Crop Science 42:71-75 (2002)
© 2002 Crop Science Society of America
CROP BREEDING, GENETICS & CYTOLOGY
Seed Coat Cracking in Soybean Isolines for Pubescence Color and Maturity
Daijun Yanga,
Takehiro Nakamurab,
Norihiro Ohtsuboa,
Koji Takahashia,
Jun Abec and
Ryoji Takahashi*,a
a R. Takahashi, National Institute of Crop Science, Kannondai, Tsukuba, Ibaraki, 305-8518 Japan
b Faculty of Agriculture, Tamagawa University, Machida, Tokyo, 194-8610 Japan
c Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589 Japan
* Corresponding author (masako{at}narc.affrc.go.jp)
 |
ABSTRACT
|
|---|
Seed coats of soybean crack under various stress conditions. Cracking of seed coats degrades the external appearance of soybean seeds and reduces their commercial value. Previous studies revealed that the T gene responsible for pubescence color, and the maturity genes, E1 and E5, had inhibitory effects on low-temperature induced seed coat cracking. The objective of this study was to evaluate the effects of the T gene and five maturity genes (E1 to E5) on the intensity of seed coat cracking induced by pod-removal treatments. Soybean cv. Harosoy (te1e2E3E4e5) and its near-isogenic lines for T and E1 to E5 loci were used in the experiment. Cracking was induced by removing the upper 50% of pods on the plant 40 d after anthesis. Frequency and degree of cracking were not different among the isolines in the control group. In contrast, there were significant differences among isolines subjected to the pod-removal treatment. Frequency and degree of cracking was low in Harosoy, Harosoy-E1, e3, and e4, and high in Harosoy-T and E2. The results suggest that genotypes at T and E2 loci were associated with severity of seed coat cracking induced by pod-removal. There was a positive correlation (r = 0.90**) between individual seed weight and frequency of cracking among isolines in the pod-removal treatment. Seed coat cracking was probably exacerbated in part by the genes that allow enlargement of individual seeds in response to pod-removal. The differences among isolines suggest that the mechanism of seed coat cracking induced by pod removal may differ from that induced by low temperature treatment.
Abbreviations: DAO, days after opening of individual flowers • NIL, near-isogenic line
|
INTRODUCTION
|
|---|
SEED COAT CRACKING adversely affects external appearance and reduces the commercial value of soybean. Seed coat cracking results from the separation of epidermal and hypodermal tissues that exposes the underlying parenchyma tissue (Wolf and Baker, 1972; Yaklich and Barla-Szabo, 1993). Liu (1949) designated two types of cracking that occur in some cultivars regardless of environmental conditions: Type I with irregular cracks and Type II with net-like cracks. Type I cracking is controlled by genes T and I (Stewart and Wentz, 1930). Soybean lines with double recessive genotypes (i and t, or i–k and t) produce severely cracked seeds, while no symptom is observed with the other allelic combinations (Nicholas et al., 1993). Type II cracking also seems to be controlled by two genes (Nagai, 1926; Liu, 1949) that are independent of T and I (Liu, 1949). Although the physiological mechanism of cracking is still unclear, quantitative differences in proline-rich cell wall proteins in seed coats were found within the subsets of near-isogenic lines (NILs) for both Type I and II cracking (Nicholas et al., 1993; Percy et al., 1999).
On the other hand, most soybean cultivars occasionally exhibit seed coat cracking to some extent when exposed to adverse environmental conditions such as alternate wetting and drying (Wolf et al., 1981), or chilling temperatures at flowering (Sunada and Ito, 1982; Hara et al., 1993; Takahashi, 1997). However, the causes of seed coat cracking occurring under field conditions have been difficult to identify in most cases.
Substantial genotypic differences in tolerance to seed coat cracking have been observed (Okabe, 1996). Three kinds of treatments to promote seed coat cracking have been used in soybean breeding in Japan: pod-removal (Maruyama and Mikoshiba, 1976), drying of imbibed seeds (Murata et al., 1991), and application of an ethylene generating reagent, ethychlozate (Figaron, Nissan Chemical Industry Inc., Japan) (Okabe, 1996). These treatments induce reproducible genotypic differences in tolerance to seed coat cracking that occur under field conditions. Removal of the upper 50 to 75% of pods on a plant at 35 to 48 d has been effective in enhancing frequency of cracked seeds (Sasaki and Nakamura, 1981), and the results have been reproducible across the years (Okabe, 1996). Although results of drying treatments of imbibed seeds also are reproducible, frequency of cracked seeds is generally high even in tolerant cultivars and humidity control during drying which is essential to accurately evaluate genotypic differences (Murata et al., 1991). In ethychlozate treatments, uniform application is difficult and a large number of plants should be treated to obtain an accurate evaluation of genotypic differences (Takahashi, unpublished results, 2000).
Cultivars with yellow hilum color are preferred in Japan to those with brown hilum for confectionery use because of their better external appearance and higher protein content. However, chilling temperatures (about 15°C) during flowering induce seed coat cracking as well as browning around the hilum region (Sunada and Ito, 1982). Seed coat deterioration occurs only in yellow hilum cultivars and is absent in cultivars with brown hilum (Sunada and Ito, 1982). Usually, Japanese cultivars with brown hilum have brown hilum (i-i) and brown pubescence (T), while yellow hilum cultivars generally have yellow hilum (I) and gray pubescence (t).
To clarify the genetic basis of genotypic differences in seed coat deterioration, Takahashi and Asanuma (1996) and Takahashi (1997) evaluated the roles of gene T (responsible for pubescence and hilum color) and gene I (responsible for distribution of seed coat color) using NILs for the two loci. Independent of the genotypes at the I locus, the dominant allele T completely suppressed the development of pigmentation around the hilum region and substantially reduced seed coat cracking. Dominant allele I also suppressed seed coat deterioration under the genotype of tt, although its inhibitory effect was not as obvious as gene T. Genes T and I are involved in flavonoid biosynthesis (Palmer and Kilen, 1987). On the basis of the similar inhibitory effects of these genes, Takahashi (1997) presumed that low temperature-induced cracking and Type I cracking were derived from an identical physiological mechanism(s).
In addition to genes T and I, there is genetic variation in the tolerance to pigmentation and cracking among cultivars with yellow hilum (I) and gray pubescence (t) (Takahashi and Abe, 1994). On the basis of genetic analysis, Takahashi and Abe (1994) found that one of the genes responsible for tolerance was closely associated with a dominant gene for late maturity, the recessive allele of which was involved in floral induction under the artificially induced long days by means of incandescent lamps. Eight loci have been reported to control time to flowering and maturity in soybean: E1, E2 (Bernard, 1971); E3 (Buzzell, 1971); E4 (Buzzell and Voldeng, 1980); E5 (McBlain and Bernard, 1987); E7 (Cober and Voldeng, 2001) and J for long juvenility (Ray et al., 1995), and an unnamed gene that was reported to be involved in insensitivity to incandescent long daylength (Abe et al., 1998). Takahashi and Abe (1999) treated NILs for maturity genes (E1 to E5) with chilling temperatures and found that some of the maturity genes exhibited inhibitory effects on low-temperature induced seed coat deterioration.
The present study was conducted to investigate the effects of T, and maturity genes, E1 to E5, on seed coat cracking induced by pod-removal treatments using NILs for the respective genes.
|
MATERIALS AND METHODS
|
|---|
‘Harosoy’ (te1e2E3E4e5) and its NILs for T and E1 to E5 loci, Harosoy-T (L66-707: Te1e2E3E4e5), Harosoy-E1 (L68-694: tE1e2E3E4e5), Harosoy-E2 (L64-4584: te1E2E3E4e5), Harosoy-e3 (L62-667: te1e2e3E4e5), Harosoy-e4 (OT94-41: te1e2E3e4e5), and Harosoy-E5 (L64-4830: te1e2E3E4E5) were used (Table 1). Seeds of L66-707, L68-694, L64-4585, L62-667, and L64-4830 were obtained from the USDA Soybean Germplasm Collection. The lines were produced by crossing Harosoy with lines having the respective genes and backcrossing the progenies to Harosoy up to BC6 (Bernard et al., 1991). Seeds of OT94-41 were obtained from Plant Res. Ctr., Agric. Agri-Food Canada, Ottawa, ON, Canada. OT94-41 was selected from a cross between Harosoy isolines, OT89-5 (te1e2e3e4e5) and L67-153 (te1e2E3E4e5) (Cober et al., 1996).
View this table:
[in this window]
[in a new window]
|
Table 1. Harosoy and near-isogenic lines used to study the relationship between maturity genes and seed coat cracking by soybean pod-removal treatments.
|
|
Experiments were conducted from June to October 2000 at the National Institute of Crop Science, Tsukuba, Japan (36°06'N, 140°05'E). Five seeds were planted in pots (12.5 cm diam.) filled with 2.5 kg soil (low-humic andosols) supplemented with ammonium sulfate (0.8 g), monocalcium phosphate (1.6 g), fused magnesium phosphate (3.2 g), and potassium sulfate (0.8 g) on June 12. One week after emergence, seedlings were thinned to one per pot and grown in an unheated vinyl plastic greenhouse throughout their life cycle. Harosoy and its early-maturing NILs (Harosoy-T, Harosoy-e3, and Harosoy-e4) generally produce fewer seeds than late-maturing NILs (Harosoy-E1, Harosoy-E2, and Harosoy-E5). Therefore, 15 plants of the former three lines and 10 plants of the latter three lines were used. Nine plants of the former and six plants of the latter lines were subjected to the pod-removal treatment and the others were used as controls.
Pots were randomized and repositioned twice a week. The number of days from planting to opening of the first flower (R1) (Fehr et al., 1971), and from planting to maturity (R8), when 95% of pods had mature color, were recorded for individual plants. Days from planting to flowering and maturity were subjected to analysis of variance and the treatment means were compared among NILs with extended Tukey's HSD test (Spjotvoll and Stoline, 1973).
Cracking was induced by removing the upper 50% of pods on the plant 40 d after anthesis. Pods were removed on the basis of the position of individual pods, regardless of the position of pods in branch, or position of the branch in plants. Flowers were individually labeled with the date of opening from 0 to 15 d after anthesis of plants. As a result, the pod-removal treatment was conducted from 25 to 40 DAO (days after opening of individual flowers) depending on the date of opening of each flower. The degree of cracking of each seed was scored by a cracking index (0 = not cracked to 4 = severely cracked) as previously described (Takahashi, 1997; Takahashi and Abe, 1999). The effect of the pod-removal treatment initiated at different flower stages from 25 to 40 DAO on seed coat cracking was evaluated by averaging the indices of seeds obtained from flowers at the same developmental stage. Frequency of cracking, cracking indices, and characters concerning productivity on an individual plant basis were subjected to analysis of variance. Means of these values were compared with extended Tukey's HSD test. Statistic analysis was conducted using the Statistica Software ver. 4.1 (StatSoft Inc., Okalahoma).
|
RESULTS AND DISCUSSION
|
|---|
The dominant alleles, E1 to E5, delay the transition from vegetative to reproductive growth in soybean. Harosoy-e3 matured earlier than Harosoy in this experiment (Table 2). Similar to our previous report (Takahashi and Abe, 1999), the time to flowering and maturity of Harosoy-e4 did not differ from that of Harosoy. Harosoy-E1, Harosoy-E2, and Harosoy-E5 flowered 14, 6, and 9 d later, and matured 18, 11, and 10 d later than Harosoy, respectively. Flowering of Harosoy-T was not delayed, but its maturity was delayed by 7 d compared with Harosoy. Pod-removal delayed maturity by 2 to 6 d in Harosoy, Harosoy-T, Harosoy-E1, Harosoy-e3, Harosoy-e4, and Harosoy-E5, and by 11 d in Harosoy-E2. Chilling treatment delayed the maturity of Harosoy-E2 as well (Takahashi and Abe, 1999).
View this table:
[in this window]
[in a new window]
|
Table 2. Days to flowering (R1) and maturity (R8), and frequency and intensity of seed coat cracking in Harosoy and its near-isogenic soybean lines at Tsukuba, Japan in 2000.
|
|
Frequency of seed coat cracking ranged from 0.2 to 4.7% in the control groups. The analysis of variance revealed that the differences among NILs were not significant. Frequency of cracking and average cracking indices were increased by the pod-removal treatment. Frequency of cracked seed and average cracking index ranged from 45.3 to 92.2% and from 0.78 to 2.16, respectively. The analysis of variance revealed that the differences among NILs for both traits were significant at the 1% level. Pod removal caused the frequency of cracked seeds to be 
45.3% in all the lines. Harosoy-T and Harosoy-E2 had the highest and most severe seed coat cracking, while Harosoy, Harosoy-E1, Harosoy-e3, and Harosoy-e4 showed low scores for both traits.
Takahashi (1997), Takahashi and Abe (1999), and Takahashi and Asanuma (1996) evaluated NILs and found that T, and genes for delayed maturity, E1 and E5, had strong inhibitory effects on low-temperature induced seed coat cracking. However, the dominant allele T intensified seed coat cracking under the pod-removal treatment in this experiment. Furthermore, the delaying effects of the maturity genes E1 and E5 were not associated with the intensity of cracking under the pod-removal treatment. Thus, the interaction of these genes with the pod-removal treatment is different from their effects on low temperature-induced seed coat cracking.
Previous results obtained from the low temperature treatment showed that the intensity of seed coat cracking was dependent on the developmental stage of each flower at the time of treatment (Takahashi, 1997; Takahashi and Abe, 1999). In contrast, the intensity of seed coat cracking was not dependent on the developmental stage of flowers at the time of treatment from 25 to 40 DAO except for Harosoy-E5, which exhibited a higher seed coat cracking index from 29 to 36 DAO (Fig. 1)
. On the basis of these results, the developmental stage of seeds seems to be independent of the intensity of seed coat cracking induced by the pod-removal treatments.
View larger version (30K):
[in this window]
[in a new window]
|
Fig. 1. Effects of the soybean pod-removal treatment on seed coat cracking in Harosoy, early-maturing isolines (Harosoy-T, Harosoy-e3, Harosoy-e4; upper), and late-maturing isolines (Harosoy-E1, Harosoy-E2, Harosoy-E5; lower). Cracking index (0, not cracked to 4, severely cracked), bars indicate standard error of the mean, numbers at each data point represent the number of seeds obtained from flowers exposed to treatment at a similar stage.
|
|
The analysis of variance revealed that the number of pods and seeds, total seed weight, and individual seed weight were different among NILs at the 1% level (Table 3). Of these, seed size represented by individual seed weight increased in response to the pod-removal treatment. Individual seed weight was correlated with frequency of seed coat cracking (r = 0.90**) and with average seed coat cracking index (r = 0.86*).
View this table:
[in this window]
[in a new window]
|
Table 3. Pod and seed number, total and one seed weight in Harosoy and its near-isogenic soybean lines at Tsukuba, Japan in 2000.
|
|
It has been observed that seed coat cracking occurs under environments that promote seed size enlargement (Maruyama and Mikoshiba, 1976). Seeds with cracked coats were generally larger than seeds with normal coats (Yaklich and Barla-Szabo, 1993). Sasaki and Sakai (1981) presumed that cracking was caused by an imbalance between the thickness of the seed coats and enlargement of the cotyledons. Under low temperature treatment, one seed weight did not increase in NILs for E1 to E5 exposed to low temperature stress (Takahashi, 1998, unpublished result). Therefore, it appears that there may be at least two environmental mechanisms that induce seed coat cracking. Low temperature stress promotes seed coat cracking without embryo enlargement whereas pod-removal induces seed coat cracking and embryo enlargement.
The present and previous studies suggest that the T locus and some of the maturity genes play a role in the occurrence of seed coat cracking. The present study suggests that genotypic differences in seed enlargement might be closely associated with those of seed coat cracking under the pod-removal treatment. Time to maturity in Harosoy-E2 was extended by 11 d without significant differences in pod or seed numbers (Tables 2, 3). It may have increased seed size and eventually affected severity of seed coat cracking. Pod number and total seed weight were similar between Harosoy and Harosoy-T. The decrease in seed numbers in Harosoy-T may have contributed to higher seed weight. Alleles at the T locus, interacting with alleles at the I locus, affect the structural integrity of the seed coat (Stewart and Wentz, 1930; Nicholas et al., 1993). In addition to seed enlargement, it is possible that the T locus had some qualitative effects on defectiveness of the seed coats in response to pod-removal.
It is uncertain whether seed enlargement might be a direct cause of seed coat cracking, or whether both seed enlargement and seed coat cracking might be concomitant events derived from some other physiological disturbance(s). Biochemical analysis especially on cell wall proteins of seed coats under cracking treatments or genetic analysis using DNA markers may help clarify the mechanisms of seed coat cracking induced by environmental stresses.
|
ACKNOWLEDGMENTS
|
|---|
We thank Dr. R.L. Nelson at USDA-ARS Univ. of Illinois, and Dr. H.D. Voldeng and Dr. E.R. Cober at Plant Res. Ctr., Agric. Agri-Food Canada for supplying the seeds of the isolines. We are grateful to Dr. Joseph G. Dubouzet (JIRCAS) for critical reading of the manuscript.
Received for publication January 31, 2001.
|
REFERENCES
|
|---|
- Abe, J., K. Komatsu, and Y. Shimamoto. 1998. A new gene for insensitivity of flowering to incandescent long daylength (ILD). Soybean Genet. Newsl. 25:92.
- Bernard, R.L. 1971. Two genes for time of flowering in soybeans. Crop Sci. 11:242–244.[Abstract/Free Full Text]
- Bernard, R.L., R.L. Nelson, and C.R. Cremeens. 1991 USDA soybean genetic collection: isoline collection. Soybean Genet. Newsl. 18: 27–57.
- Buzzell, R.I. 1971. Inheritance of a soybean flowering response to fluorescent-daylength conditions. Can. J. Cytol. 13:703–707.
- Buzzell, R.I., and H.D. Voldeng. 1980. Inheritance of insensitivity to long daylength. Soybean Genet. Newsl. 7:26–29.
- Cober, E.R., J.M. Tanner, and H.D. Voldeng. 1996. Genetic control of photoperiod response in early-maturing, near-isogenic soybean lines. Crop Sci 36:601–605.[Abstract/Free Full Text]
- Cober, E.R., and H.D. Voldeng. 2001. A new soybean maturity and photoperiod-sensitivity locus linked to E1 and T. Crop Sci. 41: 698–701.[Abstract/Free Full Text]
- Fehr, W.R., C.E. Caviness, D.T. Burmood, and J.S. Pennington. 1971. Stage of development descriptions for soybeans, Glycine max (L.) Merrill. Crop Sci. 11:929–931.[Abstract/Free Full Text]
- Hara M, T. Hayashi, and K. Suzuki. 1993. Occurrence and its varietal difference of soybean seed coat cracking by low temperature treatment at flowering stage (in Japanese). Tohoku Agric. Res. 44: 119–120.
- Liu, H.L. 1949. Inheritance of defective seed coat in soybeans. J. Hered. 40:317–322.[Free Full Text]
- Maruyama, N., and K. Mikoshiba. 1976. Seed coat cracking in soybeans (in Japanese). Proc. Crop Sci. Soc. Jpn. (Sup. 2) 45:45–46.
- McBlain, B.A., and R.L. Bernard. 1987. A new gene affecting the time of flowering and maturity in soybean. J. Hered. 78:160–162.[Abstract/Free Full Text]
- Murata, K., A. Kikuchi, and S. Sakai. 1991. Soybeans seed coat cracking test by soak and drying method (in Japanese). Rep. Tohoku Br., Crop Sci. Soc. Jpn. 34:57–58.
- Nagai, I. 1926. Inheritance in soybeans. Part 2 (in Japanese). Agriculture and Horticulture 1:107–118.
- Nicholas, C.D., J.T. Lindstrom, and L.O. Vodkin. 1993. Variation of proline rich cell wall proteins in soybean lines with anthocyanin mutations. Plant Mol. Biol. 21:145–156.[ISI][Medline]
- Okabe, A. 1996. Inheritance of seed coat cracking and effective selection method for the resistance in soybean. Jpn. Agric. Res. Q. 30:15–20.
- Palmer, R.G., and T.C. Kilen. 1987. Qualitative genetics and cytogenetics. p. 135–209. In J.R. Wilcox (ed.) Soybeans: Improvement, production, and uses, 2nd. ed. Agron. Monogr. 16. ASA, CSSA, and SSSA, Madison, WI.
- Percy, J.D., R. Philip, and L.O. Vodkin. 1999. A defective seed coat pattern (Net) is correlated with the post-transcriptional abundance of soluble proline-rich cell wall proteins. Plant Mol. Biol. 40:603–613.[ISI][Medline]
- Ray, J.D., K. Hinson, J.E.B. Manjono, and M.F. Malo. 1995. Genetic control of long-juvenile trait in soybean. Crop Sci. 35:1001–1006.[Abstract/Free Full Text]
- Sasaki K., and S. Nakamura. 1981. Test for resistance to seed coat cracking by pod removal in soybeans (in Japanese). Tohoku Agric. Res. 29:107–108.
- Sasaki, K., and S. Sakai. 1981. Seed coat cracking in a soybean cultivar, Toyosuzu (in Japanese). Hokunou 48:1–14.
- Spjotvoll, E., and M.R. Stoline. 1973. An extension of the T-method of multiple comparison to include the cases with unequal sample sizes. J. Am. Stat. Assoc. 68:975–978.
- Stewart, R.T., and J.B. Wentz. 1930. A defective seed-coat character in soybeans. J. Am. Soc. Agron. 22:658–662.
- Sunada, K., and T. Ito. 1982. Soybean grain quality as affected by low temperature treatments in plants (color of hilum, seed coat cracking) (in Japanese). Rep. Hokkaido Branch, Crop Sci. Soc. Jpn. and Hokkaido Branch, Jpn. Soc. Breeding 22:34.
- Takahashi, R. 1997. Association of soybean genes I and T with low-temperature induced seed coat deterioration. Crop Sci. 37:1755–1759.[Abstract/Free Full Text]
- Takahashi, R., and J. Abe. 1994. Genetic and linkage analysis of low temperature-induced browning in soybean seed coats. J. Hered. 85: 447–450.[Abstract/Free Full Text]
- Takahashi, R., and J. Abe. 1999. Soybean maturity genes associated with seed coat pigmentation and cracking in response to low temperatures. Crop Sci. 39:1657–1662.[Abstract/Free Full Text]
- Takahashi, R., and S. Asanuma. 1996. Association of T gene with chilling tolerance in soybean. Crop Sci. 36:559–562.[Abstract/Free Full Text]
- Wolf, W.J., and F.L. Baker. 1972. Scanning electron microscopy of soybeans. Cereal Sci. Today 17:125–130.
- Wolf, W.J., F.L. Baker, and R.L. Bernard. 1981. Soybean seed-coat structural features: Pits, deposits and cracks. Scanning Electron Microscopy 3:531–544.
- Yaklich, R.W., and G. Barla-Szabo. 1993. Seed coat cracking in soybean. Crop Sci. 33:1016–1019.[Abstract/Free Full Text]
TOP
ABSTRACT
INTRODUCTION
