Standards Development

Scope

This document provides requirements and recommendations to laboratories that perform PCR analyses of cottonseed, leaf, cotton fibre and cotton fibre-derived materials.

The following are within the scope of this document:

a) identifying the materials to be assessed, based on the probability of obtaining good quality, fit for purpose DNA from the materials in subsequent steps in the cotton cloth production process;

b) specifying a method for efficient DNA isolation from cotton and cotton-derived materials;

c) specifying the cotton-specific method(s) to be used as control for amplifiable DNA;

d) specifying the screening procedure that provides optimal chances to detect the target DNA as a result of the performance of the lowest number of element screening assays.

This document does not cover sampling of the seed, bale or fabric, or preparation of the laboratory sample. The standards sets describes the conditions for obtaining DNA for the screening of specific DNA elements and is set up in a way that offers the probability of also detecting unknown GM cotton events that possibly contain similar DNA sequences. A recommended sampling method is given in ISO 6497. General guidance for the sampling of bulk materials or for cotton-based products is available in standards such as ISO 1130, ASTM D1441-12 ISTA rules . TENTATIVE TEXTILE STANDARD No. 77. (1963)

Purpose

The purpose of this document is to provide guidance to laboratories worldwide to assess, in a standardized way, whether cotton, cotton fibre and/or cotton-derived materials contain a specific DNA sequence or sequences. It can be applied to, for example, detection of genetically modified (GM) cotton in non-GM cotton and textile production, detection of a specific genetically modified (GM) cotton in other GM cotton and for confirming or tracing a particular species, variety or genetic marker.

The DNA sequence screening approach described in this document is based on Polymerase Chain Reaction (PCR)-based methods. The standard is designed to work on all four of the major commercial cotton species: Gossypium hirsutum, G. barbadense, G. arboreum, G. herbaceum. Cotton (Gossypium spp.) has been cultivated for lint for over 8 000 years. There are over 50 species in the Gossypium genus (Wendel et al., 2009).

The Gossypium genome is complex, containing 2,25 to 2,43 gigabasepairs (Arumuganathan and Earle, 1991). One primary purpose of this standard is to provide standardisation of detection of GM cotton in non-GM cotton. While GM-cotton cultivation covers a large percentage of global cotton production today (ISAAA, 2016), there are countries where the cultivation of GM cotton is not allowed by law as well as voluntary private and/or public standards that do not allow the intentional use of genetically modified organisms (GMOs) in the cotton and textile production process.

This creates a need for an adequate and harmonized protocol on the screening of cotton and cotton-derived materials for the potential presence of GM-cotton related sequences. Due to asynchronous regulatory approvals, a GM cotton that is approved for growth and/or import in one country may not be approved in another country. There is therefore a need for detection of a specific GM cotton event in GM (or non-GM) cotton.

The detection methods submitted and approved by global regulatory agencies are available for that purpose and this method does not supplant those nor the results of those analyses. Some supply chains are tracking specific cotton varieties as part of for example sustainability efforts. Therefore, there is a desire to be able to detect specific varietal-specific markers in cotton and cotton products.

This document describes the key factors necessary to screen seed, leaf and fibre samples at different stages of textile development in the cotton production chain for the potential presence of specific DNA elements.

The protocol describes three major steps:

a) an effective way to isolate DNA from cotton materials;

b) a  method to confirm that the isolated DNA consists of amplifiable cotton DNA, i.e. suitable for PCR, preferably a low copy nuclear target;

c) A screening method or GM event specific cotton detection, or molecular markers to be performed on the cotton DNA isolate. If the results of the screening methods described in this protocol are ‘not detected’, the likelihood that the cotton sample is (at least partly) derived from GM cotton is minimal, based on the ability of the screening methods to detect elements and constructs of the GM cotton events. GM cotton levels below the detection limit of the method or unknown GM cotton events that do not contain any of the elements or the construct tested cannot be determined by this detection method.

When one or more screening method indicates that GM elements are present, the sample may be considered as derived from GM cotton and confirming detection methods may be applied.

In analysis of GM cotton seed and cotton products, the screening approach is designed so as to be able to differentiate the intended target(s) from the bulk background material. Similarly, molecular markers that are intended to identify specific markers will be designed to do so in a background of other varieties.