The Use of Maleic Hydrazide for Effective Hybridization of Setaria viridis

April The Use of Maleic Hydrazide for Effective Hybridization of Setaria viridis Govinda Rizal 0 1 2 Shanta Karki 0 1 2 Richard Garcia 0 1 2 Nikki Larazo 0 1 2 Michael Alcasid 0 1 2 William Paul Quick 0 1 2 0 1 C4 Rice Center, International Rice Research Institute (IRRI), Los Banos, Laguna, the Philippines, 2 University of Sheffield , Sheffield , United Kingdom 1 Funding: This work was supported by the Bill & Melinda Gates Foundation, the UK AID, the C4 Rice Consortium; and the International Rice Research Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Academic Editor: Manoj Prasad, National Institute of Plant Genome Research , INDIA An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis is considered to be the new model plant for the study of C4 system in monocots and so an effective crossing protocol is urgently needed. S. viridis is a small grass with C4-NADP (ME) type of photosynthesis and has the advantage of having small genome of about 515 Mb, small plant stature, short life cycle, multiple tillers, and profuse seed set, and hence is an ideal model species for research. The objectives of this project were to develop efficient methods of emasculation and pollination, and to speed up generation advancement. We assessed the response of S. viridis flowers to hot water treatment (48C) and to different concentrations of gibberellic acid, abscisic acid, maleic hydrazide (MH), and kinetin. We found that 500 M of MH was effective in the emasculation of S. viridis, whilst still retaining the receptivity of the stigma to pollination. We also report effective ways to accelerate the breeding cycle of S. viridis for research through the germination of mature as well as immature seeds in optimized culture media. We believe these findings will be of great interest to researchers using Setaria. - Competing Interests: The authors have declared that no competing interests exist. Green foxtail (Setaria viridis) is one of the most widely used genetic systems for research on C4 photosynthesis and Panicoid grasses; in addition it is the ancestral wild relative of S. italica, an important cultivated millet [1, 2]. With the increased interest and funding for C4 photosynthesis research related to the wider aim of the introduction of C4 characteristics into C3 cereals to enhance yield, S. viridis has been widely used for basic research for the identification of genes related to C4 biochemistry and leaf anatomy [35]. It is also an emerging model system for C4 monocots, weeds, and biofuel research because of its short life cycle, small plant stature, small genome of about 515 Mb (2n = 18), simple cultivation requirement, and profuse seed set [6]. A significant number of the available literature on Setaria species focus on the optimization of its growth conditions, seedling establishment and germination [710]. However, reports on hybridization strategies are few [11, 12], and they are far from being standardized and efficient [5]. For genetic studies such as gene identification and population improvement, efficient hybridization techniques are necessary so that different types of populations can be developed. S. viridis has perfect hermaphrodite flowers with three stamens and a bifurcated pistil inside each small spikelet. It is self-pollinating and also cross-compatible with natural cross pollination rates of up to 4% [13]. The precise crossing of S. viridis for basic and applied research is limited by a few major barriers such as complex flowering pattern and long flowering period. Spikelets open during the cool hours of the day and anthesis is neither uniform nor follows a pattern [4]. It takes many days to complete flowering within a panicle. Manual emasculation is tedious and labor consuming. The spikelets are prone to shattering even with minor disturbances like shaking; hence manual emasculation is not preferred in large scale crossing programs. Several methods of emasculation have been reported in different crops which include plastic bag emasculation in sorghum and warm water emasculation in buckwheat [14, 15]. Even in a single crop like rice, several emasculation techniques are reported such as a mechanical method, a hot water method, manual emasculation, a suction method, alcohol treatment, cold treatment, genetic emasculation, and the use of gametocides [1624]. However, efficient methods for emasculation of S. viridis are not available. A range of chemicals are used as gametocides, like gibberellic acid (GA3), in different genotypes of German chamomile (Matricaria recutita) and in oil crops [25, 26]. Kalidasu et al. (2009) [27] reported an assessment of five gametocides including GA3 and maleic hydrazide (MH) in coriander flowers (Coriandrum sativum). However, there are no previously identified chemical gametocides suitable for the efficient emasculation of S.viridis. The use of such chemical gametocides could make emasculation of S. viridis much more efficient. This research, therefore, aimed to (1) identify an appropriate chemical for emasculation, optimize the method of emasculation, and cross-pollination; (2) confirm crosses through molecular methods; and (3) find means to accelerate the advancement of generations. We tested the effectiveness of different chemicals for emasculation and here, we report that MH was the most effective gametocide. We also report a simplified method of manual emasculation that is suitable in small experiments and when chemicals are not readily available. We also confirm the ability of S. viridis seeds (immature and mature) and F1 seeds to germinate in plant growth medium and we report the optimum condition that helps shorten the life cycle of S. viridis. These findings will greatly complement the efforts of researchers working on this C4 monocot. Materials and Methods Cultivation of plants The seeds of S. viridis were obtained from North Central Regional Plant Introduction Station of the United States Department of Agriculture and were multiplied at IRRI, Philippines. S. viridis accessions A10.1 and PI 202407 were used for the development and optimization of emasculation and crossing techniques. The seedlings were grown in plastic cups or root trainers inside contained greenhouse conditions. Soil was mixed with fertilizers nitrogen, phosphorous and potassium (NPK, 1:1:1) at a rate of 10 g per 100 kg of soil. Seedlings were watered twice a day for the first week and once a day thereafter to maintain about 50% soil moisture content. Succession planting was scheduled to coincide with the flowering times of the different accessions. Chemical solution preparation and treatments We prepared 100, 200 and 500 M each of GA3, MH, abscisic acid (ABA), and kinetin (KNT). All four chemicals were dissolved in water at room temperature. In case of MH, the pH dropped to 4.5 which inhibited complete dissolution, so 1N NaOH (...truncated)