Maize pollen mediated gene flow in the Po valley (Italy): Source–recipient distance and effect of flowering time

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Europ. J. Agronomy 28 (2008) 255–265

Maize pollen mediated gene flow in the Po valley (Italy): Source–recipient distance and effect of flowering time Giovanni Della Porta a,1 , Davide Ederle b,1 , Luca Bucchini c , Matteo Prandi c , Alberto Verderio a , Carlo Pozzi b,∗ b

a CRA-Stezzano, via Stezzano 24, Bergamo, Italy Fondazione Parco Tecnologico Padano, via Einstein loc. Cascina Codazza, 26900 Lodi, Italy c Hylobates Consulting SrL via Gaggiano 42, 00135 Roma, Italy

Received 14 December 2006; received in revised form 24 July 2007; accepted 30 July 2007

Abstract Gene flow in maize can be monitored by measuring the cross-fertilization rate from a pollen source to a pollen recipient plot. According to European Commission Recommendation 2003/556, co-existence measures should allow non-GM crops to be grown and marketed so that the adventitious presence of GM material does not exceed the labelling threshold of 0.9% set by EC Regulation 1829/2003. Using dominant phenotypic markers we have investigated in farm scale fields in the Po Valley (Italy) the effect of: distance between the pollen source and recipient plants, with and without pollen competition; wind; synchrony in flowering times, in determining cross-fertilization. To this purpose, three types of experimental fields were designed: in type 1, a block of pollen source was planted in the middle of a recipient field; in fields of type 2, the source was separated from the recipient maize by fallow soil and/or maize buffer zones of variable shape and dimension; in type 3 experiments, the pollen source was planted within a recipient field of maize hybrids having different growing cycle lengths (and, hence, differing flowering synchrony). The following conclusions could be drawn: (1) the 0.9% cross-fertilization threshold was reached within, on average, 10 m in type 1 experiments (but exceptionally at 25 m); 17.5 m in type 2a experiments; 1.5 m for areas contiguous to pollen source or to recipient in type 2b experiments; (2) the influence of wind was minor compared to distance between pollen source and recipient; (3) buffer maize plants that shed non source pollen, rather than fallow land, were the most efficient barrier against cross-fertilization. Type 3 experiments allowed to conclude that: (1) little or no reduction in pollen flow was observed if there were only up to 3 days of difference in flowering time between pollen source and recipient; (2) when the time interval was 4–5 days a 25% reduction of pollen flow was recorded; (3) when the time interval was 6 days, the reduction was 50%, reaching levels close to 0% when the off-set was higher than 7 days. © 2007 Elsevier B.V. All rights reserved. Keywords: Coexistence; Maize; Pollen flow

1. Introduction Maize (Zea mays) is a monoecious allogamous species increasingly improved by the expression of technological traits, such as resistance to pests and production of vaccines, industrial enzymes and plant-made pharmaceuticals (Bates et al., 2005). Under such circumstances, the establishment of robust measures to assure appropriate confinement of transgenic crops is a recognized necessity (Ervin et al., 2003). In fact, foreign genes can be transmitted via pollen dispersal to conven-

∗ 1

Corresponding author. Tel.: +39 0371 4662200; fax: +39 0371 4662349. E-mail address: [email protected] (C. Pozzi). These authors made equal contribution to this work.

1161-0301/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.eja.2007.07.009

tional genotypes adjacent to GM fields (Eastman and Sweet, 2002). Pollen-mediated gene flow to non-GM plants of the same species is particularly relevant in maize due to its biology and to the characteristics of its pollen. A single maize inflorescence produces from 2 to 5 million pollen grains (Goss, 1968). Pollen shedding may last for 5–6 days (Westgate et al., 2003) and is normally not synchronous with silking, conditions favouring a high level of cross-fertilization. Gene flow can be monitored by assessing the crossfertilization rate from a pollen source field to a recipient plot. Several studies have considered the dispersal of pollen under natural conditions, revealing a substantial decrease of pollen grains within the first meters from the source: the maximum recorded distances average 200 m (Luna et al., 2001) although no clear

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cut-off distance, beyond which the level reaches zero, has been identified (Sears and Stanley-Horn, 2000; Pleasants et al., 2001). A number of factors – particularly air humidity and temperature – are important in determining pollen viability (reviewed in Ma et al., 2004). Since the late 1940s and 1950s (Bateman, 1947; Jones and Brooks, 1950, 1952), a number of experiments have been carried out to evaluate gene flow in maize. Most of the studies were conducted by using a pollen source carrying a dominant kernel marker and determining the rate of cross-fertilization in the surrounding or adjacent fields, characterized by the recessive allele (reviewed in Devos et al., 2005). The rate of appearance of such markers in the progeny of a sampled plant is the measure of pollen dispersal, which also depends on the dilution effect due to the pollen produced by the recipient. More recently, studies have been carried out using GM traits as genetic markers, with source plots as large as 0.2 and 0.4 ha (reviewed in Devos et al., 2005), and analysing gene flow on-farm in Spain (Brookes and Barfoot, 2003). Rate of cross-fertilization depends on several factors: distance between pollen source and recipient; size, shape and orientation of pollen source and recipient; wind intensity and direction; quantity of rain; pollen viability and its water content; other climatic conditions; degree of male fertility. In all studies, the decrease of cross-fertilization below the 1% threshold occurred at distances of 30 m or less (Ma et al., 2004). Male and female flowering synchrony has an important effect on the rate of cross-fertilization (Duplessis and Dijkhuis, 1967): the closer the synchrony between pollen source and recipient, the higher the probability of cross-fertilization (Angevin et al., 2001; Bock et al., 2002; Westgate et al., 2003). Silks are receptive for about 5 days and senesce within 8 days if not fertilized (Anderson et al., 2004). The existence of different degrees of synchrony accounts for the impossibility to directly relate pollen dispersal with gene flow. Experiments to evaluate differences in flowering time between pollen source and recipient as a mean to increase genetic isolation have been conducted indirectly (i.e. evaluating the importance of factors affecting synchronous flowering, Ma et al., 2004) or directly (i.e. studying the deliberate temporal separation, Halsey et al., 2005). US guidelines for complete isolation (i.e. absence of gene flow) of GM maize require 1600 m for fields planted at the same time, but half the distance if there is a 4-week time-shift (USDA-APHIS). For hybrid production the same guidelines suggest an isolation of 185–200 m (Luna et al., 2001). Conclusions inferred from studies carried out in specific experimental or climatic situations should be applied with caution to different environments (Luna et al., 2001; Aylor, 2004). Thus, further studies are needed to determine to what extent the available data can be applied to local situations, where GM and non-GM varieties are sown at different dates and with different separation measures, all conditions that could influence pollen flow. According to European Commission Recommendation 2003/556 co-existence measures should allow non-GM crops to be grown and marketed ensuring that the adventitious presence of GM material does not exceed the labelling threshold of

0.9%. This work is devoted to study, for the first time in the Po valley, the effects on the rate of maize pollen mediated gene flow of separation distances, barriers, and flowering asynchrony. 2. Materials and methods 2.1. Field experiments Three types of field experiments were carried out: the first to study cross-fertilization as a function of distance and wind; the second to evaluate the effect of isolation and buffer zones (border rows); the third mainly to evaluate the effect of flowering synchrony. Experiments were conducted in the growing season 2005 at six sites in Lombardy, Italy, reported in Table 1 according to their eastwards geographical coordinates. Structure and orientation of the experimental fields are reported in Fig. 1. Soil texture was silty-loam at all locations. Around the fields at least 100 m in all directions were free of any kind of barriers opposing to wind flow. After thinning a density of approximately 7 plants/m2 was attained (distance between rows: 0.75 m in type 3; 0.7 m in others). Sites are indicated by the acronyms of the locations, followed by the indication of the type of experiment, as specified in Table 1. Dates of sowing were March 25th at site Ma/1; 21st at Pi/1; April 4th at Ca/2a; May 21st at Ti/2b; April 26th at As/3 and April 20th at Ta/3. During the growing season irrigation was supplied to avoid drought effects on pollen viability. Table 1 and Fig. 1 summarize agronomic characteristics of the experimental sites, including information on hybrids used and kernel colour. Recipient maize had white kernels at sites Ca/2a, Pi/1, Ti/2b and Ma/1 (with a yellow source) and yellow at sites Ta/3 and As/3 (with a purple source). Yellow is dominant over white; purple over yellow. Purple kernel B73xMo17 hybrid was obtained by crossing the two lines B73 ACR and Mo17 ACR developed at the Istituto Sperimentale per la Cerealicoltura, Bergamo (Italy). In type 1 experiments, a block of yellow maize was planted at the centre of the field and surrounded by at least 70 m of recipient white maize (Fig. 1A). In the type 2a experiment, a block of yellow (source) maize was sown in the centre of the field. The field was divided in three parts, along the longer side. In the first part, the recipient white variety was planted contiguously to the source. In the second part, the recipient was at 17.5 m from the source (i.e. 25 maize rows, given an inter-rows distance of 0.7 m). In the third part the recipient was planted at 34.3 m from the source (Fig. 1B). The type 2b field consisted of three parts, separated by 9.1 m empty corridors. The central part was planted with yellow maize (pollen source); the two lateral parts were planted with white maize (recipient). Buffer zones of twelve rows of white maize were planted within the source or within the recipient plots (Fig. 1C). The set-up of type 3 fields (sites As/3 and Ta/3) was as follows: a 6 m × 17.5 m block of purple maize (pollen source) was planted at the centre of a 87-row yellow maize recipient field. Growing degree units (GDU) were the same for the source and most of the recipient hybrids. Four-row plots of 78 yellow maize

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Table 1 Experimental sites: dimensions, replicates and genetic material used as pollen source and pollen recipient Site

Location

Design

Geographical coordinates

Dimensions (m)

210 × 260

Replicates

Size of source plot (m)

Hybrid namea Pollen source

Pollen recipient

190 × 73

Costanza (600) yellow Eleonora (700) yellow

PR32B10 (600) white

B73xMo17 (600) purple

PR32A46 (500) yellow Costanza (600) yellow PR33506 (500) yellow Eleonora yellow

Ca/2a

Carpiano

2a

45◦ 19 N; 9◦ 16 E

Ta/3

Tavazzano

3

45◦ 19 N; 9◦ 23 E 66 × 70.3

3

Pi/1

Pizzighettone

1

45◦ 11 N; 9◦ 46 E

260 × 320

1

65 × 88

Lolita (600) yellow Costanza (600) yellow PR31K18 (700) yellow

PR32B10 (600) white Damiana (600) white

Ti/2b

Ticengo

2b

45◦ 21 ; 9◦ 49

120 × 190

1

125 × 85

Costanza (600) yellow Eleonora (700) yellow

PR32B10 white

As/3

Aspice

3

45◦ 12 N; 9◦ 54 E 66 × 70.3

3

B73xMo17 (600) purple

DKC6040 (500) yellow DKC6530 (600) yellow DKC440 (300) yellow Eleonora yellow

Ma/1

Marcaria

45◦ 06 N; 10◦ 31 E

1

Costanza (600) yellow Eleonora (700) yellow

PR32B10 (600) white Damiana (500) white

260 × 320

1

6 × 17.5

6 × 17.5

65 × 95

For different hybrids, FAO maturity class is in brackets; kernel colour is in italics. a Hybrids Costanza, Lolita, Eleonora, PR31K18, PR32B10, PR33A46 and Damiana were provided by Pioneer High-bred, Cremona, Italy; hybrids DKC6040 and DKC6530 were provided by Monsanto, Lodi.

Fig. 1. Field design of the experiments. Dimensions of fields (m) and North (arrow) are indicated. (A) Type 1 experiments (Pi/1 and Ma/1). (B) Type 2a (Ca/2a). (C) Type 2b (Ti/2b) and (D) Type 3 (Ta/3 and As/3). In (D) colours/patterns indicate within-field plots of recipient hybrids with a shift in flowering time compared to the pollen source (white and grey, recipient hybrid 1 and 2, near isogenic with pollen source; dotted boxes, late flowering recipient hybrids; black boxes, early flowering recipient hybrids; central rectangle, pollen source).

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plants had different GDUs (Fig. 1D). Type 3 experiments at sites Ta/3 and As/3 were replicated three times, with replicates placed at approximately 200 m from each other.

(20% of total estimated plants). In type 3 fields, quality control was performed by randomly recounting sampled ears (20 per replicate).

2.2. Wind determination

2.4. Statistical analysis

Meteorological information, including wind speed and direction, was recorded by on-site weather stations mounted on a 10 m post, set up at the canopy top during pollen shed in all experimental fields. Wind directions and intensities were measured continuously and reported as half-hour averages. In the time range which covered the beginning of flowering for the earliest hybrid until the end of flowering of the latest one, wind measurements from 9 a.m. to 2 p.m. were used for analysis. In the text, S indicates wind from south; E = wind from east, etc.

Cross-fertilization percentages and distances from source were fitted to an exponential equation as in Ma et al. (2004). Log-transformation and subsequent linear regression were also used, as described by Gustafson et al. (2006), i.e. 10 logarithms of both percentage cross-fertilization and distance (log/log); the logarithm of cross-fertilization and the square root of distance (log/square). The resulting equations are: y = cekx (exponential), √ k x k y = cx (log/log) or y = c10 (log/square). In all cases, y is the modelled estimate of cross-fertilization, x the metric distance from the pollen source, c the estimated cross-fertilization for x = 0 and k is the shape coefficient of the equation. For the same equation, a lower k indicates a curve which decreases less markedly with distance. Coefficients of determination, r2 , were used to select the best equation to fit the data; fitted lines were also visually checked against the observed data. All data, or data from the first 15 m, were used to fit the equations; data from the first 15 m provided a better fit.

2.3. Sampling In this paper, the term “threshold” has been used to indicate 0.9% cross-fertilization, taking into account the maximum level set by EC Regulations for adventitious presence of GMOs in conventional maize. Visual inspection of recipient maize ears and xenia counting were used to calculate cross-fertilization. Selected ears from rows on each side of the pollen source were sampled at all sites (quadrants III and IV; Fig. 3, inset); sampling above and below the pollen source (quadrants I and II; Fig. 3, inset), within the same source/recipient row, was conducted for fields of type 1 and 3. The procedure involved an initial estimate of the average total kernel number, in each recipient field or replicate, per hybrid; subsequently, depending on the type of experiment, purple or yellow xenia kernels were counted on ears sampled according to the plan described below. Distances from source and quadrants were recorded. Therefore, at a given distance and in a specific quadrant, percentage of cross-fertilization could be estimated by dividing the sum of xenia kernels counted by the total number of kernels examined. Recipient genotypes in type 1 fields were sampled as follows: in quadrants III and IV (part ␣ in Fig. 1A), one row every two was sampled in the first 11 rows from the source; one row every four was sampled in rows 13–33 and one every eight from row 34 to the end of the field. In each sampled row, one ear every 10 m was collected. In quadrants I and II (area ␤ in Fig. 1A), the sampling was carried out every eight row, collecting one ear every 5 m. Overall 3172 ears were assessed (0.3% of total estimated ears). In type 2a, ears were sampled every 5 m in the first row and then every 10 m in every second row, in rows 3–9 starting from the source. Sampling was carried out every fourth row in rows 10–17, every eight row from row 18 to the end of the field (␣, ␤ and ␥ in Fig. 1B). Ears were collected, on each row, every 10 m (except for the first row: every 5 m) for a total of 713 ears. In type 2b fields, the sampling protocol was as for type 2a, and sampling was carried out only in parts ␣ to ␥ (Fig. 1C). In type 3 fields, ␣, ␤ and ␥ parts included only the isogenic recipient (Fig. 1D); sampling density varied with distance. Total number of ears sampled was 14,600

3. Results Wind speed and direction were monitored during tasseling and silking, as indicated in Section 2. Precipitations during the flowering period were in the 0.33–1.67 mm/day range, and occurred almost always at night, at all locations. Hailing was observed at site Ta/3 but tassels of the pollen source hybrid were unaffected. Wind direction and speed, monitored at each site, are reported in Fig. 2: shaded areas represent the “amount” (frequency and speed) of wind in a given direction, i.e. the “windiness” of a location. At site As/3, prevailing SW, moderate winds were noted; individual peaks did not exceed 7 km/h (data not shown). At site Ta/3, the windiest site, SW winds prevailed in terms of direction and speed (peak speed ranged from 2 to 10 km/h). At site Ma/1 prevailing winds blew from NE. Overall, this site was far less windy than Ta/3, although peak speed was in the 5–14 km/h range. At site Pi/1, wind direction was variable and winds weak. As a consequence, on one hand, based on windiness, at site Ta/3 and at As/3, we could define NE (in practice, given the field orientation N and E) as downwind and SW (in practice, S and W) as upwind, and expected well defined directional wind effects on pollen dispersal were expected; at Ma/1, only NE was evidently upwind and a weaker effect was predicted. At Pi/1 we could not define upwind or downwind; little wind effect was predicted. On the other hand, wind peaks during pollen shed, rather than average wind, have been indicated as the force affecting pollen flow (Halsey et al., 2005), and these were particularly high at Ma/1. Where no study of the effect of wind was planned (sites Ca/2a and Ti/2b), wind was considered only when present at speeds potentially affecting the experiments; only at Ti/2b relatively high speed winds (up to 10 km/h) from W, WNW were observed (data not shown).

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Fig. 2. Distance travelled by wind at sites As/3, Ta/3, Ma/1 and Pi/1 (shared area) in km, for each wind direction. Distance travelled is obtained by multiplying half-hour intervals during which wind blew in that particular direction times the wind speed during the interval. North is indicated with “N”.

The rate of cross-fertilization recorded varied considerably at the different sites. Data will be presented according to the different experiments, as defined in Section 2 and summarized in Fig. 1. Type 1 fields were designed to monitor pollen dispersal taking into account the influence of wind. At site Ma/1, experimental fields lied within commercial maize fields of approximately 20 ha. At Ma/1 and Pi/1, average cross-fertilization in the first row adjacent to the source was as high as 49.54% (Ma/1, quadrant I or SE) and as low 3.55% (Pi/1, quadrant IV, SW) (Table 2).

At Ma/1, the first-row cross-fertilization rate in the upwind quadrant (NE) was markedly lower than in other downwind quadrants (Table 2); association with wind direction was, as expected, poor at Pi/1. Cross-fertilization declined rapidly in subsequent rows, with values of about 0.02–0.2% towards the margin of the field (90–95 m from the source). Below-threshold cross-fertilization was observed as close as 4 m from the source. On average, a decrease from 20.05% (first row from the source) average cross-fertilization to 2.95% was observed at 4 m from the source (Table 2). The longest distance to reach the threshold

Table 2 Rate (%) of cross-fertilization recorded in type 1 experiments at Ma/1 and Pi/1 Distance from the sourcea (m)

0–2 2–4 4–6 6–8 8–10 10–12 14–16 20–25 40–45 60–65 90–95

Average

20.05 2.95 2.58 1.40 0.78 0.87 0.47 0.30 0.20 0.14 0.13

Site Ma/1

Site Pi/1

SW

SE

NW

NE UW

SE

SW

NW

NE

23.33 4.94 3.85 1.80 1.21 0.59 0.71 0.36

49.54

22.08

3.55 1.60

21.31

8.53

4.13 1.80

8.22

0.83

10.73 3.44 NS 0.91 0.68 0.48 0.09 0.16 0.27 0.09

0.52

0.21

0.11

3.47 1.59 0.87 0.54 0.49 0.10

0.41 0.33 0.32 0.16 0.10 0.21

SW indicates south–west, etc.; NS, not sampled. Only NE at Ma/1 could clearly be defined as upwind (UW). a The complete table is available at the website www.tecnoparco.org.

0.30 0.28 0.05 0.09 0.02 0.07

0.32 0.15 0.28 0.05 0.02 0.02

0.07 0.12 0.06 0.03 0.01 NS

2.60 0.95 0.39 0.19 0.12 0.19 0.11

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Table 3 Rate (%) of cross-fertilization recorded for white pollen recipient plants when fertilized by yellow pollen Distance from the source (m)

0.7 2.1 3.5 4.9 6.3 9.1 11.9 17.5 18.9 20.3 21.7 23.1 25.9 28.7 34.3 35.7 37.1 38.5 39.9 42.7 45.5 48.3 51.1 53.9 56.7

Source and recipient plants contiguous

Source and recipient plants separated by a 17.5 m empty corridor

Source and recipient plants separated by a 34.3 m empty corridor

Average

SE

NW

Average

SE

NW

4.46 1.73 1.18 1.24 0.76 0.7 0.61 0.27

23.13 13.45 1.64 1.18 1.24 0.97 0.85 0.82

1.62 1.44 0.38 0.34 0.19 0.27 0.64

3.95 0.82 0.41 0.24 0.39 0.24 0.30

Average

SE

NW

31.45 10.53 5.03 3.88 3.31 1.08 1.29 0.81

31.40 2.99 1.17 4.77 1.25 0.87 1.06 0.49

31.50 18.07 8.90 2.99 5.38 1.29 1.52 1.14

0.7

0.68

0.72

0.66 0.34

0.64 0.45

0.68 0.23

13.79 7.59 1.41 1.21 1 0.83 0.73 0.54

0.55

0.91

0.19

0.31

0.27

0.36

0.36

0.23

0.49

0.43

0.39

0.48

2.78 1.13 0.39 0.29 0.29 0.25 0.47

0.53

0.53

0.53

0.40

0.48

0.33

0.29

0.23

0.35

0.59

0.27

0.91

0.28

0.15

0.42

0.27

0.15

0.39

Type 2a experiment at Ca/2a. SE means south–east; NW means north–west.

was observed at site Ma/1 where the threshold was reached at approximately 25 m. Type 2 fields were designed to evaluate the effect on the level of cross-fertilization of fallow land as a barrier (type 2a); type 2b permitted the appraisal of the effect of buffer areas (i.e. parts of the field sown with maize producing pollen competing with

the source). In the type 2a experiment at Ca/2a (Table 3), the threshold of 0.9% was reached at an average distance of 17.5 m in rows sown contiguously to the source (Fig. 1B, part ␣; Fig. 4) and cross-fertilization declined from 31.45 to 1.08% in approximately 8 m (corresponding to 12 rows of plants). When the recipient plot was separated from the source plot by 17.5 m of

Table 4 Rate (%) of cross-fertilization recorded for white pollen recipient plants fertilized by yellow pollen Distance from the source (m)c

0.0 1.5 3.0 4.5 6.0 9.0 12.0 15.0 18.0 21.0 24.0 27.0 30.0 33.0 36.0 39.0

Buffer contiguous to the recipienta

Buffer area contiguous to the pollen source

Average

DWb

UW

Average

DW

UW

6.63 0.90 0.48 0.46 0.46 0.21 0.09 0.15 0.09 0.16 0.22 0.21 0.15 0.43 0.79 0.17

12.87 1.56 0.89 0.69 0.48 0.33 0.15 0.23 0.06 0.25 0.31 0.36 0.23 0.79 1.39 0.15

0.39 0.24 0.07 0.22 0.44 0.09 0.02 0.07 0.11 0.07 0.13 0.06 0.06 0.07 0.18 0.18

1.16 0.63 0.26 0.24 0.12 0.13 0.05 0.05 0.04 0.05 0.05 0.19 0.04 0.26 0.03 0.18

1.86 0.55 0.20 0.15 0.12 0.08 0.03 0.10 0.00 0.07 0.05 0.35 0.05 0.50 0.03 0.23

0.45 0.71 0.32 0.32 0.12 0.18 0.07 0.00 0.07 0.02 0.05 0.03 0.03 0.02 0.02 0.13

Type 2b experiment at Ti/2b. a Indicated as ␣ (upwind) and ␦ (downwind) in Fig. 1C. b UW, Upwind; DW, downwind. c Values at distances (m) 40–63 were used to derive the modelling equations. The complete table is available at the website www.tecnoparco.org.

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fallow land (as defined in Section 2; Fig. 1B, part ␤), a rapid decline was observed, on average, from 13.79 to 1.21% crossfertilization in approximately 3 m, corresponding to 5 maize rows. On average, the 0.9% threshold was reached at 27 m. When the fallow land gap was of 34 m (Fig. 1B, part ␥), the cross-fertilization declined from 2.8 to 1.13% from the first row of recipient plants to the second, and the 0.9% threshold was reached, on average, at 36 m from the source. In type 2b fields (Table 4), rows of white buffering maize were planted at variable distances and positions from the yellow maize source plot. When the buffering area was within the pollen source plot (Fig. 1C, ␣ and ␦), 1.16% cross-fertilization was observed in the first row of recipient plants (average of both sides of the field) and the 0.9% threshold was reached in the third row from the source. When the buffering area was within the pollen recipient (Fig. 1C, ␤ and ␥), the threshold was reached already in the second row of maize after the corridor. In this case, the 12 buffering rows were able to lower cross-fertilization from an average of 6.63% in the first row to 0.46% in 4.5 m. Type 3 experiments were designed to monitor the effects of flowering synchrony of the pollen source with pollen recipient hybrids. Sowing was delayed to the last days of April to maximize the density of pollen (sowing delay in the environment of the Po Valley increases tassel fertility). As expected, an almost complete flowering synchrony was observed between the pollen source and those hybrids known to be nearly isogenic (Table 1; hybrids DKC6530 and Costanza). An interval of 4–5 days was observed between pollen source and late recipient hybrids (with the highest FAO class of maturity corresponding, in our experiment, to the 700 class). An anticipation of 4–6 days in flowering time was recorded when comparing the flowering time of the pollen source and of recipient hybrids defined as early maturing. Peak silking at 50% pollen shed was observed around the 8 or 9 August. Overall, flowering lasted for about 7–8 days. At sites As/3 and Ta/3 the 0.9% threshold was reached 4.5–6 m from the source in those parts of the field sown with nearly isogenic hybrid (Table 5). For late hybrids, at Ta/3 a threshold decrease from 24.96 to 3.29% cross-fertilization was observed in 2 m from the source. In the case of early hybrids, in the first

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row, the cross-fertilization was 6.04 and 2.09% at sites As/3 and Ta/3, respectively. 3.1. General considerations Cross-fertilization at variable distances from the source plot can be described by empirical models. Data from type 1 and 3 experiments (Fig. 3) have been used to compare three different regression models; predicted values and parameters from the best fitting model (the log/log model, a power-law equation), are detailed in Tables 6 and 7. None of the models tested could account for the abrupt decline in cross-fertilization observed in the first 3 m of the recipient, although they performed well further from the source (Fig. 3). Interestingly, there was little association between the shape coefficients, k, and c, cross-fertilization at 0 m, suggesting that first-row cross-fertilization may be partially influenced by factors that differ from those affecting subsequent rows (Table 6). Higher values of c were associated with downwind, as expected, except that in Ma/1; on the other hand, lower k values, indicating a smoother decline in cross-fertilization rates, were not associated with downwind at Ma/1 and As/3 (Table 6). Estimated distance to reach the 0.9% threshold was in the 1.28–11.00 m range, with the exception of SE at Ma/1 (extrapolated distance was 27.33 m; Table 6) (Fig. 4). When all models were used to extrapolate distances needed to reach the 0.1% threshold, these exceeded 100 m in the case of SE at Ma/1; in two other cases, the threshold was reached between 50 and 100 m and between 20 and 50 m in four more cases. Distances below 20 m were estimated for other quadrants. Similar conclusions were obtained without extrapolation, using the log/log model fitted to all the data. It appears that 0.1% threshold distances cannot be reliably established within 100 m from the pollen source. Considering type 2a and type 2b experiments together, based on modelling (data not shown) and observed data, the conclusion on the effect of barriers is that the presence of maize plants producing a competitive pollen cloud (i.e. of recipient genotype) is very effective in reducing cross-fertilization.

Table 5 Rate (%) of cross-fertilization recorded for yellow pollen recipient plants when fertilized by purple pollen Flowering

Site As/3

Site Ta/3

Distance from the source (m)

Synchronous (hybrid 1)

Synchronous (hybrid 2)

3–4 days delayed flowering

3–4 days anticipated flowering

Synchronous (hybrid 1)

Synchronous (hybrid 2)

5 days delayed flowering

6 days anticipated flowering

0.75 1.5 2.25 3.0 4.5 6.0 7.5 9.0 12.75

20.05 14.55 4.65 1.99 1.27 0.52 0.31 0.16 0.05

34.07 15.87 8.27 3.07 1.52 0.68 0.38 0.13 0.10

23.31 13.41 5.07 1.98 0.88 0.36 0.12 0.04 0.02

6.04 4.15 1.75 1.44 0.12 0.09 0.08 0.04 0.01

19.46 12.60 6.46 2.97 1.00 0.50 0.17 0.36 0.15

26.00 12.37 7.17 2.63 0.94 0.49 0.11 0.13 0.07

24.96 7.93 3.29 1.31 0.35 0.44 0.70 0.67 0.08

2.09 0.87 0.96 0.21 0.15 0.06 0.05 0.02 0.00

Type 3 experiments at As/3 and Ta/3. Delays and anticipation of flowering in comparison to the source. a Values at distances (m) 15–27 were used to derive the modelling equations. The complete table is available at the website www.tecnoparco.org.

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Fig. 3. Type 1 and 3 experiments. The four quadrants describe the parts of the field indicated in the small inset diagram. Observed (histograms) and model-estimated (lines) rates of cross-fertilization as a function of distance (m) from the pollen source, per quadrant. (Histograms) Site As/3, black; site Pi/1, dark grey; site Ta/3, light grey; site Ma/1, grey. (Lines) Site Ta/3, dotted line; site Pi/1, dashed line; site As/3, solid line; site Ma/1, dot-dashed line.

Experiments conducted in types 1, 2a, and 2b fields agreed in supporting (1) the 0.9% cross-fertilization threshold within, on average, 10 m in type 1 experiments, but exceptionally at 25 m; 17.5 m in type 2a experiments; 1.5 m for areas contiguous to pollen source or to recipient in type 2b experiments; (2) although

the examined measures of wind do not fully explain directional differences in cross-fertilization rates, wind significantly affects pollen flow, even under the moderate wind conditions observed; (3) the containment role of barriers, which proved most effective if consisting in maize plants shedding non-source pollen.

Table 6 10-log transformed linear regression or log/log model (y = cxk ), of percentage of cross-fertilization and distance (fitted data up to 15 m from source only) Site

Estimated cross-fertilization at 0 m (c)

Shape coefficient (k)

I II III IV

0.53 0.18 0.20 0.10

−1.23 −1.61 −1.30 −1.37

0.99 0.96 0.98 0.89

Pi/1

I II III IV

0.17 0.06 0.01 0.04

−1.81 −1.74 −0.89 −0.83

0.97 0.90 0.25 0.68

As/3

I II III IV

0.31 0.17 0.26 0.24

−2.25 −2.34 −1.58 −1.87

0.97 0.97 0.94 0.92

4.84

I II III IV

0.33 0.10 0.38 0.10

−1.99 −2.29 −1.76 −2.06

0.96 0.95 0.96 0.92

6.10

Ma/1

Ta/3

Coefficient of determination (r2 )

Estimated distance at which cross-fertilization is 0.9% (m)

Position from pollen source as in quadrants in Fig. 3

DW

Type 1 and 3 experiments. Downwind could not be defined for Pi/1 (see text).

UW

27.33 6.44 11.00 5.73 5.10 3.04 1.28 6.22 3.46 8.42 5.82 2.81 8.32 3.23

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263

Table 7 10-log transformed linear regression or log/log model (y = cxk ), of cross-fertilization and distance (fitted data up to 15 m from source) in type 2a experiments Site

Empty space between source and recipient (m)

Estimated cross-fertilization (%) at 0 m (c)

Shape coefficient (k)

Coefficient of determination (r2 )

Estimated distance at which cross-fertilization is 0.9% (m)

Ca/2a

0 17 34

0.29 0.18 0.02

−1.18 −1.33 −0.94

0.88 0.91 0.83

18.70 9.38 (after 17.5 m)a 2.19 (after 34.3 m)a

a

Corresponding, respectively, to the linear measures of the empty corridors in ␤ and ␥ (Fig. 1).

Type 3 experiments, allowed to conclude that: (1) there was no considerable reduction in pollen flow when the difference in flowering time between pollen source and recipient was up to 3 days; (2) when the difference in flowering time was of 3–4 days, a reduction of 75% of pollen flow was recorded; (3) when

the difference was of 5 days, the reduction was 50%. In addition, the data supported the conclusion that earlier hybrids were more efficient in pollen flow containment, probably due to silk saturation with self pollen before the source pollen was made available.

Fig. 4. Type 2a experiment. Observed (histograms) and model-estimated (lines) rates of cross-fertilization as a function of distance (m) from the pollen source, per quadrant. Model estimated rates for empty spaces may not be visible when very low. Arrows point to distances where the 0.9% threshold is reached.

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4. Discussion We report cross-fertilization rates for maize as affected by distance from pollen source, wind direction, size of buffer zones and effect of differences in flowering time between pollen source and recipient. Type 1 experiments evaluated the cross-fertilization rate at different distances and in different directions from the source plot. Type 2 experiments assessed the influence of fallow land and of buffering zones on the same parameters. Type 3 fields addressed the effect of time of flowering and of distance. Results of previous studies (reviewed in Devos et al., 2005) were obtained at least in part with individual plants or small recipient plots, planted at various distances from the pollen source, conditions which do not reflect field situations. In such cases, due to the small and diluted self pollen cloud hanging over individual plants or small recipient plots, the incoming pollen may have had a probability of a successful fertilization which differs from in farm situations. In this sense, our large field experiments were designed to simulate real-world scenarios of coexistence of GM and nonGM crops. Commercial hybrids were sown on large surfaces to mimic the farm situation also in terms of pollen cloud size, a condition not tested before in Italy (Sorlini, 2004). Moreover, this design is comparable with experiments carried out in France, Germany, Spain, and the UK, where worst-case commercial on-farm situations were mimicked (e.g. pollen source next to or completely surrounded by a recipient field; Schiemann, 2003; Messeguer et al., 2003; Henry et al., 2003; Stevens et al., 2004; Weber et al., 2005; Messean et al., 2006; Messeguer et al., 2006). Care was taken to use phenotypic markers of proven marking capacity. Moreover, all experiments (but type 3) were conducted using pollen recipient hybrid varieties having maturation classes coincident with the average maturation class of the source. As expected, the highest level of cross-fertilization was observed within rows contiguous to the source plot. An incomplete association between wind direction and cross-fertilization rates was observed: wind peaks in critical days could provide an explanation, as discussed in Halsey et al. (2005), and Feil and Schmid (2002), but we could not consistently confirm this trend. As compared to Ma et al. (2004), when using the exponential equation, we have found (data not shown) lower modelled cross-fertilization levels near the pollen source (1–37%) and also lower shape (k) coefficients of our exponential equations (between −0.19 and −0.53). Other empirical models (Gustafson et al., 2006) have been tested, that seem to provide a better fit, especially in the 0–15 m range. Specifically, a power-law equation (which often corresponds to an inverse square law, with a power ranging from −0.61 to −2.31), did provide the best fit among the models tested, perhaps unsurprisingly (Aylor et al., 2003). However, all the equations tested did not model well the decline in the first 3 m, where our sampling was actually rather accurate (Fig. 3) compared to other studies (e.g. Ma et al., 2004). The presence of zones of empty spaces proved to be ineffective if smaller than 30 m. When corridors were more than 30 m wide, even with a directional effect of wind, the gene flow

decreased below the 0.9% threshold already in the first row of the pollen recipient field. Synchronization of pollen shedding of the source plot with the silking of the recipient plants, and the amount of pollen available from the source, are also influencing the extent of pollen transfer and cross-fertilization, as evaluated in type 3 experiments. Pollen viability is quickly lost if silk are not receptive at the moment of fertilization (Uribellarea et al., 2002; Westgate et al., 2003; Brookes et al., 2004). Interestingly, the same level of cross-fertilization (2%) was reached after 0.7 m in type 3 experiment when using an early hybrid, as compared to the average value of 6 m in type 1 fields. This result should be attributed to the anticipation of silking of just 6 days and it is even more dramatic than the one recorded by Halsey et al. (2005). From our data, it appears that a 6-day shift causes approximately a 50% reduction in cross-fertilization, while this parameter is reduced by 25% with a 4–5-day shift. Earlier varieties are apparently more efficient in gene flow containment since they probably can saturate receptive silks with self pollen well before the flowering of the source. These data are in agreement with the findings of Brookes et al. (2004). Based on pollen source size, higher levels of crossfertilization were expected in type 1 fields at sites Pi/1 and Ma/1 when compared to sites Ta/3 and As/3: the latter had a source plot extending on ca. 100 m2 , while the dimension of the source in the other sites was 6000 m2 . The source to recipient ratio was therefore 2% at sites Ta/3 and As/3 and 7% at sites Pi/1 and Ma/1. The data support the observation that it is not the amount of foreign pollen in the recipient crop per se but rather the ratio of foreign pollen to the crop’s self pollen which determines the rate of cross-fertilization (Feil and Schmid, 2002). Data of site As/3 recorded in the rows adjacent to the pollen source, and explainable as cases of precise synchrony in flowering, indicate a rapid shift in cross-fertilization rate already after 1.5–2.0 m (data not shown). Ears showing the absence of cross-fertilization were found more far away from the source in type 1 fields with large source plots consistently with the observations of Klein et al. (2003) and Ma et al. (2004). Hot spots (i.e. ears with an exceptionally high number of xenia kernels) were recorded in type 1 fields, at unexpected distances from the source, consistently with Ma et al. (2004). They could be due to the fact that the pollen source had a wider flowering time than the pollen recipient plants, thus allowing late flowering plants of the recipient to be more exposed to the pollen source. Generally, they appeared to be offtypes. Procedures defined as “identity preserving” have been defined for protection against the possibility of mixing seeds derived from fields that have to be preserved in purity (for example, planted with non-GM varieties) with seeds from fields not needing identity certification (for example, GM). Field designs such as those of experiments of type 2a and b could address this problem: it would surely be easier to manage the harvest of GM crops with buffering non-GM maize within the GM field. The buffering plants could be harvested together with the GM crop, minimizing losses, while obtaining an efficient non-GM pollen barrier.

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