Enlightening Medicago truncatula transformation and shading GFP fluorescence

摘要

Medicago truncatula (M. truncatula) has been proposed as a model legume formolecular and genetic studies of legumes. While many genetic resources have beendeveloped for this model legume, genetic transformation of the M. truncatula line A17proved to be a problem. A reproducible transformation method is described for M.truncatula A17. Procedures are detailed that yielded an average regeneration frequencyof 35% for recovery of transgenic shoots from cotyledonary node explants. Previously,rooting of transgenic shoots of this line has proven difficult, but media and cultureprocedures are described that yielded an average frequency of 39% for root inductionfrom 419 phosphinothricin-resistant shoots. Fertile M. truncatula A17 plants transgenicfor 35S-GFP, phas-GUS or phas-GFP were obtained. The presence of transgenes wasconfirmed by expression of transgenes and by genomic DNA blots. Interestingly,although GUS and GFP driven by the phas promoter were very strongly and uniformlyexpressed in seed cotyledons of most transgenic M. truncatula lines, silencing of theGUS expression from the phas promoter was observed in several lines, indicating the occurrence of novel epigenetic events.The diminution of GFP fluorescence in transgenic M. truncatula occurs despitethe presence of GFP transcript and protein. To evaluate the generality and causes of thisphenomenon, fluorescence during leaf development from the same 35S-GFP transgenewas compared in M. truncatula, rice and Arabidopsis. A substantial decrease influorescence early in the development of M. truncatula and rice leaves was found tocorrelate with chlorophyll accumulation. Several approaches showed that chlorophyll iscausally involved in the loss of GFP fluorescence. Removal of chlorophyll from leavesof transgenic M. truncatula, rice or Arabidopsis through etiolation or by extraction withethanol yielded up to a tenfold increase in fluorescence. Direct evidence that chlorophyllis implicated in the loss of fluorescence from GFP was obtained by mixing solutions ofchlorophyll and GFP. At low concentration, fluorescence loss was fourfold greater forchlorophyll b than for chlorophyll a, reflecting their relative interference with GFPexcitation and emission. Thus, substantial errors in estimating promoter activity fromGFP fluorescence can occur if pigment interference is not considered.