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.

Bacterial wilt-resistant banana

Crop scientists have successfully transferred genes from green pepper to banana that enable the crop to resist the Banana Xanthomonas Wilt (BXW). BXW or bacterial wilt is one of the most devastating diseases of banana in the Great Lakes region of Africa. It causes about half a billion dollars worth of damage yearly.

The transformed banana, infused with plant ferredoxin-like amphipathic protein (Pflp) or hypersensitive response-assisting protein (Hrap) from green pepper, have exhibited strong resistance to BXW in the laboratory and screenhouses.

The Hrap and Pflp are novel plant proteins that give crops enhanced resistance against deadly pathogens. They work by rapidly killing the cells that come into contact with the disease-spreading bacteria, preventing them from spreading any further. They can also provide effective control against other BXW-like bacterial diseases in other parts of the world such as “Moko”, Blood, and “Bugtok”. The genes used in this research were acquired under an agreement from the Academia Sinica in Taiwan.

The mechanism is known as hypersensitivity response and activates the defense of surrounding and even distant uninfected banana plants leading to a systemic acquired resistance.

Scientists from IITA and the National Agricultural Research Organization of Uganda, in partnership with African Agricultural Technology Foundation, would soon be evaluating these promising resistant lines under confined field trials after the Ugandan National Biosafety Committee recently approved the conduct of the tests.

Presently, there are no commercial chemicals, biocontrol agents, or resistant varieties that could control the spread of BXW. Developing a truly resistant banana through conventional breeding would be extremely difficult and would take years, given the sterile nature and long gestation period of the crop.

Growing cassava in cold Denmark

Kirsten Jørgensen, kij@life.ku.dk, and Birger Lindberg Møller, blm@life.ku.dk

cassava1Cassava, that tropical tuber that is a staple to millions in sub-Saharan Africa, is being grown in freezing Denmark—at the University of Copenhagen. The precious plants are grown in the greenhouse during the dark winter days when the snow is lying on the roof.

The crop has been a prime focus of the University’s research because of its importance as a food security crop and commodity in economic development. It has a high content of cyanogenic glucosides, toxic substances which may constitute a nutritional problem in regions where cassava is the dominant or sole staple food. During processing, cyanogenic glucosides are converted into cyanohydrins, ketones, and hydrogen cyanide. These are all toxic and should not be consumed in excessive amounts.

Cyanogenic glucosides are an ancient group of bioactive natural products present in crop plants, forage plants, and important trees. More than 3,000 plant species are cyanogenic, including cassava, apricot, cherry, clover, flax, barley, sorghum, wheat, bamboo, eucalypt, and poplar. We study most of these plant species to understand these compounds in terms of their synthesis, turnover and regulation, and their biological function. In these studies, we use model plants such as Lotus japonicus. Lotus contains linamarin and lotaustralin, the very same cyanogenic glucosides found in cassava. The genome sequence of Lotus has been sequenced, and the use of transgenic model plants is a key tool in our studies.

Our laboratory was a world-first in isolating all three genes for cyanogenic glucoside synthesis. This work was done in sorghum. We were also a world-first in isolating the genes responsible for cyanogenic glucosides synthesis in cassava.

Embryo culture of Kibaha. Photo by K. Jørgensen
Embryo culture of Kibaha. Photo by K. Jørgensen

Our research aims to control the level of cyanogenic glucosides in different parts of the cassava plant. It involves understanding the regulation of the biosynthetic genes and the turnover and transport processes. We use tools such as the relevant omics platforms for these studies, including metabolomics where we use high-pressure liquid chromatography and mass spectrometry to determine the constituents found in the different cassava tissues as a result of environmental challenges. A second important tool is transcriptomics. In collaboration with IITA researchers, such as Dr Ivan Ingelbrecht, we have designed a cassava DNA chip that allows us to monitor the profile of gene expression and how these profile changes for individual genes respond to plant development, nutritional status, and environmental challenges.

In the long term, we want to produce a virtual model of the cassava plant that would enable us to predict responses during growth and development, and to environmental stimuli. In these studies, changes in cyanogenic glucoside content, in the levels of the enzymes and genes controlling their synthesis, breakdown, and transport are given special attention.

Our studies will provide information on the level of natural variation from one plant to another in terms of interesting characteristics. To facilitate identification of individual plants with interesting properties, high throughput screening technologies have also been implemented. Cassava is a tetraalloploid plant. This makes traditional breeding very time consuming and complicated. The development of transgenic approaches where multiple gene copies may be “knocked out” in a single step offers great opportunities to develop varieties with an optimal content and distribution of cyanogenic glucosides.

Progress in cassava research is slow, because few research groups in the world are working on cassava. Typically, the tool boxes successfully used in wheat, maize, and rice breeding are not available in cassava. This makes breeding of new varieties cumbersome and time-consuming because many of the techniques have to be set up from scratch. One such example is the development of a transformation protocol for cassava that would enable us to knock out or insert new genes in elite cassava cultivars.

Transgenic shoots from TME12. Photo by K. Jørgensen
Transgenic shoots from TME12. Photo by K. Jørgensen

We have devoted a lot of effort to develop such a transformation system. This includes tissue culture work to establish protocols enabling us to produce embryogenic cultures and to regenerate plantlets from these. Likewise, reliable procedures for Agrobacterium-mediated gene transfer for elite cultivars had to be optimized. In this research we took advantage of the pioneering work on cassava transformation carried out at the Swiss Federal Institute of Technology (ETH) in Zürich. The system we now use is robust and we are able to transform elite lines from IITA that are high yielding and that have optimal resistance to disease and pest attack. Our transformation technology has been transferred to IITA. In 2007, we also transferred the technology to obtain cassava transformants to the Danforth Plant Science Centre, USA.

It is important to select the optimal cassava lines for our research. We want to produce cassava lines appropriate for end-users. There is not much value in engineering interesting agronomical traits into lines that have no value to the end users, i.e., the farmers and consumers. This is yet another example where close collaboration with IITA researchers has greatly benefited us. Collaboration with IITA helped us to focus our work on agriculturally important lines.

In the initial phase of our work to reduce cyanogenic glucoside synthesis, we used constitutive promoters such as that from the 35S cauliflower mosaic virus. We have now shifted our focus to using native cassava promoters that more efficiently target the cells in the tubers where, for example, synthesis of cyanogenic glucosides takes place, thus providing better control. The content of cyanogenic glucosides varies among vegetatively propagated and thus genetically identical plantlets. Accordingly, multiple tests at different growth stages have to be carried out to determine the degree of downregulation (reduction) of cyanogenic glucoside synthesis. This makes the procedure to find the right lines time-consuming.

The protein content in current elite cassava cultivars is very low partly perhaps because of the continued breeding for high starch yield. In the past, breeding for high protein content would be at least partly in vain because a significant proportion of the protein is lost anyway during processing when the cyanogenic glucosides and their toxic degradation products are removed to provide food safe for consumption.

Using molecular breeding, there are several ways to achieve transgenic lines with enhanced protein levels in the tubers. One way is to identify specific storage proteins from wild cassava varieties that exhibit a high content of essential amino acids, such as methionine and lysine, and then express these nutritionally beneficial proteins in the elite lines. Another way would be to use storage proteins from other known species, such as patatin from potato. We are currently transforming African elite lines with constructs encoding patatin that will be incorporated into the starch grains in the cassava tuber.

Transgenic acyanogenic cassava (TME12) in greenhouse. Photo by K. Jørgensen
Transgenic acyanogenic cassava (TME12) in greenhouse. Photo by K. Jørgensen

The original focus on the molecular breeding of elite cassava cultivars with a controlled and reduced content of cyanogenic glucosides and a higher protein content has recently been expanded to incorporate varieties carrying yellow tubers. These have increased levels of carotenoids that are precursors of vitamin A. IITA provided the cassava lines with high carotenoid content obtained by classical breeding.

The long-term aim of our research is to improve the nutritional value of cassava tubers from African elite cultivars by blocking the accumulation of cyanogenic glucosides, enhancing the protein content, and increasing the pro-vitamin A content. Future goals involve engineering resistance to important pests and diseases in the very same lines.

Climate change has spurred a worldwide demand for drought-tolerant plants, such as cassava. Likewise, as Western industrialized countries move towards a bio-based society less dependent on fossil fuels, starchy plants that can produce very high yields under optimal growth conditions become key targets for research. As part of these efforts, the cassava genome is now being sequenced in the US. When the genome sequence is available research on cassava will be that easier and may be the first step in developing the crop as an efficient environmentally benign “green factory” for producing valuable chemicals and pharmaceuticals.

The cassava group at the University of Copenhagen is headed by Professor Birger Lindberg Møller and principal investigator Associate Professor Kirsten Jørgensen, together with Assistant Professor Rubini Kannangara, Technicians Charlotte Sørensen, Evy Olsen, and Susanne Bidstrup, and gardener Steen Malmmose.


http://www.proactiveplants.life.ku.dk