Crop Science 42:1483-1487 (2002)
© 2002 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Coleoptile Length of Dwarf Wheat Isolines
Gibberellic Acid, Temperature, and Cultivar Interactions
M. J. Pereiraa,
P. L. Pfahler*,b,
R. D. Barnettc,
A. R. Blountc,
D. S. Woffordb and
R. C. Littelld
a Biology Dep., Univ. of North Carolina at Pembroke, Pembroke, NC 28372
b Agronomy Dep., Univ. of Florida, Gainesville, FL 32611
c North Florida Research and Education Center, Univ. of Florida, Quincy, FL 32351
d Statistics Dep., Univ. of Florida, Gainesville, FL 32611
* Corresponding author (plp{at}gnv.ifas.ufl.edu)
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ABSTRACT
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The dwarfing alleles in wheat (Triticum aestivum L.) are associated with shorter coleoptile length that can produce unacceptable and erratic stands. The combined effect of temperature (2, 10, 18°C) and five gibberellic acid (GA) concentrations [0, 5, 50, 100, 500 mg L-1 of potassium gibberellin A3 (C19H21O6K)] in the germination medium on the coleoptile length of four homozygous isolines [normal or T-0 (Rht-B1a/Rht-D1a), semidwarf-1 or SD-1 (Rht-B1b/Rht-D1a), semidwarf-2 or SD-2 (Rht-B1a/Rht-D1b), dwarf or D-12 (Rht-B1b/Rht-D1b)] in ‘Marfed’ (spring) and ‘Burt’ (winter) was studied. With each decrease in temperature, the coleoptile length in each isoline and cultivar increased, with the increase greater in those isolines and cultivars having the shortest length at 18°C and containing at least one dwarfing allele (Rht-B1b and/or Rht-D1b). At 2°C, higher GA concentrations increased coleoptile length over 0 GA mg L-1 in all isolines, with the greatest increase at the 500 mg L-1 concentration. The three midconcentrations (5, 50, 100 mg L-1) resulted in an intermediate but almost equal increase. Increasing temperatures decreased the response to GA so that at 18°C, only T-0 and SD-1 responded to GA applications. The reported "GA insensitivity" was found to be highly temperature dependent, with the Rht-B1b allele having a wider temperature response range than Rht-D1b. The results suggested that differences in genetic background, possibly related to the winter–spring growth habit, could influence the effect of the dwarfing alleles.
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INTRODUCTION
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GENETIC DWARFING IN WHEAT influences many seed, seedling, and plant characteristics (Gale and Youssefian, 1985). Dwarf genotypes carrying the dwarfing Rht-B1b and/or Rht-D1b alleles (McIntosh, 1988; Borner et al., 1996) have, in addition to shorter culms, significantly less lodging, higher harvest index, greater yield per plant and grain number per ear, higher tiller number per square meter, and longer spikes (Allan, 1989; McClung et al., 1986). However, lower test and seed weight, lower seed protein content, and considerably shorter coleoptile length in the seedlings are also associated with dwarfing. The shorter coleoptile length in some genetic backgrounds or cultivars, causes emergence problems and erratic stands (Allan, 1980), especially when low soil moisture conditions are present at planting and deeper planting depths are used in an attempt to overcome the soil moisture problem.
Gibberellic acid (GA) is one of the most important plant growth regulators or hormones. GA promotes 
-amylase production for endosperm starch hydrolysis during seed germination and facilitates cell elongation in different organs and tissues throughout plant growth and development (Graebe, 1987; Karssen et al., 1989). The first leaf of dwarf isolines of wheat was reported to be insensitive to exogenous GA application (Gale and Marshall, 1973, 1975; Keyes et al., 1989) and this insensitivity was suggested as a method to identify dwarf genotypes in segregating populations (Gale and Gregory, 1977). The effect of exogenous GA application on the coleoptile length of dwarf isolines has not been studied extensively but, at germination temperatures above 15°C, coleoptile length was reported to be GA insensitive (Allan et al., 1959, 1961; Pinthus and Abraham, 1996). Apparently, the GA response in dwarfs is influenced by temperature at least in some tissues.
Additional information on the effects of various genetic, chemical, and environmental factors on coleoptile length in dwarfs would be very advantageous in developing procedures to minimize the dwarfing effect on emergence and stand establishment and, in so doing, improve the commercial usefulness of this trait. The purpose of this study was to determine the combined effects of GA in the germination medium and temperature on the coleoptile length of four dwarf isolines in two cultivars or genetic backgrounds.
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MATERIALS AND METHODS
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Seeds of four homozygous isolines (I) developed from two cultivars (C), Marfed (spring growth habit) and Burt (winter growth habit), were used. Each I in each C was derived after seven backcrosses to the recurrent parent and should be over 99% isogenic at those loci not linked to the dwarfing loci (Allan and Pritchett, 1975, 1980, 1982). The designation [phenotype (genotype)] for the four I within each of the two C, are as follows: (i) T-0 [normal height or tall (Rht-B1a Rht-B1a/Rht-D1a Rht-D1a)], (ii) SD-1 [semidwarf-1 (Rht-B1b Rht-B1b/Rht-D1a Rht-D1a)], (iii) SD-2 [semidwarf-2 (Rht-B1a Rht-B1a/Rht-D1b Rht-D1b)], and (iv) D-12 [dwarf (Rht-B1b Rht-B1b/Rht-D1b Rht-D1b)]. The above genotypic designations follows the gene symbolization recently recommended for wheat (McIntosh, 1988; Borner et al., 1996).
Seeds of each of the four I in each C were soaked for 4 d at 2°C in 10 mL of five gibberellic acid (GA) concentrations [0, 5, 50, 100, 500 mg L-1 of potassium gibberellin A3 (C19H21O6K)], plus 0.2 mL L-1 Vitavax 200 (Gustafson, McKinney, TX) fungicide. After soaking, 10 seeds were enfolded in germination paper inserted in a 16.5- by 17.8-cm plastic growth pouch (Mega International of Minneapolis, Minneapolis, MN) containing 25 mL of the same GA solution used in soaking. Each seed was oriented in the germination paper with its embryo down. Positioning of the embryos and the vertical orientation of the pouches in the containers promoted straight coleoptile growth for accuracy in measurement.
Four pouches (each containing 10 seeds) of each of the 40 combinations (GA–I–C) at each of three temperatures (18, 10, and 2°C) were randomly placed in vertical partitions of closed transparent rigid plastic containers with 100% relative humidity maintained during the germination process. The containers were placed randomly inside a dark constant temperature chamber at one of three temperatures (T). The coleoptile length of five seedlings from each of the four pouches was measured in millimeters.
In this study, the coleoptile length was measured after reaching the maximum length as indicated by the emergence of the primary leaf from the coleoptile tip. Since T influenced the seedling growth rate, the coleoptile length at each T was measured on different days after germination initiation. At 18, 10, and 2°C, the coleoptile measurements were made at 9, 26, and 68 d after germination initiation, respectively.
The experimental design was a split-plot with temperature as the main plot and the remaining variables as subplots. Two replications (R) were used. Twenty plants which were measured in each R, were nested within each of the 120 experimental units (5 GA x 2C x 4I x 3T).
A complete factorial analysis of variance including all main effects (GA, T, I, C, R), all possible interactions, and plants nested within all main effects was performed.
Individual analyses of variance including GA and the appropriate variables were performed to test for linearity of response to GA. Also, certain interactions of interest involving the C and I response to GA were examined from these analyses. An analysis of variance based on 400 measurements was performed on each I and T combination and included GA, C, R, and the nested plants within each of these variables. From these analyses of variance, the effect of C on the GA response in each I was indicated by the significance of the GA x C interaction. An analysis of variance based 800 measurements was performed on each C and T combination and included GA, I, R, and the nested plants within each of these variables. From these analyses of variance, the effect of I on the GA response in each C was indicated by the significance of the GA x I interaction.
The minimum differences for significance presented in the tables were obtained with the Duncan's range values for the maximum number of means to be compared (Harter, 1960).
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RESULTS
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The F values for the main effects (GA concentration, temperature, isoline, cultivar) and all their possible interactions were significant at the 1% level except for the main effect, cultivar (Table 1)
. The interactions of greatest interest, I x GA x T and C x GA x T, were significant at the 1% level.
The means showing the effect of GA concentration and temperature on the coleoptile lengths in each isoline and cultivar, are presented in Table 2
. The longest lengths and the greatest response to GA in all isolines and cultivars occurred at 2°C. The shortest lengths in all isolines and cultivars were present at 18°C with the least response to increasing GA concentrations. The response at 10°C was somewhat intermediate, but closer to the response at 18°C. In general, a linear response to increasing GA concentrations was found but was less pronounced at 18°C with both isoline and cultivar effects apparent. In Burt, the response to GA was not significant for D-12 at any temperature and was not significant for SD-2 at 10 or 18°C.
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Table 2. Coleoptile length means of each isoline (I) in each cultivar (C) at each gibberellic acid (GA) concentration and temperature (T).
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The effect of GA concentration and temperature on the coleoptile length of each isoline over both cultivars is shown in Table 3 . From 18 and 2°C with 0 GA concentration, T-0, SD-1, SD-2, and D-12 increased 26 (102 mm at 2°C–76 mm at 18°C), 47, 41, and 46 mm, respectively. The increase was more pronounced in isolines which had shorter lengths at 18°C and contained at least one allele (Rht-B1b and/or Rht-D1b) for dwarfing. Temperature also had a pronounced effect on the response of the isolines to increasing GA concentrations. At 2°C, T-0 responded the most and D-12 the least to GA with the response linear in all isolines. At all temperatures, the single gene dwarfs, SD-1 and SD-2, had an intermediate response and were quite similar to each other. However, SD-1 responded to GA over a wider temperature range than SD-2. The intermediate GA concentrations (5, 50, 100 mg L-1) produced a substantial, but almost equal increase in length, with the 500 mg L-1 concentration producing an additional increase over these intermediate concentrations. In general, the response of all isolines was linear at all temperatures except for D-12 at 18°C. The significance of the GA x C interaction indicated that the response of T-0 depended on the cultivar only at 2 and 10°C.
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Table 3. Coleoptile length means of each isoline over both cultivars (C) at each gibberellic acid (GA) concentration and temperature (T).
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The means showing the effect of GA concentration and temperature on the cultivars over all isolines, are presented in Table 4
. In general, increasing temperatures reduced coleoptile length regardless of cultivar or GA concentration. However, the response of the cultivars to increasing GA concentrations was extremely temperature dependent. At 0 GA concentration at all temperatures, the differences between Marfed and Burt were relatively small. At 2 and 10°C, increasing GA concentrations resulted in a much greater increase in Marfed than Burt. At 18°C, increasing GA concentrations had only a slight effect in increasing the length of Marfed compared to Burt. As shown in the analysis of the effect of GA concentration and temperature on isolines, the intermediate GA concentrations (5, 50, 100 mg L-1) produced a substantial but almost equal increase in length with the 500 mg L-1 concentration producing an additional increase over the intermediate concentrations. At 2 and 10°C, Marfed had the greater response to increasing GA concentrations. At 18°C, the only difference between Marfed and Burt was found at the 500 mg L-1 concentration. The response of both cultivars was linear at all temperatures. The significance of the GA x I interaction indicated that the response of the isolines depended on the cultivar.
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Table 4. Coleoptile length means of each cultivar over four isolines (I) at each gibberellic acid (GA) concentration and temperature (T).
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DISCUSSION
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The results of this study indicated that the effect of GA concentration was enhanced in each isoline and cultivar by lowering temperature. At 0 GA concentration, the increase in each isoline and cultivar at 2°C compared with 10 or 18°C was more pronounced in those isolines which had shorter coleoptile lengths at 18°C and contained at least one dominant allele for dwarfing. Apparently, the presence of Rht-B1b and/or Rht-D1b resulted in greater sensitivity to lower germination temperature.
When GA concentration is considered at 2°C, the greatest increase over 0 GA concentration in T-0, SD-1, and SD-2 was found at 500 mg L-1 with the intermediate (5, 50, 100 mg L-1) GA concentrations having an intermediate but almost identical increase in length over the 0 concentration. With increasing temperature, the same pattern was present, but the increase was reduced so that at 18°C, only T-0 and SD-1 responded to GA application. However, SD-1 responded to GA over a wider temperature range than SD-2 suggesting that the Rht-B1b allele would probably be more desirable in improvement programs than Rht-D1b. Many characters (height, coleoptile length, internode number and length, dry weight, flowering time) in semidwarfs and dwarfs were reported to be "GA insensitive" compared with the normal tall genotypes when the plants were grown at 18°C (Allan, 1989; Gale and Marshall, 1973). A study examining the T x GA x I interaction comparing 11 and 25°C indicated that the coleoptile length at the higher temperature was reduced in T-0, SD-1, SD-2, and D-12 but SD-1 was slightly sensitive to GA application at 11°C, but not at 25°C (Pinthus and Abraham, 1996). Apparently, the lower germination temperatures magnifies the effect of increasing GA concentrations on the isolines containing Rht-B1b or Rht-D1b with Rht-B1b being more temperature sensitive.
The morphological and/or physiological mechanisms associated with the effect of temperature on the expression of the dwarfing alleles at the Rht-B1 and/or Rht-D1 loci on coleoptile length are not completely understood, but GA metabolism and/or function is almost certainly involved (Gale and Marshall, 1975). An early study (Allan et al., 1962) concluded that coleoptile length was related to both cell length and number, but only length was inhibited by higher temperatures. GA-stimulated elongation studies with various species including wheat dwarfs have indicated that increased coleoptile length was correlated with increases in cell wall extensibility, indicating that temperature effects were associated with the GA action on the cell wall (Keyes et al., 1990; Taiz, 1984). Using deembryonated seeds of dwarf wheat, low temperatures also were found to increase endogenous levels of GA released from ‘Kite’ (Rht-B1a/Rht-D1b) aleurone tissue (Singh and Paleg, 1984). Apparently, lower temperatures increase not only GA sensitivity, but also the amount of GA3 released and the GA3 receptor sites available.
In this study, the effect of temperature and GA application on the coleoptile length of the isolines was greatly influenced by cultivar, with the spring cultivar Marfed showing a greater response than Burt, the winter cultivar. A number of studies (Allan et al., 1959, 1961; Gale and Youssefian, 1985; Radley, 1970) have reported cultivar differences in the expression of dwarfism. In general, the spring types were generally more responsive than the winter but no distinct relationship with growth habit was confirmed.
The reduction of the stand problem associated with the shorter coleoptile length in semidwarfs which limits their commercial acceptance can be approached in a number of ways. The most reliable and inexpensive method would be incorporating Rht-B1b (rather than Rht-D1b) into genetic backgrounds which produce longer coleoptile length. From the results presented here, the spring cultivar was more responsive, suggesting that spring growth habit may influence the expression of the dwarfing character. The negative relationship between coleoptile length and temperature indicates that temperature at planting and seed germination is an important factor and should be considered if practical. The increase in coleoptile length indicates that GA applications and possibly other growth-stimulating substances to seeds before planting may be effective. Additional research especially in the area of the effect of temperature and a broader array of growth-stimulating substances which may increase coleoptile length during germination and seedling growth would be desirable.
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ACKNOWLEDGMENTS
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Appreciation is expressed to Dr. R. E. Allan of the Crops Research Division, United States Department of Agriculture, Washington State University, Pullman, WA, for generously supplying the seeds of the four isolines in each cultivar used in this study.
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NOTES
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Part of a dissertation submitted by the senior author for the partial fulfillment of the requirements of the Ph.D. degree at the Univ. of Florida. Florida Agric. Exp. Stn. J. Series No R-07680.
Received for publication April 30, 2001.
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ABSTRACT
INTRODUCTION
