Pattern of grain set in boron-deficient and coldstressed wheat (Triticum aestivum L.)

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1 Journal of Agricultural Science, Cambridge (2), 134, Cambridge University Press Printed in the United Kingdom 25 Pattern of grain set in boron-deficient and coldstressed wheat (Triticum aestivum L.) K. D. SUBEDI *, P. J. GREGORY, R. J. SUMMERFIELD AND M. J. GOODING Plant Environment Laboratory, Department of Agriculture, University of Reading, Cutbush Lane, Shinfield, RG2 9AD, UK Department of Soil Science, University of Reading, Whiteknights, PO Box 233, Reading, RG6 6DW, UK (Revised MS received 2 July 1999) SUMMARY When crops of wheat (Triticum aestivum L.) are stressed, grain set in potentially fertile florets is reduced. Cold temperatures and boron (B) deficiency during reproductive development cause grain set failure in wheat. Patterns of grain set in cold-stressed and B-deficient wheat ears were studied under field conditions in Nepal and in controlled environments in the UK. In both B-deficient and cold-stressed circumstances, ear fertility was reduced by up to 98% but the pattern of grain set within an ear was similar. Under cold-stressed conditions, florets in the uppermost of the ear were 41 to 53% less fertile than those located in the middle and basal regions. Even in the unstressed crops, the top of the ear was less fertile than below by as much as 8 13%. Similarly, within a spikelet, the distal florets always had fewer grains than the proximal ones. Decreased grain set following stress markedly reduced yield per ear. We conclude that fertility should be assessed on the entire ear. The determination of competent florets should be based on the presence of well-developed ovaries, feathery stigmas and the structures of anthers (which can still be seen in the sterile florets at maturity) rather than on the length of the lemma or on judgements based on visual appearance or other subjective criteria. INTRODUCTION Failure of grain set (i.e. sterility) in fully developed florets of wheat (Triticum aestivum L.) has been reported from several countries. The problem is particularly troublesome in subtropical Asia (e.g. India, China, Nepal, Bangladesh and Thailand) where under severe conditions complete crop failure can occur (Subedi et al. 1996). Grain set can be reduced by either adverse environmental or edaphic conditions prior to and during anthesis, through effects on floral development and fertilization (Evans et al. 1972). Those environmental stresses which cause sterility when experienced during the reproductive development include boron (B) deficiency (Rerkasem & Loneragan 1994; Subedi et al. 1997a), cold (Langer & Olugbemi 197; Subedi et al. 1998a), heat (Saini & Aspinall 1982), drought (Saini & Aspinall 1981) and * To whom all correspondence should be addressed at: Sustainable Soil Management Programme, GPO Box 688, Bakundol, Kathmandu, Nepal. psussmp wlink. com.np limited radiation (Demotes-Mainard et al. 1995). Florets may also fail to set grains if the supply of assimilate to the ear is limited during and just after anthesis (Rawson & Ruwali 1972; Warrington et al. 1977). Boron deficiency and cold temperature-induced sterility are especially common constraints in field grown wheat in subtropical Asia (Rawson 1996; Subedi et al. 1996) and severe yield losses have been reported (e.g. Subedi et al. 1997b, 1998b). Each spikelet of the wheat spikelet contains eight to ten florets but only four to six are potentially fertile (Kirby & Appleyard 1987). The distal florets do not grow sufficiently large enough to contain an ovary or any anthers (Rawson 1996). However, seldom if ever, do all the fertile florets set grains (Evans et al. 1972; Subedi et al. 1996) so that in practice wheat ears are at least partially sterile. The pattern of grain set has important implications on the quantification of sterility or fertility in an ear and the actual yield losses due to sterility. There are no detailed reports showing how the pattern of grain set within an ear is affected by B-deficiency and cold temperature. Different workers have defined sterility in various ways and because there is not a single standardized

2 26 K. D. SUBEDI ET AL. method for estimating it, the impact on seed yield can be assessed quite differently. For example, Saini & Aspinall (1982) used the term percentage grain set which they estimated as the proportion of the florets with grains, having first visually determined the number of fertile florets. In contrast, da Silva & de Andrade (1983) assessed the percentage male sterility and Marcellos & Single (1984) quantified ear fertility as the number of grain-set per spikelet. Sthapit (1988) characterized sterility based on the number of florets without grains in an ear although the number of florets per ear was only calculated as three times the numbers of spikelets. Mishra et al. (1992) estimated sterility by hand feeling of the ear at maturity. The definition of sterility given by Rawson (1996) as the absence of grain in any floret that grows sufficiently large to contain a grain is an improvement on earlier definitions but the term sufficiently large needs to be specified precisely. The method of estimating sterility used by Sthapit et al. (1989) is quite common, whereby: Number of florets per ear Number of grains per ear Sterility (%) Number of florets per ear 1 and is designated hereafter as the Lumle method (LM). This method has the drawback that the competent florets are not defined. There is a risk therefore of overestimation of sterility if the presence of naturally sterile florets is not taken into account. A similar method has been used by Rerkasem & Loneragan (1994) who defined a grain set index (GSI) as the percentage (%) of grain set in the two basal florets (F and F ) of ten central spikelets: Number of gains in F and F GSI of 1 central spikelets 2 1 This method is designated hereafter as GSI, and gives sterility (%) as (1 GSI). When Subedi et al. (1993, 1996) compared these two methods in various field experiments in Nepal, the GSI method underestimated sterility by 8 to 15% compared to LM. However, there was a chance of overestimation by LM if the competent florets were not determined accurately. Furthermore, in the surveys of Subedi et al. (1993), neither method was related to the number of grains per ear nor to total grain yield per ear so that the ability of the methods to quantify yield losses remains unclear. In the research reported here the pattern of grain set under stress conditions was studied and the LM and GSI methods of estimating sterility were compared. The objectives were to examine the pattern of grain set in wheat ears when plants were B-deficient and cold-stressed and to compare and evaluate the reliability of the two aforementioned methods. MATERIALS AND METHODS The work reported here involved measurements from two separate field studies in Nepal and from one controlled environment experiment undertaken in the UK. Field observations under cold-stressed conditions Twenty advanced accessions of spring wheat were grown at the Lumle Agricultural Research Centre Farm in Nepal at 1675 m a.s.l., N and E, where the problem of cool temperature induced sterility is severe. During the flowering period (January) of the October sown wheat, the night temperatures fall between 1 C and 5 C and the diurnal mean temperatures remain 1 C (Subedi et al. 1998a). The experiment was sown on 2 October 1994 in three randomized complete blocks and all plants had matured by the middle of April The land used was a rainfed hill terrace. The crop was fertilized with 2 t ha farmyard manure and 8:4: 2 kg ha N, P O and K O. One half of the N and all of the P and K were applied as a basal dose and the remaining N was side-dressed 3 d after sowing. Each experimental plot was 2 m long with 12 rows each 25 cm apart. Seeds were sown in furrows at 12 kg ha. At the beginning of anthesis (GS 6; Zadoks et al. 1974), symptoms of non-fertilization were observed in ten cultivars as transparent and gaping glumes. These accessions (see Table 1) as well as Annapurna-3, a reputedly cold-tolerant cultivar used as a standard check, were chosen for further investigation. At GS 6, three main shoot ears of each of the eleven accessions were tagged at random in each plot, which were then harvested at physiological maturity (GS 93). In each ear, the total number of spikelets was counted. The spikelets were then divided equally into three groups (basal, middle and upper) within the intact ear and the numbers of competent florets and grains in each floret position within a spikelet were recorded separately for each group. Sterility was calculated by the LM. Field observations under boron-deficient conditions Six commercial spring wheat cultivars (Table 2) grown under farmers management at Rishingpatan (42 m a.s.l.) in Nepal (28 1 N and 84 2 E) were studied on soils where B-deficiency had been confirmed by soil analysis and measurement of B- deficiency induced sterility in previous years (Subedi et al. 1993, 1996, 1997b). Boron concentrations in the soils were mg kg (Subedi et al. 1997b). The night temperature during heading to anthesis

3 Pattern of grain set in boron-deficient and cold-stressed wheat 27 Table 1. Patterns of grain set in the basal, middle and uppermost portions of ears of different cultivars of wheat grown under cold conditions at Lumle, Nepal. Figures in parentheses are numbers of competent florets S.E.M. (means of nine observations) Percentage of competent florets with grains Cultivar Basal Middle Uppermost Entire ear Annapurna (25 2 ) 88 5 (26 1 7) 78 9 (2 1 6) 84 WK (15 1 2) 62 5 (16 1 4) 33 3 (18 1 1) 54 2 WK (18 1 5) 61 8 (17 1 3) 44 (18 1 7) 53 8 NL (21 1 ) 54 5 (22 1 4) 44 4 (18 1 ) 51 9 BL (16 1 3) 44 4 (18 1 2) 17 6 (17 1 2) 33 2 WK (2 1 5) 15 (2 1 7) 5 (2 7) 15 JS-5 25 (18 1 ) 13 5 (19 1 2) 4 7 (18 1 1) 14 4 RR (13 5) 14 (14 7) 7 7 (13 5) 12 2 HD (14 8) 12 5 (16 9) 7 7 (13 1 ) 9 1 NL (18 1 ) 7 5 (2 1 ) 1 5 (17 1 ) 5 3 NL (19 1 ) 5 (2 9) 1 8 (19 9) 1 6 Mean 34 8 (18 1 1) 34 (19 1 3) 22 4 (17 8) 3 4 Table 2. Patterns of grain set in the ears of six cultivars of wheat grown under B-deficient and B-sufficient conditions at Rishingpatan (42 m), Nepal. Figures in parentheses are numbers of competent florets per ear S.E.M. (mean of 1 observations) Percentage of competent florets with grains (%) B-deficient B-sufficient (control) Cultivar Basal Middle Uppermost Basal Middle Uppermost RR-21 NL-297 BL-122 BL-166 BL-1135 Annapurna-3 Mean 71 4 (14 3) 58 7 (16 3) 47 4 (19 3) 36 1 (18 3) 2 5 (19 3) 13 9 (18 3) 41 (17 2) 71 3 (15 3) 53 1 (16 3) 36 8 (19 4) 33 3 (18 2) 18 9 (19 3) 12 2 (18 2) 37 6 (18 2) 6 9 (11 4) 3 (12 3) 9 3 (15 3) 21 4 (14 3) 1 3 (14 4) 7 1 (14 3) 23 (13 1) 76 5 (15 4) 75 5 (17 3) 87 3 (19 3) 8 (19 4) 86 4 (2 4) 81 8 (19 4) 77 9 (18 2) 82 4 (15 4) 77 8 (17 3) 73 3 (2 3) 7 7 (19 3) 87 (21 4) 8 9 (19 4) 8 2 (19 2) 69 2 (13 4) 65 4 ( (15 4) 6 (15 4) 78 3 (16 5) 68 4 (15 4) 68 7 (15 1) stage (January and February) ranged between 7 4 C and 9 9 C and mean temperature ranged between 14 2 C and 16 5 C. One hundred ears of each cultivar were sampled at random from each of the B-sufficient plots (where no symptoms of sterility had been observed) and B-deficient plots where transparent and gaping glumes symptoms were observed. The pattern of grain set was determined as previously described. Controlled environment investigation Two cultivars of spring wheat from Nepal (Annapurna-3 and NL-683) were sown on 1 April 1996 into 18 cm diameter plastic pots containing crushed gravel, washed sand and loamless peat compost (4:2:1 v v) supplied with nutrient solution with or without 2 µm B l at the Plant Environment Laboratory at the University of Reading, UK (Subedi et al. 1998b).

4 28 K. D. SUBEDI ET AL. Plants were grown outdoors until the flag leaf ligule was visible (GS 39) when they were transferred to growth cabinets maintained at 8 2 C day night temperatures for 14 1 h d with a photoperiod of 16 h d. This temperature treatment was given during three stages: from GS 39 to awn first visible (GS 49), from GS 49 to GS 59 or from GS 59 to when anthesis was complete (GS 69). One half of the plants remained outdoors throughout. The diurnal mean outdoor temperature during the period from GS 39 to GS 69 was C. Immediately after ear emergence, ears were bagged with glassine paper bags to prevent any chances of out-crossing. The main shoot ears were harvested at crop maturity and the pattern of grain set was measured (i.e. the number of spikelets per ear, number of competent florets per spikelet, and grain set in each competent floret position). A floret was considered as competent if it contained a welldeveloped ovary, feathery stigma and anthers. This mapping protocol also enabled the number of grains in the two basal florets of the ten central spikelets to be determined. Sterility was estimated by both of the methods described previously. The weight of grains in each ear was recorded after drying at 85 C for 72 h. Analysis of variance was performed and correlations were determined between the total grain weight per ear and percentage sterility as estimated by the two methods. RESULTS AND DISCUSSION Cultivar responses to cold and boron Cultivars tested in the field differed greatly in their response to both cold temperature stress (Table 1) and B-deficiency (Table 2). In the controlled environment conditions, cold had more effect on sterility (Table 3) than B-deficiency. Detailed results on the effects of cold and B-deficiency have already been published in Subedi et al. (1998b). Sterility was as high as 98% under either stress. There were also contrasting responses of cultivars to stress: for example, in the fields, Annapurna-3 was little affected by cold but was the most susceptible to B-deficiency, whereas the response of RR-21 was the opposite. In the controlled environment study, NL-683 was more severely affected by cold than Annapurna-3. The pattern of ear fertility under both cold stress and B- deficiency was similar. Even on the unstressed crops, between 16 and 23% of competent florets did not contain grain (Tables 1 and 2). Spikelet position and fertility On average spikelets were present in each ear in the different cultivars when grown under field conditions compared with 17 spikelets per ear under controlled environment conditions. The percentage of competent florets with grains in the basal and middle regions of the ear recorded in the field were similar (34 to 41%) whereas the uppermost portion had less (22 to 23%) (Tables 1 and 2). Averaged over the two field experiments, floret fertility in the uppermost onethird portion of the ear was 53% less than in the middle portion and 41% less than in the basal portion. Even when B-sufficient, the spikelets in the uppermost of the ear were reduced by 13% more than those in the middle and basal parts of an ear (Table 2). These field observations were confirmed in controlled environments where the fertility of the apical spikelets was substantially less than that in the middle and basal spikelets (Fig. 1). Under cold stress, few if any grains were set in the apical spikelets, especially in NL-683. The basal spikelets were also less fertile than those in the middle third although not to the same extent as in the distal region. Marcellos & Single (1984) also found that the fertility of spikelets in the middle third of the ear was greater in both frosted and non-frosted ears. During ear development, the most advanced spikelets are always found in the middle third of the ear whereas those in the apical third are the last to appear and their formation coincides with the elongation of the main culm internodes and the enlargement of the upper leaves (Rawson & Evans 197). Floret position and fertility The numbers of competent florets were similar in the basal and middle regions of each ear but there were fewer in the uppermost region because apical spikelets usually have fewer florets than Table 3. Differences in sterility (%) of wheat ears from plants grown under ambient temperatures and cold stress conditions (8 2 C, day night temperatures) estimated by the LM and GSI (averaged over two wheat cultivars, three stages of exposure to cold stress and three boron nutrition regimes) Sterility (%) Treatment LM GSI Difference Control (plants in the outside) C day night temperature

5 Pattern of grain set in boron-deficient and cold-stressed wheat 29 Proportion of florets with grains (%) Spikelet position Fig. 1. Floret fertility (%) in relation to position of the spikelet within the ear, from the base (1) to the most distal apical region (17), in each of two cultivars of wheat ( Annapurna-3 and NL-683) grown under ambient temperatures (solid symbols ) and cold stressed (open symbols) at the Plant Environment Laboratory, Reading, UK. Proportion of florets with grains Floret A Floret B Floret C Floret D Floret position in a spikelet Floret E Fig. 2. Fertility (%) of different competent florets according to position within the spikelet in two cultivars of wheat ( Annapurna-3 and NL-683) grown with (solid symbols) or without (open symbols) 2 µm B l. The bars are the S.E.M. proximal ones (Subedi et al. 1998b). Within a spikelet, the fertility of the distal florets was lower in both stressed and unstressed crops (Fig. 2). Rawson & Evans (197) found that the development of grains in the earliest florets to reach anthesis restricted grain set in later florets. Evans et al. (1972) confirmed that the position of florets within an ear was important for grain set. Rawson & Ruwali (1972) suggested that limited carbon availability from just before to just after anthesis can lead to sterility in distal florets and especially in those located towards the apex of the ear. In this study too, the failure of grain set in the competent florets was greater in the distal florets than in the basal ones. Relative sterility (%), GSI Relative sterility (%), LM Fig. 3. Relation between the estimates of ear sterility (%) made by the LM and GSI methods. The data are from the combinations of two cultivars, three boron nutrition, two temperature treatments and three stages of the exposure to cold temperature in the controlled environment study. The diagonal line represents y x. Estimated sterility and grain yield Comparisons between the LM and GSI methods in the controlled environment study showed that the GSI method underestimated sterility by 7% (32 c.f. 39%) compared with the LM (Table 3). When sterility was high, the difference between estimates was small and vice versa. In those ears which had moderate or relatively little sterility, the LM estimated slightly greater sterility. When the GSI indicated no sterility (i.e. %), the LM showed as high as 2% sterility (Fig. 3). This is not surprising given that the chances of failure of grain set are greater in the apical and basal spikelets (Tables 1 and 2), which this method takes into account but which the GSI method does not. However, if ears are severely sterile, then the spikelets in the middle and basal florets are also affected and so the estimates based on both methods are similar. Not only did the GSI method underestimate actual sterility in an ear but the standard errors of the means were also almost twice as large as those for the LM. This reflects the fact that under stress, the numbers of grains were reduced mostly in the apical spikelets (Tables 1 and 2, Fig. 1) and distal florets rather than in the proximal ones (Fig. 2). Because the GSI method is only based on the basal two florets of the 1 central spikelets it does not represent the actual grain set in an entire ear; thus it invariably underestimates sterility, typically by about 8 to 15% (Subedi et al. 1993). Individual grain weight decreased as the position of the florets became more distal but the weights of such grains are excluded if sterility is estimated by the GSI. Rawson & Evans (197), Rawson & Ruwali (1972) and Warrington et al. (1977) have found that distal grains were smaller than proximal ones. Bremner &

6 3 K. D. SUBEDI ET AL. Sterility (%) Sterility (%) Grain wt (g) No. of grains per ear Grain weight per ear (g) Fig. 4. Relationship between the number of grains per ear with estimated sterility (LM) and grain yield per ear. The data are from the combinations of two cultivars, three boron nutrition, two temperature treatments and three stages of the exposure to cold temperature in the controlled environment study. Rawson (1978) described how grains in the terminal spikelets were the smallest ones and that those in the basal one-half of the ear were generally the largest. Within the central spikelets, at least, grain weight increases from the basal floret to the second floret and decreases thereafter towards apical florets. Therefore, if only the ten central spikelets and their basal florets are assessed, the distal parts which always have lower fertility and lighter grains will be under-represented. Hence, the GSI method did not accurately represent the actual yield loss due to sterility or the true distribution of sterility within an ear. In contrast, the LM (i.e. assessment of the whole ear) represented the actual sterility in an ear, as well as the variations in the weight of grains per ear. Therefore, the actual yield loss due to sterility will be correspondingly better explained by the LM. The relationship between the number of grains per ear, grain yield per ear and corresponding sterility percentage by LM is shown in Fig. 4. Given that the LM seems to provide a better representation of the actual yield loss due to sterility, the accuracy of this method nevertheless depends on how the competent florets are determined. Rawson & Bagga (1979) defined a competent floret based on the length of lemma: a fertile floret, they judged, is one having lemma 3 mm. However, this criterion does not directly assess the fertility status of a floret and such measurements are not practical in the counting of fertile florets. The assessment of a fertile floret should therefore be judged by the presence of welldeveloped anthers, stigma and ovary structures because these floral parts can still be seen in sterile florets even at crop maturity. We conclude that whatever the causes of sterility, the whole ear should be assessed. Once the competent florets are determined, the assessment of ear fertility by the LM is simple, accurate and widely applicable for agronomic and physiological research and for plant breeding. We thank Lumle Agricultural Research Centre, Nepal and the Department for International Development (DFID) for the study leave and financial support for K. D. Subedi. 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