Seedlings to be grafted were germinated on 100 × 15-mm plates containing 0.5× MS Macro- and Micronutrients, 0.5× MS Vitamins (Caisson Laboratories, Inc.), 3 mg/L Benomyl [methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate; Sigma, http://www.signaaldrich.com ), 0.04 mg/L BA (6-benzylaminopurine; Sigma), 0.02 mg/L IAA (indole acetic acid; Sigma), and 12 g/L agar. Hormone treatment was found to greatly improve grafting efficiency due to at least three factors: enhanced callusing at the graft union, retardation of shoot growth which maintained contact at the graft union, and an approximately 90% reduction in adventitious root formation from the graft union. Benomyl virtually eliminated fungal contamination [26 (link)]. Plates containing the stratified seeds were placed vertically under controlled environmental conditions (16 h light:8 h dark and 25 °C). Five-day-old seedlings were grafted on the plate by the 90-degree blunt end technique with a 15-degree Stab Knife (Fine Scientific Tools, North Vancouver, British Columbia, Canada) without collars [27 (link)]. The grafted seedlings remained on the plate for an addition 5 d to allow the formation of the graft union. Successfully unified seedlings were transplanted directly to soil (as described above in “General plant growth conditions”). Plants were harvested for Na+ analysis 4 wk after transfer to soil. Postharvest analysis of graft unions was performed under the stereoscope to identify any adventitious root formation from grafted individuals. Individuals with adventitious roots emerging at or above the graft union were eliminated from subsequent analyses.
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