The zucchini squash industry in Queensland is valued at approximately $10M. Zucchini yellow mosaic virus (ZYMV) and or papaya ringspot virus type W (PRSV-W) often cause severe losses of marketable yield of zucchini (Cucurbita pepo) in all production areas with annual losses of $3.8M.

Because no cultural practice effectively controls the viruses, resistant cultivars are urgently needed. However, these are unavailable. We had previously produced partially resistant breeding lines from the resistant gourd Cucurbita ecuadorensis but this material required further assessment, improvement and stabilisation (by inbreeding).

In traditional plant breeding inbreeding is the procedure whereby the genetic variability within each plant is reduced by successive self-pollinations until there is essentially no difference between the parent(s) and all the progeny. This provides plants suitable for use as parents of hybrid cultivars. The process usually takes between six and eight generations of self-pollination.

However more recently ovule and anther culture methods which develop plants from unfertilised ovules and pollen have been used in breeding programmes. Production of haploid plants with the subsequent doubling of chromosome number has facilitated the rapid development of diploid inbreds from segregating populations of plants and thereby the release of cultivars.

The objective of our HRDC and QFVG (COD) funded project was therefore to develop ovule (and anther) culture in zucchini so as to hasten the production of inbred, virus resistant lines using C. ecuadorensis as the source of resistance.

The programme at the Department of Primary Industries Queensland’s (DPIQ) Redlands Research Station involved determining the conditions which would allow the production of large numbers of plants from ovules and subsequently determining the tissue from which these plants originated. (The programme was limited to ovule culture because the success of this technique was considered more likely than that of anther culture.) A backcrossing programme was conducted concurrently to improve resistance, quality and fertility.

A series of experiments investigated the influence on ovule germination of combinations of growth regulators (IAA, NAA, 24D, Kinetin, BAP, and 2IP at 0 to 103 M) with different durations (0-13 days) of treatment and lighting. Correlations between ovule germination and fruit, plant and ovule characters were assessed. The origin of resultant plants was also determined by evaluating progeny.

Some development occurred even in the absence of growth regulators but the best occurred when material was taken from vigorous plants at early flowering when 0.6 – 0.8mm long ovules were extracted from fruit two days before flower opening and inoculated in darkness on growth regulators IAA, NAA (each 125 x lO^M), 24D (30 x 10-*M),

Kinetin (25 x lO^M), BAP, 2IP (each 3.125 x 10-*M) on MS media plus de Vauix vitamins, 3% sucrose, 0.8% agar for 24 – 48 hours, followed by incubation on similar media without growth regulators in a 16 hr day.

One haploid plant in 26 was successfully produced using this ovule culture protocol but the efficiency needs to be increased to allow routine usage. It is postulated that the recovery of haploids can be increased by subcultures and further media modification however further investigations in such areas could require substantial resources. Other fertile plants produced from ovules, although of somatic origin and not the more desirable spontaneously doubled haploids, were virus free. The protocol therefore also provides a means of readily developing virus free breeding stock.

A semi-quantitative ELISA technique, based on biotinylated antibody and streptavidin conjugate, was developed at DPIQ’s Plant Pathology Branch, to permit better evaluation of resistance to PRSV-W and ZYMV. The technique allows the quantification of the differences between plants which express only moderate levels of resistance; a situation which occurs in backcrossing.

Highly resistant but poor quality, low fertility plants were developed through a series of crosses in the backcrossing programme. This material represents a marked improvement in resistance over the original partially resistant plants which expressed increased susceptibility in the first spring after selection. With much further selection and backcrossing this highly resistant material should improve in fertility and quality. Such efforts are warranted.

Genetic transformations which may confer resistance, for example incorporation of the viral coat protein gene, should also be considered in future.