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Cotton Ovules Culture and Analysis
棉花胚珠的培养和分析

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Abstract

The cotton ovules culture was innovated by Beasley and Ting (1973), and named after them. It is a convenient system to analyze the effect of chemical or environmental treatment on fiber development directly on ovules. This protocol was generated according to previous published papers and our practical experience.

Keywords: Cotton(棉), Ovule culture(胚珠培养), Plant hormone(植物激素), Fiber(纤维)

Materials and Reagents

  1. Flowers (-1 to 1 DPA of flowers are easy to manipulate)
  2. 0.1% (w/v) HgCl2 (caution: HgCl2 is very dangerous. Please select an advantageous reagent to sterilize according to your lab condition. Otherwise 75% ethanol and N,N'-Dicyclohexylcarbodiimide (DCCS) could be the alternatives)
  3. Toluidine blue O (Sigma-Aldrich, catalog number: T3260 )
  4. Glacial acetic acid-ethanol-water (10:95:5, v/v)
  5. Vitamins:
    VB1(Vitamin B1, Thiamine) (Sigma-Aldrich, catalog number: T3902 )
    VB6 (Vitamin B6, Pyridoxine) (Sigma-Aldrich, catalog number: P8666 )
    VB3 (Vitamin B3, Nicotinic acid) (Sigma-Aldrich, catalog number: N0765 )
  6. Inositol (Sigma-Aldrich, catalog number: I3011 )
  7. IAA (Sigma-Aldrich, catalog number: I2886 )
  8. GA3 (Sigma-Aldrich, catalog number: G7645 )
  9. KH2PO4 (Sigma-Aldrich)
  10. CaCl2.2H2O (Sigma-Aldrich)
  11. MgSO4.7H2O (Sigma-Aldrich)
  12. KNO3 (Sigma-Aldrich)
  13. H3BO3 (Sigma-Aldrich)
  14. Na2MoO4.2H2O (Sigma-Aldrich)
  15. KI (Sigma-Aldrich)
  16. CoCl2.6H2O (Sigma-Aldrich)
  17. MnSO4.H2O (Sigma-Aldrich)
  18. ZnSO4.7H2O (Sigma-Aldrich)
  19. CuSO4.5H2O (Sigma-Aldrich)
  20. FeSO4.7H2O (Sigma-Aldrich)
  21. Na2EDTA (Sigma-Aldrich)
  22. BT medium preparation (see Recipes)
  23. Hormone preparation (see Recipes)
  24. Working BT medium preparation (see Recipes)

Equipment

  1. Erlenmeyer flask (50 ml)
  2. 1.5 ml microcentrifuge tubes
  3. Spectrophotometer (Beckman Coulter, model: DU 800 ) or microplate reader (Tecan Trading AG, model: infinite® M200 )
  4. Clean bench laminar air-flow-hood with burner (Harbin East Electronic Technology, model: HD-1360 )
  5. Common plant tissue culture equipment (Measuring cylinder/volumetric flask, Beaker, Weighing machine, Autoclave, pH-meter, Scalpel, Fine forceps)

Software

  1. ImageJ

Procedure

  1. Ovary sterilization
    1. Flowers (Figure 1) are collected with 1 to 3 cm anthocaulus from plants (the length of anthocaulus is usually 1 to 3 cm in our used cotton species, if some others is longer, you can cut as you wish; but the shorter ones is not recommended to choose unless there is no alternative). Remove the petals, stamens and bracts with hand carefully and thoroughly (or you can do it with some special accessory appliances but make sure do not hurt the cotton boll).


      Figure 1. The organ of cotton flower

    2. If the pistils are vigorous, such as those from in or before blooming flowers in which the pistils are linked with the cotton boll, while they will be physiological abscission after 2 day post anthesis (DPA), they should be kept to avoid liquid permeating into the cotton boll through the wound when sterilized.
    3. Soak the whole remaining tissue (cotton boll linked with anthocaulus and/or pistils) into 0.1% (w/v) HgCl2 for 15 min immediately (occasional shaking/stirring is needed to keep all the tissue fully soaked), discard HgCl2 and rinse with sterilized ddH2O three times. Each for 2 to 5 min.

  2. Ovules preparation
    1. Hold the tissue with the anthocaulus, and remove the shuck of the ovary (or the young cotton boll) with a sterilized forceps carefully, then float and disperse the intact ovules on the liquid BT medium gently. Make sure the ovules are not injured, broken or attached the brown gossypol from the broken shuck.
    2. For same batch culture, equal ovules should be put in each flask and usually less than 20 ovules for each batch.
    3. If the ovules are going to culture for more than 10 days, the ovules sum in each flask should be less than 10 at the beginning.
    4. For young ovules that have not grown fiber, they could be separated easily, while for ovules with longer fiber that are sticking together, such as the ovules in 2 day post anthesis (DPA), they need to be carefully separated one by one with forceps before floating on the medium.
    5. Label the flask with the date, the age of the ovules and the hormones contained.

  3. Ovule culture
    1. The floating ovules are incubated in 30 °C without light and shake.
    2. The fiber should be easily visible after 4-5 days of culture for the 0 DPA ovules (Figure 2).


      Figure 2. 0 DPA ovules after 5 days culture

    3. The ovules or fiber can be used for further analysis, such as fiber production measurement, RNA extraction and biochemical analysis etc.

  4. Fiber measurement
    There are two methods to measure fiber production.
    1. Total fiber units (TFU) measurement, which was also innovated by Beasley et al. (1974).
      1. For 12 days cultured ovules, 10 ovules for each assay. Ovules are dried out of medium with filter paper. Sunk in boiling water for 2 min, then dried again with filter paper for 2 to 3 min.
      2. Stain the ovules in a small beaker with 20 ml 0.02% Toluidine blue O for 30 sec, then discard the liquid with a fine sieve and wash in running water for 1 min immediately to remove the non-absorbed dye.
      3. Dry the ovules, then destain in flask with 20 ml glacial acetic acid-ethanol-water (10:95:5) for 2 h. The flask should be sealed to avoid liquid evaporation which can cause differences in concentration of destaining liquid.
      4. Measure the absorbance of the destaining liquid in 624 nm and count as the relative yield of fiber.
    2. Fiber length measurement (Figure 3)
      1. Ovules are sunk in boiling water for 2 min or in 75% ethanol for 15 min, then put the ovules in slowly running water to make the fiber flow to one side and measured with a ruler.
      2. Or put the ovules on a glass slide, comb the fiber gently with a dissecting needle, then take photos and measure the length with a ruler or you can use software for automation of the measurement (e.g. Image J).


        Figure 3. Cultured ovule (1) is soaked in 75% ethanol (2) to separete fiber and measured on a glass slide (3)

Notes

  1. This protocol can be used for analyzing the effects of dozen of chemicals, including hormones, plant growth regulators, plant growth inhibitors and flavonoids (Tan et al., 2013) etc., on fiber development in our lab. We found it functioned similarly in Gossypium hirsutum (more than 4 cultivars were applied), G. arboreum and G.herbaceum. But for G. barbadense, there will be several differences in the fiber growth rate and hormones response. The fiber will grow later and fewer, and GA3 can’t promote fiber grow for the 0 DPA ovules independently.
  2. While it is a very sensitive system, many factors are involved in a success assay, especially for that of ovule developmental stage. According to our unpublished data, there are dramatic differences for 0 DPA ovules from 7 am to 12 am in response to IAA and GA3. And GA3 could promote fiber growth independently on the 0 DPA ovules post 12 am except for that from G. barbadense. The ovules before 0 DPA may be not easy to float on the liquid medium, should be taken more gently. And ovules of different developmental stages are also response differentially to chemical treatment (Tan et al., 2012). So for the same assay, the ovules should be from the same stage and all the manipulations should be strictly consistent and control should be set up for each. The samples from different batches could not analyze together.
  3. CaCl2.2H2O should be separately prepared and stored with KH2PO4 and MgSO4.7H2O, be carefully mixed and diluted before use.
  4. Other chemical should be sterilized first and added into the medium with IAA and/or GA3. Each new chemical should be undergone a dose test to determine the optimal dose.
  5. Infection rate of ovules from the glasshouse is less than that from the field.
  6. Take a simple plan before start. It will take a lot of time to release the ovules from the ovary (approximately 10 ovules per hour for freshman).
  7. For TFU assay, it is 10 ovules for each assay usually, while it should be changed depending on the days of culture and the bulk of ovules.

Recipes

  1. BT medium preparation
    Preparation of 20x Macro element stock solution
    (stored in 4 °C)
    Chemicals (company)
    Mol.wt
     Final conc.
    (mg/L)
    Final conc.
    (mM)
    Conc. stock
    (mM)
    mg to take
    Final volume of stock
    KH2PO4
    136.09
    272.18
    2
    40
    5,443.6
    1,000 ml
    CaCl2.2H2O
    147.01
    441.06
    3
    60
    8,821.2
    MgSO4.7H2O
    246.47
    493.00
    2
    40
    9,860
    KNO3
    101.10
    5,055.50
    50
    1,000
    101,110
    Preparation of 100x Micro element stock solution
    (stored in 4 °C)
    H3BO3
    61.83
    6.183
    0.1
    10
    618.3
    1,000 ml
    Na2MoO4.2H2O
    241.95
    0.242
    0.001
    0.1
    24.2
    KI
    166
    0.830
    0.005
    0.5
    83
    CoCl2.6H2O
    237.93
    0.024
    0.0001
    0.01
    2.4
    MnSO4.H2O
    169.02
    16.902
    0.1
    10
    1,690.2
    ZnSO4.7H2O
    287.56
    8.627
    0.03
    3
    862.7
    CuSO4.5H2O
    249.69
    0.025
    0.0001
    0.01
    2.5
    Preparation of 100x Fe salt stock solution
    (stored in brown bottle and 4 °C)
    FeSO4.7H2O
    278.01
    8.341
    0.03
    3
    834.1
    1,000 ml
    Na2EDTA
    372.24
    11.167
    0.03
    3
    1,116.7
    Preparation of 1,000x Vitamins mixture stock solution
    (stored in 4 °C)
    VB1
    337.27
    1.349
    0.004
    4
    1,349
    1,000 ml
    VB6
    205.64
    0.822
    0.004
    4
    822
    VB3
    123.11
    0.492
    0.004
    4
    492
    Preparation of 100x Inositol stock solution
    (stored in 4 °C)
    Inositol
    180.16
    180.160
    1
    100
    18,016
    1,000 ml

  2. Hormone preparing
    The general hormones for cotton ovules culture are IAA and GA3.The stock solutions of IAA and GA3 are 5 mM and 0.5 mM, respectively. Both are 1,000x solutions, pre-dissolved in a small volume (500-1,000 μl) of 95% ethyl alcohol, then brought to volume with sterilized double-distilled H2O water, aliquoted into 1.5 ml microcentrifuge tubes, sealed and stored in -20 °C. Usually 10 to 50 ml stock solution is prepared for each time.
  3. Working BT medium preparation
    Stock
    Conc. of stock
    Amount of stock soln to take
    Final volume of media
    Macro-element mixture
    20x
    50 ml
    1,000 ml
    Micro-element mixtures
    100x
    10 ml
    Fe salt solution
    100x
    10 ml
    Vitamin mix
    1,000x
    1 ml
    Inositol
    100x
    10 ml
    Glucose

    24 g
    pH 5.0

    Medium is prepared in 100, 200 ml or other certain volumes before used, after sterilized, added suitable hormones, mixed and aliquoted into sterilized 50 ml flasks with about 10 ml each. Label the flask with what kinds of hormones contained, preparation and expiration dates (the medium should be used within less than 15 days).

Acknowledgments

The protocol was based on Beasley and Ting’s original work (Beasley and Ting, 1973), and adapted from two of our previous published work (Tan et al., 2012; Tan et al., 2013). This work was supported by the University Scientific and Technological Self-innovation Foundation, the National Natural Science Foundation of China (grant no. 30871560 and 31230056) and the National High-Tech Program of China (grant no. 2012AA101108).

References

  1. Beasley, C. and Ting, I. P. (1973). The effects of plant growth substances on in vitro fiber development from fertilized cotton ovules. Am J Bot 130-139.
  2. Beasley, C. A., Birnbaum, E. H., Dugger, W. M. and Ting, I. P. (1974). A quantitative procedure for estimating cotton fiber growth. Stain Technol 49(2): 85-92.
  3. Tan, J., Tu, L., Deng, F., Hu, H., Nie, Y. and Zhang, X. (2013). A genetic and metabolic analysis revealed that cotton fiber cell development was retarded by flavonoid naringenin. Plant Physiol 162(1): 86-95.
  4. Tan, J., Tu, L., Deng, F., Wu, R. and Zhang, X. (2012). Exogenous jasmonic acid inhibits cotton fiber elongation. J Plant Growth Regul 31(4): 599-605.

简介

棉花胚珠文化由Beasley和Ting(1973)创新,并以它们命名。 它是一种方便的系统,用于分析化学或环境处理对直接对胚珠的纤维发育的影响。 该协议是根据以前发表的论文和我们的实践经验生成的。

关键字:棉, 胚珠培养, 植物激素, 纤维

材料和试剂

  1. 花(-1至1 DPA的花容易操作)
  2. 0.1%(w/v)HgCl 2(警告:HgCl 2)是非常危险的,请根据实验室条件选择有利的试剂进行灭菌,否则75%乙醇和 N,N'-二环己基碳二亚胺(DCCS)可以是替代物)
  3. 甲苯胺蓝O(Sigma-Aldrich,目录号:T3260)
  4. 冰乙酸 - 乙醇 - 水(10:95:5,v/v)洗脱
  5. 维生素:
    VB1(维生素B1,硫胺素)(Sigma-Aldrich,目录号:T3902) VB6(维生素B6,吡哆醇)(Sigma-Aldrich,目录号:P8666) VB3(维生素B3,烟酸)(Sigma-Aldrich,目录号:N0765)
  6. 肌醇(Sigma-Aldrich,目录号:I3011)
  7. IAA(Sigma-Aldrich,目录号:I2886)
  8. GA 3(Sigma-Aldrich,目录号:G7645)
  9. (Sigma-Aldrich)
  10. (Sigma-Aldrich公司)。
  11. MgSO 4·7H 2 O(Sigma-Aldrich)
  12. KNO 3(Sigma-Aldrich)
  13. (Sigma-Aldrich)
  14. (Sigma-Aldrich公司),其具有以下结构:其中R 1,R 2,R 3,R 4,R 5,R 6,
  15. KI(Sigma-Aldrich)
  16. (Sigma-Aldrich公司)。
  17. (Sigma-Aldrich)
  18. ZnSO 47H 2 O(Sigma-Aldrich)
  19. (Sigma-Aldrich)制备的CuSO 4·6H 2 O(Sigma-Aldrich)
  20. (Sigma-Aldrich)
  21. Na 2 EDTA(Sigma-Aldrich)
  22. BT介质准备(参见配方)
  23. 激素准备(见配方)
  24. 工作BT介质准备(参见配方)

设备

  1. 锥形烧瓶(50ml)
  2. 1.5 ml微量离心管
  3. 分光光度计(Beckman Coulter,型号:DU 800)或酶标仪(Tecan Trading AG,型号:infinite M200)
  4. 带燃烧器的洁净台层流风罩(哈尔滨东电子科技,型号:HD-1360)
  5. 普通植物组织培养设备(量筒/容量瓶,烧杯,称重机,高压灭菌器,pH计,手术刀,精钳)

软件

  1. ImageJ

程序

  1. 卵巢灭菌
    1. 花(图1)收集与植物1至3厘米的Anthocaulus(Anthocaulus的长度通常是1至3厘米在我们使用的棉花种类,如果一些其他的更长,你可以根据你的意愿削减;但较短的是不推荐选择,除非没有其他选择)。用手小心地彻底去除花瓣,雄蕊和苞片(或者你可以用一些特殊的辅助器具,但确保不伤害棉铃)。


      图1.棉花花卉

    2. 如果雌蕊是活力的,例如来自开花期之前或之前的雌蕊,其中雌蕊与棉铃连接,而它们在开花后2天(DPA)后将是生理脱落的,则应当保持它们以避免液体渗入棉铃通过伤口灭菌时。
    3. 将整个剩余的组织(与孔雀和/或雌蕊相连的棉铃)立即浸泡在0.1%(w/v)HgCl 2中15分钟(需要偶尔摇动/搅拌以保持所有组织完全浸泡),弃去HgCl 2并用灭菌的ddH 2 O 3冲洗三次。每次2至5分钟。

  2. 胚珠制备
    1. 保持组织与Anthocaulus,并删除卵巢(或年轻棉铃)小心用无菌镊子,然后漂浮和分散完整的胚珠在液体BT培养基轻轻地。 确保胚珠没有受伤,破碎或附着棕色棉酚从破碎的shuck。
    2. 对于相同的批次培养,每个烧瓶中应该放入相等的胚珠,每个批次通常少于20个胚珠。
    3. 如果胚珠培养超过10天,每个烧瓶中的胚珠总量应该在开始时小于10。
    4. 对于没有生长纤维的幼小胚珠,它们可以容易地分离,而对于粘在一起的具有较长纤维的胚珠,例如开花后2天(DPA)中的胚珠,它们需要用镊子 之前漂浮在介质上。
    5. 给烧瓶标明日期,胚珠的年龄和所含的激素
  3. 胚珠培养
    1. 将浮动胚珠在30℃无光照和温育。
    2. 在0 DPA胚珠培养4-5天后,纤维应该是容易看到的(图2)。


      图2.培养5天后的DPA胚珠

    3. 胚珠或纤维可用于进一步分析,例如纤维生产测量,RNA提取和生物化学分析等。。

  4. 光纤测量
    有两种方法来测量纤维生产
    1. 总光纤单元(TFU)测量,其也由Beasley等人(1974)创新。
      1. 对于12天培养的胚珠,每个测定10个胚珠。 用滤纸从培养基中干燥胚芽。 浸泡在沸水中2分钟,然后用滤纸再次干燥2至3分钟
      2. 在一个小烧杯中用20ml 0.02%甲苯胺蓝O染色胚珠30秒钟,然后用细筛筛除液体,在流水中洗1分钟,立即除去未吸收的染料。
      3. 干燥胚珠,然后用20ml冰醋酸 - 乙醇 - 水(10:95:5)在烧瓶中脱色2小时。 烧瓶应密封以避免液体蒸发,这可能导致脱色液体浓度的差异。
      4. 测量脱色液在624nm处的吸光度,并计算为纤维的相对产率
    2. 光纤长度测量(图3)
      1. 将胚珠在沸水中浸泡2分钟或在75%乙醇中浸泡15分钟,然后将胚珠置于缓慢流动的水中以使纤维流向一侧并用尺子测量。
      2. 或者将胚珠放在载玻片上,用解剖针轻轻梳理纤维,然后用尺子拍照并测量长度,或者可以使用软件进行测量的自动化(例如图像J) 。


        图3.培养的胚珠(1)在75%乙醇(2)中浸泡至分离纤维并在载玻片(3)上测量

笔记

  1. 该方案可用于分析十几种化学物质的影响,包括激素,植物生长调节剂,植物生长抑制剂和类黄酮(Tan等人,2013)。 ,在我们实验室的纤维发展。我们发现它在陆地棉中同样地起作用(应用了超过4个品种),G。 arboreum 和 G.herbaceum 。但是对于巴巴多登(G.barbadense)来说,纤维生长速率和激素反应将存在一些差异。纤维将生长更晚和更少,并且GA <3>不能独立地促进0 DPA胚珠的纤维生长。
  2. 虽然它是一个非常敏感的系统,许多因素参与成功测定,特别是对于胚珠发育阶段。根据我们未发表的数据,对于响应于IAA和GA 3的从上午7点到12点的0DPA胚珠存在显着差异。和GA 3可以促进纤维生长独立于0 DPA胚珠上午12时,除了来自G。 barbadense 。在DPA之前的胚珠可能不容易漂浮在液体介质上,应该更温和地采取。不同发育阶段的胚珠也对化学处理有不同的响应(Tan等人,2012)。因此对于相同的测定,胚珠应当来自相同的阶段,并且所有的操作应该严格一致,并且应该为每个设置控制。来自不同批次的样品不能一起分析
  3. 应该分别制备并与KH 2 PO 4反应来储存CaCl 2 2+ 2H 2 O 2。在使用前仔细混合并稀释。
  4. 其它化学品应首先灭菌并加入具有IAA和/或GA 3的培养基中。每种新化学药品都应进行剂量测试以确定最佳剂量
  5. 来自温室的胚珠的感染率小于来自田间的胚珠的感染率。
  6. 开始之前先做一个简单的计划。从卵巢释放胚珠需要很多时间(对于新生,每小时大约10个胚珠)。
  7. 对于TFU测定,通常每个测定为10个胚珠,而其应根据培养日期和胚珠体积而改变。

食谱

  1. BT培养基制备
    制备20x微量元素储液
    (储存在4℃)
    化学品(公司)
    mol.wt
     最终浓度。
    (mg/L)
    最终浓度。
    (mM)
    浓缩。 股票
    (mM)
    mg服用
    库存最终数量
    KH 2 PO 4
    136.09
    272.18
    2
    40
    5,443.6
    1000 ml
    CaCl 2 2H O
    147.01
    441.06
    3
    60
    8,821.2
    MgSO 4。 。 O
    246.47
    493.00
    2
    40
    9865
    KNO 3
    101.10
    5,055.50
    50
    1,000
    101,110
    制备100x微量元素储液
    (储存在4℃)
    H 3 BO 3
    61.83
    6.183
    0.1
    10
    618.3
    1000 ml
    241.95
    0.242
    0.001
    0.1
    24.2
    KI
    166
    0.830
    0.005
    0.5
    83
    CoCl <2> 6H <2> O
    237.93
    0.024
    0.0001
    0.01
    2.4
    MnSO 4 H O
    169.02
    16.902
    0.1
    10
    1,690.2
    ZnSO 4 。 7H O
    287.56
    8.627
    0.03
    3
    862.7
    CuSO 4 5H 2 O
    249.69
    0.025
    0.0001
    0.01
    2.5
    100×Fe盐储备溶液的制备
    (储存在棕色瓶中,4℃)
    FeSO 4 7H <2> O
    278.01
    8.341
    0.03
    3
    834.1
    1000 ml
    Na 2 EDTA 372.24
    11.167
    0.03
    3
    1,116.7
    1,000x维生素混合物储备液的制备
    (储存在4℃)
    VB1
    337.27
    1.349
    0.004
    4
    1,349
    1000 ml
    VB6
    205.64
    0.822
    0.004
    4
    822
    VB3
    123.11
    0.492
    0.004
    4
    492
    制备100x肌醇储备液
    (储存在4℃)
    肌醇
    180.16
    180.160
    1
    100

    1000 ml

  2. 激素准备
    棉花胚珠培养物的一般激素是IAA和GA 3。IAA和GA 3的储备溶液分别为5mM和0.5mM。 两者都是1,000x的溶液,预先溶解在小体积(500-1,000μl)的95%乙醇中,然后用无菌的双蒸水H 2 O水补足体积,分成1.5ml 微量离心管,密封并储存于-20°C。 通常每次制备10至50毫升储备溶液
  3. 工作BT介质制备
    库存
    浓缩。 的股票
    取得的股票金额
    最终卷 of media
    宏元素混合物
    20x
    50 ml
    1000 ml
    微元素混合物
    100x
    10 ml
    Fe盐溶液
    100x
    10 ml
    维生素混合物
    1,000x
    1 ml
    肌醇
    100x
    10 ml
    葡萄糖

    24克
    pH 5.0

    在使用前,在100,200ml或其它一定体积中制备培养基,灭菌后,加入合适的激素,混合并等分到灭菌的50ml烧瓶中,每个约10ml。 标记烧瓶中含有什么类型的激素,准备和有效期(培养基应在不到15天内使用)。

致谢

该协议基于Beasley和Ting的原始工作(Beasley和Ting,1973),并且根据我们先前发表的两篇文章(Tan等人,2012; Tan等人, 2013)。 这项工作得到了大学科技自主创新基金会,中国国家自然科学基金(拨款号30871560和31230056)和中国国家高技术计划(拨款号2012AA101108)的支持。

参考文献

  1. Beasley,C。和Ting,I.P。(1973)。 植物生长物质对来自受精棉胚珠的体外纤维发育的影响。 a> 130-139。
  2. Beasley,C.A.,Birnbaum,E.H.,Dugger,W.M.and Ting,I.P。(1974)。 估算棉纤维生长的定量程序 染色技术 49(2):85-92。
  3. Tan,J.,Tu,L.,Deng,F.,Hu,H.,Nie,Y.and Zhang, 遗传和代谢分析显示,棉花纤维细胞发育被类黄酮柚皮素阻滞。 162(1):86-95。
  4. Tan,J.,Tu,L.,Deng,F.,Wu,R。和Zhang,X。(2012)。 外源茉莉酮酸抑制棉花纤维伸长。植物生长调节 31(4):599-605。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Tan, J., Deng, F., Tang, W., Han, J., Kai, G., Tu, L. and Zhang, X. (2013). Cotton Ovules Culture and Analysis. Bio-protocol 3(22): e972. DOI: 10.21769/BioProtoc.972.
  2. Tan, J., Tu, L., Deng, F., Hu, H., Nie, Y. and Zhang, X. (2013). A genetic and metabolic analysis revealed that cotton fiber cell development was retarded by flavonoid naringenin. Plant Physiol 162(1): 86-95.
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