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

Summary

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

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.

Introduction

Tree stems comprise a significant amount of the planets biomass and are of immense biological, cultural and commercial importance. Secondary stems create habitat by providing resources and shelter for many other life forms. They deliver many other services to the ecosystems they inhabit and act as a renewable resource for the production of timber, pulp and paper and other wood and non-wood products. Secondary stem development and more specifically wood formation is governed by complex molecular system that regulate the development of specific cell types, the biochemical composition of their cell walls and how they are arranged to form tissues and organs. Dissecting th....

Protocol

1. Preparation of Plant Material

1. Prior to experimentation, raise new seedlings of the preferred tree species from seed or cutting and grow tree/s until the diameter of the stem in the area intended for experimentation is approximately 1 cm in diameter.
   Note: The time needed may vary due to plant growth rates therefore allow between three to nine months for this step.

2. Binary Vector Creation

1. Conduct work from this section to section 7 in a laboratory or greenhouse where Agrobacterium tumefaciens can be handled and that appropriate personal protective equipment prescribed for the laboratory is ....

Results

Using this protocol all live secondary stem cell and tissue types have been shown to be susceptible to A. tumefaciens transformations and have been defined into sector types based on the cell type initially transformed and it subsequent developmental growth pattern. Sector types include periderm, phloem, cambial, wound parenchyma and tylose (Figure 1b, 1c, 1d) and can be found in consistent locations describe in the remainder of .......

Discussion

The ISSA protocol is a relatively simple and efficient method for the creation of transgenic stem tissues in tree species in the space of a few months for analysis of genes and promoters of interest involved in wood and stem formation. Little effort, beyond keeping plants alive, is required to grow transgenic stem tissue following inoculation which stands in contrast to in vitro methods where extensive culturing is required to maintain tissue or plants, where wood production can take up to years to commence or w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plants	NA	NA	Please consult local nursery suppliers for plants as needed
Agrobacterium strain	NA	NA	There are many possible avenues to obtain Agrobactrium strains. We suggest you follow up within your local research community as there may be restrictions in obtaining the bacteria in your country and region.
Binary vector (gene and promoter)	NA	NA	We have developed a range of vectors to suite the ISSA protocol using a the Gateway Recombinase system. This include overexpression, RNAi knockouts and promoter fusion vectors based on modified pCAMBIA vectors and happy to provide as needed. In addition, there are many vectors avialable to the research community.
LB media	Sigma	L3022	The same product could be sourced from another company
LB media with agar	Sigma	L2897	A like product could be sourced from another company
Antibiotics	Sigma	NA	The catalog number will be dependent on the antibiotic you require as a range of antibiotic are used for bacterial selection in binary vectors. This product could be sourced from a  range of companies
50 ml Screw top tubes	Fisher Scientific	14-432-22	The same product could be sourced from another company
2 ml Microtube	Watson Bio Lab	132-620C	The same product could be sourced from another company
MS Media	Sigma	M9274	The same product could be sourced from another company
Scalpel blade no. 11	Sigma	S2771	The same product could be sourced from another company
Parafilm "M"	Bemis	PM996	This is the best product to use to bind the cambial window post creation
14 ml round bottom tubes	Thermo Scientific	150268	The same product could be sourced from another company
EDTA	Sigma	E6758	The same product could be sourced from another company
Triton	Sigma	X100	The same product could be sourced from another company
X-Gluc	X-GLUC direct		You will need to go to the website to order - http://www.x-gluc.com/index.html
Potassium Ferricyanide(III)	Sigma	244023	The same product could be sourced from another company
Potassium Ferrocyanide(II)	Sigma	P9387	The same product could be sourced from another company
Litmus paper	Sigma	WHA10360300	The same product could be sourced from another company
Single edge razor blade	ProSciTech	L055	The same product could be sourced from another company
Double edge razor blade	ProSciTech	L056	The same product could be sourced from another company
SEM Pin Stub	ProSciTech	GTP16111	The same product could be sourced from another company
Sample vial with screw cap	ProSciTech	L6204	The same product could be sourced from another company
Ethanol	sigma	E7023	The same product could be sourced from another company
LR white	ProSciTech	C025	The same product could be sourced from another company
Embedding Mould	ProSciTech	RL090	We recommend this variety, however there are plenty of options available
Water Soulable mounting media	ProSciTech	IA019	One example of a mounting media that could be used however other options do exist and could be explored.
Hydrogen peroxide	Sigma	216763	A like product could be sourced from another company
Glacial acetic acid	Sigma	A9967	A like product could be sourced from another company
Safranin O	ProSciTech	C138	A like product could be sourced from another company
Quanta Environmental Scanning Electron Microscope	FEI		This is the instrument used at part of this study but any other SEM that has a low vacuum mode could be utilised
ImageJ imaging software			can be sourced from the following URL http://rsbweb.nih.gov/ij/