Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

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

BJ Bettina Johst
MR María del Carmen Dacasa Rüdinger

Published: Jul 20, 2018 DOI: 10.21769/BioProtoc.2939 Views: 4366

download pdf Download PDF
ask Ask a question
Favorite
Cited by

Abstract

This protocol presents a method to test the effect of sorbitol in vitro on hybrid larch plants derived from somatic embryogenesis. We have tested four different media variants, one control variant without sorbitol and three variants of decreasing water potential corresponding to sorbitol concentrations in the culture medium of 4%, 10%, and 20%. We cultured two hybrid larch clones on these media during 35 days and assessed their vitality and weight growth after this time.

Keywords: In vitro stress test search Larch search Tree breeding search Sorbitol search

Background

Forest tree breeding can benefit from methods that permit a high-throughput screening of plant lines in early stages of its development. Such methods help to delimitate the number of genotypes to be tested later in the field, an issue that in case of forest tree species requires considerable amounts of space. One possibility is to test young plantlets in vitro, a procedure that has the additional advantage of facilitating homogeneous and well-controlled experimental conditions. Aspects of tolerance against biotic and abiotic stress factors can be for instance screened in vitro. Among them, drought stress is receiving special attention in improvement programs. A common way to mimic water deficit is to add an osmoticum to the root supporting medium which reduces water availability. The osmoticum can be ionic like NaCl or non-ionic like mannitol, sorbitol or PEG (polyethylene glycol) (Singh and Singh, 2015). We have developed a protocol that allows testing the effect of sorbitol in vitro on hybrid larch emblings derived from somatic embryogenesis.

Materials and Reagents

  1. Filter paper (90 x 90 mm) (sterile)
  2. Aluminum foil
  3. Weighing pan
  4. Disposable syringe (20 ml DiscarditTM 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. GelriteTM (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

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant physiology > Plant growth

Plant Science > Plant physiology > Abiotic stress

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

0/150

tip Tips for asking effective questions

+ Description

Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.

post Post a Question
0 Q&A