Case Study: Gardner’s grid system and plant selection efficiency in cotton (Verhalen et al., 1975)
5.6 Genetic Manipulation
content is increased above 1.2% (Schuster, 1980). The combination of a high bud gossypol with glabrous cotton strains can result in as much as a 60–80% reduction in Helicoverpa zea and Heliothis virescens larval populations (Lukefahr et al., 1975;
Niles, 1980).
These examples illustrate both the poten- tial uses of biochemical bases for resistance, in that they can be highly effective and could potentially provide a fast means of screening segregating plant populations, and the reality of the practical situation where reliable and fast analytical methods are required if such an approach is to be incorporated into a breeding programme.
5.5.4 Diagnostic characters and genetic
5.6.1 Techniques in genetic manipulation Genetic manipulation is the process by which potentially useful characteristics of one organism are transferred in the form of recombinant DNA to another organism in order that the new organism has enhanced value such as insect resistance.
Transferring genes from one organism to another requires the availability of: (i) a DNA vector, which can replicate in living cells after foreign DNA has been inserted into it; (ii) a DNA donor molecule to be transferred; (iii) a method of joining the vector and donor DNA; (iv) a means of introducing the joined DNA molecule into the recipient organism in which it will replicate; and (v) a means of screening for recombinant lines that have replicated the desired recombinant molecule (Lindquist and Busch-Petersen, 1987).
The two basic methods for plant trans- formation currently in use are: (i) Agrobacterium mediated transfer; and (ii) direct gene transfer (the DGT technique).
The Agrobacterium technique makes use of the characteristic of Agrobacterium to deliver a segment of extra chromosomal plasmid (the Ti plasmid) called the T-DNA genes which cause host plant tissue to pro- liferate to form a tumour (galls) and to syn- thesize novel metabolites which are used by the pathogen as carbon and nitrogen sources (Lazzeri, 1998). This naturally occurring transformation system has been modified to enable almost any gene to be transferred into plant cells. Horsch et al.
(1985) developed a simple and general pro- cedure for transferring genes into plants with Agrobacterium Ti vectors. Until recently, however, Agrobacterium transfer techniques were confined to the natural hosts of Agrobacteriumwhich restricted its value. This led to the development of DGT methods which include: microinjection of DNA into cells; electroporation of cells or protoplasts; puncturing cells with micro- scopic silicon carbide fibres; and particle bombardment (Barcelo and Lazzeri, 1998).
Each of these techniques have been used to manipulate crop species but because most of them require complex cell culture and
regenerative procedures to function effi- ciently, their broad range application has been limited (Lazzeri, 1998).
5.6.2 Genetic manipulation for insect resistance
Two classes of genes have received most attention for enhancing crop plant resis- tance to insects: (i) genes encoding for plant genes; and (ii) those encoding for Bt endotoxins or other non-plant ‘toxins’.
The main plant proteins that have been investigated for development of resistance in crop plants are inhibitors of digestive enzymes, in particular protease inhibitors, lectins and enzymes (Gatehouse et al., 1998). In addition there is some potential in the manipulation of plant secondary metabolites to produce crops resistant to insects (Hallahan et al., 1992).
The first example of a foreign plant gene conferring resistance to insects was the transfer of a trypsin inhibitor (CpTi) gene from cowpea in tobacco Nicotiana tabacum (Hilder et al., 1987). The trypsin inhibitors affect insect digestive enzymes and the cowpea trypsin inhibitors provide protection against species of Lepidoptera, Orthoptera and Coleoptera (Gatehouse et al., 1992) including the vine weevil Otiorhynchus sulcatus in strawberry (Graham et al., 1996). The tomato pro- teinase inhibitors expressed in transgenic tobacco have been shown to confer resis- tance against tobacco hornworms Manduca sexta(Johnson et al., 1989) and the potato proteinase inhibitor PPi-11 is effective against Chrysodexis criosoma(McManus et al., 1994). The a-amylase inhibitor (from the common bean Phaseolus vulgaris) in seeds from the transgenic pea plants and Adzuki bean have been shown to confer bruchid resistance (Schroeder et al., 1995;
Ishimoto et al., 1996).
Lectins are carbohydrate-binding pro- teins found in many plant tissues and are abundant in the seeds of some plant species (Panda and Khush, 1995). The first lectin to be expressed in transgenic plants was the pea lectin in tobacco which resulted in enhanced resistance against
Heliothis virescens (Boulter et al., 1990).
Since then the snowdrop lectin (GNA) has been transferred to potato and tomato where it has reduced damage by Lepidoptera (Gatehouse et al., 1997) and aphids (Down et al., 1996). Despite these successes and the wider potential for use of lectins (e.g. Allsopp and McGhie, 1996) the use needs to be tempered by the possibility of toxicity to consumers and hence the need for vigorous testing for potential human toxicity.
The potential of plant derived genes encoding insecticidal proteins for resis- tance against insects has as yet only been demonstrated in the laboratory and glasshouse trials. They are yet to undergo the large scale trials and commercialization process of Bt transgenic crops (Gatehouse et al., 1998).
The bacterium Bacillus thuringiensis (Bt) produces a number of insect toxins which are protein crystals formed during sporulation (Section 6.8). Preparations of Bt spores and crystals have been used as commercial biopesticides for over 20 years.
The first isolation and cloning of a Btgene was achieved in 1981, the transformation
of the endotoxin into tobacco occurred in 1987 (Vaeck et al., 1987) and the first field trials with transgenic crops began in 1993 in the USA and Chile (Merritt 1998). Bt transformed agricultural crops now include tomato (Delannay et al., 1989), cotton (Perlack et al., 1990), maize (Koziel et al., 1993) and potato (Merritt 1998), while in forestry, transformed Populus has also been shown to be effective (McCown et al., 1991).
Transgenic cotton containing Bt genes has been field tested in replicated field crops in 1987, and in 1995 bulking of seed for commercial sale was undertaken (Harris, 1997a). Transgenic Bt cotton strains produced enough toxin to provide excellent control of Heliothis zea, Trichophlesia ni and Spodoptera species.
The Bt was first commercialized in the USA in 1986 on 729,000 ha, then increased to just over 1 million ha in 1997 and 2 mil- lion ha in 1998 (Table 5.10) (Merrit, 1998).
The growth in Btmaize is no less spectacu- lar with five companies supplying seed for the 1998 season for a total area of 14 mil- lion acres in the USA (Anon., 1997). The Bt maize is aimed at control of the European
Table 5.10.Approximate total areas of commercial plantings (thousands of hectares) of insect protected crops containing Bt genes from Monsanto (from Merrit, 1998).
Country Year Cotton* Corn Potato
USA 1996 729
1997 1000+ 1300 10
1998 2025+ 4000 20
Canada 1997 4 2
1998 122 4
China 1998 53
Mexico 1997 15
1998 40.5
Argentina 1998 8 1.6
Australia 1997 60
1998 81
South Africa 1998 12
*Figures for cotton in the USA in 1997 and 1998 include a proportion of herbicide tolerant and combined insect protected and herbicide tolerant cotton.
corn borer which annually costs between
$10 and $30 million to control (Ostlie, 1997). The use of Bt maize should reduce the number of chemical applications and provide additional control from preserved natural enemies.
Insect protected crops using Bt genes have shown rapid uptake by growers which will encourage further development and investment into this technology by the commercial companies. Btcrops and those modified to express other proteins, etc. are going to have a significant impact on the pest management in the next 20 years.