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.

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.