Gene constructs generally contain a coding sequence for a gene of interest coupled with a promoter. May et al (1995) proposed the incorporation of a promoter on one transgene construct and the gene of interest on another gene construct. Using a trans-activation system from yeast they combined promoters and coding sequences by sexual hybridisation. Thus a wide array of promoters could be tested with particular coding sequences following hybridisation without having to prepare separate constructs for every promoter/coding sequence combination.
One of the major limiting factors of using transformation for modifying crop plants in the future will be the availability of cloned genes for agronomically important plant characters. Endogenous transposable elements have been used to isolate genes from maize and have now been introduced into several plant species for the isolation of their genes. The well characterised Ac/Ds element system from maize has been found to transpose in various species, including tomato, potato, rice and Brassica.
Separate constructs containing an immobilised Ac element and others with a Ds element have been introduced into different B. napus plants. The reason for having the constructs in different plants is to enable the two elements to be combined in the same plant (activates transposition) and separated again into different plants (inactivates transposition) by sexual hybridisation.
Chitin is a major component of the cell walls of most fungi. Plants frequently produce hydrolytic enzymes, such as chitinase as a defence mechanism against fungal diseases. Many attempts have been made to determine whether the over production of chitinase in plants will give enhanced levels of resistance to fungal diseases. A chitinase gene with a 35S promoter and a nopaline synthase terminator has been incorporated into an inbred line of B. napus using A. rhizogenes based transformation. Progeny from the T3 generation were subjected to four fungal pathogens:
When compared with the non-transgenic controls there was an increased resistance to all four pathogens, thereby leading to large scale field trials.
The incorporation of a viral coat protein gene can give protection against the same, or related virus and it is now being applied to transgenic plant production. The mechanism by which the resistance operates is not fully understood but it is believed to involve a process similar to cross protection that has been used in virus disease control for many years, whereby inoculation with an innocuous virus offers protection against the pathogenic virus.
This type of resistance was studied in the widely occurring beet yellows virus (BWYV), the coat protein gene along with other relevant sequences have subsequently been transformed into Nicotiana benthimiana and B. napus.
Various strategies are being considered for making plants more resistant to insect pests. Before transgenic plants can be used in field trials (then commercially) it is essential to assess whether a pest resistance strategy has any impact on non-target organisms. For example, as oilseed rape crops are often a major source of nectar and pollen used by the honey bee, it is imperative to determine the impact of insect resistance strategies on honey bees.
Protease inhibitors (PI) are known to provide plants with enhanced resistance to feeding insects. In an attempt to determine whether PI's had any impact on honey bees a cysteine PI from rice has been introduced into B. napus under the control of the 35S promoter. Transgenic plants along with non-transgenic control plants were exposed to honey bees (Apis melifera melifera) in various ways. No differences were observed in the numbers of visits by honey bees between the transgenic and non-transgenic control lines (variety 'Drakkar'). These results showed that the PI gene in B. napus has an insignificant effect on bees.
Anti-sense strategies are being evaluated for modifying a wide range of characters. mRNA coded by the anti-sense version of a particular gene is thought to bind with the mRNA coded by the sense version of the resident gene, and cause reduction in the specific protein gene product. This doesn't provide a complete explanation, however the introduction of the sense version of a transgene has also been observed to result in a reduction in the corresponding protein gene product.
eg. To study the effect of anti-sense DNA on protein production, an anti-sense gene for cruciferin was inserted into B. napus. The transgenic lines resulted in a reduction in the amount of cruciferin in seeds, accompanied by an increase in napin and three essential amino acids (ie. improved seed quality):
