Objective 4. Double Haploid Trials.

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Medicinal and aromatic plants share common breeding objectives with most traditional crops – the industry is striving for increased yields and superior quality. Traditional breeding programs are costly and take years to achieve results. This is particularly true in self pollinating species and in situations where extensive backcrossing is required to introduce a new trait into a crops with complex quality and yield expectations.

dillIn the double haploid (DH) technique used by PBI/NRC (Alison Ferrie) immature pollen grains (haploids) are exposed to treatments that result in doubling of the existing genetic material – resulting in homozygous, true breeding material in a single generation. This technique has been used to reduce the length of the crop improvement cycle of crops like canola and wheat by several years. Dr. Ferrie has also applied the DH technique to a wide range of medicinal and nutraceutical plant species.

The objective of this aspect of the program was to evaluate the field performance of the double haploids of various herb and spice crops that have been created by Dr. Ferrie.

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2003 Trials

2004 Trials

2006 to 2008 – Dill Trials

2006 to 2008 – Caraway Trials

Field Evaluation of Doubled Haploid Plants in the Apiaceae: Dill, Caraway, and Fennel


2003 Trials

In 2003, seed of double haploids of dill and fennel were obtained from Dr. Alison Ferrie of PBI/NRC. Due to limited seed supplies, the double haploid lines were grown from transplants. Seed was pre-germinated on petri dishes and then transferred to transplant flats held in the Dept of Plant Sciences greenhouses. The transplants were moved into the field when they were 6 weeks old.

The field plots were located at the Department of Plant Sciences, Horticultural Crops Research Station in Saskatoon. The features of the site were previously described in the Cumin Germplasm Evaluation Section.

The seedlings were planted out in early June. Rows were spaced 0.5 m apart, with 15 cm between seedlings within a row. The number of plants of each line varied according to the % of the seeds planted that actually germinated. Weeds were controlled by hand removal. The plots were watered as needed. No significant problems with insect pests or disease were observed. Plant heights were recorded at several points during the growing season. The crops were harvested in late September, after the first killing frosts but before significant shattering occurred.

Results

Germination percentages varied greatly between crops and DH lines. In general, the parental lines of dill had a higher germination % than the DH, but some of the DH lines of dill showed excellent seed viability. A few of the DH lines of dill completely failed to germinate. The parental material in the fennel trial had excellent seed viability. This was matched by about 50% of the DH lines; others had a lower % germination and a few DH lines of fennel failed to germinate. .

Plants heights also varied between DH lines and the parental strains. Eight of the nine DH lines of dill tested were substantially shorter than the parental line. However, one line (DH-1) was significantly taller than the parental line. The DH lines of dill were also much more uniform in height from plant to plant than the parental lines. Heights of the DH fennel were either very similar to the parental line or were nearly double the norm. There were too few DH anise plants to effectively judge plant heights.

Plant height of double haploid dill - graph Germination % for double haploid dill - graph
Plant heigts of doubled haploid fennel - graph Germination % for doubled haploid fennel - graph

Seed yields for a number of the DH lines of dill were substantially greater than yields from the parental lines. The three most productive DH lines averaged 61% higher yields than the parental line. There was no apparent relationship between the height of the DH plants and their productivity.

The quantity and quality of the oil produced by the DH dill lines was evaluated by solvent extraction following by gas chromatography.

Plant heigts of double haploid dill - graph Seed yields of double haploid dill - graph

The essential oil content of many of the DH lines was lower than the parental line (3.3%)(Table 4.1). However, DH lines DH-1 and DH-12 had substantially higher essential oil content than the parental line. DH-12 also produced higher yields than the parental line. The oil extracted from dill should contain > 50% carvone. All of the DH lines except for DH-1 met this quality criterion.

Table 4.1. Essential oil content and quality for double haploid lines of dill – 2003.

Sample % Essential Oil % Carvone
Mammoth (Parental) 3.3 52.8
DH-1 3.9 44.2
DH-4 1.3 57.5
DH-7 2.6 51.8
DH-12 3.8 51.1
DH-19 1.6 51.8
DH-24 1.8 55.7
DH-35 1.9 52.5
DH-42 2.1 51.9
DH-45 1.5 51.0


2004 Trials

In 2004, the main trial involved direct seeding the DH dill lines tested the previous season using transplants. The trials were run at the Department of Plant Sciences, Horticultural Crops Research Station in Saskatoon. This features of the site were previously described.

The site was prepared by rotovating prior to seeding. The plts were seeded on May 14 using a pushtype precision small plot seeder. The trial was seeded with 5 cm between seeds within a row and 50 cm between rows. Each plot of each line consisted of four 6 m long rows. Weeds were controlled by hand removal. The plots were watered as needed. No significant problems with insects pests or disease were observed. Plant heights were recorded at intervals during the growing season. The crops were harvested on October 13, after the first killing frosts but before significant shattering had occurred. The plots were harvested using a Wintersteiger Nursery Master Elite small plot combine. The harvested material was air dried at 40 C for 5 d, further threshed and then cleaned using a dockage tester. Seed yields and 1000 seed weights were determined at this point.

Results

Direct seeded dill trial.

As the trial was seeded quite heavily, all lines produced a complete stand. Line DH-12 emerged exceptionally quickly, established a very strong stand and was the first line to begin flowering. At the time of evaluation in late July, DH-12 was the tallest line and was furthest advanced in terms of flower development (Figure and Table 4.2). Line DH-12 also showed great uniformity of plant height at that time. However, by the final harvest line DH-12 was the shortest line but its seed yields exceeded all others, including the parental line by a factor of 300 % (Figure and Table 4.2).

The precocious characteristics exhibited by DH-12 appeared highly beneficial in the relatively cool and short 2004 growing season. None of the lines were actually very mature by the time of harvest, but line DH-12 was more advanced than the others. Seed size for DH-12 was comparable to the parental line. None of the other DH lines produced seed yields that exceed the parental line. Although line DH-7 had yielded well in the 2003 trial, it performed poorly in 2004. Line DH-1 was slowest to develop but by the en d of the growing season it was substantially taller than the parental line. Seed yields from DH-1 were poor as it is slow to develop, however the seeds that it did produce were exceptionally large (Table 4.2). The unusually large size of the DH-1 plants may make it desirable for production of dill oil which can be extracted from both the seed and the herbage.

Seed yields of doubled haploid dill - graph Plant heights of doubled haploid dill

Table 4.2 Plant heights, yields and seed size for various lines of direct seeded double haploid dill in 2004.

July 26 Final Harvest
Height
(cm)
Height
(cv %)
Stage Height
(cm)
Height
(cv %)
Seed Yield (kg/ha) 1000 seed wt (g)
DH 1 51 e 22 0 175 a 29 76 c 3.55 a
DH 7 64 d 21 1.5 144 cd 29 54 c 2.67 ab
DH 12 88 a 5 5 125 e 29 1205 a 1.94 b
DH 24 72 c 9 2 146 c 29 230 bc 2.12 b
DH 35 82 b 12 2 136 cd 30 481 b 1.70 b
DH 45 72 c 14 1.5 135 de 30 286 bc 2.09 b
Parental 78 b 9 2 159 b 30 344 bc 3.02 b

Values within columns followed by the same letter are not significantly different p = 0.05

 

Table 4.4. Essential oil content and quality for double haploid lines of dill – 2004.

Sample % Essential oil % Carvone % Limonene Oil yield (kg/ha)
Mammoth (Parental) 2.91 57.4 38.5 1001
DH 1 2.38 55.0 40.7 180
DH 7 2.48 51.8 40.0 133
DH 12 2.63 53.4 43.9 3169
DH 24 2.38 51.2 44.1 547
DH 35 2.12 49.6 45.8 1019
DH 45 2.45 59.0 46.4 700
DH 47 *** 2.51 46.8 46.3 8117
DH 53 *** 3.11 52.9 43.3 7501

*** = transplanted single row plots

Oil yields for double haploid dill - graphOn average, essential oil content in 2004 was somewhat lower than in 2003; this likely reflects differences in the growing season. In 2003, several of the DH lines had a higher essential oil content than the parental line – but in 2004 the parental line had the highest oil content. Most lines met quality standards for % carvone (50%). Oil yield (kg/ha) for line DH 12 was 300% of the parental line. This reflects both its high seed yield and relatively high oil content. Oil yields for lines DH 47 and DH 53 were both exceptionally high – but as previously mentioned, this may reflect the advantage provided by transplanting and wide row spacings.

 

Fennel

As in the 2003 trial, the germination % of the DH fennel lines was quite variable. Many lines appeared to be non-viable while a couple showed excellent vigor.

Plant heights for the DH fennel lines in 2004 did not show the extreme line to line variability that was observed in the 2003 trial. Due to the short, cool growing season, no mature seed was harvested from this trial.

 

Germination % for double haploid fennel - graph Plant heights of double haploid fennel - graph


2006 – DH Dill Trials

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In 2006, the main testing trial utilized the DH lines of dill generated by PBI/NRC that had shown the most promise in small scale screening trials conducted in 2004 and 2005. Lines were selected and multiplied for further testing if they showed promise in any of the following areas;

a) plant vigor, uniformity of height, uniformity of maturity
b) seed yield
c) oil content of the seed
d) chemical composition of the seed oil.

Key objectives were to identify lines that retained the seedling vigor of the parental lines, but were shorter and more uniform in stature. Improved uniformity of seed set was a highly desirable characteristic. The presently available commercial dill lines have a very indeterminate growth habit resulting in uneven seed maturity. With highly indeterminate seed set it is inevitable that a portion of the seed is lost to shattering before the crop is ready to harvest … while a portion of the seed is also still immature at harvest. Immature dill seed does not have the flavor profile required by the industry.

All field trials were conducted at the University of Saskatchewan Horticulture Research Facility in Saskatoon (2909-14th Street East). This field site features a Sutherland Series heavy clay soil. This soil type is less than ideal for establishment of small seeded crops – but once the crops get established, its moisture and nutrient retention characteristics result in excellent yields. The test site had been in summer fallow in 2005.

The field was rotovated prior to planting to provide as uniform a seed bed as possible. No supplemental fertilizers were required. The trial was seeded on May 23. This is a later planting date than most commercial growers would use – but it reflects the fact that the clay soil at the test site is very slow to dry and warm in the spring. The crop was seeded with a push-type small plot seeder. Each line was planted out in 3 rows (0.5 m apart) with each row being 8 m long. Each plot was separated by a 1 m buffer. The trial was laid out in a randomized complete block design with 3 replicates.

Most of the seed used in the 2006 trials was generated in 2004 and 2005 in small plot observation trials conducted by the U of S. Because of adverse growing conditions in 2004 and problems with herbicide injury in 2005, the viability of the seed was relatively low and also varied between lines. This was confirmed in germination tests conducted prior to seeding the 2006 crop (Table 2006-1). To compensate for the low seed viability, we opted to use an exceptionally high seeding rate – roughly 4X normal. This resulted in a very dense stand in the lines with the highest germination % , but only a spotty stand in the lines with the poorest germination %. Like canola, dill is an indeterminate crop. Dill plants are capable of altering their branching habit to fully utilize the available growing space. Consequently, once the plants reached maturity, the plots for the various lines were relatively uniform in terms of canopy density.

Warm weather coupled with timely rainfall in early June resulted in rapid and uniform crop emergence but also produced near-ideal conditions for the germination of weeds. The herbicide linuron (Lorox) was applied (200 g a.i./a) once the crop reached the 3 true leaf stage. The herbicide was lightly watered in 4 days after application. Linuron is cleared for use on a wide range of Umbelliferous crops (ie; coriander and carrot) – but it is not presently registered for use on dill. Rick Holm (weed scientist at the U of S) has previously tested linuron on dill and found it to be quite crop safe. However, when this product was used on the 2005 seed increase of the DH dill, it caused significant crop damage (yellowing and some seedling death). This problem was initially attributed to adverse conditions following application of the herbicide. However, similar problems occurred again in 2006 – and in that case conditions at and following application of the linuron were near-ideal. The degree of damage was similar in both the parental lines and the double haploids. Relatively few plants actually died as a result of the herbicide treatment in 2006, but growth of the crop was set back by at least 3 weeks. The linuron provided excellent season-long weed control in this trial.

In a presentation made to the 2006 Annual Meeting of the Canadian Society for Agronomy, Zheljazkov et al showed that while application of linuron to dill caused about 30% plant mortality, this degree of damage had no impact on yields. This illustrates the yield plasticity of this crop. They identified a number of herbicides that were less toxic to the crop than linuron – these products should be considered for use in future work with dill.

By early July, the dill crop had out-grown the effects of the herbicide – except that the sprayed plants were considerably shorter than plants in areas missed by the spray. This is illustrated in Figure 2006-1. The crop was irrigated once in late July.

By the 3rd week in August, the flowers and some of the upper leaves on some of the lines had begun to yellow. Initially this was attributed to these lines being early maturing – but it turned out that the yellowing was largely due to disease. The disease started in a couple of localized points, but spread throughout the plot with a one week period. Although the causal organism was not isolated, the symptoms are typical of blossom blight. This disease complex has become fairly widespread in commercial plantings of coriander and caraway in Saskatchewan – but dill is usually not as severely affected. Distribution of the disease was not uniform across the plots – but whether this reflects differences in disease sensitivity of the various DH lines – or was due to differences in distribution on the initial disease inoculum could not be determined. Because of the rapid spread of the disease, no chemical control was attempted. Trials conducted by the University of Saskatchewan with blossom blight of coriander suggest that this disease cannot be controlled utilizing commercially available foliar-applied fungicides once it has become established within the canopy. Fungicidal seed treatments followed by a preventative spray program may be more effective – and this practice was followed in subsequent trials.

Each line was straight cut combined as it matured using a WinterSteiger Plot Master combine. Due to the indeterminate nature of dill, some pre-harvest shattering loss of the first seed set is inevitable. In 2006, the onset of wet, cool weather in mid-September resulted in a substantial delay in the harvest of many of the later maturing lines (September 26). Unfortunately, once dill is ready to the crop is ready to harvest, anything delay can result in substantial losses to shattering. Although swathing may reduce problems with shattering, most commercial growers try to avoid swathing dill because of its rank growth habit. It may be useful to try to chemically desiccate the later maturing lines.

The seed was air dried after harvest and then cleaned using a dockage tester.

Table 2006-1. Agronomic Observations on Double Haploid and Parental lines of Dill (2006).

Yields per unit area were below the average of 1000 kg/ha seen in most commercial dill fields – this likely reflects the fact that the rows were very widely spaced to facilitate roguing and crop evaluation. Of more importance is the fact that several of the DH lines had yields that were substantially greater than the industry standards (Mammoth and Giant). It is noteworthy that the yields exceeded that of Mammoth – as it was Mammoth that was used to generate the DH lines included in this test.

Oil content and quality were evaluated utilizing standard methods (Arganosa et al 1999). Briefly, a 5 g sample of cleaned seed was steam distilled to extract the essential oils and then the composition of the oil is determined by HPLC.

The results of the oil analysis and the corresponding oil yields are presented in Table 2006-2. Overall, the oil content was slightly higher than normal – this likely reflects the warm growing conditions through July and August of 2006. Although there was a significant degree of variation in the essential oil content of the various lines tested, the composition of the oil (limonene : carvone ratio) was quite similar (Table 2006-2).

Table 2006-2. Oil content and composition for double haploid and parental lines of dill (2006).

The essential oil content of DH 12 was superior to all other DH lines, as well as the industry standard lines.

Notable performance, yields and quality characteristics of the lines were;

a. Line DH-12 had performed very well in previous trials – but in this trial it appeared to experience a greater degree of damage by the herbicide than the other lines. This may reflect the fact that DH-12 emerges very quickly – resulting in this line being at a more advanced stage of crop development at the time of the herbicide was applied. This may have rendered it more susceptible to herbicide damage. DH-12 is also very uniform in height, with a short stature and early maturity (Table 2006-1). Figure 2006-3 shows that on July 20, DH-12 was in full bloom – whereas most of the other lines were just starting to bud out at that time. In the 2006 trial this meant that this line was flowering at a time when weather conditions appeared to be most suitable for development of the blossom blight. As a consequence many of the seeds formed by this line were shrunken and sterile. This is reflected in the exceptionally high grade out and low 1000 seed weight for DH-12. Yields for this line were consequently lower than normal. However, DH-12 is very early maturing. This is reflected in the exceptionally high oil content of seed of this line.

b. Line DH-47 is another short stature line that had performed well in previous trials. Although this line showed excellent seedling vigor, it was quite severely affected by blight, resulting in low seed yields and a high % seed grade out due to shrunken (blight affected) seeds (Table 2006-1). The poor quality of the seed harvested for this line was also reflected by its low oil content (Table 2006-2).

c. CDC Giant – experienced some of the same herbicide toxicity problems as D12. This line also appeared to be particularly sensitive to the blossom blight. The essential oil content of CDC Giant was quite high, reflecting the strong selection pressure for this trait in dill breeding programs.

d. Lines DH-7 and DH-53 – in this trial, as in previous trials, these lines were very vigorous, producing plants that were taller than the parental line (cv Mammoth) but much more uniform in height. Both of these lines were relatively late maturing. Harvest of these lines was delayed due to bad weather from mid-September onwards. This resulted in extensive seed losses to shattering. Despite this loss, these lines were the highest yielding in the trial. In part, this could be attributed to the fact that these lines were relatively unaffected by the blight. Whether this reflects inherent disease resistance or simply reflects differences in growing conditions at the key stages of development of both the crop and the disease infection could not be determined. Although these lines were both quite late, the essential content of the seed was acceptable – with DH 53 having a seed oil content superior to CDC Giant. Overall oil yield/unit area of Lines DH-7 and DH-53 were respectively 58% and 116% greater than the industry standard Mammoth – this reflects both the high yields and relatively high seed oil content of these lines.

e. Line DH-1 – in previous trials this line had been extremely late but had produced very large plants with very large seeds. Seed viability was very poor – likely reflecting problems with getting mature seed. The poor seed viability led to a poor stand and seed yields were consequently low. The seed oil content was also quite low.

Evaluation of New DH Dill Lines

Over the winter of 2005/2006 the program generated seed of the DH lines that had produced the best results in the 2004 and 2005 field trials (DH 12, 47 and 53). This step was considered prudent given the poor viability of the seed generated in the 2004 and 2005 field trials. This seed was used to establish a nursery for seed generation of these DH lines. Using transplants to establish the nursery insured that high quality mature seed could be harvested in a timely manner. It also allowed for evaluation of the germination %, field performance and seed quality of the selected lines against the parental lines. Two new DH lines were also included in this trial.

Results – The germination % of the previously selected DH lines was not as good as the parental types, but once in the field these DH lines again out-performed their corresponding parental lines, producing uniform stands of vigorous plants. DH 12 and 47 were far earlier to mature than the parental lines, while DH 53 produced a very vigorous, uniform stand of late maturing but high yielding plants. It is interesting to note that there was very little disease in this trial – while these same lines (DH 12 and DH 47) had been severely affected by disease in the adjacent direct seeded trial. This suggests that disease losses in this crop are strongly influenced by interactions between disease, crop development stage and environmental conditions. The two new DH lines included in this trial failed to germinate.

2007 – DH Dill Trials

The 2007 trial utilized the most promising DH lines selected from previous trials. Lines were included in these trials if they had demonstrated promise in any of the following areas;

a) plant vigor, uniformity of height, uniformity of maturity
b) seed yield
c) oil content of the seed
d) chemical composition of the seed oil.

Key objectives were to identify lines that retained the vigor of the parental lines, but were shorter and more uniform in stature. Improved uniformity of seed set was a highly desirable characteristic. The presently available commercial dill lines have a very indeterminate growth habit resulting in uneven seed maturity. With highly indeterminate seed set it is inevitable that a portion of the seed is lost to shattering before the crop is ready to harvest, while a portion of the seed is also still immature at harvest. Immature dill seed does not have the flavor profile required by the industry.

Unless otherwise specified, the procedures utilized in the 2007 DH dill trials corresponded to the procedures previously described for the 2006 trials. The trial was again conducted at the University of Saskatchewan Horticulture Research Facility in Saskatoon. The test site had been in pumpkins in 2006 which resulted in greater weed pressure than in previous years. The field was prepared as previously described. The trial was seeded on May 23. This is a later planting date than most commercial growers would use – but it reflects the fact that the clay soil at the test site is very slow to dry and warm in the spring. The crop was again seeded with a push-type small plot seeder. Each line was planted out in 3 rows (0.5 m apart) with each row being 8 m long. Each plot was separated by a 1 m buffer. The trial was laid out in a randomized complete block design with 4 replicates.

The seed used in the 2007 trials was generated in the 2006 trial. Although the germination % of the seed was relatively good, we still opted to seed relatively heavily because of the heavy nature of the soil at the test site. This resulted in a dense stand for most lines except DH 45 and 47 (Table 2007-1). By July, the crop canopy for the various DH dill lines appeared quite uniform – despite the difference in initial plant stand. This reflects dill’s indeterminent growth habit.

Warm weather coupled with timely rainfall in early June resulted in rapid and uniform crop emergence but also produced near-ideal conditions for the germination of weeds – particularly red root pigweed and common groundsel. Weed pressure was particularly heavy in the 4th block of this trial. In the 2006 trial the herbicide linuron (Lorox) applied at 200 g a.i./a once the crop reached the 3 true leaf stage had caused significant crop damage (yellowing and some seedling death). In the 2007 trial we again opted to use Linuron, but at a lower rate (150 g a i./a). The lower rate of linuron reduced but did not eliminate herbicide damage to the crop (see Table 2007-1) and also provided less effective weed control – especially in the heavy weed pressure in the 4th block. The 4th block had to be hand weeded on several occasions in 2007.

In the 2006 trial, the DH lines as well as the industry standard and parental lines had appeared uniformly susceptible to the relatively high rate of linuron. In the 2007 trial when the lower linuron rate was used there appeared to be some variability in relative sensitivity – with several of the DH lines showing more damage than the established lines (Table 2007-1). Whether these differences reflects actual differences in herbicide sensitivity or are simply related to differences in crop developmental stage at the time of exposure to the linuron could not be determined. The fact that CDC Mammoth seemed particularly resistant to linuron in 2007, yet in the 2006 trial it was amongst the most sensitive lines, suggests that the effect was mediated by environmental conditions or developmental stages.

By early July, the dill crop had out-grown the effects of the herbicide, but development and vigor of the 4th block was delayed by weed pressure. The crop was irrigated once in late June and again two weeks later. Although the soil was quite dry from mid-July onwards we opted to not irrigate because of the problems with blossom blight observed in the 2006 trial.

Because of the problems with blossom blight observed in 2006, we opted to implement a preventative spray program in 2007. The crop was sprayed with azoxystrobin (Quadris) or chlorthalonil (Bravo) every 10 days from mid-July as the crop began to come into flower through until mid-August by which time the crops had begun to mature. There were no obvious signs of blossom blight in 2007 – whether this indicates the spray program was effective could not be determined as there were no untreated areas for comparison. Although there was no disease apparent, some lines again flowered well yet set relatively few seeds and many of those seeds were shrunken and of low quality. Whether this problem reflects a disease or is a varietal response to adverse growing conditions at flowering could not be determined.

Seed loss due to shattering, both prior to harvest and during combining, is a major issue during the harvest of dill. In previous years the crop was straight cut combined once it had matured – but this resulted in significant shattering loss, particularly in early maturing lines or in situations where die-down was delayed by cold, wet weather. In 2007 we opted to chemically desiccate the crop with diquat (Reglone at 1.0 l/a) as soon as it began to mature, followed by hand swathing 5 days later to minimize shattering due to wind action. One week later the swaths were picked up and combined using a standard WinterSteiger small plot combine. Although this harvest procedure did not completely eliminate shattering loss, the losses were far lower than in previous years. This is reflected by the fact that yields in 2007 were almost 10X higher than in 2006 (Table 2007-1). Although the altered harvesting procedures may have made a significant contribution towards the higher yields observed in 2007, near ideal growing conditions through September followed by very favorable harvest conditions were also helpful.

Yields per unit area in 2007 were all well above the average of 1000 kg/ha seen in most commercial dill fields – this likely reflects the near ideal conditions in 2007, coupled with the extra care taken to reduce seed loss due to shattering.

In the 2006 trial several of the DH lines had yields that were substantially greater than the industry standards (CDC Giant) or the parental line (Mammoth). In 2007, none of the DH lines outperformed the standards. This may reflect the fact that growing conditions through the fall of 2007 were near ideal – this would have been advantageous for the relatively late maturing lines like CDC Giant and Mammoth. In years with an earlier or less favorable fall the earlier maturing DH lines would likely produce better yields and as the seeds would be more mature, the oil content would also expected to be higher.

The seed was air dried after harvest and then cleaned using a dockage tester. Quality analyses of the essential oils extracted from the seed were conducted as previously described.

Table 2007-1. Agronomic observations on double haploid and industry standard lines of dill in 2007).

Notable performance and yields characteristics of the DH dill lines tested in 2007 were;

a. Line DH-12 produced yields that were within 10% of the industry standard lines, yet it was ready for harvest more than a month earlier (August 23 versus October 6) than the standard lines. DH 12 again produced a vigorous stand of fast growing uniform plants. While it had showed significant herbicide damage in the 2006 trial, there was relatively little damage in 2007. This suggests the importance of developmental stage in determining sensitivity to linuron. DH 12 is far shorter in stature than the standard lines. A shorter plant stature may be desirable in rank crops like dill as it reduces the mass of material to be combined. In the 2006 trial, line DH 12 experienced severe yield loss to what was thought to be blossom blight. In the 2007 trial no blossom blight was observed, yet there was again a fairly high incidence of aborted or shrunken seed in DH 12. Flowering of DH 12 is quite synchronous and occurs when the plants are relatively small. It is possible that the apparent issues with seed set simply reflect the plants’ inability to fill the huge number of seeds that set at one time. Selecting for synchronous flowering at an early growth stage may be a yield limiting strategy – however it tends to guarantee at least some yield. Seed of DH 12 was quite large, again suggesting a high degree of maturity.
b. Line DH-47 is another short stature line that had performed well in early trials. However the seed of DH 47 generated in 2006 had been of poor quality and that resulted in a poor stand and limited yields in the 2007 trial.
c. CDC Giant – had problems with herbicide toxicity and blight in 2006, but in 2007 it produced the highest yields of the lines tested.
d. Lines DH-7 and DH-53 – in 2007 these lines again produced vigorous stands of uniformly tall plants. Both of these lines were relatively late maturing and their yields were substantially lower than industry standards which are also late maturing. DH-7 produced the largest seeds of any line tested in 2007.

Seed Quality in 2007 (see Table 2007-1)

Overall the essential oil content of the seed harvested in 2007 was slightly higher than in 2006, reflecting the greater degree of crop maturity achieved in 2007. The seed harvested in 2007 had a higher limonene content than seen in 2006 – the reason for this quality shift is not clear. Within the lines tested in 2007, there was less variability in the oil composition (% limonene) than in the oil content. Oil extracted from CDC Giant appeared to have a lower limonene content than any of the other lines tested in 2007. Oil yields (kg/ha) were far higher in 2007 than in 2006. This reflects the higher seed yields in 2007 rather than any improvement in seed oil content.

While line DH-12 had the highest seed oil content in the 2006 trial, its seed oil content was amongst the lowest of the lines tested in 2007. This emphasizes the importance of multi-year trials when evaluating any germplasm. DH-53, which is quite late maturing, had the highest seed oil content in both the 2006 and 2007 trials.

2008 – DH Dill Trials

The 2008 trial focused on the DH lines tested in 2007 as no new lines tested in 2007 had shown sufficient promise to merit inclusion in this trail.

Unless otherwise specified, the procedures utilized in the 2008 DH dill trials corresponded to the procedures previously described for the 2007 trials. The trial was again conducted at the University of Saskatchewan Horticulture Research Facility in Saskatoon. The test site had been in lettuce and onions in 2007 – this resulted in relatively limited weed pressure – except for common groundsel which has become problematic throughout the test plots. The field was prepared for planting as previously described. The trial was seeded in the 3rd week of May. This is a later planting date than most commercial growers would use – but it reflects the fact that the clay soil at the test site is very slow to dry and warm in the spring. The crop was again seeded with a push-type small plot seeder. Each line was planted out in blocks of 4 rows, with each row being 6 m long. The between row spacing in the 2008 trial was reduced to 0.3 m as compared to 0.5 M in previous years. The tighter row spacing was used to produce a thicker crop canopy earlier in the season, thereby potentially reducing problems with weed competition. Each plot was separated by a 1 m buffer. The trial was laid out in a randomized complete block design with 4 replicates.

The seed used in the 2008 trials was generated in the 2007 trial. Although the germination % of the seed was relatively good, we still opted to seed relatively heavily because of the heavy nature of the soil at the test site. This resulted in a dense stand for most lines except DH 7 and 45. By July, the crop canopy for the various DH dill lines appeared quite uniform – despite the difference in initial plant stand. This reflects dill’s indeterminent growth habit.

Cool weather through late May and most of June resulted in extremely slow and uneven crop emergence. Some plants came up within 2 weeks of planting, but the majority took over a month to emerge. This delayed emergence response appeared to be consistent across lines and replicates. Although conditions were not favorable for emergence of the dill crop, they were suitable for the germination of weeds – particularly common groundsel. A healthy weed population coupled with slow crop emergence resulted in heavy weed pressure throughout the plot area. In the 2006 trial the herbicide linuron (Lorox) applied at 200 g a.i./a once the crop reached the 3 true leaf stage had caused significant crop damage. In the 2007 trial using Linuron at a lower rate (150 g a i./a) had reduced crop damage but also provided less effective weed control. Because of the heavy weed pressure in 2008 we opted to return to the higher rate of Linuron. This resulted in little crop damage and decent weed control, except for the common groundsel. The groundsel was so advanced by the time that the dill crop was finally ready to spray that the groundsel went to seed before the herbicide became effective. As these weed plants collapsed and died they released their seeds. These seeds were incorporated into the soil during the supplementary tillage operations required to clean up weed escapes. These weed seeds germinated following each rain event – creating flush after flush for the duration of the growing season. These weeds were controlled by hand tillage until the dill crop grew to the point where it shaded out the small stature groundsel plants.

Because of the problems with blossom blight observed in 2006, we again opted to implement a preventative spray program in 2008. The crop was sprayed with azoxystrobin (Quadris) or clorthalonil (Bravo) every 10 days from mid-July as the crop began to come into flower through until mid-August by which time the crops had begun to mature. There were no obvious signs of blossom blight in 2008 – whether this indicates the spray program was effective could not be determined as there were no untreated areas for comparison. Although there was no disease apparent, some lines again flowered well, yet set relatively few seeds and many of those seeds were shrunken and of low quality. Whether this problem reflects a disease or is a varietal response to adverse growing conditions at flowering could not be determined.

Seed loss due to shattering, both prior to harvest and during combining, is a major issue during the harvest of dill. In 2006 the crop was straight cut combined once it had matured – but this resulted in significant shattering loss, particularly in early maturing lines or in situations where die-down was delayed by cold, wet weather. In 2007 we had tried chemically desiccating the crop, followed by swathing 5 days later and then combining after another week of dry down. Although this harvest procedure did not completely eliminate shattering loss, the losses were far lower than in previous years. In 2008 we tried to further refine this process. We chemically desiccated the crop as before, but allowed it to dry standing and then straight combined the crop. The thought was that the standing crop would dry out more quickly and that elimination of the swathing step might also reduce total loss to shattering. Unfortunately conditions in the fall were less than ideal for this approach to crop management. All lines were slow to mature in 2008 and therefore the desiccation and dry down step occurred during relatively cool weather. This slowed the drying process, leaving the standing crop exposed to shattering loss for extended periods of time. This problem was most severe on the late maturing lines (DH 7 and 53) which stood for more than 3 weeks before they were dry enough to combine. Although it was not possible to quantify shattering losses, visual inspection of the field suggested that it was substantial.

Despite problems with weed competition and shattering losses, seed yields in 2008 were all well above the average of 1000 kg/ha seen in most commercial dill fields.

In the 2006 trial several of the DH lines had yields that were substantially greater than the industry standards (CDC Giant) or the parental line (Mammoth). In 2007, none of the DH lines outperformed the standards. In the 2008 trial, the early maturing DH lines (DH 12 and 35) had yields that were significantly higher than the standards. Yields of the later maturing lines (DH 7 and 53) were reduced due to shattering loss.

The seed was air dried after harvest and then cleaned using a dockage tester. Quality analyses of the essential oils extracted from the seed were conducted as previously described.

Table 2008-1. Agronomic observations on double haploid and industry standard lines of dill in 2008).

Notable performance and yield characteristics of the DH dill lines tested in 2008 were;

a. DH 35 has emerged as the line that most consistently delivers yields superior to the existing standard lines. It produces a vigorous stand of moderate stature plants that are ready to harvest about 1 week before the standard lines.
b. Line DH-12 produced yields in 2008 that exceeded the industry standard lines, and it was ready for harvest two weeks earlier (Sept 10 versus October 1) than the standard lines. In previous trials DH 12 had shown superior vigor early in the season. This was also apparent in 2008 – where a portion of the seeds germinated within 2 weeks of planting despite less than ideal field conditions. Although these plants got off to an early start, the majority of the DH seedlings took over a month to emerge. This resulted in a very uneven crop for DH 12 in 2008 – whereas in other years this line had been exceptionally uniform. Seed from the early emerging plants of DH 12 was lost to shattering before the rest of the plants in the DH 12 plots were ready to be desiccated.
c. Line DH-47 is another short stature line that had performed well in 2008. This line had produced a poor stand in the 2007 trial but produced an acceptable stand in 2008.
d. As usual, the DH-7 and DH-53 lines produced vigorous stands of uniformly tall plants. Both of these lines were relatively late maturing and their yields were reduced due to shattering loss in 2008.

Seed Quality in 2008 (see Table 2008-1)

Overall the essential oil content of the seed harvested in 2008 was slightly lower than in 2007, but was comparable to 2006 – this reflects the relatively greater degree of crop maturity achieved during the exceptionally long and warm 2007 growing season. The seed harvested in 2008 had a higher limonene content than seen in 2006 or 2007 – the reason for this shift is not clear. Within the lines tested in 2008, there was again less variability in the oil composition (% limonene) than in the oil content.

Oil extracted from CDC Giant appeared to have a lower limonene content than any of the other lines tested in both 2007 and 2008. Oil yields (kg/ha) in 2008 were lower than in 2007 but higher than in 2006. Year to year differences in seed yield have a greater impact on oil yields/unit area than differences in seed oil content. . DH-12 had the highest seed oil content in the 2006 and 2008 trials, but its seed oil content was amongst the lowest of the lines tested in 2007. DH-53, which is quite late maturing, had the highest seed oil content in both the 2006 and 2007 trials, but in the 2008 trial its seed oil content was near the mean. These year to year differences in performance emphasize the importance of multi-year trials when evaluating any germplasm.

Conclusions for DH Dill and Caraway Trials

In replicated field trials of DH dill conducted from 2006 through 2008 this project identified several lines that differed greatly from the parental lines in important agronomic characteristics such as speed of crop emergence, crop stature and time to maturity. These traits may be of value in a breeding program, especially as they are present in a homozygous form in the DH lines. We also identified several DH lines that were superior in seed and seed oil yield relative to the parental line and the most widely grown commercial dill variety. These lines may have immediate market value.

Opportunities for commercial release of the most promising DH lines identified in this project are being pursued by the Crop Development Center of the University of Saskatchewan working in collaboration with PBI/NRC and the Saskatchewan Herb and Spice Growers Association. The DH dill lines with traits of potential value in crop improvement programs will be preserved and made more widely available through the Plant Gene Resource Center of Agriculture Canada.


2006 – DH Caraway Trials

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 A total of 25 DH lines of caraway were available for the 2006 trials – along with 3 parental lines. Because of limited amounts of seed available for the DH lines, all lines were seeded in the Dept of Plant Sciences greenhouses and then transplanted out in early June. The seedlings were raised in 144 cell transplant flats filled with Sunshine Mix #4 media. The flats were maintained under near optimum conditions for germination – a 24/18 C temperature regime with a 16h photoperiod. When the seedlings were 4 weeks old they were transplanted into the previously described field plot area (15 cm apart with 0.5 m between rows). The growth habit of the plants was observed. The trial was hand harvest in early October and then threshed using a stationary combine. The seed was cleaned using a dockage tester. Because of the uneven numbers of plants no yield comparisons were possible.

General observations

The parental material showed relatively poor seed viability even under the near-ideal conditions of the greenhouse (Table 2006-3). Once transplanted into the field, the parental lines performed relatively well – they appeared vigorous and were quite uniform in stature and maturity. No problems with disease were observed in the parental or the DH lines.

As has been seen in other crops, many of the DH caraway lines had serious agronomic drawbacks – ie; many had low germinate % even under ideal greenhouse conditions (Table 2006-3), or they had very poor vigor or an exceptionally short or distorted growth habit. Although some of these poorly adapted lines did produce seed, they will likely be omitted from future trials. Some of the DH lines (ie; DH-10 and 29) showed much better germination % than the corresponding parental lines. None of the DH caraway lines were obviously superior to the parental lines in terms of vigor, uniformity of plant configuration or yield.

Seed quality assessments were conducted on 5 g samples of clean seed, utilizing the procedures outlined in the dill section. The essential oil content of line DH-21 was significantly higher than the parental line, while for lines DH-14 and 29 the essential oil content of the seed was far lower than the parent. The oil composition of these lines was also quite different from the parent. The combination of low oil content and different oil composition may reflect the fact that the seed of these lines was not fully mature at harvest.

Sufficient seed was generated in this trial to allow a replicated yield trial to be conducted in 2007.

2007 – DH Caraway Trials

The 2006 field trials with DH annual caraway had produced very limited seed yields, and the seed was of poor quality. This strongly suggests that the growing season in Saskatchewan is not long enough to consistently mature the presently available lines of annual caraway. It is noteworthy that the 2006 crop failed to mature despite the fact that the crop received a head-start by transplanting coupled with the fact that 2006 was an exceptionally warm year.

The 2007 field trial with DH caraway was conducted adjacent to the DH dill trial, at the previous described University of Saskatchewan research site. All variables and procedures were identical to those described in the dill trial except;

- the trial was seeded using the DH caraway lines that had produced appreciable amounts of seed in 2006. This process strongly selected for fast maturing lines.
- due to limited quantities of available seed. the trial was only planted out in three replicates and each replicate consisted of only two rows rather than the three rows used in the dill trial.
- as annual caraway is extremely late maturing, the crop was left in the field until mid-October. It was then direct combined. The delayed harvest may have resulted in some seed loss to shattering. Delaying the harvest until this point in the season would be extremely risky for a commercial grower.

Results

The caraway was slower to emerge than the dill and the stand quality of the various lines was highly variable (Table 2007-2). Stand counts for the parental lines were not appreciably different than for DH 10 and 29. The caraway appeared to suffer fewer ill effects from the linuron spray than the dill. This may reflect the smaller size of the caraway plants at the time of spraying.

In mid-June it became apparent that there was a severe problem with aster yellows in the entire research plot area – including the caraway plots. We had seen increasing levels of this disease in 2006, but did not suspect that the problem would persist into 2007. Nonetheless, the caraway crop was heavily infected and the effect on the plants was much more severe than in dill or coriander – and much more like the devastating effect seen in carrots. The plants became yellow and produced multitudes of small distorted leaves and stems. None of the infected plants survived to produce seed and as a consequence, seed yields in this trial were compromised. There did not appear to be any significant difference in the incidence or impact of aster yellows for the various DH lines or the parental lines of caraway.

Given the problems with stand establishment, and aster yellows, seed yields were actually unexpectedly high – exceeding by a substantial margin the yields typically seen under commercial production. It is risky to extrapolate small plot yields to field scale operations – but the results are promising. Of particular interest is the fact that line DH 10 out-yielded the line it was derived from (NN-2) by a factor of 5 fold. In both the 2006 and 2007 trials, DH 10 had produced a better stand than its parent. In the 2007 trial, this stand advantage was further enhanced by the fact that DH 10 appeared to be less susceptible to aster yellows than NN-2. Of note is the fact that the seed of DH 10 is substantially smaller than the seed of any of the other DH lines or the parental lines. This may be undesirable for sales direct to the consumer where large seed size is equated with quality – however it would be of little importance to the processing sector.

Quality analyses of the 2007 caraway crop were completed as planned. The seed oil content was much higher in 2007 than in 2006 – this likely reflects the fact that the crop was allowed to mature late into the fall of 2007. The seed oil content of the DH lines was comparable to the parental lines – although the variability between lines was greater than that seen in the dill trial. Again the oil composition (% limonene) was more stable across the lines tested than the actual oil content. The highest yielding DH caraway line (DH 10) had a moderate seed oil content – but for total oil produced/ha it would clearly have exceeded all other lines, including its parent.

2008 – DH Caraway Trials

The 2008 field trial with DH caraway was conducted adjacent to the DH dill trial, at the previous described University of Saskatchewan research site. All variables and procedures were identical to those described in the dill trial except;

- the trial was seeded using the DH caraway lines that had produced appreciable amounts of seed in 2006 and 2007. This process strongly selected for fast maturing lines.
- as annual caraway is extremely late maturing, the 2008 caraway crop was left until mid-September before it was desiccated and harvest was delayed until mid-October. This process may have resulted in some seed loss to shattering. Delaying the harvest until this point in the season would be extremely risky for a commercial grower.

Results

The caraway was even slower to emerge than the dill in 2008 and as noted with the dill this led to problems with weed competition. Stand counts for the parental lines were not appreciably different than for DH 10 and 29 (data not shown). As noted in previous years, the caraway appeared to be more tolerant of the linuron spray than dill. This may reflect the smaller size of the caraway plants at the time of spraying.

As with the 2008 dill crop, the linuron treatment of the 2008 caraway plot came too late to provide adequate control of the dominant weed in the plot area – common groundsel. While the dill crop outgrew the relatively short stature groundsel plants by mid-July, the shorter caraway plants had to be hand weed for the duration of the 2008 crop season.

Aster yellows was less of a problem in the 2008 caraway crop than in 2007, with only about 10 % of the plants showing signs of infection. As the infected plants are too small and chlorotic to produce a significant number of seeds, yields may have been reduced due to this problem.

Given the problems with stand establishment and weed control, seed yields in 2008 were actually unexpectedly high – exceeding by a substantial margin the yields typically seen under commercial production. Line DH 10 again out-yielded the line it was derived from (NN-2), although by a far smaller amount than was observed in 2007.

Quality analyses of the 2008 caraway crop were completed as planned. The seed oil content was higher in 2008, than in either 2006 or 2007 – this likely reflects the fact that the crop was allowed to mature late into the fall of 2008. The seed oil content of the DH lines was higher than the parental line (NN-2). Again the oil composition (% limonene) was more stable across the lines tested than the actual oil content. The highest yielding DH caraway line (DH 10) in the 2008 trial also had the highest seed oil content. Oil yield (kg/ha) for the best DH caraway line (DH10) was 50% higher than for the parental line (NN-2).

Conclusions for DH Caraway Trials

In replicated field trials of DH caraway conducted from 2006 through 2008 this project identified one line of DH annual caraway (DH-10) that was clearly superior to presently grown varieties for both seed yields and quality. However this line still required a longer growing season than is available in Saskatchewan – and therefore the market for this improved DH line of annual caraway lies elsewhere. Opportunities for commercial release of the most promising DH lines identified in this project are being pursued by the Crop Development Center of the University of Saskatchewan working in collaboration with PBI/NRC and the Saskatchewan Herb and Spice Growers Association. The DH caraway lines with traits of potential value in crop improvement programs will be preserved and made more widely available through the Plant Gene Resource Center of Agriculture Canada.


Field Evaluation of Doubled Haploid Plants in the Apiaceae: Dill, Caraway, and Fennel

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Field evaluation of doubled haploid plants in the Apiaceae: dill (Anethum graveolens L.), caraway (Carum carvi L.), and fennel (Foeniculum vulgare Mill.)

A. M. R. Ferrie • T. D. Bethune
G. C. Arganosa • D. Waterer

Abstract

The Apiaceae family includes vegetables, as well as herb and spice crops. Compared to major crops, there have been few breeding or genetic improvement programs for any of the Apiaceae, especially the herb and spice species. Haploidy technology can be used to develop uniform, true-breeding lines, as well as to accelerate breeding programs.
Field trials of dill (Anethum graveolens L.), caraway (Carum carvi L.), and fennel (Foeniculum vulgare Mill.) doubled haploid (DH) lines were conducted over 2–5 cropping seasons. Several of the DH dill lines had desirable agronomic characteristics such as short uniform stature along with early maturity that would be useful for crop improvement. Seed yields and the essential oil content of the seed harvested from the best performing DH dill lines were either equal to or higher than the parental line.

A DH annual caraway line was identified that produced higher seed yields than the industry standard. The main constituents of the essential oil for the DH lines of both dill and caraway were similar to the parental lines. Fennel DH lines exhibited differences in height but were too late in maturity for seed production under prairie conditions. The results indicate that not only were we able to generate DH lines that could be used in a crop improvement program, but we developed DH lines that could be used directly as cultivars as these lines performed better than the industry standard (parental line).

Introduction

The Apiaceae include a number of economically important vegetable, herb, and spice crops used as food, flavourings, perfumes, cosmetics, and medicinals for humans and animals (French 1971). They are grown worldwide, but are most common in the temperate regions of the northern hemisphere. There is an economic need to develop new cultivars of Apiaceae species better suited to Canadian prairie conditions. Earlier maturity coupled with increased yields of fruit (commonly known as seed), higher seed oil content, and better oil quality are the main objectives of improvement programs for Apiaceae.
Dill (Anethum graveolens L.) is an annual or biennial herb used primarily as a condiment. Dill seed and leaves are used as flavouring in sauces, vinegars, pastries, and soups. Dill has medicinal value as a diuretic, stimulant, and a carminative (Peirce 1999). The main constituents of the oil extracted from dill seed are D-carvone (35–60%) and D-limonene (35–50%) (Weiss 2002). Caraway (Carum carvi L.) is an annual or biennial herb native to Europe or western Asia. Caraway seed is used whole as a spice or crushed to produce caraway oil. The seeds have a licorice flavour and are used in breads, soups, spreads, salad dressings, and liqueurs. The leaves can be used in cooking, as can the roots. Caraway seed and seed oil have medicinal applications for disorders such as rheumatism, eye infections, toothaches, and nausea (Peirce 1999). The main constituents of the seed oil of caraway are similar to dill with D-carvone (45–60%) and D-limonene (35–55%) (Weiss 2002). Carvone has been used as a flavour additive in foods, a sprouting inhibitor for potatoes (Beveridge et al. 1981), a growth inhibitor for fungi (Smid et al. 1995) and bacteria (Helander et al. 1998; Oosterhaven et al. 1996) and as an insect repellent (Salom et al. 1996; Lee et al. 1997). The oil can also be used as a fragrance component in cosmetics (e.g., soaps, creams, lotions, and perfumes). Fennel is an erect, umbelliferous herb of the same family (French 1971). There are three subspecies of Foeniculum vulgare ssp. capillaceum: azoricum, also known as bulb fennel, Italian fennel, or Florence fennel; dulce, also known as sweet fennel, French fennel, or oilseed fennel; and vulgare (bitter fennel), which is perennial, and has an essential oil content higher than that of dulce. Bulb fennel is used as a vegetable, whereas sweet fennel is used as a condiment. Fennel seed is used in the food industry to flavour meats, vegetables, fish, soups, salad dressings, stews, breads, pastries, teas, and alcoholic beverages. Trans-anethole is the dominant component in the essential oil extracted from fennel seeds (Weiss 2002). This oil is used in condiments, soaps, creams, and perfumes. The medicinal or nutraceutical applications of fennel include use as an antispasmodic, carminative, diuretic, expectorant, laxative, and stomachic (Peirce 1999). Doubled haploid (DH) technologies, to produce embryos/plants from haploid immature pollen grains (microspores), are being used around the world to develop new cultivars and uniform lines in many plant species (Thomas et al. 2003). The main advantage to plant breeders of using doubled haploid breeding lines is the reduction in time required to achieve homozygosity. True-breeding lines can be generated in one generation rather than several years of backcrossing or selfing. This is also beneficial for producers and consumers as high yielding cultivars with improved agronomic and quality traits can be developed more rapidly. The objective of this project was to compare the agronomic performance of DH dill, caraway, and fennel lines derived via microspore culture against their corresponding parental lines under field conditions. Materials and methods The DH lines were derived from locally adapted cultivars using the doubled haploidy methodology outlined in Ferrie et al. (2010). As seed supply of the DH lines was limited, lines were initially grown in small plot non-replicated trials. The seed obtained from these trials was used in subsequent larger scale replicated trials. The agronomic characteristics of the DH lines were evaluated in field trials conducted at the University of Saskatchewan Horticulture Research Facility in Saskatoon, Saskatchewan, Canada. Unless otherwise specified, the crops were managed using standard commercial production practices. Soil fertility in the plot area was adjusted prior to planting to meet industry recommendations. Weeds were controlled using tillage and herbicides. The plots were only irrigated under extreme drought conditions to improve crop emergence or as required to activate soil-applied herbicides. No insect or disease control measures were employed.

Dill

A total of 41 DH lines of dill were evaluated from 2003 to 2008. Any DH line with poor seed viability (\20% germination) was eliminated from further field testing. In 2003, viable seed of 12 DH lines was available to conduct a field trial. Due to the limited seed supply and unknown vigor of the DH lines, the seedlings were initially grown for 6 weeks under greenhouse conditions before being moved to the field. The seedlings were planted out in the field in late May in rows spaced 0.5 m apart, with 15 cm between plants within the row. Seedlings of the parental line (‘Mammoth’) were included in this trial for comparison purposes. In 2004, the DH dill lines that had produced the most complete stand and the highest seed yields in the 2003 field trial were evaluated in a direct-seeded field trial that again included the parental line. This trial was initiated using seed harvested from the field trial conducted in the previous year. The trial was planted in mid-May using a small plot seeder. Each plot consisted of four 8 m long rows of each DH line. The trial was laid out in a randomized complete block design with three replicates. Seed was harvested as each line matured using a small plot combine. The 2005 trial was lost to flooding. Field evaluations were repeated in 2006, 2007, and 2008 using the same procedures, except in 2007 when each line was chemically desiccated with diquat (Reglone) applied at 2.2 l/ha as soon as the line began to mature. The lines were hand swathed 5 days later, allowed to dry in the field for 1 week, and then combined as previously described. This modification to the harvest method was implemented in an effort to reduce the shattering losses that occur as the dill crop begins to ripen. In each year of testing, the DH lines and the parental line were compared for time of bloom, plant height at maturity, seed yields, seed oil content, and the relative amounts of the main constituents in the oil.

Caraway

A total of 25 DH lines of annual-type caraway and three parental lines (NN-1, NN-2, and Moran) were available for field testing, beginning in 2006. As with dill, the first year of testing of caraway involved a limited number of plants established as transplants in a non-replicated trial. The 4-week old transplants were set out in rows spaced 0.5 m apart with 15 cm between plants within each row. The trial was hand-harvested in early October and threshed using a stationary combine. Because of the highly variable numbers of plants for each line, no yield comparisons were possible in 2006. The three DH caraway lines that had produced significant quantities of seed in the 2006 trial were evaluated in a replicated direct-seeded field trial in 2007 and 2008. These trials were planted and maintained as described in the dill trial. The trials were chemically desiccated in early October at which time all of the lines were still growing vigorously. Due to the short stature of the caraway plants, they had to be hand harvested and then threshed using a stationary combine.

Fennel

The fennel trials were conducted in 2003 and 2004. Due to limited seed supplies, all lines were grown from transplants rather than by direct seeding. In late May, six-week old fennel seedlings were transplanted into the field. Plants for each line were spaced 30 cm apart within the row, with 50 cm between rows. Each line was grown in a single 10 m long row. In the 2003 trial, 14 DH oilseed fennel lines were evaluated, along with the parental line. In 2004, another 11 DH oilseed fennel lines were evaluated along with the parental line. Germination percentages and plant heights were recorded. In both years, the growing season was too short to allow development of seed in any of the fennel lines tested.

Chemical analyses: dill and caraway

Essential oils were extracted from the harvested seed of dill and caraway by hydro-distillation using a Clevenger-type apparatus. Five grams of clean seed was ground for 40 s in a Krups coffee grinder, transferred into a 1,000 ml round bottom flask, after which 500 ml distilled water was added. Distillation was allowed to proceed for 2 h after the first few drops of distillate were obtained. After cooling the apparatus for 20 min, the length of the essential oil column in the side-arm of the Clevenger apparatus was measured and the volume of essential oil calculated. The results are expressed in percent essential oil (v/w) based on the weight of the air-dried seed. The oil was stored at -18_C in the dark prior to subsequent chromatographic analysis. A Hewlett-Packard gas chromatograph (Series Model 5880A) equipped with a flame ionization detector and a capillary column containing 95% dimethyl-5% diphenyl polysiloxane (DB-5; 10 m 9 0.25 mm i.d.; film thickness: 0.25 lm; J&W Scientific, Folsom, CA) was used. Helium was the carrier gas with a column inlet pressure of 2.1 kg/cm2. The injector and detector temperatures were set at 275 and 300_C, respectively. The oven temperature was programmed as follows: [a] initial temperature: 75_C; [b] initial time: 5.0 min; [c] program rate: 8_C/min; [d] final temperature: 200_C; and [e] final time: 2 min. Ten microliters of essential oil were diluted to 500 ll with chromatographic grade hexane prior to gas chromatography. One microliter was injected at a split ratio of 1:50. The individual peak areas were quantified using a Hewlett-Packard 3396 Series II integrator with a chart speed of 1.0 cm/min and an attenuation of 2. The retention times of the compounds of interest, carvone and limonene, were compared to the retention times of a pure carvone and limonene standard runs under identical conditions. The results were expressed as a percentage of the total peak area.

Statistical analysis

All data from the replicated trials were analyzed using analysis of variance procedures. Where significant (P\0.05) F-test values were observed, means were compared using Fishers protected least significant difference test (P\0.05).

Results and discussion

Dill

The presently available commercial dill lines have an indeterminate growth habit resulting in uneven crop maturity and challenging harvest conditions. With highly indeterminate seed set, it is inevitable that a portion of the dill seed is lost to shattering before the crop is ready to harvest, while another portion of the seed is still immature at harvest. Immature dill seed does not have the oil content or flavor profile required by the industry. The development of an early, uniform-maturing dill line would be beneficial for producers.
For 23 of the 41 DH dill lines tested, seed viability was extremely low (\20%) and those lines were eliminated from further testing (data not shown). A few of the DH lines showed a moderate degree of reduction of seed viability (40–60% germination) relative to the parental line ([90% germination). The poor germination rates of these lines limited their yield potential and these lines were also removed from the trial. The germination rates of the remaining DH lines were comparable to the parental line in all years of testing. Differences in plant height at maturity were observed between the parental line and the dill DH lines.

Over 4 years (2003, 2004, 2006, 2007), five of the DH lines (DH-7, DH-12, DH-35, DH-45, DH-47) were shorter than the parental cultivar Mammoth by 12–32%. Two lines (DH-1, DH-53) were similar in height to the parental cultivar. Irrespective of the average height, the DH lines were more uniform in stature than the parental line (Fig. 1). The height of the umbels in the DH dill lines also tended to be more uniform than in the parental line, which should improve the efficiency of mechanical harvesting. The shorter stature DH lines could be useful in situations where lodging leads to harvest problems; however, a taller stature might indicate greater yield potential if the leafy portion of the crop were destined for processing as dill weed. The dill doubled haploid line DH-1 is a very vegetative plant with large seeds. Seed production by this line was low because of the late maturity (Table 1), and the vegetative growth of the plant. This line might prove useful for dill weed and/or dill weed oil production, however the yield and oil content of the leaves were not analyzed in this project.

This line was not evaluated in 2007 and 2008 because of the low seed yield. Our selection focus was on early maturing lines with high seed yield. Differences were observed among the DH dill lines and the parental cultivar for date of flowering. On July 20, 2006, DH-12 was in full bloom, whereas most of the other lines were just starting to flower (3% flowering with DH-53 to 47% flowering in DH-45; Table 1). The parental line was 78% flowering. There was year to year variation in time of flowering. On July 18, 2007, only two lines had started flowering, DH-12 and the parental line. In 2008, 60% of DH-12 was flowering, whereas other lines had not begun to flower (DH-7, DH-53), even though data was collected 8–10 days later than the previous years (Table 1). Early flowering and early maturity are beneficial for prairie climatic conditions, however it can also be detrimental as the earlier flowering lines appeared more prone to infection with blossom blight, as was the situation in 2006. As a consequence of this disease, many of the seeds were shrunken or non-viable. Relative seed yield of the DH dill lines, compared to the parental line, varied from year to year. In 2003, seed yields from the most productive DH lines (DH-7, DH-12, DH-35, and DH-45) were on average 61% higher than for the parental line (data not shown). In 2004, seed yields of DH-12 were far higher than the parental line (350% of Mammoth), with DH-35 also producing a high yield (139% of Mammoth) (data not shown). A frost in mid-August of 2004 severely damaged the later maturing DH lines, as well as the parental line. Both DH-12 and DH-35 are relatively early maturing, and they largely avoided the damaging effects of the frost. In 2006, several of the DH lines again had seed yields that were similar to or greater than the parental line; however, seed yields for DH-12 were poor in 2006 as this line was severely infested with blossom blight (Table 2). Seed yields in the 2007 trial were almost 10 fold higher than in previous years, indicating the magnitude of shattering loss that occurred when the dill was directly combined. None of the DH lines had seed yields that exceeded the parental line in 2007, although yields for DH-7, DH-12, and DH-35 were not significantly different from the parental line. The combination of more careful harvest management practices, coupled with near ideal conditions through the fall of 2007 was advantageous to the relatively late maturing parental line. In 2008, DH-35 was the highest yielding line and was ready to harvest 1 week before the parental line. DH-12 also produced a yield which exceeded Mammoth in 2008 and it was ready to harvest 2 weeks earlier than the parental line. In years with an earlier or less favorable fall, the earlier maturing DH lines would likely produce better yields and as the seeds would be more mature, higher oil content would be expected.

Over the 3 years of replicated trials, DH-12 and DH-35 had an average seed yield greater than the parental line. The seed yields obtained in these experiments were substantially greater than those obtained in previous studies. Bailer et al. (2001) reported yields of 400–600 kg/ha in 1 year and less than 200 kg/ha in another year. These low yields were due to seed shattering, a common problem with dill cultivars. A uniformly maturing line would alleviate some of the seed shattering associated with this species. Essential oil content of dill seed typically ranges from 1.2 to 7.7%, depending on the variety, growing conditions, and method of oil extraction (Weiss 2002). In Canada, seed oil content for dill typically ranges from 2 to 4%. Seed oil content of the DH dill lines evaluated in this project ranged from 2.2 to 3.7% over 3 years of replicated testing (2006–2008), with limited year to year variability in the oil content (Table 2). Over the 3 years, DH-12 consistently had similar or greater percent essential oil content compared to the parental line Mammoth. For most lines, the seed oil contained about equal amounts of carvone and limonene (Table 2) and this ratio was also consistent across production years. The North American dill seed oil industry is based on carvone content and stipulates that dill oil must contain at least 30% carvone; all DH dill lines tested in this trial exceeded this standard.

Caraway

In western Canada, caraway is typically grown as a biennial crop, as the growing season is too short for most presently available annual-type lines. The current annual types mature in about 120–130 days. Growing caraway as a biennial effectively ties up the field for two seasons, at significant cost to the grower. If annual types suited to short seasons were to become available they would represent a means to double the growers’ land use efficiency. A total of 25 DH lines and three parental lines of annualtype caraway were evaluated in field trials conducted in 2006. Seed viability was poor for most DH lines, with only three of the DH lines (DH-10, DH-21, and DH-29) showing more than 25% emergence of the planted seed.

However, it should be noted that the parental lines also had poor emergence percentages resulting in poor stand (Table 3). In 2007, the only DH caraway seed available for larger scale field trials was from the three DH lines that had performed relatively well in 2006. In both the 2006 and 2007 trials, DH-10 produced a better stand than its parent (NN-2; Table 3). In mid-June 2007, a problem with aster yellows disease developed in the DH caraway plots. The extent of the infection and the impact on the plants was much more severe than in adjacent plots of DH dill. None of the infected caraway plants survived to produce seed and consequently, seed yields were potentially compromised. There did not appear to be any significant difference in the incidence or impact of aster yellows on the various DH lines or the parental lines of caraway. Given the limited stand and the problems with aster yellows, seed yields were still unexpectedly high in the 2007 trial (Table 3). DH-10 produced 500% more seed than its parental line (NN-2) in 2007. In 2008, DH-10 again outperformed the parental line.

Seed of DH-10 is substantially smaller than the seed of any of the other DH lines or the parental lines (data not shown). This may be undesirable for direct sales to the consumer where large seed size is equated with quality, however seed size would be of little importance to the processing sector. Essential oil content of annual caraway seed typically ranges from 1.5 to 5.0%, depending on the variety, growing conditions, and method of oil extraction (Weiss 2002). The seed oil content of the caraway DH lines was much higher in 2007 and 2008 than in 2006 (Table 4); this likely reflects the fact that the crop was allowed to mature late into the fall of 2007 and 2008. The seed oil content of the parental line Moran was higher than any of the DH lines in 2007, however, this was not the case in 2006 or 2008. Over 90% of caraway seed oil consists of two monoterpenes, carvone and limonene. For the caraway DH lines, the ratio of carvone to limonene varied more between years than between lines in a given year. In Saskatchewan, carvone levels are around 46–50% in annual caraway and 54–57% in biennial caraway (Arganosa et al. 1998). This can also vary according to the genotype, seeding date, and location. Our DH lines show a lower carvone content, however the parental lines were also similar. The industry prefers caraway oil to have a higher carvone content (50–60%) (Weiss 2002) than was observed in any lines tested in this trial, including the parental lines. Carvone content increases as the seed ripens, and consequently, biennial types of caraway tend to have a higher carvone percentage than the annual types used in this project.

Fennel

In both 2003 and 2004, seed germination of the DH lines of oilseed fennel was quite variable. In 2003, four of the 14 lines were non-viable, while in 2004, five of the 11 lines tested did not germinate.

Of the 16 viable DH lines (10 lines from 2003 and 6 lines from 2004), five had 100% germination while the germination percentages of the other lines varied from 25 to 60% (data not shown). The parental line consistently had a high germination percentage (95–100%). There was significant variation from line to line in plant height of the fennel at the end of the growing season (ca 120 days after planting). In 2003, two of the DH fennel lines (DH-37 and DH-90) were approximately twice the height of the parental line (60 cm; Fig. 2a). The heights of the DH lines tested in 2004 more closely resembled the parental lines (Fig. 2b). Within the DH lines, plant heights were more uniform than the parental material, likely reflecting the genetic homogeneity characteristic of DH. Aside from differences in height, the morphology and rate of development of the DH fennel lines were similar to the parental line. In both years, the growing season was too short to allow development of seed in any of the fennel lines tested.

Conclusion

The Apiaceae species have been considered recalcitrant when it comes to androgenesis, especially microspore culture. Very little research has been published in this area and there were no efficient protocols available for generating doubled haploids prior to the methods developed by Ferrie et al. (2005, 2010). The objective of this project was to compare the agronomic performance of DH lines of dill, caraway, and fennel relative to the parental lines, while also surveying the DH material for traits that could be useful in subsequent crop improvement. Field comparisons of DH lines and their parental controls have not been previously reported in the Apiaceae, but there are reports in the Brassica species and barley (Hordeum vulgare) (Park et al. 1976; Friedt and Foroughi-Wehr 1983; Powell et al. 1986). In those species differences were observed between the DH lines and the parental line for a number of characteristics (e.g. yield, height, maturity). Generally, the DH lines were inferior (Palmer et al. 1996), but superior lines could be identified (Friedt and Foroughi-Wehr 1983). In our study, a significant proportion of the DH lines of dill, caraway, and fennel proved to have limited seed viability. Generally, this has not been observed in DH lines of other species. However, many of the Apiaceae DH lines that were viable showed promise in field trials. DH lines of dill were identified that were earlier maturing, more uniform in stature, and higher yielding than the parental lines. A single DH line of caraway with exceptional yield potential was also identified. Seed oil content and composition of the DH lines of dill and caraway were generally comparable to the parental lines, suggesting that the improvement in field performance was achieved without compromising crop quality. Our results show that Apiaceae doubled haploidy techniques can generate lines that have the potential for commercial production as well as lines that can be incorporated into a breeding program. Further efforts to create and characterize DH lines of dill and caraway with superior agronomic or quality characteristics are ongoing.

Acknowledgments

The authors thank Jackie Bantle and William Hrycan for their technical assistance.

Financial assistance from Saskatchewan Agriculture, Food and Rural Revitalization, Agriculture Development Fund was greatly appreciated.

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