PROMOTING GERMINATION OF FRESHLY HARVESTED BARLEY GRAIN WITH ALKALINE SOLUTIONS. By J. Q. Hou and G. M. Simpson

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1 J. Inst. Brew., November-December, 1994, Vol. 100, pp PROMOTING GERMINATION OF FRESHLY HARVESTED BARLEY GRAIN WITH ALKALINE SOLUTIONS By J. Q. Hou and G. M. Simpson Department of Crop Science and Plant Ecology, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 0WO, Canada Received 3 February 1994 Effects of immersing freshly harvested barley {Hordeum vulgare L.) grains in concentrated KOH and NaOH solutions on germination were studied in barley cv. Harrington. Promotion of germination up to 100% was achieved by alkaline treatments with a wide range of solution concentrations and immersion time. The effectiveness of alkaline treatments was dependent on maturity, presence of the hulls and moisture content of the grains. While alkaline treatments were generally not damaging to the grain, excessive exposure to the chemicals can harm the grains and adversely affect germination and sub sequent growth of seedlings. Alkaline hydrolysis of the cuticles in the covering layers of the grain is at least partially responsible for the promotive effects on germination. Key Words: Hordeum vulgare. barley, dormancy, germination, potassium hydroxide, sodium hydroxide, hulls, seed coat. Introduction Many studies have focused on developing industrial technology to overcome grain dormancy of barley (Hordeum vulgare L.) that causes difficulty for maltsters4. The dormancy problem is especially evident in freshly-harvested grains. Evidence accumulated over the past indicates that dormancy in barley as well as in other grass species can be imposed by covering layers (hulls, pericarp and testa)2'5'14'16. Proposed mechanisms of the imposed dormancy include physical restraint of embryo growth and impermeability to water or gases. Removal of, or mechanical injury to, the covering layers can often induce germination in otherwise dormant seeds '15. A previous study demonstrated that immersion of dry dormant caryopses in alkaline (KOH and NaOH) solutions can promote germination in wild oat (Avena fatua L.)8. The alkaline treatment was investigated because it has been suggested that the cuticular layers in the seed coat may form permeability barriers13, and the cuticle can be depolymerized by alkaline hydrolysis". Modification of the seed coat and enhanced water uptake were observed in the treated caryopses8. For similar reasons, the alkaline treat ments should be able to influence germination of barley grains which have cuticles in the covering layers14. The purpose of this study was to test effects of immersing freshly-harvested dormant barley grains in concentrated alkaline solutions on germination. Such a study has not been previously reported in major cereal crops to the best of our knowledge although dilute sodium hydroxide has been reported to have no effect on dormancy or germination4. Experimental Plants of barley cv. Harrington were grown in an experi mental field at the Department of Crop Science and Plant Ecology, University of Saskatchewan, Canada. Three lots of grains with different maturity and moisture content were collected by hand. The hulls of lot-1 grains had not com pletely lost their chlorophyll. The moisture content of whole grain was ±0.19% (wet basis); The hulls of lot-2 grains had lost their chlorophyll, but some awns were still greenish. The moisture content was ±0.4%; The hulls and awns of lot-3 grains were yellow. The moisture content was ± 0.75%. Collected grains were air-dried at room temperature for one day, then kept in a desiccator at 90% RH for the period of the study that lasted for about four weeks. The RH was maintained with 1.58MCaCU (BDH, Edmonton, Canada) solution according to Stokes and Robinson17. Keeping the grains at the high RH was an attempt to delay their recovery from dormancy through the action of desicca tion14. The matter is discussed later. Some grains were dehulled by hand. Potassium hydroxide, sodium hydroxide and sodium carbonate (BDH-ACS certified grade; Edmonton, Canada) solutions were freshly prepared and cooled to room tempera ture. Purities of 85% for KOH, 97% for NaOH and 98% for Na2CO3 were used to calculate the normality concentrations. Fifty grains were immersed in a 50 ml solution in a 250 ml beaker. The grains were initially gently stirred with a pair of forceps to get rid of air bubbles on the grain surface. Then the beaker was shaken on a reciprocating shaker at a rate of 150 strokes/min. The solution concentrations and length of immersion are indicated in the results section. Grains used for control were immersed in distilled water and shaken as above. Grains, after chemical treatments, were washed three times with 125 ml aliquots of distilled water, then soaked in 125 ml distilled water for 10 min, then washed three times again. Washed grains were briefly blotted dry on filter paper. Fifteen grains were placed in a 6-cm Petri dish lined with two layers of 6-cm Whatman No. 1 filter paper wetted with two ml distilled water. For each treatment three Petri dishes (replicates) were placed in a germination box lined with wetted paper towels to maintain a high relative humidity. The germination box was incubated in darkness at 20 ± 1 C. Ger mination (visible extrusion of a colcorhiza or coleoptile) was assessed every 12 hours for seven days. Viability of ungerminated grains after seven days of incubation was evaluated with 0.5% tetrazolium (2,3,5-lriphenyl-tetrazolium chloride, Sigma, St. Louis, USA) solution according to the procedure recommended by the International Seed Testing Association9. All experiments were repeated twice. Germination values were transformed into angles by the arcsin transformation. The angles were analyzed with the analysis of variance and Tukey's studentized range test. Differences in germination after different days of incubation were examined with the contrast (see SAS/STAT Guide, SAS Institute Inc., Cary, NC, USA, for the statistical procedures). Significance of difference is declared at a= Results Effects of immersing hulled or dchullcd grains in 5 N KOH solutions on germination were tested in the three lots of samples. Immersing time was 5, 10, 15 or 20 min for dehulled grains, and 15, 20 or 25 min for hulled grains. Final ger mination of controls was low in dehulled grains at early

2 422 GERMINATION OF BARLEY [J. Inst. Brew Fig. 1. Effects of immersing freshly harvested barley grain in 5 N KOH solutions for different times on germination. A: Dehulled lot-l grain; B: Hulled lot-l grain; C: Dehulled lot-2 grain; D: Hulled lot-2 grain; E: Dehullcd lot-3 grain; F: Hulled lot-3 grain; Each point is the mean±se of six replicates. Germination temperature was 20 ± I C. See materials and methods for grain conditions of different lots.

3 Vol. 100, 1994] GERMINATION OF BARLEY (D CD ffl 03 S (D 0 0 [j] [j] [}] [fa cfa EJ3 fo 4 30 min 20 min D 10 min Fig. 3. Germination responses of the hulled lot-3 grain to immersion in 5 N NaOH solutions for different times. Each point is the mean±se of six replicates. Incubation temperature was 20 ± PC. a a a a a d S S88 8 fribats KOH D NaOH Fig. 4. Comparison of effects of KOH and NaOH immersion on germination of the hulled lot-3 grain. Solution concentration was 4N, and immersion time was IS min. Incubation temperature was 20 ± PC. Each point is the mcanise of six replicates. Fig. 2. Germination responses of the hulled lot-3 grain to KOH solutions of reduced concentrations. A: lomin immersion; B: IS min immersion; C: 20 min immersion. Each point is the mean±se of six replicates. Incubation temperature was 20±PC. 6 maturation stages (about 10% in lot-! grains, Fig. I A; 20% in lot-2, Fig. 1C). All KOH treatments eventually promoted germination to 100%. Initial germination rate, however, was significantly different for the different treatment times. For example, germination after long immersion times (IS and 20 min) was lower than after short immersion times (5 and 10 min) when measured at 60 hours of incubation. Germin ation of control in dehulled grains at the later maturation stage (lot-3) was basically the same pattern as the grains

4 424 GERMINATION OF BARLEY [J. Inst. Brew. 100 EJUULJULJULJLJLJ 20 min 2 10 min Fig. 5. Germination responses to 4 N KOH immersion of the hulled lot-2 grains stored at 90% RH (A) and air-dried (B) for two weeks. Immersion time is indicated by the legends. Incubation temperature was 20±l*C. Each point is the mcan±se of six replicates. treated with KOH for five min (Fig. IE). Long treatment times, especially IS and 20 min, delayed germination in the early stages of incubation. There was no germination of controls in all hulled grains (Fig. IB, D, F). Treatments with KOH promoted final ger mination to about 73% in lot-1, 85% in lot-2, and 100% in lot-3 grains. Germination after 23 min of treatment with KOH was higher than 15 and 20 min treatments in lot-1 grains in the early stage of incubation (Fig. IB), but lower in lot-3 grains (Fig. IF). Treatments with reduced KOH concentrations (1, 2.5, 4 N) for 10, 15 and 20 min were tested in hulled lot-3 grains. Germination of controls was below 10% (Fig. 2). Treatments with 2.5 and 4 N KOH generally promoted final germination to above 90%. However, treatment with N KOH promoted germination to above 90% only after 20 min immersion (Fig. 2C vs. 2A, B). To test effects of other alkaline materials on germination, hulled lot-3 grains were treated with 5 N NaOH or Na,CO3 for 10, 20 and 30 min respectively. Germination did not occur after Na2CO3 treatments (data not shown). By contrast with about 2% germination in the controls, final germination was about 95% after 30 min, 84% after 20 min, and 40% after 10 min treatments with NaOH (Fig. 3). Fig. 6. Effects of 5 N KOH immersion on the growth of germinated dehulled lot-2 grains after 65 hours of incubation at 20±l C. The grains were from the experiment in Fig. 1C. A: Control; B: 5 min immersion; C: 10 min immersion; D: 15 min immersion; E: 20 min immersion. To directly compare effects of KOH and NaOH on germination, hulled lot-3 grains were treated with their 4 N solutions for 15 min respectively. This experiment was con ducted about 20 days later than the one in Figure IF. About 23% of the control germinated after seven days of incubation (Fig. 4). Both alkaline treatments promoted germination to 100% after 60 hours of incubation. Before 60 hours, germin ation of NaOH-treated grains was significantly lower than the KOH-treated. To test whether responses to KOH treatments in the barley grain change with grain moisture content, hulled lot-2 grains stored at 90% RH or air-dried for two weeks were treated with 4 N KOH for 10 or 20 min. The moisture content at the time was 21.9 ±0.14% for grains stored at 90% RH, and ±0.23% for air-dried. Grains stored at 90% RH did not germinate without KOH treatment (Fig. 5A). Treatment with KOH for 10 min induced about 22% final germination, and treatment for 20 min induced about 90% germination. About 52% air-dried grain germinated without KOH treat ment (Fig. 5B). Germination was promoted to 100% by the KOH treatments after 48 hours of incubation. Abnormal growth of the primary roots was observed in germinated grains treated with KOH for relatively long periods of time. The photograph of Figure 6 was taken after 65 hours of incubation of dehulled lot-2 grains. Note the absence of growth of incubation of the two primary roots in the grains treated with 5 N KOH for 15 or 20 min despite the elongation of the coleoptiles (Fig. 6D, E). Such abnormal growth was limited in dehulled grains treated with KOH for 15 or 20 min, or in hulled grains treated for 25 min. Another noticeable feature was the development of red patches on the seed coat after alkaline treatments. The colour change was more obvious in dehulled grains than in hulled ones. The tetrazolium tests made after seven days of incubation revealed more than 80% death in hulled ungerminated lot-1 and lot-2 grains treated with 5 N KOH for 25 min. A grain was considered dead if the root region was not stained9. The viability of other samples was generally above 90%. Discussion and Conclusion Immersing grains in concentrated KOH or NaOH solution is an effective method to promote germination in freshly har-

5 Vol. 100, 1994] GERMINATION OF BARLEY 425 vested barley grains. The effectiveness of alkaline treatments depends upon maturity, existence or the hulls, moisture content or the grains, as well as chemical concentration and immersion time. For the lot-3 grain that was at a later maturation stage, one hundred percent germination was induced with a wide range or chemical concentrations and treatment times (Figs. IF, 2, 4). In contrast, the alkaline treatments may not be 100% eltectivc in hulled grains at early maturation stages (Figs. IB, D). Increased germination after removal or the hulls (Figs. I A, C, E) has been described in earlier reports5. This study indicates that extra promotive effects can be achieved with alkaline treatment that cannot be achieved with physically removing the hulls alone (Figs. 1A, C). Desiccation can accelerate recovery from dormancy in barley grains (Reference 14; Fig. 5, this study). Desiccated grains are also more responsive to alkaline treatment than those kept at high RH (Fig. S). When the concentration of alkaline solution or immersion time is increased, germination may vary over a relatively large scale (Figs. 2, 3). Further increase in concentration or immer sion time makes no difference once the effect on germination reaches the maximum. Excessive exposure to the chemicals, however, may cause damage to the grains, and adversely affect germination and subsequent growth of seedlings (Figs. 1.6). Seed germination responses to environmental conditions usually change with progress of storage period How storage period affects germination behaviour is not a topic in this study. Storage period, however, obviously created certain variations in the results, for example, in early (Fig. 1F, zero germination in the control) and later (Fig % germin ation in the control) experiments. Although keeping the grains at high RH is considered satisfactory for maintaining dormancy status in barley grains in this study, maintaining a uniform state of dormancy may be a challenge to laboratory workers if a study lasts too long. Freezing, as an accepted way of maintaining dormancy, may not be suitable when grains with high moisture content are used. The fact that both KOH and NaOH can induce germin ation indicates that the promotive effects are not cation specific. KOH is more effective than NaOH in both barley and wild oat8, probably because the former chemical is a stronger alkali than the latter. The colour change on the grain surface after alkaline treatments probably arose from changes in the bonding of bound phenolics as suggested by Jones10. This colour change may also serve as an indication that the alkaline hydrolysis of the cuticles is at least partially res ponsible for the promotive effects on germination. A maximum promotion of germination apparently requires that the alkalies work their way through the cuticles in the covering layers of the barley grain. The modification of the covering layers may not only accelerate water uptake" and germination, but also help the grains to overcome the stress caused by oxygen depletion during steeping. This is supported by the following preliminary observation; When after-ripened (non-dormant) grains were immersed in water, germination of alkaline-treated grains was faster and more uniform than the non-treated (data not shown). Prolonged immersion in water suppressed germination (water dormancy) in non-treated grains but not in treated ones. In conclusion, although more chemical and physiological studies are needed to elucidate the mechanism of the alkalies action, the alkaline treatment provides a fast, simple and reliable method to promote germination in barley. Other mechanisms of inducing dormancy such as the presence of surface micro-organisms that create anaerobic conditions may interact with alkaline treatment4. References 1. Baxter. E. D., Booer, C. D. & Palmer. G. H. Journal of the Institute of Brewing, , Briggs, D. E. In Barley: Genetics, biochemistry, molecular biology and biotechnology. (Shewry, P. R. edit.) p Walingford, CAB International. 3. Brown. C. R. Journal of the Institute of Brewing , Dor.in, P. J. & Briggs. D. E. Journal of the Institute of Brewing, 1993, 99, Dunwell, J. M. Annals of Botanv, 1981, 48, Ellis, R. H., Hong, T. D. & Roberts. E. H. Seed Science and Technology IS, Hou, J. Q. & Simpson. O. M. Phvsiohgia Plantarum , Hou, J. Q. & Simpson, G. M. Canadian Journal of Plant Science, (in press, 1994). 9. International Seed Testing Association. Seed Science and Technology, 1985, 13, Jones. J. H. Plant Physiology, , Kolattukudy. P. E. Annual Review of Plant Physiology, , Mclntyre, G. I. & Hsiao. A. I. Botanical Gazette , Morrison, I. N. & Dushnicky. L. Weed Science. 1982, 30, Palmer, G. H. In Cereal science and technology. (Palmer. G. H. edit.), 1989, p. 61. Aberdeen, Aberdeen University Press. 15. Palmer, G. H.. Barrett. J. & Kirsop. B. H. Journal of the Institute of Brewing. 1970, 76, Simpson, G. M. Seed dormancy in grasses, 1990, 297 pp. Cambridge. Cambridge University Press. 17. Stokes, R. H. & Robinson. R. A. Industrial and Engineering Chemistry , 2013.