Tomato ‘Avigail’ )870) was used as the background line for both transgenic root lines: mj-far-1 OE and the control Kan, as described previously [26 (link)]. Both root lines were subcultured on standard-strength Gamborg’s B5 salt medium (Duchefa, Haarlem, The Netherlands), supplemented with 2% (w/v) sucrose and solidified with 0.8% (w/v) Gelrite agar (Duchefa). Roots were subcultured on B5 medium, with one root section per petri dish (Miniplast, Ein Shemer, Israel), and incubated horizontally in a growth chamber at 26°C in the dark for 1 week to allow root branching before nematode inoculation.
Gelrite agar
Manufactured by Duchefa Biochemie
Gelrite agar is a gelling agent used in microbiological and plant tissue culture applications. It is a high-purity polysaccharide derived from the bacterium Sphingomonas paucimobilis. Gelrite agar forms a transparent, firm gel at low concentrations and is commonly used as a substitute for traditional agar in various laboratory procedures.
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4 protocols using gelrite agar
1
Tomato Root Transgenic Line Protocol
2
Characterization of Arabidopsis ALKBH9 Mutants
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All Arabidopsis thaliana (L.) Heynh. plants studied in this work were homozygous for the mutations indicated in each case. The Nottingham Arabidopsis Stock Center provided seeds of the Col-0 accession (N1092; referred to herein as WT), as well as of the alkbh9a (N693727), alkbh9b (N515591) and alkbh9c (N521775) mutants. For rosette morphology analyses and to obtain multiple mutant genetic combinations, plants were grown under sterile conditions on 150-mm diameter Petri dishes, containing 100ml half-strength Murashige and Skoog agar medium, 0.6% Gelrite Agar (Duchefa), and 1% sucrose, at 20°C±1°C, 60–70% relative humidity, and continuous illumination of ≈ 75μmol·m−2·s−1, and transferred into pots 21days after stratification (das) as previously described (Ponce et al., 1998 (link)). Sixteen evenly spaced seeds were sown per plate. Crosses were performed as previously described (Quesada et al., 2000 (link)). Photographs of rosettes were taken on a Nikon SMZ1500 stereomicroscope equipped with a Nikon DXM1200F digital camera. Rosette leaf area was calculated using Phenovein (version r214) of the MeVisLab software (version 3.1.1; Bühler et al., 2015 (link)).
3
Tomato Protoplast Isolation and Co-Incubation
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Sterilized tomato seedlings were grown at 26 °C under 16 h daylight in sterile plates on standard-strength Gamborg's B5 Salt medium (DUCHEFA, Haarlem, The Netherlands), supplemented with 2% (w/v) sucrose and solidified with 0.8% (w/v) Gelrite agar (DUCHEFA). Roots were subcultured on the same medium with one root section per petri dish (MINIPLAST, Ein Shemer, Israel) and incubated in a growth chamber at 26 °C in the dark for 2 weeks. Protoplasts were released and isolated from about 40 plates of roots using Demidchik and Tester's116 (link) protocol. Fresh protoplasts were incubated with 60,000 freshly hatched sterilized J2 at 26 °C in the dark for 3 h.
4
Tomato and Arabidopsis Transformation Protocol
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Tomato (Lycopersicon esculentum) cv Avigail 870 was used as the background line for the transformation in experiments involved tomato plants. For the transformation protocol, tomato seeds were treated with 1.6% NaOCl for 10 min with continuous shaking, washed with sterile water for 5 min, and placed on standard-strength Gambourg's B5 medium supplemented with 2% sucrose and 0.8% Gelrite agar (Duchefa, Haarlem, The Netherlands). The plates were kept in darkness at 26°C for 2 days, followed by 2 weeks under a 16/8-h photoperiod until cotyledons emerged. They then were used immediately for cocultivation as described by Chinnapandi et al. (2017 (link)). Similarly, tomato roots sections were subculture, by placing one root per Petri dish (Miniplast, Ein Shemer, Israel), containing B5 medium. Branching roots were used for inoculation as described below. For experiments conducted on Arabidopsis, A. thaliana WT and transgenic lines were grown in a growth chamber at 24°C on MS medium (Murashige and Skoog, 1962 (link)) or in pots filled with cocopeat. The plants were maintained under a 14 h/10 h day/night cycle at a light intensity of 50–60 μmol/m2s. Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used as the genetic background for all transgenic lines.
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