Published in Agron J 91:840-844 (1999)
© 1999 American Society of Agronomy
677 S. Segoe Rd., Madison, WI 53711 USA
Agronomy Journal 91:840-844 (1999)
© 1999 American Society of Agronomy
SOYBEAN CULTIVAR DEVELOPMENT
Soybean PI 416937 Root System Contributes to Biomass Accumulation in Reciprocal Grafts
Vincent R. Pantalonea,
Gregory J. Rebetzkeb,
Joseph W. Burtonc,
Thomas E. Carter, Jr.c and
Daniel W. Israeld
a Dep. of Plant and Soil Sciences, Univ. of Tennessee, Knoxville, TN 37901-1071 USA
b CSIRO, Div. of Plant Industry, P.O. Box 1600 Canberra, ACT 2601, Australia
c Jr., USDA-ARS and Dep. of Crop Science, North Carolina State Univ., Raleigh, NC 27695-7631 USA
d USDA-ARS and Dep. of Soil Science, North Carolina State Univ., Raleigh, NC 27695-7619 USA
vpantalo{at}utk.edu
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ABSTRACT
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Soybean [Glycine max (L.) Merr.] plant introduction PI 416937 (PI4) has an extensive fibrous-like root system that contributes to enhanced drought and Al tolerance. The root system of PI4 appears to be more highly nodulated than standard southern U.S. cultivars, and thus has potential for enhanced N2 fixation. Genetic transfer of PI4 root system to soybean cultivars may lead to increased seed N at harvest through increased biomass or seed protein concentration. This hypothesis has not been tested. The objective of this study was to determine the influence of PI4 root system on plant productivity and protein accumulation in soybean seedling reciprocal grafts grown to maturity in the field. In three experiments, grafts were initiated 5 d after greenhouse planting by transversely severing the hypocotyl 2 cm below the apical meristem and transferring wedge-cut scions to severed root stock. Plants were then transplanted and grown in the field. PI 416937 maintained its superior root fibrosity in graft combination with other genotype scions. In Exp. 2, at the end of the season, plants of non-PI4 scions grafted to PI4 root stock averaged significantly higher in root fibrosity score (8.2) than the mean of their self-grafts (6.0); however, when PI4 scions were grafted to root stock from other genotypes, the root fibrosity score decreased significantly (6.6) compared with PI4 self graft (8.4). Thus, grafting revealed that the root system itself, rather than the scion of PI4, regulates expression of the fibrous-like rooting trait. Seed protein concentration did not increase significantly for genotype scions grafted to PI4 root stock. In Exp. 3, `Lee 74' or N85-492 grafted to PI4 root stock had significantly higher seed dry weight (161.1 g plant-1 for Lee 74 grafted to PI4 vs. 96.4 g plant-1 for the self-graft; 129.5 g plant-1 for N85-492 grafted to PI4 vs. 79.4 g plant-1 for the self-graft). The fibrous-like root system of PI4 enhances seed biomass when grafted to some non-PI4 genotypes. The genetic transfer of the PI4 rooting trait to elite germplasm through applied breeding may lead to the development of more productive soybean lines.
Abbreviations: PI4, PI 416937
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INTRODUCTION
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SOYBEAN PI 416937 (PI4) has an unusual, extensive, fibrous-like root system that appears to contribute to drought and Al tolerance (Goldman et al., 1989; Sloane et al., 1990; Hudak and Patterson, 1995). This plant introduction also has greater biological N2 fixation activity during mid-pod fill than Forrest, a southern U.S. cultivar (Marlow, 1993). The extensive root system of PI4 may contribute to increased N2 fixation by providing more sites for nodulation. Pantalone et al. (1996a) found that, compared with Lee 74, PI4 had significantly higher root surface area, nodule number, nodule dry weight, and root fibrosity score (a phenotypic measure of fibrous-like root area). In other work, Pantalone et al. (1996b) found a positive genotypic correlation between root fibrosity score and seed protein concentration. They observed some progeny lines from a cross between Lee-74 and PI4 that had high seed protein concentrations (>440 g kg-1) with seed yields equivalent to that of the productive cultivar Brim. The favorable attributes of the fibrous-like root system suggest that PI4 may become an important breeding line in the southeast region of the United States. The extensive fibrous-like root system of PI4 is a heritable trait (Pantalone et al., 1996b) and is being transferred by breeders in an effort to improve drought tolerance. This transfer may also enable growers to increase the total harvestable protein per hectare through increases in yield potential or seed protein concentration.
At present, the effect of the PI4 root system on biomass and protein concentration is unknown. Grafting experiments have been useful in discerning the effects of root and shoot systems in regulation of soybean nodulation and the accumulation of protein. For example, Caldwell and Hanson (1968) revealed through a grafting experiment that seed protein accumulation is governed by the aboveground portion of the plant. Delves et al. (1986) used reciprocal grafts to determine that autoregulation of nodulation in soybean is shoot controlled. This finding was later confirmed in other grafting experiments (Delves et al., 1987; Cho and Harper, 1991; Delves et al., 1992; Mathews et al., 1992). Cho and Harper (1991), and Mathews et al. (1992) further showed that nonnodulation is strictly root controlled, whereas supernodulation is shoot controlled.
The purpose of the present study was to use reciprocal grafting experiments to (i) ascertain whether the fibrous-like root trait of PI4 maintains its integrity of expression in graft combination with other genotype scions, (ii) determine the influence of PI4 root stock on seed protein concentration, and (iii) determine the influence of PI4 root stock on plant biomass and total protein.
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Materials and methods
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Grafting Procedure
For Exp. 1 and 2, soybean seeds were planted in greenhouse flats consisting of 32 cells filled with 131 cm3 of soilless growth medium (PRO-MIX BX; Premier Brands, Stamford CT). For Exp. 3, seeds were planted in compressed peat pots with growth medium (Jiffy pellets; Jiffy Products, Batavia, IL) as described by Pantalone et al. (1996c). Grafts were initiated 5 d after planting, when the apical meristem of seedlings had reached a height of approximately 4 cm above the soil surface. The hypocotyl was transversely severed with a razor approximately 2 cm below the cotyledon. The lower portion remained in the soil to become the root stock and the upper portion became the scion. A vertical incision was made with a razor to a depth of approximately 0.75 cm into the top center of the root stock. The scion was razor-trimmed to a V-wedge, which was inserted into the root stock incision. The graft union was wrapped with paraffin film (Parafilm; American National Can Co., Greenwich, CT). A 12-cm-length bamboo skewer was set into the soil to provide support. Additional paraffin film was used to secure the graft to the bamboo skewer. Grafts were placed under greenhouse benches, out of direct sunlight. The seedling grafts were hand-watered through a fine-mist nozzle every hour for the first 12 h, then four times daily for 4 d. Grafts were then brought to the top of the greenhouse benches for 3 d, at which time the plants were transplanted to the field.
Exp. 1: Root Fibrosity Graft Evaluation
A randomized complete block design with two replications was used on a Dothan loamy sand (fine-loamy, siliceous, thermic Plinthic Kandiudults) at the Central Crops Research Station in Clayton, NC, in 1992. Moisture was not a limiting factor, because the study was irrigated when necessary to alleviate water stress. Entries consisted of two genotypes, Lee 74 and PI4. Lee 74 was developed from `Lee' (Bernard et al., 1988). Lee is a common ancestor in southern U.S. soybean cultivars (Carter et al., 1993). In other experiments, Lee 74 had significantly lower root score, root surface area, nodule number, and nodule dry weight than PI4 (Pantalone et al., 1996a). PI 416937 is a Maturity Group VI plant introduction, Houjaku Kuwazu, from Japan (Hartwig and Edwards, 1980).
Nongrafted, self-grafted, and reciprocally grafted seedlings were transplanted to field plots in June. The center row of a three-row plot consisted of six plants, spaced 10 cm apart. Border rows consisted of the same genotype as the center row for self-graft or nongraft entries and a uniform mixture of scion and root genotype for reciprocal graft combinations. Roots were excavated to a depth of approximately 45 cm using a tractor-drawn peanut (Arachis hypogaea L.) inverter (Hobbs Engineering, Suffolk, VA) 68 d after transplanting to the field. A peanut inverter is a common piece of field equipment in the southeastern United States. It severs the taproot, lifts the plant out of the soil, and inverts it, laying the roots on the surface of the soil. Plants were scored individually for root fibrosity, a phenotypic measure, on a scale of 0 to 10 (low to high), based on percentage of fibrous-like root area, using the method of Pantalone et al. (1996a). The plot mean root fibrosity score was used for data analysis.
Exp. 2: Root Fibrosity and Seed Protein Concentration Graft Evaluation
A randomized complete block design with seven replications of plants of the genotypes Lee 74, NC 111, and N85-492 (one plant per replication) were used as nongrafts, self-grafts, and in reciprocal graft combination with PI4. Plants were spaced at 1-m intervals on a Dothan loamy sand at the Central Crops Research Station in Clayton, NC, in 1992. Moisture was not a limiting factor because the study was irrigated when necessary to alleviate water stress. NC 111 is a breeding line with high protein concentration (489 g kg-1, Carter et al., 1986); N85-492 is a high-yielding breeding line with low protein concentration (390 g kg-1) (J.W. Burton, unpublished data).
Seed were harvested at maturity and roots were excavated with a peanut inverter to determine root score. A 20-g seed sample from each plant was ground in a coffee grinder for 30 s, with agitation, and the tissue was analyzed for N to determine protein concentration using the micro-Kjeldahl procedure (Jones and Case, 1990).
Exp. 3: Whole-Plant Biomass and Protein Concentration Graft Evaluation
A randomized complete block design with four replications was used. A plot consisted of three plants per replication. In addition to the genotypes in Exp. 2, NC 103 was used. NC 103 is a breeding line with intermediate seed protein concentration (449 g kg-1; Carter et al., 1986). Self-grafts and reciprocal grafts were effected and transplanted to a Candor sand (sandy, si0liceous, thermic Arenic Paleudults) at 1-m intervals at the Sandhills Research Station in Jackson Springs, NC, in 1993. Moisture was not a limiting factor because the study was irrigated when necessary to alleviate water stress.
At the R7 growth stage (Fehr and Caviness, 1977), plants were surrounded by a bird netting perimeter to capture fallen leaves, which were removed at weekly intervals. Plants were excavated with a shovel at maturity and transported to the lab for processing. Plants were separated into roots, stems plus petioles (hereafter referred to simply as stems), leaves, podwall, and seed. Plant parts were dried, weighed, and ground in a Wiley Mill through a 1-mm screen. Protein concentration was determined by the Kjeldahl procedure at the USDA-ARS National Center for Agricultural Utilization Research at Peoria, IL. Plot means were used for data analysis for all traits.
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Results and discussion
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Exp. 1: Root Fibrosity Graft Evaluation
The grafting methodology presented in this paper and in our previous report (Pantalone et al., 1996c) was highly successful (90% over all graft combinations), and facilitated transplanting in the field. Similar success rates for grafting soybean have been achieved by Bezdicek et al. (1972), Sanders and Brown (1973), and Delves et al. (1986). White and Castillo (1989) pointed out that a potential weakness in grafting studies is the occurrence of graft-induced changes, which remain unreported because of failure of the studies to include nongrafted controls and reciprocal graft combinations. In our study, the grafting procedure per se had no effect on root fibrosity score, as evidenced by nonsignificant differences between self-grafted and nongrafted root scores. Nongrafted Lee 74 vs. self-grafted Lee 74 and nongrafted PI4 vs. self-grafted PI4 had nonsignificant contrasts in the analysis of variance (data not shown). Self-grafts were therefore used as controls to analyze the effect of PI4 root stock on other genotype scions.
It is important to know whether or not PI4 root stock maintains its fibrosity when grafted to Lee 74 scion. Root score was significantly (P < 0.05) higher for Lee 74 grafted to PI4 root stock than for self-grafted Lee 74 (Table 1)
. Furthermore, the root score for Lee 74 grafted to PI4 root stock was not significantly (P > 0.05) different from that of self-grafted PI4, suggesting that the PI4 type root system maintains its integrity in graft combination with a genotype that normally confers a lower root score. This observation will be of importance to breeders who wish to genetically transfer the fibrous-like root system to currently adapted cultivars.
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Table 1 Mean root fibrosity score for nongrafted, self-grafted, and reciprocal-grafted soybean grown for 68 d at Clayton, NC, in 1992
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Exp. 2: Root Fibrosity and Seed Protein Concentration Graft Evaluation
The fibrous-like root system of PI4 may confer benefits to the plant through enhanced N2 fixation. This experiment evaluated whether some genotype scions would increase their seed protein concentration when grafted to PI4's fibrous-like root stock. As in Exp. 1, self-grafting did not affect expression of visual root score. This was true for the PI4 genotype as well as the non-PI4 genotypes (Table 2)
. A lack of influence of PI4 scion on root score was shown by the nonsignificant contrast in the analysis of variance between the mean of PI4 scions grafted to root stock from genotypes other than PI4, versus the mean of self-grafts of genotypes other than PI4 (data not shown). This verifies that it is the root system rather than the scion of PI4 that confers fibrosity.
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Table 2 Mean root fibrosity score and protein concentration for nongrafted, self-grafted, and reciprocal-grafted soybean grown for 128 d at Clayton, NC, in 1992
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In graft combination with all genotype scions, PI4 root stock had a high root score that was not significantly different from the self-grafted PI4. That PI4 maintains a high root score in graft combination with other genotype scions is further evidenced by a significant contrast between the mean of non-PI4 scions grafted to PI4 root stock versus the mean of their self-grafts (data not shown).
Specifically, when Lee 74 and NC 111 scions were grafted to PI4 root stock, root scores increased significantly by 2.2 and 3.7 units, respectively, relative to their self-grafts (Table 2). And when N85-492 (which had a relatively high root score of 7.7 when self-grafted) was grafted to PI4 root stock, root score also increased, although this increase was not significant. Seed protein concentration was not significantly increased by grafting scions to PI4 root stock (Table 2).
Exp. 3: Whole-Plant Biomass and Protein Concentration Graft Evaluation
This experiment included all of the graft combinations used in Exp. 2. Analysis of variance combined over two years indicated that the observed increase in seed protein concentration for genotypes grafted to PI4 root stock was not significant. When biomass of plant parts was examined in Exp. 3, significant effects due to PI4 root stock were apparent.
Lee 74 and N85-492 scions grafted to PI4 root stock significantly increased in podwall and seed dry weight compared with self-grafts (Table 3)
. NC 103 showed a nonsignificant increase and NC 111 showed a nonsignificant decrease in podwall and seed dry weight for scions grafted to PI4 root stock compared with their self-grafts. All non-PI4 genotypes grafted to PI4 root stock showed nonsignificant increases in leaf dry weight compared with their self-grafts. All non-PI4 genotypes (except NC 111) grafted to PI4 root stock showed nonsignificant increases in stem and root dry weight compared with their self-grafts. It is interesting to note that non-PI4 scions grafted to PI4 root stock did not significantly increase root dry weight. A possible explanation for this observation is that, in this experiment, roots were sampled at the end of the season only to a depth of approximately 30 cm, representing a partial excavation of the soybean root mass, which may have accumulated a lower depths. Other researchers found that the root mass of PI4 was about twice that of Forrest during vegetative growth stages (Hudak and Patterson, 1995).
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Table 3 Mean root, stem, leaf, podwall, and seed dry weight for self-grafted, and reciprocal-grafted soybean grown to maturity at Jackson Springs, NC, in 1993
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A nonsignificant increase in seed protein concentration was observed for every non-PI4 genotype grafted to PI4 root stock, compared with the self graft (Table 4)
. The addition of the fibrous-like root stock generally appears to enhance plant productivity and thereby, total protein. In previous work, PI4 was shown to have significantly higher root score, root surface area, nodule number, and nodule dry weight than Lee 74 (Pantalone et al., 1996a). Moreover, in a population derived from a cross of Lee 74 and PI4, a significant but weakly positive genotypic correlation between root score and seed protein concentration was observed (Pantalone et al., 1996b). Because the root system of PI4 has greater root surface area and nodulation capacity, it may promote vigorous plant growth through increased water and nutrient uptake and N2 fixation. Supporting evidence is seen in the present study, in that Lee 74 increased significantly in biomass and total protein compared with self grafts when Lee 74 scions were grafted to PI4 root stock. A negative genotypic correlation between seed protein concentration and yield has often been reported for soybean populations (Burton, 1987). However, populations derived from PI4 parentage show neutral genotypic correlations between seed protein concentration and yield (Pantalone et al., 1996b). Our present work is unusual because it indicates that, for some genotypes, incorporation of a fibrous root system can enhance productivity with no decrease in seed protein concentration, thereby providing the potential for more total harvestable protein per hectare.
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Table 4 Mean root, stem, leaf, podwall, and seed protein concentration for self-grafted, and reciprocal-grafted soybean grown to maturity at Jackson Springs, NC, in 1993
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Root systems have been shown to play an important role in N2 fixation (Vincent, 1982; Darcy Lameta and Jay, 1987; Bowen and Rovira, 1991; Vance, 1991). It is therefore not entirely surprising to find that increases in total protein could be achieved by incorporating the enhanced root system of PI4. However, the increases in total protein observed in our work seem to be largely driven by increased plant biomass.
Traditional plant breeding methods are currently being used to transfer the extensive fibrous-like root system of PI4 to potential soybean cultivars. Carter (1989) suggested that enhanced rooting, associated with increased ability to extract soil water, is the most promising genetic mechanism for improving soybean drought tolerance. The root fibrosity scoring methodology (Pantalone et al., 1996a), the observation of high protein high yielding lines derived from Lee 74 x PI 416937 reported by Pantalone et al. (1996b), and the findings of the present study all suggest that breeders will be able to incorporate the fibrous-like root trait into the genetic background of some elite material to produce breeding lines with higher seed yield and greater total harvestable protein.
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ACKNOWLEDGMENTS
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Warren Rayford, Donna Thomas, and the staff at the USDA-ARS National Center for Agricultural Research are gratefully acknowledged for their assistance in conducting N analysis of plant tissue samples.
Received for publication November 9, 1998.
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ABSTRACT
INTRODUCTION
