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.

 

Novel yam propagation technologies: the aeroponics system

Norbert Maroya, n.maroya@cgiar.org, Morufat Balogun, Lava Kumar, Robert Asiedu, and Beatrice Aighewi

Norbert Maroya, Project Coordinator, YIIFSWA; Morufat Balogun, Agronomist (YIIFSWA); Lava Kumar, Virologist and Head, Germplasm Health Unit; Robert Asiedu, R4D Director for West Africa, IITA; and Beatrice Aighewi, Seed Systems Specialist, YIIFSWA

The multiplication ratio of yam in the field is known to be very low (less than 1:10). The methods developed to address this limitation include the minisett technique, vine propagation, and micropropagation using in vitro culture of apical meristems and nodal cuttings. These methods are well suited to rapid multiplication of seed tubers for new and other recommended varieties, and are also amenable to the application of sanitary methods that ensure high seed quality. Other methods of rapid propagation developed at IITA include production of microtubers from plantlets in vitro, and the production of seed tubers using slips (sprouts) and peels. Other technologies also exist but are not yet being used for yam.

Three new technologies targeted to be implemented for seed yam propagation are aeroponics system (AS), temporary immersion bioreactor system (TIBs), and photoautotrophic culture (PC). These technologies are being tested under the Bill & Melinda Gates Foundation-funded project “Yam Improvement for Income and Food Security in West Africa (YIIFSWA)”. These technologies are known to be effective for other vegetatively propagated and horticultural crops for high ratio propagation and assurance of high seed quality. However, their cost-effectiveness for yam propagation is yet unknown. Very recently two of these technologies, AS and TIBs, have been established at IITA-Ibadan, Nigeria. Progress achieved with AS is summarized in this article.

What is an aeroponics system?
The basic principle of AS is growing plants in air in a closed or semi-closed environment without the use of soil or an aggregate media and spraying the plant’s roots with a nutrient-rich solution (mist environment). The techniques of growing plants without soil were first developed in the 1920s by botanists who used primitive aeroponics to study plant root structure. The aeroponics system has long been used as a research tool in root physiology (Barker 1922). Carter (1942) was the first researcher to study air culture growing and described a method of growing plants in water vapor to facilitate examination of roots. Went (1957) named the air-growing process in spray culture as “aeroponics”.

The International Union of Soil-less Culture defines aeroponics “as a system where roots are continuously or discontinuously in an environment saturated with fine drops (a mist or aerosol) of nutrient solution” (Nugali et al. 2005). AS has been used successfully in producing several horticultural and ornamental crops (Biddinger et al. 1998). It has also been applied successfully in Korea for potato seed tuber production (Kang et al. 1996; Kim et al. 1999). At the International Potato Centre (CIP) in Peru, yields of over 100 tubers/plant were obtained using aeroponics technology (Otazu 2010). Aeroponics technology is also being tested in several African countries for the production of potato mini tubers (Lung’aho et al. 2010).

IITA’s experience in propagating yam in AS

Seed yam production using aeroponics was initiated recently. A consultant from Kenya helped to establish an AS of 14 boxes of four tables each in an existing screenhouse at IITA, Ibadan, Nigeria, with an adjacent powerhouse as a source of the spray of nutrient-rich solution to the roots.

From yam seedlings transplanted in July 2012, vine cuttings were made on 6 to 19 December 2012 and planted in black plastic pots for pre-rooting. The pre-rooted vines were transplanted in AS on 26 to 28 February 2013. Vine cuttings were collected from other seedlings transplanted on 28 August 2012 and planted directly in AS on 1 March 2013.

Both pre-rooted and direct-planted vines have continued to grow normally in AS with the development of new shoots and roots. The two types of plants produced viable minitubers which were harvested in June 2013. The key finding in this experiment is the ability to root vine cuttings in AS. Within 10 days more than 50% of the vines produced roots and in 3 weeks 85–100% of the direct-planted vine cuttings produced roots in AS. If a yam plant is certified clean, one can directly collect vine cuttings from such plant for propagation in AS through vine cuttings.

This is the first report of successful yam propagation in AS. Also all previous studies on AS for potatoes or horticulture crops used transplants of rooted plantlets and not unrooted vine cuttings. This is the first experience using yam vine cuttings in AS. The minitubers harvested in June 2013 were planted in August 2013 and sprouted well.

Many of the farmer-preferred yam genotypes are also being evaluated in AS. Direct vine cutting of variety Puna—a popular cultivar in Ghana—was planted in AS on 10 July 2013 and harvested on 6 November 2013 (4 months).

Production of bulbils of yam in AS

The second set of experiments was done using only vine cuttings of plants produced in a glasshouse. To increase the size of mini tubers, two new fertilizers—potassium sulfate (K2SO4) and Triple Super Phosphate—were added to the existing nutrient solution. Between 45 and 60 days after vine cuttings were planted in AS we observed that many varieties of both D. rotundata (white yam) and D. alata (water yam) had produced bulbils. All the bulbils produced by D. rotundata were growing with new shoots and roots; it was the same for D. alata with most bulbils increasing in size. Bulbils mainly harvested from D. rotundata were planted in plastic bags, sprouted, and are growing normally.

Percentage of bulbils formed per genotype on AS

Genotypes

Number of plants

% of plants with bulbils

TDa 291

32

9.4%

TDa 98/01176

53

18.9%

TDr 02475

47

42.6%

TDr 89/02665

18

72.2%

TDr 95/18544

50

22.0%

TDr 95/19158

12

16.7%

TDr 95/19177

26

11.5%

Total

238

26.1%


Challenges

Ideally the AS environment should be kept free from pests and diseases so that the plants will grow healthier and quicker than plants grown in a soil medium. However, current arrangements do not provide an ideal environment due to lack of control on temperature and pest and disease infestation. Plants generated in AS were frequently infested (19 to 29%) by Colletotrichum sp. (both leaves and stem), Sphaerosporium sp. (stems) (typically saprophytic), and Fusarium sp. (stems). Steps are being taken toward reducing the heat inside the screenhouse with industrial fans and providing adequate shade. Measures are also being implemented to control infestation of fungal pathogens.

Conclusion

Despite the relatively recent (less than one year) attempt to propagate yam in AS, some of the results obtained so far are very encouraging and impressive. They have clearly shown that AS does not necessarily need rooted plantlets/vines for yam propagation. Micro-tubers, bulbils, and mini-tubers can be produced respectively within 2 and 4 months after vine cuttings are planted in AS.

References

Barker BTP. 1922. Studies on root development. Long Ashton Res. Station Ann. Rep. 1921: 9-57.

Biddinger E.J., Liu C.M.. Joly R. J, Raghothama K.G. 1998. Physiological and molecular responses of aeroponically grown tomato plants to phosphorous deficiency. J. Am Soc. Hortic. Sci. 123: 330-333

Carter W.A. 1942. A method of growing plants in water vapor to facilitate examination of roots. Phytopathol. 732: 623-625.

Lung’aho C., Nyongesa M., Mbiyu M.W., Ng’ang’a N.M.. Kipkoech D.N., Pwaipwai P., Karinga J. 2010. Potato (Solanum tuberosum) minituber production using aeroponics: another arrow in the quiver? In: Proceedings of the 12th Biennial Conference of the Kenya Agricultural Research Institute.

Kang J.G., Kim S.Y.; Om Y.H., Kim J.K. 1996. Growth and tuberization of potato (Solanum tuberosum L.) cultivars in aeroponic, deep flow technique and nutrient film technique culture films. J. Korean Soc. Hort. Sci. 37: 24-27.

Kim H.S., Lee E.M., Lee M.A., Woo I.S., Moon C.S., Lee Y.B., and Kim S.Y. 1999. Production of high quality potato plantlets by autotrophic culture for aeroponics systems. J. Korean Soc. Hort. Sci. 123: 330-333.

Nugali Yadde M.M., De Silva H.D.M., Perera, R., Ariyaratna D., Sangakkara U.R. 2005. An aeroponic system for the production of pre-basic seed potato. Ann. Sri Lanka Department Agric. 7: 199-288.

Otazú V. 2010. Manual on quality seed potato production using aeroponics. International Potato Center (CIP), Lima, Peru. 44 p. ISBN 978-92-9060-392-4. Produced by the CIP Communication and Public Awareness Department (CPAD)

Went F.W. 1957. The experiment control of plant growth. New York.

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.

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.


From yam production and postharvest constraints to opportunities

D.B. Mignouna, d.mignouna@cgiar.org, T. Abdoulaye, A. Akinola, and A. Alene

Food insecurity remains a huge concern in West Africa. Agriculture, without doubt remains the main source of food and livelihood. Over the past two decades, agricultural yields have stayed the same or declined. Although there has been a recent rise in agricultural productivity, it derived more from expanded planting areas for staple crops than from yield increases. Thus, increasing and sustaining agricultural productivity should be a critical component of programs that seek to reduce poverty and attain food security in the region.

Yam (Dioscorea spp.), a vegetatively propagated crop cultivated for its underground edible tubers, is the mainstay for about 300 million people in West Africa. It is a very important food and income source for millions of producers, processors, and consumers in the region. About 48 million tons are produced annually in this subregion on 4 million ha. The five major yam-producing countries (Bénin, Côte d’Ivoire, Ghana, Nigeria, and Togo) account for 93% of the world’s production, with Nigeria alone accounting for 68% of global production (36 million t on 3 million ha) with 31.8% of the population depending on yam for food and income security. The crop contributes substantially to the amount of protein in the diet, ranking as the third most important source, much more than the more widely grown cassava, and even higher than some sources of animal protein. Hence, yam is important for food security and income generation with a domestic retail price of US $0.49/kg. Yam is also integral to the sociocultural life in the subregion.

In present-day Nigeria, yam is still culturally significant because it plays an important role in betrothal ceremonies or traditional marriages. It is one of the significant items a suitor presents to his in-laws to obtain their approval to marry their daughter. Some grooms are compelled to present as many as 40 pieces of long and fat yam tubers, aside from gallons of palm oil, baskets of kola nuts, bags of salt, and other sundry items, the nonprovision of which could invalidate the union. The cultural importance of yam is higher in some regions in Nigeria as it is a crop celebrated annually during the New Yam Festival, with rituals to thank the god of agriculture, to seek its blessings for a bumper harvest in the forthcoming years. Yam is produced more in the middle belt zone of Nigeria and is consumed more in the South, but those making commercial gains from its sales are core northerners from the North West, the North Central, and the North East.

Despite its importance in the economy and lives of many people, the crop faces several constraints that significantly reduce its potential to support rural development and meet consumers’ needs for improved food security and enhanced livelihood. Constraints limiting yam production and postharvest handling need to be identified to provide a basis for appropriate interventions. This was the reason behind the interventions through the Yam Improvement for Income and Food Security in West Africa (YIIFSWA) project. YIIFSWA was initiated to work with other stakeholders in West Africa to identify the opportunities of interventions that could potentially help to increase productivity in the region. This report documents production and postharvest constraints and opportunities in yam.

Using Nigeria and Ghana as cases, important worldwide yam-producing countries, a study was carried out using a multistage, random sampling procedure in selecting a total of 800 and 600 households, respectively. All surveyed households were interviewed using a structured questionnaire.

Survey results indicated that a range of factors limited yam production and storage. These include insect pests, diseases, water-logging, drought, rodents, low soil fertility, shortage of staking material, inadequate input supply and storage facility, land shortage, high cost of labor, lack of improved varieties, and others such as theft (Fig. 1).

High cost of labor stands out as the most pressing problem in all the surveyed zones, both in Nigeria and Ghana. For instance, mounding as a seedbed preparation method, is laborious, and hence expensive. But apart from mound making all yam production operations are labor intensive because they are performed with hand hoes, machetes, and digging sticks without any form of a labor-saving technology.

Another main constraint are insect pests and diseases. The unavailability and high cost of good quality disease-free seed yam had been on one hand a result of pests and diseases and on the other hand a serious hidden constraint due to the fact that farmers do not purchase seed yam. Other important constraints mentioned were the inadequate input supply that was very pronounced in Ghana, low soil fertility more reported in Nigeria, rodents and drought (Ghana), water-logging (Nigeria), lack of improved varieties more prominent in Ghana, shortage of land and staking material (Ghana), and others such as theft that were not negligible in both countries.

It is clear that there are shared priority constraints in the two countries, indicating no specificity of problems by country. The YIIFSWA research agenda needs to be informed by the constraints facing yam farmers and based on these the following interventions were identified: (i) Key investments for lowering farmers’ production cost using agricultural research (breeding, agronomy) and extension (improved agronomic and management practices; and (ii) Managing pests and diseases.

As regards opportunities, yam could be be a formidable force in the fight against poverty, hunger, and deadly diseases if research and development measures are implemented to develop and disseminate technologies that can bring the crop into central focus in national food policies. This will enable it to benefit from policy programs that can drive down production costs. Yam is a preferred food in the region; some varieties, especially yellow varieties, are sources of betacarotene. The crop is produced mostly for sale, and it is increasingly becoming a major source of foreign exchange in the region as an export crop.

Therefore, YIIFSWA, through its initiatives, should ensure that all constraints are turned into opportunities for all the yam value chain players in general and farmers in particular.

Enhancing yam improvement for West Africa

Hiroko Takagi

EDITS Project: JIRCAS International Collaborative Research for West African crops

In the past, most agricultural investments and international agricultural research in Africa were focused on developing major cereals and crops for export. Recently, however, the focus has shifted to approaches to diversify agricultural innovations in defined locations to contribute to productivity and profitability increase and achieving sustainable food security to overcome poverty and malnutrition. In addition to so-called “major global crops”, attention has also been placed on many more crops that are regionally or locally important for nutrition and income and that are often underresearched but are nutritious, valued culturally, adapted to local environments, and contribute to diversifying regional agriculture systems.

The Japan International Research Center for Agricultural Sciences (JIRCAS), together with several Japanese research institutions and IITA, initiated in 2011 a 5-year collaborative research project called “Evaluation and Utilization of Diverse Genetic Materials in Tropical Field Crops (EDITS)”. The project focuses on yam (EDITS-Yam) and cowpea (EDITS-Cowpea), and aims to generate a solid understanding of the available wide genetic resources in these West African traditional crops, and develop efficient evaluation techniques for effective crop improvement. The outputs from these collaborative efforts are expected to contribute to breeding programs in West Africa.

JIRCAS is playing a key role by linking the Japanese scientific capacities to African communities through IITA, which is the entry point for many overseas research institutions to overcome the various constraints in African agriculture. The knowledge and techniques gained from the collaborative research project is expected to enhance the development of improved yam and cowpea varieties that can help promote rural livelihoods in West Africa.

EDITS-Yam

Yam is a traditional staple crop of significant economic and sociocultural importance in West Africa. The demand for yam is projected to increase, mostly due to population growth in the region. However, little improvement of farm yields has been registered in this crop in the last few decades, indicating an urgent need for more investment in yam research and development. To increase its productivity and enhance the income generation capacity of small-holder farmers, research-for-development should focus on increasing productivity through improved varieties and production technologies to meet the regional needs.

The last couple of years saw a breakthrough in genome sequencing technologies, and in the application genomic information to plant breeding. Genome analysis and improved molecular techniques would tremendously facilitate germplasm characterization, genetic mapping and tagging, and functional genomics of yam. These new tools, if incorporated into the breeding program, will pave the road for effective genetic improvement of yam. Since April 2011, JIRCAS together with the Iwate Biotechnology Research Center (IBRC) and IITA, has been implementing EDITS-Yam to develop and use advanced genomic and molecular tools to enhance germplasm evaluation and improvement for D. rotundata in West Africa.

EDITS-Yam is designed to strengthen genotyping using molecular tools and develop phenotyping protocols to facilitate yam breeding. The project aims to (1) generate the first reference genome of D. rotundata (Guinea yam), (2) develop and apply genomic information and molecular tools in yam breeding, (3) provide improved tools for biodiversity analysis and identification of potentially useful germplasm, and (4) develop phenotyping protocols for important agronomic traits. The outputs from this collaborative research are expected to contribute to the enhancement of yam breeding activities in the region. Consequently, new improved varieties will provide better food security and income for the small-holder farmers in West Africa and beyond.

Progress in 2011-2013

Sequencing of Guinea yam genome

To enhance Guinea yam breeding by fully exploiting modern genomics tools, generating a reliable reference sequence is a prerequisite. To this end, we have been gathering efforts to obtain the first whole genome sequence (WGS) of D. rotundata. The de novo assembly is currently in its final stage. The reference of genome will be completed soon, and the finding will be shared with the global yam community (Fig. 1).

Whole-genome sequencing-based analysis of diversity in Guinea yam

Next generation sequencing (NGS) allows large-scale genome-wide discovery of genetic markers that are important for genomic and genetic applications such as construction of genetic and physical maps, and analysis of genetic diversity. As a component of the on-going effort to construct the first draft sequence of D. rotundata and accelerate the breeding program, WGS-based genetic diversity analysis of D. rotundata accessions is under way. So far, 10 D. rotundata breeding materials, including five landraces and five breeding lines, have been resequenced. These materials are diverse with respect to traits such as maturity time, yield, tuber quality, and resistance to nematode and Yam mosaic virus (YMV), and have been extensively used as parental lines in the IITA yam breeding program.

Aligning the Illumina paired-end short reads obtained from resequencing of the breeding materials to D. rotundata scaffold sequence allowed genome-wide extraction of single nucleotide polymorphism (SNP) and insertion/deletion (indel) markers, which are being used to estimate the genetic relatedness among the lines/accessions studied and reveal the genetic diversity available to breeders. Findings of this study will have huge implications for genetic and genomic studies in yams, including among others, the application of SNPs, the most abundant genetic markers in genomes, for the development of high throughput genotyping platforms and for marker-assisted breeding. More accessions will be considered for resequencing in the future to mine the diversity in D. rotundata in detail.

Diversity Research Set (DRS) as a tool for diversity evaluation of D. rotundata germplasm

The availability of genotypic and phenotypic tools is critical to understand the diversity present in germplasm collections and enhance the active use of genetic resources. IITA currently holds over 2,000 accessions of D. rotundata. Of these, we selected a subset of experimental materials called Diversity Research Set to develop genotyping and phenotyping tools and protocols for germplasm evaluation.

In principle, DRS should be small in size for ease of handling and to allow a detailed analysis of diversity, but retain most of the diversity present in the original collection both at molecular and morphological levels. Accordingly, 106 accessions have been selected as the DRS-EDITS based on 21 key morphological traits, ploidy level, and SSR polymorphisms. The materials are currently being used for (1) detailed genotyping using DNA markers generated from the ongoing WGS, (2) morphological characterization and identification of key descriptors for regional D. rotundata collection, and (3) detailed phenotyping of economically important traits (Fig. 2)

Developing phenotyping protocols

In yam, as well as other root and tuber crops, phenotyping remains the major bottleneck to fully use genotyping information in germplasm evaluation and breeding. EDITS-Yam is also aiming to develop phenotyping protocols on key traits such as tuber yield, earliness of tuber growth and maturation, starch content and properties in collaboration with agronomists and food science specialists. These protocols, once developed, will be used for large-scale phenotyping applied to genetic and diversity studies (Fig. 3).

The information generated and tools developed in the framework of the EDITS-Yam project are expected to contribute immensely to broadening the knowledge base in yam, thereby facilitating the management of available genetic resources and aiding efficient use of yam germplasm for future improvement of the crop. This project and the collaboration it forged are expected to contribute to raising the profile of yam, and trigger the initiation of more and concerted international approaches to yam research for development. The preliminary outputs from the EDITS-Yam project suggest that there is a need for complementary studies to effectively use genetic and genomic tools being generated for yam improvement. To this effect, possibilities for additional resources are being explored.