Kirsten Jørgensen: Research to help sub-Saharan Africa

Kirsten Jorgensen with her transgenic cassava plants
Kirsten Jorgensen with her transgenic cassava plants

Kirsten Jørgensen obtained her MSc in biology at the University of Copenhagen in 1989. The focus of her Ph.D studies was the identification of auxin-binding proteins in Brassica napus. The work was carried out at the Danish Institute of Agricultural Sciences in Roskilde. Following her Ph.D. she was employed in Danisco Biotechnology, Holeby, Lolland, Denmark as responsible for the plant biotechnology R&D laboratory. This laboratory bred new varieties of sugar beet, rapeseed, sunflower, and potatoes using biotechnological approaches. The main techniques implemented were transformation, double haploid formation, and micropropagation.

In 2000 she was employed as Associate Professor at the Plant Biochemistry Laboratory, Department of Plant Biology, University of Copenhagen where her work focused on molecular breeding of cassava to achieve acyanogenic-transformed lines high in protein and vitamin content. As an expert in imaging techniques used for tissue, cell, and organellar localization of gene expression, enzymes, and enzyme activities, her network of collaborators is extensive.

She is married with three grown-up daughters, and is now a grandmother to three boys aged 1-3.

Please describe your work on acyanogenic cassava and its importance. What is the status of the research?
I first worked on cassava in 2000 when I started to work in the group of Prof. Birger Lindberg Moeller, together with part-time technician, Christina Mattson. Today, the group consists of Asst Prof. Rubini Kannangara, who takes care of the molecular biology; three technicians: Charlotte Sørensen, Evy Olsen, and Susanne Bidstrup, who assist in all aspects of this project from producing the transgenic plants, analyzing them, and helping in the greenhouse, where our gardener Steen Malmmose takes care of the plants.

The cassava group is a part of a larger group with a focus on cyanogenic glucosides—from the regulation of these compounds in the plant to their end use as a defense system. In cassava, our emphasis is now the “when”, “where”, and “why” the cyanogenic glucosides are found in the plant. We also work on producing an acyanogenic cassava.

We are currently working on producing the third generation of genetically modified organisms (GMOs) and analyzing the second generation in the greenhouses. The first generation was based on antisense technology and the background of the transformed plants was the South American model line Mcol22. When the RNAi technology became available for downregulation (reduction) of specific genes, we used this technique to obtain second generation plants, exhibiting a more complete downregulation of cyanogenic glucoside content (second generation). Eventually we started to work with African elite lines from IITA to be closer to the product that could be used directly after testing the GMO lines in their appropriate environment. In the third generation we have been fine-tuning the downregulation of cyanogenic glucosides to assure that this takes place in the specific cells which express the enzymes involved in their synthesis.

Plants from the first generation, based on Mcol22, have limited utility for field testing as they are far from the cultivars grown today. Our focus has shifted to African lines, either those used today or promising breeding lines from IITA. By now, we have African elite lines (e.g., TME12) downregulated to contain less than 10% of the cyanogenic glucoside content in tubers measured in wild type TME12 growing in the greenhouse. Several lines are completely devoid of cyanogenic glucosides in their leaves.

Kirsten Jorgensen and Rubini Kannangara, Plant Biochemistry Laboratory
Kirsten Jorgensen and Rubini Kannangara, Plant Biochemistry Laboratory

Our next goal is to produce cassava lines with enhanced nutritional value. We have focused on using a storage protein from potato (patatin) and are currently transforming African elite lines with this trait provided by IITA’s Dr Alfred Dixon. Two of these lines have been bred to contain an enhanced amount of carotenoids, the precursor of vitamin A. Our dream is to assemble all these traits—producing cassava that is acyanogenic and nutritious.

What are some of the important tools you use on the job? How would genetic engineering help you meet your research goals?
The tools are all the techniques currently used today in a modern, biotechnology oriented laboratory. The basic knowledge of the synthesis of cyanogenic glucosides gives us the opportunity to strengthen work for an improved cassava. Our group works with basic science, which is then converted to applied research, and ends up, for example, in new cassava lines improved by molecular breeding—another word for genetic engineering. Genetic engineering is just a tool which can be used where it is difficult or impossible to achieve the improvements wanted in a variety. So far, no one has succeeded in obtaining acyanogenic cassava by classical breeding methods. Here genetic engineering comes in as an important tool.

What are some of the challenges in working on this area?
Working with a crop which has limited focus from breeding companies makes it difficult to obtain funding in a nontropical country such as Denmark. Because cassava is a tropical crop, it is difficult to mimic tropical conditions—however, we are pleased that we are able to grow the cassava plants in our greenhouse under conditions where they do produce tubers. So our data are based on measurements on real tubers.

As the scientific community working on cassava is small, we need to share knowledge. On our part we have been open in sharing our techniques. So far Dr Ivan Ingelbrecht, IITA, and Dr Sareena Sahab, Danforth Plant Science Centre, have visited us and been trained on how to carry out cassava transformation using our protocols and regeneration systems.

Who are your partners in this collaborative effort and what are their roles? Who funds the research?
Our collaborator for more than a decade has been IITA with whom our group has collaborated on various projects mostly funded by Danish International Development Agency (DANIDA). IITA has also provided important financial support. In the same period we have collaborated with CIAT, Columbia, on molecular markers for the genes encoding the enzymes involved in the synthesis of cyanogenic glucosides. A newer collaboration is with Kenyatta University, Kenya, with whom we have collaborated on the latest DANIDA project “Improvement of the nutritional value of cassava: high storage protein content and no cyanide liberating toxins”.

In addition to the funding from DANIDA we had a project on “Biofortification of Cassava” funded by the Research Council for Technology and Production.

The funding and generous sharing of elite lines from IITA have strengthened the ties between our laboratory and IITA.

How would you describe the collaboration with IITA and other partners working on the project? Any insights on collaboration and partnership?
The close and fruitful collaboration with Dr Ivan Ingelbrecht and Dr Alfred Dixon has helped us a lot, for example, with respect to choosing optimal cassava lines for our transformation work. We really want to work with lines that are of value to African end users. In addition to the collaboration on producing GMO cassava, we have collaborated on the bioinformatics and logistics to design and build a cassava microarray DNA chip. Our collaborations have been very open and enjoyable. For us, it is very important to keep close contact with scientists working in an African environment. This helps us to set the right research priorities.

How would you measure the impact of your work on cassava in SSA?
Our aim is to improve the nutritional value of cassava. This includes reducing its content of cyanogenic glucoside and introducing a higher content of proteins and vitamin A precursors. In our lab we can only go as far as producing these lines and testing them in our greenhouse facilities. Although the lines behave well there, we cannot mimic real tropical conditions and cannot expose the plants to the environmental challenges they encounter when grown in African soils. So we really want to collaborate to have these lines grown in their real environment to observe how the plants behave.

Any personal information or other insights that you want to share with our readers?
Throughout my working life, the emphasis has been to produce new improved crops—both for the European market and now for the African continent in the case of cassava—using biotechnological techniques. It is important always to use the appropriate techniques to reach the goal most efficiently. I am driven by a strong desire to show that high quality basic research provides the way to obtain improved crop plants for the future.

One of my main interests is working with plants—both at work and at home, where I spend a lot of time in the garden and in our summerhouse. The rest of the time is for the family—I look forward at one point to visit Africa and especially IITA.

In my career I have wanted to use my knowledge in applied science. Tissue culture fascinates me—to start from such small pieces of tissue and end up with plants in the greenhouse—I am still amazed at what plants can do.

CBSD: Enemy number 1

Caroline Herron,

Tertiary vein chloroses of CBSD. Photo by C. Herron
Tertiary vein chloroses of CBSD. Photo by C. Herron
A major disease is ravaging cassava production in the lowland areas of Eastern Africa. The culprit is the cassava brown streak disease (CBSD), which has been spreading to higher altitude areas.

CBSD was first described in detail in Tanzania in the early 20th century and for decades, has been causing severe economic losses in coastal and low altitude Eastern Africa where cassava mosaic disease (CMD) is endemic.

CBSD symptoms are not as distinct in cassava foliage as those of CMD. The main differences are that in CBSD the leaves are not distorted in shape (no epinasty); and the myriad of chlorotic mosaics and blotches in the leaves commonly start as tertiary vein chloroses and are also often very insignificant compared with those from CMD.

Two symptoms in the same plant: CMD (top left) and CBSD (bottom right). Photo by C. Herron
Two symptoms in the same plant: CMD (top left) and CBSD (bottom right). Photo by C. Herron
CBSD leaf symptoms may be limited to the lower leaves only, depending on the cultivar. If the above-ground symptoms are in the lower leaves only, then during plant maturity these often senesce, and thus symptoms can be missed. Other CBSD symptoms in very susceptible cultivars are necrotic longitudinal streaks on the stem (from where the disease derives its name)—which eventually coalesce from the shoot downwards, plant shoot dieback, and petiole necroses.

In the tuberous storage roots of susceptible cultivars, CBSD’s dry necrotic lesions may develop from approximately 5 months onwards, depending on the cultivar, with or without any external root symptoms. Root lesions vary widely; in a cross-section of roots these symptoms may range from dry dark brown radially aligned lesions to lesions with a dry white firm interior and a necrotic exterior. The time of first appearance of root symptoms and the tissues in which the symptoms appear are also variable.

External symptoms in some cultivars appear as radial root constrictions, creating abnormal root shapes. Root symptoms may also be irregularly distributed across fields planted with the same cultivar and also within roots of the same plant. CBSD in the roots can lead to significant reductions in quality and yield, or complete spoilage.

Various CBSD root symptoms on cultivars from Tanzania. Photo by C. Herron, IITA
Various CBSD root symptoms on cultivars from Tanzania. Photo by C. Herron, IITA
Economic costs
Alleviating the CBSD problem would reduce human burden and toil during the growing season and during processing, and enhance livelihoods.

On average, cassava yields are approximately 3.5 t/ha in CBSD-infested fields compared to 10 t/ha in fields without CBSD. The estimated economic loss due to CBSD is about US$130/ha, based on a sample in eastern Tanzania. Extrapolating to the entire cassava-producing regions of the country shows a loss of $45 million/year based on a conservative 14% yield loss from early harvesting to escape heavy CBSD losses. If the farmers’ estimated yield loss of 64% at full physiological maturity is taken into consideration, the estimated annual loss for the country is as high as $202 million (Manyong et al. 2008).

Current management strategies
The use of field-resistant cultivars is the recommended disease management strategy. Many field-resistant cultivars should be deployed at any one time in every cassava cultivation area where CBSD is endemic. Susceptible cultivars are not recommended for these areas.

Across the region, many national agricultural research systems (NARS) partners are involved in ongoing cassava breeding programs and multilocational field trials. True seeds from lines of resistant materials identified in CBSD-infested areas (coastal Tanzania) over many growing seasons have been distributed to surrounding countries for incorporation of resistance in farmer-acceptable cultivars. This management strategy has allowed farmers in some of the worst-affected CBSD areas, who had given up growing cassava, to grow and thrive from cassava once more.

Annual monitoring and evaluation of the CBSV presence and CBSV strains over production areas should be combined with this approach. Other management strategies are also being evaluated currently for usefulness, such as use of virus-free propagative materials.

Sustaining disease management
Sustaining CBSD management through planting field-resistant cultivars means that scientists cannot afford to be complacent. While these cultivars are proving to be an excellent and the sole current first line of defense against the pathogen, the situation is dynamic, and CBSD damage may be expected over time. This is because genetically diverse pathogens, if allowed to exist widely and in large numbers, can eventually cause damage to earlier tolerant cultivars. Certain pathogen strains or isolates become more “fit” within the population, eventually leading to disease damage or “resistance breakdown”.

Ideally, a continuous “pipeline” of cultivars may be needed in every cassava-growing area, together with large-scale detection and monitoring of CBSV strains predominant in the population in any given area on an annual basis. This will provide an early warning detection system for the CBSV strain population shift or resistance breakdown. Long-term management strategies and the development of true resistant cassava cultivars are still desirable.

To date, all new cassava breeding lines tested in Tanzania in field trials have CBSV in many tissues. This indicates that CBSV immunity may not be present within currently used materials. Other attempts must thus be made to provide useful and different mechanisms of resistance to the pathogen. Hybridization between cassava and wild relatives and use of pathogen-mediated resistance could provide various types of resistance to CBSV.

cassava_tissue_cultureThe transformation approach
Constraints with the traditional approach make the use of transformation a viable alternative in incorporating virus resistance to cassava. The Mikocheni Agricultural Research Institute (MARI) under the Ministry of Agriculture, Food Security and Co-operatives, and IITA are undertaking cassava genetic transformation in Tanzania. The project is funded by the Rockefeller Foundation, but will continue to be funded in 2009 by the Bill and Melinda Gates Foundation. Other partners include the Plant Biosafety Office, Tropical Pesticides Research Institute; Agricultural Biosafety Scientific Advisory Committee; National Biosafety Focal Point; and National Biotechnology Advisory Committee.

To date, work on incorporating resistance to the East African cassava mosaic virus and CBSV in cassava cultivars is ongoing. Twenty-six farmer-preferred cultivars from Tanzania that are high yielding but susceptible to CMD and CBSV have been micropropagated. From these cultivars, a total of 1014 plantlets had been produced by end-September 2008. So far, cassava cultivar Kibandameno produced the highest number of plantlets (282) followed by Karatasi (58), while Pikipiki and Katakya from the Lake Victoria zone produced the least (4). These tissue culture plantlets will be used for producing embryogenic cultures for transformation.

Manyong V.M, E.E. Kanju, G. Mkamilo, H. Saleh, and V.J. Rweyendela. 2008. Baseline study on livelihood status and technology adoption levels in Cassava Brown Streak Disease (CBSD)-infected areas of Eastern Tanzania and Zanzibar. Technical Report. IITA-Tanzania. 37 pages.