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Abstract As a popular leafy vegetable, lettuce(Lactuca sativa L.) has been considered by scientists to be an ideal platform for plant bioreactor. In the present study, 2 lettuce cultivars(Grand Rapid and Hong Kong Glass)commonly cultivated in the production were evaluated which one was feasible to be served as explants for chloroplast transformation. To do this, the tissue culture regeneration systems of both kinds of explants were developed separately in which adventitious shoot buds were induced using young leaf explants of 2 cultivars placed on induction media supplemented with different concentration of hormone and plantlets were obtained on rooting medium. The Grand Rapid cultivar was chosen to provide explants for subsequent chloroplast transformation approach because of its higher frequencies of induction, regeneration and rooting. According to the published sequence of 16S-trnI-TrnA-23S region located in lettuce chloroplast genome, the homologous recombination sequence covering most of 16S-trnI-TrnA-23S region was obtained by PCR amplification using lettuce-specific designed primers, and then inserted T vector to form an intermediate cloning vector. Chemically synthesized expression cassette harboring gfp reporter gene and aadA resistant gene were inserted to the recognizing locus of Ecl136Ⅱ restriction enzyme located in above-mentioned homologous recombination sequence so as to finally finish the construction of specific expression vector PTLE. In the expression cassette, gfp gene represents the green fluorescent protein was used as a visible marker, and aadA gene confered the resistance to spectinomycin and streptomycin by adenylylation. The PTLE vector was transferred into chloroplast genome via particle bombardment, after 2 rounds of selection on medium supplemented with spectinomycin at concentration of 30 mg/L, the surviving plants were obtained on the resistant medium. PCR amplification and green fluorescence analysis confirmed that the regenerated plants were still heteroplastomic, meaning more works of selection were needed to reach the homoplasmy. Taking together, these results suggested that the basic approach of lettuce chloroplast transformation was established and pave the road for further achieving homoplastomic plants and extending to comprehensive understanding of plant bioreactor.
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Received: 16 October 2013
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