溶解 将体积调整为10 ml
    过滤灭菌,贮存于-20°C
  12. 2mg/ml 6-BA储备液
    将20mg 6-BA粉末溶解在10ml DMSO中 过滤灭菌,贮存于-20°C
  13. 1 M KOH
    将5.61g KOH粉末溶解在100ml去离子水中
    在室温下贮存
  14. 15%漂白剂
    将7.5ml次氯酸钠溶液加入42.5ml去离子水中
  15. 10%漂白剂
    将5毫升次氯酸钠溶液加入45毫升去离子水中
  16. 感染缓冲区
    将30g葡萄糖粉末溶解在1L去离子水中

致谢

Wang实验室的工作得到国家自然科学基金(31430013; 31222029; 912173023),中国国家重点基础研究计划(2013CB127000),上海优秀学术带头人计划(15XD1504100)和NKLPMG主要研究项目。该方案改编自我们最近的出版物(Zhang等人,2015a; Zhang等人,2015b)。

参考文献

  1. Duclercq,J.,Sangwan-Norreel,B.,Catterou,M。和Sangwan,R.S.(2011)。 重新拍摄器官发生:从艺术到科学。 Trends Plant Sci 16(11):597-606。
  2. Toledano,H.,D'Alterio,C.,Czech,B.,Levine,E.and Jones,D.L。(2012)。 let-7-Imp轴调节了果蝇睾丸干的老化-cell niche。 Nature 485(7400):605-610。
  3. Valvekens,D.,Van Montagu,M。和Van Lijsebettens,M。(1988)。 根瘤土壤杆菌 - 介导的拟南芥的转化 >使用卡那霉素选择的根外植体。美国国家科学院院刊85(15):5536-5540。
  4. Weigel,D。和Glazebrook,J。(2006)。 使用冻融法转化土壤杆菌。 CSH Protoc em> 2006(7)。
  5. Zhang,T.,Wang,J.and Zhou,C。(2015a)。 miR156在 Nicotiana tabacum的发育转换中的作用。 Sci China Life Sci 58(3):253-260。
  6. Zhang,TQ,Lian,H.,Tang,H.,Dolezal,K.,Zhou,CM,Yu,S.,Chen,JH,Chen,Q.,Liu,H.,Ljung,K.and Wang,JW (2015b)。 内在微小RNA定时器调节植物的苗再生能力的进行性下降。 植物细胞 27(2):349-360。
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引用:Zhang, T. and Wang, J. (2016). Shoot Regenerative Capacity Assays in Arabidopsis and Tobacco. Bio-protocol 6(5): e1753. DOI: 10.21769/BioProtoc.1753.
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