Optimization of elm regeneration in vitro using leaf explants and evaluation of the process in transformation experiments

Abstract

We set up a regeneration process for the elm hybrid ‘Sapporo’ resistant to Dutch Elm Disease, using leaf discs from in vitro grown plants and tested it in transformation experiments. Two steps were needed : bud induction (3 weeks) followed by bud elongation (4 weeks). Bud meristems initiated near the cut ribs, from several tissues (phloem, procambium, rib upper parenchyma) except epidermis. Regeneration depended on the use of agarose (6 g/l LSM) and diluted MS medium (1/2). The best results (11-14 buds/explant) were obtained in presence of 0.1 μM TDZ and 0.06 μM IAA and when maltose (55-110 mM) or sorbitol (110 mM) were used as carbohydrate sources. During shoot elongation TDZ should be decreased to 0.01 μM or replaced by 1-2 μM BAP and 1.4 μM of GA3 added. At this stage, a mixture of 55 mM sorbitol and 27.5 mM maltose was required. Up to 7 shoots / explant developed and were easily rooted (96%) and acclimatized when sorbitol (27.5 mM) and active charcoal (2 g/l) were added to the rooting medium. Transformation experiments were performed with Agrobacterium tumefaciens disarmed strain GV3101-pMP90 (pKyGUSintron). Regeneration zones exhibited stable GUS expression. However, all shoots grown in vitro died on a selective rooting medium (50 mg/l kanamycin). When attempts for in vitro selection were made (12.5 mg/l kanamycin), some buds were initiated but elongation was prevented. Susceptibility to neomycin seemed very high, therefore, selection markers and selection steps must be revised carefully.