Development of tissue culture and transformation systems for garlic, (Allium sativum L.)

Tissue culture systems were developed and utilized in the development of a stable transformation system in garlic, Allium sativum L. A diverse germplasm collection was evaluated to determine the plant growth regulator treatment that would produce abundant, friable callus using the first leaf primordia surrounding the meristem. The addition of 2,4-D (4.5 {dollar}mu{dollar} sc M) to a modified Gamborg's B-5 medium for callus initiation followed by transfer to medium with picloram (4.7 {dollar}mu{dollar} sc M) + 2iP (0.49 {dollar}mu{dollar} sc M) stimulated production of friable abundant callus while limiting the exposure of callus to 2,4-D. Long-term regeneration of garlic clones was evaluated since selection of putative transformants may require prolonged exposure to selective agents. The addition of picloram (1.4 {dollar}mu{dollar} sc M) + BA (13.3 {dollar}mu{dollar} sc M) to the basal medium stimulated the highest rate of shoot production and regeneration of plantlets occurred on callus up to 16 months old. A continuous callus production system was developed using root segments from shoot-tip derived plantlets because availability of fresh cloves year round is limited and meristem culture is labor intensive. The best callus production was observed on root segments initially grown on 2,4-D and transferred to picloram (4.7 {dollar}mu{dollar} sc M) + 2iP (0.49 {dollar}mu{dollar} sc M). This callus was highly regenerable (85.3%) and embyrogenic friable callus was used to establish suspension cultures. These tissue culture systems were used to produce target tissue for the optimization of transformation parameters and development of a stable transformation system for garlic using particle bombardment as the method of gene delivery. CaMV35S promoter activity was much higher for garlic than either the maize Adh1 or rice Act1 monocot promoter. Osmoticum did not enhance promoter activity. Increased shaker speeds (240 rpm), produced a more friable, embyrogenic and highly transformable callus type. Selection of putative transformants occurred on medium solidified with 0.35% low gelling temperature agarose supplemented with parmomycin sulfate (100 mg L{dollar}sp{lcub}-1{rcub}).{dollar} Incorporation of foreign genes into the genome of sixteen month old transgenic plants was confirmed by PCR with specific primers for GUS and NPT II and Southern blot analysis. GUS assays of leaves and meristematic regions from stably transformed plants revealed no GUS activity.