Evaluation of Agronomic Performance and Seed Oil Composition of 15 Sunflower Genotypes in South Madagascar

Sunflower (Helianthus annuus L.) is one of the world’s most important oilseed crops together with oil palm (Elaeis guineensis Jacq.), soybean (Glycine max (L.) Merr.) and rapeseed (Brassica napus L.). Despite the 8.5% of world sunflower cultivation is found in Africa, just few studies on oil seed performances and their chemical composition were carried out in tropical countries, thus reducing the knowledge on the adaptability and performances of this crop in humid areas. In this study the agronomic performance, environmental adaptability, oilseed production and fatty acid composition of 15 sunflower varieties cultivated in two underexploited areas of the Plateau de l’Horombe in southern Madagascar were evaluated. Results of this study indicated that: 1) sunflower has well performed in sub-arid localities thanks to its adaptability to harsh conditions, with similar performances to those obtained in other worldwide countries; 2) the well-structured and fertile soil resulted to be the key driver of sunflower performances; 3) the most productive hybrids between the sites were PR63D82 (conventional typology) and Klarika for yield and oil content, respectively; 4) the oleic/linoleic ratio of both HO and conventional sunflowers was influenced by changes in temperature.


Introduction
Sunflower (Helianthus annuus L.) is one of the world's most important oilseed crops together with oil palm (Elaeis guineensis Jacq.), soybean (Glycine max (L.) as climate (air temperature, rainfall, soil water regime, intercepted solar radiation, etc.) and agriculture management can affect the grain filling phase, thus modifying the final oil profile [3] [8] [9] and yield [6].
Sunflower oil is considered a high quality oil because of being rich in monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) [3] [10]. These acids are indeed indispensable for human health, playing a protective role for humans by reducing plasma cholesterol and the risk of cardiovascular disease [11] [12]. Also, sunflower seeds are essential for the human diet since they are full of vitamins (e.g. E, B1, B5, B6) and specific acids (chlorogenic and folic) [11]. However, vegetable oils rich in PUFA are susceptible to lipid oxidation, which can originate from cytotoxic and genotoxic compounds that negatively affect the nutritional value and shelf-life of food products [13].
At the end of the last century, new genotypes called High-Oleic sunflower (HO) with higher MUFA content were developed [14] and became available on the market [15]. A diet rich in MUFA provides similar benefits than those provided using conventional sunflower oil (PUFA), avoiding the problem of lipid oxidation in the food oil [10] [16]. Particularly, oil seeds from HO genotype have a greater oxidative stability than conventional oils, which is desirable in cooking applications, refining process and storage [3] [5]. Nowadays sunflower hybrids can be divided into two principal groups based on oleic acid content: 1) conventional sunflower (15.0% -50.9% oleic acid) and 2) high-oleic sunflower (over 87.4% oleic acid) [15].
Despite the 8.5% of world sunflower cultivation is found in Africa [17], just few studies on oil seed performances and their chemical composition can be currently found in tropical areas [1] [2], since in many African countries, such as Madagascar, this crop is cultivated only in limited surfaces [17]. However, thanks to well-developed root system, allopathic potential for weed control, etc., [18] [19], its cultivation may be enclosure within agronomic rotation by farmers, with a consequent introduction of a product able to provide healthy nutritional compounds for locals.
Given the large number of genotypes currently present in the global market,

Study Area
The field experiments were carried out over the Plateau de l'Horombe, in the

Field Set Up and Practices
Experimental material included 13 commercial sunflower hybrids (nine conventional, and four high-oleic sunflower genotypes) and 2 Tanzanian populations ( Table 2). Specific crop characteristics such as length of cycle or oil seed profile were known only for commercial hybrids, whilst no information was available for the populations.  The seed-bed was prepared using a spike-tooth harrow (30 -35 cm depth) followed by a disc-harrow (10 cm depth) before the seeding (December 2012).
Fertilization was applied at a seed-bed time using 500 kg/ha of NPK (11:22:16) and in January 2013 using Urea (46% N) in a dose of 100 Kg/ha. Sowing was done in December 2012 when minimum soil temperature was 13 degrees [21].
Harvest was done at the end of April (2013) when plants turned bracts in the capitula into brown color [22]. Achenes collected were dried, cleaned by broken seeds and impurities and finally (i.e. 200 g from each test) analyzed.

Data Collection
The different phenological phases were monitored during the trials, following the methodology proposed in by [23] (Schneiter and Miller, 1981). During the growing stages the following parameters were collected analyzing five plants randomly chosen on each plot: plant height (cm), stem diameter (mm), leaves per plant and at harvest time also the plant dry weight (g) capitula diameter (cm), seeds number and weight per capitula (g) (Table S1). Finally, data about total yield and biomass (t/ha) and the 1000 seeds weight (g) were collected from each plot ( Table 2).

Oil Analysis
Seeds subsamples from each block of the same experimental site were mixed together and pressed for oil extraction using a mechanical press (IBG Monforts Oekotec GmbH&Co, CA 59G-2008). The extracted oil, expressed as a weight percent relative to the initial weight of sunflower seeds, was maintained in a drying oven at 105˚C for one hour. A specific amount of oil (i.e. 10 g) was soaked with 0.3 methanol-sodium methylate and then held at 90˚C for 2 h to convert the Fatty Acids (FA) into its methyl derivatives (Fatty acid methyl esters, FAME). Oil fatty acid composition was determined using Shimadzu GC-FID (model GC2010 Plus), equipped with a capillary column (wax-58 CB) and flame ionizing detector. FAME was identified by comparing retention times with those of well-known commercial standards and quantified as a relative percentage area.

Statistical Analysis
Morphological and productive traits were analyzed with a generalized linear mixed model (GLMM) with localities as random factor using the software IBM SPSS Statistics v.25 [24]. Multiple comparisons between genotypes were carried out using Tukey test. The correlation between all the collected variables was as- acids as independent ones. Then we carried out a multiple regression model between the dependent and independent variables to evaluate the path coefficients, that are the standardized β weights of the regression analyses. These coefficients are important for path analysis because they represent the direct impact of an independent variable on dependent ones. A correlation analysis between the independent variables was performed to evaluate and explain their reciprocal effect. All these coefficients were then put in the hypothesized input path diagram to explain the connection between variables and finally convalidate an output path diagram. All analysis was carried out with SPSS software.

Biomass and Yield
Analysis of variance allowed a general understanding of the productive and morphological response to locality and genotype (Table 3). For productive data, yield and total biomass resulted to be statistically affected by locality and the   (Table 2).
Between the sites, the most productive hybrid was PR63D82 (0.94 t/ha), while the less productive was Heliawin (0.60 t/ha), both belonging to conventional group. Globally, the average yield from conventional hybrids was 0.77 t/ha whilst that from HO hybrid was 0.71 t/ha.

Oil Quality
The 13 different FA showed a carbon structure from C14 (myristic acid) to C24 (lignoceric acid) (Table S2). Oil content was affected only by locality (p < 0.01).
More specifically, oleic, linoleic and gondoic acids showed high statistical significance only to genotype (p < 0.01); margaric acid was influenced only by locality (p < 0.05), whilst myristic, palmitic, palmitoleic, linolenic, arachidic and behenic acids were influenced by both locality and genotype. For the remaining acids (i.e. erucic and lignoceric acids), none statistical significance has been found.
The average oil content ranged from 34.7% at Andiolava to 31.06% at Satrokala ( acids were also reported more in detail since they globally constitute the 98.1% ± 0.4% of the total oil fatty acid composition (Table 2). Overall, no great differences were found between the sites, with average higher values for palmitic, stearic and linoleic acids at Satrokala and for oleic acid at Andiolava. Looking at the different genotypes, the average highest palmitic and stearic acids were found in Durban (6.42%) and PR63D82 (4.21%). The oleic acid showed the highest content in PR64H41 (85.28%) and generally in all HO hybrids, which showed an oil content almost two times greater than conventional. Linoleic acid showed the average highest content in Durban (53.53%) and generally in all conventional group.

PCA and Correlation Analysis
The results of PCA were graphically represented in a two-dimensional plot ( Figure 1). The first two components explained more than 60% of the variability between the samples. Genotypes were clearly subdivided into two groups de- Stearic acid was also negatively correlated to gondoic (r = −0.584), but positively with arachidic (r = 0.722) and margaric (r = 0.452) acids.
Path coefficient was calculated to obtain further information about the relation between oleic acid content and the other FA engaged in its biosynthetic pathway ( Figure 2). Analysis has been carried out separately for HO and conventional genotypes. Results indicated that HO genotypes showed a negative relation between oleic and linoleic acid (−0.941), very close to that found for conventional (−0.975). Finally, stearic and palmitic acid were negatively correlated with oleic acids both in HO and conventional genotypes.

Discussion
In this study the agronomical performances, yields and oil content of 13 hybrids and 2 varieties of sunflower cultivated in two different areas of southern Madagascar were investigated. Firstly emerged as the different characteristics of the two study areas strongly affected crop performances. Morphological parameters resulted to be mainly influenced by soil characteristics such as texture, pH and fertility. The high influence of soil characteristics on sunflower performances was partly expected since several studies indicated that even in different climatic areas they play the key role on vegetative performances [3] [27] [28]. The soil characteristics at Andiolava likely favored plant growth and development, strongly increasing the sunflower biomass. In particular, the higher SOM content at Andiolava may have improved soil physical properties such as texture, structure, bulk density as well as soil nutrient availability, thus favoring the plant growth and dry matter accumulation [29]. Another key aspect was the soil pH.
Looking at oil production, the oil content in the two sites ranged from 31% to 34.7%. These values agree with those found by [ [3]. This was probably due to a specific gene that increases oleic acid stability as observed by several studies [27] [37] [38]. Globally, the correlations found in this study reflected those highlighted in other countries such as Nigeria [39], South Africa [36], Brasil [34], Argentina [15], and Italy [3], and confirmed the biosynthesis mechanism of FA proceeds in seed storage lipids as reported by [40]

Conclusions
The studies of sunflower cultivation in tropical areas are quite lacking since mostly lands are used for staple food crop. This lacking, however, strongly reduced the understanding of sunflower biomass and oil production performances as well as the knowledge on possible chemical changes in oil seed due to climate or soil characteristics in tropical areas. To our knowledge, this is one of the few studies which assessed the agronomic performance, environmental adaptability, oilseed production and fatty acid composition of sunflower by comparing 15 different varieties in a tropical environment such as Madagascar. Results of this study indicated that: 1) sunflower has well performed in sub-arid localities thanks to its adaptability to harsh conditions, with similar performances to those obtained in other worldwide countries; 2) the well-structured and fertile soil, resulted to be the key driver of sunflower performances; 3) the most productive hybrids between the sites were PR63D82 (conventional typology) and Klarika for yield and oil content, respectively; 4) the oleic/linoleic ratio of both HO and conventional sunflowers was influenced by changes in temperature.
These results should be considered as a first step to improve the knowledge and use of sunflower in Madagascar. Information retrievable from this study can indeed encourage changes in local market perspectives beside to provide suggestions to local farmers for choosing the best genotypes for any specific cultivation areas. The spreading of sunflower cultivation may also allow increasing the consumption of healthy vegetable oils compared to those currently used in Madagascar. However, further researches are needed to fill the gap of the sunflower response in tropical countries compared to the US or Europe.