- Email: [email protected]
Morphological and anatomical modifications in winter barley culm after late plant growth regulator treatment
European Journal of Agronomy 11 (1999) 45–51
Morphological and anatomical modifications in winter barley culm after late plant growth regulator treatment Patricia Sanvicente a, Sviatoslav Lazarevitch b, Andre´ Blouet a, *, Armand Guckert a a Laboratoire Agronomie et Environnement, ENSAIA-INRA, 2 avenue de la foreˆt de Haye, F-54505 Vandoeuvre le`s Nancy, France b Chaire de Ge´ne´tique et Botanique de l’Acade´mie Agronomique de Bie´lorussie, 213410 Gorki, Byelorussia
Accepted 22 January 1999
Abstract One of the major factors limiting yield production in barley (Hordeum vulgare L.) has been its poor straw strength. Consequently, a greater stiffness of the upper part of the culm was desirable. So, a late application of plant growth regulator could be interesting for weak-strawed barley cultivars. The objective of these experiments was to investigate the effect of a late application of a combination of chlormequat chloride (2-chloroethyl-trimethyl-ammonium chloride), ethephon (2-chloroethyl phosphonic acid ) and imazaquin (2-[4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl ] nicotinic acid ) on the morphological and anatomical characteristics of barley main stems. In a field trial and a greenhouse experiment, a foliar treatment was applied to a two-row winter barley (Labe´a) at growth stage 7 (Feekes, 1941). Measurements of plant height, internode lengths, dry weights and microscopic examination of cross-sections of the top three internodes were made at anthesis. The plant growth regulator (PGR) decreased significantly plant height by reducing the length of the upper internodes. The PGR did not modify the total weight of the upper internodes, but induced an increase of the dry weight per unit length of the main stem. The shortening of the internodes combined with the higher density of the tissues contributes to the stiffness of the stem. This was confirmed by anatomical studies which showed the modifications of the supporting tissues. The application of the plant growth regulator did not affect grain yield. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Anatomy; Barley; Chlormequat chloride; Ethephon; Imazaquin; Stem shortening
1. Introduction In many countries lodging is one of the major factors causing yield loss in cereal production. Barley (Hordeum vulgare L.) is more susceptible to lodging than other cereals because of its characteristic weak stems. It has long been recognised that, in barley, the length of the upper portion of the plant and the weight of the ear are associated * Corresponding author. Fax: 33-3-83-59-57-99. E-mail address: [email protected] (A. Blouet)
closely with lodging (Pinthus, 1973). Although breeders have developed short or semi-dwarf cultivars, the problem of lodging has still not been eliminated. For this reason, the plant growth regulators (PGRs) are of interest in preventing yield losses due to lodging. When applied early (during tillering), PGRs reduce lodging in barley by shortening the culm base (Jung, 1964; Kust, 1985). Some information concerning the morphological and anatomical modifications of basal internodes of PGR-treated plants is available (Jung and Sturm, 1966; Zaher et al., 1973). However, there
1161-0301/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S1 1 6 1 -0 3 0 1 ( 9 9 ) 0 0 01 7 - 9
46
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
is currently little information on these aspects when PGRs are applied at late growth stages (stem elongation). The objective of this study was to determine whether significant differences in culm morphology and anatomy developed in response to a late application of a combination of chlormequat chloride, ethephon and imazaquin.
2. Materials and methods 2.1. Field experiment A field experiment with a two-row malting barley (cv. Labe´a) was conducted in 1996 near Nancy (France) on a clay loam soil. The plots were 8 m×2 m and laid out in a randomised complete block design with three replicates. The previous crop was oilseed rape (Brassica napus). Nitrogen was applied as ammonium nitrate at a rate of 160 kg ha−1 in three split applications: 40 kg ha−1 at growth stage 3 (GS ), 50 kg ha−1 at GS 5 and 70 kg ha−1 at GS 7. Foliar treatment consisted of a combination of chlormequat chloride applied at 600 g ha−1 a.i., ethephon at 300 g ha−1 a.i. and imazaquin at 2 g ha−1 a.i., when two nodes were visible (GS 7). The growth regulators were applied with a hand-held sprayer in 3 l of water per plot at a pressure of 300 kPa. 2.2. Greenhouse experiment Seeds of winter barley (cv. Labe´a) were vernalized at 4°C on blotting paper in dark, moist conditions. After four weeks, seedlings were planted out in PVC pots (8 cm diameter, 40 cm long) containing a mixture of agricultural soil, sand and perlite (50/30/20, v/v/v) and then grown in a greenhouse in a randomised design. Plants were watered three times a week before tillering and every day thereafter. Each pot received a nutrient solution providing 40 mg of nitrogen at GS 5, and 20 mg at GS 7. PGR application was performed at GS 7 with a three nozzle spraying system on plants previously selected with the same morphology (number of tillers, number of main stems leaves) as the controls. The application rate of the PGRs was the same as that used in the field
trial. The treatment was applied with a spray volume equivalent to 350 l ha−1 at a pressure of 300 kPa. 2.3. Plant measurements At anthesis, 14 main stems from each treatment in the greenhouse experiment and 20 stems per block in the field trial were randomly selected. The following measurements were taken from each sample: (i) total height (cm), measured from plant base (soil level ) to ear tip excluding awns; (ii) internodes (IN ) length (cm), considering the most apical one as the first internode; (iii) dry weights (mg) of individual internodes, only for the greenhouse experiment. For anatomical study, six stems per treatment in the field trial and four stems per treatment in the greenhouse experiment were sampled at anthesis. Main stem internodes were separated and the three upper ones were used for analysis. The samples were fixed in ethanol 96% and acetic acid (3/1, v/v). Two days later, the samples were transferred to 70% ethanol and stored at 4°C until analysis. Cross-sections, approximately 50 mm thick, were cut by hand in the middle of each internode for microscopic investigation. The sections were stained in a combination of carmine alum and iodine green (Gurr, 1956). This double staining brought out the lignified elements in green and the cellulose in pink. After 30–40 min., stained sections were mounted on glass slides in glycerol. The following measurements were made on each crosssection with a microscope linked to an image processing system: (i) culm diameter (mm); (ii) culm wall thickness (mm) from the epidermis to the innermost edge of parenchyma cells; (iii) number of vascular bundles in an internode; (iv) mean diameter of each vascular bundle; (v) diameter of metaxylem, protoxylem and phloem of each vascular bundle. 2.4. Statistical analysis Data were analysed using an analysis of variance procedure () in Systat©. Means were compared by Tukey’s test.
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
47
3. Results
3.3. Dry weight per unit length
3.1. Plant height
At anthesis, the dry weight of each internode of treated plants grown in greenhouse conditions was not altered in comparison with the control, with the exception of the upper one which had a higher biomass. The result was an increase of the dry weight per unit length of the internodes of treated plants ( Table 1).
Plant height decreased significantly with the application of PGRs (Fig. 1). The reduction of main stem height was 7% in field conditions and 16% in the greenhouse experiment.
3.2. Internode length
3.4. Anatomy of the main stem
The PGRs decreased plant height by reducing the length of the internodes ( Fig. 1). In field conditions, the length reduction of each internode was about 5–7%, but this effect was not significant. In the greenhouse, the length of the upper three internodes of treated plants decreased significantly (22–29%) in comparison with the control plants. The fourth internode showed the same response to the PGRs but it was not significant statistically. There was no PGR effect on the length of the fifth internode of the main stems of plants grown in the greenhouse.
3.4.1. Culm diameter and wall thickness Culm diameter varied among experiments in all of the studied internodes ( Table 2). This parameter decreased from the lower to the upper internodes. In field conditions, the culm diameter of the three studied internodes was unchanged after PGR treatment. By contrast, greenhouse results showed a significant decrease for internodes 2 and 3 diameter of the PGR-treated plants. As for culm diameter results, inconstancy of significant treatment differences according to experimental device was noted for the wall thickness of the culm ( Table 2). In both experiments, the first internode of untreated plants had a thicker culm than the PGR-treated plants. Conversely, the wall thickness of internodes 2 and 3 in treated plants was greater than in the control. The differences, however, were only significant under field conditions.
Fig. 1. Plant height and internode lengths.
3.4.2. Diameter of small vascular bundles, metaxylem, protoxylem and phloem in the hypoderm In the hypoderm, the mean diameter of vascular bundles in treated plants was smaller than in untreated plants ( Table 3). However, in the greenhouse experiment the difference was not significant for internode 3. The first internode was more affected by the PGRs than the second one in the greenhouse. In field conditions, the third internode was altered more than the others. The diameter of the metaxylem was significantly greater in the control plants in all cases except for internode 2 (field and greenhouse) and for internode 3 (greenhouse). The same trend was observed for the diameter
48
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
Table 1 Dry weight per unit length (mg cm−1) of main stem internodes in greenhouse
Control PGR
Internode 1
Internode 2
Internode 3
Internode 4
Main stem
3.6 3.5 NSa
7.7 8.6 NS
11.5 14.3 11b
16.4 21.7 111
7.2 8.5 1
a NS: not significant. b 1, 11, 111 Significant at the 0.1, 0.05 and 0.01 probability levels.
Table 2 Culm diameter (mm) and culm wall thickness (mm) Internode 1
Culm diameter
Wall thickness
Internode 2
Internode 3
Treatment
Field
Greenhouse
Field
Greenhouse
control PGR NSa control PGR 1
2.60 2.53 NS 254.3 245.8 1
2.28 2.09 NS 261.6 239.7 1
3.37 3.65 11b 311.0 352.3 NS
3.76 3.38 NS 306.8 318.0 1
Field
Greenhouse
3.67 3.48 1 422.1 444.1 NS
4.03 3.63 355.8 401.7
a NS: not significant. b 1, 11 Significant at the 0.1 and 0.05 probability levels.
Table 3 Diameter (mm) of the small vascular bundles, metaxylem, protoxylem and phloem in the hypoderm Internode 1 Treatment Field
Greenhouse
bundles metaxylem protoxylem phloem bundles metaxylem protoxylem phloem
Internode 2
Control
PGR
65.8 13.6 8.3 29.8 59.8 12.9 6.5 32.8
59.9 12.2 8.2 27.8 48.7 11 4.9 30.1
11a 111 NS NS 1 ° 1 NS
Internode 3
Control
PGR
57.4 12.2 8.9 24.8 42.2 12.5 7.9 27.2
54 11.5 8.1 23.2 38.0 11.4 5.9 26.2
11 NSb 11 NS 1 NS 1 NS
Control
PGR
56.6 13.6 9.5 27.1 38.3 12.3 5.5 24.5
50.3 11.8 8.0 24.9 37.9 13.7 6.4 26.3
11 111 11 NS NS NS NS NS
a °, 1, 11, 111 Significant at the 0.1, 0.05, 0.01 and 0.001 probability levels. b NS: not significant.
of the protoxylem, except for internode 1 (field) and for internode 3 (greenhouse). In contrast, there was no significant effect of the PGRs on the diameter of the phloem in all internodes, irrespective of the treatment.
3.4.3. Diameter of large vascular bundles, metaxylem, protoxylem and phloem in the soft parenchyma Cross-sections in the three internodes showed that the PGRs reduced significantly the soft paren-
49
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51 Table 4 Diameter (mm) of the large vascular bundles, metaxylem, protoxylem and phloem in the soft parenchyma Internode 1 Treatment Field
Greenhouse
bundles metaxylem protoxylem phloem bundles metaxylem protoxylem phloem
Internode 2
Control
PGR
110.3 22.8 15.0 47.5 116.6 20.6 13.0 53.3
97.2 20.0 13.4 45.1 102.9 17.5 9.8 44.1
111a 11 1 1 11 1 11 11
Internode 3
Control
PGR
112.8 22.6 15.8 39 119.2 24.4 13.9 52.4
103.9 20.4 14.6 36.3 107.3 22.1 11.3 46.7
11 11 ° NS ° ° 1 °
Control
PGR
111.9 25.6 17.8 41.9 123.8 27.6 15.7 48.8
99.9 25.0 16.0 41.6 118.3 25.1 14.1 44.3
111 NSb 1 NS NS ° 1 NS
a °, 1, 11, 111 Significant at the 0.1, 0.05, 0.01 and 0.001 probability levels. b NS: not significant.
chyma vascular bundles diameter except for internode 3 in the greenhouse (Table 4). In general, this reduction was combined with a decrease of metaxylem and protoxylem diameter. The same trend was observed for phloem diameter, but was significant for the first internode (both experiments) and for internode 2 in the greenhouse. The PGRs preferably affected the parenchyma structure of the peduncle more than all the other internodes in both experiments.
modified. Indeed, most of this main stem shortening was explained (77% in the field and 67% in the greenhouse) by the reduction in length of the upper two internodes. These findings are in agreement with other studies, which have shown that the height reduction was obtained in peduncle length after ethephon application at late booting or early heading stage (Dahnous et al., 1982) or at flag leaf stage (Bridger et al., 1995). The application of chlormequat chloride during internode elongation reduced the length of the upper two or three internodes (Lowe and Carter, 1972).
4. Discussion 4.2. Culm stiffness These experiments showed the effects of a late application of the PGR on the morphology and some anatomical characteristics of a two-row winter barley at anthesis. 4.1. Plant height and internode lengths Many authors have reported that a late application of PGRs causes a decrease in final plant height (Bulman and Smith, 1993; Caldwell et al., 1988; Steen and Wu¨nsche, 1990). In our experiments, the PGRs decreased significantly plant height by reducing the length of the upper internodes. When the treatment was applied the lower culm internodes have yet completed their elongation and were lignified. So, they could not be altered by the PGR treatment. Consequently, the growth of the elongating internodes alone was
Plant growth regulator did not modify the dry weight of the internodes. As the length was reduced the dry weight per unit length increased. The culms of treated plants had a higher dry weight per unit length than the control plants. PGR-treated plants condensed the same amount of biomass into shorter stems. Our results are in agreement with those reported for barley cultivars differing in lodging resistance (Tandon et al., 1973; White, 1991): the strongstrawed barley cultivars have a higher dry weight per unit length than the weak-strawed cultivars. In the same way, Leitch and Hayes (1989) noted an increase of dry weight per unit length for oats treated with chlormequat chloride. Effects of mepiquat chloride and ethephon application at GS 7 on winter barley stem charac-
50
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
Table 5 Culm density (mg mm−3) of main stem internode in greenhouse
Control PGR
Internode 1
Internode 2
Internode 3
0.222 0.271
0.237 0.294
0.277 0.344
teristics ( White, 1995) are in contrast to our results: treated plants had a lower dry weight per unit length of stem than untreated ones. In the literature, dry weight per unit length was regarded as a surrogate for ‘tissue density’ which was associated with stiffness (Pinthus, 1973; White, 1995). However, the expression of dry weight per unit length of stem takes into consideration both height and weight of the stems. As the stem has a hollow structure, it is not easy to appreciate the difference between untreated and treated plants. To estimate the internode strength we used the parameter: ‘culm density’ (specific weight= mg mm−3). The result was an increase (+22–24%) of the density of the studied internodes ( Table 5). This result could suggest that tissue density might be part of the mechanism of stem strength influenced by the studied PGR. 4.3. Anatomy of the main stem The results of this study have shown interesting differences in a number of anatomical characteristics of the culm between PGR-treated plants and untreated plants. The PGR treatment reduced culm diameter in greenhouse conditions, only for internodes 2 and 3. In contrast, the PGRs increased wall thickness of internodes 2 and 3. Consequently, tissue area was more important in treated plants than in control plants, especially for the middle part of the culm (internodes 2 and 3). This observation explains, to a certain extent, the increase in dry weight per unit length of the internodes that was noted in the greenhouse experiment. So the PGRs increased culm stiffness of the main stem. Therefore, a combination of chlormequat chloride, ethephon and imazaquin could be recommended for cultivars considered most sensitive to breakdown of ears. Several studies on barley cultivars showed the importance of short, thick-walled
culms for lodging resistance (Dunn and Briggs, 1989; Jezowski, 1981; Tandon et al., 1973). Thus, we can draw a parallel between the anatomical structure of lodging-resistant barley genotypes and the PGR-treated plants. However, there was no clear relationship between thick-walled culms and break-resistance of plants treated with PGRs. On the one hand, Stanca et al. (1979) pointed out that no consistent relationship was found between lodging susceptibility and anatomical character. On the other hand, a study with chlormequat chloride, applied at the beginning of stem elongation, has shown that PGRs might help in increasing lodging resistance by thickening the culm wall, especially by increasing the supporting tissue (Zaher et al., 1973). In the present study, the PGRs did not affect the number of vascular bundles in the internodes (data not shown). These results suggest that there was no relationship between the number of vascular bundles and the density of straw. Many authors have reported that the number of vascular bundles alone was not a good indicator of lodging resistance (Dunn and Briggs, 1989; Stanca et al., 1979). According to Stanca et al. (1979) it is possible that the number of vascular bundles becomes significant for lodging resistance in combination with short internodes. The application of the PGRs decreased the mean diameter of each vessel, and its effect was more pronounced in the greenhouse experiment than under field conditions. In greenhouse conditions the reduction of protoxylem diameter was higher than the reduction of metaxylem diameter in internodes 1 and 2. This result can be explained by environmental conditions. In the field, conditions were dry and cold (mean temperature was 10°C ) during the week after treatment. On the contrary, greenhouse conditions were quite humid and warmer than field conditions. As the metaxylem appears after the protoxylem we can suppose that there was a compensation effect in treated plants (homeostasis phenomenon). Indeed, the metaxylem diameter/protoxylem diameter ratio increases in treated plants. Moreover, the diameter of phloem decreased in treated plants, especially in the large vascular bundles. This result could suggest a modification in efficiency of the biochemical processes of loading, translocation in phloem
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
elements or unloading, by impairing the movement of metabolites in sieve elements all along the path of the phloem (Ma and Smith, 1992). However, the PGR treatments did not affect grain yield in both experiments (data not shown).
Acknowledgements The authors would like to thank Cyanamid Agro France for making available the plant growth regulators. Professor A. Souvre´ (Laboratoire de Biotechnologie et Ame´lioration des plantes, ENSAT, Toulouse, France) is gratefully acknowledged for allowing us to use the image processing system.
References Bridger, G.M., Klinck, H.R., Smith, D.L., 1995. Timing and rate of ethephon application to two-row and six-row spring barley. Agron. J. 87, 1198–1206. Bulman, P., Smith, D.L., 1993. Yield and grain protein response of spring barley to ethephon and triadimefon. Crop Sci. 33, 798–803. Caldwell, C.D., Mellish, D.R., Norries, J., 1988. A comparison of ethephon alone and in combination with CCC or DPC. Can. J. Plant Sci. 68, 941–946. Dahnous, K., Vigue, G.T., Law, A.G., Konzak, C.F., Miller, D.G., 1982. Height and yield response of selected wheat, barley and triticale cultivars to ethephon. Agron. J. 74, 580–582. Dunn, G.J., Briggs, K.G., 1989. Variation in culm anatomy among barley cultivars differing in lodging resistance. Can. J. Bot. 67, 1838–1843. Feekes, W., 1941. Detarwe en haar milien. Versl. Techn. Tarwe Commun. 12, 523–888. Gurr, E., 1956. in: A Practical Manual of Medical and Bio-
51
logical Staining Techniques. Leonard Hill, London, pp. 413–420. Jezowski, S., 1981. Variation, correlation and heritability of characters determining lodging of spring barley (Hordeum vulgare L.). II. Analysis of relationship between lodging grade and some morphological characters of spring barley varieties. Genet. Pol. 22, 45–61. Jung, J., 1964. Experimental results on the action of CYCOCEL in wheat. CCC Research Symposium, Geneva, pp. 43–71. Jung, J., Sturm, H., 1966. CCC, its combination with certain herbicides and special considerations of dosage rate as related to disease incidence in cereals. Proc. Weed Conference, pp. 245–256. Kust. C.A., 1985. Cycocel plant growth regulant: uses in small grains. Cyanamid document. Leitch, M.H., Hayes, J.D., 1989. Effects of chlormequat chloride application on stem characteristics, yield and panicle conformation of winter oats. J. Agric. Sci. 113, 17–26. Lowe, L.B., Carter, O.G., 1972. The influence of CCC (2-chloroethyltrimethylammonium chloride) and temperature on dry matter and assimilate accumulation in wheat. Aust. J. Agric. Res. 23, 573–583. Ma, B.L., Smith, D.L., 1992. Growth regulator effects on aboveground dry matter partitioning during grain fill of spring barley. Crop Sci. 32, 741–746. Pinthus, M.J., 1973. Lodging in wheat, barley and oats: the phenomenon, its causes, and preventive measures. Adv. Agron. 25, 210–256. Stanca, A.M., Jenkins, G., Hanson, P.R., 1979. Varietal responses in spring barley to natural and artificial lodging and to a growth regulator. J. Agric. Sci. 93, 449–456. Steen, E., Wu¨nsche, U., 1990. Root growth dynamics of barley and wheat in field trials after CCC application. Swedish J. Agric. Res. 20, 57–62. Tandon, J.P., Jain, K.B.L., Singh, S.B., 1973. Relationship between lodging resistance and some morphological characters in barley. Indian J. Genet. 33, 347–354. White, E.M., 1991. Response of winter barley cultivars to nitrogen and a plant growth regulator in relation to lodging. J. Agric. Sci. 93, 449–456. White, E.M., 1995. Effect of management and development on stem characteristics related to lodging in winter barley. Eur. J. Agron. 4, 327–334. Zaher, A., Foad, M.K., El-Shaarawi, A., El-Fouly, M., 1973. Morphological and anatomical modifications in wheat after treatment with chlormequat chloride (CCC ). Egypt. J. Bot. 16, 125–136.
Morphological and anatomical modifications in winter barley culm after late plant growth regulator treatment Patricia Sanvicente a, Sviatoslav Lazarevitch b, Andre´ Blouet a, *, Armand Guckert a a Laboratoire Agronomie et Environnement, ENSAIA-INRA, 2 avenue de la foreˆt de Haye, F-54505 Vandoeuvre le`s Nancy, France b Chaire de Ge´ne´tique et Botanique de l’Acade´mie Agronomique de Bie´lorussie, 213410 Gorki, Byelorussia
Accepted 22 January 1999
Abstract One of the major factors limiting yield production in barley (Hordeum vulgare L.) has been its poor straw strength. Consequently, a greater stiffness of the upper part of the culm was desirable. So, a late application of plant growth regulator could be interesting for weak-strawed barley cultivars. The objective of these experiments was to investigate the effect of a late application of a combination of chlormequat chloride (2-chloroethyl-trimethyl-ammonium chloride), ethephon (2-chloroethyl phosphonic acid ) and imazaquin (2-[4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl ] nicotinic acid ) on the morphological and anatomical characteristics of barley main stems. In a field trial and a greenhouse experiment, a foliar treatment was applied to a two-row winter barley (Labe´a) at growth stage 7 (Feekes, 1941). Measurements of plant height, internode lengths, dry weights and microscopic examination of cross-sections of the top three internodes were made at anthesis. The plant growth regulator (PGR) decreased significantly plant height by reducing the length of the upper internodes. The PGR did not modify the total weight of the upper internodes, but induced an increase of the dry weight per unit length of the main stem. The shortening of the internodes combined with the higher density of the tissues contributes to the stiffness of the stem. This was confirmed by anatomical studies which showed the modifications of the supporting tissues. The application of the plant growth regulator did not affect grain yield. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Anatomy; Barley; Chlormequat chloride; Ethephon; Imazaquin; Stem shortening
1. Introduction In many countries lodging is one of the major factors causing yield loss in cereal production. Barley (Hordeum vulgare L.) is more susceptible to lodging than other cereals because of its characteristic weak stems. It has long been recognised that, in barley, the length of the upper portion of the plant and the weight of the ear are associated * Corresponding author. Fax: 33-3-83-59-57-99. E-mail address: [email protected] (A. Blouet)
closely with lodging (Pinthus, 1973). Although breeders have developed short or semi-dwarf cultivars, the problem of lodging has still not been eliminated. For this reason, the plant growth regulators (PGRs) are of interest in preventing yield losses due to lodging. When applied early (during tillering), PGRs reduce lodging in barley by shortening the culm base (Jung, 1964; Kust, 1985). Some information concerning the morphological and anatomical modifications of basal internodes of PGR-treated plants is available (Jung and Sturm, 1966; Zaher et al., 1973). However, there
1161-0301/99/$ – see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S1 1 6 1 -0 3 0 1 ( 9 9 ) 0 0 01 7 - 9
46
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
is currently little information on these aspects when PGRs are applied at late growth stages (stem elongation). The objective of this study was to determine whether significant differences in culm morphology and anatomy developed in response to a late application of a combination of chlormequat chloride, ethephon and imazaquin.
2. Materials and methods 2.1. Field experiment A field experiment with a two-row malting barley (cv. Labe´a) was conducted in 1996 near Nancy (France) on a clay loam soil. The plots were 8 m×2 m and laid out in a randomised complete block design with three replicates. The previous crop was oilseed rape (Brassica napus). Nitrogen was applied as ammonium nitrate at a rate of 160 kg ha−1 in three split applications: 40 kg ha−1 at growth stage 3 (GS ), 50 kg ha−1 at GS 5 and 70 kg ha−1 at GS 7. Foliar treatment consisted of a combination of chlormequat chloride applied at 600 g ha−1 a.i., ethephon at 300 g ha−1 a.i. and imazaquin at 2 g ha−1 a.i., when two nodes were visible (GS 7). The growth regulators were applied with a hand-held sprayer in 3 l of water per plot at a pressure of 300 kPa. 2.2. Greenhouse experiment Seeds of winter barley (cv. Labe´a) were vernalized at 4°C on blotting paper in dark, moist conditions. After four weeks, seedlings were planted out in PVC pots (8 cm diameter, 40 cm long) containing a mixture of agricultural soil, sand and perlite (50/30/20, v/v/v) and then grown in a greenhouse in a randomised design. Plants were watered three times a week before tillering and every day thereafter. Each pot received a nutrient solution providing 40 mg of nitrogen at GS 5, and 20 mg at GS 7. PGR application was performed at GS 7 with a three nozzle spraying system on plants previously selected with the same morphology (number of tillers, number of main stems leaves) as the controls. The application rate of the PGRs was the same as that used in the field
trial. The treatment was applied with a spray volume equivalent to 350 l ha−1 at a pressure of 300 kPa. 2.3. Plant measurements At anthesis, 14 main stems from each treatment in the greenhouse experiment and 20 stems per block in the field trial were randomly selected. The following measurements were taken from each sample: (i) total height (cm), measured from plant base (soil level ) to ear tip excluding awns; (ii) internodes (IN ) length (cm), considering the most apical one as the first internode; (iii) dry weights (mg) of individual internodes, only for the greenhouse experiment. For anatomical study, six stems per treatment in the field trial and four stems per treatment in the greenhouse experiment were sampled at anthesis. Main stem internodes were separated and the three upper ones were used for analysis. The samples were fixed in ethanol 96% and acetic acid (3/1, v/v). Two days later, the samples were transferred to 70% ethanol and stored at 4°C until analysis. Cross-sections, approximately 50 mm thick, were cut by hand in the middle of each internode for microscopic investigation. The sections were stained in a combination of carmine alum and iodine green (Gurr, 1956). This double staining brought out the lignified elements in green and the cellulose in pink. After 30–40 min., stained sections were mounted on glass slides in glycerol. The following measurements were made on each crosssection with a microscope linked to an image processing system: (i) culm diameter (mm); (ii) culm wall thickness (mm) from the epidermis to the innermost edge of parenchyma cells; (iii) number of vascular bundles in an internode; (iv) mean diameter of each vascular bundle; (v) diameter of metaxylem, protoxylem and phloem of each vascular bundle. 2.4. Statistical analysis Data were analysed using an analysis of variance procedure () in Systat©. Means were compared by Tukey’s test.
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
47
3. Results
3.3. Dry weight per unit length
3.1. Plant height
At anthesis, the dry weight of each internode of treated plants grown in greenhouse conditions was not altered in comparison with the control, with the exception of the upper one which had a higher biomass. The result was an increase of the dry weight per unit length of the internodes of treated plants ( Table 1).
Plant height decreased significantly with the application of PGRs (Fig. 1). The reduction of main stem height was 7% in field conditions and 16% in the greenhouse experiment.
3.2. Internode length
3.4. Anatomy of the main stem
The PGRs decreased plant height by reducing the length of the internodes ( Fig. 1). In field conditions, the length reduction of each internode was about 5–7%, but this effect was not significant. In the greenhouse, the length of the upper three internodes of treated plants decreased significantly (22–29%) in comparison with the control plants. The fourth internode showed the same response to the PGRs but it was not significant statistically. There was no PGR effect on the length of the fifth internode of the main stems of plants grown in the greenhouse.
3.4.1. Culm diameter and wall thickness Culm diameter varied among experiments in all of the studied internodes ( Table 2). This parameter decreased from the lower to the upper internodes. In field conditions, the culm diameter of the three studied internodes was unchanged after PGR treatment. By contrast, greenhouse results showed a significant decrease for internodes 2 and 3 diameter of the PGR-treated plants. As for culm diameter results, inconstancy of significant treatment differences according to experimental device was noted for the wall thickness of the culm ( Table 2). In both experiments, the first internode of untreated plants had a thicker culm than the PGR-treated plants. Conversely, the wall thickness of internodes 2 and 3 in treated plants was greater than in the control. The differences, however, were only significant under field conditions.
Fig. 1. Plant height and internode lengths.
3.4.2. Diameter of small vascular bundles, metaxylem, protoxylem and phloem in the hypoderm In the hypoderm, the mean diameter of vascular bundles in treated plants was smaller than in untreated plants ( Table 3). However, in the greenhouse experiment the difference was not significant for internode 3. The first internode was more affected by the PGRs than the second one in the greenhouse. In field conditions, the third internode was altered more than the others. The diameter of the metaxylem was significantly greater in the control plants in all cases except for internode 2 (field and greenhouse) and for internode 3 (greenhouse). The same trend was observed for the diameter
48
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
Table 1 Dry weight per unit length (mg cm−1) of main stem internodes in greenhouse
Control PGR
Internode 1
Internode 2
Internode 3
Internode 4
Main stem
3.6 3.5 NSa
7.7 8.6 NS
11.5 14.3 11b
16.4 21.7 111
7.2 8.5 1
a NS: not significant. b 1, 11, 111 Significant at the 0.1, 0.05 and 0.01 probability levels.
Table 2 Culm diameter (mm) and culm wall thickness (mm) Internode 1
Culm diameter
Wall thickness
Internode 2
Internode 3
Treatment
Field
Greenhouse
Field
Greenhouse
control PGR NSa control PGR 1
2.60 2.53 NS 254.3 245.8 1
2.28 2.09 NS 261.6 239.7 1
3.37 3.65 11b 311.0 352.3 NS
3.76 3.38 NS 306.8 318.0 1
Field
Greenhouse
3.67 3.48 1 422.1 444.1 NS
4.03 3.63 355.8 401.7
a NS: not significant. b 1, 11 Significant at the 0.1 and 0.05 probability levels.
Table 3 Diameter (mm) of the small vascular bundles, metaxylem, protoxylem and phloem in the hypoderm Internode 1 Treatment Field
Greenhouse
bundles metaxylem protoxylem phloem bundles metaxylem protoxylem phloem
Internode 2
Control
PGR
65.8 13.6 8.3 29.8 59.8 12.9 6.5 32.8
59.9 12.2 8.2 27.8 48.7 11 4.9 30.1
11a 111 NS NS 1 ° 1 NS
Internode 3
Control
PGR
57.4 12.2 8.9 24.8 42.2 12.5 7.9 27.2
54 11.5 8.1 23.2 38.0 11.4 5.9 26.2
11 NSb 11 NS 1 NS 1 NS
Control
PGR
56.6 13.6 9.5 27.1 38.3 12.3 5.5 24.5
50.3 11.8 8.0 24.9 37.9 13.7 6.4 26.3
11 111 11 NS NS NS NS NS
a °, 1, 11, 111 Significant at the 0.1, 0.05, 0.01 and 0.001 probability levels. b NS: not significant.
of the protoxylem, except for internode 1 (field) and for internode 3 (greenhouse). In contrast, there was no significant effect of the PGRs on the diameter of the phloem in all internodes, irrespective of the treatment.
3.4.3. Diameter of large vascular bundles, metaxylem, protoxylem and phloem in the soft parenchyma Cross-sections in the three internodes showed that the PGRs reduced significantly the soft paren-
49
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51 Table 4 Diameter (mm) of the large vascular bundles, metaxylem, protoxylem and phloem in the soft parenchyma Internode 1 Treatment Field
Greenhouse
bundles metaxylem protoxylem phloem bundles metaxylem protoxylem phloem
Internode 2
Control
PGR
110.3 22.8 15.0 47.5 116.6 20.6 13.0 53.3
97.2 20.0 13.4 45.1 102.9 17.5 9.8 44.1
111a 11 1 1 11 1 11 11
Internode 3
Control
PGR
112.8 22.6 15.8 39 119.2 24.4 13.9 52.4
103.9 20.4 14.6 36.3 107.3 22.1 11.3 46.7
11 11 ° NS ° ° 1 °
Control
PGR
111.9 25.6 17.8 41.9 123.8 27.6 15.7 48.8
99.9 25.0 16.0 41.6 118.3 25.1 14.1 44.3
111 NSb 1 NS NS ° 1 NS
a °, 1, 11, 111 Significant at the 0.1, 0.05, 0.01 and 0.001 probability levels. b NS: not significant.
chyma vascular bundles diameter except for internode 3 in the greenhouse (Table 4). In general, this reduction was combined with a decrease of metaxylem and protoxylem diameter. The same trend was observed for phloem diameter, but was significant for the first internode (both experiments) and for internode 2 in the greenhouse. The PGRs preferably affected the parenchyma structure of the peduncle more than all the other internodes in both experiments.
modified. Indeed, most of this main stem shortening was explained (77% in the field and 67% in the greenhouse) by the reduction in length of the upper two internodes. These findings are in agreement with other studies, which have shown that the height reduction was obtained in peduncle length after ethephon application at late booting or early heading stage (Dahnous et al., 1982) or at flag leaf stage (Bridger et al., 1995). The application of chlormequat chloride during internode elongation reduced the length of the upper two or three internodes (Lowe and Carter, 1972).
4. Discussion 4.2. Culm stiffness These experiments showed the effects of a late application of the PGR on the morphology and some anatomical characteristics of a two-row winter barley at anthesis. 4.1. Plant height and internode lengths Many authors have reported that a late application of PGRs causes a decrease in final plant height (Bulman and Smith, 1993; Caldwell et al., 1988; Steen and Wu¨nsche, 1990). In our experiments, the PGRs decreased significantly plant height by reducing the length of the upper internodes. When the treatment was applied the lower culm internodes have yet completed their elongation and were lignified. So, they could not be altered by the PGR treatment. Consequently, the growth of the elongating internodes alone was
Plant growth regulator did not modify the dry weight of the internodes. As the length was reduced the dry weight per unit length increased. The culms of treated plants had a higher dry weight per unit length than the control plants. PGR-treated plants condensed the same amount of biomass into shorter stems. Our results are in agreement with those reported for barley cultivars differing in lodging resistance (Tandon et al., 1973; White, 1991): the strongstrawed barley cultivars have a higher dry weight per unit length than the weak-strawed cultivars. In the same way, Leitch and Hayes (1989) noted an increase of dry weight per unit length for oats treated with chlormequat chloride. Effects of mepiquat chloride and ethephon application at GS 7 on winter barley stem charac-
50
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
Table 5 Culm density (mg mm−3) of main stem internode in greenhouse
Control PGR
Internode 1
Internode 2
Internode 3
0.222 0.271
0.237 0.294
0.277 0.344
teristics ( White, 1995) are in contrast to our results: treated plants had a lower dry weight per unit length of stem than untreated ones. In the literature, dry weight per unit length was regarded as a surrogate for ‘tissue density’ which was associated with stiffness (Pinthus, 1973; White, 1995). However, the expression of dry weight per unit length of stem takes into consideration both height and weight of the stems. As the stem has a hollow structure, it is not easy to appreciate the difference between untreated and treated plants. To estimate the internode strength we used the parameter: ‘culm density’ (specific weight= mg mm−3). The result was an increase (+22–24%) of the density of the studied internodes ( Table 5). This result could suggest that tissue density might be part of the mechanism of stem strength influenced by the studied PGR. 4.3. Anatomy of the main stem The results of this study have shown interesting differences in a number of anatomical characteristics of the culm between PGR-treated plants and untreated plants. The PGR treatment reduced culm diameter in greenhouse conditions, only for internodes 2 and 3. In contrast, the PGRs increased wall thickness of internodes 2 and 3. Consequently, tissue area was more important in treated plants than in control plants, especially for the middle part of the culm (internodes 2 and 3). This observation explains, to a certain extent, the increase in dry weight per unit length of the internodes that was noted in the greenhouse experiment. So the PGRs increased culm stiffness of the main stem. Therefore, a combination of chlormequat chloride, ethephon and imazaquin could be recommended for cultivars considered most sensitive to breakdown of ears. Several studies on barley cultivars showed the importance of short, thick-walled
culms for lodging resistance (Dunn and Briggs, 1989; Jezowski, 1981; Tandon et al., 1973). Thus, we can draw a parallel between the anatomical structure of lodging-resistant barley genotypes and the PGR-treated plants. However, there was no clear relationship between thick-walled culms and break-resistance of plants treated with PGRs. On the one hand, Stanca et al. (1979) pointed out that no consistent relationship was found between lodging susceptibility and anatomical character. On the other hand, a study with chlormequat chloride, applied at the beginning of stem elongation, has shown that PGRs might help in increasing lodging resistance by thickening the culm wall, especially by increasing the supporting tissue (Zaher et al., 1973). In the present study, the PGRs did not affect the number of vascular bundles in the internodes (data not shown). These results suggest that there was no relationship between the number of vascular bundles and the density of straw. Many authors have reported that the number of vascular bundles alone was not a good indicator of lodging resistance (Dunn and Briggs, 1989; Stanca et al., 1979). According to Stanca et al. (1979) it is possible that the number of vascular bundles becomes significant for lodging resistance in combination with short internodes. The application of the PGRs decreased the mean diameter of each vessel, and its effect was more pronounced in the greenhouse experiment than under field conditions. In greenhouse conditions the reduction of protoxylem diameter was higher than the reduction of metaxylem diameter in internodes 1 and 2. This result can be explained by environmental conditions. In the field, conditions were dry and cold (mean temperature was 10°C ) during the week after treatment. On the contrary, greenhouse conditions were quite humid and warmer than field conditions. As the metaxylem appears after the protoxylem we can suppose that there was a compensation effect in treated plants (homeostasis phenomenon). Indeed, the metaxylem diameter/protoxylem diameter ratio increases in treated plants. Moreover, the diameter of phloem decreased in treated plants, especially in the large vascular bundles. This result could suggest a modification in efficiency of the biochemical processes of loading, translocation in phloem
P. Sanvicente et al. / European Journal of Agronomy 11 (1999) 45–51
elements or unloading, by impairing the movement of metabolites in sieve elements all along the path of the phloem (Ma and Smith, 1992). However, the PGR treatments did not affect grain yield in both experiments (data not shown).
Acknowledgements The authors would like to thank Cyanamid Agro France for making available the plant growth regulators. Professor A. Souvre´ (Laboratoire de Biotechnologie et Ame´lioration des plantes, ENSAT, Toulouse, France) is gratefully acknowledged for allowing us to use the image processing system.
References Bridger, G.M., Klinck, H.R., Smith, D.L., 1995. Timing and rate of ethephon application to two-row and six-row spring barley. Agron. J. 87, 1198–1206. Bulman, P., Smith, D.L., 1993. Yield and grain protein response of spring barley to ethephon and triadimefon. Crop Sci. 33, 798–803. Caldwell, C.D., Mellish, D.R., Norries, J., 1988. A comparison of ethephon alone and in combination with CCC or DPC. Can. J. Plant Sci. 68, 941–946. Dahnous, K., Vigue, G.T., Law, A.G., Konzak, C.F., Miller, D.G., 1982. Height and yield response of selected wheat, barley and triticale cultivars to ethephon. Agron. J. 74, 580–582. Dunn, G.J., Briggs, K.G., 1989. Variation in culm anatomy among barley cultivars differing in lodging resistance. Can. J. Bot. 67, 1838–1843. Feekes, W., 1941. Detarwe en haar milien. Versl. Techn. Tarwe Commun. 12, 523–888. Gurr, E., 1956. in: A Practical Manual of Medical and Bio-
51
logical Staining Techniques. Leonard Hill, London, pp. 413–420. Jezowski, S., 1981. Variation, correlation and heritability of characters determining lodging of spring barley (Hordeum vulgare L.). II. Analysis of relationship between lodging grade and some morphological characters of spring barley varieties. Genet. Pol. 22, 45–61. Jung, J., 1964. Experimental results on the action of CYCOCEL in wheat. CCC Research Symposium, Geneva, pp. 43–71. Jung, J., Sturm, H., 1966. CCC, its combination with certain herbicides and special considerations of dosage rate as related to disease incidence in cereals. Proc. Weed Conference, pp. 245–256. Kust. C.A., 1985. Cycocel plant growth regulant: uses in small grains. Cyanamid document. Leitch, M.H., Hayes, J.D., 1989. Effects of chlormequat chloride application on stem characteristics, yield and panicle conformation of winter oats. J. Agric. Sci. 113, 17–26. Lowe, L.B., Carter, O.G., 1972. The influence of CCC (2-chloroethyltrimethylammonium chloride) and temperature on dry matter and assimilate accumulation in wheat. Aust. J. Agric. Res. 23, 573–583. Ma, B.L., Smith, D.L., 1992. Growth regulator effects on aboveground dry matter partitioning during grain fill of spring barley. Crop Sci. 32, 741–746. Pinthus, M.J., 1973. Lodging in wheat, barley and oats: the phenomenon, its causes, and preventive measures. Adv. Agron. 25, 210–256. Stanca, A.M., Jenkins, G., Hanson, P.R., 1979. Varietal responses in spring barley to natural and artificial lodging and to a growth regulator. J. Agric. Sci. 93, 449–456. Steen, E., Wu¨nsche, U., 1990. Root growth dynamics of barley and wheat in field trials after CCC application. Swedish J. Agric. Res. 20, 57–62. Tandon, J.P., Jain, K.B.L., Singh, S.B., 1973. Relationship between lodging resistance and some morphological characters in barley. Indian J. Genet. 33, 347–354. White, E.M., 1991. Response of winter barley cultivars to nitrogen and a plant growth regulator in relation to lodging. J. Agric. Sci. 93, 449–456. White, E.M., 1995. Effect of management and development on stem characteristics related to lodging in winter barley. Eur. J. Agron. 4, 327–334. Zaher, A., Foad, M.K., El-Shaarawi, A., El-Fouly, M., 1973. Morphological and anatomical modifications in wheat after treatment with chlormequat chloride (CCC ). Egypt. J. Bot. 16, 125–136.