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.

Developing genomic resources for banana

Jim Lorenzen, j.lorenzen@cgiar.org

jim_lorenzen-looking-at-banana-flower
Jim Lorenzen checking a banana flower. Photo by IITA

Banana and plantain (Musa sp.) are a very important staple food and cash crop in Africa. Although the principles of banana breeding and genetics were established decades ago, it is still a time-, land-, and resource-intensive process. A crew of several persons collects male flowers and pollinates female flowers while perched on ladders. When successful, seeds must be surface-sterilized and embryos removed for germination in test tubes (or else most won’t germinate), multiplied, and carefully “weaned” for field planting.

Large-sized plants require much field space, and new hybrids must be evaluated through two or three production cycles (about 3 years) before being selected for further testing, such as for disease resistance. Some essential attributes, such as resistance to disease or nematodes may also take several years to assess properly. It would be a huge advantage if early selection could be done, based on some associated marker or rapid test, to eliminate susceptible individuals without wasting resources on them. For other complex traits, it would be useful to have markers based on component genes to be able to select ideal “genotypes”.

One way to do early selection is to use molecular markers that are linked to the target traits (molecular-assisted breeding). This method is becoming common in cereal breeding, yet should be even more cost-effective for a large perennial crop such as banana that requires so much time and space to evaluate. The tools of DNA fingerprinting are applied, and by knowing which DNA markers lie near genes of interest, selecting for the markers will be equivalent to selecting for the trait a year or more later.

The problem is that we lack enough information on the banana genome to have molecular tools to map traits. One of our activities has been to map and characterize new molecular markers for use in banana breeding and genetics. PhD student Gaby Mbanjo from the University of Yaoundé, Cameroon, has been working in Uganda and Kenya to characterize and map a large new set of simple sequence repeat (SSR) markers, often called microsatellite markers. She is a scholar of the Biosciences for Eastern and Central Africa (BecA) program, with funding provided by the Canadian International Development Agency (CIDA).

Gaby is also working to develop other types of molecular markers based on small genetic differences (single nucleotide polymorphisms = SNPs) between alleles of genes involved in controlling plant defensive reactions. These will be used to try to map the genetic loci responsible for resistance to the burrowing nematode (Radopholus similis) and banana weevil (Cosmopolites sordidus) in the population she is studying. Markers of both types can be converted to semi-automated assays for hundreds or thousands of assays. This effort is expected to result in a DNA fingerprinting assay in which we can select the associated DNA markers and thus also select the target resistance without spending as many resources on susceptible plants.

The molecular markers will have other practical uses. Unfortunately, sometimes varieties get distributed with wrong names, or a batch of plants supposedly of a single variety may actually contain a mixture of varieties. Molecular markers can be used to “fingerprint” mother plants used to produce new planting material to ensure that they are of the proper variety. They can also be used to select diverse parents for maximizing the heterozygosity of offspring. Some of the markers are being transferred to a national research program for assessing varietal purity in their advanced selections.

Molecular markers are a way in which biotechnology and the rapidly expanding knowledge of DNA sequences in plant genomes can be used to make classical breeding more efficient. This should be especially helpful for large perennial crops such as banana and plantain.

Biotechnology and nematodes

Leena Tripathi, l.tripathi@cgiar.org

Banana and plantain (Musa spp.) are major staple foods and a source of income for millions in tropical and subtropical regions. Most of the banana grown worldwide are produced by small-scale farmers for home consumption or sale in local and regional markets.

Leena Tripathi inspecting a diseased banana leaf. Photo by IITA
Leena Tripathi inspecting a diseased banana leaf. Please note that this picture does not relate to nematode damage or diseased by nematodes as correctly pointed out by Danny Coyne, IITA, thanks!Photo by IITA

Many pests and diseases significantly affect banana cultivation. 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 plants.

Pest management in banana is based on several principles, primarily through the use of clean, healthy planting material, crop rotation, and in commercial settings, chemical control. However, crop rotation is often impractical, especially for small-scale farmers, while nematicides are locally unavailable or not affordable for subsistence farmers. These pesticides are also highly toxic, environmentally unacceptable, and increasingly being withdrawn from use.

Limited sources of nematode resistance and tolerance are present in the banana gene pool. Some resistance has been identified against one of the most damaging nematode species, the burrowing nematode (Radopholus similis), but this needs to be combined with consumer-acceptable traits. Furthermore, several species of nematodes are often present together, requiring a broad spectrum resistance able to control not just Radopholus but other damaging nematodes, such as species of Pratylenchus, Meloidogyne, and Helicotylenchus.

Enter biotechnology. Biotechnology offers sustainable solutions to the problem of controlling plant parasitic nematodes. Several approaches are possible for developing transgenic plants with improved resistance; these include strategies against invasion and migration and against nematode feeding and development.

Woman selling banana in a local market. Photo by IITA
Woman selling banana in a local market. Photo by IITA

Some successes in genetic engineering of banana have been achieved, enabling the transfer of foreign genes into the plant cells. An efficient transformation protocol for African banana cultivars has been established at IITA using meristematic tissues. The protocol avoids the callus and cell suspension culture requirements of other approaches. It is rapid, genotype independent, and avoids the somaclonal variation that often results from regenerating embryogenic cell suspensions.

IITA, in partnership with the University of Leeds, UK, is exploring the potential of biotechnology to develop plantain resistant to nematodes with funds from the Department for International Development/ Biotechnology and Biological Sciences Research Council.

Prof. Howard Atkinson’s group in Leeds has demonstrated that more than one independent basis for transgenic resistance provides an additive effect for nematode control. Our use of three independent additive approaches is designed to ensure a resistance level that prevents the buildup of damaging populations, even if virulent individuals completely challenge one line of defense or partially compromise them all. We intend to demonstrate that this additive approach can provide durable resistance.

nematodes
nematodes, photo by IITA

The three approaches are a cysteine proteinase inhibitor (a cystatin), a potato tuber serine/aspartic proteinase inhibitor, and a repellent peptide. Cysteine proteinases are used by a wide range of plant parasitic nematodes to digest dietary protein. The cystatin prevents this digestion and slows nematode growth. Transgenic expression of both proteinase inhibitors provides effective control of both cyst and root-knot nematodes and cystatin has also been shown to be effective against Radopholus.

Cysteine proteinases are not present in mammals and those we will use lack toxicity or allergenicity for humans. They occur in common foods, such as the seeds of maize, rice, and cowpea, and people rapidly digest them. The other, very distinct, novel approach is the use of a repellent. This is also not lethal to nematodes or other organisms. Nematodes do not invade roots applied with repellent because they fail to detect the host’s presence. This approach is effective against a wide range of nematode species.

We will also be using a novel RNA interference (RNAi) approach. The use of RNAi for functional analysis of plant parasitic nematode genes was first established in the University of Leeds. The approach relies on the production of double-stranded RNA molecules by banana cells. When they are ingested by the nematode, they specifically interfere with the expression of the essential nematode gene they target. The advantage of the RNAi approach is that no novel protein production is required to achieve resistance to nematodes. This offers a considerable biosafety advantage, given that RNA molecules represent no food risk and there is little likelihood of nontarget effects. The challenge is to provide an effective level of resistance to all banana nematodes by this approach. Genetic transformation of plantain using these approaches is in progress at IITA.

Banana field trials in Rwanda. Photo by IITA
Banana field trials in Rwanda. Photo by IITA

Gene flow is not an issue for this crop, making the transgenic approach even more attractive. Banana and plantain lack cross-fertile wild relatives in many production areas. Most edible banana are male- and female-sterile and depend on vegetative propagation. The new defense will be integrated with other pest management strategies already developed at IITA to maximize resistance levels and safeguard durability. This work is part of a new, interdisciplinary research partnership between IITA and the University of Leeds, directed at enhancing human health and food security in sub-Saharan Africa.

We also plan to stack genes for Xanthomonas wilt and nematode resistance into one line to produce a high-value product for farmers. Gene stacking is becoming common, adding multiple traits at once into the plant genome. Resistance to diseases and pests can be achieved by integrating several genes with different targets or modes of action into the plant genomes. We already have promising results with genes for resistance to banana Xanthomonas wilt (BXW) (R4D Review Edition 1), which we would like to combine with nematode resistance. Banana cultivars with resistance to multiple diseases and pests will be a breakthrough in banana improvement.