Vol. 76, No. 1, 1999 139 Variation in Grain Mass, Grain Nitrogen, and Starch B-Granule Content Within Wheat Heads F. L. Stoddard 1 ABSTRACT Cereal Chem. 76(1):139-144 Grain mass (mg) and grain nitrogen concentration (%) were determined in 3,278 individual grains from eight cultivars with reference to the posi- tion of the grain on the head (spikelet number and floret number). Selected grains were removed from certain heads at anthesis. Grain nitrogen con- tent (mg) was determined as the product of grain nitrogen concentration and grain mass. Grain mass and starch B-granule content were determined in 3,030 grains from a further 12 cultivars with reference to grain position, and selected grains were removed from certain heads at anthesis. Grains from distal florets were always smaller and had lower B-granule contents, nitrogen contents, and nitrogen concentrations than those from the two proximal florets on each spikelet, which were not significantly different from each other. Grains in the basal two spikelets of the head were smaller with lower nitrogen contents and higher B-granule contents than those in most of the head. Their nitrogen concentration, however, did not differ from that in the rest of the head. All four traits declined in the grains in the top four to five spikelets of the head. The differences in grain mass, nitrogen, and B-granule content between florets within spikelets and between spike- lets within the head varied with cultivar. Grains on treated heads were larger, higher in nitrogen and slightly lower in B-granule content than those on untreated heads. The effects of floret and spikelet on grain mass and nitrogen were not significantly changed by the treatments. Grains from florets 1 and 2, excluding those from the bottom three and top five spike- lets, therefore represented a particularly uniform population for grain mass, protein nitrogen and starch granule composition. Genetic analyses based on samples of these grains will be more robust and repeatable than those based on unselected samples. Implications for improving seedling vigor, plant yield, and grain protein concentration in breeding programs are discussed. It has widely been observed in wheat, as in other crops, that large grains grow into more vigorous seedlings and higher yielding plants than small ones (Ries and Everson 1973, Evans and Bhatt 1977). Similarly, grains with a higher protein concentration (GPC) at a given size produce plants that outperform those with lower GPC (Millet and Zaccai 1991). It therefore appears that grain protein content (in milligrams per grain rather than percent) is an important contrib- utor to the subsequent seedling vigor. Nevertheless, modern cultivars often have smaller grains (Fjell et al 1985) and lower GPC (Stoddard and Marshall 1990) than old varieties. This effect has been attributed to the combination of gains in yield and a common negative correlation between yield and GPC (Simmonds 1995). A major step in yield has been attrib- uted to the Rht genes, which in turn have been associated with a direct reduction in both grain mass and GPC in near-isogenic lines (Allan 1986, 1989; Pinthus and Gale 1990). It has therefore been proposed that restoring grain mass would move the yield-protein relationship to a higher level (Fjell et al 1985; Stoddard and Mar- shall 1990). The mechanism behind the relationship between grain mass and grain nitrogen concentration is one subject of this article. New technologies offer opportunities for looking with greater detail at variation among individual grains. While grain mass could be determined with a micro-balance some years ago, the nitrogen content could not be determined until relatively recently. Micro- Kjeldahl methods required the pooling of several grains to get enough material for analysis. Questions about the partitioning of assimilate within the head, or about the contributions of maternal, embryo, and endosperm genotype to the composition of a grain, could be addressed only indirectly and with difficulty. Single-grain analysis for a number of components is becoming more popular (Delwiche 1995). If we are to apply such methods in a breeding pro- gram we need to have a map of the distribution of the quality factors within the head. We would then be able to focus on the grains that provide the best expression of the plant’s potential without the confounding influence of poorer grains. MATERIALS AND METHODS Grain Mass and Nitrogen Eight cultivars of wheat were sown in four replicates, one plant per 20 cm pot, in an unheated glasshouse. The potting mix was three parts sand to two parts peat by volume, with lime and a complete fertilizer mix added. Plants were fed weekly with half-strength com- mercial water-soluble fertilizer (N:P:K 23:4:18, Aquasol, Hortico Ltd., Laverton North, Vic., Australia). One head on each plant was left as a control. In another, the middle florets were removed from each spikelet within two days of anthesis, leaving only the first and second florets. In a third head, the first floret was removed from each spikelet, leaving all distal ones. The two treatments and control were applied in random order to the first three heads on each plant. Heads were harvested intact at maturity. Each grain was removed from the head, its spikelet number and floret number were recorded, and it was weighed to the nearest 0.1 mg. The grain’s nitrogen con- centration (GNC%) was then determined by the Dumas total com- bustion method using an elemental analyzer (CHN-1000, Leco Inc., St. Joseph, MI). The grain nitrogen content (GNmg) was then calculated from the product of GNC and grain mass. In total, 3,278 grains were analyzed. Starch B-Granule Content Twelve cultivars of wheat were grown in a controlled-environ- ment chamber, one plant per 15 cm pot, with three replicates. The chamber was set to a 14 hr day at 18°C and a 10 hr night at 13°C. The potting mix and fertilizer application were the same as in the previous experiment. In four heads on each of five cultivars, the bottom two and top four spikelets and the distal florets from the remaining spikelets were removed, the glumes were trimmed and a paper bag was placed over the head. Heads were harvested intact at maturity. Two untreated heads per plant were analyzed. Each grain was removed from the head, its spike- let number and floret number were recorded, and it was weighed to the nearest 0.1 mg. The starch was extracted and cleaned for par- ticle size analysis. Each grain was crushed with a pair of smooth- jawed pliers, put into a 2-mL Eppendorf microfuge tube with 0.5 mL of 0.5M NaCl, and soaked overnight at 4°C. The next morning it was ground with an Eppendorf pestle attached to a drill press until the gluten formed a tight ball. The starch slurry was decanted through a 0.2-mm mesh sieve into a fresh microfuge tube, and the residue 1 Plant Breeding Institute, Woolley Bldg A20, The University of Sydney, NSW 2006, Australia; Quality Wheat CRC Ltd, Locked Bag No. 1345, North Ryde, NSW 2113, Australia. Phone: +61 2 9351 4594. Fax: +61 2 9351 4172. E-mail: Stoddard@mail.usyd.edu.au Publication no. C-1999-0107-07R. © 1999 American Association of Cereal Chemists, Inc.