Bioreactors for the rapid mass micropropagation of yam

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Morufat Balogun, m.balogun@cgiar.org

The tissue culture technique using meristems followed by serial nodal cultures can be effective for producing high quality seed yam but its use is limited by the slow rate of regeneration and propagation in a conventional semi-solid culture medium. Conventional tissue culture employs manual introduction into culture vials. However, the slowness of yam propagation in vivo also occurs in vitro where cultures for some genotypes can take more than 1 year to regenerate from meristems. This low multiplication rate limits the use of in-vitro produced plantlets; there are also losses during acclimatization and transplanting. Other limitations resulting in low propagation rates are frequent sub-culturing which increases labor costs, culture container size (hence nutrients), and sub-optimal culture aeration and uptake (Cabrera et al. 2011).

As part of its objective to develop technologies for the high ratio propagation of high quality seed yam, YIIFSWA is set to standardize in vitro propagation techniques using conventional and temporary immersion technologies. In most crops tested (pineapple, cocoa, potato, and others), the Temporary Immersion Bioreactor system (TIB) increased propagation rates (Watt 2012) through culture aeration combined with automation, both of which increase productivity.

The TIB technology involves the timed immersion of plant tissues in a liquid medium to allow for the aeration of cultures. Each unit is a bioreactor—an enclosed sterile environment provided with inlets and outlets for air flow under pressure—and therefore circumvents the limitations associated with conventional tissue culture. Although the TIB system requires the interplay of plant physiology and the chemical and physical sciences, growth rate is significantly enhanced therein since gas exchange is guaranteed (Watt 2012).

IITA’s TIB system is a “twin flask” type (Adelberg and Simpson 2002), having 1 container for the medium and the other for the cultures. It has potentials for both plantlet and yam microtuber production which will facilitate the production of quality breeders’ seed yam from which healthy foundation and certified seed yam will be multiplied. IITA’s TIB is established with 128 units and, when running at full capacity, can produce at least 12,000 seed yam in 1 year. It is programmable and remotely controlled online. It can also be used to fast-track genetic improvement through accelerated in-vitro variations and selection. Seed yam from this technology will be bulked in IITA’s aeroponics facility; other end-users include researchers, farmers, and public/private seed companies.

References

Adelberg, J.W. and E.P. Simpson. 2002. Intermittent Immersion Vessel Apparatus and Process for Plant Propagation. Internl. S/N: PCT/US01/06586.

Cabrera, M., R. Gómez, E. Espinosa, J. López, V. Medero, M. Basail and A. Santos. 2011. Yam (Dioscorea alata L.) microtuber formation in Temporary Immersion System as planting material. Biotecnologia Apl. 28: 4.

Watt, M.P. 2012. The status of temporary immersion system (TIS) technology for plant micropropagation.African Journal of Biotechnology 11: 14025-14035.

 

Prof Felix Nweke: Staying true to the course

Prof Felix Nweke about himself: I was born in Eastern Nigeria as an Igbo man but I consider myself an African right now. My training background is agricultural economics. I am a professor by occupation, retired some years back. Just call me “Prof.”

How did you get into root and tuber crops research & development?
I like that question because yam and cassava are the rhythm of my life from the beginning; I was born growing and eating them. When I was born where I was born we woke up in the morning, ate cassava fufu, then went to the field. Later in the afternoon we ate yam and continued to work in the field until dusk. We went home and ate cassava again for dinner; we did this day in, day out and it was good at that time.

As I grew up and went to school I was attracted to agriculture by the then Government of Eastern Nigeria which offered scholarships. My interest was in mathematics but my parents could not pay my university education costs from growing yam and cassava. At the University of Nigeria, Nsukka, which is in the yam belt of the world, what was taught was not called yam and cassava but those crops were still part of everyday life.

When I completed the undergraduate program and after my Biafran experience, I went to Michigan State University (MSU) for postgraduate studies. There, I was spared working on and eating cassava and yam but on return to the University of Nigeria, Nsukka, as a teacher I got immersed in the influence of those crops once again. The first research program I conducted was a project titled “Yam-Based Cropping System of Eastern Nigeria”; it was as if I had no choice but work on yam.

Can you explain the benefit and value of yam to you.
I envy my parent’s generation and rhythm of life for its routine and stable life pattern. That is the positive side. But there is a negative side to the life of that generation; I told you we ate cassava in the morning, yam in the afternoon, and cassava again at night. That story is true but yam was not always available, especially during the hungry season (after planting yam). During the hungry season we ate cassava morning, afternoon, and night. At the time, it meant nothing to me; but today if I have to worry about what my grandchildren would eat the next day, I would probably go what some people of the Caribbean describe as “separat”, i.e., mind and body going separate ways.

My parents worried about what we would eat the next day. Today, not everybody can afford even to eat cassava three times a day. I know families that live under leaking roofs, if you can call it a roof at all; I know families that cannot afford painkillers when a member is sick. When I walk in the streets I see beggars all the time. I do not give to them because private charity does not solve the social inequality problem. I pursue social justice by doing my work with honesty, courage, and commitment; in that way everybody can benefit from my work. If successful, my work on yam and cassava research will benefit everybody. That is what I got from my childhood experience of poverty and deprivation, which are still the experience of many people today.

What make yam and cassava so interesting?
Cassava and yam are interesting to me because they are rooted in my blood; if you cut me, I shall bleed cassava and yam. I could have migrated to the US and worked on wheat or corn but that will be a betrayal; by working on yam and cassava I am staying true to the course; I am giving back to what made me what I am and I feel good doing that.

Those crops are important to people of sub-Saharan Africa as a whole. There is a lot of value in these crops; we know that about cassava in Africa because that is a crop that is now well studied. It is clear that cassava has a lot of food and monetary values while its value as feed and industrial raw material remains potentials as far as Africa is concerned.

On the other hand, yam is not studied and people do not understand the crop. The monetary value to farmers who produce yam is quite high; when farmers grow yam they can sell all of it because they cannot afford their own yam. The money they get from it is more valuable to them than the yam; they use that money to buy cheaper foods like cassava.

It is often said that yam has cultural values, but people have a superficial understanding of that value. When a farmer distinguishes himself in yam production, he becomes a reference point in his community; when he speaks, people listen. He plays a key role in community mobilization and leadership. Rites of thanksgiving, passage, appeasement, and petition that are performed with yam as a ritual object among several yam-producing people of West Africa sustain the traditional social values in which the existence of the people, individually and communally, is rooted.

What does the future of yam look like to you?
The future of yam looks bright to me. Today, yam is costly to produce because of Stone Age technologies that dominate the yam crop sector. Yam production, harvesting, and storage technologies are primitive. Why? The answer is that there has not been significant investment in yam research and development. Yam is produced and consumed in West Africa, mostly, that is. West African governments do not care and in that case the Western world does not bother.

But the situation is beginning to change with the funding of YIIFSWA by the Bill & Melinda Gates Foundation. In a regional agricultural research on a crop produced and consumed with rudimentary technology, US$12 million over five years is seed money. But it is an important seed money because it is the first of its kind and it will grow to help break the low technology bottleneck in the yam crop sector.

People have talked about the extinction of yam because of its primitive production and handling technologies. Such people should understand that as long as there are yam eaters, yam would be produced. Yam has a bright future because in spite of high costs, West Africans have continued to produce and eat yam. More importantly, with the US$12 million funding for YIIFSWA, the international donor community is beginning to appreciate the various values of yam.

What is yam’s added value?
It is not easy to think clearly of those added values with the present high cost of yam production. Converting yam to starch, liquor, ethanol, etc., will be irrational behavior because there are cheaper sources of those products. If yam is discovered to possess some high medicinal value, which no other plant has, such as a substance that can cure common diseases that have so far defied cure such as diabetes, various cancers, HIV/AIDS, etc., then the crop can be rationally diverted to such use.

How would you then describe the yam of the future?
This is a good question but the answer is not direct because of the different purposes that yam serves which may be conflicting in terms of the nature of yam that serves each purpose. Long ago, I think it was in 1980, the future of yam was the subject of a panel discussion at the Triennial Conference of the ISTRB-African Branch at IITA. The answer to the question ought to consider the different requirements for the various uses for yam. But there is a bottom line and that is cost; the yam of the future must be delivered for the various uses at reduced cost. Yam has no rival as a ritual object in cultural rites in producing communities, but that use alone cannot sustain yam in the future. Yam as food has a wide range of competitors some of which are produced at very low costs following high levels of investments in research and development in the Western countries. As those alternative foods become cheaper, people will switch to them.

You have been associated with IITA for so many years. What do you see as its strengths and what areas need more focus?
I have been associated with IITA since 1977. When I returned to Nigeria from graduate school at Michigan State University, the first place I had a job was the University of Nigeria, Nsukka, as a teacher. A university teacher in agriculture has responsibility for teaching, research, and extension. But the university did not have money for research and I did not want my research responsibility to suffer.

I had a senior colleague in graduate school who was a scientist at IITA, his name was Fred Winch. Fred passed away a few years ago; I do want to remember him. I used Fred’s facilities to carry out the study of “Yam-Based Cropping Systems of Eastern Nigeria” which I referred to earlier. Kun Tekail, who was Director of the Farming Systems Program at IITA, asked me to come to IITA as a full Scientist. I declined because I was enjoying what I was doing in Eastern Nigeria. He then appointed me as a Honoris Causa Scientist at IITA. I continued to work with IITA resources as an honorary scientist until 1987 when I caved in to pressure from Larry Stiffel to join IITA as a full scientist.

I was employed at IITA as a yam economist to work with yam agronomists. Dunstan Spencer was the director of the Resource and Crops Management Program in which I was based. A few months after I came to IITA Dunstan assigned the COSCA project leadership to me and I moved from working on yam to working on cassava. I left IITA in 1997 but I have continued to work on cassava.

There is an agricultural problem that IITA needs to address. That problem is neglected perhaps because of the assumption that Africa’s agricultural development will necessarily follow the path of other regions’ agriculture. For example, in Africa R and D effort is focused on achieving a Green Revolution because of the success of Green Revolution in Asia and South America. But are Africa’s needs and circumstances the same as those of Asia and South America? In Africa, a different kind of revolution is needed to pave the way for a Green Revolution; that is Mechanical Revolution. In the 21st century African agriculture based on the hand hoe cannot compete with the rest-of-the-world agriculture. I am not talking of tractor mechanization but improved farm tools that are designed by engineers working in Africa and maintainable by local artisans.

How do you perceive the impact of IITA’s work on roots and tubers for farmers in Africa?
Whoa! Tremendous! In Nigeria, scientifically determined yield of cassava was 15 tons per hectare in the 1990s following wide adoption of IITA’s high-yielding mosaic resistant TMS varieties. There was an estimate that in Nigeria alone, the additional value in terms of gari from these high-yielding mosaic resistant TMS varieties was enough to feed 29 million people annually. We have information which shows that the price of cassava products relative to the price of other commodities dropped in the 1990s, which meant increased income to consumers who paid less for cassava food products such as gari. At the same time, because of reduced cost, farmers are making more money. IITA’s effort on cassava including the biological control, mosaic disease control, and the high-yielding varieties produced tremendous value in terms of income to millions of cassava producers and consumers.

How do you picture Africa in the next 50 years?
Income will improve in Africa and people will be better off materially. The meaningfulness of that in terms of improved welfare depends on how much the measures that create the wealth interfere with the fundamental values of the African people. I wish to see a significant decline in the present high levels of poverty, deprivation, and inequality in Africa. Retaining African social values while improving the economic conditions of the masses of the people will be the better of two worlds.

How will agriculture play a role in doing that?
Improved agricultural productivity will mean improved income for farmers through reduced production costs and for consumers through reduced food prices. Improved productivity in agriculture will generate feed and industrial raw materials and help expand employment opportunities in the industrial sector. Large farms could be depended on to improve agricultural productivity but they can convert small farmers into farm laborers. Measures to improve agricultural productivity should protect small farms to allow even distribution of increased farm income from improved productivity.

In the ISTRB symposium 2 years ago, you were given the Lifetime Achievement award. What does that award mean to you?
The Award for Lifetime Achievement in research on roots and tuber crops was given to me by peers in the ISTRC and that makes it satisfying. One of the reasons I was given the award is the pan-African cassava research project in which I served as Project Leader while I was a scientist at IITA, i.e., the Collaborative Study of Cassava in Africa or COSCA study. I do not claim the award for myself alone even though it was given in my name.

The COSCA study involved 63 scientists from all over the world not just Africa. The study was the idea of Dunstan Spencer, John Lynam, and others whom I do not even know. Soon after I came to IITA in 1987 as a Yam-Based Systems Economist, the new COSCA study was assigned to me to execute. CIAT, NRI, International Child Health Institute, and MSU are among collaborating institutions from outside Africa. National agricultural research centers of Cote d’Ivoire, Ghana, Nigeria, Democratic Republic of the Congo, Tanzania, and Uganda played critical roles.

What is the value of a medal like this award? Igbo wisdom says that “if you say thank you to somebody, the person will do more.” Saying thank you is inexpensive but it is an inspirer. Besides, awards such as this one can open doors; Prof Felix Nweke, winner of Lifetime Achievement Award, is at the door, please let him in. I am going to make effective use of this effect to do more work on cassava and yam in Africa. That is what the award means to me.

What would you say is the highlight of your career?
This question can hardly be answered with dispassion; self-assessment is more often than not underrated or overrated depending on one’s level of humility. Nevertheless, I consider that the highlight of my career is demonstrated in the accomplishments of the people with whom I have grown up professionally. These are not only students whom I taught in the classrooms or those whose higher degree dissertations I supervised; there are several of those. But having worked closely with Prof Carl Eicher of Michigan State University for the past 50 years beginning in 1963 at the University of Nigeria, Nsukka, I assimilated what I consider his finest professional value. Carl Eicher is like a magnet that draws young professionals to him and he grows with them, in some cases for life. Many of the people I have grown with that way are highly accomplished professionally and they are all over the world, not just in Africa. That is the highlight of my career and it could not have been better.

Tony Sikpa: Commercializing yam in Ghana

Anthony Sikpa is the president of the Federation of the Associations of Ghanaian Exporters (FAGE). His group includes producers, exporters, and farmers. The federation currently has 13 associations with up to a couple of hundred members each. He also works with people in the horticulture sector producing vegetables, papaya, pineapple, and yam. He has been involved in organizing the group and doing advocacy work as president. He is an exporter of commodities such as cotton seed, cashew, coffee—both processed and raw materials, and other products.

How did you get involved in yam development?

Yam has been of interest to us and Ghana has been exporting yam for many years. So when the government approached IITA and ITC to help them develop a sector strategy for yam, it was interesting. Initially I was not involved because I did not handle yam but the association members asked me to lead them from the private sector angle. So I worked together with Dr Antonio Lopez (IITA) and Nelson (ITC), and we designed a participatory approach in crafting the strategy and in implementing and evaluating the whole process. This meant getting everybody in on one house: farmers, researchers, policy makers, exporters, traders, and financiers, under one roof. We talked, addressed issues, and came out with six broad objectives; how to improve the planting material, how the research and private sector will support the crop, how to help yam farmers in producing products using improved technologies, and how to market yam? Because we can’t just take anything into the market, we introduced ‘quality certification’ along the way to test the product.

The exercise was very useful. For the first time the misconceptions along the value chain were addressed. A farmer had the opportunity to ask scientists “why don’t you produce this type of material for me?” The approach really addressed the need of each person in the room.

For me, it helped to tell the public sector to create an environment for the private sector to lead and work with them.

Where are we going with the strategy?
This strategy is very important. The document provides a future road map, priorities and areas for investment/resource allocation, including milestones to assess the progress. The beauty of the strategy which makes me happy is that it is not dependent on one person to make it work. The farmers can go to the researchers to get the varieties. Fortunately we have IITA also to approach for new varieties. The exporter now clearly knows that he has work from the market; what the market requires and its standard. So he should prepare himself for the market.

We have used the strategy to position yam as an input for the industry. We should not just see yam as a food for the table. Yam can be used for different types of products including wine, in pharmaceuticals, etc. These are the ways we want to project yam so that we create a bigger demand for yam, and researchers would have to produce different varieties to suit the different needs of people. We will then be making yam as an industrial crop and create a bigger demand for it.

How are you involved with YIIFSWA?

I took advantage of the YIIFSWA meeting in Kumasi to go and tell them what we are doing in the yam sector and to also get their support for what we are doing. Maybe some of the products such as the new seeds and technology that would come out of the project could be made available to us in executing our strategy. I also told them about the gap that I saw: the emphasis on seed production was too much. We need to go beyond that into processes, coming out with new varieties for different uses. That is where they thought I could be useful and they invited me to join the technical advisory for the first time.

What are the lessons for other countries in Ghana’s experience of developing a yam sector strategy?

Others can learn from us. You cannot go into export without knowing which market you want. The export market has different strings; you need to look at the size of the market and use that to determine your production methods and even the varieties you want to produce. The variety that is in big demand is Pona. It has a very short shelf life and is delicate. You have to put all this into consideration when transporting it. You must package it well. The researchers need to take their time to study this. This project would help us to collaborate with, for example, our Nigerian brothers for us to be able to show them a few things we are doing that they can do. We know the way we use yam is different from the way they use yam. If they want to export yam, they need to go for smaller sizes for easy packaging because we measure in weight and not in hip or sizes.

What are the challenges in commercialization of yam?

The major challenge is in numbers and regularity. If you look at the trend in marketing now, in supermarkets they don’t want to buy small quantities because the supermarket has a chain, so you need to produce the volume required. If you can do this, you won’t have problem.

Aggregation. For instance you have so many farmers producing yam, you need someone to aggregate and make sure the quality is the same. The supermarket would not buy from you again if you get it wrong because they have a responsibility to their consumers to assure their safety. I will keep on saying certification. You cannot put any product in the European market without certification. They would look for international certification like the rainforest alliance as this would give them access to the international market. Another thing is transportation. You need to transport your product directly because if you don’t it will get cooked.

What is your vision for yam in Africa?

My vision for yam is to put it where potato is. Potato is everywhere. When people bring yam to town and package it into yam flour, then we’ll have done what this project is set out to do.

Would you have any advice to young farmers?

The young farmers should be happy and should grasp this opportunity of coming into agriculture now. It is a new learning. They should not be afraid of scientists; they should go to them with trust and patience.

Embryo rescue and anther culture for breeding new yam varieties

Yukiko Kashihara, y.kashihara@cgiar.org

To contribute to reducing poverty and increasing food security, IITA is breeding new varieties of yam based on demand and value addition. This is usually done by crossing two parents of the same species (intraspecific breeding, e.g., Dioscorea rotundata clones, TDr Ehuru × TDr Ehobia) or different species (interspecific hybridization, e.g., D. rotundata clone, TDr Ufenyi × D. alata clone, TDa 85/00250) to transfer useful traits. However, intraspecific or interspecific yam breeding is still being constrained by poor flowering, low seed-setting, a low rate of seed germination, and differences in flowering periods as male and female parents flower at different times.

Interspecific hybridization is particularly useful to produce additional variations in Dioscorea species. For instance, interspecific hybridization of D. alata and D. rotundata can contribute to the transfer from D. rotundata to D. alata of genes conferring tolerance to anthracnose disease. However, interspecific hybridization is still a challenge. One example in Japan dealt with the hybridization between D. japonica and D. opposita produced with the aid of the embryo rescue technique (Araki et al. 1983). In addition to interspecific hybridization, the use of homozygous parents (having the same pairs of genes) could add efficiency to breeding. Anther culture can also produce homozygous plants through chromosome doubling of haploid plants. Establishing an anther culture system will further diversify breeding approaches. Therefore, current studies are focusing on (i) refining the embryo rescue technique to improve the efficiency of interspecific hybridization, and (ii) establishing a method for producing haploid yam which saves several generations in the breeding program.

Using ovule culture (excised ovules from the ovary and cultured on media), we obtained one plant which resulted from a cross in 2012. However, after flow cytometric analysis, we found that the plant was derived from an ovule parent. For anther culture, more investigation is needed to find out the optimum medium and conditions. The establishment of reliable tissue culture protocols and efficient working schemes is essential. The success of this will contribute to allowing wide hybridization and saving time and space for IITA’s yam improvement program. Moreover, having haploids will be advantageous for yam research, especially in gene mapping, genomics, and other applications.

Reference

Araki, H. et al. 1983. Some Characteristics of Interspecific Hybrids between Dioscorea japonica Thunb. and Dioscorea opposite Thunb. Japanese Society for Horticultural Science 52:153-158.

Genetic transformation of yam

Leena Tripathi, l.tripathi@cgiar.org

Yam (Dioscorea spp.) is a multi-species, polyploid and vegetatively propagated tuber crop in the tropics and subtropics that provides food security and income to over 300 million people. There are 600 Dioscorea species; however, only a few of them are regularly cultivated for food. Dioscorea rotundata and D. cayenensis (both known as Guinea yam) are most popular and economically important in West and Central Africa where they are indigenous, while, D. alata (known as water yam) is the most widely distributed species globally. Yam is the second most important root and tuber crop in sub-Saharan Africa after cassava in terms of production with about 57 million metric tons. Over 95% of world yam production occurs in the yam belt of West and Central Africa with Nigeria alone accounting for about 66% of the world’s total. Some wild yam species are also known to produce secondary metabolites of pharmaceutical importance such as steroidal sapogenin, diterpenes, and alkaloids.

Despite the crop’s economic and sociocultural importance, its cultivation is generally limited by high costs of planting material and labor, decreasing soil fertility, low yield potential of varieties, and increasing levels of field and storage pests (nematodes) and diseases (anthracnose, tuber rots, and yam virus complex). In West Africa, about 11 million tons of yam are lost annually because of damage in storage initiated by nematodes. Plant-parasitic nematode damage is a critical factor in tuber quality reduction and yield loss in yam, both in the field and in storage, which is perpetuated over seasons through infected seed material. Nematodes also facilitate fungal and bacterial attacks.

Nematodes can be managed by nematicides, but are not commonly used due to their high cost. As yam is vegetatively propagated, nematode-affected tubers are transferred in infected seed yam material. Yam nematodes reproduce and build up large populations in stored tubers, causing severe damage and facilitating fungal and bacterial attacks that cause anthracnose disease, dry rot, soft rot, and wet rot. It is therefore necessary to control plant-parasitic nematodes to increase or at least maintain reasonable yields of yam and protect susceptible germplasm from total loss. Nematode-resistant varieties of yam can be very effective for nematode control.

Genetic transformation is an alternative tool for developing nematode-resistant varieties. This option is also important because host plant resistance has not yet been found in the major cultivated species (D. rotundata, D. cayenensis, D. alata) or close relatives with which they can be crossed by conventional breeding and selection for the trait. It is therefore necessary to control plant-parasitic nematodes to increase or at least maintain reasonable yields of yam and preserve susceptible germplasm.

Genes conferring nematode resistance are already available from the University of Leeds for plantain (Musa spp.) transformation at IITA, which can easily be made available and assessed against nematodes in yam. This can only be possible after the yam transformation system is established. To date, only a single report has been published on the stable transformation of D. alata by particle gun using reporter gene (Tor et al. 1993), and this still needs improvement. Tor et al. (1998) also reported transient gene expression in protoplast of Dioscorea spp. using polyethylene glycol (PEG)-mediated direct uptake method. The stable transformation of yam using PEG-mediated uptake still needs to be developed.

There is no report on Agrobacterium-mediated transformation of D. alata and D. rotundata, which is the preferred method for genetic engineering of plants, offering several advantages over direct gene transfer methodologies (particle bombardment, electroporation), such as the possibility of transferring only one or a few copies of DNA fragments carrying the genes of interest at higher efficiencies with lower cost and the transfer of very large DNA fragments with minimal rearrangement.

The development of stable transgenic plants requires an efficient regeneration system amenable to genetic transformation and stability of transgene under field conditions. Regeneration systems from meristems of D. rotundata and D. alata have recently been established at IITA (Adeniyi et al. 2008; Tripathi et al. unpublished). Recently, direct shoot organogenesis was also reported in petiole explants of D. rotundata, D. cayenensis, and D. alata (Anike et al. 2012). These regeneration systems are yet to be evaluated for their amenability to transformation. Regeneration through callus or somatic embryogenesis, which is ideal for transformation, remains to be established. There are only a few reports on plant regeneration from embryogenic cell cultures of Chinese yam (D. opposita), D. alata, and D. cayenensis (Belarmino and Gonzales 2008; Nagasawa and Finer 1988; Twyford and Mantell 1996; Viana and Mantell 1989). However, there is no report of regeneration from somatic embryogenesis of D. rotundata. We have obtained embryogenic callus but further research is needed to develop an efficient regeneration protocol using callus.

As a transformation system for yam is currently not available, therefore, IITA, with support from the Bill & Melinda Gates Foundation, is conducting research to develop a regeneration and transformation system for yam varieties most preferred by farmers. Once a transformation system for yam is established, the protocol will then be used for producing nematode-resistant varieties for effective control of this major pest in yam production systems.

References

Adegbite AA et al. 2006. Survey of plant-parasitic nematodes associated with yams in Edo, Ekiti and Oyo states of Nigeria. African J Agric Res 1: 125-130.

Adeniyi OJ et al. 2008. Shoot and plantlet regeneration from meristems of Dioscorea rotundata poir and Dioscorea alata L. African J Biotechnol 7: 1003-1008.

Anike FN et al. 2012. Efficient shoot organogenesis in petioles of yam (Dioscorea spp.). Plant Cell Tiss Organ Cult 111:303–313.

Belarmino MM et al. 2008. Somatic embryogenesis and plant regeneration in purple food yam (Dioscorea alata L.). Ann Trop Res 30:22-33.

Bridge J et al. 2005 Nematode parasites of tropical root and tuber crops. In: Luc, M., Sikora, R.A. and Bridge, J. (eds) Plant Parasitic Nematodes in Subtropical and Tropical Agriculture, 2nd edn. CABI Publishing, Wallingford, UK, pp. 221-258.

Nagasawa A, Finer JJ. 1988. Plant regeneration from embryogenic suspension cultures of Chinese yam (Dioscorea opposite thumb.). Plant Sci 60:263–271.

Tor M et al. 1993. Stable transformation of the food yam Dioscorea alata L. by particle bombardment. Plant Cell Rep 12: 468-473.

Tor M et al. 1998. Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies. Plant Cell Tiss Organ Cult 53:113–125.

Twyford CT, Mantell SH. 1996. Production of somatic embryos and plantlets from root cells of greater yam. Plant Cell Tissue Organ Cult 46:17–26.

Viana AM, Mantell SH. 1989. Callus induction and plant regeneration from excised zygotic embryo of the seed propagated yams Dioscorea composite Hemsl. and D. cayenensis Lam. Plant Cell Tissue Organ Cult 16: 113–122.

Novel approaches for the improvement of yam germplasm conservation and breeding

Gezahegn Girma, g.tessema@cgiar.org

IITA, Ibadan, Nigeria

Genetics and Biotechnology Lab, Plant and AgriBiosciences Research Centre (PABC), School of Natural Sciences, National University of Ireland Galway, Ireland

Efforts are ongoing at IITA, in collaboration with the National University of Ireland Galway, to understand the genetic diversity, evolutionary relationship of yam species, flowering and sex-related genes, polyploidy and its effect on phenotypic performance and somaclonal variation in in vitro regenerated plants. This work is expected to contribute to the development of new tools for assessing yam diversity, and understanding genetic and phenotype linkages for improving yam breeding and germplasm conservation. The status of thrust areas being pursued to address these gaps is summarized here.

Identification of chloroplast or nuclear regions that can help discriminate species

Developing molecular tools supported by taxonomic identification is very important for unambiguous species naming or classification. A DNA barcode aids taxonomic identification, which uses a standard short genomic region that is universally present in target lineages and has sufficient sequence variation to discriminate among species. The single locus rbcL, matK, combination of rbcL+ matK, the noncoding intergenic spacer, trnH-psbA of chloroplast regions, and ITS of nuclear region were evaluated using a criterion for candidate DNA barcode for Dioscorea species identification. All the sequences were assessed for universality (ease of PCR amplification and sequencing), sequence quality, and species discriminatory power.

Genetic polymorphism of cultivated guinea yams and their relationship with wild relatives

Next generation-based genotyping procedures such as genotyping by sequencing (GBS) is now considered as an excellent tool in plant genetics and breeding (Poland and Rife 2012) due to its genome-wide molecular marker discovery, genotyping for multiplexed samples, flexibility, and low cost. A study was conducted to understand the genetic diversity within and between two cultivated guinea yams and five of its wild relatives (D. praehensilis, D. mangenotiana, D. abyssinica, D. togoensis, and D. burkilliana) using GBS, morphology, and ploidy analysis.

In general, the genetic contribution of wild relatives, origin of cultivated species, ongoing domestication practices, potential polyplodization process and utility of GBS in generating genotypic information were demonstrated. Similarly, GBS could further be used for understanding of genetic relationship studies of other species within the genus Dioscorea that holds a large number of species in addition to the guinea yams. Due to the cost effectiveness of GBS, there is major potential for the yam genebank collection in IITA (and other yam germplasm collections) to be genotyped using the GBS procedure. GBS can help to indicate the level of genetic diversity and guide the need for more germplasm collection, duplication, and mismatch identification.

Understanding the molecular genetics of flowering

D. rotundata are mostly dioecious, with separate male and female plants, although a few lines are identified as monoecious. It is also common to find nonflowering, which is perhaps dominant. The dioecy of the crop makes the synchronization of flowering time very difficult. For the efficient improvement of yam crops through conventional breeding, understanding better the genetic mechanisms of flowering in Dioscorea is essential. The tiny and large numbers of Dioscorea chromosomes is also a challenge to make critical observations. Therefore identification of the sex-determining chromosomes is difficult at cytological level. In addition, the genes that control the flowering in yam are not yet known. A study is being conducted to identify gene expression patterns in relation to different flowering habits (male, female, and monoecious) of D. rotundata accessions using the SuperSAGE technique. The study outcome is expected to help understand the flowering biology of the crop in general and once confirmed, identified sex-determining candidate genes can be incorporated in varieties with inconsistent nonflowering to produce regularly flowering cultivars, and hence, improve yam production.

Morphological, ploidy and molecular diversity

The section Enantiophyllum of the genus Dioscorea is generally known to produce only 1-3 underground tubers. Hence, there is a need for exploitation of other options of planting materials such as aerial tubers as an alternative planting material to underground tubers. The study was conducted to investigate the molecular, morphological, and ploidy variation across Dioscorea alata accessions producing aerial tubers in comparison with accessions without aerial tubers. The aerial tuber production of accessions was found correlated with ploidy level, distinct morphological characteristics, and SSR analysis discriminated according to its pattern of aerial tuber production.

Evaluating somaclonal variation under in vitro regeneration

In spite of the several advantages from the tissue culture system in plant ex situ conservation, somaclonal variation is regarded as one of the major problems of many tissue-cultured plants (Bordallo et al. 2004). On the other hand, somaclonal variation is known for its usefulness in crop improvement by creating novel sources of variability that could result in improved yield, resistance to diseases, and quality improvement. Detection and elimination of undesirable variants and spotting variants with useful agronomic traits is therefore essential. The study on meristem derived in vitro clones of D. rotundata accessions with their original genotypes is ongoing to evaluate and verify somaclonal variation using AFLP markers.

References

Bordallo PN, Silva DH, Maria J, Cruz CD, Fontes EP. 2004. Somaclonal variation on in vitro callus culture potato cultivars. Horticultura Brasileira 22:300-304.

Poland JA, Rife TW. 2012. Genotyping-by-Sequencing for Plant Breeding and Genetics. Plant Gen 5:92-102.

Yam genetic resources conserved at IITA

Badara Gueye (B.Gueye@cgiar.org) and Michael Abberton

Within CGIAR, IITA has the mandate for the collection, characterization, and exchange of yam species. IITA’s Genetic Resources Center (GRC) thus holds a major yam international germplasm collection in trust under the Multilateral System of the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA).

With 3,872 accessions, the IITA yam collection is the world’s largest, including nine of the major cultivated species: Dioscorea rotundata, D. alata, D. bulbifera, D. cayenensis, D. dumentorum, D. esculenta, D. preussii, D. manganotiana, and D. praehensilis. Conserved in the field and through an in vitro genebank, it represents a large genepool for yam crop improvement to help the crop reach its full potential for food and income for poor farmers. To meet this objective, GRC is working on many themes for harnessing yam genetic resources in collaboration with a range of partners. The first one is the filling of genetic gaps to ensure the availability of a broader yam gene pool. In 2014, we are carrying out germplasm collection in Nigeria and Benin Republic. The entire yam collection is conserved in the IITA field bank at Ibadan, Nigeria, and the use of outstation sites offers different conditions for the regeneration of recalcitrant lines, which reduces germplasm losses. More than one-third of the yam collection is duplicated in the in vitro medium-term storage facility at IITA-Ibadan. The development of a yam cryopreservation protocol will allow long-term conservation.

To promote the use and distribution of yam germplasm, especially in breeding, the entire yam collection was characterized using agromorphological descriptors, leading to the identification of a core collection. Increasing collaboration with the yam breeders, germplasm health specialists, and national partners is ongoing to carry out genetic resources evaluation including for market demanded traits. Molecular tools and advanced phenotyping methods are also being employed to further characterize the germplasm and further promote its use. All the data are made available and accessible worldwide and efforts are ongoing to increase the proportion of quarantine pathogen free yam genetic resources for more distribution across borders. Viruses are important pathogens of yam, therefore germplasm use will also be promoted through production of virus-free material for exchange and production of planting material. Virus elimination methods (heat treatment, cryotherapy and chemotherapy) are being explored to establish a reliable protocol for yam virus cleaning.

GRC plays also a major role in training and capacity building of national programs with respect to collection, conservation and characterization of genetic resources of yam.

For further information visit: http://www.iita.org/genetic-resources.

Mobilizing yam farmers in Ghana and Nigeria to produce good quality seed yam

Beatrice Aighewi, a.aighewi@cgiar.org

The availability and quality of seed determine the success of any farmer and this has been an issue because the seed sector is poorly developed in many African countries. The challenges that result from poor seed are particularly serious with regard to vegetatively propagated crops such as yam. Several reasons are responsible for this situation: the resultant unavailability and poor quality of seed yams is compounded by the following reasons:

· Seed yam is mostly produced by traditional methods which are relatively slow.

· The high cost of seed yam causes most farmers to rely mainly on seed tubers saved from previous harvests.

· Lack of quality control and absence of formal seed yam production systems result in scarcity of genetically uniform and disease-free seed in markets.

· Poor understanding of the quality attributes of seed yam entrenches poor quality.

· Lack of capacity/knowledge on rapid multiplication methods to obtain clean planting material.

In trying to improve the current situation of seed yams, the Yam Improvement for Income and Food Security in West Africa (YIIFSWA) project is placing emphasis on improving the quality of seed yam on farmers’ fields and initiating the development of a formal seed yam system since ensuring quality is a hallmark of any formal seed system.

In most yam-producing systems in West Africa, seed yam production is not separated from ware yam production. Traditionally, a farmer plants setts to produce ware yams and in the process obtains seed by double harvest (milking), cutting some large tubers or using those that are too small for food. Due to the low rate of multiplication, the seed produced is rarely enough in quantity for expansion of yam cultivation and because the same seed is recycled over many generations, a build-up of disease on seed tubers reduces the productivity. In many seed yam markets, it is the left-over seed after the farmer has planted his field that is sold, and chances are high that the quality of such seed will be poor (Fig. 1) because a farmer will normally reserve the best to plant in his field).

The only region where relatively good quality seed yam is found in market is in parts of the River Niger area of Nigeria, particularly at Illushi and Otuocha Markets, where there are farmers who specialize in seed yam production. Seed yam in the market is sorted by size and variety and they look good in terms of quality (Fig. 2). Discussions with yam farmers in Nigeria and Ghana revealed that for most of them, the only reason why good seed yam is sold is if they are faced with a financial crisis and need immediate cash. Seed yam which is an inheritance for most farmers is a major component of their financial and food security, and in some instances anyone who takes seed yam to the market to sell is considered a thief. In Salaga District of Ghana, no seed yam is sold at any market; it can only be bought from a farmer on his farm.

Observations at Illushi market shows that even without the formal seed system, farmers can produce better quality seed if they are enlightened and specialize in seed yam production. If such seed yam markets could be developed in major yam-producing areas, most of the problems associated with unavailability and poor quality will be sorted out.

To improve the quality of seed yams on farmers’ fields and develop the seed yam market, YIIFSWA has targeted the small-holder farmer who forms the majority of producers. A multidisciplinary YIIFSWA team uses the platform of cross-objective workshops and capacity building to train farmers in some communities of major yam-producing regions of Nigeria and Ghana to produce and market good quality seed yam (Figs. 3 and 4). Many of the farmers trained belong to farmers’ groups that have already had some training on producing seed yams from minisetts on demonstration plots in their localities. Some of those who participated in previous training exercises had an opportunity to explain to other farmers what they had learned and encouraged other farmers to try the minisett technique to produce their seed yam, and remove barriers that have prevented them from getting more seed.

YIIFSWA also provided training for extension agents on high ratio propagation methods—minisett technique and use of vine cuttings which can produce more tubers of better quality than what farmers are currently producing (Fig. 5). It is expected that they will train farmers in their areas of operation, and sustain the campaign for production of better quality seed yam.

Countering yam anthracnose threat in West Africa

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Nematode pests of yam

Danny Coyne

Plant parasitic nematodes are ever-present and incidental with plant growth and crop production, occurring on just about every crop or plant known.

Nematodes are mostly microscopic and thus unseen; and the symptoms of nematode infection are difficult to determine in the field, as these are often nonspecific.

Yam farmers are very much aware of the physical damage that nematodes cause to the tubers, but are mostly completely unaware of what causes the damage. In the field, nematodes reduce crop vigor and performance, leading to lower yields. They cause significant damage to the tubers, resulting in deformed, unsightly tubers or tubers with cracked and flaking skin that conceals an underlying rot. Such symptoms have an immediate and direct impact on the marketability of tubers, but they also relate to reduced crop productivity. Infected tubers, when unwittingly used as planting material, due to low, unnoticeable levels of infection, affect the ability of seed to produce—or even to germinate.

A wide range of nematodes are associated with yam, but only two ‘types’ are of concern: root-knot nematodes (Meloidogyne spp.), which are evident by the disfigurement they cause to tubers (Figs. 1 and 2), and lesion nematodes (Scutellonema bradys, Pratylenchus spp.), which result in ‘dry rot’ and cracked tubers. Infected tubers can also develop erratically growing roots, referred to as ‘crazy roots’ (Fig. 1A).

Meloidogyne spp., are an especially damaging group of pests for numerous crops, which are becoming an increasingly serious problem on yam, likely due to gradual intensification of cropping practices. For example, a recent 2013 survey by IITA in Nigeria found that approximately a quarter of all harvested tubers have some level of tuber damage by Meloidogyne spp. (Fig. 1B).

The same survey also discovered, for the first time, a particularly aggressive species, M. enterolobii, infecting yam, among a number of other Meloidogyne species, which may occur simultaneously in the same field, and on the same plant. This has implication in breeding for resistance, and requires that the screening process takes into account the variety of species affecting the crop, such as is undertaken at IITA. Once infected by Meloidogyne spp., tubers become galled and disfigured. Symptoms will vary depending on conditions, nematode species, and yam variety. Tubers normally look ‘knobbled’ due to the development of galls on the surface, the severity of which depends on the level of infection.

Farmers do not understand how this disfigurement occurs, believing it to be a supernatural occurrence in some cases. Tuber galling damage will not generally develop further once harvested. During storage, galled tubers lose weight and deteriorate much faster than healthy tubers. Galled tubers used for seed will (if they survive) result in the development of more heavily damaged (galled) tubers at harvest.

Dry rot, caused by lesion nematodes, results from their feeding action as they ‘migrate’ from cell to cell, destroying them as they pass through the yam tissue. This damage occurs first in the subsurface tissue, just below the tuber skin, moving deeper with time. A relatively healthy looking skin can also often mask the underlying damage, which may not be visible until the surface is damaged or cut back (e.g., Fig. 2A), to reveal the brown, discolored, necrotic tissue beneath. Surface cracking is also a typical symptom of lesion nematode infection, but the relation between the nematodes and cracking is less clear-cut, with cracked tubers occurring in the absence of nematodes in tubers.

The species of nematodes responsible for dry rot and cracking on yam is intriguing for several reasons. One is that S. bradys is a key species, which feeds as an endoparasite, while the remaining species in the genus are better known as ectoparasitic feeders, remaining in the soil and feeding on the outside of roots. The other is that at least two species of Pratylenchus (P. coffeae and P. sudanensis) also cause the same symptoms as S. bradys. The species distribution and occurrence is geographically related; the more research we undertake, the more we unravel the story. We have determined the center of origin for S. bradys, for example, as the central Nigeria/Benin area, although the nematode has since been distributed around the globe on infected yam tubers. At harvest, severely affected tubers may be obvious, but lower levels of infection may go unnoticed. However, during storage the nematodes continue to feed, which is a major consequence of lesion nematode infection. Heavy infections result in the complete deterioration of tubers, while less damaged tubers can, to some extent, be consumed after removing the rotted sections. Low levels of infection may not be detected, and can contribute to the disease cycle when tubers are used as seed material.

A particular characteristic of yam is that the nematode problem is perpetuated through the use of infected planting material. Heavily damaged tubers are obvious, and are not generally used as seed, and usually consumed at home. Less visibly damaged tubers may be sold in the market or stored, for sale or consumption later or for use as a planting material in the next season. The use of poor quality planting material thus serves to maintain the disease cycle, by returning inoculum from the store back to the field. This adversely affects crop establishment, yield, and storability of harvested tubers, ensuring a continued and negative impact on quality, especially of highly susceptible cultivars. It is likely even, that nematode damage, in particular, has been a major contributing factor to the loss of some traditional (susceptible) varieties.

To overcome nematode problems on yam, a key area of focus is to target the seed system, and farmer awareness and understanding of the problem. This has become an increasingly important focus for IITA and its partners in recent years. It also forms the central pillar for a large IITA-led yam project. By generating and maintaining sustainable healthy seed systems, farmers will have greater access to seed material that will result in more productivity that will be less likely disfigured at harvest. In-field infection will occur, especially by root-knot nematodes, but damage will be less severe and yields higher.