Transgenic broccoli (Brassica oleracea var. italica) with antisense chlorophyllase (BoCLH1) delays postharvest yellowing

Postharvest yellowing in broccoli is known to result from chlorophyll degradation, with chlorophyllase the first enzyme to degrade chlorophyll. In broccoli, three putative chlorophyllase genes (BoCLH1, BoCLH2, and BoCLH3) were cloned using degenerate primers from the conserved regions of known chlorophyllases. Among these three genes, only BoCLH1 is transcribed during the course of broccoli postharvest senescence. A chimeric construct with the antisense BoCLH1, driven by the CaMV 35S promoter and Nos-terminator and harboring the hygromycin resistance gene, was used for Agrobacterium-mediated transformation to study the effects of the antisense BoCLH1 gene on the postharvest senescence of broccoli. From a total of about 90 primary transformants, 35 individuals were selected and grown for further studies, with 22 of these grown to maturity. Based on the Chl retention rate on Days 4 and 5, respectively, 45% of the detached florets and over 60% of the detached leaves of the selected transformants exhibited slower yellowing when stored at 20 °C in darkness. Southern blot analyses were conducted to eliminate the possible non-independent transformants and investigate the insertion patterns and copy numbers. Only a few lines with simple insertion site and copy number and postharvest yellowing retardation effects were self-pollintated for further evaluation. Northern analyses showed antisense BoCLH1 mRNA transcripts on the day of harvest, the levels of which gradually decreased 4–5 days postharvest when stored at 20 °C in darkness. Positive correlations between the antisense BoCLH1 transcripts and slower postharvest yellowing were noted in some selected lines. Only 1–2 days delay in yellowing was observed in the selected antisense BoCLH1-positive transformants. No individual antisense BoCLH2 or BoCLH3 transformants showed significant slowing of postharvest yellowing. The results suggested genes other than the BoCLH-Chlases obtained in the present study might also be essential in the yellowing process.