Northern Australia has long presented a series of problems in terms of sustainable cotton insect management. This is exemplified by the failure of the Ord cotton industry in the early 1970's under extreme insect pressure. Numerous studies and reports have suggested that for cotton growing to be successful in Northern Australia considerable changes to the production system were required (Yeates 2001). The advent of transgenic cotton has provided the impetus for renewed research interest in cotton in northern Australian. The production system proposed involves growing transgenic varieties in the winter or dry season. The move to the dry season is largely in response to research findings showing that insect pressure from Helicoverpa armigera, Spodoptera litura, and Pecinophora gossypiella are lower in this period. Although preliminary research has shown this to be true, a number of additional problems have become apparent. This paper discusses some of the entomological issues associated with cotton production in northern Australia and the research being undertaken to address these problems

The 2015-16 CRDC Researchers' Handbook is a key resource for all researchers working with, or interested in applying for funding from, the CRDC.

Updated annually, the Handbook outlines the key information researchers need to know, including key dates, the application process, funding and stipends available, the payment, evaluation and reporting processes and the CRDC’s intellectual property policy. These, and other critical details needed by researchers are provided in the Handbook. In 2015-16, the Handbook has undergone a complete update, to ensure it remains a critical reference tool for researchers.

When faced with attack by disease-causing organisms, plants rely on an elaborate surveillance system that detects pathogens and triggers a battery of defences to protect the host. Recent studies on the DNA of plants have uncovered an extensive collection of genes that direct this recognition of harmful organisms. These disease resistance genes (R genes) are present in hundreds to thousands of copies, and generally reside in large clusters on the plant genome. SurprisingIy, all of these R genes possess regions of similar DNA sequence that encode highly-conserved

protein structures essential for effective plant defence. Despite this similarity,

different genes can provide resistance to pathogenic organisms as diverse as

bacteria, viruses, fungi and nematodes (see our background story in the January

2001 issue of The Australian Cottongrower). In earlier work we successfully cloned a

small selection of R gene-like DNA sequences, known as resistance gene analogues

or RGAs, from cotton. In this project we proposed to extend the existing work, and as

a result we have now targeted the two major classes of R-genes in plants (NBS-LRR

and STK types). We also proposed to characterise these different genes, and have

attempted to link DNA polymorphism within the genes with Verticillium disease resistance in Australian cotton cultivars.

The floral organs and wounded leaves of Nicotiana alata, the ornamental tobacco, produce a serine protease precursor molecule (NaPI) which is cleaved into six individual inhibitors of 6 kDa. Two of these inhibitors have chymotrypsin activity and four have trypsin activity. These proteinase inhibitors inhibit trypsins and chymotrypsins in the gut of Lepidopteran

pests, and when incorporated into artificial diets or transgenic plants they have a detrimental effect on growth and development of Hellcoverpa punctigera larvae, but some larvae are unaffected. This study describes the characterisation of one of the major targets for NaPI, the chymotrypsins and their potential role in the tolerance of larvae to ingestion of Napl. Chymotrypsin CDNAs were isolated from a CDNA library made from the gut H. punctigera larvae. Phylogenetic analysis of the encoded protein sequences indicated there were six major families of chymotrypsin genes in Lepidoptera.

Cotton is currently the only crop whose transgenic varieties are used in large-scale production in Australia. Transformation of cotton is therefore of special importance for the healthy development of plant biotechnology in Australia.

Unfortunately, cotton is also one of the most difficult plants to transform and regenerate from tissue culture. Currently, transformation of elite Australian cottons is through backcross to Coker varieties, which are no longer used in production. The main obstacle for direct transformation of Australian varieties, as well as most other non-Coker varieties around the world, is the lack of a reliable plant regeneration system for these varieties. CSIRO Plant Industry has had some success with one Australian variety, Siokra 1-4, and a breeding line, Siokra I-3, but only at low frequency in both cases. There are no published reports about embryogenesis or regeneration of other Australian cotton varieties.

Even in the Coker varieties, which have been used as the model varieties in cotton transformation studies, transformation efficiency is low. To make things worse, this process is very time consuming, requiring more than 9 months to obtain transgenic plantlets from Coker varieties. In addition, it has been difficult to regenerate normal plants from cotton embryogenic callus, so the frequency of healthy transformed plants is very low even in the best available system.

One way to speed up the genetic transformation of cotton is to substantially improve the regeneration of plants from tissue culture. The initial phase of our research focussed on applying techniques that have proven successful in lower plants and in carrot tissue cultures to induce elite lines of cotton to regenerate from tissue culture.

In the course of this project, we concluded that a systematic study of the response of

cotton to tissue culture conditions was necessary for development of reliable

regeneration systems for cotton transformation. Most published reports are about

success with specific genotypes, and the protocols outlined are essentially identical

to earlier published protocols with small modifications. The paucity of information

about culture conditions favouring cotton regeneration may be due to difficulties in

collecting sufficient solid data for publication, as cotton tissue culture requires much

more time and space than other commercial crops because the success rate is so low.

Cotton provides about 55% of the fibre in textile manufacturing globalIy and is an important contributor to the Australian economy. The cotton fibre is a single cell hair (trichome) growing out from the surface of the cotton seed epidermis. Little is known about the molecular basis for the control of which cells initiate fibre growth and how that growth is regulated to produce fibres of a desired quality. We have initiated a project to study the genes expressed during the early stages of fibre cell initiation in cotton using both directed approaches to target genes that might be expected to have a role in fibre initiation, as well as a genomics approach to look for novel genes expressed in the early stages of fibre growth. cDNA libraries from early stage cotton ovules have been produced and we have started sequencing clones at random from these libraries (supplemented by a one year grant from CRDC - CSPllOC) and we will use bioinformatics techniques(computer database searching) to try to identify clones that might have a role in the control of fibre development processes (particularly genes known to control other genes - transcription factor genes). This can generate thousands of candidates and the next stage of the process is to narrow these genes down to aworkable number based on their patterns of expression in plants. To do this we can use micro-arrays - these are glass slides onto which small amounts of each of many thousands of the genes are spotted in an ordered sequence. By then using fluorescently labelled probes of the total RNA from particular stages or conditions we can use a dedicated scanner and computer software to measure the level of expression of all 10,000 or more genes under different conditions, such as at different stages of fibre initiation and development or between cultivars with different quality attributes. Genes whose expression goes up, down or stays the same can be identified and by doing different comparisons we can hope to identify those genes controlling the initiation process or quality traits.

The focus of much of the research in the Australian Cotton industry is to reduce the industry's dependence on synthetic insecticides. There has therefore been a strong push by the industry towards the successful deployment of transgenic cotton crops and the implementation and adoption of a true Integrated Pest Management(IPM) program. The use of transgenic cotton variety has become the key element in future ERM strategies for the industry. The proposed project endeavours to quantify the Envirologix kit for use in Australia.

The fibre length and cellulose content are the two determinates of fibre yield and quality. The rapid elongation process and massive cellulose biosynthesis in fibre cells must depend on sufficient supply of photoassimilate. Sucrose is the predominant form of photoassimilate imported to fibres for cellulose synthesis and for generating turgor pressure to drive the fibre elongation. Sucrose and other solutes may move into fibres either symplastically through PIasmodesmata or apoplastically via the plasma membrane of the fibre cells. The cleavage of sucrose by sucrose synthase (SuSy)in the fibre generates UDPglucose, the immediate substrate for fibre cellulose biosynthesis. In developing countires, unloaded sucrose is utilized in diverse pathways for cellulose, starch and lipid biosynthesis by the fibre, seed coat and cotyledons, respectively. The strong competition by the latter two sink tissues could limitthe availability of photoassimilate for cellulose biosynthesis in the fibre, and hence reduce the fibre yield. Previous studies sugget that SuSy expression in fibres is important for mobilising sucrose into this tissue. However, conclusive evidence is lacking regarding the role of SuSy in fibre development. Little is known on the cellular pathway of import of sucrose and other solutes into the developing cotton fibre. A clear understanding of the pathway and regulation of sucrose import into fibres is essential for designing appropriate molecular approaches to enhance photoassimilate mobilization to this biosyntheticly active sink tissue for elongation and cellulose synthesis.

Productivity of the Australian cotton industry is constantly threatened by reduced availability of irrigation water in eastern Australia. Consequently there has been considerable interest from cotton growers, with the possibility of re-establishing cotton in the Ord River and other areas of NW Australia, where extensive supplies of water (> 20% of Australia's annual surface water runoff) and land are available. The development of a cotton industry in NW Australia would complement production in eastern Australia, providing reliability of supply to maintain valuable markets for high quality cotton lint. In the next 4 - 5 years about 50,000 ha of irrigation land is to be released in stage 2 of the Ord. A further 20-30,000 ha of irrigation land has recently been identified in the Katheririe-Daly basin of the NT. There is also some 60- 70,000 ha of potentially itrrigable black soil on the Bains-Angallari rivers 150 km E of Kununurra and considerable areas on the Fitzroy River to the west.

Research during the 3 seasons previous to this project produced promising results: small plot yields were similar to eastern Australia; pest numbers were lower in the dry (winter) season and Ingard cotton was effective on relevant Lepidoptera pests. The installation of a 'research gin', at a cost of $800,000 by Colly Farms in partnership with the Ord River District Co-operative in 1997 has facilitated the expansion of integrated pest management studies (IPM) to commercial scale areas on-farm (250ha to 900 ha during 1997 to 2001). 'Best bet' agronomic practices, were tested in these on-farm IPM areas. This helped to fine~tune practices, but most importantly the on-farm research identified knowledge gaps and areas of further research. For example, a greater understanding of the water usage and ripening processes in this climate is required. Other important knowledge gaps included:the need to understand compensatory growth mechanisms in response to mirid damage; the need for varieties that produce a longer fibre when grown in the dry season; and nutritional requirements. This project aimed to address these and other questions as part of a broader objective to assess the feasibility of sustainable cotton production in NW Australia.

Fungal diseases of cotton, in particular Fusarium oxysprum f.sp. vasinfection, are important in Iimiting the yield and production of cotton in Australia. Improvement of crop resistance is a major strategy in the control of the impacts of disease on cotton production. We have therefore conducted a long-term program to incorporate transgenes for putative antifungal proteins into cotton germplasm.

The aim of the project is to improve cotton's tolerance to fungal pathogens. We have targeted the vascular, root-invading pathogens, Verticillium and Fusarium, but one of the advantages of antifungal proteins is that they may be effective against a range of pathogens. Therefore the material we have generated has the potential to have improved tolerance to several fungal diseases. Our original focus was on Verticillium wilt, and our testing of transgenic lines was conducted using this pathogen. However, with the increasing importance of Fusarium, in wilt to the industry, we decided to shift to using Fusarium as our test organism.