Transgenics in crop improvement research

Leena Tripathi (l.tripathi@cgiar.org)
Biotechnologist, IITA, Nairobi, Kenya

Biotechnology has opened unprecedented avenues for exploring biological systems. Transgenics is one of the key techniques particularly useful for the genetic improvement of crops that are not amenable to conventional breeding, such as those that are vegetatively propagated. In IITA, transgenic technologies are being used for improving banana/plantain (Musa sp.), cassava (Manihot esculenta), and yam (Dioscorea sp.).

Harvested bunch of transgenic banana, Kampala, Uganda. Photo by L. Tripathi.
Harvested bunch of transgenic banana, Kampala, Uganda. Photo by L. Tripathi.
Genetic transformation platform
An efficient protocol for plant regeneration and transformation is a prerequisite for the successful use of transgenic technologies. Despite the technical difficulties in transforming monocot species, efficient transformation protocols that are embryogenic cell suspension based and Agrobacterium mediated have been established for many cultivars of banana/plantain. This system, however, is a lengthy process and cultivar dependent. Therefore, a transformation protocol using meristematic tissues was also established which is rapid and genotype independent. These protocols have paved a way for the genetic manipulation of banana/plantain by incorporating agronomically important traits such as those conferring resistance to diseases or pests as well as tolerance to abiotic stress factors.

Agrobacterium-mediated transformation protocols for three popular cassava varieties preferred by African farmers were established through somatic embryogenesis. A regeneration and transformation protocol is also established for yam (Dioscorea rotundata and D. alata) using nodal explants, but transformation efficiency needs to be improved. A transformation protocol using somatic embryogenic callus for yam is under development.

Development of disease- and pest-resistant transgenic crops
Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum (Xcm), is the most devastating disease of banana in the Great Lakes region of Africa. In the absence of natural host plant resistance, IITA, in partnership with NARO-Uganda and the African Agricultural Technology Foundation, has developed transgenic banana by constitutively expressing the Hypersensitive Response Assisting Protein (Hrap) or plant ferredoxin-like protein (Pflp) gene from sweet pepper (Capsicum annuum). The transgenic plants have exhibited strong resistance to BXW in the laboratory and screenhouse tests. The best 65 resistant lines were planted in a confined field trial at the National Agricultural Research Laboratories (NARL), Kawanda, Uganda, for further evaluation.

Transgenic technologies provide a platform for controlling diseases in banana, cassava, and cowpea. Photo by IITA.
Transgenic technologies provide a platform for controlling diseases in banana, cassava, and cowpea. Photo by IITA.
Based on results from mother plants and their first ratoon plants, 12 lines were identified that show absolute resistance. The plant phenotype and the bunch weight and size of transgenic lines are similar to those of nontransgenic plants. These lines will be further tested in a multilocation trial in Uganda. They will be evaluated for environmental and food safety in compliance with Uganda’s biosafety regulations, risk assessment and management, and procedures for seed registration and release, and are expected to be released to farmers in 2017.

Cassava brown streak disease (CBSD) has emerged as the biggest threat to cassava cultivation in East Africa. As known sources of resistance are difficult to introgress by conventional methods into the cultivars that farmers prefer, the integration of resistance traits via transgenics holds a significant potential to address CBSD. Of the available transgenic approaches, RNA silencing is a very promising strategy that has been successfully employed to control viral diseases. IITA, in collaboration with Donald Danforth Plant Science Centre (DDPSC), USA, is developing CBSD-resistant cassava for East Africa.

Nematodes pose severe production constraints, with losses estimated at about 20% worldwide. Locally, however, losses of 40% or more occur frequently, particularly in areas prone to tropical storms that topple the banana plants. IITA, in collaboration with the University of Leeds, UK, has generated transgenic plantain using maize cystatin that limits the digestion of dietary protein by nematodes, synthetic peptide that disrupts chemoreception, or both of these traits. These lines expressing the transgenes were challenged in a replicated screenhouse trial with a mixed population of the banana nematodes, Radopholus similis and Helicotylenchus multicinctus. Many lines were significantly resistant to nematodes compared with nontransgenic controls. The promising transgenic lines showing high resistance will be planted in confined fields in Uganda for further evaluation in mid-2012.

Transgenic technologies for abiotic stress tolerance
Cassava roots undergo rapid deterioration within 24–48 hours after harvest, the so-called postharvest physiological deterioration (PPD), which renders the roots unpalatable and unmarketable. IITA, in collaboration with the Swiss Federal Institute of Technology (ETH) Zurich, is developing cassava tolerant of PPD through the modification of ROS (reactive oxygen species) scavenging systems. The potential is being assessed of various ROS production and scavenging enzymes, such as superoxide dismutase, dehydroascorbate reductase, nucleoside diphosphate kinase 2, and abscisic acid responsive element-binding protein 9 genes, to reduce the oxidative stress and the extent of PPD in transgenic cassava plants.

Future road map
Efforts at IITA over the last 10 years to establish transformation protocols for all the IITA crops have been paying off and have led to the establishment of a genetic transformation platform for cassava, banana/plantain, and yam―the three most important food crops in sub-Saharan Africa. These technologies have contributed to significant advances in incorporating resistance to pests and diseases in banana and cassava. Some of these technologies have the potential to offer additional benefits. For instance, the transgenic technology to control Xanthomonas wilt may also provide an effective control of other bacterial diseases of banana (Moko, blood, and bugtok diseases), and of bacterial blight in other crops such as cassava and cowpea.

Christian A. Fatokun: The guardian of biosafety

Fatokun
Christian Fatokun. Photo by IITA

Christian Fatokun is a plant breeder/molecular geneticist in charge of biosafety at IITA. He attended the University of Ibadan where he obtained BSc and PhD degrees in agriculture and plant breeding, respectively. He was a lecturer in plant breeding and genetics at his alma mater before joining IITA as a scientist in September 1993. He was also a postdoctoral research fellow in IITA’s farming systems program between 1976 and 1977. According to him, his active working career will end at IITA when he retires this year.

Tell R4D Review about your work.
I work on cowpea improvement. In Nigeria, we eat a lot of cowpea, a cheap source of protein. Before joining IITA I had worked on exploring the genetic potentials of wild cowpea relatives and on the genomics of cowpea. I continued research in both areas when I joined IITA. Now my research is on improving cowpea for enhanced levels of drought tolerance. Compared with many other crops, cowpea is drought tolerant. Our efforts are geared towards increasing the level of tolerance in existing improved and farmers’ varieties. The new varieties would be better adapted to drought stressed environments.

What are some of the highlights of IITA’s work on cowpea?

IITA has a global mandate to study cowpea. It has conducted research, including agronomy, pathology (bacteriology, virology, nematology, Striga and Alectra biology), entomology, physiology, microbiology, socioeconomics, technology transfer, seed storage of germplasm lines, breeding and genetics, and attempts to genetically modify the crop.

Making wide crosses, studying the genome structure, identification of markers with effects on traits of importance, and evaluating germplasm lines for their drought tolerance are all aimed at genetic improvement of cowpea.

We are now developing varieties with enhanced drought tolerance, resistance to Striga and Maruca vitrata, and possessing consumer-preferred traits, such as higher nutritional levels and easier processing. The Bacillus thuringiensis gene which was recently introduced, through bioengineering, into cowpea in Australia can be transferred, using conventional breeding techniques, to lines that are popular with farmers and consumers if the gene is found to be effective in controlling Maruca. I foresee increased research on the contribution of cowpea consumption to human health.

What impact does IITA’s work on cowpea research have on producers and consumers in Africa?
The average cowpea grain yield for Africa up to the 1990s was around 300 kg/ha. According to FAO, average cowpea grain yield for West Africa in 2006 increased from 240 kg/ha to 470 kg/ha. For Nigeria, the world’s number one producer and consumer, grain yield increased from about 470 kg/ha to 670 kg/ha. IITA research on cowpea contributed to these increases.

cowpea-plants
Cowpea. Photo by IITA

There are now new varieties that mature within 60 days from planting. These varieties suffer less damage from drought and pests than traditional varieties that remain much longer in the field before flowering and setting pods. Some improved breeding lines with resistance to field pests such as aphids and flower thrips and the parasitic weeds, Striga gesnerioides and Alectra vogelii are also available. Farmers who adopt these new improved varieties apply insecticides less often. These have reduced production cost, less damage to the environment, and provide less exposure to chemicals. Improved production practices have also increased yield. Improved dual-purpose varieties are particularly beneficial to farmers in the dry savanna regions because the haulms are a very good source of quality fodder for livestock.

What is your work philosophy?
It is to do my very best and leave the rest for others along the chain of workers. The primary school that I attended has as its motto: ”what is worth doing is worth doing well”. We were made to repeat this statement regularly.

In addition, I am convinced that the most benefit can be derived from one’s income only when one always has put in one’s best efforts. Essentially, the nature of the harvest accruing to an individual cannot be different from what has been sown.

How did you get involved in biosafety work?
I participated in the preparation of the Nigeria Biosafety Guidelines that IITA initiated in 1993 and published in 1994. Later, I was invited by the Federal Ministry of Environment to participate in preparing the National Biosafety Framework.

IITA has a biosafety containment facility built next to the biotechnology laboratory. It is at a biosafety level (BL2-P standard), which is appropriate for growing bioengineered crops. We also have a confined field trial site on campus at Ibadan that has not been used since it was constructed. IITA is therefore committed to ensuring compliance with international standards in biosafety.

What are some of the biggest constraints to the adoption of biotechnological tools or products in Africa?
The use of these relatively new tools in research in Africa is in its infancy. There is great promise in the new technology. However, the capacity to carry out research using these new tools is very limited in most African countries. The critical mass of scientists in the field is nonexistent in several African countries. In addition, the needed infrastructure is also lacking. A few countries, such as South Africa and Egypt, have been exemplary in developing scientific capacity and providing resources to support research in this field. Their farmers, both commercial and small-scale, have seen some of the benefits that the technology has to offer. Burkina Faso has recently joined these countries to commercialize a genetically modified crop. Kenya has likewise given approval for the growing of a bioengineered crop in the country.

Biosafety regulatory regimes would be needed for the technology to prosper in Africa.

Fatokun shows off cowpea plants in the screenhouse. Photo by O. Adebayo
Fatokun shows off cowpea plants in the screenhouse. Photo by IITA

What could be done to take advantage of opportunities that current agricultural technologies provide and harness them for the development of African agriculture or the improvement of food security in SSA?
Governments in Africa need to show more commitment to agriculture by addressing the problems that limit productivity. For example, crop production is mostly rainfed and this exposes crops to terminal drought, especially in the savanna region where there is large potential farmland. There are very limited irrigation facilities where year-round farming could be practiced.

Inadequate fertilizer supply to farmers is a major problem. Many of the newly developed crop varieties are responsive to good soil fertility. They cannot attain their optimum yield where soil fertility is low. There should be commitment on the part of governments to ensure availability of this important input. The private sector should be encouraged to get more involved with production, importation, and distribution of agricultural inputs.

In a nutshell, expansion of irrigation facilities, better supply of fertilizers and other needed inputs, such as improved seeds, etc., and the construction of motorable roads to ensure easy transportation of farm produce would, among others, enable farmers to take advantage of available technologies. If these and other identified problems faced by farmers could be addressed, agricultural productivity would be increased and food security ensured. When farmers come across good technologies, they embrace them.

Any lessons or insights that you want to share with colleagues?
We should see ourselves as members of a community of scientists working to solve problems of food security and poverty in Africa. There is a great need for harmoniously working together since we are all working toward the same objectives of increased food production, ensuring food security, and regeneration of income for our clientele. The Yoruba people in Southwestern Nigeria have a saying. When translated, it means that if hunger is eliminated from among the problems associated with poverty, then the burden of poverty is remarkably reduced. As IITA staff, we should always remember that our little individual contributions add up and could make a difference to many people.

Ensuring biosafety

Christian Fatokun, c.fatokun@cgiar.org

For thousands of years, developing improved crop varieties has depended on conventional plant breeding methods.

With developments in scientific research and technologies, it is now possible to ”design” crops with improved characteristics within a shorter time and with more precision using biotechnology. Through transformation, genetic information (genes) can be transferred between distantly related species, which would not happen in nature (transgenes). This allows novel and unique characteristics to be incorporated into crop varieties. Through this technology it is possible to develop new crop varieties (genetically modified organisms, GMOs) with higher yield, adaptation to variable environments, resistance to pests and diseases, enhanced storage time, and improved nutritional values, among others.

Cowpea plants in the IITA screenhouse. Photo by O. Adebayo
Cowpea plants in the IITA screenhouse. Photo by IITA

Concerns have been expressed about the impact on human and animal health of these transgenes. Concerns also revolve on their possible movement from the bioengineered crop to other cross-compatible crops, and in particular, to wild relatives growing in regions where the crop has its origin or center of diversity, and on the impact of products of transgenes with pesticidal activities on nontarget organisms The concern is that transgenes could confer “fitness” on the crop’s wild relatives, thus making such plants develop into “super weeds,” especially when they become resistant to herbicides. There are also concerns that the protein in the transgenes could be allergenic to people. Outcrossing is a common occurrence between compatible plants and the degree of outcrossing depends on the crop species.

To regulate the release of GMOs to the environment in any country, a set of biosafety regulations are put in place. The International Biosafety Protocol (Cartagena Protocol) places emphasis on the transboundary movements of GMOs and offers a set of guidelines on their safe handling and use. It has been adopted by several countries, including 38 in Africa.

Biosafety is generally defined as “policies and procedures adopted to ensure the environmentally safe applications of modern biotechnology in medicine, agriculture, and the environment, so as to avoid endangering public health or environmental safety.”

IITA’s research on genetic engineering is in consonance with the CGIAR’s guiding principles on the application of modern biotechnology in the improvement of any of its mandate crops. We do not have a separate set of biosafety guidelines. The Institute has worked very closely with agencies of the Federal Government of Nigeria to establish biosafety guidelines for the country. The Federal Ministry of Environment is responsible for regulating the release of bioengineered products, and reports that a Biosafety Bill has been prepared. The document will soon be presented to the National Assembly for deliberation prior to being passed into law. With the existing biosafety guidelines that became operational in 2001, it is possible to carry out research on genetic engineering and test products of the technology under confinement in Nigeria.

Technician examines banana cultures, IITA genebank. Photo by O. Adebayo
Technician examines banana cultures, IITA genebank. Photo by IITA

Uganda and Tanzania are two countries where IITA is undertaking transformation research. In Uganda, work on transforming banana resistant to banana Xanthomonas wilt (R4D Review Edition 1) and nematodes is ongoing. In Tanzania, transformation research on incorporating resistance to cassava brown streak disease is being undertaken with partners. Both Uganda and Tanzania are signatories to the Cartagena Protocol, which requires signatory countries to develop a regulatory framework and the capacity (in terms of people, expertise, and technology) to undertake risk assessments in developing and using GMOs.

The Government of Uganda recognizes biotechnology as a tool that can be used to help stimulate economic development and meet national goals for improving the standard of living for the poor. Biotechnology is specifically included in the Poverty Eradication Action Plan as a component in the Program for the Modernization of Agriculture.

Recently Uganda’s cabinet has approved its first National Biotechnology and Biosafety Policy after 8 years of deliberation. The policy provides objectives and guidelines for promoting and regulating biotechnology use in the country, and contains the guidelines on the legal, institutional, and regulatory framework. The guidelines cover tissue and cell culture, medical diagnostics, industrial microbiology, and biochemical engineering.

For the policy to be implemented, there must be a law. At the moment, a draft bill has been presented to Parliament. The commercialization of GM crops in any country requires this law.
Tanzania released its National Biosafety Framework in 2005. An Institutional Biosafety Committee addresses biosafety activities within any institution conducting genetic modification. The Division of Environment is currently the National Biosafety Focal Point, which is responsible for overseeing the review and approval of applications, and implementation of biosafety issues.

In vitro yam seedlings. Photo by O. Adebayo
In vitro yam seedlings. Photo by IITA

An interim biosafety regulatory process exists for permitting small-scale confined research/field trials of plant and plant products. Applications are reviewed by the Agricultural Biosafety Scientific Advisory Committee and the National Biotechnology Advisory Committee. The Tropical Pesticides Research Institute and the Plant Biosafety Office require risk management measures to ensure that the field trial does not adversely affect the environment or human health.

The first application using the interim measures was for the MARI-IITA project on cassava genetic transformation for virus resistance in Tanzania.

With contributions from Leena Tripathi, IITA–Uganda, and Caroline Herron, IITA–Tanzania.

Guiding Principles
1. In keeping with its mission, IITA will continue to engage in research designed to produce international public goods appropriate for use by resource-poor farmers. In doing so, it will typically use a range of technologies, including in some cases modern biotechnological methods, to produce breeding and planting materials containing traits important to and useful for resource-poor farmers. It follows that IITA believes that genetically modified organisms (GMOs) that contain traits beneficial to small farmers and have been fashioned carefully, with due regard to the range for social, economic, biosafety, and environmental concerns, are a legitimate subject for its research and development.

2. For sound scientific and practical reasons, IITA will continue to work with the gene pools of cultivated species and their wild or weedy relatives as the first and often most effective means of bringing benefits to resource-poor farmers. The formulation of these Guiding Principles is therefore not intended to be, nor should it be interpreted as signaling a shift in emphasis or priorities in IITA research programs: conventional breeding techniques will continue to be used widely in all crop improvement programs. Indeed, they are likely to remain the dominant approach for some time to come.

3. IITA will continue to monitor, research, and assess the possible social and environmental implications of the use of genetically transformed plant varieties in the ecological regions in which they might be used and, especially, in the centers of origin or of diversity of the species that may be genetically transformed. As in other subject areas, these activities will routinely be carried out in cooperation with national agricultural research systems, farmers, and other partners.

In all its genetic engineering-related research, IITA will observe the highest scientifically accepted standards of safety in the conduct of laboratory and field experiments.

Yam cultures in IITA genebank. Photo by O. Adebayo
Yam cultures in IITA genebank. Photo by IITA

4. IITA will comply with relevant national or regional biosafety, food, environmental and policy regulations for the deployment of genetically engineered organisms. IITA will not deploy genetically engineered organisms in any country lacking such regulations. In certain circumstances, IITA may voluntarily adhere to higher or more stringent standards than the minimums imposed by national legislation and regulation. IITA will not make GMOs or other such products available in a country without that country’s prior informed knowledge, consent, and support.

5. IITA will work with national partners, using the best expertise available, to address potential risks and ensure confidence in the product. If a recipient country lacks the expertise to conduct its own risk assessment,

6. IITA will work with national partners to help develop this capacity, and to develop appropriate strategies and methodologies.

7. Currently, IITA adds a modest number of plant genetic resource accessions each year to those it already conserves under long-term, ex-situ conditions. Under proper management, geneflow between accessions is essentially nonexistent, and thus the presence of GMOs within IITA’s collection is not considered to undermine or pose a significant danger to the goal of its long-term conservation of genetic diversity. When circumstances so indicate, however, IITA will screen incoming (and/or already-held) accessions for the presence of promoters or other indications of the presence of GMOs. IITA will make the resulting data available to anyone requesting samples of these accessions, and IITA will take sufficient measures to ensure the appropriate and safe management and use of such materials.

Designer (cowpea) plants

Christian Fatokun, c.fatokun@cgiar.org

Plants can be designed to order. Science has long found a way to combine good and useful characteristics in a plant by studying the genes for such traits, and putting them together in a process called “genetic engineering.”

Caterpillar boring into a cowpea pod. Photo by S. Muranaka
Caterpillar boring into a cowpea pod. Photo by S. Muranaka, IITA

Cowpea is grown mainly for its protein-rich grains and quality fodder for livestock. At present, biological control and conventional breeding methods are proving inadequate in developing cowpea varieties resistant to destructive pests, such as the legume pod borer Maruca vitrata.

M. vitrata is the most widespread cowpea pest. The adult moth lays eggs on the plant. The larvae that emerge from the eggs damage plants in the field, particularly during the reproductive stage, through feeding on young succulent shoots, flowers, pods, and seeds. This pest can cause significant grain yield reduction, between 20% and 80% if not controlled with insecticides.

Farmers usually spray insecticides to protect the cowpea crop from Maruca and other pests. Purchasing chemicals, however, adds to the production cost, thus reducing the farmers’ profit. Also, farmers are not well equipped to protect themselves when using such toxic chemicals. In some farming communities, adulterated chemicals that do not control the pests are sold to farmers. The development of cowpea varieties with resistance to Maruca and other insect pests would benefit the most resource-poor African farmers who grow the crop.

Cowpea is grown extensively in the savanna region of sub-Saharan Africa (SSA). At least one major insect pest attacks cowpea at every stage in the life cycle, including seeds in storage. These pests are significantly responsible for the low grain yield in farmers’ fields.

Through conventional breeding, some varieties have been developed that show resistance to some of the pests, such as aphids and flower thrips, and low levels of resistance to the storage weevil. However, not much progress has been made in host plant resistance, especially M. vitrata.

Efforts continue to identify parasites and predators that could be used as biocontrol agents. When deployed, such agents would greatly reduce the population of the Maruca larvae in the field, giving the cowpea plant some respite for the production of flowers and pods containing whole and well-formed seeds.

Using conventional breeding, several hundreds of accessions of cultivated cowpea and its wild relatives have also been screened for resistance to this pest. Accessions belonging to Vigna vexillata were found to be resistant to M. vitrata. These accessions were found to be closest to cowpea in a phylogenetic study of diversity in the Vigna species. The study was based on data obtained after DNA genotyping. Efforts were made to cross cowpea with V. vexillata but without success.

This strong cross-incompatibility makes gene exchange between the two species impossible. This is where biotechnology comes to the rescue. Two major steps are needed to develop genetically modified cowpea with resistance to M. vitrata. First is developing a transformation system and the second is identifying the transgene that would be effective against the pest when introduced into cowpea. Since Maruca is a Lepidopteran, some of the genes from Bacillus thuringiensis (Bt) should be effective against the insect’s larvae. IITA screened several Bt protoxins on Maruca by incorporating different concentrations in the diet fed to the larvae. The protoxin of Bt gene ”Cry1ab” was found to be most effective even at very low concentrations in the artificial diet. This Bt gene (Cry1ab) was therefore selected as the candidate gene for designing Maruca-resistant cowpea.

Scientists at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Australia developed the transformation system using an IITA-developed breeding line ”IT86D-1010” derived from a cross between ”TVx4659-03E” and ”IT82E-60”. CSIRO scientists have now obtained cowpea lines containing the Bt gene. Monsanto donated the Bt gene used to transform cowpea by licensing it to the African Agricultural Technology Foundation (AATF) for use in Africa. The Rockefeller Foundation and USAID funded this cowpea transformation project.

Farmers transporting cowpea harvest. Photo by S. Muranaka
Farmers transporting cowpea harvest. Photo by S. Muranaka, IITA

The Bt cowpea had been tested in the CSIRO laboratory in Australia and found to be effective against the larvae of another Lepidoptera, Helicoverpa armigera. The Bt gene in cowpea is expected to be effective against M. vitrata, the cowpea pest, but needs to be tested in an environment where Maruca thrives. Apart from Burkina Faso, none of the African countries where cowpea is an important crop has a biosafety law in place. A few lines of the Bt cowpea were, therefore, taken to Puerto Rico for field testing. The field trial was carried out in late 2008. If the Bt gene in the cowpea lines is found to be effective against Maruca, the next step would be to transfer the Bt gene into popularly grown cowpea varieties selected from interested countries. The line presently containing the Bt gene is not high yielding, and farmers are not likely to accept it readily.

Under the international biosafety protocol (Cartagena protocol on biosafety) it is necessary to carry out risk assessment on the Bt cowpea before it is introduced to another country. The data obtained from risk assessment form part of the dossier that accompanies applications requesting for importation to any country. Risk assessment would entail studies on gene flow, the effect of the transgene on nontarget organisms, food safety, and resistance management strategies.

A meeting of experts in these various fields is planned in March 2009 at the Donald Danforth Plant Science Center, St. Louis, Missouri, USA. The experts would design studies to address the different questions that may arise from biosafety regulators in the countries where the Bt cowpea is meant to be grown. Many of the proposed studies are necessary, because cowpea is an indigenous food crop in SSA where cross-compatible wild relatives are found growing in agroecologies similar to farmers’ fields. Biosafety reviews in the African countries would, therefore, be rigorous.