Isolating and Measuring the Growth and Morphology of Pro-embryogenic Masses in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)
灯台南洋杉(南洋杉科)中原胚团的分离及生长和形态测定   

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PLOS ONE
Apr 2016

 

Abstract

Embryogenic suspension cultures of Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae) can be used as a model to test the effects of compounds added to the culture medium on the cellular growth and morphology of Pro-Embryogenic Masses (PEMs). PEMs are formed by embryogenic and suspensor-type cells. To measure changes in the cellular growth of embryogenic cultures, we performed sedimented cell volume (SCV) quantification, which is a non-destructive method. Morphological analysis by microscopy allowed for the observation of growth and development of PEMs and the alterations in embryogenic and suspensor-type cells. The methods used here provide an efficient means for monitoring the cellular growth of PEMs and identifying morphological changes during the development of embryogenic cultures. These studies can also be combined with biochemical and molecular analyses, such as proteomics, to further investigate embryo growth and morphology.

Keywords: Somatic embryogenesis (体细胞胚胎发生), Size (大小), Sedimented cell volume (沉降细胞体积)

Background

Silveira et al. (2006) used SCV measurements to analyze the effects of exogenous polyamines on the morphological changes of A. angustifolia PEMs and Osti et al. (2010) tested the effect of different nitric oxide donors on cellular growth and PEM morphology. Recently, Douétts-Peres et al. (2016) studied the effect of a cellular growth inhibitor on cellular growth and PEM morphology using SCV, fresh and dry weight, PEM area, and individual diameters of embryogenic-type cells, including the length and width of the suspensor-type cells. In addition, alterations to cellular growth and morphology in response to endogenous compounds, such as polyamines, nitric oxide and specific proteins have been evaluated using this method (Silveira et al., 2006; Osti et al., 2010; Douétts-Peres et al., 2016).

Materials and Reagents

  1. Falcon tube rack (Kasvi, catalog number: K30-1552 )
  2. 12-well cell culture plates - disposable (TPP, catalog number: 92012 )
  3. Manual pipette 200 µl tips (Corning, Axygen®, catalog number: T-200-Y )
  4. Manual pipette 1,000 µl tips (Corning, Axygen®, catalog number: T-1000-B )
  5. Aluminum foil
  6. Glass slides (Kasvi, catalog number: K5-7101 )
  7. Cover slips (Kasvi, catalog number: K5-2450 )
  8. Falcon tubes, 50 ml (Kasvi, catalog number: K19-0050 )
  9. Embryogenic suspension cultures of A. angustifolia, induced according to the methodology established by Steiner et al. (2005)
  10. Cellulase (Sigma-Aldrich, catalog number: 22178 )
  11. Potassium nitrate (KNO3) (Sigma-Aldrich, catalog number: V000944 )
  12. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: V000199 )
  13. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: V001861 )
  14. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: V000104 )
  15. Potassium dihydrogen phosphate (KH2PO4) (EMD Millipore, catalog number: 104873 )
  16. MnSO4·H2O (Labsynth, catalog number: S2036 )
  17. ZnSO4·7H2O (Labsynth, catalog number: S1072 )
  18. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: 31146 )
  19. Potassium iodide (Kl) (Sigma-Aldrich, catalog number: V000130 )
  20. Cobalt(II) chloride hexahydrate (CoCl2·6H2O) (Sigma-Aldrich, catalog number: V000213 )
  21. CuSO4·5H2O (Labsynth, catalog number: S1054 )
  22. Sodium molybdate dihydrate (Na2MoO4·2H2O) (Sigma-Aldrich, catalog number: M1651 )
  23. FeSO4·7H2O (Labsynth, catalog number: S1057 )
  24. Na2EDTA (Labsynth, catalog number: E2005 )
  25. Myo-inositol (Sigma-Aldrich, catalog number: I17508 )
  26. Nicotinic acid (Labsynth, catalog number: A1043 )
  27. Pyridoxine (Sigma-Aldrich, catalog number: P9755 )
  28. Thiamine (Sigma-Aldrich, catalog number: T4625 )
  29. L-glutamine (Labsynth, catalog number: G1011 )
  30. Sucrose (Labsynth, catalog number: 2731 )
  31. MSG culture medium (see Recipes)

Equipment

  1. Cell dissociation sieve-screens, 150 mesh (Sigma-Aldrich, catalog number: CD1-1KT ) sterilized by autoclave (121 °C, 30 min)
  2. Chamber flow (or its equivalent) (Pachame, model: PA 220 )
  3. Adapted glass Erlenmeyer flasks (custom-made) (Figure 1), sterilized by autoclave (121 °C, 30 min). This adjustment to the flask can be performed by a company that produces laboratory glassware, fusing a glass tube to an Erlenmeyer flask
  4. Ruler
  5. Orbital shaker (or its equivalent) (Cientec Equipamentos para Laboratório, model: CT-165 )
  6. Analytical balance (or its equivalent) (Shimadzu, model: BL3200H )
  7. Spatula sterilized by autoclave (121 °C, 30 min) (VWR, catalog number: 231-2233 )
  8. AxioPlan light microscope (Carl Zeiss, model: AxioPlan )
  9. Manual pipettes (or their equivalent) (Eppendorf, catalog numbers: 3120000062 and 3120000054 )
  10. Forced air circulation drying oven (or its equivalent) (Ethik Technology, model: 420-6D )
  11. AxioCam MRC5 digital camera (Carl Zeiss, model: AxioCam )
  12. Desktop computer


    Figure 1. Adapted Erlenmeyer flasks (100 and 50 ml) used in SCV analyses. For an adapted Erlenmeyer flask of 100 ml capacity, we used 25 ml of culture medium and 500 mg of fresh cells. For an adapted Erlenmeyer flask with 50 ml capacity, we used 10 ml of culture medium and 200 mg of fresh cells, with a ratio of 20 mg fresh cells to 1 ml of culture medium.

Software

  1. AxioVision Rel. 4.8 software (Carl Zeiss, AxioVision)

Procedure

  1. SCV analysis
    1. First, in the flow chamber, separate the embryogenic suspension cultures from the medium using the cell dissociation sieve (150 mesh screens) (Figure 2A).


      Figure 2. SCV analysis procedures. A. Separation of cells from the culture medium using the sieve; B. Sampling the cells (in terms of fresh weight) for the SCV analyses; C. Adapted Erlenmeyer flask containing the culture medium with inoculated cells; D. Transferring the cells and culture medium to the lateral side tube of the adapted Erlenmeyer flask for SCV measurement; E. Erlenmeyer flask held upright for 15 min; F and G. Details of SCV measurement using the ruler.

    2. Then, place 200 mg of culture (fresh weight) consisting of separated cells in an adapted Erlenmeyer flask (Figure 1) containing 10 ml of the culture medium containing the required testing treatments (Figures 2B and 2C).
    3. To measure the initial SCV, transfer the entire contents of the flask (culture medium and cells) to the adapted tube outside of the Erlenmeyer flask (Figure 2D). Keep upright in a Falcon tube rack (Figure 2E) for 15 min.
    4. After this time, measure the initial SCV using a ruler (Figures 2F and 2G).
    5. Later, return the entire contents (culture medium + cells) into the Erlenmeyer flask by carefully inverting to make sure to return most of the cells into the Erlenmeyer flask (Figure 2C). Keep the adapted Erlenmeyer flasks on a horizontal shaker (100 rpm in the dark at 25 ± 2 °C).
    6. To determine PEM growth, repeat the measurement procedures every three days until the PEMs reach the decline phase.

    Notes:

    1. A cellular growth curve (Figure 3) is performed to identify the phases of cellular growth of the embryogenic cultures in suspension. This identification can be used to establish the sampling points for the assessment of morphology and for biochemical studies.
    2. If an Erlenmeyer flask of 100 ml capacity is used, we suggest the following proportion: 25 ml of culture medium used for every 500 mg of fresh cells.


      Figure 3. Graphic representation of the cellular growth by SCV. A cellular growth curve by SCV analysis from embryogenic suspension cultures of A. angustifolia in MSG culture medium showing the different phases during incubation.

  2. Fresh and dry weight measurement
    1. Use 12-well cell culture plates for this analysis.
    2. In a flow chamber, separate the cells from the suspension cultures using a sieve to collect only the cells; discard the culture medium (Figure 2A).
    3. Next, use the analytical balance (Figure 2B) to weigh the cells and place 60 mg fresh cells into each well of the cell culture plate, which should contain 2 ml of culture medium per well (Figure 4A).
    4. Close the cell culture plates and keep on a horizontal shaker for incubation (100 rpm in the dark at 25 ± 2 °C).
    5. To measure the fresh weight on specific days (established by the growth curve, for example), take the cell culture plate and carefully remove the liquid culture medium using a pipette (Figure 4B). Repeat this procedure for each individual well.
    6. Use a spatula to collect all the solid contents of the cell culture from each well (Figure 4C), separately, on a piece of pre-weighed aluminum foil.
    7. Measure the fresh weight of cells using an analytical balance (Figure 4D). Dry the samples at 70 °C for 48 h. Then, measure the samples plus aluminum foil again on the analytical balance. It is necessary to subtract the weight of the aluminum foil (pre-weighed) to obtain the dry weight.


      Figure 4. Measurement of fresh weight using 12-well cell culture plates. A. 12-well cell culture plates with cellular culture; B. Removal of liquid culture medium using a pipette; C. Collection of the cells from each well using a spatula; D. Measurement of the fresh weight with an analytical balance using pre-weighed aluminum foil.

  3. Slide preparation for PEM morphology studies
    1. First, in the flow chamber, separate the embryogenic suspension cultures from the medium using the cell dissociation sieve (150 mesh screens) (Figure 2A).
    2. Then, 1,200 mg of separated cells, fresh weight (Figure 2B), should be placed into an Erlenmeyer flask (250 ml capacity) containing 60 ml of the culture medium containing the required testing treatments. Three flasks for each treatment should be maintained for three biological replicates.
    3. Place the flasks on a horizontal shaker during incubation (100 rpm in the dark at 25 ± 2 °C).
    4. For analyzing the incubation results on specific days, in a flow chamber, take a sample of PEMs from the liquid culture medium using the modified pipette tip. It is necessary to remove the end of the pipette tip (Figures 5A and 5B) to prevent the disruption of PEMs. The pipette tips must be sterilized before use.
    5. Put a drop of the PEM culture on a glass slide (Figure 5C), and cover with a cover slip.
    6. Observe under an optical microscope.

    Notes:

    1. The remaining cells in the flask could be returned to the horizontal shaker for use on the next day of analysis. If you do not continue the incubation, and the cells will be discarded, it is not necessary to use a sterilized pipette tip for sampling.
    2. The PEMs can alternatively be incubated in an Erlenmeyer flask with a smaller volume (keeping the ratio of 20 mg fresh cells for 1 ml of culture medium).


      Figure 5. Slide preparation of PEMs from embryogenic suspension culture. A. Removal of the end of a tip; B. Comparison of the tip before and after cutting; C. Plating with a drop of PEMs in suspension culture.

  4. Slide preparation for isolated embryogenic and suspensor-type cells
    1. First, dissociate the PEMs to obtain embryogenic and suspensor-type cells by incubating the PEM suspension culture in culture medium with 0.1% (w/v) cellulase (Sigma-Aldrich).
    2. Keep the samples incubating in cellulase for 3 h on a rotary shaker (100 rpm) in the dark at 25 ± 2 °C.
    3. After incubation, transfer the cells to a Falcon tube and wash with a new culture medium that is cellulose free. Remove the culture medium carefully with a pipette and repeat this procedure three times.
    4. To visualize the cells with an optical microscope, follow the procedure described in Procedure C used to prepare slides using a modified pipette tip.

  5. Image capture
    1. To capture images for morphological analysis, we use AxioVision Rel. 4.8 software according to the following procedures (other comparable image analysis software should suffice):
      1. Open the software AxioVision Rel. 4.8, and in the Tool Bar, select: View>Toolbars>Standard.
      2. To view the image on the screen: click the ‘Live’ option.
      3. Set in the software the specifications of the objective and ocular you are using to capture the image using the ‘Snap’ option.
      4. When saving the image, choose the .zvi format.

  6. Procedure for morphological measurement and analysis
    1. Area measurement
      1. Open the image in the microscope software.
      2. Choose the options: View>Toolbars>Measure.
      3. To measure the area, use the tool options, ‘outline’ or ‘outline spline’.
      4. Then, draw a closed shape around the embryonal head (Figures 6A and 7). When the form is closed, the area appears on the figure in µm2. This value is used for area analyses.
      5. Repeat this operation with several samples of embryonal head and suspensor-type cell images.


        Figure 6. Measurement scheme of PEMs and isolated cells. A. Area analysis of embryonal head and suspensor-type cells from non-dissociated PEMs; B. Diameter, length and width analysis of dissociated embryogenic and suspensor-type cells.


        Figure 7. Area measurement procedure for non-dissociated PEMs using AxioVision software. Arrows (red) indicate the tool options used.

    2. Diameter, width and length measurement
      1. Open the image in the microscope software.
      2. Choose the options: View>Toolbars>Measure
      3. To measure the area, use the tool option ‘length’.
      4. Then, using the ‘length’ tool, measure the embryogenic-type cells to obtain the diameter data (see red line in Figure 6B and black line in Figure 8). 
      5. Using the ‘length’ tool, measure the length and width in suspensor-type cells, as shown by a black line in Figure 8.
      6. Repeat this operation with several samples of dissociated embryogenic and suspensor cells.

    Notes:

    1. In Araucaria, as embryogenic-type cells are isodiametric, the size was measured based on the diameter, and as suspensor-type cells are elliptic and elongated, the size was measured using the length and width (at the middle of the cell) (Figure 8).
    2. If the suspensor-type cells are curved, the ‘curve spline’ tool can be used as shown in Figure 9.


      Figure 8. Measurement of diameter, width and length of embryogenic and suspensor-type cells using AxioVision LE software. The red arrow indicates the tool used for A. diameter analysis of embryogenic-type cells, B. length and C. width in suspensor-type cells.


      Figure 9. Width and length measurements of suspensor-type cells using AxioVision software. The red arrow indicates the ‘Curve spline’ tool used for length of curved suspensor-type cells, and the black arrow indicates the ‘Length’ tool used for width of these cells. 

Data analysis

  1. For the analysis of area, diameter, length and width, the values of different samples can be processed and presented in a graph format. Additionally, the data can be subjected to analysis of variance (ANOVA) and mean separation tests (such as Tukey or SNK) by using an appropriate statistical software.
  2. SCV analysis: examples of results obtained from SCV analysis can be observed in Figure 1 of Silveira et al. (2006), Figure 2 of Osti et al. (2010) and Figure 4 of Douétts-Peres et al. (2016).
  3. Fresh and dry weight analysis: Figure 5 of Douétts-Peres et al. (2016) is an example of fresh and dry weight measurement.
  4. Cell size determination: Area measurement of the embryonal head and suspensor-type cells described here (Figure 6) can be observed in Figure 7 of Douétts-Peres et al. (2016). In addition, the diameter of embryogenic-type cells and the width and length of suspensor-type cells can be observed in Figure 8 of Douétts-Peres et al. (2016).

Recipes

  1. MSG culture medium (1 L) (Becwar et al., 1989)


    1. Preparation of liquid MSG culture medium
      1. To prepare 1 L of liquid MSG culture medium, weigh 30 g of sucrose and dissolve in ~500 ml of distilled water.
      2. Add 100 ml solution A, 5 ml solution B, 10 ml solution C and 2 ml solution D. Add 1,460 mg of L-glutamine.
      3. Dissolve all reagents. Adjust the volume with distilled water to 1 L. Mix the culture medium and adjust the pH to 5.7. Sterilize in an autoclave for 15 min at 121 °C.
    2. Preparation of stock solutions
      1. To prepare stock solutions A, B and D, weigh the reagents in an analytical balance, dissolve in distilled water, and make up the final volume with distilled water.
      2. To prepare stock solution C, weigh each reagent and add separately to ~150 ml of heated distilled water (~45 °C). Then, mix the two solutions and adjust the final volume to 500 ml. Protect this solution from light.








Acknowledgments

This work was supported by FAPERJ (Foundation for Research Support of the State of Rio de Janeiro) and CNPq (National Counsel of Technological and Scientific Development). The VCS method presented was derived from Silveira et al. (2006).

References

  1. Becwar, M. R., Noland, T. L. and Wyckoff, J. L. (1989). Maturation, germination, and conversion of Norway spruce (Picea abies L.) somatic embryos to plants. In vitro Cell Dev 25(6): 575-580.
  2. Douétts-Peres, J. C., Cruz, M. A., Reis, R. S., Heringer, A. S., de Oliveira, E. A., Elbl, P. M., Floh, E. I., Silveira, V. and Santa-Catarina, C. (2016). Mps1 (Monopolar Spindle 1) protein inhibition affects cellular growth and pro-embryogenic masses morphology in embryogenic cultures of Araucaria angustifolia (Araucariaceae). PLoS One 11(4): e0153528.
  3. Osti, R. Z., Andrade, J. B. R., Souza, J. P., Silveira, V., Balbuena, T. S., Guerra, M. P., Franco, D. W., Floh, E. I. S. and Santa-Catarina, C. (2010). Nitrosyl ethylenediaminetetraacetate ruthenium(II) complex promotes cellular growth and could be used as nitric oxide donor in plants. Plant Sci 178(5): 448-453.
  4. Silveira, V., Santa-Catarina, C., Tun, N. N., Scherer, G. F. E., Handro, W., Guerra, M. P. and Floh, E. I. S. (2006). Polyamine effects on the endogenous polyamine contents, nitric oxide release, growth and differentiation of embryogenic suspension cultures of Araucaria angustifolia (Bert.) O. Ktze. Plant Sci 171(1): 91-98.
  5. Steiner, N., Vieira, F. D. N., Maldonado, S. and Guerra, M. P. (2005). Effect of carbon source on morphology and histodifferentiation of Araucaria angustifolia embryogenic cultures. Braz Arch of Bio Technol 48(6): 895-903.

简介

可以使用南洋杉(Araucaria angustifolia)(Bertol。)Kuntze(Araucariaceae)的胚胎悬浮培养物作为模型来测试添加到培养基中的化合物对前胚胎形成质量的细胞生长和形态的影响PEM)。 PEM由胚胎形成和悬浮细胞形成。为了测量胚胎发生培养物的细胞生长的变化,我们进行沉降细胞体积(SCV)定量,其是非破坏性方法。通过显微镜进行的形态学分析允许观察PEM的生长和发育以及胚胎形成和悬浮体型细胞的改变。这里使用的方法提供了一个有效的手段监测PEM的细胞生长和识别胚胎发育培养发展过程中的形态变化。这些研究还可以与生物化学和分子分析例如蛋白质组学结合,以进一步研究胚胎生长和形态。
关键词:体细胞胚胎发生,大小,沉淀细胞体积
< br /> [背景] Silveira 等。 (2006)使用SCV测量来分析外源多胺对A的形态学变化的影响。 angustifolia PEMs和Osti等人 。 (2010)测试不同一氧化氮供体对细胞生长和PEM形态的影响。最近,Douétts-Peres等人。 (2016)研究了细胞生长抑制剂对细胞生长和PEM形态的影响,使用SCV,新鲜和干重,PEM面积和胚胎型细胞的个体直径,包括悬挂型细胞的长度和宽度。此外,已经使用该方法评价了对内源化合物(例如多胺,一氧化氮和特异性蛋白质)的细胞生长和形态的改变(Silveira等人,2006; Osti等人Al ,2010;Douétts-Peres 。,2016)。

关键字:体细胞胚胎发生, 大小, 沉降细胞体积

材料和试剂

  1. Falcon管架(Kasvi,目录号:K30-1552)
  2. 12孔细胞培养板 - 一次性(TPP,目录号:92012)
  3. 手动移液器200μl提示(Corning,Axygen ®,目录号:T-200-Y)
  4. 手动移液器1000μl提示(Corning,Axygen ®,目录号:T-1000-B)
  5. 铝箔
  6. 玻璃载玻片(Kasvi,目录号:K5-7101)
  7. 盖玻片(Kasvi,目录号:K5-2450)
  8. Falcon管,50ml(Kasvi,目录号:K19-0050)
  9. 胚胎发生悬浮培养物。 angustifolia,根据Steiner等人建立的方法诱导的 。 (2005)
  10. 纤维素酶(Sigma-Aldrich,目录号:22178)
  11. 硝酸钾(KNO 3)(Sigma-Aldrich,目录号:V000944)
  12. 氯化钙二水合物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:V000199)
  13. 硫酸镁七水合物(MgSO 4·7H 2 O)(Sigma-Aldrich,目录号:V001861)
  14. 氯化钾(KCl)(Sigma-Aldrich,目录号:V000104)
  15. 磷酸二氢钾(KH 2 PO 4)(EMD Millipore,目录号:104873)
  16. MnSO 4 H·H 2 O(Labsynth,目录号:S2036)
  17. ZnSO 4 7H·7H 2 O(Labsynth,目录号:S1072)
  18. 硼酸(H 3 BO 3)(Sigma-Aldrich,目录号:31146)
  19. 碘化钾(K1)(Sigma-Aldrich,目录号:V000130)
  20. 氯化钴(II)六水合物(CoCl 2·6H 2 O)(Sigma-Aldrich,目录号:V000213)
  21. CuSO 4 H 5·5H 2 O(Labsynth,目录号:S1054)
  22. 钼酸钠二水合物(Na 2 MoO 4·2H 2 O)(Sigma-Aldrich,目录号:M1651)
  23. FeSO 4 7HH 2 O(Labsynth,目录号:S1057)
  24. Na 2 EDTA(Labsynth,目录号:E2005)
  25. 肌醇(Sigma-Aldrich,目录号:I17508)
  26. 烟酸(Labsynth,目录号:A1043)
  27. 吡哆醇(Sigma-Aldrich,目录号:P9755)
  28. 硫胺素(Sigma-Aldrich,目录号:T4625)
  29. L-谷氨酰胺(Labsynth,目录号:G1011)
  30. 蔗糖(Labsynth,目录号:2731)
  31. MSG培养基(参见配方)

设备

  1. 通过高压灭菌器(121℃,30分钟)灭菌的细胞离解筛网(150目(Sigma-Aldrich,目录号:CD1-1KT))
  2. 室流(或其等效物)(Pachame,型号:PA 220)
  3. 适配的玻璃锥形瓶(定制的)(图1),通过高压灭菌器(121℃,30分钟)灭菌。对烧瓶的这种调节可以由生产实验室玻璃器皿的公司进行,将玻璃管熔合到锥形烧瓶。
  4. 标尺
  5. 轨道振动器(或其等同物)(Cientec Equipamentos paraLaboratório,型号:CT-165)
  6. 分析天平(或其等价物)(Shimadzu,型号:BL3200H)
  7. 通过高压灭菌器(121℃,30分钟)灭菌的小铲(VWR,目录号:231-2233)
  8. AxioPlan光学显微镜(Carl Zeiss,型号:AxioPlan)
  9. 手动移液器(或等效物)(Eppendorf,目录号:3120000062和3120000054)
  10. 强制空气循环干燥炉(或其等同物)(Ethik Technology,型号:420-6D)
  11. AxioCam MRC5数码相机(Carl Zeiss,型号:AxioCam)
  12. 桌面计算机


    图1.用于SCV分析的改型Erlenmeyer烧瓶(100和50ml)。对于100ml容量的改造的Erlenmeyer烧瓶,我们使用25ml培养基和500mg新鲜细胞。对于具有50ml容量的改造的锥形瓶,我们使用10ml培养基和200mg新鲜细胞,比例为20mg新鲜细胞与1ml培养基。

软件

  1. AxioVision Rel。 4.8软件(Carl Zeiss,AxioVision)

程序

  1. SCV分析
    1. 首先,在流动室中,使用细胞离解筛(150目筛)从培养基中分离胚胎发生悬浮培养物(图2A)。


      图2. SCV分析程序。 A.使用筛子从培养基中分离细胞; B.对SCV分析对细胞进行取样(以鲜重计);适用于带有接种细胞的培养基的锥形瓶; D.将细胞和培养基转移到适应的Erlenmeyer烧瓶的侧面管中用于SCV测量; E.锥形瓶直立保持15分钟; F和G.使用标尺进行SCV测量的详细信息。

    2. 然后,将200mg由分离的细胞组成的培养物(鲜重)置于含有10ml含有所需测试处理的培养基(图2B和2C)的适应的Erlenmeyer烧瓶(图1)中。
    3. 为了测量初始SCV,将烧瓶的全部内容物(培养基和细胞)转移到锥形瓶外面的适应管(图2D)。在Falcon管架中保持直立(图2E)15分钟。
    4. 此后,使用尺子测量初始SCV(图2F和2G)。
    5. 然后,通过小心地倒转以确保将大部分细胞返回到锥形瓶中(图2C),将全部内容物(培养基+细胞)返回到锥形瓶中。保持适应的锥形瓶在水平摇床(100 rpm在黑暗中25±2℃)。
    6. 为了确定PEM生长,每三天重复测量程序,直到PEM达到下降阶段。

    注意:

    1. 进行细胞生长曲线(图3)以鉴定悬浮的胚性培养物的细胞生长的阶段。这种鉴定可用于建立形态评估和生化研究的取样点。
    2. 如果使用100ml容量的锥形瓶,我们建议以下比例:每500毫克新鲜细胞使用25毫升培养基。


      图3.通过SCV的细胞生长的图示。通过SCV分析从胚胎发生悬浮培养的细胞生长曲线。 angustifolia 在MSG培养基中显示孵育期间的不同阶段
  2. 新鲜和干重测量
    1. 使用12孔细胞培养板进行此分析。
    2. 在流动室中,使用筛子从悬浮培养物中分离细胞以仅收集细胞;丢弃培养基(图2A)
    3. 接下来,使用分析天平(图2B)称重细胞并将60mg新鲜细胞置于细胞培养板的每个孔中,每孔应含有2ml培养基(图4A)。
    4. 关闭细胞培养板并保持在水平振荡器上孵育(在25±2℃下在黑暗中100rpm)。
    5. 为了测量特定天的鲜重(例如通过生长曲线确定),取细胞培养板并使用移液管小心地移除液体培养基(图4B)。对每个井重复此过程。
    6. 使用刮刀从每个孔中收集细胞培养物的所有固体成分(图4C),单独地,在一块预先称重的铝箔上。
    7. 使用分析天平测量细胞的鲜重(图4D)。在70℃下干燥样品48小时。然后,在分析天平上再次测量样品加上铝箔。必须减去铝箔的重量(预称重)以获得干重。


      图4.使用12孔细胞培养板测量鲜重。A:具有细胞培养物的12孔细胞培养板; B.使用移液管去除液体培养基; C.使用刮刀从每个孔收集细胞; D.使用预称重的铝箔,用分析天平测量鲜重。


  3. 的幻灯片准备
    1. 首先,在流动室中,使用细胞解离筛(150目筛)从培养基中分离胚胎发生悬浮培养物(图2A)。
    2. 然后,将1200mg分离的细胞,鲜重(图2B)放入含有60ml含有所需试验处理的培养基的锥形烧瓶(250ml容量)中。每个处理的三个烧瓶应保持三次生物学重复
    3. 在孵育期间将培养瓶置于水平摇床上(100rpm,在25±2℃的黑暗中)
    4. 为了分析在特定天的孵育结果,在流动室中,从改进的移液管尖从液体培养基中取得PEM的样品。必须移除移液管尖端的末端(图5A和5B),以防止PEM的破裂。移液器吸头在使用前必须进行灭菌。
    5. 将一滴PEM培养物放在载玻片上(图5C),并用盖玻片覆盖。
    6. 在光学显微镜下观察。

    注意:

    1. 可以将烧瓶中的剩余细胞返回到水平摇动器,以在分析的第二天使用。如果不继续孵育,细胞将被丢弃,则无需使用消毒的移液器吸头进行取样。
    2. 或者,可以在具有较小体积(保持20mg新鲜细胞与1ml培养基的比例)的锥形瓶中孵育PEM。



      图5.从胚胎发生悬浮培养物中制备PEM的载玻片。 A.去除尖端的末端; B.切割前后刀尖的比较; C.用悬浮培养物中的一滴PEM涂覆。

  4. 分离的胚胎形成细胞和悬浮细胞的载玻片制备
    1. 首先,通过将培养基中的PEM悬浮培养物与
    2. 为了捕获图像进行形态分析,我们使用AxioVision Rel。 4.8软件根据以下程序(其他可比较的图像分析软件应该足够):
      1. 打开AxioVision Rel软件。 4.8,然后在工具栏中选择:查看>工具栏>标准
      2. 要查看屏幕上的图像:单击"Live"选项。
      3. 在软件中设置您使用"捕捉"选项捕获图像的目标和目标的规格。
      4. 保存图像时,选择.zvi格式。


  5. 形态测量和分析程序
    1. 面积测量
      1. 在显微镜软件中打开图像。
      2. 选择选项:查看>工具栏>测量。
      3. 要测量区域,请使用工具选项"outline"或"outline spline"。
      4. 然后,围绕胚胎头绘制闭合形状(图6A和7)。当窗体关闭时,图中的区域以μm 2 显示。此值用于区域分析。
      5. 重复这个操作几个胚胎头和悬吊型细胞图像样品

        图6.PEM和分离的细胞的测量方案 A.来自非解离的PEM的胚胎头部和悬挂器型细胞的面积分析; B.解离的胚发生和悬浮细胞的直径,长度和宽度分析

        图7.使用AxioVision软件的非离解PEM的面积测量程序箭头(红色)表示使用的工具选项。

    2. 直径,宽度和长度测量
      1. 在显微镜软件中打开图像。
      2. 选择选项:查看>工具栏>测量
      3. 要测量面积,请使用工具选项"长度"。
      4. 然后,使用"长度"工具,测量胚胎型细胞以获得直径数据(参见图6B中的红线和图8中的黑线)。
      5. 使用"长度"工具,测量悬挂器型单元格的长度和宽度,如图8中的黑线所示。
      6. 对解离的胚胎发生细胞和悬浮细胞的几个样品重复该操作

    注意:

    1. 在南洋杉中,因为胚发生型细胞是等比体的,所以根据直径测量大小,并且作为悬吊型细胞是椭圆形和伸长的,使用长度和宽度测量大小(在细胞中间)(图8)。
    2. 如果悬挂式单元格是弯曲的,则可以使用"曲线样条"工具,如图9所示。


      图8.使用AxioVision LE软件测量胚胎形成细胞和悬浮细胞型细胞的直径,宽度和长度。红色箭头表示用于胚胎形成细胞的直径分析的工具B.长度和悬挂器型细胞中的C.宽度

      图9.宽度和长度测量 使用AxioVision软件悬挂细胞型细胞。红色箭头表示 用于弯曲悬挂器型细胞长度的"曲线样条"工具, 黑色箭头表示用于这些单元格宽度的"长度"工具。

数据分析

  1. 对于面积,直径,长度和宽度的分析,可以以图形格式处理和呈现不同样品的值。另外,可以通过使用适当的统计软件对数据进行方差分析(ANOVA)和平均分离测试(例如Tukey或SNK)。
  2. SCV分析:获自SCV分析的结果的实例可以在Silveira等的图1中观察到。 (2006),Osti等人的图2。 (2010)和Douétts-Peres等人的图4。 (2016)。
  3. 新鲜和干重分析:图5的Douétts-Peres等人。 (2016)是新鲜和干重测量的一个例子
  4. 细胞尺寸测定:这里描述的胚胎头和悬吊型细胞的面积测量(图6)可以在Douétts-Peres等人的图7中观察到。 (2016年)。此外,胚胎型细胞的直径和悬吊型细胞的宽度和长度可以在Douétts-Peres等人的图8中观察到。 (2016)。

食谱

  1. MSG培养基(1L)(Becwar等人,1989)


    1. 制备液体MSG培养基
      1. 为了制备1L液体MSG培养基,称取30g蔗糖并溶解在〜500ml蒸馏水中
      2. 加入100ml溶液A,5ml溶液B,10ml溶液C和2ml溶液D.加入1,460mg L-谷氨酰胺。
      3. 溶解所有试剂。用蒸馏水调节体积至1L。混合培养基并调节pH至5.7。在高压灭菌器中在121℃灭菌15分钟
    2. 储备溶液的制备
      1. 准备储备溶液A,B和D,在分析天平中称量试剂,溶于蒸馏水中,并用蒸馏水补足最终体积。
      2. 为了制备储备溶液C,称重每种试剂,并分别加入〜150ml加热的蒸馏水(〜45℃)。然后,混合两种溶液并将最终体积调节至500ml。保护此溶液免受光照。








致谢

这项工作得到FAPERJ(里约热内卢州研究支持基金会)和CNPq(国家技术和科学发展委员会)的支持。所提出的VCS方法源自Silveira等人。 (2006)。

参考文献

  1. Becwar,MR,Noland,TL和Wyckoff,JL(1989)。  L.)体细胞胚胎到植物。体外细胞发育25(6):575- 580.
  2. Douglas-Peres,JC,Cruz,MA,Reis,RS,Heringer,AS,de Oliveira,EA,Elbl,PM,Floh,EI,Silveira,V.and Santa-Catarina, (单极纺锤体1)蛋白质抑制作用影响细胞生长和前胚胎发生的质量(Araucariaceae)的胚胎发生培养物中的形态。 11(4):e0153528。
  3. Osti,RZ,Andrade,JBR,Souza,JP,Silveira,V.,Balbuena,TS,Guerra,MP,Franco,DW,Floh,EIS和Santa-Catarina,C。(2010)。< a class = ke-insertfile"href ="http://dx.doi.org/10.1016/j.plantsci.2006.02.015"target ="_ blank">亚硝酰基乙二胺四乙酸钌(II)络合物促进细胞生长,可用作一氧化氮植物科学 178(5):448-453。
  4. Silveira,V.,Santa-Catarina,C.,Tun,NN,Scherer,GFE,Handro,W.,Guerra,MPand Floh,EIS(2006)。  多胺对内生多胺含量,一氧化氮释放,南洋杉胚胎悬浮培养物的生长和分化的影响angustifolia (Bert。)O.Ktze。 Plant Sci 171(1):91-98。
  5. Steiner,N.,Vieira,FDN,Maldonado,S.和Guerra,MP(2005)。 
  • English
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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。
Copyright: © 2016 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. Douétts-Peres, J. C., Silveira, V., Cruz, M. L. and Santa-Catarina, C. (2016). Isolating and Measuring the Growth and Morphology of Pro-embryogenic Masses in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae). Bio-protocol 6(23): e2031. DOI: 10.21769/BioProtoc.2031.
  2. Douétts-Peres, J. C., Cruz, M. A., Reis, R. S., Heringer, A. S., de Oliveira, E. A., Elbl, P. M., Floh, E. I., Silveira, V. and Santa-Catarina, C. (2016). Mps1 (Monopolar Spindle 1) protein inhibition affects cellular growth and pro-embryogenic masses morphology in embryogenic cultures of Araucaria angustifolia (Araucariaceae). PLoS One 11(4): e0153528.
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