Effect of Cultivars and Planting Date on Yield , Oil Content , and Fatty Acid Profile of Flax Varieties ( Linum usitatissimum L . )

1 Instituto Nacional de Tecnoloǵıa Agropecuaria (INTA), Estación Experimental Paraná, Ruta 11, Km. 12.5, Entre Rı́os, 3100 Paraná, Argentina 2 Instituto de Tecnologı́a de Alimentos, Facultad de Ingenieŕıa Quı́mica, Universidad Nacional del Litoral, Santiago del Estero 2829, 3000 Santa Fe, Argentina 3 Consejo Nacional de Investigaciones Cient́ıficas y Técnicas (CONICET), Avenida Rivadavia 1917, C1033AAJ Buenos Aires, Argentina


Introduction
Flaxseed, derived from the seeds of the flax plant Linum usitatissimum, belongs to the Linaceae family.Flaxseed is one of the most important cultivated plants concerning its linen and oil.This species is planted mostly for oil and textile industry.Fibre flax is mainly grown in some regions of northern Europe, Russia, and China, while oilseed flax is widely grown in cool temperate regions of Argentina, India, China, Russia, USA, and Canada.However, the flax is mostly being harvested for oil so, it is an economically important oilseed crop [1].
Flax industrial quality is attributable to the oil concentration of its seeds, which may vary from 38 to 46 g/100 g [2], from 30 to 45 g/100 g [3] and from 36 to 48 g/100 g [4][5][6].It also has a high content of total dietary fiber and proteins.
Linolenic acid (18:3n-3) and linoleic acid (18:2n-6) are considered to be indispensable for human beings and must be obtained from food oils and fats.They are important nutrients for nervous system and the intake of linolenic acid being particularly helpful for a good blood circulation and a heart protection.
At present, nutritionists consider that the ideal edible oils should contain about 3.0 g/100 g linolenic acid [8].
The 18:3n-3 FA is essential for normal growth and has biological effects very useful in the prevention and treatment 2 International Journal of Agronomy of coronary artery disease, hypertension, diabetes, arthritis, other inflammatory and autoimmune disorders, and certain types of cancers [9,10].
FA profile makes flax very attractive as a functional food.Therefore, flax addition to the diet would be a healthy option, being particularly attractive from the viewpoint of food development.
It has been suggested that in order to obtain high yields and a good FA profile, plants must be handled in such a way that periods for yield determination coincide with favourable conditions for growth.
Oil synthesis in flax begins almost immediately after flowering and it progresses slowly during the 11 following days.After that time, a rapid accumulation of oil occurs for 10 days, achieving 3 g/100 g a day and reaching a maximum at day 21 after flowering.Oil formation ceases as soon as chlorophyll has been completely removed, the subsequent variations being practically negligible [13].
Flax is adapted to many environments mainly in temperate climates [14,15].It has been demonstrated that seed yield and seed yield components, plant height, time to reach harvest maturity, oil content, and oil composition and depend mainly on the temperature during plant development [14][15][16].Other factors such as seed moisture at harvest may also influence the final seed oil content and the oil composition [15].
Experiments carried out in controlled conditions showed that high temperatures (>30 ∘ C) during the ripening phase reduce the number of seeds per capsule and the seed weight and decrease oil yield and quality [17].
Seed production and oil production are known to be conditioned by the optimum planting date and the technological level used.Because of its geographical location, weather conditions, and soil characteristics, Entre Rios province of Argentina can be included within the subregion IIB, where the optimum planting date ranges from late May to late June, when temperatures are more conducive to the growth and development of the crop allowing a proper flowering to maturity period.
The aim of this work was to evaluate the effect of cultivars and planting date on the fatty acid profile and oil content of flax (Linum usitatissimum L.) in order to get information about agronomic conditions useful for flax functional food development.Flaxseed was planted, on June 11, 2008 (1st dateoptimum planting date) and July 30 (2nd date-late planting date for this region) using a randomized complete block design with three repetitions, parcels having a surface of 3.42 m 2 .The crop was carried out without water and nutritional constraints and freely of weeds, insects, and diseases, applying all the necessary preventive and curative measures.

Materials and Methods
Seed samples were taken from each parcel at harvest time (November and December 2008).

Methods of Analysis
2.3.1.Seed and Oil Yield.Seed yield was estimated at harvest time and expressed as kg⋅ha −1 (d.b.).Oil content was measured by Nuclear Magnetic Resonance (NMR) Analyzer (Newport 4000, Oxford Institute Limited, Oxford, England) by Robertson and Morrison [18] and expressed as kg⋅ha −1 (d.b.).

Fatty Acid Oil
Profile.FA content was determined according to Bannon et al. method [19] with modifications.
A sample of 10 ground flaxseed was added with 0.5 mL of NaOMe and stirred for 2 minutes.After that, 2.0 mL of isooctane ca. and 3.0 mL of saturated sodium chloride were added.The mixture was vigorously stirred for 20 min, and, after phase separation, 1 mL of the top phase containing fatty acid methyl esters was collected for gas chromatography analysis, using a gas chromatograph (HRGC-3000C Konik Instruments, Barcelona, Spain) equipped with a flame ionization detector and Restek Stabilwax-DA capillary column (30 m × 0.25 mm i.d., 0.50 m film thickness).The carrier gas was nitrogen with a constant column flow rate of 2.0 mL/min.The injector temperature was 250 ∘ C and the detector temperature was 250 ∘ C. The column temperature program was as follows: the initial column temperature was 220 ∘ C, which was maintained for 0.3 min and then to a final temperature of 240 ∘ C at a rate of 10 ∘ C/min and hold for another 1 min.Fatty acid methyl esters were identified by comparison with retention times and peak areas of the standard fatty acid methyl esters (Sigma, Chemical Co., St. Louis, MO, USA).
The amount of each sample injected was 1.0 L.Data were processed by means of specific software (IST-Crom).FA content was calculated as area (g/100 g), measuring the peak area corresponding to each FA and expressing the result as g of FA/100 g of oil (d.b.).

Statistical Analysis.
The effect of factors (cultivars and planting date) on the fatty acid profile, seed yield, and oil content was evaluated by multifactorial analysis of variance, using the INFOSTAT statistical package [20].Significant differences among factors were determined at the significance level of  < 0.05.When this analysis showed statistical differences between factors, the averages were compared through Tukey's test.The accumulated rainfall, during the period from flowering to harvest, for the first planting date, was 135.2 mm, while the second planting date was 132.6 mm.March and April rainfalls, usually characterized by high precipitation, which recharges the soil profile, were 60% lower than the historical average recorded at INTA-AES Paraná.This fact, together with the scarce rains recorded subsequently until October, resulted in a dry campaign which probably limited the crop yields.

Results and Discussion
Table 1 shows seed yield (kg⋅ha −1 ), oil yield (kg⋅ha −1 ), and FA (g/100 g) profile in the seven analyzed cultivars in the two planting dates.

Effect of Cultivar and Planting Date on Oil and Seed Yield.
Table 2 shows oil and seed yield content (kg⋅ha −1 ) of the analyzed cultivars.
A significant effect of cultivars on oil yield was observed (P: 0.0082).The oil yield from Tape INTA cultivar was found to be noticeably lower than that of the rest of cultivars.By contrast, cultivars Baikal, Panambí INTA, Prointa Ceibal, Curundú INTA, Carapé INTA, and Prointa Lucero showed no significant differences among them, with oil yield between 728 kg⋅ha −1 and 842 kg⋅ha −1 .
The delay in planting date also displaces seed filling to periods of high temperature, which affect their oil content and yield.In agreement with Lafond et al. [21], oil yield showed a significant response (P: 0.0001) to planting date, decreasing from 844.75 kg⋅ha −1 to 644.35 kg⋅ha −1 in the late date (Table 3).
The seed yield per hectare obtained in this study was significantly affected by planting date (P: 0.0001).The highest yields were obtained at the early date, with an increase of 434.35 kg⋅ha −1 .While Lafond et al. [21] did not find effects of planting date on flaxseed yield, other authors [22][23][24][25] found that early seeding produces the highest flax yields.The reasons for this behavior could be the same as those mentioned when evaluating the effect on oil yield.Delayed planting date causes the seed fixation and filling period to be located within environmental conditions with higher temperatures, thus resulting in yield reductions.

Effect of Cultivar and Planting Date on Fatty Acid
Profile.Table 4 shows concentration of palmitic, stearic, oleic, linoleic, and linolenic acids, content of saturated and unsaturated FA, and their relationship as a function of the different cultivars.
Concentrations of oleic and linolenic acids showed no significant differences between cultivars (P: 0.3142 and P: 0.6522, resp.), the average for the seven cultivars under study being 30.19 g/100 g and 47.96 g/100 g, respectively.Cultivars showed to have an effect on palmitic (P: 0.0060), stearic (P: 0.0092), and linoleic (P: 0.0001) acids.
Regarding FA, significant differences were found between saturated and unsaturated ones among cultivars but were not found between the saturated/unsaturated ratio (P: 0.026; P: 0.0089; P: 0.0209, resp.).
The highest content of saturated FA was found in Prointa Ceibal (15.27 g/100 g) and the least in Baikal cultivar (11.11 g/100 g), the rest having intermediate values.
Unsaturated FA, on the other hand, showed Prointa Ceibal as the cultivar with the least content (92.36 g/100 g) and Baikal with the highest (96.20 g/100 g), with a difference of 3.84 g/100 g between them.
The extreme values for both saturated and unsaturated FA differ from those reported by [26] and [27] since our results exceed them in almost 3 g/100 g and 5 g/100 g for saturated and unsaturated, respectively.
References [26] and [27] found that the amount of total unsaturated fatty acids in flax cultivar was 87-91 g/100 g, whereas for total saturated fatty acids were 9-12 g/100 g.
The average for the seven cultivars under study was 6.94.Average for seven cultivars of palmitic, stearic, oleic, linoleic, and linolenic acids, content of saturated and unsaturated FA, and unsaturated/saturated ratio, at two planting dates, is shown in Table 4.
FA composition, saturated and unsaturated content, and saturated/unsaturated ratio were not statistically affected by planting dates.
Although not significant, data showed that late planting date, associated with higher daily temperatures, increased the oleic acid content from 28.95 g/100 g to 31.43 g/100 g in the second planting date and decreased linolenic acid from 48.68 g/100 g to 47.24 g/100 g.The decrease in the linolenic acid could be attributed to the fact that its accumulation ceases at 30 ∘ C [28].
Considering flax for use as functional food in the early date, Prointa Lucero had the highest content of linolenic acid while in the late date Prointa Ceibal had the highest content.
Although no significant effects of planting date were found on linolenic acid during this campaign, a marked effect had been in fact reported by [19] in three planting dates (June 1, 12 July, and 4 August).Probably, a later planting date had been optimal for their effect on the content of linolenic acid (Table 5).

Interactions between the Studied Factors.
When considering the results, the interaction cultivar versus planting date was not significant in none of the five FA under studies, as well as on the saturated and unsaturated FA, the saturated/unsaturated ratio, oil content, and seed yield.

Conclusions
Regarding the FA profile, significant differences were found between cultivars for palmitic, stearic, and linoleic acids content.The planting dates did not significantly affect palmitic, stearic, oleic, linoleic, and linolenic acids.However, results suggest that the flowering to maturity period adopted in this study was not the most appropriate; that is, temperatures recorded during such period did not favor seed formation and oil accumulation and the planting date subsequent to the second half of July reduced significantly the seed and oil yields.
No cultivar versus planting date interaction was observed in fatty acid composition, content of saturated and unsaturated FA, and saturated/unsaturated ratio, oil content, and seed yield.
Taking into account the effects of different planting dates on linolenic acid, it can be concluded that Prointa Lucero variety should be used when planting at an early date, whereas Ceibal INTA should be considered if planting at late dates.In this way, the highest content of linolenic acid could be obtained, and flaxseed could be used as functional food.

Figure 1 :
Figure 1: Rainfalls recorded during 2008 at Paraná AES and differences with the average in period 1934-2008.

3. 1 .
Climatic Conditions.Figures 1 and 2 show rainfall and temperature conditions during the campaign, which proved to be adequate for temperature.The average temperature, from flowering to harvest (harvesting dates were November 25, 2008-1st date and December 23, 2008-2nd date), for the first planting date was 20.7 ∘ C, while for late planting date was 23.4 ∘ C.

Table 4 :
Average concentration of palmitic, stearic, oleic, linoleic, and linolenic acids, and content of saturated, unsaturated (g/100 g), and unsaturated/saturated ratio in the analyzed cultivars in the two planting dates.Different letters in the same column indicate significant difference according to Tukey's test ( < 0.05) between cultivars.

Table 5 :
Average among the seven cultivars of palmitic, stearic, oleic, linoleic, and linolenic acids, content of saturated and unsaturated fatty acids (g/100 g), and unsaturated/saturated ratio at two planting dates.