Determina)on of LD50 in two cassava landraces using Ethylmethane sulphonate (EMS)
SP 09-‐11
Biotechnology and Nuclear Agriculture Research Ins6tute, P.O. Box LG 80, Legon, Accra, Ghana
Introduc)on:
Plant biotechnology can be coupled with muta6on induc6on to produce new cassava varie6es with desired traits. In the IAEA database the two mutant cassava released were induced by gamma rays, physical mutagens (IAEA,2015: Asare, 1995). The use of chemical mutagens has not yet been reported in cassava yet probably due to the bulky nature of stem cuPngs or its toxicity on plant 6ssues. To overcome the limita6on of bulky stem cuPngs, in vitro generated nodal cuPngs can be treated with known chemical mutagens such as Ethylmethane sulphonate (EMS) or dimethyl sulphonate (DMSO) for mutant induc6on. Chemical mutagens allows for easy detec6on of frequency of muta6ons using TILLING in many plant species (Jain,2005). A prerequisite for muta6on induc6on is the determina6on of the sensi6vity of the 6ssues to the mutagen or the lethal dose (LD50). In this report we determined the LD50 of nodal cuPngs using EMS in two landraces of cassava. Also, the effect of the mutagen on shoot regenera6on is reported.
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Figure 1: Effect of EMS on shoot growth (A) control and (B) 0.06% treated EMS in Ankrah aBer 14 days of culture
Shoot emergence reduc6on in Ankrah ranged from 33.3 to 75% and 58.3 to 60% for 4 and 6 hours respec6vely. Contrarily, in Madam it ranged from 13.3 to 40% and 33.3 to 60% for 4 and 6hr indica6ng that Ankrah is more sensi6ve than Madam. The effect of EMS on shoot reduc6on in other plant species is well documented. Talebi et al observed shoot reduc6on using 0.25-‐2% EMS in rice while Omar recommended an op6mum concentra6on of 0.23% for 3hr. According to FAO (2009) any dose causing a dose reduc6on of more than 30% is considered too high for large scale muta6on induc6on. Considering this as a general rule, the LD50 for Ankrah was 0.06 for 4 or 6hr while in madam LD50 was 0.04 for 6hr.
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Figure 2: Effect of EMS on shoot regeneraHon and growth reducHon of two cassava accessions (A and C) Ankrah and (B and D ) Madam aBer six weeks of culture. In Ankrah Pearson correla6on coefficient (r) was calculated so that the LD50 can be determined using shoot emergence. In Ankrah, r was 9.3 and 8.5 for 4 and 6 hr respec6vely while for Madam it was 8 and 8.6. Using percentage emergence the LD50 was determined as 0.07 and 0.05% for 4 and 6hr respec6vely for Ankrah and 0.19 and 0.11% for Madam
Conclusion Chemical mutagens can be used to complement physical mutagens for muta6on breeding programmes in cassava. It can be combined with in vitro regenera6on methods. In vitro propagules such as nodal cuPngs can easily absorb the solu6on and thus cause gene6c changes in the plant. The LD50 depended on the dura6on of immersion in the aqueous solu6on of the mutagen. Of the two landraces, Ankrah was more sensi6ve to the chemical mutagen than Madam. References Asare, E., Safo-‐ Kantanka, O (1995).Improvement of cassava cooking quality through muta6on breeding. Interna6onal Atomic Energy Agency-‐TECDOC-‐951 Proceedings of a final Research Co-‐ordina6on Mee6ng of a FAO/IAEA Co-‐ordinated Research Programme, held in Naples, Italy, 30 October-‐November 1995 FAO (2009) Plant breeding and farmer par6cipa6on.Methodologies for generaHng variability Part 4: MutaHon techniques by Maluszynski, M, Szarejko I, BhaHa C.R,Nichterlein K and Lagoda P.J.L. FAO, Rome IAEA. IAEA mutant database. Vienna: Interna6onal Atomic Energy Agency; c2015 [accessed November2015].Available from:hhp://mvd.iaea.org/ Jain, S.M.,(2005). Major muta6on-‐assisted plant breeding programs support by FAO/IAEA. Plant cell Tissue org. cult 82:113-‐123 Omar, M.S., Novak, F.J. and Brunner, H. (1989).In vitro ac6on of ethyl methanesulphonate on banana shoots 6ps. Sci.Hor6c 40: 283– 295. Talebi, A. B.Talebi, A. B. &Shahrokhifar, B.(2012) Ethyl Methane Sulphonate (EMS) Induced Mutagenesis in Malaysian Rice (cv. MR219) for Lethal Dose Determina6onAmerican Journal of Plant Sciences;3(12):1661-‐1665
Materials and Methods: Flow chart of methods Selec6on of landraces ↓ Prepara6on and Plan6ng of stem cuPngs ↓ Harves6ng of shoots ↓ Prepara6on of nodal cuPngs
↓ Treatment of cuPngs with EMS
↓ (Conc. 0.02, 0.04, 0.06, 0.08, 0.1) Culture on standard cassava regenera6on medium
↓ Observe for shoot regenera6on
Result and Discussion : Accurate determina6on of LD50 is prerequisite for successful muta6on in plant species. In the present study EMS had effect on shoot emergence as well as growth (height) in both landraces but this effect dependent on the concentra6on and dura6on of immersion in the chemical mutagen (Figure 1). Immersion in the mutagen for longer 6me (6hr) had more effect on shoot reduc6on than 4hr. The sensi6vity of the two landraces Ankrah and Madam also differed (Figure 2) but at the highest concentra6on of 0.1% there were no differences between shoot emergence at 4 and 6 hours.
0
20
40
60
80
0 0.02 0.04 0.06 0.08 0.1Shoot em
ergence red
uction %
Concentration of EMS
4hr 6hr
0
20
40
60
80
0 0.02 0.04 0.06 0.08 0.1Sho
ot emergence redu
ction %
Concentration of EMS
4hr 6hr
0
20
40
60
80
0 0.02 0.04 0.06 0.08 0.1
Shoo
t heigh
t reductio
n %
Concentration of EMS
4hr 6hr
0
20
40
60
80
0 0.02 0.04 0.06 0.08 0.1
Shoot height redu
ction %
Concentration of EMS
4hr 6hr
y = -‐488x + 78.567 R² = 0.8734
0
20
40
60
80
100
0.00 0.02 0.04 0.06 0.08 0.10 0.12
Percentage shoot
emergence
Concentration of EMS
y = -‐485.86x + 67.31 R² = 0.7334
0
20
40
60
80
100
0.00 0.02 0.04 0.06 0.08 0.10 0.12
Percentage shoot
emergence
Concentration of EMS
y = -‐248.14x + 92.424 R² = 0.6902
0
20
40
60
80
100
0.00 0.02 0.04 0.06 0.08 0.10 0.12
Percentage shoot
emergence
Concentration of EMS
y = -‐362.29x + 84.781 R² = 0.738
0
20
40
60
80
100
0.00 0.02 0.04 0.06 0.08 0.10 0.12
Percentage shoot
emergence
Concentration of EMS
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