Production Of Transgenic Early Flowering Lemon New Germplasm With GFP Gene

Citrus is one of the most important fruits in the world.But citrus always have a long juvenility,which seriously affect the efficiency of conventional breeding methods.In recent years,the continuous development of transgenic technology provides a more rapid method for citrus research,combined with the short juvenility materials can accelerate the research process.This experiment used short juvenile Early flowering lemon tube seeding plant epicotyl as explant,based on Agrobacterium-medium transformation system to transfer green fluorescent protein(gfp)gene,finally got 8 transgenic regenerated plants.At the same time,this experiment studied the effect factors of transformation efficiency of Early flowering lemon.The main results are as follows:1.Regeneration of transgenic plants.This research included 7 batches of transformation experiments altogether 2215 epicotyls,the average regeneration rate was 60.6%,the highest rate was 75%.Testing by Stereo Fluorescence Microscope indicates the average GFP-positive rate was 1.8%,the highest rate was 2.8%.In the end,8 GFP-positive buds took roots and transplanted into greenhouse;fluorescence of regenerated plants leaf and stem was obvious,in addition of PCR identification,shown the regenerated plants were GFP-positive.2.Factors influenced transformation efficiency.This experiment sets up 3 AS and Agrobacterium tumefaciens concentration,found that AS concentration had little effect to efficiency;nevertheless,the efficiency was lower when Agrobacterium tumefaciens concentration gone high,OD600 0.6 was the best concentration.As it's shown in the transformation process,this genotype form callus after a week dark culture,usually one adventitious bud regenerated on epicotyl incision,in very rare instances both incisions regenerated buds of one epicotyl.Epicotyls etiolated easily after co-cultivation,the potential of regenerated callus is not very high,part of epicotyls browning after several days cultivation,which likely adverse to bud regeneration even under good condition.