Genetic engineering is one of the key techniques for improving crops particularly those that are vegetatively propagated or not amenable to conventional breeding. At IITA, the technique is being used for the improvement of crops that are economically important to Africa, such as banana and plantain (Musa sp.), cassava (Manihot esculenta), and yam (Dioscorea sp.).
Genetic transformation platform for banana and cassava
Genetic engineering of any crop requires efficient transformation protocols. However, many African laboratories lack the capacity and expertise to carry out the genetic transformation of staple crops and this work has been limited to advanced laboratories. There is a need to build the capacity of researchers in Africa to carry out the genetic transformation of staple crops such as banana and cassava.
Despite the technical difficulties of transforming a monocot species, we have developed an efficient transformation system for several banana and plantain cultivars using embryogenic cell suspensions. This, in turn, has paved the way for the genetic manipulation of banana and plantain by incorporating agronomically important traits such as those conferring resistance to diseases or pests as well as tolerance to
abiotic stress factors.
The Transformation Laboratory at IITA in Nairobi, Kenya, has also successfully developed an effective transformation platform for farmer-preferred varieties of cassava that can be used to develop improved varieties with desired traits. This is the first-ever report of the successful Agrobacterium-mediated transformation of African farmer-preferred cassava varieties in a laboratory based in sub-Saharan Africa.
Banana Xanthomonas Wilt (BXW) caused by Xanthomonas campestris pv. musacearum has caused estimated economic losses of between US$2 and 8 billion over the last decade in Africa. In the absence of natural host plant resistance, researchers at IITA and National Agricultural Research Laboratories [NARL] in Uganda, have developed transgenic banana by inserting Hypersensitive Response-Assisting Protein gene (Hrap) and Plant Ferredoxin-Like Protein gene (Pflp) from sweet pepper. These genes were licensed by the African Agricultural Technology Foundation (AATF) on a royalty basis from the Academia Sinica in Taiwan. The 11 transgenic lines selected for field trials, after exhibiting strong resistance in the laboratory and greenhouse, have been shown to be 100% resistant to BXW through three successive crop cycles.
They will be further tested at multiple locations to capture the effects of different environmental conditions on disease resistance.
It is well known that pathogens can evolve and “break down” resistance to disease. To avoid this, we have also developed transgenic banana by stacking the two genes together in the same line to enhance durability of resistance. An ex-ante impact analysis conducted last year in Uganda has clearly shown that if this new technology is successfully adopted in the region, both consumers and producers will benefit. The greatest benefits would be in countries that have experienced large production losses from BXW. These transgenic lines are also currently under testing for food and environmental safety in compliance with biosafety regulations.
Based on success with transgenic banana, we are trying to transfer transgenic technology from banana to enset in partnership with the Ethiopian Institute of Agricultural Research. Enset, closely related to banana, is a staple food source for over 15 million people in Ethiopia. Its production has also been severely threatened by BXW in all the enset-growing areas. We have established a protocol for enset transformation and we are currently developing transgenic enset using Hrap and Pflp genes.
We are also identifying additional resistance genes for use in gene stacking or pyramiding strategies. We tested the potential of rice pattern recognition receptor (PRR), Xa21 for providing resistance against X. campestris pv. musacearum. Our results confirmed that the constitutive expression of the rice Xa21 gene in banana results in enhanced resistance to BXW disease.
The Banana Bunchy Top Disease (BBTD) is a serious threat to banana across the world. It is extremely difficult to control and is continuing to spread in many countries
where banana are primarily produced by smallholder farmers. BBTD has already moved into Nigeria and is causing major losses in plantain, the country’s third most important starchy staple. The disease has also been reported in several Central and East African countries and there is a risk of it spreading into Uganda and Tanzania where banana is a key staple crop. The virus infects all types of banana including East African Highland banana, plantain, and dessert varieties.
Host plant resistance is the most appropriate form of control. However, there is no known resistance against the Bunchy top virus in the Musa germplasm. Therefore, IITA and Queensland University of Technology (QUT), Australia, are developing transgenic banana and plantain with resistance to BBTD using the RNAi approach. About 50 transgenic lines of plantain cultivar Gonja manjaya have been developed in the laboratory at IITA-Kenya. These lines will be characterized at molecular level and then sent to IITA-Nigeria for glasshouse evaluation for
resistance to BBTD.
Plant parasitic nematodes can cause losses of up to 70% on plantain and cooking banana in Africa. Application of nematicides is inappropriate and resistant cultivars are not available. IITA in partnership with the University of Leeds, UK, has developed transgenic plantain using an anti-feedant cysteine proteinase inhibitor (cystatin) from maize and an anti-root invasion, non-lethal synthetic peptide, either singly or by stacking these genes. The glasshouse study showed that both genes are capable of providing resistance in plantain to concomitant
infection by different nematode species. Confined field testing of 12 promising lines demonstrated that transgenic expression of maize cystatin and synthetic peptide confers resistance against key nematode pests Radopholus similis and Helicotylenchus multicinctus. The best peptide transgenic line improved agronomic performance compared with non-transgenic controls and provided about 99% resistance to nematodes at harvest of the mother crop. Its yield was 186% of the nematode-challenged controls, based on its larger bunches and reduced plant toppling in storms because roots were less damaged.
Double haploid banana
Banana is a slow breeding crop and developing a pure breeding line can take up to several years. Haploid inducers are used in breeding to hasten the process. Haploid breeding could, therefore, revolutionize the improvement of slow-cycling crops. IITA and the University of California at Davis (UC Davis) are trying to develop double haploid banana. A transgenic approach developed at UC Davis previously in the model plant Arabidopsis thaliana was transferred to banana in an effort to develop a haploid inducer. The approach involves silencing an endogenous histone protein CENH3 and replacing it with a modified version. When plants with the modified CENH3 of the protein are crossed to the wild type (with no modification in CENH3), haploids are obtained. Under this project, we developed a genetic transformation system for the diploid banana cultivar Zebrina GF, which is a fertile parent used in breeding programs. The haploid inducers for the diploid banana cultivar Zebrina GF were developed and transferred to the glasshouse for flowering. Wild type plants of two diploid parents (Zebrina GF and Calcutta 4) were also planted in the field to be crossed to transgenic haploid inducer Zebrina GF for haploid induction. The transgenic line flowered in the glasshouse, was crossed with pollens of wild type Zebrina GF, and is currently under seed setting. Once the seeds are set, they will be tested for haploidy.
Genetic transformation of yam
Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, its production remains constrained by increasing levels of field and storage pests and
diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient transformation and regeneration system for the crop. Recently, IITA has developed an efficient, fast, and reproducible protocol for Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. This provides a useful platform for future GE studies in this economically important crop. This is the first report of the Agrobacterium-mediated transformation of yam with experimental
evidence of stable integration of T-DNA in D. rotundata genotypes. This protocol opens up an avenue for future genetic improvement of D. rotundata with candidate genes of proven agronomic importance to attain sustainable production.