Stimulation of Short-term Plant Growth by Glycerol Applied as Foliar Sprays and Drenches under Greenhouse Conditions

The effects of aqueous solutions applied as foliar spray and drench applications of glycerol were tested on the ‘Chantenay’ carrot (Daucus carota L.) family Apiaceae, corn (Zea mays L.) family Poaceae, and spearmint (Mentha spicata L.) family Lamiaceae under greenhouse conditions. Foliar sprays and drenches were administered to carrots at concentrations of 0, 1, 3, 5, 10, 25, or 50 ml L. Fresh weights, dry weights, and taproot diameter from carrot seedlings sprayed with a solution containing 5 mL L (50 mM) glycerol increased 105.6%, 158.4%, and 53.8%, respectively, when compared with untreated carrots. Foliar sprays were administered to corn at concentrations of 0, 0.1, 0.3, 0.5, and 1 ml L and spearmint at concentrations of 0, 1, 5, and 10 mL L. Growth responses increased in corn and spearmint by using certain glycerol concentrations. Fresh weights, dry weights, and shoot length from corn seedlings sprayed with a solution containing 0.5 mL L (5 mM) glycerol increased 83.5%, 154.6%, and 90.9%, respectively, when compared with untreated corn. Fresh weights, dry weights, and shoot length from mint plants sprayed with a solution containing 5 mL L (50 mM) glycerol increased 46.6%, 68.7%, and 102.5%, respectively, when compared with untreated plants. Glycerol applications can stimulate growth responses in diverse plant species. The U.S. Federal mandates affecting biodiesel production include the Energy Policy Act, which requires specific levels of alternative fuel use in federal automobile fleets, and the Clean Air Act, which requires all diesel fuel sold for on-road use to be ultralow in sulfur emissions by 2006 (Demirbas, 2009; Ebert, 2007; McIntyre, 2007). This legislation has resulted in a number of governmental biodiesel incentive programs, which contributed toward a U.S. biodiesel market of 150 million gallons production per year in 2006 (double the 75 million gallons per year produced in 2005) and have an anticipated future of 2 billion gallons per year production expected by the year 2015 (Ebert, 2007; McIntyre, 2007). The U.S. biodiesel industry vegetable feedstocks current output is 3.8 billion gallons of fats and oils annually. For every 10 gallons of biodiesel produced, 1 gallon of glycerol is generated through transesterification of animal and vegetable oils (Demirbas, 2009; Kenar, 2007). This dramatic biodiesel production increase over the past 5 years has resulted in decreasing glycerol prices resulting from excessive production. Commercialization of the glycerol generated by this industry is therefore becoming increasingly important (Ebert, 2007; Lines, 2009; McIntyre, 2007; Pachauri and He, 2006). Developing higher-valued products from glycerol would improve investment returns to the biodiesel industry. The historic markets for glycerol (e.g., antifreeze agents, food additives, cosmetics, and health products) are already limited; therefore, new products are needed (Ebert, 2007; Lines, 2009). Glycerol is a nontoxic, edible, biodegradable sugar alcohol making it an ideal ‘‘environmentally friendly’’ agricultural chemical. The influence of glycerol on plant tissue cells has been repeatedly demonstrated in its use as a cryoprotective agent used in cell and tissue cyrogenetic storage (Kim and Oh, 2009; Panis and Lambardi, 2005; Silayoi, 2001; Towill and Mazur, 1976; Withers and King, 1979). Glycerol has also been used in tissue culture studies using numerous plants—seaweed [Grateloupia filicina (J.V. Lamouroux) C. Agardh], kelp (Ecklonia radiata J. Agardh), morning glory (Pharbitis nil L.), Orchid tree (Bauhinia forticata Link), zedoary (Curcuma zedoaria Roscoe), bean (Phaseolus vulgaris L.), sweet orange [Citrus sinensis (L.) Osbeck] (Baweja and Sahoo, 2009; Hisajima and Thorpe, 1985; Lawlor et al., 1989; Mello et al., 2001; Vu et al., 1993). Glycerol has also been used as a carbohydrate supplement to stimulate algae tissue culture growth (Baweja and Sahoo, 2009; Kaczyna and Megnet, 1993; Lawlor et al., 1989; Marián et al., 2000; Robaina et al., 1990). For example, red algae [Gracilaria verrucosa (Hudson) Papenfuss] grown on 217 mM (i.e., 20 mL L) glycerol with plant growth regulators (2 mg L indole-3propionic acid, 2 mg L indole-3-acetic acid, 1 mg L N-isopentenyladenine) stimulated callus growth (Kaczyna and Megnet, 1993). Glycerol has been used in higher plant tissue cultures with mixed results (Aubert et al., 1994; Bellettre et al., 2001; Gautheret, 1955). Generally, glycerol is a poor substitute for sucrose or the reducing sugars (glucose or fructose) as a carbon source of plant cell cultures (Gautheret, 1955; Hisajima and Thorpe, 1985; Mello et al., 2001; Vu et al., 1993; Vuke and Mott, 1987). Aubert et al. (1994) studied the influence of glycerol on cell metabolism of sycamore (Acer pseudoplatanus L., family Sapindaceae) whereby callus was removed from sucrose and grown solely on glycerol for short durations. Chicory hybrid ‘474’ (Cichorium intybus L. var. sativum · C. endivia L. var. latifolia, family Asteraceae) leaf tissues cultured on a combination of sucrose and glycerol manifested somatic embryogenesis (Bellettre et al., 1999, 2001). Several citrus tissue culture investigators have reported stimulation of somatic embryogenesis using glycerol (Ben-Hayyim and Goffer, 1989; Ben-Hayyim and Neumann, 1983; Singh et al., 1992; Tomaz et al., 2001; Vu et al., 1993). For example, Vu et al. (1993) reported that ‘Hamlin’ sweet orange nucellar callus grown on medium substituting 2% glycerol for 5% sucrose promoted more somatic embryogenesis, chlorophyll formation, and RuBisCO activity. According to a report issued by the Glycerin Producers’ Association (1964), glycerol has several potential uses in the agricultural, floricultural, and horticultural fields (Anonymous, 2011). Glycerol (0.1 to 10%) can be substituted for water to moisten peatmoss around roots before shipping resulting in more successful transplanting. Narcissus sp. bulbs grown in gravel with diluted glycerol solutions exhibited greater growth and flowering (Anonymous, 2011). Glycerol has also been used to prevent freezing of branches, leaves, buds, and blossoms if used as a spray consisting of water, glycerol, a spreader, and alcohol (Anonymous, 2011). In this case, the protective coating that formed on the plant surfaces insulated plant tissues from the freezing temperatures. Soaking seeds in glycerol can counteract the adverse effect of salinity on growth and secondary metabolism (Ali et al., 2008). Seeds of Castor bean (Ricinus communis L.) soaked for 48 h in 5, 25, or 50 mM glycerol or a mixture of 10:5, 25:10, and 50:15 mM glycerol:aspartic acid, respectively, resulted in seedlings exhibiting higher fresh and dry weights than seedlings obtained from watersoaked seeds. In addition, essential oil and alkaloid contents were higher in plants derived from glycerol treated seeds than controls in saline environments (Ali et al., 2008). Despite these positive reports, glycerol is not commercially used to promote plant growth Received for publication 14 June 2011. Accepted for publication 15 Sept. 2011. We thank Debra Palmquist for statistical analysis. Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer. To whom reprint requests should be addressed; e-mail [email protected]. 1650 HORTSCIENCE VOL. 46(12) DECEMBER 2011 for agricultural or horticultural systems. The purpose of these studies was to conduct shortterm experiments with various plants to evaluate the merits of using glycerol as drenches or foliar sprays within the relatively uniform environment of a greenhouse. Materials and Methods Plant materials. Carrot seeds (Daucus carota L. ‘Chantenay’), Dwarf Corn (Zea mays L. cv. Gaspé Flint) seeds, and spearmint (Mentha spicata L. PI # ‘294099’) plantlets as sterile shoot cuttings were used. ‘Chantenay’ carrot was selected for its rapid root formation, dwarf corn because it could accommodate the limited greenhouse space, and cloned spearmint plantlets to ensure a uniform response. Greenhouse experiments. All plants were grown on a soilless medium formulated with 1 Canadian sphagnum peatmoss (Ferti-lome, Cheek Garden Products, Austin, TX):1 coarsegrade vermiculite (Premium Grade; Sun-Gro Horticulture Dist. Inc., Bellevue, WA), and amended with 10.9 g kg Micromax (Scotts Co., Marysville, OH) of 14S–15Fe–2.5Mn– 1.5Mg–1Cu–1Zn–0.2B–0.05Mo, and 62.2 g kg Osmocote (Scotts Co.) of 14N–4.2P– 11.6K. Carrots were grown in containers (3.8 cm diameter · 21 cm high) containing 50 g medium. Spearmint and corn were grown in 41⁄2$ rectangular pots (10.8 cm wide · 9.5 cm high · 11.4 cm deep) containing 100 g medium. Aqueous glycerol solutions were prepared with distilled water and food-grade glycerol (99.7%) (Soap Goods, Atlanta, GA). To determine the effect of glycerol on carrot growth and morphogenesis, 20 2-week-old seedlings were treated with foliar sprays at concentrations (Table 1). Foliar sprays were administered with handheld trigger spray bottles (24 oz. cap.) (U.S. Plastic Corp., Lima, OH) until liquid began to drain off the leaves ( 2.5 mL/plant). The same concentrations were also administered to seedlings as a soil drench using aliquots of 25 mL/container (Table 1). Glycerol treatments were given to carrot plants twice, first at the initiation of the experiment and a second time 4 weeks later (Table 1). Experiments were ended 8 weeks past application of the first glycerol treatment. Two-week-old corn seedlings were sprayed with 25-mL aliquots of glycerol twice, first at the initiation of the experiment and 2 weeks later (Table 1). Experiments were ended at the end of 4 weeks from the time of the first foliar application. Spearmint plants were maintained on agar Murashige and Skoog medium containing 3% sucrose in vitro as shoot cuttings. Eight-weekold plantlets were removed from agar medium, rinsed with water to remove excess agar and sucrose, and then established in 41⁄2$ pots and kept under high humidity conditions for 2 weeks to become acclimated to greenhouse conditions. Soil-established plantlets were then sprayed once with 25-mL aliquots of glycerol (Table 1). Experiments were ended at the end of 4 weeks from the time of the first foliar application. Plants were watered four times per week. Experiments were repeated in the greenhouse at least twice using 20 replications per treatment between 2008 and 2010. Average daily temperature was 25 C and varied from 20 to 29 C. Illumination during experiments was provided by natural and artificial lighting as necessary with an average daily photosynthetic photon flux of 630 to 700 mmol m s. Statistical analysis. Ten seedlings for each treatment were analyzed for shoot and root fresh weights and dry weights, tap root diameters (carrot only), and shoot and root lengths. Best fit regression equations were calculated with Table Curve 2D software Version 5.0 (Systat Software, Inc., San Jose, CA) for each response variable as a function of glycerol concentration. Regression model analyses and 95% confidence limits on the mean predicted Table 1. Glycerol treatments and concentrations applied to plants. Plant Treatment Glycerol concn [mL L (mM)] Carrot Spray 0 (0), 1 (11), 3 (33), 5 (50), 10 (109), 25 (271), 50 (543) Carrot Drench 0 (0), 1 (11), 3 (33), 5 (50), 10 (109), 25 (271), 50 (543) Corn Spray 0 (0), 0.1 (1.1), 0.3 (3.3), 0.5 (5), 1 (11) Spearmint Spray 0 (0), 1 (11), 5 (50), 10 (109) Fig. 1. (A–D) Influence of aqueous glycerol spray concentrations applied twice over an 8-week growth period on carrot growth parameters. All correlation coefficients are significant. Table 2. Influence of aqueous glycerol spray concentrations on carrot taproot color parameters from internal carrot cut 1 cm below shoot. Glycerol concn (mL L) Color parameters