Biotechnology

The Biotechnology Division of the Agricultural Research Council’s Vegetable and Ornamental Plant Institute is situated at Roodeplaat, near Pretoria. The Division specializes in research on vegetables, ornamental plants, indigenous and grain crops. The Biotechnology Division offers expertise in research, development, and transfer of biotechnology products and services to resource poor and commercial farmers, plant breeders and other agricultural organizations. Research projects have been identified and implemented by the Biotechnology Division with the aim of developing new cultivars better suited to South African conditions.

The research group of the Biotechnology Division actively promotes collaboration with other ARC institutes, the Council for Scientific and Industrial Research (CSIR), universities, agricultural industries and the National Department of Agriculture.

There are several integrated research programs within the Biotechnology Division:

Abiotic stress

• Drought and heat are some of the main factors limiting agricultural productivity world-wide. The aim of this program is to improve agricultural crops through identification and evaluation of traits associated with drought and heat tolerance in higher plants. Currently, drought screening is performed on crops such as potato, soybean, Vigna, Amaranthus and sweetpotato. Drought screening on sweet potato is particularly done between 3 different research sections to achieve maximum input on cultivation and evaluation. The screening of the sweet potato does not only entitles the physiological screening of the plant itself but also the physical plant growth performance as well as the yield of tubers are taken into account for the calculation of adaptability. Physiological and biological screening methods are applied to select for drought and heat tolerance / sensitivity traits, and results are used to rate crops for adaptation to extreme conditions.
  The responses of plants to the environment determine the adaptation of such plants and influence their behaviour during stress periods.  An understanding of the plant's response to environmental stress will enable the development of methods for achieving efficiency increases. The most prevalent environmental stress on plants involves water status.  Plant species vary in their sensitivity and response to the decrease in water potential caused by drought. Genetic engineering and conventional plant breeding have the potential to enhance the efficiency of crop production when certain stresses are present by providing new cultivars with greater resistance to abiotic stresses.
  This information is applied in strategies for the selection of drought tolerance in breeding programs. Genes associated with these traits have been incorporated into crops using transformation techniques. Potato was transformed to increase expression of the CuZn superoxide dismutase gene, and soybean with the P5CR (proline biosynthesis) gene. Ultimately the aim is to progress towards the improvement of crop productivity, quality and food security in arid and semi-arid areas of the world.

GM Plant Evaluation (P5CR Soybean)

• A non-tissue culture Agrobacterium-mediated vacuum infiltration transformation system for soybean (patent SA 2001/3076 in the name of ARC/PRF) was developed.  The role of proline biosynthesis in response to drought stress in soybean was investigated by the use of sense and antisense gene technology. The Arabidopsis L-D1-Pyrroline-5-carboxylate reductase (P5CR) gene was cloned in the sense and antisense orientation into a heat shock cassette containing an inducible heat shock promoter (IHSP). The construct allowed the gene's expression and consequent proline production to be manipulated in plants subjected to environmental stress conditions. The construct was used in the production of transgenic soybean plants in cultivar Ibis. Analysis indicated that the transgenic genotypes contained at least 5 copies of the P5CR gene with at least three integrations. Thorough evaluation for drought tolerance in the greenhouse and laboratory were performed using physiological, anatomical and molecular techniques over a 4 year period. Six genotypes were planted in the rainout shelter trial upon selection of their improved drought performance in the greenhouse experiments, together with one antisense line and three wild type cultivars.  Results from the field trial indicated that all six sense transgenic lines were more heat tolerant than the three wild type cultivars.  Four of these lines produced very high yields under the drought conditions.

Bt Potato

• Cultivated potato (Solanum tuberosum) is one of the world’s major food crops, following rice, wheat and maize.  Potato tuber moth hampers potato production in many parts of the world, including S.A.Potato production in developing countries is, however, plagued by the potato tuber moth (Phthorimaea operculella; PTM).  Currently, the only available control measure is the use of chemical pesticides.  A more desirable alternative is the development of potato tuber moth resistant varieties through conventional breeding, but this process would require a 10 – 12 year period and is hampered by a lack of resistant germplasm.  Therefore, introducing resistance to the PTM through biotechnology is the only alternative in order to develop resistant varieties.  Potato farmers and the consumers will benefit through lower input costs as well as reduced post-harvest losses. This product will lead to an increased marketable yield for both small-scale and commercial farmers due to the improved quality of their product.  Further advantages are that human exposure to chemicals and pesticides residues on potatoes will be reduced substantially.

Plant Molecular Biology

• Plant resistance to fungi is being studied by determining the role of polygalacturonase inhibiting proteins (PGIP).  The apple pgip1 and apple pgip2 genes have both been isolated from apple, expressed in transgenic tobacco, and the proteins purified to homogeneity for use in PGIP: fungal polygalacturonase (PG) inhibition studies.  These studies give a clear indication as to the inhibitory abilities of the apple PGIPs, and gives researchers at ARC-VOPI clearer understanding of the relevant apple pgip gene to be used in the production of fungal resistant transgenic crop lines. 
• The apple pgip1 gene has also been expressed in transgenic potato and evaluated in the greenhouse for increased resistance to Verticillium dahliae. 
• The project “Genetic Improvement of maize to enhance food safety by introducing resistance to Fusarium verticillioides” was funded by the European Union and the partners were: Biotechnology Division; ARC-VOPI; CSIR Bio/Chemtek; Department of Botany, FABI, University of Pretoria; Plant Research International, The Netherlands; Crop Science Department, University of Zambia and the Department of Plant Biology, University of Rome, Italy.  The main objective of this research was to develop maize with durable resistance to F. verticillioides.  The research targeted Zambia and South Africa in order to establish a core regional expertise in both the pathogen and host.  A further objective was to test the hypothesis that engineered resistance is enhanced through synergistic interaction between antifungal proteins with different modes of action.  Four anti-fungal defense genes derived from plants, namely a polygalacturonase-inhibitor protein (pgip), a chitinase, a b-glucanase and a maize ribosome-inhibiting protein (b-32) were chosen.  
• Lupin anthracnose represents a major threat to the South African lupin industry and in an effort to combat lupin anthracnose a project was initiated at ARC-VOPI to introduce defense genes into a white lupin (Lupinus albus) and a narrow leaf lupin (Lupinus angustifolius) cultivar in collaboration with Bio/Chemtek at the CSIR, Pretoria, South Africa.  The project was funded by the Protein Research Foundation (PRF).  The aim of the project was to develop a strategy towards anthracnose resistance in lupin using molecular techniques. Colletotrichum species are considered to be major plant pathogens of cereals and legumes around the world, causing significant crop losses.  Colletotrichum acutatum causes anthracnose disease on lupin.
• A major point of contention around agricultural biotechnology in developing countries involves intellectual property issues.  To this end, researchers at ARC-VOPI have undertaken to isolate their own promoters, as well as drought tolerance and plant defense genes. 
• An inducible promoter has been isolated from lupin, and biolistic transformation of various plant tissues has shown that the isolated fragment has promoter activity.  Tobacco plants transformed with the promoter: gus constructs have also been produced, and promoter activity has been demonstrated in planta. Furthermore, the promoters of several of the pgip genes are also being isolated.
• A new project has been initiated that aims to identify and isolate the genes conferring drought tolerance in cowpea for use in vegetable crops of importance to resource poor farmers in South Africa.  In recent years several techniques have been developed for the isolation of differentially expressed genes, namely suppression subtractive hybridization (SSH) and DNA microarray analysis. These methods will thus be used to identify and isolate the gene/s leading to drought tolerance in cowpea. 

Transformation

Plant transformation forms an integral part of the Biotechnology Division of ARC-VOPI.  Agrobacterium tumefaciens-mediated transformation is mainly being utilized, but success has been obtained with biolistic transformation.  Ornithogalum, a bulbous ornamental plant, has been successfully transformed with a herbicide resistance gene using biolistics.  Since Ornithogalum is important to the South African pot plant industry, it has also been transformed with a virus resistance gene using Agrobacterium-mediated transformation.  Another important focus is the transformation of vegetables with a wide array of genes.  Tomato has been transformed with genes expressing resistance to fungal and viral infections, e.g. tomato spotted wilt, and potato has been transformed with genes rendering resistance to potato leaf roll virus, potato virus Y as well as the Bt (Bacillus thuringiensis)gene rendering resistance to the potato tuber moth. Crops other than vegetables such as lupin have been transformed with fungal resistance genes and soybean has been transformed with drought resistance genes.

Virus elimination

• Virus elimination techniques have been developed for potato, sweetpotato and cassava to provide meristem-derived disease free material for use in propagation, breeding, in various genebanks, and to provide certified virus-free planting material to farmers and the commercial sector.

Molecular markers

• A plant molecular marker laboratory has been established to support the ARC-VOPI breeding programs. It provides a service to clients for seed lot purity testing, trueness-to-type confirmation, cultivar identification and confirmation of parentage. The main techniques employed are random amplified polymorphic DNA (RAPDs) and simple sequence repeats (SSRs). Marker assisted selection using PCR forms part of the services offered. Marker technology is also used for estimation of variation in Protea populations, germplasm management of sweetpotato as well as macadamia’s and onions.