Vanilla protoplasts: isolation and electrofusion

Abstract

The vanilla plant is an orchid mainly commercialized for the production of vanillin. This compound is considered the second largest natural flavoring source in the world. In recent years, price of vanillin has increased considerably, which has generated the necessity of improving the quality of the plant materials. There are few works related to the isolation, fusion and regeneration of protoplasts in vanilla, not allowing to boost the benefits that this technique can generate for crop breeding. This work presents efficient protocols for protoplast isolation and fusion from leaf and protocorm like bodies (PLBs) of vanilla (Vanilla planifolia and Vanilla pompona) in order to contribute to the genetic improvement of the genus. A three-week pretreatment in the dark was standardized before placing the explants in an osmotic solution (0.06 M MES, 0.4 M mannitol, pH 5.7) for one hour at 50 r.p.m. This solution was then replaced with different enzymatic solutions for three hours at 25 ± 1 ºC and 50 r.p.m. The isolated protoplasts were filtered (320 mesh), centrifuged (100 xg for 5 min) and re-suspended in a 0.6 M sucrose solution. Subsequently, a washing solution (50% MS salts with 0.03 M MES and 0.2 M mannitol, pH 5.7) was added to separate protoplasts by flotation-centrifugation. Protoplasts` viability was evaluated with 0.01% Evans Blue. Enzymatic solution containing 1% cellulase, 1% pectolyase and 0.5% hemicellulase (pH 5.7) yielded the highest amount of protoplasts from V.planifolia leaf explants (2,9 x 10 5 ± 0,7 x 10 5 protoplasts/g fresh weight, with a viability of 81%) and PLBs (2,8 x10 5 ± 0,7 x 10 5 protoplasts/g fresh weight, viability 80%). In V.pompona, yields of 2,8 x 10 5 ± 0,8 x 10 5 protoplasts/g fresh weight from leaf explants (viability 79%) and 2,5 x 10 5 ± 0,8 x 10 5 protoplasts/g fresh weight from PLBs (viability 79%) were obtained. For electrofusion, a hypoosmolar solution (Eppendorf®, HA, AL) was used, and the alignment and fusion parameters were standardized. The fusion parameters U1 = 8 V, 60 s; A = 170 V, 30 μs, n 3; U2 = 8 V, 60 s generated the highest number of fusion events (8.9%). Highest number of microcalli (plating efficiency 9.4%) was observed on media containing 50% MS salts supplemented with MS vitamins, 1% CaCl2, 1mg/L benzyladenine, 1 mg/L 2,4 - dichlorophenoxyacetic acid, 0.2 M mannitol, 0.03 M 2-(N-morpholino)ethanesulfonic acid, 1 g/L hydrolyzed casein, 20g/L sucrose, and 6.2 g/L agar as a gelling agent (pH 5.7) in diffuse light (16 hours to 1000 lux).