Phenotypic and data analysis
After co-cultivation with bacteria for an indicated time period, the
length of the newly elongated PR grown during the treatment was
quantified and emerged LRs in the whole PR were counted. The plates with
seedlings were scanned with EPSON XL11000 for the measurement of PR
elongation with Fiji software
(http://fiji.sc/) and emerged LRs were recorded under a microscope.
The LR density was determined by
dividing the LR number by the whole PR length for each seedling
analyzed.
The total biomass production of each seedling was measured on an
analytical balance. LRPs were quantified 6 d after co-cultivation.
The pCYCB1:GUS seedlings
were stained and cleared to
visualize
the LRPs at early stages of development and each
LRP developmental stages were
classified according to Malamy and Benfey (1997) as follows. Stage I,
LRP initiation, in the longitudinal plane, approximately 8 to 10
“short” pericycle cells are formed. Stage II, the formed LRP is
divided into two layers by a periclinal division. Stage III, the outer
layer of the primordium divides periclinally, generating a three-layer
primordium. Stage IV, LRP with four cell layers. Stage V-VII, from the
LRP is midway through the parent cortex to the LRP appears to be just
about to emerge from the parent root.