U usporedbi s kukuruzom standardnog tipa, kemijski sastav zrna kukuruza šećerca karakterizira povećani sadržaj šećera i smanjeni sadržaj škroba. Dosadašnja istraživanja u svijetu pokazuju da kemijski sastav zrna kukuruza šećerca prvenstveno ovisi o tipu mutacije endosperma, dok u Hrvatskoj nije bilo ovakvih istraživanja. Stoga su glavni ciljevi ovog doktorskog rada bili istražiti promjene kemijskog sastava zrna tijekom dozrijevanja domaće germplazme kukuruza šećerca koja obuhvaća hibride tipa sugary (su1) i shrunken (sh2) mutacija kao i utjecaj okolinskih čimbenika (vegetacijske sezone i roka sjetve). Dva pokusa (1. i 2.) provedena su na pokusnom polju Poljoprivrednog instituta Osijek u Osijeku (PIO) uz primjenu standardnih agrotehničkih mjera za proizvodnju kukuruza šećerca. U pokusu 1., tijekom dvije vegetacijske sezone (2008. i 2009.) uzgojeno je devet hibrida kukuruza šećerca tipa sugary mutacije endosperma, a koji su kreirani na PIO. Sedam hibrida priznato je u Hrvatskoj (OS 250su, OS 254su, OS 253su, OS 255su, OS 256su, OS 247su, OS 258su) dok su dva hibrida eksperimentalni (Alpos su i OS 332su exp). U pokusu 2. tijekom vegetacijske sezone 2011., u tri roka sjetve posijano je pet sugary hibrida (OS 254su, OS 255su, OS 256su, OS 247su i OS 258su) izabranih na osnovu prethodnog dvogodišnjeg istraživanja, te pet shrunken hibrida i to Superslatki (Bc Institut za oplemenjivanje i proizvodnju bilja d.d.), jedan priznati (OS 244sh) i tri eksperimentalna (OS exp 1sh, OS exp 2sh, OS exp 3sh) hibrida PIO. Tijekom istraživanja na svim biljkama provedena je kontrolirana (ručna) oplodnja i uzeti uzorci za kemijske analize u pet stadija zrelosti zrna i to 17., 19., 21., 23. i 25. dan nakon oplodnje (DNO). Na uzorcima pokusa 1. utvrdio se sadržaj vode, saharoze, glukoze, fruktoze, ukupnih šećera, vodotopivih polisaharida, škroba, te parametri boje (L* i b*) u promatranim stadijima zrelosti zrna. Na uzorcima zrna iz pokusa 2. dodatno su utvrđeni ukupni fenoli i antioksidacijska aktivnost. U pokusu 1. optimalni stadij zrelosti zrna za berbu su1 hibrida nastupio je unutar istraživanog perioda zrelosti zrna, a hibridi su se međusobno razlikovali u dinamici otpuštanja vode tijekom dozrijevanja. Najdulji optimalni period za berbu obzirom na sadržaj vode u zrnu imao je hibrid OS 250su. Unutar optimalnih stadija zrelosti zrna za berbu, određene hibride poput OS 250su, OS 253su i OS 254su karakterizirao je i najveći sadržaj saharoze i ukupnih šećera. U pokusu 2. svi hibridi sh2 tipa mutacije imali su značajno viši sadržaj ukupnih šećera i značajno niži sadržaj vodotopivih polisaharida u zrnu u odnosu na su1 hibride. Kod su1 skupine mutanata najveći sadržaj ukupnih šećera utvrđen je za hibrid OS 256su, a kod sh2 skupine za hibrid OS 244sh. Najveći sadržaj ukupnih fenola unutar su1 skupine imao je hibrid OS 258su, a unutar sh2 skupine hibrid OS exp 2sh. Hibride sh2 mutacije karakterizirao je veći porast antioksidacijske aktivnosti u kasnijim stadijima zrelosti zrna u odnosu na su1 hibride. Vegetacijska sezona je značajno utjecala na većinu istraživanih svojstava su1 hibrida kukuruza šećerca. U usporedbi sa 2008., u toplijoj i sušnijoj 2009. vegetacijskoj sezoni utvrđen je značajno veći sadržaj pojedinačnih i ukupnih šećera te parametara boje (žutoća i intenzitet) zrna, dok je reakcija za sadržaj vode, vodotopivih polisaharida i škroba bila obrnuta. U pokusu 2. utvrđen je i značajan utjecaj roka sjetve na većinu kemijskih svojstava zrna istraživanih su1 i sh2 hibrida kukuruza šećerca. Rezultati višegodišnjih istraživanja jasno ukazuju da se hibridi domaće germplazme kukuruza šećerca međusobno značajno razlikuju po svim kemijskim svojstvima zrna tijekom dozrijevanja i da na specifičnu reakciju hibrida mogu utjecati i okolinski čimbenici (vegetacijska sezona i rok sjetve).
Introduction: Sweet corn (Zea mays L. var. saccharata Sturt.) is an important vegetable crop. Although it is grown on much smaller areas than field corn, recently the production and consumption of sweet corn has been growing both in Europe and in the Republic of Croatia. The main reason for its popularity and growing consumers interest is conditioned by the pronounced trend of healthy human nutrition, and sweet corn is characterized by numerous highly nutritious properties. In comparison to field corn, sweet corn endosperm is characterized by an increased content of sugars and decreased content of starch. Previous studies are showing that the chemical content of kernels mostly depends on the type of endosperm mutation, but also on the genetic basis. In Croatia there is no comprehensive research in this area. Consequently, the main objective of this dissertation was to study domestic germplasm of sweet corn which includes hybrids of su1 (sugary-1) and sh2 (shrunken-2) types. Differences in chemical composition of kernels were assessed between and within groups of sugary and shrunken types of sweet corn hybrids based on three year field research. In trials with controlled pollination the content of water, sucrose, glucose, fructose, total sugars, water-soluble polysaccharides, starch, total phenols content in kernels, antioxidant activity and colour of kernels at various stages of kernel development were determined. In addition, the influence of environmental factors, year and sowing date, on kernel chemical composition of su1 and sh2 sweet corn hybrids during various stages of kernel development have been evaluated.
Materials and methods: Field trials were on the experimental fields of the Agricultural Institute Osijek in Osijek (45°33'N, 18°41'E) during 2008, 2009 and 2011. During growing seasons, standard agricultural measures were applied. The first trial was set up over two years (2008 and 2009) and nine maize hybrids with sugary endosperm mutation (standard sugar) were sown. Hybrids in the trial are created at the Agricultural Institute Osijek. Seven hybrids are registered in the Republic of Croatia, and two hybrids are new, experimental hybrids, translated hybrids of the standard kernel type. Registered hybrids are OS 250su, OS 254su, OS 253su, OS 255su, OS 256su, OS 247su, OS 258su, and the two experimental hybrids are Alpos su and OS 332su.
The trials were sown by hand, basic plot of each treatment was sown in two rows with a row spacing of 0.7 m and a length of 6.0 m. Controlled pollination was performed on all plants of the basic plot. It was ensured that sampling is done on certain days after pollination. Samples for chemical analysis of kernels were harvested five times during various stages of kernel development, on the 17th, 19th, 21st, 23rd and 25th day after pollination (DAP). Samples were harvested early in the morning, not later than 8 am and delivered to the laboratory at maximum of 10-15 min after harvest to avoid sugar loss. The content of water, sucrose, glucose, fructose, total sugars, water-soluble polysaccharides and starch were determined on a sample of five ears.
Preparation of samples for chemical analysis began immediately upon reception of harvested ears. Kernels of sweet corn were separated from the cob by cutting with a sharp knife from the top to bottom of ear. A sample of 100 g of kernels was prepared, frozen, and frozen sample lyophilized. Lyophilization lasted for 48 h. Lyophilized samples were stored at -20 °C and used to determine starch content. The rest of the separated kernels were crushed and homogenized using a stick mixer, blender. Homogenized sample was used to determine content of water, sugar and water-soluble polysaccharides. Determination of water content was carried out by drying of homogenized sample weighing 5 g in an oven at 105 °C to constant weight. Sugars and water-soluble polysaccharides extractions were conducted with ultrapure water according to a modified method (Sturm et al., 2003). Determination of sugars and water-soluble polysaccharides was performed on a high-performance liquid chromatographic system (HPLC) series 200. The standard solution consisted of sucrose, glucose, galactose (internal standard) and fructose. Sugars from the water extract were identified by their retention times and quantified by the peak area by the method of internal standard. Total sugars are expressed as the sum of sucrose, glucose and fructose. Other peaks on the chromatograph were used to calculate water-soluble polysaccharides. The content of sugars and water-soluble polysaccharides is expressed in % in dry matter. The starch content was determined in a lyophilized sample by the polarimetric method according to Ewers (1997). The parameters of colour (L* and b*) in the ground samples were determined according to the CIELAB method (CIE, 1976) with a three-filter colorimeter Chroma meter Konica Minolta CR-400. Colours are described through three components. L* (luminance) represents the colour intensity (brightness) and the coordinates a* and b* colours. In this study, values of the L* and b* (ratio of yellow and blue colour) components were monitored.
Second trial was set up in 2011. Five sugary hybrids (selected on the basis of a previous two-year study) and five shrunken hybrids (supersweet sweet corn) were sown in three sowing dates. Selected sugary hybrids were selected based on the results of chemical analyses but also on the base of real needs of producers and the possibility of commercialization of selected hybrids.
Selected sugary hybrids were OS 254su, OS 255su, OS 256su, OS 247su and OS 258su. Shrunken hybrids represented in the trial were one registered hybrid of Agricultural Institute Osijek (OS 244sh), one registered hybrid of Bc Institute Zagreb (SUPERSLATKI) and three experimental hybrids of Agricultural Institute Osijek (OS exp 1sh, OS exp 2sh, OS exp 3sh). The first sowing date was 14th April and this sowing date reflects the optimal sowing date in most years in the area where the trial was conducted. The second sowing period was two weeks after the first sowing period and two weeks before the third sowing period. These sowing dates were selected as the optimal sowing days to be applied in production of sweet corn with a distance between sowing dates sufficient to ensure successive harvesting and chemical analysis in the laboratory. Second trial was sown as Trial 1 and controlled pollination was performed on all plants of the basic plot as in Trial 1. Samples for chemical analysis of kernels were harvested five times during various stages of kernel development. Beside the parameters analysed in Trial 1, the chemical analysis of the kernel included the analyses of total phenols and the antioxidant activity. As in Trial 1 after receiving the samples, colour of kernels was determined on the ear and sample was prepared for chemical analysis. Sample of 100 g of kernels was frozen and lyophilized. Lyophilized samples were stored at -20 °C and used for determination of starch, total phenols and antioxidant activity.
The content of total phenols in kernel of sweet corn was determined spectrophotometrically by modified semimicro method according to Singleton and Rossi (1965). The concentration of total phenols was calculated from a standard curve with gallic acid as standard and expressed as gallic acid equivalent in dry matter (mg GAE/100 g DW). Total antioxidant activity was determined spectrophotometrically by DPPH method according to Brand-Williams et al. (1995) and expressed as gallic acid equivalent in dry matter (mg GAE/100 g DW). The GENSTAT 7.2 program was used to process the results.
Results and conclusions: The results of this research show that relatively small changes in the water content in the kernel during the observed stages of kernel development are associated with significant changes in the chemical composition of kernel. The decrease in water content in kernel during maturation is significantly associated with a decrease in the content of single and total sugars (from r=0.61 to r=0.72) as well as a decrease in the content of total phenols (r= 0.50). The decrease in kernel water content during kernel
development is significantly associated with an increase in water soluble polysaccharides (r=-0.70), starch (r=-0.76), antioxidant activity (r=-0.43) and kernel colour (r=-0.39). During the observed maturation period, the metabolic processes in the kernel are very intensive, so the harvesting of sweet corn in the appropriate stage of kernel development is crucial to ensure high nutritional quality.
For all observed characteristics, significant differences were found between the tested hybrids, where the type of endosperm mutation is the most important factor for the content of sucrose, total sugars, water soluble polysaccharides and starch in kernel. The change in water content, single and total sugars, water-soluble polysaccharides and starch during kernel development is greater in su1 hybrids compared to sh2 hybrids. Greater increase in antioxidant activity during maturation was found for sh2 hybrids compared to su1 hybrids.
Significant differences were found between hybrids of the same type of mutation in content and dynamics of change of content during kernel development for all observed characteristics. Considering the duration of the optimal harvest period, sugar content and reduction of sugar content by kernel development, and the lowest starch content hybrids OS 254su and OS 256su can be distinguished as the best in the group of su1 hybrids, The new sh2 hybrid OS exp 2sh had a slightly lower initial content of total sugars compared to the standard hybrid OS 244sh, while in the hybrid OS exp 2sh the content of total sugars did not change significantly during the optimal harvest period and had a significantly higher sugar content at the last stage of maturity compared to other sh2 hybrids. For the OS exp 2sh hybrid, very little variation was found between different sowing dates both in the content of total sugars and in the change of total sugars during maturation, which is very important considering that the selection of a hybrid for commercial production depends on the stability of its performance through environments.
The results of this research for total phenol content and antioxidant activity show a high part of genotype × maturity stage x sowing date interaction in total variability. Although significant differences were found between the examined hybrids for both characteristics, the dynamics of change during kernel development as well as the variation between sowing dates prevent the emphasis of hybrid with potentially higher functional value. In any case, the identified differences between genotypes indicate that research needs to be continued in a way to include more genotypes and involve more groups of bioactive compounds (especially carotenoids) in order to isolate hybrids with potentially higher health benefits.