A simple hydroponic hardening system and the effect of nitrogen source on the acclimation of in vitro cassava (Manihot esculenta Crantz)
Plant tissue culture technology is being widely used for large-scale, rapid, clonal multiplication and genetic transformation in cassava. The main limitation of this technology is the period of acclimation of the fragile in vitro plants after they have been multiplied or regenerated. Most losses of in vitro plants occur when the plantlets are moved directly from the test tubes to the ex vitro conditions. Our aim was to design a simple, rapid, low-maintenance hydroponic system to improve survival rate of transplanting to the ex vitro conditions through the rapid acclimation process of in vitro plants. In this paper, we have developed a simple hydroponic system to accelerate the cassava acclimation and multiplication process. This system considerably increased the survival percentage of in vitro and/or transgenic lines and reduces the time requirement for multiplication by hydroponic acclimation. In order to assess the effectiveness of the acclimation of seedlings on their establishment, we analyzed plant growth and field survival rate with response to different nitrogen (N) sources using different cassava accessions. Nitrogen sources of NO3 − and NH4NO3 increased plant growth and root length compared to NH4 + alone, or water treatments. The greenhouse and field survivability of N-hardened plants, including transgenic lines, were significantly different in growth and development. We present a simple NO3 − hydroponic acclimation system that can be quickly and cheaply constructed and used by the cassava community around the world. The efficiency of our proposed N hydroponic acclimation system is validated in the transgenic development pipeline which will enhance the cassava molecular breeding.
KeywordsIn vitro cassava Hydroponic system N sources Survival rate Rooting
We thank Dr. Manabu Ishitani, Dr. Joe Tohme, and Dr. Hershey Clair from Agrobiodiversity Research Area-CIAT for their continuous support. We also thank Dr. Kensuke Okada from the University of Tokyo; Alfredo Delgado, a research scholar from Texas A&M; Dr. T. Ramasubramanian, a senior scientist from SBI, ICAR, India, for their critical evaluation and suggestions for the manuscript. We appreciate Milton Valencia and Didier Marin from CIAT for their technical assistance. SO conducted this study as a research fellow of the Japan Society for the Promotion of Science (JSPS) and also has received financial support from the University of Tokyo.
MGS, SO, and OC designed the study. OC, SO, and AM implemented the experiments. OC and SO performed the statistical analysis. OC, SO, and MGS drafted the manuscript. All authors read and approved the final manuscript. This work was performed in partial fulfillment of the requirements for the master degree of Mr. Oscar Castañeda-Méndez under the guidance of Dr. Michael Gomez Selvaraj, CIAT.
- Ahloowalia BS, Prakash J, Savangikar VA, Savangikar C (2004) Plant tissue culture. In “Low cost options for tissue culture technology in developing countries” Proceedings of a Technical Meeting organized by the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture held in Vienna, 26–30 August 2002″: 1–15. (Also available at www-pub.iaea.org/mtcd/publications/pdf/te_1384_web.pdf).
- Aladele SE, Kuta DK (2008) Environmental and genotypic effects on the growth rate of in vitro cassava plantlet (Manihot esculenta Crantz). Afr J Biotechnol 7:381–385Google Scholar
- Cuesta B, Villar-Salvador P, Puértolas J, Jacobs DF, Rey-Benayas JM (2010) Why do large, nitrogen rich seedlings better resist stressful transplanting conditions? A physiological analysis in two functionally contrasting Mediterranean forest species. For Ecol Manag 260:71–78. doi: 10.1016/j.foreco.2010.04.002 CrossRefGoogle Scholar
- Jorge MAB (1996) Cassava acclimatization in Mozambique. Afr J Root Tuber Crops 2:216–219Google Scholar
- Kassianof EP (1992) Biomass partitioning characteristics, tissue culture and transformation for the genetic improvement of cassava (Manihot esculenta Crantz). Dissertation, University of ZimbabweGoogle Scholar
- Koehorst-van Putten HJJ, Sudarmonowati E, Herman M, Pereira-Bertram IJ, Wolters AMA, Meima H, de Vetten N, Raemakers CJJM, Visser RGF (2012) Field testing and exploitation of genetically modified cassava with low-amylose or amylose-free starch in Indonesia. Transgenic Res 21:39–50. doi: 10.1007/s11248-011-9507-9 CrossRefPubMedGoogle Scholar
- Mabanza J, Rodriguez-Andriyamasi AF, Mahouka J, Boumba B (1995) Evaluation of cleaned cassava varieties in Congo. In: The Cassava Biotechnology Network: proceedings of the Second International Scientific Meeting, 22–26 August 1994, Bogor, Indonesia. Centro Internacional de Agricultura Tropical, Cali, 194–201 (Working Document no 150)Google Scholar
- Ospina PB, Segovia RJ, Bedoya A (2007) Micro-propagation of cassava plants through the temporary immersion system and hardening of massive numbers of cassava in vitro plants. CIAT http://ciat library.ciat.cgiar.org/Articulos_ciat/asia/proceedings_workshop_02/161.pdfGoogle Scholar
- Roca, WM (1984) Cassava. In: Handbook of plant cell culture. Crop Species. pp. 269–301. Chapt. 10. Vol. 2. Sharp, W.R., Evans, D.A., Ammira to, P.V. and Yamada, Y. Eds. Macmillan, New York, USAGoogle Scholar
- Roca WM, Mroginski L (1991) Cultivo de tejidos en la Agricultura: Fundamentos y aplicaciones. (CIAT) No. 151.Google Scholar
- Salifu KF, Timmer VR (2003) Nitrogen retranslocation response of young Picea mariana to nitrogen-15 supply. Soil Sci Soc Am J 67:309–317Google Scholar
- Ubalua AO (2015) Agrobacterium-mediated transformation of two cassava cultivars (Nwibibi and TMS 60444). KMITL Sci Tech J 15:47–54Google Scholar
- Wang W, Feng B, Xiao J, Xia Z, Zhou X, Li P, Zhang W, Wang Y, Møller BL, Zhang P, Luo MC, Xiao G, Liu J, Yang J, Chen S, Rabinowicz PD, Chen X, Zhang HB, Ceballos H, Lou Q, Zou M, Carvalho LJCB, Zeng C, Xia J, Sun S, Ful Y, Wang H, Lu C, Ruan M, Zhou S, Wu Z, Liu H, Kannangara RM, Jørgensen K, Neale RL, Bonde M, Heinz N, Zhu W, Wang S, Zhang Y, Pan K, Wen M, Ma PA, Li Z, Hu M, Liao W, Hu W, Zhang S, Pei J, Guo A, Guo J, Zhang J, Zhang Z, Ye J, Ou W, Ma Y, Liu X, Tallon LJ, Galens K, Ott S, Huang J, Xue J, An F, Yao Q, Lu X, Fregene M, Becerra López-Lavalle LA, Wu J, You FM, Chen M, Hu S, Wu G, Zhong S, Ling P, Chen Y, Wang Q, Liu G, Liu B, Li K, Peng M (2014) Cassava genome from a wild ancestor to cultivated varieties. Nat Commun 5:5110 . doi: 10.1038/ncomms61101-9CrossRefPubMedPubMedCentralGoogle Scholar
- You W, Barker AV (2002) Herbicidal actions of root-applied glufosinate ammonium on tomato plants. J Am Soc Hortic Sci 127:200–204Google Scholar
- Zimmerman TW, Williams K, Joseph L (2007) Rooting and acclimatization of cassava (Manihot esculenta) ex vitro. IS on Biotechnol. Temp. Fruit Crops & Trop. Species Eds. R.E. Litz and R. Scorza Acta Hortic 738Google Scholar
- Zok S, Nyochemeing L, Tambong J, Watoch T (1992) Rapid seed stock multiplication of improved clones of cassava through shoot tip culture in Cameroon. Proceed Cassava Biotechnology Network 17:96–98Google Scholar