An in vitro mutagenesis protocol for the production of sugarcane tolerant to the herbicide imazapyr

Field and laboratory assessments of sugarcane mutants selected in vitro for resistance to imazapyr herbicide

“…The Mut1-7 obtained by Koch et al (2012) and N12 control plants were bulked-up in vitro (Ramgareeb et al 2010). After 3 months (30 -40 cm in height), they were planted in field areas A, B and C. Stalks from 11-month-old plants from area A were cut into 3-budded setts and planted in area D. The field (lat 29° 42' 24.5585'' S, long 31° 02' 45.1735 E''; long-term mean annual rainfall 1023 mm) was divided into four areas (A-D); A, B and C were subdivided into three replicated plots (1 x 3.5 m row, 10 plants per row) in a randomized complete block design.…”

“…Leaf ALS enzyme activity (μmol acetoin h -1 mg -1 protein) was determined colorimetrically (530 nm) by acetoin formed , Koch et al 2012) and total protein the method of Bradford (1976).…”

“…Similar to Mut1, Mut5 and Mut6, Mut4 and Mut7 were significantly better than the control N12 in terms of stalk survival and height when sprayed with imazapyr (Table 2), and in % germination and shoot height when planted in imazapyrtreated soil (Table 4). Reduced absorption through the cuticle or other physical barrier and reduced translocation are unlikely to be involved as imazapyr resistance was generated in callus cells (Koch et al 2012). However, in phase I of detoxification, herbicide molecules are activated and functional groups are exposed to phase II conjugation enzymes, and often to oxidation by cytochrome P450 monooxygenases known to participate in IMI metabolism (Manabe et al 2007).…”

“…Because of the lengthy plant breeding and selection process in sugarcane, legislative restrictions, licensing costs and public opposition to transgenesis, our preferred approach is the generation of herbicide-resistant variants of proven elite genotypes using mutagenesis. Hence, cultivar N12 was selected for generating variants resistant to the herbicide imazapyr (Koch et al 2012). N12 is known to be hardy (McIntyre and Nuss 1998) and is a favored cultivar of emerging small-scale farmers in South Africa, who operate under RS Rutherford et al low input conditions, on predominantly strongly acid (pH<5) soils, with weed pressure as a major production constraint (Cockburn et al 2012).…”

“…Seven putatively imazapyr-resistant sugarcane mutant plants (Mut1-Mut7) were generated from N12 by callus exposure to 16 mM ethyl methanesulfonate, and selection on imazapyr-containing medium (Koch et al 2012). This report describes subsequent studies on these mutants grown in the field addressing their imazapyr resistance, basal activity of the ALS enzyme and the imazapyr concentration required to inhibit it by 50 % (IC 50 ), and plant characteristics and yield, compared with the 'parent' N12.…”