Protocol to Test the Effect of Sorbitol in vitro on Hybrid Larch (Larix x eurolepis Henry) Emblings   

Abstract

Materials and Reagents

1. Filter paper (90 x 90 mm) (sterile)
   
2. Aluminum foil
   
3. Weighing pan
   
4. Disposable syringe (20 ml Discardit^TM II) (BD, catalog number: 300330 )
   
5. Syringe filters (0.2 μm, cellulose acetate membrane, VWR, catalog number: 514-0061 )
   
6. Glass pipettes (sterile)
   
7. 2x Scalpel (sterile)
   
8. Permanent marker
   
9. Steri Vent Container HIGH (sterile) (107 x 94 x 96 mm) with lid (sterile) (Duchefa Biochemie, catalog numbers: S1686 and S1681 respectively)
   
10. Plant material
    Note: Produced via somatic embryogenesis (larch emblings) and delivered into sterile culture medium. We used two different clones of hybrid larch and 12 ramets per medium variant. The experiment was repeated twice. A total of 192 emblings were needed.
    
11. Potassium hydroxide (KOH)
    
12. Hydrochloric acid (HCl)
    
13. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Duchefa Biochemie, catalog number: M0513 )
    
14. Potassium dihydrogenphosphate (KH₂PO₄) (Duchefa Biochemie, catalog number: P0574 )
    
15. Calcium chloride dihydrate (CaCl₂·2H₂O) (Duchefa Biochemie, catalog number: C0504 )
    
16. MS micro salt mixture (Duchefa Biochemie, catalog number: M0301 )
    
17. MS vitamin mixture (Duchefa Biochemie, catalog number: M0409 )
    
18. Calcium nitrate tetrahydrate (Ca(NO₃)₂·4H₂O) (Duchefa Biochemie, catalog number: C0505 )
    
19. Casein-Hydrolysate (Duchefa Biochemie, catalog number: C1301 )
    
20. Thiamine hydrochloride (C₁₂H₁₇ClN₄OS·HCl) (Duchefa Biochemie, catalog number: T0614 )
    
21. Sucrose (C₁₂H₂₂O₁₁) (Duchefa Biochemie, catalog number: S0809 )
    
22. Gelrite^TM (Duchefa Biochemie, catalog number: G1101 )
    
23. L-Glutamine (C₅H₁₀N₂O₃) (Duchefa Biochemie, catalog number: G0708 )
    
24. D-Sorbitol (C₆H₁₄O₆) (Duchefa Biochemie, catalog number: S0807 )
    
25. Sterile 0.2 M L-Glutamine stock solution (see Recipes)
    
26. BM5-medium (Medium composition) (see Recipes)
    
27. Sorbitol medium (see Recipes)

Equipment

1. 5x Duran glass bottle 1 L (sterile)
   
2. 3x Tweezers (sterile)
   
3. Instrument stand (sterile)
   
4. 4x Erlenmeyer flask 1 L
   
5. Volumetric flask 50 ml
   
6. Ruler (sterile)
   
7. pH meter
   
8. Magnetic stirrer
   
9. 4x Stir bars
   
10. Precision balance
    
11. Rapid sterilizer for laboratory instruments (Simon Keller, model: Steri 250, catalog number: 31100 )
    
12. Autoclave (steam sterilizer, Tuttnauer, model: 3150 EL )
    
13. Osmometer (Wescor, model: 5500 Vapor Pressure Osmometer )
    
14. Laminar flow bench (EUROCLONE, model: AURA HZ 48 )
    
15. Plant culture room (Equipped with Philips lamps, Philips, model: Master TL-D 58W/840, catalog number: 927922084023 )
    

Software

1. IBM^® SPSS^® Statistics

Procedure

1. Preparation of culture medium in four different variants of water potential
   1. Sterilize the required material.
      
   2. Prepare a sterile 0.2 M (29.2 g/L) L-Glutamine stock solution (see Recipe 1).
      
   3. Prepare four 1 L Duran glass bottles with 3 g of Gelrite^TM each.
      
   4. Prepare four 1 L Erlenmeyer flasks, each of them with all chemicals of Recipe 2 except L-glutamine and Gelrite^TM. Add sorbitol to the flasks as indicated in Recipe 3, a portion of high-purity water and a stir bar. Place the flasks on a magnetic stirrer until components are dissolved. The resulting water potential of the media is given in Recipe 3. The presence of Gelrite^TM in medium also reduces its water potential but only slightly and its effect can be neglected (George et al., 2008). Complete the volume of high-purity water in each Erlenmeyer flask to 1 L and adjust pH to a value of 5.7 using HCl or NaOH.
      
   5. Transfer adjusted media, including the stir bar to the Duran glass bottles containing 3 g Gelrite^TM and sterilize the bottles in the autoclave.
      Note: The following steps have to be conducted on a laminar flow bench previously surface sterilized. Every object to be placed on the laminar flow bench must have been previously autoclaved or surface sterilized.
      
   6. Let autoclaved medium from Step A5 slightly cool and add 25 ml/L filter sterilized L-Glutamine to each Duran glass bottle.
      
   7. Place the filter paper with the help of tweezers on the Steri Vent container lid so that approximately one centimeter jut out of the lid. Note that a very small amount of culture medium previously poured on the lid can help you to better place the filter paper onto it. Pour a small part of the culture medium onto the filter paper (Figure 1A). This step will result in a thin slice of semi-solid medium (Figure 2A). Pour the rest of 125 ml culture medium into the Steri Vent container and let it solidify. Mark each container with the corresponding sorbitol concentration.
      
      
      Figure 1. Schematic illustration of the container preparation. All steps are carried out in sterile conditions. A. Semi-solid medium slice on container lid and container with medium; B. Transfer of emblings to culture medium; C-D. Placement of cover slice; E-F. Removement of filter paper; G. Container with plants ready to be placed in the culture room.
      
   
   
2. Transfer of larch emblings to the culture medium
   1. Take larch emblings carefully out of their sterile culture medium (Figure 3A) with tweezers. Before transferring emblings to the test medium, you may want to record some parameters like weight, root length or total plant length (Figures 3B and 3C). Note the results.
      
   2. Place larch emblings on the culture medium (three per Steri Vent container) and take care of arranging the roots parallel to the longest side of the container (Figures 1B and 2C).
      
   3. Pull the filter paper by picking it with tweezers at its jutting side in order to take the slice of semi-solid medium out from the lid (Figures 1C and 2B) and cover the roots with this slice starting from the root neck (Figures 1D, 2D and 2E). Remove the filter paper (Figures 1E, 1F and 2F). Close the container with the lid (Figure 1G) and label the container with date, plant number, clone number and experiment number. If you want to record root growth, mark the start and end points of the root with a permanent marker on the container bottom (Figure 4A).
      
   4. Place the Steri Vent containers in a plant culture room for 35 days under short-day conditions (8 h light /16 h darkness), 15 μmol quanta m^-2 sec^-1 of photosynthetically active radiation and 24 °C (Figure 4B).
      
      
      Figure 2. Detailed view of larch emblings transferred to the culture medium. A. Semi-solid medium cover slice (arrow) on the container lid; B. Pulling filter paper with semi-solid slice; C. Emblings on the culture medium; D-E. Covering roots with the medium slice; F. Removement of filter paper (arrow); G-H. Successful root covering with a thin slice of semi-solid medium (arrow).
      
      
      Figure 3. Larch emblings. A. Larch emblings delivered into sterile culture medium; B-C. Weighting and measuring emblings under sterile conditions.
      
      
      Figure 4. Steri Vent containers. A. Marks on the bottom of the container. Culture medium is solid so that the container can be turned around. Nevertheless be careful with this step and do not let the container vertical for too long. B. Steri Vent containers in the plant culture room at 24 °C under short-day conditions (8 h light/16 h darkness) by 15 μmol quanta m^-2 sec^-1 of photosynthetically active radiation.
      

Data analysis

1. Rating schema for the classification of plant vitality
   We used a five-level rating schema (Figure 5) to classify plant vitality after growing larch emblings for 35 days on the different culture media. Vitality levels are defined as follows:
   Level 0 Plant fully vital with green needles
   Level 1 Plant almost fully vital with green needles and sporadic dry needles
   Level 2 Clearly affected plant with around 50% yellow to dry needles
   Level 3 Plant with clear damages and only scattered green to yellow needles
   Level 4 Dead plant
   
   
   Figure 5. Vitality phenotypes. Level 0 corresponds to a fully vital plant, level 4 to a dead plant.
   
   
2. Results
   We found a decay of plant vitality clearly associated with an increase of sorbitol concentration in medium (Figure 6).
   
   
   Figure 6. Distribution of vitality levels per clone after 35 days of growth on culture medium. The percentage of damaged plants increases proportionally to the increase of sorbitol concentration in the culture media.
   
   To determine plant weight growth, we weighted plants again after the culture period of 35 days. Whereas plant weight increased on control medium, the reduced water potential in the sorbitol medium variants led to a loss of weight as indicated by negative values of weight increase (Figure 7).
   
   
   Figure 7. Average weight increase of emblings after 35 days of growth on culture medium. Values were negative for medium with sorbitol indicating a desiccation of the plants. Bars indicate standard error.
   
   
3. Conclusion
   We expected sorbitol to diminish the water potential of culture media when added to them and cause therefore symptoms of drought stress on plants growing on these media. Our protocol permits to test this effect in vitro on plants in a very early ontogenetic stadium under sterile conditions. The results show a reduction in plant vitality and a loss of weight associated to the addition of sorbitol confirming the expectations. The use of a semi-solid medium slice to cover roots after extracting plants from one culture media and transferring them to another makes unnecessary to push roots in the second medium lowering therefore the risk of root damage during transfer, especially when these are very long. We conclude that this protocol is well suited to test the effect of sorbitol in vitro on small plants under sterile conditions and presume that it can be adapted to test other types of abiotic stress.
   

Notes

1. We tested the same protocol using 10 g agar instead of 3 g Gelrite. The resulting medium cover slice was too hard and broke easily when handled. Gelrite in low concentration results in a stable and flexible slice easy to handle and place over the roots. In addition, the cover slice with Gelrite is more transparent than with agar which eases the observation of root development. Finally, there are some reports about potential negative effects of agar on cultured tissue but we didn’t study this in our experiment.
   
2. Instead of using the ready-made MS mixtures of microelements and vitamins you can also use the individual components contained in these ready-made mixtures to prepare the BM5 medium.
   

Recipes

1. Sterile 0.2 M L-Glutamine stock solution
   1. Prepare a 0.2 M (29.2 g/L) L-Glutamine stock solution by dissolving L-Glutamine into distilled water
      
   2. Filter-sterilize the solution with the disposable syringe and the syringe filter into a sterile Duran glass bottle
2. Adapted BM5-Medium (1 L)
   
   *Filter sterilized (add after autoclaving).
   
   **BM2-5 Macro Stock solution 20x
   
   
   
3. Sorbitol medium
   Note: Sorbitol concentration and corresponding water potential.
   
   *Measured with a Wescor 5500 Vapor Pressure Osmometer
   

Acknowledgments

We thank Kurt Zoglauer and his team at the Humboldt-Universität Berlin, Department ‘Biology, Botany & Arboretum’ for delivering all in vitro plant material. We also would like to thank Ute Tröber and Ursula Franke from the Division of Forest Genetics and Forest Tree Breeding at the Wood and Forestry Competence Centre of the Public Enterprise Sachsenforst for their help in the laboratory and Marianne Kadolsky for facilitating sterile work. We are very grateful to the head of the Division Heino Wolf for lending the required unit facilities and making this project possible. This work has been supported by the German Federal Ministry of Food and Agriculture by decision of the German Bundestag (Grant No. 22034914). There are no conflicts of interest or competing interests.

References

1. George, E. F., Hall, M. A. and De Klerk, G. J. (2008). Plant propagation by tissue culture. Volume 1. The background. Berlin. Springer Science & Business Media pp: 134.
   
2. Singh, B. D. and Singh, A. K. (2015). Marker-assisted plant breeding: Principles and practices. New Delhi. Springer India pp: 514.