Green Gene Technology (GGT), an integral part of modern biotechnology, has steadily progressed over the last decade. Countries such as India, United States, China, Canada, and Brazil have all turned towards GGT in recent times. Besides staple foods rice and wheat, fruits and vegetables were bred in order to generate quantitative and qualitative resistance traits. The recombinant technology has been applied to apple for more than 20 years now.
Scientists aimed at developing a disease-resistant variety of apple with an improved shell life and rooting ability of rootstocks.
Apple- The Early Target
Apple was one of the early targets of the emerging DNA technology. The materials that were used in the original experiments were callus cultures or leaf fragments and a disarmed Ti-binary vector (Agrobacterium tumefaciens). The first transgenic apple plant contained a gene cassette for neomycin phosphotransferase (npt II) and nopaline synthase.
The transformed cells were selected, regenerated, and rooted under the presence of antibiotic kanamycin. Genes were incorporated into the genome by the technique Southern blot analysis. In this experiment, the transgenic apple plant exhibited a normal phenotype.
Parallel to this, many laboratories performed various researches to show that apples can be genetically transformed and enhanced.
Developing the Technology
These researches used not only Agrobacterium tumefaciens but also Agrobacterium rhizogenes. To activate the gene, many researchers relied on the well-characterized 35S promoter derived from the cauliflower mosaic virus (CaMV 35S). Attempts are being made to find other possible promoters too. Although not specifically expressed, promoters such as 940 extA (found active in young tissue), SRS1, and RBCS3C, which are suitable for specific expression patterns and transgene expression in the apple's green photosynthetic tissues, are still under characterization and development. Currently, the most acceptable and promising system developed as an alternative to antibiotic resistance is the gene phosphomanose isomerase (PMI).
PMI-transformed cells have the ability to use mannose as their primary carbon source, which is otherwise not possible for untransformed apple cells. The ultimate idea of the research was to generate a 'clear vector technology' for market-free transgenic apples.
Plant Research International, the Netherlands based organization, proposed such a technology and as a result, researchers were able to generate transgenic apple plants in which selection marker genes were removed.
Resistance to Insects Pests
Resistance to primary insect pests in apples is regulated through quantitative, additive genes and single major genes. Only the aphid gene sd1 (rosy leaf curling apple) has been well-characterized and mapped till date; however, sd1 has not been cloned. Therefore, apple's resistance to insect pests relies on the genes of other species. The gene cryIA (c) and cowpea trypsin inhibitor (CpTI) from Bacillus thuringiensis that encodes an intracellular protein were used in apple transformation and was tested against Cydia pomonella (codling moth). In the beginning only CryIA (c) was expressed in low-levels, but when the gene's synthetic version and the promoter CaMV 35S were used in combination, the problem was almost overcome. Currently, research is being conducted on generating codling moth resistant GM apples.
Genes expressing biotin proteins, strepavidin and avidin, were used to induce resistance to the transformants found in the cultivar Royal Gala against Epiphyas postvittana (light brown apple moth). It was found in these experiments that around 90 percent of the larvae were destroyed within a 3-week period.
Self-Incompatibility- Wild Malus
The Malus fertility has always interested scientist because each genotype, that is, each cultivar is self-infertile. Since the flowers are infected with foreign pollens, orchard planning has to include plants from the wild Malus or other cultivars at regular intervals.
Climatic conditions may hamper the pollination, thus leading to irregular fruit production. Single multiallelic locus (S) controls the self-incompatibility (SI). The growth of the pollen tube is blocked when an S allele matches the other partner's allele. Four years of continuous field trials have showed that producing apples from two transgenic sequences can be compared to the heights of cross-pollination.
In the upcoming years, resistance genes for apples will be thoroughly sequenced and transferred to some test cultivars. The RNA interference technology aims to block certain unwanted traits. With the help of 'clean vector technology', which allows the removal of selection gene marker, the possibility to produce cisgenic apples is wide open. DNA recombinant technology aims to generate GM apples resistant to various diseases for real.
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