Transgenic banana for Africa

Leena Tripathi, l.tripathi@cgiar.org

Banana (Musa spp.) are one of the most important food crops after maize, rice, wheat, and cassava. Annual production in the world is estimated at 130 million t, nearly one-third of it grown in sub-Saharan Africa, where the crop provides more than 25% of the food energy requirements for over 100 million people. East Africa is the region that produces and consumes the most banana in Africa. Uganda is the world’s second largest producer after India, with a total of about 10 million t.

Banana plantation damaged by Xanthomonas wilt. Photo by IITA.
Banana plantation damaged by Xanthomonas wilt. Photo by IITA.

The banana Xanthomonas wilt (BXW) disease caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm) was first reported about 40 years ago in Ethiopia on Ensete spp., a close relative of banana. Outside Ethiopia, BXW was first identified in Uganda in 2001, subsequently in the DR Congo, Rwanda, Kenya, Tanzania, and Burundi. The disease is highly contagious and is spread plant-to-plant through the use of contaminated agricultural implements. It is also carried by insects that feed on male buds, and is present on plant material, including infected debris. The rapid spread of the disease has endangered the livelihoods of millions of farmers who rely on banana for staple food and cash.

Infection by Xcm results in the yellowing and wilting of leaves, uneven and premature ripening of fruits, and yellowish and dark brown scars in the pulp. Infected plants eventually wither and die. The pathogen infects all varieties, including East African Highland Banana (EAHB) and exotic types, resulting in annual losses of over US$500 million across East and Central Africa.

Options for BXW control using chemicals, biocontrol agents, or resistant cultivars are not available. Although BXW can be managed by following phytosaniary practices, including cutting and burying infected plants, restricting the movement of banana materials from BXW-affected areas, decapitating male buds, and using “clean” tools, the adoption of such practices has been inconsistent. They are labor-intensive and farmers believe that debudding affects the fruit quality.

The use of disease-resistant cultivars has been an effective and economically viable strategy for managing plant diseases. However, resistance to BXW has not been found in any banana cultivar. Even if resistant germplasm is identified, conventional banana breeding to transfer resistance to farmer-preferred cultivars is a difficult and lengthy process because of the sterility of most cultivars and also the long generation times.

Transgenic technologies that facilitate the transfer of useful genes across species have been shown to offer numerous advantages to avoid the natural delays and problems in breeding banana. They provide a cost-effective method to develop varieties resistant to BXW. Transgenic plants expressing the Hypersensitive Response Assisting Protein (Hrap) or Plant Ferredoxin Like Protein (Pflp) gene originating from sweet pepper (Capsicum annuum) has been shown to offer effective resistance to related Xanthomonas strains.

Plants established in confined field trial 5 months after planting. Source: L. Tripathi, IITA.
Plants established in confined field trial 5 months after planting. Source: L. Tripathi, IITA.

IITA, in partnership with the National Agricultural Research Organization (NARO)-Uganda and the African Agriculture Technology Foundation (AATF), has developed transgenic banana expressing the Hrap or Pflp gene using embryogenic cell suspensions or meristematic tissues of four banana cultivars, Sukali Ndiizi, Mpologoma, Nakinyika, and Pisang Awak. More than 300 putatively transformed plants were regenerated and validated via PCR assay and Southern blot. Of these, 65 transgenic plants have exhibited strong resistance to BXW in the laboratory and screenhouse tests. The plants did not exhibit any differences from their nontransformed controls, suggesting that the constitutive expression of these genes has no effect on plant physiology or other agronomic traits.

The 65 resistant lines were planted in a confined field trial in October 2010 at the National Agriculture Research Laboratories (NARL), Kawanda, Uganda, after approval was obtained from the National Biosafety Committee. These transgenic lines are under evaluation for disease resistance and agronomic performance in field conditions. The transgenic lines are slated for environmental and food safety assessment in compliance with Uganda’s biosafety regulations, and procedures for risk assessment and management, and seed registration and release. After completing the necessary biosafety validation and receiving approval from the Biosafety Committee, the Xcm-resistant cultivars are expected to be deregulated for cultivation in farmers’ fields in Uganda.

We plan to stack the Pflp and Hrap genes in the same cultivars to enhance the durability of resistance against Xcm. We have developed more than 500 transgenic lines with the double genes construct (pBI-HRAP-PFLP) which are being evaluated for disease resistance under contained screenhouse conditions.

This technology may also provide effective control of other bacterial diseases such as moko or blood disease, of banana occurring in other parts of the world. The elicitor-induced resistance could be a very useful strategy for developing broad-spectrum resistance. The elicitor is a protein secreted by pathogens that induce resistance. The transgenic banana carrying these genes may also display resistance to fungal diseases such as black sigatoka and Fusarium wilt. Experiments on this are being conducted in our lab in Uganda.

Confined field trial of banana plants. Source: L. Tripathi, IITA.
Confined field trial of banana plants. Source: L. Tripathi, IITA.

We are also planning to stack genes for resistance to Xcm and nematodes into one line to produce cultivars with dual resistance that would tackle two of the most important production constraints in Eastern Africa.

The development of Xcm-resistant banana using the transgenic approach is a significant technological advance that will increase the available arsenal of weapons to fight the BXW epidemic and save livelihoods in Africa. It can become a high-value product for farmers.

This research is supported by the Gatsby Charitable Foundation, AATF, and USAID.

Note: The Pflp and Hrap genes are owned by Taiwan’s Academia Sinica, the patent holder. IITA has negotiated a royalty-free license through the AATF for access to these genes for use in the commercial production of BXW-resistant banana varieties in sub-Saharan Africa.

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.

Is genetically modified cowpea safe?

Genetically modified cowpea resistant to the cowpea pod borer (Maruca vitrata) will soon become a reality. This transgenic cowpea contains the gene from the soil microbe Bacillus thuringiensis (Bt) that is toxic to the pests. But before that happens, IITA is making sure that it addresses some of the potential risks associated with using such genetically modified organisms (GMO).

Typical damage by pod borer caterpillar. Photo by M. Tamo
Typical damage by pod borer caterpillar. Photo by M. Tamo, IITA

IITA started preliminary studies to assess concerns, including the development of resistance by the target insect pest to the insecticidal protein expressed in the plant, negative effects of the insecticidal protein on nontarget organisms present in the same agroecosystem, such as natural enemies or pollinators, the accidental introduction of the gene expressing the toxic protein into wild relatives of cowpea (referred to as “gene flow”), and negative effects on human and animal health.

In the meantime, a team of scientists headed by Dr T.J. Higgins of the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Australia, was able to transform cowpea successfully with the Bt toxin-expressing gene. The transgenic plant has been tested in Puerto Rico and is not yet available for testing in Africa.

Parasitic wasp, Phanerotoma leucobasis, laying egg into egg of pod borer. Photo by M. Tamo
Parasitic wasp, Phanerotoma leucobasis, laying egg into egg of pod borer. Photo by M. Tamo, IITA

IITA started evaluating some of the unintended effects of the purified Bt-toxin on nontarget organisms, focusing on natural enemies of the target insect pest, the caterpillar of the pod borer (M. vitrata).

In our first case study, we used a locally available natural enemy, a small parasitic wasp called Phanerotoma leucobasis, which develops by destroying caterpillars of the cowpea pod borer. This wasp has a curious biology because it can insert its small egg into the bigger egg of the pod borer, but its immature stages develop inside the caterpillar only when it starts feeding on the cowpea plant. It destroys the pod borer’s internal organs from the inside, ultimately killing it.

Following standard protocols in collaboration with Purdue University, USA, we first determined the lethal dosage of the Bt-toxin that could kill 50% and 95% of the young caterpillars. Subsequently, we let the wasp parasitize the eggs of the pod borer, and transferred the hatching caterpillars onto an artificial rearing diet contaminated with different doses of the toxin to let them feed on it.

Exotic parasitic wasp Apanteles taragamae. Photo by G. Georgen
Exotic parasitic wasp Apanteles taragamae. Photo by G. Georgen, IITA

The level of wasp mortality recorded in this experiment favorably compares with results obtained in other studies, and is primarily due to the death of the host caterpillar while feeding on the contaminated diet. Similar experiments are ongoing, using another natural enemy of the pod borer, the exotic parasitic wasp Apanteles taragamae introduced into our laboratories from the World Vegetable Center (Asian Vegetable Research and Development Center) in Taiwan.

What would then be the likely impact of Bt cowpea on these natural enemies in the field?

For now, we know from previous studies (Romeis et al. 2006) that the negative, unintended effects of Bt-transformed crops such as corn and cotton on natural enemies and biodiversity at large are far less than those caused by repeated applications of synthetic pesticides to control the same pests under conventional crop protection schemes. For the cowpea pod borer, several alternative host plants exist in the wild where the pest is exposed to the attacks of natural enemies throughout the year, hence providing natural refugia and thus avoiding being negatively impacted by the Bt-toxin present in the transformed cowpea.

Reference
Romeis, J., M. Meissle, and F. Bigler. 2006. Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nature Biotechnology. 24:1. p 63-71. January. www.nature.com/naturebiotechnology

Is biotechnology a panacea?

cover_photo1Biotechnology is often understood to mean a single technology. In reality it is a collection of technologies that can be applied to address many challenges in agriculture (crop and animal health, food production) pharmaceuticals, and medicine. Biotechnology is often seen as a panacea which is not the case; it is one more tool, albeit an important one, in the arsenal of tools used against the challenges humanity faces. In agriculture, the technology can help accelerate the development of crops resistant to insects and disease, the development of new uses for agricultural products, livestock vaccines, and improved food qualities. African institutions from Cairo to Cape Town, from Dakar to Dar-es-Salaam are using biotechnology in diverse ways.

IITA’s position on biotechnologies is similar to that on all other sciences. We think Africa, its ministries, universities, teaching hospitals, and other research institutions, should not be excluded from any science. Just the need to know, so as to advice governments on the usefulness of a technology to a country’s needs, requires their involvement and knowledge in that science. Whether a particular product of that technology, e.g., genetically modified crops, is adopted or not, is a decision made by governments and not by scientists.

A vibrant local market in Ibadan, Nigeria. Photo by IITA
A vibrant local market in Ibadan, Nigeria. Photo by IITA

Although many African governments are on the brink of embracing the promised benefits of biotechnology, they have not totally committed in terms of providing government funding for more research in agricultural and social/economic development, or policy support for science. What is needed is for R4D institutions, such as IITA and its partners to continue to provide knowledge about these important technologies and their possible impact on sub-Saharan Africa.

This issue highlights some of the cutting-edge work that IITA and its partners (AATF, NARS, donors, NGOs) are doing to help find solutions to problems in tropical agriculture, and thus provide more food and improved livelihoods for the millions of people depending on agriculture. The R4D Review welcomes feedback and comment about any of the information and work featured in this issue. We encourage you to visit the online R4D Review at www.r4dreview.org.

“IITA does not and has not approved or disapproved the use of GM crops in any country. IITA uses all available scientific tools and approaches in its attempt to address hunger and poverty, but the decision to reject or approve and adopt any GM products is the domain and responsibility of the respective national governments. IITA, and rightly so, has no say in such a decison. Any comments to the contrary misrepresent the facts.” —Hartmann, IITA Director General [updated from print version on 25/03/09 ED]