PROTEIN HYDROPHOBIC DRESSING ON SEEDS AIMING AT THE DELAY OF UNDESIRABLE GERMINATION

Polymer seed-coatings have been largely tested as an altemative method for preventing diseases and have the potential to be used to control undesirable germination and thereby increasing seed storage. Amongst these, the protein-based coatings can be applied with advantage of forming stable biodegradable and hydrophobic films. Due to their chemical structure, protein dressings act as efficient barriers for water uptake, even when seeds are unavoidable exposed to a moist environment. In this work, the effect of hydrophobic dressings was tested on the germination rate of sugar beet (Beta vulgaris L.) and broccolis (Brassica oleraceae var italic L.) seeds under laboratorial condition. Zein, natural maize (Zea mays L.) proteins extracted from gluten meal, was used as precursor polymers to form the coatings. Seeds were dressed by direct submersion into a zeinlethanol formulation of zein concentration of 3.0 g L-I, followed by natural air drying. The resultant coating has elevated the hydrophobic feature due to the high content amino acids present in the structure of the zein. For both types of seeds an overall delay in sprouting and germination was observed, with a more accentuated reduction on sugar beet germination percentage after eleven days of measurements. The effect on germination rates is understood as the efficiency of the zein coating to form a physical barrier preventing water permeation into the seeds.


INTRODUCTION
required.However, some degree of dormancy could be advantageous, at least during seed development and storage.
Ali seed surfaces are basically composed of lignocellulosics like-structures, which present a fast In a broad sense, seed dormancy can be interpreted as an undesirable characteristic for agricultural cropping since rapid germination and growth are degradation by organisms which are capable of breaking them down into digestive units.Additionally, t~e hydroxyl groups, mainly present in the cellul?sech~m, are polar groups with high reactivity, suggestmg a high affinity towards water (Kiguchi, 1996).Water uptake by itself provokes dormancy break and also accelerated growth (Goodwin, 1966), what can be a problem for long-term storage.Moisture migration within grain containers can lead to detrimental chan~es in grain appearance and quality and leads to undesirable germination.Storage procedures are normalIy regulated by monitoring temperature and air moisture (AbdalIa & Roberts, 1969) and alternatively by using polymer coatings to preserve seed features.Coatings provi~e a physical barrier between the embryo and the environment, delaying undesirable germination and under specific conditions avoiding microorganism infestation (Elzein et aI., 2006).Hydrophobic coatings on seeds can be attained either by ion implantation (Volin et aI., 2000) or by direct dressing (Chachalis & Smith, 2001;Willenborg et aI., 2004).For both treatments, chemical reactions between cellulose components are expected to take place especialIy by covalent bonds b~tween functional groups incorporating hydrophobic radicaIs (English et aI., 1994).
In this work zein, a mixture of storage proteins found in the com endosperm (Wang & Padua, 2005), is used to form hydrophobic coatings replacing the traditional chlorosilanes or vinyl based-polymers and their performance is studied in relation to the germination rate of sugar beet and broccolis seeds.

MATERIAL AND MEmODS Zein Extraction and Film Preparation
The zein used in this work was extracted frorn the gluten meal (CGM) of the com cultivar BR473.The CGM is a by-product of starch production in the wet-milling processo This product has up to 70% of zein, and some polysaccharides are not removed in this process (Wu et aI., 1997;Forato et aI., 2?03).CGM oil extraction was carried out for 24 hours m a soxlhet apparatus with hexane.The residual mass was mixed up with 70% ethanol for 24 h.Zein was obtained by evaporating the solvent and then Iyophilized.For the preparation of film precursor solution, zein was solubilized in 70% ethanol (30% water) in concentrations of 3.0 g L-I.The solutions were stirred and slight1y heated to facilitate homogenization.
At this time, polypropylene glycol was gradualIy added at the ratio of 5.0 wt % to act as plasticizer.

Seed Coating
Coatings were obtained by direct immersing of the seeds into the zeinlplasticizer solutions for 5 seco Lots of 25 samples of sugar beet and broccolis seeds were separately dipped into the solution.Excess gel was alIowed to drain off and the film was allowed to be formed by drying at room temperature.The use of ethanol as solvent performs the film formation on seed surface by a natural cross chain Iinkage due to the evaporation of volatile alcohol.

Germination Tests
To evaluate germination, seeds were placed in plastic boxes (Gerbox) with two layers of filter paper.The paper was previously pressed in a way to form smalI cavities to uniformily position the seeds which were moistened with 6 mL of distilled water.The experiments were carried out at 25°C for 11 days.A seed was scored as germinated when its radical length as superior to 1.0 mm.Germinating seeds were counted twice a day, starting on the first day and until the eleventh day of imbibition, when on both set of samples the maximum germination percentage was achieved.
Three replicates of 25 seeds were used.For a reference blank test, 25 uncoated seeds of each species also underwent a percentage germination test.The data were submitted to variance analysis, using the square root are sen transformation and the average values were compared by the Tukey studentized range test (p < 0.05).The models which better fitted the points were defined using a mathematical program (Origin 7.03), by using non-linear functions amongst pre-defined curves which resulted in the best R 2

• RESUL TS AND DISCUSSION
Differences in median germination time were evident among coated and non-coated seeds.As expected, non-coated seeds presented highe.rpercentage of germination.Zein-coated samples required a longer time for germination.
For the both species the final germination percentages was reduced (Figure 1), with a slight behavior difference between them.The sugar beet coated seeds sprouted almost simultaneously in relation to the non-coated samples.However, the number of germinated coated seeds was inferior along alI the evaluated period.After II days of measurements, 100% of the non-coated seeds germinated, against 65% of coated.
Conversely, for broccolis seeds, the first sprouts have a medium delay of three days in relation to the non-coated seeds, but subsequent1y the germination folIows similar profile, presenting after II days 90% germination compared with 100% for the noncoated.In general, this behavior differs from tests of hydrophilic dressing on broccolis seeds, which was reported as having no effect on germination rates (Almeida et aI., 2005).The direct comparisons among batches showed quite similar germination rates, no changes being detected for the replicates, which is translated into the small standard deviation (error bar) for each day.The models that best fitted the experimental points were: broccolis Y = A, ( IX) , and sugar beet The hydrophobic nature of zein is related to its high content of non-polar amino acids what reduces the water surface interaction.In this sense, the delayed germination can be understood as due to a reduction of osmotic potentials the initial solution.Generally, for good germination rates seeds must have uniform water supply.With the presence of a non-polar interface the water molecules will face difficulties in entering seeds.This situation may actually reduce permeability inhibiting germination, what becomes even more pronounced if the water supply is low.
Uptake ofwater by a mature dry seed is triphasic, with a rapid initial uptake (phase I) followed by a plateau phase.A further increase in water uptake occurs only after the germination is completed, when the embryonic axes elongate (Debeaujon et aI., 2007).The passage of water into the seed during the first phase 125 of germination is quite fundamental (Simon & Wiebe, 1975).This phase is centripetal so that the outer layers must become more hydrated than the central tissue to promote the bursting of the seed coat.In short, the zein coating reduces the necessary exposure of the seed to water until it obtains sufficient moisture to sprout .
There are some studies on the use of polymer prevention on premature germination through reducing the water uptake by the seed (Watts, 1976;Stewart, 1992).Most of them deal with temperature controlled polymers in which the seeds are prevented from imbibing the water needed for germination until a desired temperature is reached.Most of the used polymers are thermoplastics such as vinyl resin emulsions (Sauve & Shiel, 1980;Barke &. Luebke, 1981;Pires et aI., 2004) and copolymers of polyvinylacetate (Puglisi & Guth, 2001), not ali of them environmentally friendly or compatible .
Evidently a comparison between these materiaIs and zein should be worthwhile.Anyway, the zein coating showed to be efficient under wet and humid conditions in maintaining a suitable levei of seed dormancy, regulating imbibition, which can be strategically applied to reduce premature germination during storage.Additionally, zein is a natural biodegradable, quite amenable polymer to be transformed into coating films, as in conjunction or not with preventive products such as fungicides and insecticides.