Ms salts and vitamins

Calli with visible (with a height of approx. 5 mm) adventitious shoot primordia were transferred to Petri dishes 90 mm in diameter and 25 mm in height (Phoenix Biomedical) containing six types of Shoot Development Medium (SDM). All SDMs contained full-strength MS salts and vitamins (Duchefa), 3% sucrose, and 0.6% agar. SDM0 did not contain any PGRs, while SDM1 was supplemented with 0.5 mg/l of TDZ, SDM2 with 2 mg/l of 6-Benzlyaminopurine (BAP), SDM3 with 2 mg/l of kinetin (KIN), SDM4 with 2 mg/l of zeatin (ZEA), and SDM5 with 2 mg/l of meta-topolin (mT). All cultures were incubated at 25 °C with a 16-h photoperiod under cool-white uorescent tubes (60-90 µmol m -2 s -1 ) for 60 days.

Regenerated shoots with the height of approx. 2-3 cm were excised from the callus clumps. Micro-cuttings were d,e); 1 cm (f, g); 1 mm (g inset); 2 cm (h, i) sub-cultured in Magenta™ vessels (Sigma) with four types of Root Induction Media (RIM). All RIMs were provided with half-strength MS salts and vitamins (Duchefa), 2 % sucrose, and 0.6 % agar (Duchefa). RIM0 did not contain any PGRs, while RIM1 was supplemented with 0.2 mg/l of indole-3-acetic acid (IAA), RIM2 with 0.2 mg/l of indole-3-butyric acid (IBA), and RIM3 with 0.2 mg/l of 1-napthaleneacetic acid (NAA). The cultures were kept under the same temperature and photoperiod conditions as described above. Shoots with a well-developed root system were removed from the culture vessels and gently washed in a beaker with sterilized distilled water to wash out the RIM. Afterwards, the plants were placed in small pots with commercial substrate for seeding (Substral Scotts Poland, Warsaw, Poland) . The potted plants were watered and covered with a plastic bag to maintain high relative humidity. After two weeks in humid conditions, holes were made in the plastic bags to gradually reduce the humidity. Two months later the shoots were transferred from pots to field conditions in the middle of August 2019. The plants were grown under green netting to protect them from direct sunlight.

Regenerated shoots with a height of approx. 2-3 cm were excised from the callus clumps. Micro-cuttings were sub-cultured in Magenta TM vessels (Sigma) with four types of Root Induction Media (RIM). All RIMs were provided with half-strength MS salts and vitamins (Duchefa), 2% sucrose, and 0.6% agar (Plant Agar, Duchefa). RIM0 did not contain any PGRs, while RIM1 was supplemented with 0.2 mg/l of indole-3acetic acid (IAA), RIM2 with 0.2 mg/l of indole-3-butyric acid (IBA), and RIM3 with 0.2 mg/l of 1-napthaleneacetic acid (NAA). The cultures were kept under the same temperature and photoperiod conditions as described above. Shoots with a well-developed root system were removed from the culture vessels and gently washed in a beaker with sterilized distilled water to wash out the RIM. Afterwards, the plants were placed in small pots with commercial substrate for seeding (Substral). The potted plants were watered and covered with a plastic bag to maintain high relative humidity. After two weeks in humid conditions, holes were made in the plastic bags in order to gradually reduce the humidity. Two months later the shoots were transferred from pots to garden soil conditions in the middle of August 2019. The plants were grown under green netting to protect them from direct sunlight.