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© 2003 Plant Management Network.
Accepted for publication 25 June 2003. Published 4 August 2003.

Does Foliar-applied Glycine Betaine Affect Endogenous Betaine Levels and Yield In Cotton?

Cassandra Meek, Graduate Student, Crop, Soil, and Environmental Sciences, University of Arkansas, PTSC 115, Fayetteville 72701; Derrick Oosterhuis, Distinguished Professor, Crop, Soil, and Environmental Sciences, University of Arkansas, 1366 West Altheimer Drive, Fayetteville 72704; and John Gorham, Research Fellow, Centre for Arid Zone Studies, University of Wales, Bangor, Gwynedd LL57 2UW, U.K.

Corresponding author: Derrick Oosterhuis. oosterhu@uark.edu

Meek, C., Oosterhuis, D., and Gorham, J. 2003. Does foliar-applied glycine betaine affect endogenous betaine levels and yield in cotton? Online. Crop Management doi:10.1094/CM-2003-0804-02-RS.

Abstract

Glycine betaine is a quaternary ammonium compound that is naturally accumulated in many higher plants, including cotton. Field studies suggested that glycine betaine might enhance cotton (Gossypium hirsutum L.) yields under water deficient conditions, even though cotton is known to accumulate glycine betaine naturally. To test this, three field experiments in 1998 and 1999 were conducted in Arkansas to determine the effects of glycine betaine on cotton in response to water-deficit stress. In general, no significant differences were found in yield components, physiological processes, or endogenous levels in glycine betaine treated cotton. Significant differences were found in endogenous levels of glycine betaine in response to water-deficit stress. While it is possible that cotton’s drought resistance is enhanced by endogenous glycine betaine accumulation, foliar applications of the compound do not appear to improve yield.

Introduction

Water is generally considered the most limiting factor in crop production. As the human population continues to increase so does the need for agricultural products. Depletion of natural resources such as land and fresh water inevitably accompanies the population explosion; therefore it is crucial that strategies be developed to maintain yields in water-depleted situations. Most of the world cotton crop is grown under rain-fed conditions and faces some degree of water stress during development.

Recently, the naturally occurring quaternary ammonium compound, glycine betaine, has received attention as a compatible solute that may aid in drought tolerance by allowing maintenance of turgor pressure (1,2,3,16,17,18,19,23). Glycine betaine also protects physiological processes such as photosynthesis and protein synthesis from the results of water deficit and other stresses (7,9,14,24). Endogenous glycine betaine concentrations vary in plants, with some taxa naturally accumulating the compound, while others do not. Cotton has been shown to accumulate high amounts of glycine betaine compared with other taxa (4,10).

Glycine betaine has been exogenously applied to a variety of crops in an effort to improve stress tolerance and yield. However, some controversy exists as to whether or not exogenously supplied or genetically engineered synthesis of glycine betaine is capable of providing drought or stress resistance (8,13,15,25). These crops include soybean (Glycine max Merr.) (3), tomato (Lycopersicon esculenium L.) (18), maize (Zea mays L.), sorghum (Sorghum bicolor Moench) (1), and cotton (11). Field studies in Pakistan revealed increases in cotton yield with foliar applications of glycine betaine at 2.68 and 5.36 lb/acre (3 and 6 kg/ha) near the time of flowering. Results varied, however, and appeared to depend on numerous factors such as type of crop, timing and rate of application, and environmental conditions. In drought-stressed, glasshouse-grown tobacco (Nicotiana tabacum L.), significant yield increases were observed with exogenous glycine betaine applications (2). In field-grown tomato subjected to salt or high temperature stress, fruit yields increased up to 39% following exogenous glycine betaine application at mid-flowering. In a previous study by Makela et al. (16) osmotic shock to plant foliage occurred when rates of application were too high, and this was more pronounced in plants that were not natural glycine betaine accumulators. A field study in Australia conducted by Agboma et al. (1) found that exogenous applications of glycine betaine significantly improved crop yield of sorghum and maize, but had detrimental effects on wheat (Triticum aestivum L.), a natural accumulator of glycine betaine.

In soybean, exogenous foliar applications of glycine betaine decreased transpiration, increased leaf conductance, improved photosynthetic activity, and increased leaf area, but an overall increase in yield between treated and control plants was not observed (3). According to Gorham et al. (11), foliar sprays of glycine betaine improved the growth of field-grown cotton in Pakistan when 2.68 lb/acre (3 kg/ha) were applied at time of squaring. Increased boll numbers in Australia and California were observed in trials conducted by Finnsugar Bioproducts (personal communication with Kari Jokinen, 1998).

The objectives of our studies were (1) to evaluate the potential use of glycine betaine to enhance yield and drought tolerance in cotton, (2) to characterize long- and short-term responses between tolerant and sensitive cultivars treated with glycine betaine under both well-watered and water-deficient conditions, and (3) to determine endogenous levels of glycine betaine in response to water-deficit stress in cultivars considered water-deficit stress sensitive and tolerant.

Field Studies

Field studies were conducted at two locations in Arkansas during 1998 and 1999 with three cotton cultivars under well-watered and water-deficient conditions. A range of application regimes was tested, including three rates and several different application times of glycine betaine (Finnsugar Bioproducts, Helsinki, Finland), both with and without one of two adjuvants, DYNE-AMIC (Helena Chemical Co., Memphis, TN) and Monsoon (Finnsugar Bioproducts, Helsinki, Finland).

Experiment 1: Glycine betaine rate and adjuvant study. The 1998 field study was conducted at Arkansas Agricultural Research and Extension Center, Fayetteville, AR. The cotton cultivar, Sure-Grow 125, was planted into a Captina silt loam soil (fine-loamy, mixed, thermic; Rhodic Paleudalfs), on May 15, 1998. The plot size was two five-meter rows with 38-inch (0.97-m) row spacing. Irrigation was applied equally to all treatments on a weekly basis, when needed according to Arkansas Cooperative Extension recommendations (5). At two weeks after first flower (FF), irrigation was withheld from all treatments to impose mild water stress. The experiment consisted of eight treatments in a randomized complete block design with four replications. Treatments are listed below:

1. Control - sprayed with water only.¹
2. Glycine betaine applied at 2.68 lb/acre.¹
3. Glycine betaine applied at 5.36 lb/acre.¹
4. Glycine betaine applied at 2.68 lb/acre + DYNE-AMIC® (0.5% v/v).¹
5. Glycine betaine applied at 5.36 lb/acre + DYNE-AMIC® (0.5% v/v).¹
6. Glycine betaine applied at 2.68 lb/acre + MONSOON® (0.2% v/v).¹
7. Glycine betaine applied at 5.36 lb/acre + MONSOON®(0.2% v/v).¹
8. Glycine betaine applied at 2.68 lb/acre + MONSOON®(0.2% v/v).²

¹ Foliar applications made at one and two weeks after FF.

² Foliar applications made at one, two, three, and four weeks after FF.

Foliar applications were made using a CO₂ backpack sprayer calibrated to deliver a volume of 10 gallons of solution per acre in the early morning with a three-nozzle boom arrangement directed at the terminal and into the middle of the canopy. Photosynthesis, intercellular CO₂ concentration, stomatal resistance, and transpiration rates were measured at saturating photon fluxes (PAR > 1,200 µmol/m²/s) and within two hours of solar noon using a LICOR-6200 portable photosynthesis system (LICOR Inc., Lincoln, NE). These measurements were taken during the boll development period at four, five, and six weeks after FF in treatments one, two, six, and eight. Mid-season boll numbers were recorded for all treatments four weeks after FF. Yield determination was accomplished by hand-harvesting two 3.28-ft rows from each plot.

Experiment 2: Glycine betaine rate and timing study. In 1999, two field studies (rate and timing, and water-stress and cultivar studies) were planted on May 10 into a Dundee silt loam soil (fine-silty,mixed, thermic; Aeric Ochraqualfs) at the Delta Branch Research Station in Clarkedale in northeast Arkansas (5). Pest control and fertilizer management were according to Arkansas Cooperative Extension recommendations. Plots consisted of four rows, 50 ft (15.24 m) in length, spaced 38 inches (0.97 m) apart. Foliar sprays were applied with a CO₂ backpack sprayer calibrated to deliver 10 gallons of solution per acre. In both studies, the nonionic adjuvant Monsoon was used at 0.2% (v/v).

In the rate and timing study, six replications of cultivar Suregrow 125 were arranged in a randomized complete block design, with no irrigation. Foliar applications began at FF and continued for four weeks. Treatments consisted of three rates of glycine betaine, 1.79, 3.57, and 5.36 lb/acre, (2, 4, and 6 kg/ha) applied weekly, biweekly, or monthly, and an untreated control, giving ten treatments. Yields were determined from the middle two rows of each plot harvested with a mechanical picker. Boll weights and fiber quality were calculated based on four replications of 30 handpicked mid-canopy bolls per plot.

Experiment 3. Water-deficit stress and cultivar study. The 1999 water-stress and cultivar study consisted of six replications arranged in a split-split plot design. The three factors were (1) water: irrigated vs. dryland (2) foliar treatment: 3.57 lb/acre (4 kg/ha) glycine betaine + Monsoon (0.2% v/v) vs. Monsoon (0.2% v/v) only, and (3) cultivar: Siokra L-23 [considered drought tolerant (20)] vs. Stoneville 506 [considered drought sensitive]. Treatments were applied at FF and FF + 2 weeks. Physiological measurements were taken at two, four, and six weeks after FF. A LICOR 6200 (LI-COR Environmental, Lincoln, NE, USA) portable photosynthesis system was used to evaluate photosynthetic parameters, and osmotic adjustment was monitored by thermocouple psychrometry (21). Yield was determined from the inside two rows of each plot with a mechanical picker. Boll numbers, boll weights, lint percent, and fiber quality were assessed by hand-harvesting two 3.28-ft of row. Three replications of leaf samples for glycine betaine quantification were collected at PHS and FF from node 4 and 8 and FF + 4 weeks from node 4, 8, and 12. Nodes were counted beginning at the apical meristem. Samples were collected between 10:00 a.m. and 1:00 p.m. and placed on ice for transport to the laboratory in Fayetteville. Leaves were stored at -80°C until further processing could occur. Glycine betaine content was analyzed by HPLC (high-performance liquid chromatography) with a strong cation exchange column (Sarasep CAR-Na, Sarasep Inc., Santa Clara, CA) and refractive index (RI) detector (Shodex, Showa Denko Europe GmbH, Germany). The method is based on the procedure by Rajakyla and Paloposki (22) and analyses were performed at the Centre for Arid Zones Studies, University of Wales, Bangor, U.K.

Statistical analyses. Data were analyzed using, where appropriate, the GLM-univariate, one-way ANOVA (with post hoc tests), independent t-test functions of the statistical package SPSS (SPSS Science, Chicago, IL). Numbers of replicates are shown in the Tables.

Yield and Components of Yield

In the 1998 rate and adjuvant study (Experiment 1), none of the treatment lint yields were significantly different from the control lint yield according to analysis of variance and pairwise, independent samples t-tests, although control plants had the lowest lint yields numerically (Table 1). Boll weight was also not significantly affected by the treatments. In contrast, Gorham et al. (11) reported that 2.68 and 5.36 lb/acre (three and six kg/ha) of glycine betaine applied at the time of the first floral buds increased lint yields and boll numbers at two locations in Pakistan compared to the untreated control. In the 1998 study (Experiment 1), all treatments receiving applications of glycine betaine had greater boll numbers at mid-season (data not shown), with this difference being significant between treatment three (two applications of glycine betaine at six kg/ha) with 58 bolls per two 3.28-ft of row and the untreated control plants with 40 bolls per two 3.28-ft of row. Treatment eight (four applications of glycine betaine at 2.68 lb/acre + MONSOON) had the highest boll weights.

Table 1. Effects of foliar application of glycine betaine and an adjuvant on mean boll number, boll weight, and lint yield of irrigated field-grown cotton (cv. Suregrow 125) in 1998 at Fayetteville, AR (Experiment 1).

¹ Foliar applications made at one and two weeks after first flowering.

² Foliar applications made at one, two, three, and four weeks after first flowering.

No treatment had a significant effect when compared with the controls in independent samples t-tests. Values are means of 6 replicates.

Analysis of variance revealed that, in the 1999 rate and frequency study (Experiment 2), seed cotton and lint yields (Table 2) were not significantly affected (P > 0.05) by glycine betaine. Plants treated biweekly with 1.78 lb/acre glycine betaine had the numerically highest lint yields. All other glycine betaine treatments in Experiment 2 resulted in lower lint yields than the untreated control. This rate of application, given weekly or biweekly, gave significantly (P < 0.01 in t-tests) higher boll weights than the control. The rate of 5.36 lb/acre (six kg/ha) of glycine betaine also resulted in significantly (P < 0.05) higher boll weights than the controls at all frequencies of application, but the results for four kg/ha were not consistent.

Table 2. Effects of foliar application of glycine betaine on boll weight and lint yield of nonirrigated field-grown cotton (cv. Suregrow 125) in 1999 at Clarkedale, AR (Experiment 2).

Adjuvant (Monsoon at 0.2%) was added to the glycine betaine treatments, but not to control plants.

¹ Means of 4 replicates

² Means of 6 replicates

³ Significance of difference from control determined by t-test.

NS = non-significant, * P < 0.05, ** P < 0.01

In the 1999 water-stress and cultivar study (Experiment 3), no significant differences (P > 0.05) in yield components (Table 3) were observed between glycine betaine treated and control plants for any variety and watering regime combination.

Stoneville 506 had significantly (P < 0.001) higher lint yields than Siokra L-23 when averaged over water and foliar glycine betaine applications (Table 3). Because Siokra L-23 was bred for Australia, the shortened season in the U.S. Mississippi River Delta does not provide conditions for maximum yields. Exogenous glycine betaine did not affect yield in the either cultivar. The water stress treatment reduced lint yield in all treatments, but there were no significant effects of cultivar, water regime, or glycine betaine treatment on boll weight.

Table 3. Effects of glycine betaine, water stress, and cultivar on boll weight and lint yield of field-grown cotton in 1999 at Clarkedale, AR (Experiment 3).

¹ Means of 4 replicates.

² Means of 6 replicates.

Water regime and cultivar had significant effects (P < 0.001, ANOVA) on lint yield, but glycine betaine treatments did not (confirmed by t-tests). No significant water*cultivar*foliar interactions existed in either parameter. In regards to lint yields, cultivar, and water*cultivar interactions were significant. Water regime was the only factor to significantly (P < 0.01) affect boll weight.

Location of Fruit on the Plant

In the 1998 study (Experiment 1), selected treatments were mapped using the COTMAP plant mapping program (6). The majority of the glycine betaine-treated plants had more effective sympodial fruiting branches, i.e., more sympodia with bolls present at the time of mapping (data not shown). Most surveyed treatments also had significantly greater boll retention at the second position compared to untreated control plants. These data suggest that glycine betaine treatment might alleviate stress and therefore enhance subsequent boll retention, i.e., farther away from the first position on the fruiting branch.

Gas Exchange Parameters

In the 1998 rate and adjuvant experiment (Experiment 1, Table 4) and the 1999 water-stress and cultivar study (Experiment 3, data not shown), no significant differences were observed in stomatal resistance, photosynthesis, transpiration, or intercellular CO₂ (data not shown in Table 4) between glycine betaine treated and untreated plants. These results are in agreement with Gorham et al. (12), whose findings suggested that gas exchange parameters were generally not affected in glycine betaine-treated cotton.

Table 4. Gas exchange parameters recorded at Fayetteville, AR in 1998 (Experiment 1).

¹ Foliar applications made at one and two weeks after first flowering.

² Foliar applications made at one, two, three, and four weeks after first flowering.

Pn = net photosynthesis, E = transpiration, gs = stomatal conductance. Glycine betaine was applied at 3 kg/ha. Values are the means of three replicates ± standard errors.

Plant Water Status

In the 1999 water-stress and cultivar study (Experiment 3, Table 5), no significant differences were observed in leaf water potential, osmotic potential, and turgor pressure between glycine betaine treated and untreated plants. Water stress resulted in significantly lower (more negative) water and osmotic potentials in Stoneville 506, but not in Siokra L-23.

Table 5. Effects of water stress and cultivar on leaf water potential, osmotic potential, and turgor pressure of field-grown cotton four weeks after FF in 1999 at Clarkedale, AR (Experiment 3).

*No significant differences were observed between glycine betaine-treated and untreated plants.

Values are the means of four replicates.

Glycine Betaine Concentration

Glycine betaine concentrations were assessed in the 1999 water-deficit stress and cultivar study (Experiment 3) only. All data were first analyzed with general linear model (GLM). Time of sampling (P < 0.000), water regime (P < 0.002), cultivar (P < 0.016), and leaf position (P ± 0.014) significantly affected sap glycine betaine concentrations. There was also a significant interaction between time of sampling and water regime, but not between any other factors. Subsequent analyses (one-way ANOVA for time of sampling and leaf position; t-test for water regime and glycine betaine treatment) were performed separately for the two varieties (Table 6). The interaction between time of sampling and water regime was significant because at PHS there was no difference between the two water regimes, while at later harvests the leaf glycine betaine concentrations increased progressively in the non-irrigated regime. Sap glycine betaine concentrations increased with time of sampling (Table 6). At pinhead square the concentration in Stoneville 506 was significantly higher than that in Siokra L-23. When averaged over location (data not shown), Stoneville 506 had significantly (P = 0.0020) higher glycine betaine concentrations (34.5 mM) compared to Siokra L-23 (28 mM). Water stress had no significant effect on glycine betaine concentrations in Siokra L-23, but significantly increased the concentration in Stoneville 506 (P < 0.020). These findings are supported by results from Gorham et al. (12) who reported substantial increases in endogenous glycine betaine levels of field-grown cotton in Pakistan with increasing water-deficit. Glycine betaine concentrations were highest in the oldest leaf (at node 12), significantly so in Stoneville 506. This variety also had significantly higher concentrations in the youngest leaf (node 4) than in the middle leaf (at node 8). Exogenous glycine betaine application did not produce a significant increase in leaf sap glycine betaine concentrations.

Table 6. Leaf sap glycine betaine concentrations (mM) in two varieties of cotton in response to water stress and foliar spray with glycine betaine (3.57 lb/acre).

1 Probability of the factor not affecting leaf sap glycine betaine concentrations

Adjuvant (Monsoon 0.2% v/v) was applied to both control and glycine betaine treatments.

In summary, foliar applications of glycine betaine to field-grown cotton generally neither improved yields of field-grown cotton in Arkansas nor had significant effects on growth parameters. Endogenous glycine betaine concentrations were significantly higher in water-deficit stressed plants, but were unaffected by foliar applications of glycine betaine. It is possible that the high endogenous levels of glycine betaine in cotton are responsible for the lack of effects with foliar applications. Overall, foliar applications of glycine betaine to cotton did not enhance the growth or yield of cotton in Arkansas.

Acknowledgements

We would like to thank Finnsugar Bioproducts for financial support of the project. We also would like to thank Adele Steger, Dennis Coker, Scott Brown, Karen Gomez, and Ron McNew for their assistance throughout these studies.

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