Project Leader: Danny Llewellyn

Brief Summary of Project Objectives: 

The aim of this project is to provide the molecular support needed for the core cotton breeding project to deliver new varieties to the industry, to support other CBA projects needing similar molecular or transformation expertise and to serve as an incubator for new ideas and projects.

Market/ end user: Cotton growers through the Core Breeding Project as the primary end user of the research.

Estimated year to uptake by end user:

First varieties containing Bollgard 3 were released for commercial production in 2016 and progressive handoff of improved varieties have followed every couple of years since. The first varieties containing XtendFlex will be available in 2022 subject to approvals and ongoing evaluation of their economic and agronomic fit in Australia.

Markers developed in CBA04 have been in use for a number of years to screen for Cotton Bunchy Top resistance and the first CBT^R cultivars also containing B3XF traits are in seed production. However, the first CBT^R conventional variety, Sicot 620, was commercially released for the 2018 planting season.

Screening for some other non-GM traits will also contribute to new varieties as part of the normal breeding program cycle.

Executive Summary

This project supports a senior scientist and a team of skilled technical staff who provide the core molecular capabilities needed for the successful development and deployment of new commercial cultivars containing GM traits by the Core Breeding project. The Core Biotech and Core Breeding projects are very closely integrated with common goals and regular interactions.

The Core Biotech project provides all the molecular screening (DNA-based, ELISA-based and biochemical screening) needed to follow GM and non-GM traits through crossing and selection to produce new varieties and also provides any additional support for the CBA QA service project in the quality control over all steps in breeding leading to the production of seed to be handed on to CSD for commercial seed increase.

This includes molecular screening of all plants used as parents in the crossing program and any new material coming in through Quarantine that may carry unexpected traits from the country of origin that may not be approved in Australia.

The team is also applying the molecular markers developed in the Disease Marker and HPR projects as the breeding team integrates those disease and Host Plant Resistance traits into their mainstream program.

The Disease Marker project finds and validates the markers for traits of interest, and the Core Biotech project then applies them for the breeders alongside our other molecular screening for commercial GM traits. We have, for example, now been routinely using markers for Cotton Bunchy Top resistance on breeding lines and for the last two years have also tested for markers for mite resistance as they are now well integrated into core breeding activities.

More screening markers will come over the next year or two as the Disease Marker Project narrows down and validates specific Black Root Rot, Verticillium and Fusarium wilt resistance associated markers. Markers are also being used to screen for nematode resistance, whitefly resistance, imidazoline resistance, okra leaf and tufted seed traits, but on smaller scale in boutique breeding populations.

Significant Achievement for the project over the last year include:

• The team performed around 60,000 ELISA assays for the insecticidal traits on seed and leaf samples, extracted just under 50,000 DNA samples from freeze-dried leaves from the glasshouse and field, and performed over 250,000 PCR genotyping assays to provide the breeding program with presence/absence or zygosity information on the multiple GM traits needed to advance specific selections from many different breeding populations and to confirm the identity of plants used in the crossing program. This was an increase of around 30% over last year. Around 15 introgressed lines containing Bollgard 3/XtendFlex traits coming from Bayer were also put through Quarantine and screened to clear them of any contamination with other commercial GM traits that they may have been exposed to while being grown up in the US. Many more such lines are ready to go through this system, as capacity allows.
• Screening for non-GM or native traits coming out of other CBA projects or from overseas has continued, especially for CBT resistance that has become almost routine, but also for some mutant herbicide tolerance traits, okra leaf, tufted seed, nematode resistance and bacterial blight resistance traits. Testing for markers associated with mite resistance has now been transferred from the Disease Marker Project to our project now that they are well validated and breeding with those traits has become part of the core breeding program activities and similar things will happen soon for screening for whitefly resistance, BRR resistance and various Vert and Fusarium traits.
• The large volume of assays required for molecular breeding is always a significant HSE risk to staff and we have continued to explore new equipment to reduce those risks, utilising CBA and CSIRO capex funds. The increased efficiencies through automation have been critical for getting us through the coronavirus pandemic when staff numbers in the lab were often severely restricted, but reliance on a single robot made us extremely vulnerable. A second robot has now been installed and is operational and was very beneficial during the Covid Lockdowns and work restrictions when we had fewer staff able to be physically on site.
• The Core Biotech team, in between activities for the breeding program, have contributed their expertise and time towards many of the other CBA and CSIRO funded cotton projects on an ad hoc basis through molecular analysis of both conventional and transgenic plants, helping the quarantine staff process cotton material (even more so this last year when they were also severely short-staffed) coming through our quarantine glasshouses for the breeding team, making gene constructs and vectors for use by other projects, assisting with the production of transgenic cotton plants containing different experimental traits being evaluated by other projects and in gaining the necessary regulatory approvals for any field testing of those GM cottons, as well as overseeing reporting back to the Regulator and contributing to submissions for reviews of the regulatory system and Plant Breeders Rights laws. All these additional activities were reduced this last year because of the pandemic and various restrictions on working in the labs and tissue culture facilities as priority had to be given to the breeding program before any other activities.
• The Core Biotech team also has a role in investigating new technologies and this year has focussed on comparing different gene editing systems that we might eventually be able to use in cotton. The focus has been to get the freely available MAD7 editing enzyme from Inscripta to work, initially in tobacco and then cotton but has now included some comparisons with other systems like Cas 9 and Cpf I that require Licenses for commercial application. Our gene editing work is progressing, but like other areas of research has been slowed by the pandemic and related issues.