In this article we will discuss about:- 1. Introduction to Application of Biotechnology in Potato 2. Potato Improvement 3. Potato Pest and Disease Constraints 4. Potato Virus Diseases 5. Potato Insect Pests 6. Utilization of Potato Germplasm 7. Methods to Transfer Genetic Loci 8. Molecular Genetics and Potato Breeding 9. Application of Biotechnology in Potato Production.
Contents:
- Introduction to Application of Biotechnology in Potato
- Potato Improvement
- Potato Pest and Disease Constraints
- Potato Virus Diseases
- Potato Insect Pests
- Utilization of Potato Germplasm
- Methods to Transfer Genetic Loci
- Molecular Genetics and Potato Breeding
- Application of Biotechnology in Potato Production
1. Introduction to Biotechnology of Potato:
Potato is the most important non-cereal food crop in the world. The crop represents roughly half of the world’s annual output of all roots and tubers and is part of the diet of half a billion consumers in developing countries. Trends toward increasing the percentage of potato used for processing may lead to new income opportunities in developing countries.
Global potato production has nearly stagnated during the last 30 years at around 260-270 million tons and area planted declined from 22 to 18 M ha during the same period. The average yield increased by 25% from 12 to 15 t ha-1.
Differences between trends in developed countries and developing countries have been identified. Developed countries will continue to have a slow growth of potato production until the end of the century while developing countries will increase their share of the global potato output up to 34% by the year 2000.
Yields vary tremendously in potato production from lows of 6 t ha-1 in sub-Saharan Africa to 42 t ha-1 in The Netherlands. Schematically, three factors could raise yields in developing countries to the upper values – access to chemical fertilizers, pesticides and good-quality planting materials.
Another noteworthy difference between developed and developing countries’ potato trends is the negligible varietal change in developed countries, whereas improved varieties continue to play an important role in increasing yields in developing countries.
Today, potato production relies on the use of large quantities of toxic chemical pesticides to ensure stable yields. Pesticide overuse threatens not only the environment but also farmers’ health, as pesticides are often handled inappropriately and are less regulated in developing countries.
Lack of access to pesticides also has dramatic consequences on potato production, especially in areas where late blight disease is severe. Hence, pesticides play a pivotal role in the fragile economy of small potato producers due to their elevated purchase cost.
2. Potato Improvement:
Reduction of pesticide use in potato production is a first priority in the International Potato Center (CIP)’s potato improvement programmes. To that end, CIP uses a blend of conventional and molecular approaches to improve potato resistance to targeted pests and diseases. Molecular approaches are based on the utilization of defence and resistance genes from either exotic sources or from the Solanum germplasm.
Through its various institutional mechanisms to set research priorities, CIP’s research agenda was debated in 1996, and formalized in a document entitled CIP Midterm Plan for the years 1998-2000. This research agenda sets a strong focus of potato improvement projects for three constraints – late blight, viruses and potato tuber moth (PTM).
We briefly review applications of biotechnology to – (i) reduce these constraints by gene technology or genetic improvement; (ii) utilizes the genetic diversity of the Solanum germplasm; (iii) transfer new genetic loci into potato varieties.
3. Potato Pest and Disease Constraints:
Late Blight Disease:
Late blight is the main focus of our research as the disease poses a renewed threat to potato production by the new migratory wave of Phytophthora infestans, the causal agent of this fungal disease. The value of crop losses from late blight in developing countries is currently estimated at US $2.75 billion.
Disease symptoms have been delayed by engineering the expression of several genes in potato, such as osmotins and glucose oxidase (GO gene). Osmotin displays membrane-disturbing properties and has been shown to inhibit hyphal growth in vitro and to cause sporangial lysis.
Transgenic potatoes have been produced with strong constitutive expression of tobacco, tomato and potato osmotins. Delayed disease symptoms were observed after inoculating detached leaves with P. infestans. Several pathogenesis-related proteins of the PR1 class, from tomato and tobacco, have been shown to inhibit germination of P. infestans zoospore.
The GO gene from Aspergillus niger was inserted into the potato genome and led to a marked delay in appearance of late blight disease symptoms in potato leaves as a consequence of elevated levels of hydrogen peroxide. Other promising transgenic approaches involve programmed cell death systems that mimic the hypersensitive reaction to fungal invasion. In one of the systems currently under development, transgenic potatoes containing a P. infestans-dependent’ cell-suicide system develop a quantitative resistance to P. infestans. These programmed cell death systems represent a general approach for pathogen resistance.
Recent breeding for late blight resistance has favoured use of polygenic race-non-specific resistance over R genes (conditioning race-specific hypersensitive responses). This type of resistance is apparently more durable with respect to new races of the pathogen. For several years, CIP breeders have conducted recurrent selection using populations of Solanum tuberosum subsp. andigena, native to South America, to improve quantitative resistance to late blight in the tuberosum germplasm.
Beyond the tuberosum boundaries another source of high levels of quantitative resistance has been identified in the native cultivated, diploid potato S. phureja. Molecular markers have been recently correlated with late blight resistance segregation as putative quantitative trait loci (QTL) in diploid crosses from S. phureja. Once confirmed, these QTL will be introgressed into the cultivated tetraploid potato following a marker-assisted breeding scheme combined with tetraploidization via 2n gametes produced by the donor species.
4. Potato Virus Diseases:
Potato viruses are economically important not only as a direct cause of severe crop losses but also as a barrier to seed trade due to phytosanitary requirements imposed by most countries. Among the three main potato viruses, potato potyvirus X (PVX) causes only mild symptoms when infecting plants alone, but when it occurs together with potato potyvirus Y (PVY) it causes a synergistic interaction that results in significant crop losses. Infections by potato leaf roll luteovirus (PLRV) or PVY cause the two major potato viral diseases. At CIP, resistance to control virus diseases has a high priority because of their worldwide importance.
Viral sequences (e.g. coat protein and replicase) have often provided cross-protection to the corresponding virus when integrated into the potato genome. Coat-protein-mediated protection has been successful in developing PVX resistance, and combined resistances to PVY and PVX in potato variety Russet Burbank.
Several potato varieties with combined transgenic resistance to PVX and PVY have been released in Europe, Mexico and the United States. The transfer of replicase genes into potato, coding for replication-related protein of these viruses, has also produced significant levels of resistance to PVX, PVY and PLRV in variety Russet Burbank. The latter led to a variety release in 1995, by the US-based Monsanto Company.
At CIP, we work with genes for extreme resistance to PVX and PVY identified in the Solanum germplasm by conventional and molecular approaches. The dominant genes Ry and Rx from S. tuberosum subsp. andigena have been incorporated into breeding populations at high frequencies. The genetic mapping of these genes conferring extreme resistance to PVX and PVY has been achieved. Molecular cloning of Rx from S. acaule and Ry from S. stoloniferum is currently underway through a collaborative research project between CIP and the Sainsbury laboratory, UK.
In both cases, molecular markers have been identified in the vicinity of the dominant genes. Flanking markers for Rx are at 0.17 cM and at 0.06 cM, and for Ry two amplified fragment length polymorphism (AFLP) markers (M17 and M6) are at less than 0.3 cM.
Genetic resistance to PLRV always has been of high interest to breeders. Only partial resistance has been encountered despite several germplasm screening efforts. Building up durable resistance to PLRV has been hampered by the multifactorial and probably multi-genic nature of the resistance. Different resistance mechanisms (infection, replication, antixenosis and antibiosis to the vector) are conferring, individually, only moderate levels of resistance.
In addition, previous studies at CIP have shown that PVX and/or PVY reduce the durability and level of resistance against PLRV for which no ‘immunity’ genes have been found. A better understanding of the precise mechanism by which extreme resistance to PVX or PVY operate in potato could make it possible to engineer a broad-spectrum and durable resistance to virus diseases.
5. Potato Insect Pests:
The PTM (Phthorimaea operculella) is the most damaging potato insect pest in stores and fields of developing countries. PTM resistance has been obtained by transferring a Bacillus thuringiensis (Bt) gene that codes for an insecticidal protein into potato. At least two crystal proteins have proved to be effective against P. operculella – CrylA(b) and CrylB.
At CIP, we have collaborated with Plant Genetic Systems, Belgium, to transfer a modified crylA (b) gene with high levels of expression into various breeding clones and varieties with different attributes and adapted for different agro-ecologies.
High levels of resistance were obtained in both foliage and tubers. Tuber resistance has been also shown to last for more than 4 months. Protease inhibitors can be transferred to potato in combination with Bt genes to ensure durability of the engineered PTM resistance.
Tuber resistance to PTM has been found in S. sparsipilum and was used at CIP to enhance resistance to PTM in diploid breeding populations. Other insects such as aphids may be effectively controlled by glandular trichomes from wild species such as S. berthaultii. This trait was characterized both biochemically and morphologically and at the genome level by genetic mapping. As the insect pests in potato were successfully managed by integrated approaches in the case of the PTM, moderate priority is given at CIP to developing host-plant resistance to insect pests.
6. Utilization of Potato Germplasm:
Potato germplasm consists of the genus Solarium which embodies over 200 tuber-bearing species and represents a wide array of adaptation to different agro-ecologies. This genetic diversity will continue to be the focus of most of our research efforts to identify and use new genetic loci to defeat pests and diseases. Biotechnology applications offer efficiency and precision in characterization and utilization of genetic resources. The developing world that hosts the largest reservoir of genetic resources will largely benefit from the appropriate use of biotechnology to develop new products and crops.
The key question is how to effectively apply biotechnology to better exploit this germplasm to develop new products and better crops. Two interconnected routes are conceivable once specific genotypes are selected from new germplasm sources for the trait of interest. Newly identified genotypes carrying a trait of interest can be used directly to isolate genes. This is particularly needed if modification of gene expression is desirable, e.g. higher levels, new targeting for protein accumulation or novel induction patterns.
Isolated genes can be transferred directly into potato via Ag-tumefaciens-mediated transformation. The other route, valid when genes are unknown or difficult to access, involves molecular markers to tag and monitor these genes in breeding. Methods to analyse the segregation of DNA markers with the trait of interest have been developed in potato. These ‘tags’ or DNA markers associated with genes governing the trait of interest, can assist breeding by providing genotypic proof of introgression without progeny testing and by helping to more rapidly eliminate unwanted segments of the donor genome.
7. Methods to Transfer Genetic Loci:
Direct Gene Transfer in Potato:
Genetic engineering in potato already has a long history, from the production of the first transgenic potatoes about 15 years ago to new commercial products developed in the past 2 years. Gene transfer into potato was achieved nearly a decade ago, early in the development of gene transfer technology via agro-infection. Potato was one of the first crops transformed genetically. Because of its ease of transformation, potato has been often used as a model species to test the expression of foreign proteins in plants.
Genetic engineering is increasingly adopted in potato improvement programmes because of the worldwide importance of this food crop, the relative ease of potato transformation, its clonal mode of multiplication and some of the inherent complications of traditional potato breeding. The widespread use of genetic engineering for potato improvement will, however, depend largely on the availability of well-adapted potato cultivars, consumer acceptance and the successful marketing of transgenic food crops.
The most widely used system to transfer foreign genes into potato is derived from the natural DNA transfer system of Agrobacterium cells. It is an efficient, easy and inexpensive system for gene transfer and is therefore well adapted for developing-country laboratories, which do not always have the capacity to purchase sophisticated and costly equipment.
The agro-infection protocol is amenable to the large-scale production of transgenic lines which is needed to select the best-performing transgenic line. Transformation of organelles, such as the chloroplast, can also be achieved via the Biolistic Gun™ approach using a chloroplast-selectable marker.
This organelle transformation offers new possibilities for engineering crop plants and provides maternal inheritance of the transgene. Transferring an intact foreign gene into a plant genome does not necessarily imply transgene expression.
The level of expression can vary significantly among the same transgenic lines presumably because of the positional effect of an integrated transgene. Numerous lines must be produced to select for the best-performing line. This apparent disadvantage can be beneficial as breeders often seek genetic variation.
In some cases, however, when the genotype is recalcitrant to genetic transformation and as a result only a few transgenic lines are obtained, the position effect should be minimized. Possible solutions are under development with the use of the scaffold attachment region to stabilize gene expression or the targeting of the transgene to specific high-expressing genomic sites by homologous recombination.
Several phenomena that alter the expression of transgenes following their genetic integration have been observed in transgenic crop management and can be grouped into either co-suppression or gene silencing. Co-suppression of transgene expression occurs as a coordinated decrease in gene expression of two transgenes or a transgene and a homologous endogenous gene in a single plant. Gene silencing via inactivation of the transgene can occur between homologous genes by at least one mechanism – the methylation of either the transgene or endogenous gene.
The importance of these modifications of transgene expression to agricultural practices should not be overestimated in the case of potatoes because – potatoes with a low copy number (preferably one) of transgenes will be most acceptable; additionally, low homology to endogenous potato sequences will be the most judicious choice in transgene design; and above all, clonal propagation of the potato is an advantage as the transgenic potato can be maintained as a hemizygous crop plant.
Nevertheless, the phenomena of gene silencing will become more important when genetic resistance in plants is pyramided. Indeed, the extensive use of the 35S promoter and of mainly one selectable marker gene in all gene constructs may favour methylation of homologous sequences and hence reduce the expected overall expression of resistance genes. More promoters and selectable markers are needed to co-transform plants and build up quantitative traits via genetic engineering.
Transgenic potato research eventually aims at the release of new varieties with added traits that pose low risk for human or animal consumption and to the environment. Field trials are an obligatory step in the development of new transgenic potato varieties. We have already conducted such field trials for three consecutive years at the CIP experimental station of San Ramon using transgenic lines with Bt genes. We have identified good transgenic lines with persistence of the original attributes and PTM resistance.
This work follows Peruvian national regulations on field trials of genetically modified organisms. Hybridization with related or wild species of potato is possible in the Andean region and hence we have taken drastic measures to avoid rare but possible gene flow into other potato varieties or Solanum species. Flower buds are removed every day during the flowering period, transgenic foliage is destroyed after harvest and monitoring of the field over the following cropping seasons is performed.
Following recommendations of a regional workshop at Iguazú, Argentina in 1995, the fitness of the transgene in the testing and cultivation area should be determined. If a convincing fitness exists, CIP recommends the use of male sterile varieties. All these precautions were taken in CIP field trials in the mid-elevation station of San Ramon in Peru where no wild species are present.
8. Molecular Genetics and Potato Breeding:
The application of marker-assisted breeding in potato is a new challenge. The Irish potato belongs to an out-breeding tetraploid species – breeding is slow because of the large number of characters that must be included in a tetraploid crop subject to inbreeding depression. This and its clonal propagation have made potato a highly heterozygous crop. Classical potato genetics is still poorly developed because of these limitations. Hence, most of the genetics in potato is learned from studies made with diploid potatoes.
Ploidy levels can be interchanged in potato as some diploid potatoes produce 2n gametes and therefore can be brought to the tetraploid level. Molecular genetics studies have produced high-density potato genetic maps. The conservation of marker order between potato and tomato, with the exception of five large inversions of chromosome segments, is an advantage as tomato genetics is further advanced.
Molecular markers are now being used to map several traits from native and wild species with the goal of introgressing these traits into cultivated potatoes. We have developed genetic maps for late blight and insect resistance from interspecific diploid populations.
The breeding scheme at CIP to improve late blight resistance in potato essentially follows two routes. One is classical breeding and genetics that make use of tetraploid germplasm followed by recurrent and mass selection methods. The other is a molecular approach involving genetic mapping at the diploid level of loci that determine the resistance to late blight in Solanum species and the selective genotyping of phenotypic extremes.
Both approaches overlap partially in that the same material is shared. The exploitation of Solanum germplasm has led to the identification of a valuable source of resistance to late blight and of several molecular markers associated with this polygenic resistance character. Two interspecific hybrid populations were developed at CIP for this purpose from the S. phureja species.
Molecular markers, random amplified polymorphic DNA (RAPD), microsatellites and AFLP were scored at CIP with the collaboration of the Scottish Crop Research Institute and the Centro de Investigaciones en Ciencias Veterinarias molecular biology laboratory in Castelar, Argentina.
Markers flanking the QTL which account for a large variation in the resistance will be used to introgress this trait in cultivated potato. A first stage will be to breed at the diploid level with the help of markers for indirect selection. The production of 2n gametes will lead to the tetraploid level and the eventual development of improved potato varieties.
A valuable type of insect resistance from the wild Bolivian species S. berthaultii is associated with the presence of trichomes on its foliage. This resistance is polygenic and several components of this resistance have been identified and placed on the potato genetic map. At Cornell University, where this work was developed, this trait has been partially introgressed into the cultivated potato. Two types of trichomes are involved in this resistance – the A type and the B type – each with specific biochemical components.
The efforts at Cornell to breed for tuber yield and resistance mediated by both types of trichomes were hindered by the association between type B trichome biochemistry and lateness and thus poor tuber production under temperate conditions. Plants producing tubers lacked sugar droplets on their type B trichomes and plants with type B trichome products did not produce tubers early enough in the growing season.
Molecular genetic mapping in S. berthaultii X tuberosum hybrid populations demonstrated genetic linkage between lateness and type B trichome properties. Indeed, important loci controlling both characters mapped to chromosome 5 but at different map positions. Molecular markers could be useful to break this linkage by selecting recombinants between these loci and observing their type B properties as well as their lateness.
Evaluation of Solanum germplasm results also in the identification of single genes determining high levels of resistance. The addition of single traits to existing potato varieties is not easy by conventional means due to potato’s heterozygosity. Several genes have been identified in potato that confers immunity or hypersensitive reaction to PVX and PVY viruses.
Nematode resistance genes have also been identified in Solanum species. The first six genes are currently being cloned by different laboratories – the Sainsbury Laboratory in Great Britain, for the virus resistance genes; and the Max Planck Institute in Cologne, Germany, for late blight and nematode resistance genes.
Both efforts proceed via a fine-mapping of the target gene followed by map-based cloning methods. Recent developments in genomic library vectors allow the cloning of large inserts (100-150 kb) in a binary plasmid which can replicate in E. coli and A. tumefaciens. The plasmid carries all features of a plant transformation vector and hence allows the direct transfer of the large DNA insert into potato without sub-cloning steps.
The molecular isolation of these resistance genes will allow their direct transfer into susceptible potato varieties without changing their genetic backgrounds. The cloned genes will also allow the engineering of new resistance types by manipulation of genie components (promoters, enhancers, coding sequence). Eventually, abroad-based resistance to pathogens may be possible in potato by applying these molecular breeding methods.
9. Application of Biotechnology in Potato Production:
Genetic engineering for potato improvement has led to substantial achievements since the first generation of transgenic potatoes more than a decade ago. Healthy transgenic potatoes have been produced and the process is now amenable to large-scale production of hundreds of transformed plants per transgene.
The agro-infection system is particularly well suited to this purpose in developing countries. A growing pool of cloned genes is becoming available to enhance pest and disease resistance and to improve abiotic stress tolerance and tuber quality.
Genes can now be engineered for stable expression, for regulation of expression in both time (developmental regulation) and space (tissue specificity), and, soon, for insertion at specific sites in the genome. Foreign genes can be tailored so that proteins encoded by them are accumulated in almost all subcellular compartments or are secreted. Abiotic and biotic control of gene expression is progressing via the development of numerous chimeric promoters in model transgenes and plants.
This area of research has developed so much that a comprehensive list of possibilities for controlling and targeting gene expression is no longer feasible. Much research remains to be done to optimize and refine these processes.
The challenge to build durable pest and disease resistance by direct gene transfer will be to produce oligo-transgenic plants in order to pyramid resistance genes and to modify biosynthetic pathways of defence compounds as well as to introduce new ones in potato. Recent developments in engineering disease resistance in plants reveal that the combination of several transgenes in a single genotype led to a synergistic effect on disease resistance.
Therefore, an oligo-transgenic approach has to be pursued to pyramid genes for resistance. Transgenic approaches should complement natural host-plant resistance by the direct gene transfer to locally adapted varieties with the highest endogenous resistance available.
Whole-plant approaches, using the phenotypic expression of resistance genes, have recently been demonstrated through the use of molecular technology to clone several resistance genes. All these resistance genes confer a qualitative resistance that is easy to score for the presence versus absence of the resistance gene in a segregating progeny. Other types of resistance are inherited quantitatively (quantitative trait) and methods for identification of QTL have been developed.
QTL analyses allow the identification of resistance-related genes that account for a significant amount of the phenotypic variation observed in a segregating population for the trait of interest. DNA markers have allowed the exploitation of wild and native Solanum germplasm, but so far have not allowed the development of new varieties per se. The application of marker-assisted selection in potato therefore remains a major challenge, mainly because of the potato’s genetic constitution.
The growing importance of the transgenic approach in potato crop improvement makes it urgent to develop appropriate public awareness activities and reduce risks associated with transgenic potatoes. Commercial applications of potato genetic engineering have so far been restricted to developed countries, but developing countries will also likely elaborate improved locally adapted varieties and products for local or regional markets using biotechnology.
These molecular techniques and products can be efficiently transferred by international agricultural research centres to national agricultural research systems of developing countries. Farmers and small-scale industries in developing countries will be able to generate higher incomes by developing new products at lower costs. The overall research efforts in applying sophisticated technology to potato improvement aim to develop sustainable production systems that will improve the well-being of potato farmers worldwide.