The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Abstract

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree's lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant's secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

Sector analysis reveals patterns of cambium differentiation in poplar stems

“…In total, 581 sectors were identified and assessed, largely conforming to previously described sector types ( Spokevicius et al , 2016 ; Van Beveren et al , 2006 ) with few (eight) exceptions. Cells giving rise to cambial tissues, specifically cambial initials and mother cells, were by far the most susceptible to Agrobacterium transformation, accounting for >65% of all sectors identified.…”

“…After decanting liquid, bacteria were immediately suspended in 1 ml of cooled Murashige and Skoog (MS) medium and stored on ice until needed. ISSA transformation was performed as described in Spokevicius et al (2016) . In brief, a series of cambium windows or upward bark flaps were created on the stems of young poplar trees by making two parallel incisions of 20 mm length, 5 mm apart and connected on their lower end by a horizontal cut through the bark.…”

“…Approximately 4.5 months after transformation, stems were harvested and stem sections at each window were excised and further sectioned into disks of between 0.5 mm and 1 mm in thickness ( Spokevicius et al , 2016 ).…”

“…Specifically, measurements were taken of total new radial growth (including wound parenchyma and xylem) and new radial xylem only growth. GUS-positive tissues and cells were initially classified according to sector categories outlined in Spokevicius et al (2016) ( Fig. 1 ) with additional sector subtypes defined as part of this analysis.…”

Cell differentiation in the vascular cambium: new tool, 120-year debate

“…Testing these mutants with sector analysis would enable a closer look at the impact of these mutations on the vascular cambium and cell differentiation in the stem, and bring us closer to understanding the intricacies of gene function. Similarly, other studies of candidate genes involved in cambium development or wood formation that use either natural variants or transgenic lines for functional analysis could benefit from incorporating sector analysis to resolve the impact of genes on cell differentiation at the level of individual cell lineages as previously shown by Spokevicius et al (2016) . This is certainly a valuable addition to the Populus tool box for functional characterization of wood formation.…”