采用MIFE技术筛选持K+叶肉用于耐盐作物的育种

Honghong Wu; Lana Shabala; Meixue Zhou; Sergey Shabala

doi:10.21769/BioProtoc.1466

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Peer-reviewed

MIFE Technique-based Screening for Mesophyll K⁺ Retention for Crop Breeding for Salinity Tolerance

采用MIFE技术筛选持K+叶肉用于耐盐作物的育种

Honghong Wu

LS Lana Shabala

MZ Meixue Zhou

Sergey Shabala email

发布: 2015年05月05日第5卷第9期 DOI: 10.21769/BioProtoc.1466 浏览次数: 9651

评审: Tie LiuRumen Ivanov

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Cover of Plant & Cell Physiology, featuring study using the protocol.

Oct 2014

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Abstract

Potassium is known as a rate-limiting factor for crop yield and plays an important role in plants response under abiotic stresses. Recently, cytosolic K⁺ retention ability in leaf mesophyll has emerged as an important component of plant salt tolerance mechanism (Wu et al., 2013; Wu et al., 2014; Wu et al., 2015). In this protocol, the procedure for screening leaf mesophyll for K⁺ retention by the MIFE (microelectrode ion flux estimation) technique is described in detail using wheat as an example. By measuring NaCl-induced K⁺ efflux in leaf mesophyll, a large number of plant accessions can be screened and categorised according to their salinity stress tolerance. The method provides a rapid and reliable tool that targets the activity of specific membrane transporters directly contributing to salinity tolerance trait and, because of this, has a competitive advantage over traditional whole-plant phenotyping. While the focus of this protocol is on wheat, the suggested method may be adopted for screening K⁺ retention in leaf mesophyll in any other crop species.

Keywords: K+ retention (钾潴留)

Ion flux (离子通量)

Salinity stress (盐胁迫)

Mesophyll cells (叶肉细胞)

Breeding (育种)

Materials and Reagents

Two to three week old wheat seedlings
NaCl (Sigma-Aldrich, catalog number: 746398 )
KCl (Sigma-Aldrich, catalog number: 746436 )
CaCl₂ (Sigma-Aldrich, catalog number: C5670 )
K⁺ LIX (liquid ion exchanger) (Sigma-Aldrich, catalog number: 60031 )
Tributylchlorosilane (Sigma-Aldrich, catalog number: 282707 )
ddH₂O
95% ethanol (VWR International, catalog number CHESEA042-20L-P )
Commercial bleach (contains 42 g/L NaClO)
Agar (Oxoid, catalog number: LP0011 )
Parafilm
Basic salt medium (BSM) solution (mM) (see Recipes)
Backfilling solution for K⁺ ion selective microelectrode (see Recipes)
Filling solution for a reference electrode (see Recipes)
K⁺ calibration solutions (μM) (see Recipes)
Potting mix (see Recipes)

Equipment

The MIFE (microelectrode ion flux estimation) system (designed, manufactured and distributed by the University of Tasmania)
(http://www.phys.utas.edu.au/physics/biophys/mifecom/MIFEHome/Home.html)
Glasshouse
Vertical electrode puller (Narishige, model: PP-830 )
Electrode filling station (contains a three-dimensional micromanipulator and a microscope)
Laboratory fume cupboard (model: 1800)
Inverted tissue culture microscope (Radical Instruments, model: RTC-6 )
Microscope (Nikon, model: 100100 ) used in the electrode filling station
Micromanipulator (Narishige, model: MMT-5 ) used in the electrode filling station
Oven (Euromiad compact cooker, model: MC 110 T )
Distiller (Labglass, model: 03DD )
pH meter (Thermo Fisher Scientific, model: Orion 420 A +)
Burner
Magnetic stirrer (ISG^® Hotplate and Magnetic stirrer, model: 153-005 ) and stirring bars
Borosilicate glass capillaries (GC 150-10) (1.5 O.D. x 0.86 I.D. x 100 L mm) (Harvard Apparatus, catalog number: 30-0053 )
Borosilicate glass capillaries (GC 100-10) (1.0 O.D. x 0.56 I.D. x 100 L mm) (Harvard Apparatus, catalog number: 30-0016 )
Petri dishes (85 mm and 35 mm diameters)
Perspex holder for immobilization of the leaf samples
Metal electrode rack for electrode silanization
Plastic electrode holder for storing silanized electrode blanks
Standard surgical blades (Kiato stainless steel, model: BS 2982:1992, ISO 7740 )
Syringe (Terumo, catalog number: SS-10L )
Plastic needle (20 μl) (Eppendorf, catalog number: 5424 956.003 )
Plastic-coated weights
Silver wire (A-M Systems, catalog number: 787000 )
4.5 L PVC (polyvinyl chloride) pots

Procedure

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Wu, H., Shabala, L., Zhou, M. and Shabala, S. (2015). MIFE Technique-based Screening for Mesophyll K⁺ Retention for Crop Breeding for Salinity Tolerance. Bio-protocol 5(9): e1466. DOI: 10.21769/BioProtoc.1466.