Plant growth chamber

Experiments were performed by the usage of Arabidopsis thaliana of the Columbia accession grown on soil in plant growth chambers (Percival Scientific) under short-day conditions (8h light/ 16 h dark) at 22°C. Nicotiana benthamiana were grown under long-day conditions (16 h light + 8 h darkness) at 28°C. gtl1-2 (Salk_005965), gtl1-5 (Salk_044308) and mpk4-2 (Salk_056245) seeds were obtained from NASC.

All Arabidopsis thaliana lines used in this study are in Columbia-0 (Col.0) background. The IDD4 transgenic lines used are published (Völz et al., 2019b (link)). The MAPK mutant line used is mpk6–2 (SALK_073907). Prior to every experiment, seeds were surface sterilized and stratified at 4°C for 2d. The stratified seeds were plated on the ½ strength Murashige and Skoog (MS) medium with 1% agar and plants were grown in plant growth chambers (Percival Scientific) under 16h light: 8h dark condition at 22°C. Unless stated, the plants were grown for 5d on ½ MS and then to apply salt-stress, were transferred onto fresh with ½ MS ± 100mM NaCl (Sigma) and grown for another 16d.

Experiments were performed by using Arabidopsis thaliana of the Columbia accession grown on soil in plant growth chambers (Percival Scientific) under short-day conditions (8h light/ 16 h dark) at 22°C. Nicotiana benthamiana were grown under long day conditions (16 h light + 8 h darkness) at 28 °C. idd4 (Salk_148352C) seeds were obtained from NASC.

Iceberg Lettuce cv. Salinas seeds were kindly provided by Ivan Simko (USDA, Agricultural Research Service, Salinas, CA, United States). Plants were grown in Supersoil potting mix with approximately 150 mg of Osmocote Plus (15-9-12) at 22°C with a 14-h photoperiod for 6–8 weeks in a plant growth chamber (Percival Scientific, Inc.), and fertilized weekly with 20-20-20 (N-P-K) liquid fertilizer past the five expanded leaf-growth stage. For experiments with cut lettuce in modified atmosphere packaging (MAP) bags, mature Iceberg lettuce heads were acquired from local commercial sources, trimmed of outer leaves, and the leaves shredded into 2-mm wide strips with a sharp knife.
Lettuce leaf lysate was prepared as described previously with modifications (Kyle et al., 2010 (link)). Iceberg lettuce heads were obtained commercially and all portions except the outermost leaves and inner-most achlorophyllous heart were used. The leaves were homogenized in a Omega juicer (Omega Model No. 8003), with the homogenate kept on ice during production and immediately centrifuged at 7,000 rcf at 4°C for 10 min to pellet chloroplasts and plant debris. The supernatant was sterilized by passage through 0.45 μm- then through 0.2 μm pore-size filters.

Plants of S. hexandrum, collected from the natural habitat (Parashar lake, Mandi; 2,730 m asl; 30°12′ N 77°47′ E, India), and maintained at CSIR-Institute of Himalayan Bioresource Technology, Palampur (1,300 m asl; 32°06′ N, 76°33′ E, India) were used for the experimental purpose. To avoid the impact of age/rhizome size, morphologically similar plants having comparable leaf and root size without any rhizome formation were selected for this study. The Biological Diversity Act 2002 of India permits bonafide Indians to collect plant samples from native habitats for scientific investigations (Venkataraman, 2009 ).
The plants were grown in potting mix (constituted of soil, sand, and farmyard manure in a ratio of 2:1:1) under a long day (16 h) photoperiod in a plant growth chamber (Percival Scientific, Perry, IA, United States) set at 25 ± 2°C with (photon flux density of 200 μmol m^–2s^–1 and relative humidity of 70 ± 10%), as described earlier (Kumari et al., 2014 (link)).

The residual toxicity of leaf/bark ethanolic aqueous extract, SO at six concentrations (625–20,000 mg/L), and their combinations of T. sebifera (SO+LEE, SO+BEE) in a 1:1 ratio was prepared in five concentrations (125–2000 mg/L) and evaluated against A. craccivora under controlled conditions (26 ± 2 °C, 70% ± 5% relative humidity, and photoperiod of 16 h light and 8 h dark) in a plant growth chamber (Percival Scientific, Perry, IA, USA). The Phaseolus vulgaris plants were raised in plastic pots (7 × 9 cm) filled with the potting mixture (1:1:1 ratio of vermiculate:cocopeat:perlite). One-week-old plants (3–4 leaf stage) were inoculated with two-day-old nymphs of A. craccivora and allowed to settle for 24 h. After settling, test solutions/concentrations were sprayed on plants using a hand sprayer. Observations of the number of dead insects/plants were recorded at 24, 48, 72, and 96 h after treatment. There were six treatments, and each treatment was replicated five times. The fractional effect indices (FEI) were calculated as mentioned above to study the joint action of binary mixtures/combinations.