Fast-tracking yam genetic improvement using High Ratio Propagation Technologies Morufat Balogun1,2, Norbert Maroya1, Adeola Ajayi1,2, Ossai Chukwunalu1, 2, Julius Taiwo1, Lava Kumar1, Beatrice Aighewi1 and Robert Asiedu1 1International Institute of Tropical Agriculture (IITA) PMB 5320 Ibadan, Nigeria; 2Department of Crop Protection & Environmental Biology, University of Ibadan 1- INTRODUCTION In conventional yam production, whole or cut tuber pieces are used as seed, giving an average of 6 new plants per tuber per year (Aighewi et al., 2015). Seed accounts for 63% of the total variable production cost while pests cause yield reduction. Low propagation ratio and irregular flowering delays conventional hybridization and genetic improvement. In the current informal seed system, uncertified seed is used in production, which may harbor contaminants, distorting research results and causing progressive yield reduction. The project ‘Yam Improvement for Income and Food Security in West Africa (YIIFSWA)’ is developing protocols for rapid production of quality seed yam using Vine rooting (VR), Conventional tissue culture (CTC), Temporary Immersion Bioreactor (TIBs) and Aeroponics (AS) technologies. Organogenesis from preformed meristems and somatic embryogenesis has been reported to be useful in cell selection programs, cryopreservation (Shu et al. 2005; Carlos and Martinez, 1998), micropropagation and production of ploidy-modified plants or somatic hybrids. The objective of this study was to determine the propagation ratio of clean plantlets in TIBs and assess ploidy variations among TC regenerants. 2- MATERIALS AND METHODS Plantlets of Dioscorea rotundata genotype TDr 95/18544 were regenerated on modified Murashige and Skoog medium using meristems from virus-tested in vitro plants which had been incubated at 37°C for 21 days. Multiplex-PCR was used to test for viruses. Fifty single nodes from virus-free plantlets, also negative for endophytic bacteria, were introduced into TIBs at 10hr medium immersion frequency. Number of nodes were recorded after 8 weeks. The plantlets were hardened and potted in carbonized rice husk + top soil mixture while others were planted in aeroponics. From the latter 2, more vines were produced and rooted to give more seedlings. Endosperm and embryo from zygotic seeds of 2 genotypes (TDr 97/00917 and TDr 95/18544) were used for callus induction and germination respectively in modified MS medium. Using the Olympus light microscope (oil immersion, 100x), the number of chromosomes in the roots (Adeigbe et al, 2015) of germinated plantlets, endosperm-derived callus and their tuber-raised mother plants were determined after staining. Plate 1. Up: Left: Setis type Bioreactor facility; Right: One TIB unit with 50 plantlets; Down: Left: Hardened TIB plants; Right: Seedlings from vine-rooting of TIBs plants. 1 2 4 3 Plate 2. 1: Germinating yam zygotic seeds; 2: Endosperm-derived callus forming roots; 3: Callus showing somatic embryos; 4: Regenerated plantlet 3- RESULTS AND DISCUSSION 4- CONCLUSIONS Yam mosaic and mild mosaic viruses were eliminated from at least 80% of the samples tested. In a 56-day cycle, a mean of 6.8 nodes was recorded in TDr 95/18544. This is much higher than conventional tissue culture with an average of 4 nodes. In an earlier study, up to 14 new nodes were produced in TDa 98/01167 from 1 initial node. This implies that up to 2,940 seeds can be produced in 1 year from 1 tuber. One plantlet introduced to aeroponics yielded up to 300 rooted vine seedlings in 1 year (Maroya et al., personal comm.), which is much higher than a 1:6 ratio of the minisett technique. Seedlings from TIBs or Aeroponics vines were produced irrespective of season. Callus was induced and proliferated in MS + 2mg/l of 2,4-D + 1mg/l of NAA at 16 h photoperiod at 25±1oC. Shoots were regenerated on MS with 1mg/l BAP. Ammirato (2004) reported that BAP enhanced shoot regeneration in D. bulbifera and D. alata. Roots from endosperm derived callus of TDr 95/18544 was pentaploid while the mother plant was tetraploid. The callus, plantlet and mother plant of TDr97/00917 were tetraploid. D. rotundata Poir. was reported to be diploid (2n = 40; Scarcelli et al., 2005), while Muthamia et al (2014) reported TDr 95/18544 to be tetraploid. Plantlets regenerated from endosperm tissues in D. zingiberensis (ZuoGuo, 2012) included both diploids and triploids. Triploids were shorter, with stronger stem, larger and thicker leaves with deeper colour, higher chlorophyll b and carotenoids. A combination of high ratio propagation technologies with TC at the heart of clean stocks increased the propagation ratio thousand-folds. Variations in ploidy level was observed when endosperm was used as explant and this can be explored in genetic improvement. However, the frequency of regeneration need to be increased and regenerated plantlets evaluated for desirable traits. Rapid propagation technologies will increase availability of sufficient materials for selection purposes and in multilocational trials geared towards varietal release. 5- REFERENCES 1. Adeigbe, O.O., Ilori, C.O. and Adewale, B. D. 2015. Phenotypic Diversity and Ploidy Level of Some Dioscorea Dumetorum Genotypes. IOSR Journal of Agriculture and Veterinary Science, 8(3):47-52. 2.Aighewi, B., Asiedu, R., Maroya, N. and Balogun, M. Improved propagation methods to raise the productivity of yam. 2015. Food Security. 1- 12. (ISSN: 1876-4517) 3. Balogun M.O., Maroya N. and Asiedu R. 2014. Status and prospects for improving yam seed systems using Temporary Immersion Bioreactors. African Journal of Biotechnology. 1315: 1614–1622, April 2014. 4. Carlos, M.V. and Martinez, F.X. 1998. The potential uses of somatic embryogenesis in agroforestry are not limited to synthetic seed technology. Revista Brasileria de Fisiologia Vegetal, 10(1):1-12. 5. Maroya N, Balogun M, Asiedu R, Aighewi B, Kumar PL, and Augusto J. 2014 c. Yam propagation using aeroponics technology. Annual Res & Review in Biology 4(24): 3849-3903. 6. Muthamia Z. K, Nyende A. B., Mamati E. G., Ferguson M. E. and Wasilwa J. 2014. Determination of ploidy among Yam (Dioscorea spp.) landraces in Kenya by flow cytometry Vol. 13(3), pp. 394-402, DOI: 10.5897/AJB2013.12496. ISSN 1684-5315 Academic Journals 7. Scarcelli N, Dainou O, Agbangla C, Tostain S, Pham JL (2005). Segregation patterns of isozyme loci and microsatellite markers show the diploidy of African yam Dioscorea rotundata (2n=40). Theor. Appl. Genet. 111:226-232. 8. Shu, Y., Ying-Cai, Y. and Hong-Hul, L. 2005. Plant regeneration through somatic embryogenesis from callus cultures of Dioscorea Zingiberensis. Plant-cell Tiss. Org. Cult. 80(2): 157-161(5). Table 1. Variations in chromosome numbers of root tips in different yam explants Plant source Root chromosome no. Ploidy level TDr 95/18544 tuber plant 39 Tetraploid TDr 95/18544 zygotic embryo plantlet 40 TDr 95/18544 endosperm callus 47 Pentaploid TDr97/00917 tuber plant 38 TDr97/00917 Zygotic embryo plantlet TDr97/00917 Endosperm callus 36 6- ACKNOWLEDGMENTS The Bill & Melinda Gates Foundation. YIIFSWA colleague and stakeholders.