Quality management in strawberry propagation, using DNA-genotyping · PDF file ·...

Quality management in strawberry propagation, using DNA-genotyping as a tool

Lien Ko and Amanda Westacott

Overview

The strawberry industry in Australia is dependent on the supply of about 60 million quality, bare-rooted transplants or runners each year. This material is produced by nurseries located at Stanthorpe in southern Queensland, at Toolangi in Victoria, and at Kempton in Tasmania.

Errors or mistakes can occur during the propagation of the new strawberry plants if they are mislabelled and these mislabelled plants are eventually sold to the fruit growers. Every now and then mutations can occur in tissue culture, with off-types produced. We have been interested in developing methods to minimize and manage the risks of these wrongly-labelled or mutated plants being sold to the strawberry industry. Most cultivars cannot readily be identified by vegetative characteristics alone. This means that mistakes made in the propagation system may not become apparent until the plants begin to bear on the fruit farms.

Over the past three years, we have examined the potential for the use of DNA-based tests to identify and differentiate individual cultivars. It has been demonstrated that this technology can help strawberry breeders and nurseries keep track of different cultivars and breeding lines. It can also help identify any off-types produced in tissue culture. It was anticipated that the tests would ensure that commercial strawberry growers are supplied with the correct, true-to-type nursery material.

The multiplication of new planting material

The introduction and multiplication of new cultivars for the strawberry industry in Australia follows a series of regulated steps. Local material is usually supplied as plants grown in pots, while material imported from overseas is usually supplied as plants grown in tissue culture. Both groups of plants are initially grown in tissue culture and then potted up in the glasshouse before they are released to industry as “foundation plants”. This initial process may take up to three years or more.

Strawberry plants are susceptible to a range of plant viruses that are difficult to control in the field. The main method of limiting the impact of these viruses is to make sure that only virus-free stock is released to the commercial runner industry. Thus, one of the first processes in the propagation of new material is to make sure that it is free of viruses. After registration and virus-indexing, the new material can then enter the propagation system as plants in pots or plants in tissue culture, and be labelled as “Mother” or “Daughter” plants.

Plants received in pots are kept in a “Low Health Status” glasshouse. This glasshouse contains plants received in pots or bare-rooted transplants (runners) from the field from various sources. This material has already been virus-indexed. Tips are taken from runners produced by these

Strawberry R&D Update 2009 �

plants, and grown in tissue culture as the “Repository”. These plants are cultured under sterile laboratory conditions and are free of fungi, bacteria and viruses.

Some of the plants growing in tissue culture enter the “Gene Bank Register”, and the others are planted out in pots in the “High Health Status” facility, becoming the “Nucleus Stock Plants”. The “High Health Status” facility contains plants that have been tissue-cultured and are free from fungal and bacterial diseases. These plants are planted out in sterile potting mix at the research station. Runner tips are taken from these stock plants each year and re-established in tissue culture to be multiplied-up to produce the foundation plants for the runner growers.

Each tip is transferred onto fresh agar medium every three to four weeks, resulting in an average multiplication rate of 150 within a six-month propagation period. In other words, one tip can produce 150 foundation plants.

The plants grown in tissue culture are potted-up between July and September, and hardened-off in a glasshouse in seedling trays. The new foundation plants are generally ready to be shipped to the runner growers by October or November each year.

Using DNA-based tests to ensure “trueness-to-type”

Errors can occur at any stage of the multiplication process. The end result can be that the runner and fruit growers are supplied with the wrong cultivar. DNA-tests can be used along the way to ensure that the plants remain “true-to-type”. Samples of plant material are typically collected at three stages of the propagation system. The first samples are collected from the initial Mother/Daughter plant, and are used as the “reference” for a particular cultivar. Leaf samples are also taken from the nucleus stock plants in the “High Health Status” facility and from a representative sample of the foundation plants in the seedling trays before they are sent to the runner producers.

The current laboratory analysis scans only part of the DNA molecule. The system will pick up major “mutations” or large differences in DNA; however, minor mutations or small differences in DNA will probably not be detected. New samples are compared against a reference DNA fingerprint of each cultivar kept in a “library”. The DNA fingerprint for each cultivar is unique.

To illustrate the importance of this tool, a worst case scenario is described in the event of an error occurring with the “Nucleus Stock Plants” (see Table 1). According to this analysis, if a single stock plant was wrongly-labelled and the error was not detected until the plants began to fruit, the potential financial losses to the strawberry industry could run into millions of dollars. Screening for “trueness-to-type” during the propagation process can significantly reduce the risks of such an event occurring.

Strawberry R&D Update 2009 8

No. of No. of mother No. of Value of the plants after multiplication in tissue culture

plants after the first year

on the runner farms

commercial plants after the second year on the runner

plants sold as commercial

stock

farms 50 - 350 2,500 - 70,000 62,500 - $24,375 -

using a 600,000 using $2,184,000 multiplication a multiplication rate of 50 - 200 rate of 25 - 80

Reliability of the DNA tests

Over the past three years, we have analysed over 1,000 leaf samples for DNA testing. We allocate a number to an individual sample, and the laboratory matches each individual sample to the DNA fingerprint of a known “reference” sample. The identity of the individual sample sent to the laboratory is masked.

When we receive the test results and all of the individual samples match the expected references, the matching rate is 100 percent or a “perfect match”. Apart from one instance, we have obtained a matching rate of 92 to 100 percent in the twelve batches of samples analysed. In instances where unexpected matches were found, a follow-up was required to confirm the problem. This might have occurred in the glasshouse, laboratory or in the field, or sometimes in the sampling process. In all cases where potential errors were detected, early intervention prevented the release of the “wrong” material.

Table 1. The economic costs of mislabelling a single nucleus stock plant. The analysis is based on a cost of $390 per 1000 plants.

Commercial implications

Our studies have shown that it is important to detect errors in the labelling of the plants early in the propagation process. Because most strawberry cultivars look similar, it is not easy to detect errors in the absence of a DNA test. Early detection of errors has the potential to save the strawberry industry millions of dollars in lost production, as would happen if the “wrong” cultivar is planted out on the fruit farms.

The cost of DNA testing is about $70 per sample. This cost will be included in the cost of producing the foundation plants for the commercial runner growers. Overall, the DNA test represents less than two percent of the cost of supplying the new plants, a very worthwhile insurance!

In the future, there will also be the opportunity for growers to submit samples for DNA testing if they suspect they have been supplied with the wrong planting material. The analysis would be done on a fee-for-service basis.

Currently, the DNA samples are analysed by a commercial laboratory in California. We are attempting to find local laboratories in Australia that can provide a similarly reliable service. This is expected to reduce the cost of the analysis and provide a faster turnaround.


Tissue-cultured genebank plants.

Multiplied tissue-culture plants.

A batch of tissue-cultured plants ready for planting out into trays in a glasshouse.

The transfer of tissue-cultured plants into fresh agar medium.

Foundation plants in trays in a glasshouse.

10 Strawberry R&D Update 2009

Runner quality Chris Menzel and Lindsay Smith

Overview

Research has shown that the quality of planting material has a strong effect on the profitability of strawberry growing in southern Queensland. Inferior runners grow and crop poorly after planting, reducing monetary returns to producers. A ten percent loss of production on the fruit farms on the Sunshine Coast is equivalent to a loss of about $14 million for the local strawberry industry. These issues also affect growers in the southern parts of Australia.

The main factors affecting the quality of the runners are related to plant size and the time of digging. We have been investigating the effect of these factors on fruit production and returns on the Sunshine Coast.

In the first experiment, transplants of ‘Festival’ were planted at different times from early March to late April, and the impact on fruit production determined. In other experiments, small and large runners were obtained from three different nurseries across Australia. The material was then grown in replicated experiments at Nambour to determine the relative importance of plant size and runner-production environment on productivity. This year, we also included a comparison of extra small runners with standard runners (crowns smaller or larger than 8 mm).

The quality of runners from different strawberry nurseries from southern Queensland and from southern Australia was also assessed.

In other experiments, we grew strawberry plants in glasshouses at different temperatures to study their impact on plant growth and stored reserves. We were interested in determining whether differences in temperature conditions in the nurseries in Australia are likely to affect runner quality.

Finally, we examined the relationship between runner quality and the concentration of stored reserves in the transplants. Material was sourced from different growing areas and from different times during the runner-growing period.

Optimum planting times for ‘Festival’ strawberries on the Sunshine Coast

Research conducted on the Sunshine Coast over the past two years has shown that runners of ‘Festival’ planted from mid-March to mid-April give better returns than runners planted earlier or later.

We have continued this work in 2009, with another time of planting experiment established over autumn at the research station in Nambour. Bare-rooted runners of ‘Festival’ obtained from Stanthorpe were planted at different times from early March to late April, using only sound, undamaged plants with at least three functional leaves. Information was collected on plant growth, yield and returns over the season from May to October.


There were only small differences in the average size of the different planting material (see Table 2). In contrast, average plant growth during the season was greater with the stock planted in early March and mid-March, followed by the stock planted in early April, mid-April and late April (see Table 2).

Table 2. The effect of time of planting on plant dry weight and yield of bare-rooted ‘Festival’ strawberries at Nambour in 2009. Plant dry weight includes the mass of the leaves, crowns and roots.

Time of Plant dry Average plant dry Yield planting weight at weight during the (g per plant)

planting (g) season (g)

Early March 3.1 18.0 788

Mid-March 2.8 20.1 934

Early April 3.3 12.8 727

Mid-April 3.1 12.2 606

Late April 3.2 11.2 555

In 2009, there was a broad optimum for yield with planting time from early March to mid-April, with much lower yields from a planting time in late April (see Table 2). The stock planted last returned $0.40 to $2.60 less per plant than that planted earlier. Planting time also affected fruit quality. The early and late plantings tended to produce a greater proportion of their crop later in the season when the fruit were small and prices marginal (see Figure 1).

Across the three years of the experiments, returns seem to be best with plantings from mid-March to mid-April on the Sunshine Coast. Further research is planned for 2010 to confirm these results. The first set of experiments will include small (crowns 6 to 8 mm) and large runners (crowns 8 to 18 mm) of ‘Festival’ and ‘Fortuna’ planted in mid-March, late March or in mid-April. We will also be conducting grower experiments in Western Australia to examine the impact of planting date on the performance of the short-day cultivar, Juliette, and the day-neutral cultivar, Albion.

Figure 1. The relationship between mean fruit weight and growing season in ‘Festival’ strawberries in runner quality experiments at the research station in Nambour. The data were collected from six experiments over three years, with different times of planting, different sources of runner-growing environment, and different sized runners. The early and late plantings tended to produce a greater proportion of their crop later in the season when the fruit were small and prices marginal. The small runners

also tended to produce a greater proportion of their crop later in the season.


Variations in plant quality in 2009

Runner quality was assessed again in 2009, with green-leaf plants obtained from Stanthorpe in southern Queensland (latitude 28.6°S), from Toolangi in Victoria (latitude 37.6°S), and from Kempton in Tasmania (latitude 42.4°S). The transplants were dug in early April.

The runners from Kempton had slightly thicker crowns than those from Stanthorpe and Toolangi, and slightly fewer leaves (see Table 3). Overall, the runners from Toolangi and Kempton were slightly larger than those from Stanthorpe. This was due to the larger crown or root mass of the plants from southern Australia.

There was a large variation in the quality of the plants from the same nursery (see Table 3 and Figure 2). For instance, the dry weight of some of the smaller runners was less than twenty percent of the dry weight of the larger runners. The same was true for the data collected in 2007 and 2008. Most of the runners met the minimum standard relating to the diameter of the crowns and the number of functional leaves. About fifteen percent of the transplants had crowns smaller than 8 mm, and about 40 percent had crowns smaller than 10 mm.

Table 3. A comparison of mean runner quality in ‘Festival’ strawberries obtained from Stanthorpe, Toolangi and Kempton in 2009. The plants were dug in early April. Data in parenthesis show the ranges in crown diameter, leaf production and plant dry weight. Plant dry weight includes the mass of the leaves, crown and roots.

Location Diameter of the Number of Plant dry crown (mm) leaves per plant weight (g)

Stanthorpe, Qld 8.5 (5-13) 4.0 (2-6) 3.3 (1.0-9.1)

Toolangi, Vic 9.5 (6-13) 3.9 (2-6) 3.8 (1.3-7.3)

Kempton, Tas 10.9 (7-16) 3.3 (2-6) 3.6 (1.0-9.3)

Figure 2. The variation in crown diameter found in ‘Festival’ strawberry transplants. The plants were harvested from Stanthorpe in Queensland (warm-growing area), and from Toolangi in Victoria and from Kempton in Tasmania (cool-growing areas) in early April, 2009. About 15 percent of the transplants had crowns below 8 mm, and about forty percent had crowns below 10 mm.



Plant type Plant dry weight at

planting (g)

Average plant dry weight during the

season (g)

Yield (g per plant)

Small runner 1.6 8.9 591

Large runner 4.6 14.1 715

Runner from Queensland 2.7 11.6 661

Runner from southern Australia 3.5 11.5 649

The effect of runner-growing environment and plant size on the yield of ‘Festival’ strawberries

Experiments over the past two years have shown that the size of the runners can affect the productivity of ‘Festival’ plants grown on the Sunshine Coast. Large plants with crowns above 10 mm returned up to $1.00 per plant more than small runners with crowns below 10 mm. In contrast, runners from Queensland were generally just as profitable as those from the cooler districts in southern Australia.

In the first experiment in 2009, we evaluated the productivity of small and large bare-rooted ‘Festival’ plants from Stanthorpe, and from Toolangi in Victoria, and from Kempton in Tasmania. The plants from each nursery were sorted into small and large runners. The small plants had crown diameters ranging from 6 to 10 mm, and the large plants had crown diameters ranging from 10 to 17 mm. Only sound, undamaged plants with at least three functional leaves were used.

In the second experiment, we obtained plants of ‘Festival’, ‘Rubygem’ and ‘Treasure’ from Stanthorpe. The plants were divided into extra small runners with crowns from 6 to 8 mm, and standard runners with crowns from 8 to 17 mm.

In the first experiment, the large runners had about three times the dry mass of the small runners at planting, and one and half times greater leaf, crown and root growth throughout the growing season (see Table 4). These differences in growth translated into higher productivity in the large runners, which yielded twenty percent more than the small runners. The small runners also tended to produce a greater proportion of their crop later in the season when the fruit were small and prices marginal (see Figure 1).

There were only small differences in growth, dry matter production and yield in the nursery material obtained from the different growing areas (see Table 4).

Table 4. The effect of runner size and production area on plant dry weight and yield of ‘Festival’ strawberries at Nambour in 2009. Plant dry weight includes the mass of the leaves, crowns and roots.

In the second experiment in 2009, the standard runners had higher dry matter production during the season than the extra small runners, and higher yields (see Table 5). Overall, the growth and yield of the three cultivars were similar.


Plant type Diameter of crowns

(mm)

Average plant dry weight during the

season (g)

Yield (g per plant)

Extra small runner 6-8 12.8 562

Standard runner 8-17 16.0 662

Table 5. The effect of runner size on plant dry weight and yield of ‘Festival’, ‘Rubygem’, and ‘Treasure’ strawberries at Nambour in 2009. Plant dry weight includes the mass of the leaves, crowns and roots. Data are the means of the three cultivars.

In these two experiments, the larger runners returned $0.70 to $0.85 per plant more than the smaller runners.

These results confirm previous research that large runners perform better than small runners on the Sunshine Coast. We will continue this work at Nambour in 2010, examining the performance of small and large runners planted at different times from mid-March to mid-April. We will also conduct grower experiments with the cultivars Juliette and Albion, in Western Australia.

Are temperature differences in the different runner-growing areas likely to affect runner quality?

Experiments were conducted to examine the effect of temperature on the growth and carbohydrate reserves of strawberry plants, and their possible impact on potential runner quality. We were particularly interested in determining if cool nights could counteract some of the harmful effects of warm days on runner quality (small crowns and roots).

This experiment examined the effect of warm days and cool nights on growth and carbohydrate reserves. The set-up was similar to the experiments described previously. Plugs of ‘Festival’ were grown in 2.8 litre pots of sand and peat (1:1) at day/night regimes of 20°/20°, 22.5°/22.5°, 30°/10° or 30°/15°C. The first and the third regimes had average temperatures of 20.0°C, while the second and fourth regimes had average temperatures of 22.5°C.

Table 6. The effect of day/night temperatures on dry matter production (g per plant) of ‘Festival’ strawberries grown in controlled-temperature glasshouses for 13 weeks.

Temperature Average Leaves Crowns Roots Plant regime temperature

20°/20°C 20.0°C 14.2 4.1 2.1 20.4

22.5°/22.5°C 22.5°C 18.0 4.9 2.8 25.8

30°/10°C 20.0°C 13.1 2.8 1.3 17.3

30°/15°C 22.5°C 11.6 2.6 1.3 15.5

Crown and root growth were greater at 20°/20°C than at 30°/10°C (even though both regimes had a similar mean temperature), and leaf, crown and root dry weight were greater at 22.5°/22.5°C than at 30°/15°C (see Table 6).

Plants growing under the constant temperatures also had a higher proportion of assimilates directed to the crowns and roots than plants growing under warm days and cool nights (see Figure 3), and a lower proportion directed to the leaves.

Figure 3. The effect of temperature on the distribution of plant dry weight in ‘Festival’ strawberries. The plants were grown at day/night temperatures of 20°/20°C (Regime 1), 22.5°/22.5°C (Regime 2), 30°/10°C (Regime 3) or 30°/15°C (Regime 4).

There were few differences in the concentrations of carbohydrates in the different treatments. The plants grown at a constant temperature of 22.5°C had slightly higher concentrations of reserves than the plants grown at the other temperatures (8.5 percent versus 6.6 to 6.8 percent).

These results indicate that cool nights are not likely to counteract the harmful effects of warm days on runner quality. Plants grown with warm days and cool nights generally had smaller crowns and roots than those grown at constant temperatures. It is apparent that runner-growing areas with warm days and cool nights are not likely to produce better runners than those areas with a more moderate climate.

Do plant carbohydrate reserves reflect runner quality?

Studies overseas have suggested a strong link between runner quality in strawberries and the reserves of carbohydrates stored in the transplants. Fruit yields were usually higher in runners with the highest reserves. We were interested in determining whether there was any relationship between runner quality and the concentrations of non-structural carbohydrates in ‘Festival’ strawberries grown in Australia.

It was thought that plants dug early in the season would have lower reserves than those dug later in the season. We also proposed that the


transplants from southern Queensland (warm-growing area) would have lower reserves than transplants from southern Australia (cool-growing areas).

In the first experiment, transplants were obtained from Stanthorpe on 3 March, 17 March, 1 April, 13 April, or on 28 April (in 2008). In the second experiment, transplants were sourced from Stanthorpe, Toolangi or Kempton in early April (in 2008). The plants were transported to Nambour for an analysis of growth and carbohydrate concentrations.

In the first experiment, information was collected on the amount of chilling accumulated by the plants from 1 January up to digging at Stanthorpe (see Table 7). The first two sets of runners accumulated little chilling below 10°C, and no chilling below 7.2°C. There was a steady increase in chilling below 10°C over the next three harvests, whereas the accumulation of chilling below 7.2°C slowed down after the fourth harvest.

Table 7. The accumulation of chilling below 7.2° and 10°C from 1 January 2008 until digging in the different treatments at Stanthorpe.

Time of digging Hours accumulated Hours accumulated below 10°C below 7.2°C

3 March 8 0

17 March 17 0

1 April 57 30

13 April 83 42

28 April 154 65

The main non-structural carbohydrates measured in the runners were glucose (mean concentration of 3.8 percent dry weight), fructose (2.9 percent), sucrose (0.8 percent), maltose (0.3 percent), and starch (2.3 percent), with lower concentrations of sorbitol (< 0.1 percent). The mean concentration of the sugars measured was 7.8 percent, and the mean concentration of the non-structural carbohydrates measured was 10.1 percent. Sugars accounted for more than three-quarters of the total non-structural carbohydrates analysed.

The plants dug on 1 and 13 April had greater weights of non-structural carbohydrates than the runners dug on 3 March, 17 March or on 28 April (see Table 8). This response was due to slightly smaller plants and slightly lower concentrations of non-structural carbohydrates in the first two diggings, and slightly lower concentrations of non-structural carbohydrates in the last digging. The differences in the total reserves were mainly due to differences in the carbohydrates stored in the roots.

In 2008, the highest yields were obtained when the runners were dug on 17 March (1092 g per plant). Yields were slightly lower when the runners were dug on 3 March (977 g per plant) or on 1 April (880 g per plant). Yields were much lower when the plants were dug on 13 April (675 g per plant) or on 28 April (510 g per plant).


Time of digging

Plant dry weight (g)

Average concentration

of carbohydrates (% DW)

Weight of carbohydrates

(mg per plant DW)

3 March 2.2 9.6 224

17 March 2.8 9.8 292

1 April 3.6 11.2 408

13 April 3.6 12.4 445

28 April 3.8 8.7 330

Table 8. The effect of digging date on the weight of non-structural carbohydrates in ‘Festival’ strawberry plants obtained from Stanthorpe.

Mean monthly maximum and minimum temperatures for the three runner-growing areas from January to April in 2008 are shown in Table 9. Average monthly maximum temperatures were similar in the three areas, whereas the average monthly minimum temperature was 2.9° to 3.6°C lower in the southern nurseries compared with conditions at Stanthorpe.

Table 9. Monthly maximum and minimum temperatures in the three runner-growing areas in 2008.

Jan Feb Mar Apr Average

Stanthorpe, Qld

Monthly max. temperature (°C) 24.1 23.8 23.0 20.2 22.8

Monthly min. temperature (°C) 15.5 13.9 11.2 7.6 12.1

Toolangi, Vic



Kempton, Tas



The runners from Stanthorpe had a greater weight of non-structural carbohydrates per plant than the runners from southern Australia (see Table 10). This was associated with a slightly greater plant mass in the runners from Stanthorpe, and a slightly higher average concentration of non-structural carbohydrates. The difference in the total reserves was mainly due to differences in the carbohydrates stored in the leaves and the roots.

These results show that lower yields obtained with early plantings of ‘Festival’ on the Sunshine Coast could be due to low reserves of stored carbohydrates in the new plants.

In contrast, the plants from the nursery in southern Queensland had higher reserves than the plants from the nurseries in southern Australia. This counters the idea that the transplants from the southern growing areas have greater stored reserves and greater potential productivity.


Origin Plant dry weight (g)

Average concentration

of carbohydrates (% DW)

Weight of carbohydrates

(mg per plant DW)

Stanthorpe, Qld 3.6 11.2 408

Toolangi, Vic 3.0 10.3 306

Kempton, Tas 3.2 8.8 285

In these experiments, differences in the weight of stored reserves in the various planting material was related to differences in the weight of non-structural carbohydrates stored in the leaves and the roots (see Figure 4). The reserves stored in the crowns were less important.

Table 10. The effect of origin of the nursery material on the weight of non-structural carbohydrates in ‘Festival’ strawberry plants.

Figure 4. The distribution of stored carbohydrates in ‘Festival’ strawberry transplants. Data are the means of plants from Stanthorpe in Queensland (warm-growing area), Toolangi in Victoria and from Kempton in Tasmania (cool-growing areas) dug in early April, 2008.

Fruit production in ‘Festival’ strawberries. Research has shown that the quality of planting material has a strong effect on the profitability of this cultivar when it is grown in southern Queensland. Good quality runners can yield more than 1000 g per plant.


Relative plant growth from the time of planting experiment at Maroochy in 2009: runners planted in early March (left), mid-March (centre) or in late April (right). The photograph was taken in early June.

Relative root growth in the glasshouse temperature experiment: plants grown at 22.5°/22.5°C (left) or at 30°/15°C (right). Higher temperatures significantly reduced root growth in the plants.

Relative plant growth from the size of runner experiment at Maroochy in 2009: small runner (left) and large runner (right). The small plants had crown diameters ranging from 6 to 10 mm, and the large plants had crown diameters ranging from 10 to 17 mm. The photograph was taken in early June.


Relative shoot growth in the glasshouse temperature experiment: plants grown at day/ night temperatures of 20°/20°, 22.5°/22.5°, 30°/10° or 30°/15°C (from left to right). Best shoot growth occurred at 20°/20° and 22.�°/22.�°C.

Harvesting fruit from the runner quality experiments at Maroochy.

Lindsay Smith weighing fruit from the runner quality experiments at Maroochy.


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