The majority of grasses are cross-pollinated by wind and are largely self-incompatible thus preventing their self-pollination. Individual plants in populations are highly heterozygous being hybrids of hypothetical parents. However, natural populations have substantial additive genetic variation for selection to be effective for most agronomic traits (Vogel et al., 1989). Individual plant selections, usually in thickly seeded stands or swards as forages or turf grasses, can be impractical and as such, evaluation and selection are often practiced in space-planted nurseries or small sward plots. The most effective breeding programs in forage grasses limit hand emasculation or crossing, instead utilizing recurrent selection improvement systems, which have the added benefit of retaining genetic variation in the populations. This system is continued with the random mating of selected individuals in isolated polycross nurseries to produce progenies for the next cycle of selection (Brown et al., 2014). The selected polycross progeny are then used in the development of synthetic populations. In the UK, hand emasculated pair-wise crosses between two species Lolium multiflorum and Lolium perenne are being used to make synthetic varieties from the interspecific hybrid, Lolium boucheanum. Grass breeders are now more interested in these types of hybrids, exploiting the higher heterosis of F1 hybrid varieties between two or more compatible interspecific combinations vs. more narrow based synthetic varieties. This approach, though more productive, requires modifications in the ways interspecific varieties are composed and subsequently multiplied.

Wind pollination is related to the size of the grass pollen being distributed since neither very large nor very small grains are wind pollinated. Grass pollen is divided into two types based on grain size; wild grass types range from 25 to 35 µ (with exceptions of 35 or 40 microns) and the cultivated grasses range from 35 to 50 µ with the modal peak at 40 µ (Erdtman, 1943). According to Wodehouse (1935), Lolium pollen grain size ranges from 22 to 33 µ. Geisler (1945) reported a range of 24-39 µ with modal peak of 31 µ for a group of six grass species including Poa and Festuca.

Pollination bags, isolation plots, isolation chambers and pollination control tents (PCTs) are some of the methods used in the controlled crossing of grasses. Pollination bags are only useful on a limited scale. Isolation chambers are expensive to build and run in order to maintain a controlled microclimate, and isolation plots demand very long distances between plots of the same species (305 m for Breeder Seed) thus limiting the number of entries to be multiplied. However, research on the effectiveness of PCTs is limited. This study addresses this gap by evaluating novel PCT technologies in a grass breeding program using nonwoven, re-usable synthetic tent fabrics.

The aim of this study is to examine the possibility of substituting the use of isolation and distance in small field crossing or seed production nurseries with new PCTs and extending our knowledge about the microclimate within such structures in order to obtain a healthy and high seed set while at the same time providing pollen proofing. Consequently, it lays the foundation for new research in plant breeding, investigating novel options that are potent enough to increase the efficiency of breeding operations in all crops by enabling many crosses to be made simultaneously or by increasing the number of seed multiplications of promising populations. The major objectives of the study were: (1) evaluating PCT structures for robustness, durability and strength of cover fabric materials under field conditions, (2) testing the pollen proofing ability of fabric materials, (3) comparing the microclimate within PCTs with outside control conditions, and (4) assessing the comparative seed output of healthy seeds and plant performance for biological traits.

 

DWB24

Nonwoven spun-bound polyester; thickness (mm) 0.40; mass per unit area/weight  (gm^-2) 110; air permeability (l/m²/s) 1470; light transmission (% 350 - 800 nm wavelength) 39; maximum pore size (microns) 214; fibre cross section is complex.

The following types of PCTs were used:

(a) Small PCTs tested in 2018 and 2019

• Three small PCTs, DWB10, size 1.5 m × 3 m × 2 m
• Three small PCTs, DWB24, size 1.5 m × 3 m × 2 m)

(b) Large PCTs tested in 2019

• One large PCT, DWB10, size 6 m × 6 m × 2 m
• One large PCT, DWB24, size 3 m × 12 m × 2 m).

Design

The frames of PCTs were made of PVC piping and were secured to the ground by placing two sand bags (22.6 kg) on each side on the bottom pipe of the frame. The structures were rigid once assembled. Additional dirt was placed around all sides of the PCT when it rained because the dirt tended to settle or was washed away, exposing the skirt edges. Soaker hoses were supplied to each tent for supplementary irrigation, if needed. Covers of both types of fabric fitted snuggly on the appropriate frames (Figure 1).

 

 

Smaller PCTs used in 2018 were stored for re-use in 2019. Both types of fabric were washed using a solution of Clorox® bleach (10%) and distilled water before re-use to clean and remove any contaminants. Duct tape was used on any fabric seams of the PCTs (both fabrics) to make minor repairs.

Small PCTs

During 2018 and 2019, two smaller PCTs, DWB10 and DWB24 were placed at each of three sites. Within each PCT, 15 tall fescue plants (Lolium arundinaceum (Scherb.) Darbyshire) representing three genotypes and cloned five times each were transplanted and grown.

A control isolation group (open pollinated) was planted at each location containing 15 tall fescue plants (same 3 genotypes used in tents and cloned 5 times each = 15 plants) at a minimum of 305 m away, which is the minimum distance between breeder or foundation seed increase of tall fescue as recommended by the Seed    Certification   Service   in   Oregon,   USA    (Oregon    Seed Certification Service Handbook, 2018).

In the 2019 trial, an additional control was added at each location. This ‘open control’ of 15 tall fescue plants (open pollinated) was located at least 305 m from the PCTs and the other control group of tall fescue plants.

Perennial ryegrass plants (Lolium perenne L.; 2n=4x=28) were planted around each PCT in 2018 (4 per side) to act as “pollen donors” for testing contamination in the PCTs, if any. Both species, inside and outside the PCTs, are out crossing and can hybridize (that is, Festulolium) allowing for detection of any chromosomal recombination between the tetraploid ryegrass and the hexaploid tall fescue resulting from pollen contamination. It was determined that approximately 15 plants around the PCT would generate enough pollen pressure with particular concentration to the southwest direction due to prevailing winds. All plants were grown and vernalized to induce flowering in early summer.

In 2018, PCTs were set up on 18th May at Ardmore and Gene Autry, and on 21st May at Burneyville. All plants were at the E₃ – R₀ growth stage (Moore et al., 1991) when transplanted in the field on 9-11^th April in 2018 and the 22nd April 2019 (Figure 1). All tents at all locations were removed on 25th June 2018. In 2019, small PCTs were erected on 4th June and taken down on 5th July at all three locations.

Increasing pollen flow in small PCTs in 2019

Overall, seed yields in 2018 were low across all treatments and especially inside the PCTs. It was thought that airflow within PCTs may be restricted compared with the natural environment. It was hypothesized that increasing the air circulation and hence the mobility of pollen within the PCT would aid cross-pollination and improve seed yield. Therefore, portable electric fans powered by solar panels were placed in all treatments, that is, small PCTs, control, and open control groups at Ardmore and Gene Autry locations in 2019 experiments. Fans were also placed in the middle of the open control at two locations (Table 1). However, no fans were placed in any treatment at the Burneyville site. Fans were easy to setup and there was no issue with them or the solar panels in the PCTs or the field (control or open plants). The fan effect was estimated through an analysis of variance in which locations are confounded with blocks.

 

 

Larger PCTs 2019

One PCT was placed at each location. The 6 m × 6 m PCT (DWB10) was placed at the Unit 3 Farm (34.11° N, 97.05° W; elevation 248 m) on a Windthorst fine sandy loam (fine, mixed, active, thermic Udic Paleustalf) and the 3 m × 12 m tent (DWB24) was erected at the Headquarters Farm (34.08° N, 97.12° W; elevation 252 m) on a Heiden clay (fine, montmorillonitic, thermic Udic Chromusterts). Both of these sites were located in Ardmore. Each tent contained 40 tall fescue plants (4 genotypes cloned 10 times each = 40). A control isolation (open pollinated) was planted at each location containing 40 tall fescue plants (same 4 genotypes used in the PCTs cloned 10 times each = 40) at a minimum of 305 m away. As in the small PCTs, all plants were grown and vernalized to stimulate flowering in early summer. PCTs were set up on 5th June at both locations. Both the inside and outside plants were harvested on 5th July and tents taken down on 6th July 2019.

Microclimate assessment

A HOBO MX temperature and relative humidity data logger (Onset Computer Corporation, Inc.) was placed in each PCT by suspending them from the roof (Figure 2). These data loggers are battery powered and record temperature, relative humidity and the dew point. Since the loggers have Bluetooth connection, we were able to collect data without disturbing the environment in the PCT.

 

 

Weather data were also collected from weather stations (Mesonet.org) located on the Noble Research Institute farms for comparison for the duration of the trial period. The same parameters were collected, along with the average maximum wind speed and maximum wind gusts, at each site.

Data collection

Biological traits

(1) After pollination and seed set, data on a number of biological traits were collected: plant height (cm) = PH and growth habit (1 = decumbent, 2 = semi-erect, 9 = fully erect scale) = GH. A visual disease score (1 to 5; 0 = no disease, 5 = death due to disease) = DS was also given to each plant.

(2) The seed related data were collected on: seed yield per plant (g) = SY, 1000-seed weight (mg) = SW, the presence of ergot (%), a visual seed quality score (1 to 5; 1= excellent, 5 = extremely poor) = SQ and germination rates on 7, 14 and 21 on the basis of 100 seeds from a sub-set of plants from each tent.

(3) The harvested seeds from all of the plants individually inside the PCTs were collected and assessed for various traits (Table 2).

 

 

Germination rate (%)

For germination rate 100 seeds per clone were sown for each genotype in treatments. The germinated seeds were counted on 7, 14 and 21 days after sowing to record percent germination.

Pollen contamination

For 2018, the measurement of contamination from ryegrass pollen from outside the PCTs on to the tall fescue plants was assessed  by looking at the possibility of hybridization of the perennial ryegrass and tall fescue. The hybrids are generally sterile and though fertility can be restored by chromosome doubling, such plants are unstable and experience chromosome loss (Scott and White, 1988). We examined some chromosomal pairing in the hybrids of the ryegrass and tall fescue.

To measure any contamination from outside pollen at the Research Park farm at Ardmore in the 2019 small PCT trials, an existing tall fescue population was chosen to measure for any pollen contamination due to the fabric (different nursery than the control and open nurseries at this site).  Sixty  plants  were  bagged (20 with DWB10 bags, 20 with DWB24 bags and 20 with wax coated paper bags). The type of wax-coated paper crossing bags (Lawson Bag Co.) that were used is represented in Figure 3. Plants were bagged between June 1 and 5th. The bags were sealed with weather proof tape and wooden stakes were used to support the tiller and bag in the field during the test period. The remaining heads in the plot were allowed to cross-pollinate. Bags were removed during the July 5-8 time-period. At this time, the bagged panicle was harvested. To determine if any seed were produced, panicles were later conditioned on a rubbing board. A total of four of the wax-coated bags  were  lost  due  to  the  weather. None  of  the other bag types was lost.

 

 

Statistical analysis of biological traits recorded on individual plants and germination percent per clone was performed following analysis of variance technique described by Sokal and Rahlf (2011). Fisher’s Least Significant Difference (LSD) was used for pair-wise comparison of treatment means and significantly different means were labelled with different letters.

 

 

 

 

 

 

The first year with the PCTs (2018) was more of a learning experience as to how to assemble the PCTs, how they withstand the weather and how to take corrective measures. For instance, at Gene Autry (Dupy farm) on the evening of 31st May, covers of both small PCTs were blown about 200 to 300 m from their original location during a thunderstorm, which produced a wind gust of 80.14 km/h as recorded by the farm weather station located approximately 750 m from the PCTs. However, the frames remained intact. The plants inside were pollinating at the time of the failure, but we placed the tents back on their frames and continued the experiment. No damage was observed to the fabric of PCT DWB24, but we had one small tear along the seam of the DWB10 PCT along the roof, which was repaired with duct tape. Thus, the need to improve anchoring was noted.

During 2019, there was no failure from wind at any location. However, the fabric covers, having been re-used, were starting to show wear. There was no animal damage in any year at any location observed to the fabric and no fan failures occurred in 2019. The experiments in 2019 are thus more reliable for conclusions and we would lay more emphasis on these results.

Pollen proofing

During 2018, the measure of any contamination from outside pollen in the PCTs was looked at through the possibility of hybridization of the ryegrass and tall fescue. We had grown tall fescue plants inside PCTs that were surrounded by ryegrass plants. Since we do not have SSR markers to measure the rate of contamination in the progeny due to outside pollen entry into the PCT, we tried to look at some chromosomal pairing due to hybridization of the ryegrass and  fescue.  There  was  no  evidence  of any contamination in the progeny. However, we believe that this tedious method was not very reliable. In retrospect, fabric material in the form of small pollination bags to measure any selfing by bagging individual open pollinated plants would probably have been more reliable. This technique was selected for examining pollen contamination in the following year. Therefore, in 2019 we bagged reproductive panicles in an open pollinated plot at the Ardmore location. No seeds were produced by panicles covered by DWB10 or DWB24 bags. Of the 20 wax-coated paper bags used for comparison, 16 remained intact in the field, with only one bagged panicle producing a viable seed (Table 3). Results of the pollen study across both years showed that both the DWB10 and DWB24 tents were safe from contamination of foreign pollen from other grasses.

 

 

Microclimate within PCTs vs. weather data

In general, year 2019 was better for performance of grasses than 2018. Overall, mean seed yield per plant was higher in 2019 than in 2018. It was 15.2 g in 2019 against 11.2 g in 2018 (36% increase) at Ardmore; 20.3 g in 2019 against 11.3 g in 2018 (79% increase) at Burneyville and 19.7 g in 2019 against 10.5 g in 2018 (87% increase) at Gene Autry.

Climate factors within the PCTs along with weather data collected from the adjacent weather stations for the open pollinated controls are listed in Tables 4 and 5 for smaller PCTs in 2018 and 2019, and for larger PCTs in 2019. In 2018, the average minimum temperature inside the PCTs was higher by 2 to 8 degrees than the control at various locations, but in 2019, the outside temperature was higher at Ardmore by about 2 degrees than inside the smaller PCTs (Table 4). The average maximum temperatures were higher in both large and small PCTs compared to the controls at all sites in both years by 6 to 23°. The overall average temperatures were either equal at Burneyville in 2019 or higher in all other cases than the controls by up to 9°. It was suspected a malfunctioning data logger might have recorded some erroneous values at Burneyville in 2019. We can conclude that, in general, the temperatures within smaller PCTs had a wider range from slightly lower to slightly higher temperatures compared to control (Table 4). However, the larger PCTs in 2019 showed higher minimum temperatures by 2 to 3° at both sites (Table 5).

 

 

 

The maximum temperatures in larger PCTs were higher by up to 15° and the average temperature by up to 6° (Table 5).

Average minimum relative humidity values were lower in the PCTs compared to the outside controls at all three locations in 2019, but were higher at the Burneyville site in 2018. Maximum relative humidity was higher in PCTs than controls in 2018, but was consistently equal in 2019. Overall, averages for relative humidity values were variable compared to the outside controls at all three locations in two years; in 2019, they were lower in the PCTs compared to the control at all sites, but in 2018 they were lower at the Burneyville site only. The other sites  showed   similar  results.  In  summary,  the  pattern appears that the range of temperature and relative humidity is greater inside the PCTs than outside (higher highs, lower lows), with smaller structures seeing slightly greater temperature and humidity range when compared to the larger PCTs.

There is some evidence to suggest that the PCTs made from DWB24 may record higher maximum temperatures than PCTs made from DWB10 despite being more air permeable; this may result from the more open structure, increasing light penetration in the longer wavelengths. The DWB24 also seems to have a greater range of relative humidity measurements through the day than the DWB10.

The range of average maximum gust  of  wind  was  not very different during the two year study ranging from 35 to 41 km/h in 2018 compared with 28 to 37 km/h in 2019 (Table 4). However, the maximum gust in 2019 was higher (up to 99 km/h) compared with 2018 (84 km/h). The direction of wind was generally SW in 2018 but SE at Ardmore and Gene Autry in 2019 (Table 4).

The average rainfall was low for the study period at all locations in 2018 with a minimum of 23 mm at Burneyville (Table 4). While it was low (33 mm) at Burneyville again in 2019 (Table 4), it was relatively higher at Ardmore (161 mm) and Gene Autry (152 mm).

Small PCTs-quantitative traits

Analysis of variance

Analysis of variance for 2018 and 2019 showed consistently significant differences among treatments for PH, SY and SW and among genotypes for GH only (Tables 6 and 7). The location effect was more pronounced in 2019 being significant for all traits, but only for GH in 2018. A significant interaction in 2018 was observed between treatments and genotypes (Table 6 and Figure 4) for seed yield, which arose from the reduced yield of genotype 1 in the DWB24 tent than in PCT DWB10. The other two genotypes did not change rank for seed yield between the two PCTs (Figure 4).

 

 

 

 

Interactions in 2019 were more pronounced for GH and SW in respect of locations vs. genotypes and treatments vs. genotypes, which are summarized in Figures 5 and 6. PCTs DWB24 and DWB10 showed cross over interaction at the Burneyville and Gene Autry locations for GH with higher values recorded at Gene Autry than at Burneyville (Figure 5). The location × genotypes interaction for GH was more pronounced for Genotype 2 at Ardmore than the other locations (Figure 5). Genotype 3 interacted significantly with PCTs due to its higher performance in PCT DWB10 and lower performance in the open treatment (Figure 5). All interactions for SW were similar to GH (Figure 6).

 

 

 

However, contributions of interactions SS to the total SS were very small which ranged from 0.4 to 11.4% in 2018 and from 1.1 to 12.5% in 2019 (Tables 6 and 7). On the other hand, the treatment SS was a significant contributor to the total SS in both years for most of the traits except for GH.

Mean performance

Fitted mean values for traits with significant differences were compared using Fisher’s t-test in pairwise ways. Location means showed the highest GH score at Ardmore in both years compared to other locations, which were similar (Tables 8 and 9).

 

 

 

 

 

 

 

 

 

 

 

 

 

Of the two years, 36 to 87% more seed per plant was produced in 2019 across sites compared to 2018. The two PCTs showed a 2 to 5% decrease for PH, SY and SW compared with the control treatment in  2018  (Figure 8). However, in 2019 the performance of tall fescue was superior to control for PH, SY, SW in small PCTs, and SY and SW in large PCTs. SY from DWB10, DWB24 small PCTs were 37 and 30% higher, respectively, over the control (Figure 8). Similarly, SY from large DWB24 PCTs were 13% higher and the DWB10 PCTs averaged 8% higher over the control (Figure 8). Clearly, SY returns from both PCTs were higher than open controls (Tables 8 and 9); thus both PCT materials were equally useful in this particular climate and crop combination. However, the choice of PCT fabric for other crops and other climates may be different.

 

 

The effect of introducing fans within PCTs and controls to determine if they improved pollination, fertilization and subsequent seed output were also examined. We hypothesized that static air within the PCTs might reduce the free airflow of pollen grains leading to poor seed set. However, the introduction of fans unexpectedly decreased seed yield by 23% (Figure 9). While no explanation could be evidenced, it is possible that the draft created by the fans could have interfered with the settling of pollen on receptive stigmas during pollination and fertilization However, the reduction of SY with fans established that there is no need for increased airflow within the PCTs; perhaps the porous nature of the fabrics allowed enough aeration and airflow within the PCTs eliminating the need for increasing it by other means.

 

 

An important feature of hybridization or seed multiplication in PCTs is the maintenance of genetic identity of stocks from contamination of foreign or unwanted pollen. We did not have any evidence, though preliminary, for any contamination from outside pollen in the PCTs. This is a very useful indication to build the confidence of plant breeders and seed producers for the use of nonwoven fabric PCT’s in grass breeding.

Economic implications

While a proper economic analysis was not a direct objective of the present research, we can examine the effect of various factors determining the economic impact of using PCTs in comparison with other means of isolation or self-pollination. This is a very preliminary analysis that could be used as a basis for future studies and follows a simplistic approach shown in Schaffert et al. (2016) and Gaddameedi et al. (2017) in sorghum. Our approach is based on explorative circumstantial evidence from the analyses provided by the available data that could be extrapolated for comparative assessments (Table 13).

 

 

In 2018, the first small PCT took about 1.5 h (3 people) to assemble. This included digging and anchoring of the skirting and placing a soaker hose for irrigation of the plants under the PCT. In 2019, it averaged about 30 to 40 min per small PCT for three people to complete the task. However, more labour is required with the control isolation plots from a maintenance standpoint. Maintenance around the isolation controls usually requires planting a pollen screen of cereal rye (Secale cereale L.) as well as hoeing and/or spraying to reduce weeds or insect pests. In addition, there is the added issue of maintaining the land around the isolation plot. Experience in Oklahoma shows that one full time person spends about 3 h per week working on keeping the outside control nursery clean of weeds or insect pests. About 0.5 h per week (1 employee) were spent on maintenance of each of the large PCTs. However, it could be possible to raise revenue through the sale of the grain produced by the pollen screen (cereal rye) or potentially other types of crops, to offset the cost of maintaining open type nurseries.

With the open pollinated controls, the only way to maintain genetic purity is with distance or isolation from the same species. For open pollinated species, such as tall fescue, a minimum of 305 m of distance between seed fields is required for the production of breeder or foundation certified seed as recommended by the Oregon Seed Certification Service (Oklahoma Crop Improvement Association standards are the same). If a breeding program established 20 or more open pollinated seed increases each year, the distance requirements would be demanding, requiring a spread of isolation nurseries over lots of different farms at different locations creating administrative and logistical challenges. If land space is a factor, then the number of isolations planted could be an issue, which may cost a generation of advancement. PCTs with reliable seed production would allow the planting of many isolation plots in a much smaller area. This would reduce time for traveling to and from many different locations and maintaining the space around these locations. For this purpose, researchers may prefer the larger PCTs compared to the smaller ones. Since the small PCTs are portable and easy to move they could also be used at leased offsite locations, such as private agricultural producers and universities. The PCTs would be much easier to maintain at these types of locations vs. larger open pollinated plots since travel to these sites may be many kilometres away. PCTs may reduce costs since less time is spent at the location for nursery maintenance. In addition, the production of high-grade seed  or  breeder  (nucleus)  seed  of  a  licensed cultivar normally costs a seed company around $35 to 50 kg^-1 to produce. In this scenario, pre-breeder seed would be a good target for the small PCTs, while the large PCTs would be ideal for breeder or nucleus seed production.

Since our experiments, a number of modifications for improvement of PCTs have been made. Previously the seams of the cover in the corner of the roof tended to show some wear and tear. This has been improved with the new robust frame structures and methods of fixing the cover fabric. It was also felt that some type of ‘U’ type anchor for holding the frame on ground could have been useful. This improved design is more robust and holds on the ground much more strongly than in previous versions. Options for windows are provided in the new design that allows viewing the interior of the PCT without disturbing it.

Future considerations

Although tents have been used for indoor and outdoor plant multiplications, the use of specifically developed PCTs as pollination control aids and seed increases are recent. Therefore, there is a market for the development of robust structures that can withstand high winds and bad weather, but are lightweight for transport, easy to assemble and include windows for examination and easy entry. Improvement regarding irrigation and agronomic operations within the PCTs without disturbance were needed following our experiments. Advances have been made since these trials and the PCT design has been improved to increase the benefit: cost ratio and for wider applicability to many crops. Flexibility in sizing the PCT covered area is also important for accustoming the protected area as per breeders’ requirement in any season. Advances in this area reflect development of PCTs of specific capacity that can be joined together as a modular structure to a number of independent parts to cover as large an area as required.

The second important aspect is the use of the right fabric as a cover. The fabric needs to be easily fitted, but hard enough to withstand wear and tear on the corners where it touches the frame, be pollen proof, but have sufficient aeration for temperature and humidity control.  Apart from the DWB10 and DWB24 synthetic nonwoven fabrics used in the study, there are a number of other fabrics that are available that have been tested in other crops such as sugar beet (Paul Townson Pers. Comm.) and mustard (S.S. Banga Pers. Comm.) with encouraging results. However, while the present study has established the superiority of synthetic PCTs, further studies to confirm wider utility in other crops and breeding scenarios will be needed in terms of estimating the economic implications in seed production.

 

 

 

Pollination control tents (PCTs) made from two nonwoven synthetic fabrics, DWB10 and DWB24, were tested against open controls across two years and three locations in Oklahoma for their seed production efficacies and control of pollen contamination. The two types of PCTs showed similar and higher seed yield by up to 36% compared with open control treatment. The higher average temperature and a lower to average humidity within the PCTs compared to the control across locations and years could have led to the more optimal and healthier seed set in the PCTs. The introduction of fans in the PCTs to increase pollen flow was not beneficial as it reduced seed yield by about 23% demonstrating that natural conditions in the PCTs were conducive for higher seed yield. Knowledge gained from this study is being used to improve the PCT design structure and to test newly developed fabrics in different crops. The proposed economic analysis and the generalized possibilities regarding the application of PCT technologies in plant breeding and in particular grass breeding, seems encouraging for increasing seed outputs, the hybridization process and seed multiplications.

 

The authors have not declared any conflict of interests.

 

The authors thank Rob Coy and Stuart Carter of PBS International, Salter Road, Scarborough YO11 3UP, UK for their help in standardising and providing the pollination tents.