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Hard wheat milling and bread baking traits affected by the seed-specific overexpression of puroindolines.

FOR COMMERCIAL PURPOSES, wheat grain is classified as either hard or soft based on endosperm texture. Differences in endosperm texture have far-reaching effects on numerous milling and end-use qualities in wheat. Soft-textured wheats typically have increased break flour yields, a smaller flour particle size, and less starch damage compared with hard-textured wheats (Symes, 1965; Symes, 1969). Soft-textured wheats are generally used to make cookies and pastries, while hard-textured wheats are typically used to make breads (Morris and Rose, 1996). The Ha locus located on the short arm of chromosome 5D (Mattern et al., 1973; Law et al., 1978) differentiates between soft and hard grain texture (Symes, 1965; Baker, 1977).

The discovery of a 15-kDa protein called friabilin on the surface of water-washed starch granules provided a biochemical way to distinguish between hard and soft wheats (Greenwell and Schofield, 1986). The presence of friabilin on water-washed starch is associated with soft endosperm texture, whereas water-washed starch from hard-textured wheats is essentially devoid of friabilin. Further investigation into the composition of friabilin revealed that friabilin was comprised primarily of two basic cysteine-rich proteins, PINA and PINB (Jolly et al., 1993; Morris et al., 1994), and that friabilin was under the genetic control of chromosome 5D (Jolly et al., 1993). Puroindoline A and PINB have a unique tryptophan domain (Gautier et al., 1994) that is hypothesized to be an active site for the binding of polar lipids found on the surface of starch granules (Gautier et al., 1994; Marion et al., 1994; Greenblatt et al., 1995).

The tightly linked genes puroindoline a (pina-D1a) and puroindoline b (pinb-D1a) have since been cloned and identified as residing at the Ha locus (Gautier et al., 1994; Sourdille et al., 1996; Giroux and Morris, 1997, 1998; Tranquilli et al., 1999), providing a genetic basis for the control of grain texture in wheat. Recent surveys of wheat genotypes across the world has revealed that soft wheats all have the same pin alleles (pina-D1a; pinb-D1a), while all hard wheats have a mutation in either pina or pinb (Giroux and Morris, 1998; Lillemo and Morris, 2000; Morris et al., 2001). To date, there have been seven pin mutations characterized (reviewed in Morris, 2002), with the most prevalent being a null mutation in pina (pina-D1b) and a point mutation in pinb (pinb-D1b) that results in a glycine-to-serine substitution at the 46th residue of the peptide (Giroux and Morris, 1997, 1998). To support the hypothesis that the puroindolines are the primary genetic elements controlling grain texture in wheat, the use of plant transformation has been employed in both rice (Oryza sativa L.) (Krishnamurthy and Giroux, 2001) and wheat (Beecher et al., 2002; Hogg et al., 2004). The transgenic addition of the puroindolines to both rice and a hard spring wheat cultivar possessing the pinb-D1b allele resulted in significantly softer endosperm texture, with seeds having reduced endosperm particle size, and reduced starch damage after milling. A positive association was also found between puroindoline transcript levels, friabilin accumulation, and soft grain texture in developing transgenic wheat kernels (Hogg et al., 2004).

Grain texture has been shown to affect many milling and bread baking characteristics in hard wheat (Slaughter et al., 1992; Ohm et al., 1998), as has puroindoline sequence type. Pinb was shown to be a major quantitative trait locus for flour yield, grain texture, starch damage, alkaline-water retention capacity, and dough water absorption in a recombinant inbred population segregating for the pinb-D1a (soft) and pinb-D1b (hard) alleles (Campbell et al., 2001). In a hard wheat recombinant inbred population segregating for the two most common pin mutations, it was found that lines possessing the pinb-D1b mutation had higher break flour yields, higher flour yields, lower flour ash, improved crumb grain scores, and larger loaf volumes compared with lines carrying the pina-D1b mutation (Martin et al., 2001). Cane et al. (2004) supports these findings by reporting that select Australian hard cultivars possessing the pinb-D1b mutation had higher flour yields, lower water absorption, and a smaller particle size than those hard cultivars possessing the pina-D1b mutation. The reconstitution of wheat flour with 0.1% PINA was also reported to have significant effects on both crumb grain scores and bread loaf volumes (Dubreil et al., 1998).

Our objectives here were to characterize milling and baking traits affected by the overexpression of puroin-dolines in vivo. We have transgenically created a set of truly isogenic lines in a hard spring wheat background that overexpress pina-D1a, pinb-D1a, or both pina-D1a and pinb-D1a solely in the seed endosperm. These lines were tested in two replicated experiments for seed texture and milling and bread baking characteristics.

MATERIALS AND METHODS

Transgenic lines used in this study were derived as described by Beecher et al. (2002) and Hogg et al. (2004). To achieve overexpression, the puroindoline genes (pina-D1a and pinb-D1b) were placed between the glutenin gene flanking regulatory sequences from pGlul0H5 (Blechl and Anderson. 1996). A total of 17 different homozygous transgenic lines were created which had added pma-D1a, pinb-D1a, or both pina-D1a and pinb-D1a. All lines were co-transformed with the Bar expression vector pRQ101A (Sivamani et al., 2000), which confers resistance to the herbicide glufosinate (AgrEvo USA Company, Willmington, DE). The cultivar Hi-Line (Lanning et al., 1992) was used for transformation, which carries the wild-type pina-D1a sequence and the variant pinb-D1b sequence (Giroux et al., 2000). Hi-Line and 161 (a transformed control line expressing only Bar), did not differ for any of the measured traits. Thus, these two lines without added pin were collectively considered the hard wheat control group. 'Heron', which carries the wild-type pina-D1a and pinb-D1a sequences, was used as the soft wheat control. Heron was excluded from the statistical analysis, however, since it did not share the common genetic background of the other lines.

To reconfirm genotype identity and uniqueness of each line, DNA was extracted (Riede and Anderson, 19961 from homozygous [T.sub.3] leaf samples representing each experimental line. Genomic DNA was digested with BamHI. Southern blot analysis was performed (Beecher et al., 2002) and replicate blots were probed with [P.sup.32] labeled pina-D1a and pnb-D1a sequences. Total PINA and PINB proteins in homozygous [T.sub.3] seeds were visualized for all experimental lines following methods described by Giroux et al. (2003). Visual estimation of puroindoline protein expression levels was performed using a scale ranging from 1 to 8, in increments of 1. This scale was constructed using a Hi-Line TX-114 protein extract, where 1 = 5 [micro]L of sample (120 [micro]L buffer 100 [mg.sup.-1] seed powder). As controls, seeds from the durum (Triticum turgidum L.) cultivar Langdon and the soft substitution line Langdon-5D(5B) were also analyzed for puroindoline presence and quantity.

Seventeen [T.sub.3] experimental lines, along with Hi-Line, a Bar control line, and Heron, were grown during the summer of 2003 at the Montana State University Arthur H. Post Farm using a randomized block design with two replications. Plots were four 3-m rows with 30 cm between rows. The experiment was grown in irrigated and nonirrigated environments. At maturity, grain from all four rows from each plot was harvested with a plot combine, and 200 [T.sub.4] seeds from each plot were used for grain texture [single kernel characterization system (SKCS) hardness index], kernel diameter, and kernel weight measurements using the Single Kernel Characterization System 4100 (Perten Instruments North America, Incorporated, Springfield, IL). Particle size index (PSI, %) was measured using a subsample of grain from each plot following Method 55-30 (American Association of Cereal Chemists, 2000) with the following change; a 90-[micro]m sieve was used in place of a 70-[micro]m sieve. Grain protein content was determined at a constant 12% moisture basis for each plot by near-infrared transmission using an Infratec 1225 Grain Analyzer (Foss North America Incorporated, Eden Prairie, MN).

Following kernel analysis, 500 g of seed from each plot was tempered to 13.0% moisture for 15 h. Samples were then milled using Quadrumat mills following the modifications of Jeffers and Rubenthaler (1977). Straight-grade flour was analyzed for moisture (Method 44-16), protein (Method 46-30), and ash (Method 08-01) (American Association of Cereal Chemists, 2000). Flour protein is reported at a constant 14% moisture basis. Mixograph analysis was performed using a 10-g instrument following method 54-40A (American Association of Cereal Chemists, 2000). Bread was then baked following Method 10-10B (American Association of Cereal Chemists, 2000). Loaf volume was determined using a canola seed displacement method and crumb grain score was rated from 0 (unsatisfactory) to 5 (excellent).

From the irrigated environment. 300 g of seed from each replication of Hi-Line, HGA-13, HGB-6, and HGAB-18 was milled into whole wheat flour using a UDY Cyclone mill with a 0.5-mm screen (UDY Company, Fort Collins, CO). Whole wheat flour was analyzed for protein content, ash content, and mixograph properties following the procedures described above. Whole wheat flour was then used to make bread, for which loaf volume and crumb grain score were determined following described procedures with the following exception: 300 ppm absorbic acid was used in place of 150 ppm absorbic acid.

Data were analyzed via ANOVA combined across environments. Environments and entries were considered fixed effects and replications as random effects using PROC GLM in SAS (SAS Institute, 1988). Least significant difference was computed to compare individual mean differences, and contrasts of linear combinations of means were used to compare group means. Correlations among traits were computed using entry means.

RESULTS AND DISCUSSION

To determine milling and baking traits associated with the overexpression of puroindolines in vivo, a set of 17 transgenic lines was created in a hard red spring wheat background (Beecher et al., 2002; Hogg et al., 2004).

The presence and expression of the puroindoline transgenes in these lines were reconfirmed in the [T.sub.3] generation using Southern blot analysis (Fig. 1 and Table 1) and Triton X-114 protein extracts (Fig. 2, Table 1). On the basis of Southern analysis, there were four transformed lines identified as possessing the pina-D1a transgene (HGA: 1, 3, 5, 13), eight lines positive for the pinb-D1a transgene (HGB: 2, 5, 6, 12, 16, 19, 21,35), and five lines possessing bothpina-D1a and pinb-D1a transgenes (HGAB: 2, 3, 11, 12, 18). The transgenic line 161, Heron, and untransformed Hi-Line were all negative for the presence of nonnative puroindoline genes (Table 1). All pin lines contained a unique banding pattern since BarnHI digestion cleaves the pina and pinb expression plasmids just once (Beecher et al., 2002, Hogg et al., 2004). Since biolistic transformation results in random insertion and variable copy number, all lines presented are unique and have variable levels of added pin expression.

[FIGURES 1-2 OMITTED]

Northern blot analysis was performed previously on eight of these lines (Hi-Line, 161, HGA-1, HGA-3, HGB-12, HGB-19, HGAB-2, and HGAB-3). In lines transformed with the puroindolines, there was approximately a four- to five-fold increase in the appropriate puroindoline transcript levels compared with the controls Hi-Line and 161 at 21 DAF (Hogg et al., 2004).

To establish protein expression levels for all experimental lines, the puroindoline proteins PINA and PINB were extracted from mature seeds using a Triton X-114 method (Giroux et al., 2003) and visualized via sodium dodecyl sulphate polyacrylamide gel electrophoresis and Coomassie blue staining. The durum cultivar Langdon and the substitution line Langdon-5D(5B) were used as negative and positive controls, respectively. As in all durum wheats, Langdon lacks the D-genome and consequentially the Ha locus that is found on 5DS (Mattern et al., 1973; Law et al., 1978). Langdon is therefore devoid of both PINA and PINB (Fig. 2). However, in the substitution line Langdon-5D(5B) both PINA and PINB are present (Fig. 2) (Giroux and Morris, 1997, 1998). The HGA lines exhibited an increase in PINA that was on average 7.6 times greater than the amount of PINA in Hi-Line and 161 (Fig. 2; Table 1). The HGB lines showed increased amounts of PINB that were on average 2.6 times greater than those seen in Hi-Line and 161 (Fig. 2). The HGAB lines had increased levels of both PINA and PINB, with average values of 6.8 and 3.6, respectively (Fig. 2). Hi-Line, 161, and Heron had similar puroindoline protein expression levels and profiles, with PINA being more prominent than PINB (Fig. 2). The PINB band seen for Heron represents the functional peptide, while the PINB band seen for HiLine and 161 represents the mutant peptide that is coded for by the pinb-D1b) allele (Fig. 2). This peptide is considered to be non- or partially functional due to a glycine-to-serine change at the 46th residue near the protein's unique tryptophan domain (Giroux and Morris, 1998). It is hypothesized that this tryptophan domain is an active site for the binding of phospholipids found on the surface of the starch granules (Gautier et al., 1994; Marion et al., 1994, Greenblatt et al., 1995). On the basis of this assumption, the true value of added functional PINB in all transgenic lines might be estimated by subtracting a value of 1 from their given PINB values (Table 1).

A very strong negative correlation (r = -0.94; P < 0.01) was found between SKCS hardness index and PSI, which corroborates reports by others (Pomeranz et al., 1985: Morris and Massa, 2003). The hard wheat control (HWC) group had a SKCS hardness mean of 83.6 and a PSI mean of 8.54% (Table 2). Heron had an SKCS hardness mean of 30.0 and a PSI mean of 14.9% (Table 2). As a group, the HGA lines had a SKCS hardness mean of 51.9 and a PSI mean of 13.4% (Table 2), while the HGB lines had a softer endosperm texture with a SKCS hardness mean of 29.0 and a PSI mean of 15.4% (Table 2). The HGAB lines had the softest endosperm texture with SKCS hardness and PSI means of 23.2 and 17.9%, respectively (Table 2). The HWC group, the HGA group, the HGB group, and the HGAB group were all significantly different from each other for both SKCS hardness and PSI values (Table 2). Using a relatively small number of transgenic pin addition lines, Hogg et al. (20(]4) reported that total puroindoline content did not correlate well with changes in grain hardness unless both functional PINA and PINB were present. The results here with a larger data set agree with Hogg et al. (2004). For example, even though the HGA lines have high levels of total puroindoline (Table 1), they are intermediate in grain hardness (Table 2) since they lack functional PINB, and cannot form a large amount of starch-surface friabilin (Hogg et al., 2004). However, when high levels of both functional PINA and PINB are present, the softest texture is achieved, exemplified by the HGAB lines (Tables 1 and 2).

The mean grain protein content for the HGB lines (176 g [kg.sup.-1]) and HGAB lines (177 g [kg.sup.-1]) was significantly higher compared with the HWC group (171 g [kg.sup.-1]), while the mean grain protein for the HGA lines (173 g [kg.sup.-1]) was not significantly different from the HWC group (Table 2). The increased protein content observed in the HGB and HGAB lines is likely a result of their decreased kernel diameter and weight (Table 2). There are significant negative correlations between wheat protein and kernel diameter (r = -0.75; P < 0.01), and between wheat protein and kernel weight (r = -0.45; P < 0.05). The relationship between kernel size and protein content observed here is in agreement with the findings of others (Ohm et al., 1998). The transgenic lines as a whole also had significantly lower flour protein than the HWC group (Table 2). This is not unexpected, since the outer endosperm layers, which contain most of the protein, are not as easily separated from the bran and aleurone layers during the milling process as they are in hard wheats. Accordingly, the HGA group, which is intermediate in hardness, possesses more flour protein compared with the extremely soft-textured HGB and HGAB groups (Table 2).

All lines were tempered to 13% moisture before Quadrumat milling. This tempering level is optimum for soft wheats, but below optimum for hard wheats. The overexpression of the puroindolines in all transgenic groups resulted in increased break flour yields, lower flour yields, and lower ash values compared with the HWC group (Table 3). It is worth noting that if these lines were tempered and milled under optimal conditions, the differences in flour yield between the groups would be minimized. For the baking characteristics of mixograph absorption and mix time, the HGA and HGB groups did not significantly differ from the HWC group (Table 3). This is expected since the glutenin and gliadin composition of these transgenic lines should be identical to that of Hi-Line, as demonstrated by Beecher et al. (2002). However, the HGAB group did significantly differ from the HWC group for these two characteristics for unknown reasons (Table 3). The transgenic addition of puroindolines to Hi-Line principally affected grain texture and milling characteristics, but also detrimentally affected some bread baking qualities. When compared as groups, the HGA lines, HGB lines, and HGAB lines all had significantly lower loaf volumes compared with the HWC group (Table 3). The HGAB group also had a significantly less desirable crumb grain score than the HWC group (Table 3).

Single-kernel hardness was positively correlated with kernel diameter and kernel weight, while PSI was negatively correlated with these same traits (Table 4). However, there was no significant correlation between wheat protein and either measurement of endosperm texture (Table 4), which concurs with the findings of others (Pomeranz et al., 1985; Ohm et al., 1998). Very strong positive correlations were found for SKCS hardness vs. flour yield (r = 0.82) (Fig. 3A), flour ash (r = 0.73), and flour protein (r = 0.72) (Table 4). On the contrary, a very strong negative correlation was found between SKCS hardness and break flour yield (r = 0.79) (Table 4, Fig. 2B). The inverse relationships were found for PSI vs. flour yield (r = -0.77), flour ash (r = -0.67), flour protein (r = -0.55), and break flour yield (r = 0.87) (Table 4). Flour yield, break flour yield, flour protein, and flour ash content were all highly interrelated (Table 5). These results reaffirm previous findings that variation in puroindoline sequence type and associated grain hardness differences influence many milling characteristics (Campbell et al., 2001; Martin et al., 2001: Cane et al., 2004).

[FIGURE 3 OMITTED]

Because of the differences in flour protein, it was difficult to determine if the decreased loaf volume observed in the transgenic groups was the result of increased puroindoline protein content or decreased flour protein content. To minimize the effect of differing flour protein levels, one line from each transgenic group and Hi-Line were milled into whole-wheat flours and then baked. Differences in whole-wheat flour protein of all lines were minimal (Table 6). Even with similar flour protein, HGA-13 and HGAB-18 still exhibited lower loaf volumes compared with Hi-Line (Table 6). HGB-6 exhibited no change in loaf volume, but had a poorer crumb grain score than Hi-Line (Table 6). HGAB-18 had the poorest crumb grain score, while HGA-13 showed no change in crumb grain compared with Hi-Line (Table 6).

On the basis of bake data from Quadrumat milled lines (Table 3) and whole-wheat lines (Table 6), it appears that high levels of puroindolines in vivo negatively affect both crumb grain score and loaf volume. In further support of this, there were significant positive correlations found between SKCS hardness vs. loaf volume (r = 0.43) and SKCS hardness vs. crumb grain score (r = 0.39) (Table 4). In this experiment, changes in SKCS hardness were a direct result of changes in both puroindoline sequence type and puroindoline content. These results conflict with previous findings which reported an improvement in crumb grain score and an increase in loaf volume after the reconstitution of blended flour with PINA (Dubreil et al., 1998). This apparent conflict likely results from the different genotypes, where our studies used Hi-Line (Lanning el al., 1992), a hard red spring cultivar selected for good end-product quality.

Here we have demonstrated that the overexpression of pina-D1a and pinb-D1a in the wheat seed endosperm not only causes drastic changes in grain texture, but also affects a wide range of milling and bread baking traits. Almost every aspect of the milling process is affected by grain texture, which in turn is a direct manifestation of puroindoline allele type and quantity. Very high levels of puroindolines in vivo also negatively affect bread baking traits such as loaf volume and crumb grain score.

Abbreviations: pina, puroindoline a gene; PINA, puroindoline A protein; pinb, puroindoline b gene; PINB, puroindoline B protein; PSI, particle size index; SKCS, single kernel characterization system.
Table 1. Southern blot results and puroindoline protein expression
levels for controls and lines transformed with pina-D1a
(HGA), pinb-D1a (HGB), or both pina-D1a and pinb-D1a
(HGAB).

                  Transgenes
                   ([double
                   dagger])
                                    PINA           PINB        Total
ID ([dagger])    pina    pinb    ([section])    ([section])     PIN

'Hi-Line'         -       -          1.0            1.0         2.0
161.00            -       -          1.0            1.0         2.0
HWCQ              -       -          1.0            1.0         2.0
'Heron'           -       -          1.0            1.0         2.0
HGA-1             +       -          7.0            1.0         8.0
HGA-3             +       -          8.0            1.0         9.0
HGA-5             +       -          8.0            1.0         9.0
HGA-13            +       -          7.5            1.0         8.5
HGA avg.          +       -          7.6            1.0         8.6
HGB-2             -       +          1.1            2.0         3.0
HGB-5             -       +          1.0            2.0         3.0
HGB-6             -       +          1.0            3.5         4.5
HGB-12            -       +          1.0            3.8         4.8
HGB-16            -       +          1.0            2.0         3.0
HGB-19            -       +          1.0            3.5         4.5
HGB-21            -       +          1.0            2.0         3.0
HGB-35            -       +          1.0            1.8         2.8
HGB avg.          -       +          1.0            2.6         3.6
HGAB-2            +       +          7.0            3.5        10.5
HGAB-3            +       +          8.0            5.0        13.0
HGAB-11           +       +          8.0            3.3        11.3
HGAB-12           +       +          5.0            1.5         6.5
HGAB-18           +       +          6.0            4.5        10.5
HGAB avg.         +       +          6.8            3.6        10.4

([dagger]) All homozygous [T.sub.3] lines were derived from the hard
spring wheat Hi-Line, which carries the pina-D1a and pinb-D1b alleles.
Heron is a soft wheat control cultivar.

([double dagger]) Southern blot analysis was performed on [T.sub.3]
genomic DNA extracts, which were digested with HindIII and hybridized
with pina-D1a and pinb-D1a PCR generated probes. Lines denoted as +
were PCR and Southern blot positive for the pina-D1a and pinb-D1a
transgenes.

([section]) Puroindoline A (PINA) and puroindoline B (PINB) proteins
were quantified using a scale that ranged from 1 to 8 on an acrylamide
gel. The number shown is an average of two experiments.

([paragraph]) HWC denotes average of hard wheat controls (Hi-Line and
161).

Table 2. Kernel characteristics, measurements of grain texture, and
protein content based on mean of two environments for controls and
lines transformed with pina-D1a (HGA), pinb-D1a (HGB), or both
pina-D1a and pinb-D1a (HGAB).

                                             Grain hardness

                      Wheat       Flour           SKCS
ID ([dagger])        protein     protein     (double dagger)

                               g [kg.sup.-1]

'Hi-Line'            170         145              85.0
161.00               172         145              82.2
HWC ([paragraph])    171         145              83.6
'Heron'              160         118              30.0
HGA-1                169         136              56.6
HGA-3                170         133              48.5
HGA-5                180         139              48.8
HGA-13               171         137              53.6
HGA avg.             173         136              51.9
HGB-2                182         133              37.1
HGB-5                180         127              28.2
HGB-6                172         124              23.1
HGB-12               172         119              21.7
HGB-16               179         129              35.7
HGB-19               175         123              24.2
HGB-21               168         122              35.1
HGB-35               179         127              26.6
HGB avg.             176         126              29.0
HGAB-2               197         147              28.0
HGAB-3               161         112              27.6
HGAB-11              169         115              21.1
HGAB-12              182         133              24.5
HGAB-18              175         124              14.9
HGAB avg.            177         126              23.2
LSD (0.05) (#)         0.50        0.29            2.00
LSD (0.01) (#)         0.71        0.41            2.87

                                      P

HWC vs. HGA            0.4885    <0.0001          <0.0001
HWC vs. HGB            0.0059    <0.0001          <0.0001
HWC vs. HGAB           0.0022    <0.0001          <0.0001
HGA vs. HGB            0.0192    <0.0001          <0.0001
HGA vs. HGAB           0.0067    <0.0001          <0.0001
HGB vs. HGAB           0.4464     0.2454          <0.0001

                     Grain hardness
                                        Kernel      Kernel
ID ([dagger])        PSI ([section])    weight     diameter

                            %             mg          mm

'Hi-Line'                  8.19         26.8        2.22
161.00                     8.89         28.5        2.33
HWC ([paragraph])          8.54         27.7        2.27
'Heron'                   14.9          28.6        2.18
HGA-1                     12.7          28.3        2.24
HGA-3                     14.0          27.1        2.13
HGA-5                     13.3          26.0        2.10
HGA-13                    13.5          28.1        2.19
HGA avg.                  13.4          27.4        2.17
HGB-2                     13.4          26.4        2.07
HGB-5                     16.2          25.5        2.02
HGB-6                     15.8          26.9        2.11
HGB-12                    15.9          26.6        2.07
HGB-16                    14.8          23.7        1.97
HGB-19                    16.8          25.4        1.99
HGB-21                    14.1          27.8        2.08
HGB-35                    16.0          24.6        1.99
HGB avg.                  15.4          25.9        2.04
HGAB-2                    17.8          23.8        1.99
HGAB-3                    15.8          28.2        2.07
HGAB-11                   18.3          24.9        1.95
HGAB-12                   18.9          25.0        2.00
HGAB-18                   18.7          25.7        2.00
HGAB avg.                 17.9          25.5        2.00
LSD (0.05) (#)             0.79          1.03       0.06
LSD (0.01) (#)             1.13          1.48       0.09

                                           P

HWC vs. HGA               <0.0001        0.4863    <0.0001
HWC vs. HGB               <0.0001       <0.0001    <0.0001
HWC vs. HGAB              <0.0001       <0.0001    <0.0001
HGA vs. HGB               <0.0001       <0.0001    <0.0001
HGA vs. HGAB              <0.0001       <0.0001    <0.0001
HGB vs. HGAB              <0.0001        0.2102     0.0241

([dagger]) All homozygous [T.sub.3] lines were derived from the hard
spring wheat Hi-Line, which carries the pina-D1a and pinb-D1b alleles.
Heron is a soft wheat control cultivar.

([double dagger]) SKCS = single kernel characterization system hardness
index values.

([section]) PSI = particle size index.

([paragraph]) HWC denotes average of hard wheat controls (Hi-Line and
161).

(#) LSD values compare individual transgenic lines vs. Hi-Line.

Table 3. Milling and baking traits based on mean of two environments
for controls and lines transformed with pina-D1a (HGA), pinb-D1a (HGB),
or both pina-D1a and pinb-D1a (HGAB).

                            Flour      Break flour     Flour
ID ([dagger])               yield         yield         ash

                                      g [kg.sup.-1]

'Hi-Line'                  616           688            4.93
161.00                     626           704            4.80
HWC ([section])            621           696            4.87
'Heron'                    562           794            4.40
HGA-1                      551           789            3.70
HGA-3                      573           807            4.00
HGA-5                      527           829            4.20
HGA-13                     553           823            4.30
HGA avg.                   551           812            4.05
HGB-2                      511           770            4.20
HGB-5                      487           828            3.90
HGB-6                      504           805            3.80
HGB-12                     483           838            3.70
HGB-16                     446           786            4.00
HGB-19                     486           809            3.70
HGB-21                     510           804            3.80
HGB-35                     483           789            3.90
HGB avg.                   489           804            3.88
HGAB-2                     468           882            4.10
HGAB-3                     488           826            3.30
HGAB-11                    480           853            3.50
HGAB-12                    513           832            4.20
HGAB-18                    517           849            3.80
HGAB avg.                  493           848            3.78
LSD(0.05) ([paragraph])      3.81          4.54         0.02
LSD(0.01) ([paragraph])      5.46          6.51         0.03

                                            P

HWC vs. HGA                 <0.0001       <0.0001      <0.0001
HWC vs. HGB                 <0.0001       <0.0001      <0.0001
HWC vs. HGAB                <0.0001       <0.0001      <0.0001
HGA vs. HGB                 <0.0001        0.0171       0.0127
HGA vs. HGAB                <0.0001        0.5344      <0.0001
HGB vs. HGAB                 0.6412        0.0008       0.0246

                             Mixograph      Mix         Loaf
ID ([dagger])               absorption      time       volume

                           g [kg.sup.-1]

'Hi-Line'                     676           5.03       1410
161.00                        673           5.10       1378
HWC ([section])               674           5.07       1394
'Heron'                       538           2.13       1072
HGA-1                         649           4.90       1273
HGA-3                         649           5.13       1254
HGA-5                         635           3.53       1223
HGA-13                        638           4.45       1284
HGA avg.                      643           4.50       1258
HGB-2                         643           3.68       1279
HGB-5                         633           5.05       1284
HGB-6                         631           4.27       1330
HGB-12                        617           4.98       1096
HGB-16                        631           6.48       1284
HGB-19                        615           3.43       1121
HGB-21                        614           4.20       1271
HGB-35                        634           5.78       1270
HGB avg.                      627           4.73       1242
HGAB-2                        671           3.43       1346
HGAB-3                        563           1.25        526
HGAB-11                       570           2.48        726
HGAB-12                       488           4.30       1322
HGAB-18                       630           5.15       1153
HGAB avg.                     584           3.32       1015
LSD(0.05) ([paragraph])        10.22        0.96         45.71
LSD(0.01) ([paragraph])        14.65        1.38         65.57

                                              P

HWC vs. HGA                     0.4175      0.1287       <0.0001
HWC vs. HGB                     0.1766      0.3103       <0.0001
HWC vs. HGAB                    0.0188     <0.0001       <0.0001
HGA vs. HGB                     0.6053      0.4114        0.2132
HGA vs. HGAB                    0.0813      0.0004       <0.0001
HGB vs. HGAB                    0.1254     <0.0001       <0.0001

                                    Crumb
ID ([dagger])              grain ([double dagger])

'Hi-Line'                           3.50
161.00                              3.50
HWC ([section])                     3.50
'Heron'                             2.29
HGA-1                               3.75
HGA-3                               3.50
HGA-5                               2.75
HGA-13                              3.75
HGA avg.                            3.44
HGB-2                               3.25
HGB-5                               3.75
HGB-6                               3.00
HGB-12                              3.25
HGB-16                              3.25
HGB-19                              3.50
HGB-21                              3.50
HGB-35                              3.25
HGB avg.                            3.34
HGAB-2                              1.50
HGAB-3                              1.25
HGAB-11                             2.00
HGAB-12                             2.67
HGAB-18                             3.00
HGAB avg.                           2.08
LSD(0.05) ([paragraph])             0.55
LSD(0.01) ([paragraph])             0.79

                                      P

HWC vs. HGA                         0.7782
HWC vs. HGB                         0.4288
HWC vs. HGAB                       <0.0001
HGA vs. HGB                         0.5819
HGA vs. HGAB                       <0.0001
HGB vs. HGAB                       <0.0001

([dagger]) All homozygous [T.sub.3] lines were derived from the hard
spring wheat Hi-Line, which carries the pina-D1a and pinb-D1b alleles.
Heron is a soft wheat control cultivar.

([double dagger]) Based on 0 (unsatisfactory) to 5 (excellent) scale.

([section]) HWC denotes average of hard wheat controls (Hi-Line and
161).

([paragraph]) LSD values compare individual transgenic tines vs.
Hi-Line.

Table 4. Correlations between grain hardness and kernel traits,
milling traits, and bread baking traits for 17 pin transgenic and
two hard control lines based on mean of two environments.

Variable                SKCS ([dagger])    PSI ([double dagger])

                                                     %

Kernel weight               0.46 *               -0.55 **
Kernel diameter             0.82 **              -0.83 **
Wheat protein              -0.17                  0.32
Flour yield                 0.82 **              -0.77 **
Break flour yield          -0.79 **               0.87 **
Flour ash                   0.73 **              -0.67 **
Flour protein               0.72 **              -0.55 **
Mixograph absorption        0.52 **              -0.55 **
Mix time                    0.27                 -0.25
Loaf volume                 0.43 *               -0.38 *
Crumb grain score           0.39 *               -0.44 *

* Significantly different from zero at the 0.05 probability level.

** Significantly different from zero at the 0.01 probability level.

([dagger]) SKCS = single kernel characterization system.

([double dagger]) PSI = particle size index.

Table 5. Correlations among milling and bread baking traits for
17 pin transgenic and two hard control lines based on the mean
of two environments.

                                Break
                     Flour      flour      Flour     Flour     Loaf
Trait                yield      yield       ash     protein   volume

Break flour yield   -0.68 **
Flour ash            0.72 **   -0.66 **
Flour protein        0.51 **   -0.40 *    0.75 **
Loaf volume          0.33 *    -0.39 *    0.65 **   0.75 **
Crumb grain score    0.34 *    -0.46 *    0.28      0.28      0.63 **

* Significantly different from zero at the 0.05 probability level.


** Significantly different from zero at the 0.01 probability level.

Table 6. Whole wheat bread baking traits for controls and transgenic
lines based on mean of two replications from irrigated environment.

                  Flour     Flour    Mixograph     Mix      Loaf
ID ([dagger])    protein     ash     absorption    time    volume

                         g [kg.sup.-1]             min       cc

'Hi-Line'         151       17.3        624        2.90     770
HGA-13            150       18.0        621        3.20     693 *
HGB-6             152       17.2        633        2.70     768
HGAB-18           154       18.0        624        3.05     643 *
LSD(0.05)           5.3      0.90        44.9      0.38      74.9

                         Crumb
ID ([dagger])    grain ([double dagger])

'Hi-Line'                3.50
HGA-13                   3.50
HGB-6                    3.00
HGAB-18                  2.50
LSD(0.05)                1.13

* Significantly different from Hi-Line at the 0.05 probability level.

([dagger]) All homozygous [T.sub.3] lines were derived from the hard
spring wheat Hi-Line, which carries the pina-D1a and pinb-D1b alleles.

([double dagger]) Based on 0 (unsatisfactory) to 5 (excellent) scale.


ACKNOWLEDGMENTS

We acknowledge the expert technical assistance of Deanna Nash, Harvey TeSlaa, and Jackie Kennedy in the baking analysis and the helpful comments of Art Bettge and Doug Engle.

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A. C. Hogg, B. Beecher, J. M. Martin, F. Meyer, L. Talbert, S. Lanning, and M. J. Giroux *

A.C. Hogg, J.M. Martin, F. Meyer, L. Talbert, S. Lanning, and M.J. Giroux, Dep. of Plant Sciences and Plant Pathology, Montana State Univ., Bozeman, MT 59717-3150; B. Beecher, Dep. of Agronomy and Horticulture, Univ. of Nebraska-Lincoln, Lincoln, NE 68583-0915. This research was supported by USDA-ARS National Research Initiative Competitive Grants Program grants 1999-01742, 2001-01728, 2004-01141, and by the Montana Agricultural Experiment Station. Received 23 Feb. 2004. Genomics, Molecular Genetics & Biotechnology. * Corresponding author (mgiroux@montana.edu).

doi:10.2135/cropsci2004.0113
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Title Annotation:GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY
Author:Hogg, A.C.; Beecher, B.; Martin, J.M.; Meyer, F.; Talbert, L.; Lanning, S.; Giroux, M.J.
Publication:Crop Science
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Date:May 1, 2005
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