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In vitro Floral Induction of Cuscuta reflexa
大花菟丝子的体外成花诱导   

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Abstract

Floral initiation and development in the angiosperms is the essential step on which the yield of the plant depends. Sometimes external climate or any abiotic stress hinders the floral initiation and ultimately affect the plant yield. Hence, in vitro floral induction and development can help to overcome the external climatic factor. Furthermore, the protocol for in vitro floral induction in the parasitic angiosperm like Cuscuta reflexa has not been reported yet. We have standardized the protocol for floral induction in the parasitic plant Cuscuta reflexa. In this study it is established that MMS (modified Murashige Skoog) media supplemented with 2 mg L-1 NAA (naphthalene acetic acid, plant growth regulator) and 2 mg L-1 2,4-D (2,4-dichlorophenoxy acetic acid, plant growth regulator) supported floral induction after shooting in vitro. Furthermore, we found that MMS media supplemented with 2 mg L-1 2,4-D rapidly induced floral buds directly from the nodal explants without any shoot elongation. This protocol will help the researcher to induce flower in vitro in the other angiosperm plants along with Cuscuta reflexa.

Keywords: Cuscuta reflexa(大花菟丝子), in vitro(体外), Floral induction(花诱导), NAA(NAA), 2,4-D(2,4-D)

Background

Cuscuta reflexa is a parasitic angiosperm parasitizing on a huge number of angiosperms (Kuijt, 1969). The ability to cause severe damage and loss of yield in the host plant has made this species important for scientific study (Nun and Mayer, 1999). Most of the Cuscuta species are non chloroplastic except few which have functional chloroplasts (Hibberd et al., 1998). Development of seedlings from larger embryos of C. reflexa in white’s medium has been reported (Maheshweri and Baldev, 1961). Floral induction of C. reflexa in short day period as well as dark conditions has been seen in vitro (Baldev, 1962). In vitro floral induction of Cuscuta japonica in short day conditions has also been established by Furuhashi et al. (1991).

Specifically, floral induction and effect of growth regulators on floral induction of C. reflexa has not yet been studied. A complete tissue culture system for Cuscuta trifolli in liquid MS culture has been reported so far (Bakos et al., 1995). The floral induction in C. reflexa on modified white’s medium, subjecting the plant to different light and dark conditions, has been studied years back (Baldev, 1962). Here we have shown a complete in vitro culture system for floral induction in C. reflexa. Concentration of 2,4-D played a significant role in floral induction. Supplementation of NAA along with 2,4-D induces shoot followed by flower, but supplementation of only 2,4-D induces flower directly from nodal explants without showing any stem elongation. This result is very attractive and is showing the importance of 2,4-D in floral induction of this plant.

Materials and Reagents

  1. Pipette tips (Tarsons)
  2. 5 ml syringe (Himedia)
  3. 0.45 micron filter (Himedia)
  4. Stem explants of Cuscuta reflexa
  5. Murashige & Skoog (MS) basal medium (Murashige and Skoog, 1962) (Sigma-Aldrich, catalog number: M5519 )
  6. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  7. Gamborg’s vitamin solution (Sigma-Aldrich, catalog number: G1019 )
  8. Agar (HiMedia Laboratories, catalog number: PCT0901 )
  9. 2,4-dichlorophenoxyacetic acid (2,4-D) (Sigma-Aldrich, catalog number: D7299 )
  10. Naphthalene acetic acid (NAA) (Sigma-Aldrich, catalog number: N0640 )
  11. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
  12. Mercury(II) chloride (HgCl2) (0.01%) (EMD Millipore, catalog number: 104419 )
  13. Bavistin (1% solution, Swat agro chemicals)
  14. Ethanol (EMD Millipore, catalog number: 100983 )
  15. MMS (modified Murashige Skoog) media (see Recipes)
  16. 2,4-D stock solution (see Recipes)
  17. NAA stock solution (see Recipes)
  18. Floral induction media-1 (see Recipes)
  19. Floral induction media-2 (see Recipes)
  20. HgCl2 (0.1%) (see Recipes)
  21. Bavistin solution (1%) (see Recipes)

Equipment

  1. 0.5-10 μl pipette (Transferpette®) (BRAND, catalog number: 704770 )
  2. 20-200 μl pipette (Transferpette®) (BRAND, catalog number: 704778 )
  3. 100-1,000 μl pipette (Transferpette®) (BRAND, catalog number: 704780 )
  4. Beaker (200 ml, Borosil)
  5. Laminar hood (Thermo Fisher Scientific)
  6. Conical flask (150 ml, Borosil)
  7. Culture room (Daihan LabTech India, model: LGC-S201 )
  8. Autoclave
  9. Forceps (ACE Surgical Supply Company, catalog number: 20-000-48 )
  10. Oven (Hicon India)
  11. Water distillation unit (Mars Scientific Instruments, catalog number: BASIC/pH4 & XL )
  12. pH meter (CD Hightech, model: APX 175 E )
  13. Weighing machine (Sartorius, model: BSA224S-CW )
  14. Scissor (general cutting scissor)

Procedure

  1. Explant surface sterilization
    1. Nodal region (Figure 1A) of the Cuscuta reflexa plant (2-month-old) grown naturally on bougainvillea plant was taken as the starting material.
    2. Keep small pieces (around 20 pieces taken here) of Cuscuta reflexa nodal stem explants (1-2 cm length, Figure 1A) inside a beaker. Treat (submerge) the explants in 100 ml of 1% Bavistin solution for 10 min inside a laminar hood to make those free from dust or any other external contaminations.


      Figure 1. Floral induction from Cuscuta reflexa nodal explants. A. Nodal explants inoculated in MMS media supplemented with floral induction hormone/hormones. B and C. Shoot regeneration following floral induction in Cuscuta reflexa nodal explants where the MMS media was supplemented with both 2,4-D and NAA. D. Direct floral bud initiation from Cuscuta reflexa nodal explants where the MMS media was supplemented with only 2,4-D.

    3. Rinse the explants with autoclaved distilled water inside a laminar hood by brief shaking with hand.
    4. Decant the distilled water and pour 100 ml of 0.1% HgCl2 into the same beaker for surface sterilization inside the laminar hood. Surface sterile the explants for 5 min with brief shaking by hand.

  2. Inoculation of explants for floral induction
    1. Inoculate some of the explants (stuck up to 0.5 cm of 3 to 4 explants into media of one flask, Figure 1A) in the conical flask containing floral induction media-1 and inoculate some explants in floral induction media-2 (method for preparation of floral induction media-1 and -2 is described in Recipes section).
    2. Plug all the conical flasks containing the media with the explants air tight to avoid any bacterial and/or fungal contamination.
    3. Keep the conical flasks inside a tissue culture room where controlled light (1,000 lux) and temperature condition (28 ± 1 °C, 12 h dark/light period) will be maintained.
    4. Notice the proliferation of nodal region (Figure 1B) and generation of shoots and/or floral buds. It will take around 25 to 30 days to initiate floral buds.
    5. Floral induction media-1 (200 µl 2,4-D/L and 200 µl NAA/L supplemented in MS media) results in shoot regeneration following flowering initiation (Figure 1C). But floral induction media-2 (200 µl 2,4-D/L supplemented in MS media) resulted in floral bud initiation without shoot elongation (Figure 1D).

Data analysis

The experiment has been repeated thrice independently. All the analysis has been done manually or visually.

Notes

  1. Nodal region of the plant should be taken as explants as from the nodal region new callus or branch will develop quickly in comparison with the other parts.
  2. Hands should be washed and sterilized (with 70% ethanol) properly during the whole experimental process.
  3. Hormones should be filter sterilized before use.
  4. Floral induction media should be prepared in the conical flasks or jam bottles instead of Petri plates to get some space for the development of vine shoots.
  5. Surface sterilization of the explants should not exceed more than 10 min as it may cause tissue damage.
  6. Light, temperature and humidity should be maintained properly in the culture room.
  7. If bacterial or fungal contamination is observed on the media surface, the explants should be transferred to new flasks/jam bottles containing fresh media.
  8. 2,4-D and NAA can also be added before autoclaving of media. But if filter sterilized, it can be added after autoclaving of media. The media need not to be pre-cooled for adding hormones.

Recipes

  1. MMS media (modified MS media containing basal MS media with vitamins of Gamborg’s media [Gamborg et al., 1968])
    1. Weigh 4.4 mg of readily available MS powder
    2. Dissolve 4.4 mg of MS powder in 1 L of distilled water. And then add 30 g of sucrose to 1 L of MS solution
    3. Add 1 ml of Gamborg’s vitamin solution to 1 L of MS basal solution
    4. Adjust pH to 5.8 with 1 N KOH
    5. Add 8 g of agar to the media and autoclave for 15 min at 121 °C
  2. 2,4-D stock solution
    Dissolve 10 mg of 2,4-D in 200 µl of ethanol
    Add 800 µl of distilled water to get 1 ml final volume
    Then filter sterilize.
  3. NAA stock solution
    Dissolve 10 mg of NAA in 200 µl of 1 N NaOH
    Add 800 µl of distilled water to get 1 ml final volume
    Then filter sterilize
  4. Floral induction media-1
    Add 2 mg of filter sterilized 2,4-D and 2 mg of NAA to 1 L of MMS media after autoclaving
  5. Floral induction media-2
    Add 2 mg of filter sterilized 2,4-D to 1 L of MMS media after autoclaving
  6. HgCl2 (0.1%)
    Dissolve 0.1 mg HgCl2 in 100 ml of autoclaved distilled water
  7. Bavistin solution (1%)
    Dissolve 1 g of Bavistin in 100 ml of autoclaved distilled water

Note: All the solutions can be freshly prepared or stored solutions (at 4 °C, stable for 30 days).

Acknowledgments

Authors are thankful to Department of Botany, Utkal University, Govt. of Odisha for the financial support. Authors acknowledge previous work published by Das et al. (2011), from which this protocol is adapted.

References

  1. Bakos, Á., Fári, M., Toldi, O. and Lados, M. (1995). Plant regeneration from seedling-derived callus of dodder (Cuscuta trifolii Bab. et Giggs). Plant Sci 109(1): 95-101.
  2. Baldev, B. (1962). In vitro studies of floral induction on stem apices of Cuscuta reflexa Roxb. - a short-day plant. Ann Bot 26(2): 173-174.
  3. Das, P., Kar, M. and Sahoo, S. (2011). In vitro hormone-regulated growth and floral induction of Cuscuta reflexa: a parasitic angiosperm. Acta Physiol Plant 33(3): 1031-1035.
  4. Furuhashi, K. (1991). Establishment of a successive culture of an obligatory parasitic flowering plant, Cuscuta japonica, in vitro. Plant Sci 79(2): 241-246.
  5. Gamborg, O. L., Miller, R. A. and Ojima, K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1): 151-158.
  6. Hibberd, J. M., Bungard, R. A., Press, M. C., Jeschke, W. D., Scholes, J. D. and Quick, W. P. (1998). Localization of photosynthetic metabolism in the parasitic angiosperm Cuscuta reflexa. Planta 205(4): 506-513.
  7. Kuijt, J. (1969). Cuscuta, Cassytha, Lenoaceae, and kramereaceae. In: Kuijt, J. (Ed). The Biology of parasitic flowering plants. University of California Press, pp 136-143.
  8. Maheshwari, P. and Baldev, B. (1961). Artificial production of buds fom the embryos of Cuscuta reflexa. Nature 191: 197-198.
  9. Murashige, T. and Skoog F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3): 473-497.
  10. Nun, N. B. and Mayer, A. M. (1999). Culture of pectin methylesterase and polyphenoloxidasein Cuscuta campestris. Phytochemistry 50(5): 719-727.

简介

被子植物的花卉引发和发育是植物产量依赖的关键步骤。有时,外部气候或任何非生物胁迫阻碍花卉开始,最终影响植物产量。因此,体外花卉诱导和发育可以帮助克服外部气候因素。此外,尚未报道如在睾丸反射中的寄生被子植物体外花诱导的方案。我们已经规定了寄生植物茧丝反射中花诱导的方案。在本研究中,确定补充有2mg L NAA(萘乙酸,植物生长调节剂)和2mg L -1的MMS(改良的Murashige Skoog)培养基> 2,4-D(2,4-二氯苯氧基丙酸,植物生长调节剂)在体外拍摄后支持花诱导。此外,我们发现补充有2mg L-sup-2,4-D的MMS培养基从淋巴结外植体直接快速诱导花蕾,而没有任何芽伸长。该协议将有助于研究人员在其他被子植物植物中诱导花along与镰刀菌反射。

背景 睾丸反射是寄生在大量被子植物上寄生的被子植物(Kuijt,1969)。在宿主植物中造成严重损害和产量损失的能力使得该物种对科学研究非常重要(Nun and Mayer,1999)。除了少数具有功能性叶绿体(Hibberd等人,1998)之外,大多数Cuscuta 物种是非叶绿体。从较大的胚胎开发幼苗。已经报道了白色介质中的反射(Maheshweri和Baldev,1961)。花卉诱导。反射炎在短时间内以及黑暗条件已经在体外中看到(Baldev,1962)。 Furuhashi等人(1991)也已经在短时间条件下进行猕猴桃的诱导体外诱导[em]。
 具体来说,花诱导和生长调节剂对花诱导的影响。 reflexa 尚未被研究。迄今为止已经报道了液体MS培养物中猕猴桃的完整组织培养系统(Bakos等人,1995)。花卉诱导在C.在改良的白色培养基上,对植物进行不同的光照和黑暗条件的反射,已经在几年前进行了研究(Baldev,1962)。在这里,我们已经展示了一种完整的体外文化体系,用于在C. reflexa 。 2,4-D的浓度在花诱导中起重要作用。补充NAA与2,4-D诱导芽后接花,但只补充2,4-D直接从淋巴结外植体诱导花,而不显示任何茎伸长。这个结果是非常有吸引力的,并且显示2,4-D在这种植物的花诱导中的重要性。

关键字:大花菟丝子, 体外, 花诱导, NAA, 2,4-D

材料和试剂

  1. 移液器提示(Tarsons)
  2. 5 ml注射器(Himedia)
  3. 0.45微米过滤器(Himedia)

  4. 的茎外植体
  5. Murashige& Skoog(MS)基础培养基(Murashige和Skoog,1962)(Sigma-Aldrich,目录号:M5519)
  6. 蔗糖(Sigma-Aldrich,目录号:S0389)
  7. Gambrog的维生素溶液(Sigma-Aldrich,目录号:G1019)
  8. 琼脂(HiMedia Laboratories,目录号:PCT0901)
  9. 2,4-二氯苯氧基乙酸(2,4-D)(Sigma-Aldrich,目录号:D7299)
  10. 萘乙酸(NAA)(Sigma-Aldrich,目录号:N0640)
  11. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:221465)
  12. 氯化汞(II)(HgCl 2)(0.01%)(EMD Millipore,目录号:104419)
  13. 巴维司汀(1%溶液,斯瓦特农药)
  14. 乙醇(EMD Millipore,目录号:100983)
  15. MMS(改良Murashige Skoog)媒体(见食谱)
  16. 2,4-D储备溶液(参见食谱)
  17. NAA储备溶液(见配方)
  18. 花卉诱导培养基-1(见食谱)
  19. 花卉诱导培养基-2(参见食谱)
  20. HgCl 2(0.1%)(参见食谱)
  21. 巴伐他汀溶液(1%)(见配方)

设备

  1. 0.5-10μl移液器(Transferpette ®)(BRAND,目录号:704770)
  2. 20-200μl移液器(Transferpette ®)(BRAND,目录号:704778)
  3. 100-1,000μl移液管(Transferpette ®)(BRAND,目录号:704780)
  4. 烧杯(200毫升,Borosil)
  5. 层层罩(Thermo Fisher Scientific)
  6. 锥形烧瓶(150毫升,Borosil)
  7. 文化室(Daihan LabTech India,型号:LGC-S201)
  8. 高压灭菌器
  9. 镊子(ACE Surgical Supply Company,目录号:20-000-48)
  10. 烤箱(Hicon India)
  11. 水蒸馏装置(Mars Scientific Instruments,目录号:BASIC/pH4& XL)
  12. pH计(CD Hightech,型号:APX 175E)
  13. 称重机(Sartorius,型号:BSA224S-CW)
  14. 剪刀(一般切割剪刀)

程序

  1. 外表面灭菌
    1. 以九重葛植物天然生长的镰刀菌反刍植物(2个月大)的节点区域(图1A)为原料。
    2. 将一个小块(约20片放在这里)的茧丝反射花瓶内的节茎外植体(1-2厘米长,图1A)。将100毫升1%巴维丝汀溶液中的外植体在层流罩内处理(浸没)10分钟,使其免于灰尘或任何其他外部污染物。


      图1.来自Cuscuta reflexa淋巴结外植体的花卉诱导。 A。接种在补充有花诱导激素/激素的MMS培养基中的结节外植体。 B和C.在Cuscuta reflexa nodal外植体中花诱导后拍摄再生,其中MMS培养基用2,4-D和NAA补充。 D.从Cuscuta reflexa 淋巴细胞外植体直接花芽开始,MMS培养基仅补充2,4-D。

    3. 用手轻轻晃动,用层压罩内的蒸压蒸馏水冲洗外植体。
    4. 将蒸馏水倾倒并将100ml 0.1%HgCl 2加入到同一烧杯中,以在层流罩内进行表面消毒。用手短暂摇动将外植体表面无菌5分钟。

  2. 接种外植体进行花卉诱导
    1. 接种含有花诱导培养基-1的锥形瓶中的一些外植体(将高达0.5cm的3至4个外植体插入一个烧瓶的培养基(图1A)),并在花诱导培养基-2中接种一些外植体(制备方法花卉诱导培养基-1和-2在食谱部分描述)
    2. 将包含培养基的所有锥形瓶与外植体气密密封,以避免任何细菌和/或真菌污染
    3. 将锥形瓶保存在组织培养室内,保持受控光(1,000 lux)和温度条件(28±1℃,12小时黑暗/光照期)。
    4. 注意节点区域的增殖(图1B)和芽和/或花芽的生成。花大约25到30天才能发芽。
    5. 花芽诱导培养基-1(200μl2,4-D/L和200μl在MS培养基中补充的NAA/L)导致开花后开始再生(图1C)。但是花诱导培养基-2(在MS培养基中补充的200μl2,4-D/L)导致花芽起始而没有芽伸长(图1D)。

数据分析

实验重复三次独立。所有的分析都是手动或视觉的。

笔记

  1. 植物的结节区域应该被视为外淋巴结,与其他部位相比,新的愈伤组织或分支将迅速发展。
  2. 在整个实验过程中,手应适当洗涤和灭菌(含70%乙醇)。
  3. 激素在使用前应进行过滤灭菌。
  4. 花芽诱导培养基应在锥形瓶或果酱瓶中制备,而不是培养皿,以获得发芽的一些空间。
  5. 外植体的表面灭菌不应超过10分钟,因为它可能导致组织损伤。
  6. 光,温湿度应保持在文化室内。
  7. 如果在培养基表面观察到细菌或真菌污染,外植体应转移到含新鲜培养基的新烧瓶/果酱瓶中。
  8. 2,4-D和NAA也可以在高压灭菌之前添加。但如果过滤灭菌,可以在高压灭菌后添加。媒体不需要预先加热激素

食谱

  1. MMS媒体(含有基质MS培养基的修饰MS培养基与Gamborg氏培养基的维生素[Gamborg等人,1968])
    1. 称重4.4毫克容易获得的MS粉末
    2. 将4.4mg MS粉末溶于1L蒸馏水中。然后向1升MS溶液中加入30克蔗糖
    3. 向1升MS基础溶液中加入1毫升Gamborg维生素溶液
    4. 用1N KOH将pH调节至5.8
    5. 将8g琼脂加入到培养基中,并在121℃下高压灭菌15分钟
  2. 2,4-D库存解决方案
    将10毫克2,4-D溶于200微升乙醇中 加入800μl蒸馏水,得到1 ml的最终体积 然后过滤消毒。
  3. NAA库存解决方案
    将10毫克NAA溶于200微升1N NaOH中 加入800μl蒸馏水,得到1 ml的最终体积 然后过滤灭菌
  4. 花卉诱导培养基-1
    在高压灭菌后,向1升MMS培养基中加入2毫升过滤灭菌的2,4-D和2毫克的NAA
  5. 花卉诱导媒体-2
    高压灭菌后,加入2毫升过滤灭菌的2,4-D至1升MMS培养基
  6. HgCl 2(0.1%)
    将0.1mg HgCl 2溶解于100ml的高压灭菌蒸馏水中
  7. 巴伐他汀溶液(1%)
    将1g巴伐他汀溶解在100ml的高压灭菌蒸馏水中

注意:所有的溶液都可以是新鲜的或储存的溶液(4℃,稳定30天)。

致谢

作者感谢Utkal大学植物学系。的奥迪沙财政支持。作者承认Das等人发表的以前的作品。 (2011),从该协议适应。

参考文献

  1. Bakos,Á。,Fári,M.,Toldi,O.and Lados,M。(1995)。< a class ="ke-insertfile"href ="http://www.sciencedirect.com/science/article/pi_/016894529504152K"target ="_ blank">来自幼苗衍生的愈伤组织的砧木植物再生(> C ii ii Bab Bab Bab>>>>>。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。。 (1):95-101。
  2. Baldev,B.(1962)。  Cuscuta reflexa Roxb的茎尖上的花诱导的体外研究。 - 一个短暂的工厂。 Ann Bot 26(2):173-174。
  3. Das,P.,Kar,M。和Sahoo,S。(2011)。 体外睾丸反射的激素调节生长和花诱导:寄生被子植物 Acta Physiol植物 33(3):1031-1035。
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引用:Das, P. and Sahoo, S. (2017). In vitro Floral Induction of Cuscuta reflexa. Bio-protocol 7(2): e2104. DOI: 10.21769/BioProtoc.2104.
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