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.

Since the deregistration in many countries of most cyclodiene insecticides, the ongoing availability of endosulfan has become important as an alternative option in resistance management strategies of pest species. Additionally, compared to many other available insecticides, it has low toxicity to many species of beneficial insects, mites and spiders (Goebel etc!., 1982). However, endosulfan is extremely toxic to fish and aquatic invertebrates, and it has been implicated increasingly in mammalian gonadal toxicity (Singh and Pandey, 1990; Sinha et al. , 1995; Sinha et al. , 1997; Turner et al, 1997), genotoxicity (Chaudhuri et al., 1999) and neurotoxicity (Paul and Balasubramaniam, 1997). These environmental and health concerns have led to an interest in post-application detoxification of the insecticide. The aim of the research supported by this grant was to isolate an endosulfan-degrading gene as a potential source of an enzymatic bioremediating agent to treat endosulfan contaminated waste water.

Many beneficial insects have been recorded in Australian cotton (Room (1979). These

include generalist predators and specialist parasitoids which attack key pests. The

potential value of these beneficial insects have not been widely exploited in cotton

pest management due to lack of understanding of the efficacy of these beneficial

insects, and lack of techniques to maximise both their abundance and effectiveness.

Adoption of within field monocultures in the cotton production system also

discriminate against and reduce the activity of beneficial insects because they lack

ecological diversity (Hagen and Hale, 1974). Hencooerpa spp. which are the major

pests of cotton crops in Australia are highly migratory and can therefore rapidly

infest cotton crops and lay their eggs and unless the natural enemies are present and

well established in high numbers before the pests arrive, they cannot respond

rapidly enough to control them before damage is sustained (Fitt, 1989; Gregg at at. ,

1993; Mensah and Harris, 1994, 1995). These have resulted in loss of Confidence in

these insects by growers who have opted instead to adopt chemical control.

A major focus of the Australian cotton industry is to reduce their dependence on

synthetic insecticides. One way this can be achieved is through the development and

adoption of a true integrated pest management program (ERM) which conserve and

utilise beneficial insects as a base of the program.

The research into shield bug pests addresses an emerging issue resulting from a

reduction in the use of disruptive insecticide sprays on cotton, especially Ingard

cotton. Shield bugs have caused serious losses on some properties and

impacted on profitability. Management approaches are necessary that

complement the adoption of IPM, are based on accurate, locally-developed

thresholds and are less reliant on insecticides. These developments will

contribute to the sustainability of cotton production enterprises, maintain

profitability, and benefit rural communities by reducing insecticide use.