Evaluation of new clones of Jerusalem artichoke (Helianthus tuberosus L.) for inulin and sugar yield from stalks and tubers

Industrial Crops and Products 19 (2004) 25–40

Evaluation of new clones of Jerusalem artichoke (Helianthus tuberosus L.) for inulin and sugar yield from stalks and tubers Mario Baldini∗ , Francesco Danuso, Maurizio Turi, Gian Paolo Vannozzi Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Università di Udine, via delle Scienze, 208, 33100 Udine, Italy Received 16 July 2001; accepted 6 June 2003

Abstract Six clones of Jerusalem artichoke, five new clones (coded as 17, 22, 56, 69 and 70) and the control cultivar “Violette de Rennes” were evaluated for variability in sugars yield, when the plant is utilised as stalk, tuber and “integral crop”. Among the above harvesting methods, the integral crop (stalks and tubers at flowering time), showed the highest yield potential of total sugars (fructose + glucose) and inulin (18.6 and 17.9 t/ha, respectively). This was obtained by the clone Violette de Rennes, which also had the greatest inulin chain length, in the text reported as average degree of polymerisation (DP). Clone 69 produced the highest yield of sugars and inulin, when the stalks are harvested at flowering (10.4 and 8.0 t/ha, respectively), while clone 17, with the conventional harvest of tubers at the end of crop cycle, reached 13.3 and 13.7 t/ha of total sugars and inulin, respectively. The average inulin chain length (DP) was highest at flowering time in both stalks and tubers with a range of 7.5–11.2 in the genotypes studied, while, at the final harvest of tubers, it significantly decreased reaching values ranging from 4.8 to 6.7. Among the organs analysed, the tubers at stalk harvest, showed both the highest inulin content and the longest inulin chain, expressed as DP. The genetic variability was very high among the clones for the other characters studied, such as flowering time, sugar content in different organs, photosynthesis activity, leaf chlorophyll content, etc. In particular, the “refractometrically measured” solids, in extracted tissue juice, expressed as the Brix-value (a very quick method), exhibited a significant positive relationship with the tuber inulin content (0.90∗∗ and 0.85∗∗ , n = 6, in the first and second harvest, respectively), confirming its suitability as a fast screening method in breeding, avoiding time consuming and expensive laboratory analysis. © 2003 Elsevier B.V. All rights reserved. Keywords: Jerusalem artichoke; Genetic variability; Harvest methods; Sugar; Inulin; Stalk and tuber

1. Introduction

∗ Corresponding author. Tel.: +39-0432-558663; fax: +39-0432-558603. E-mail address: [email protected] (M. Baldini).

Jerusalem artichoke (Helianthus tuberosus L.) is cultivated mainly for use as green or ensiled forage, as a cover crop in marginal areas and to produce sugars (especially fructose) and fructans (inulin), which are used as food or for various chemical, electronic

0926-6690/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0926-6690(03)00078-5

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M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

and pharmaceutical applications (Bosticco et al., 1989; Maijer and Mathijssen, 1991; Marchetti, 1993). Environmental issues and depletion of natural resources favour this latter industrial utilisation. Many researchers agree that, in the future, more agricultural feedstocks will be used for their special properties, since they are renewable resources, have better degradability and are more attractive, being natural, for consumers, than synthetic products. Jerusalem artichoke is one of the most important candidates for use as a raw material for the industrial production of biological fructose and inulin. It is a particularly interesting and suitable crop, for southern European countries and especially in low-requirement environments (Paolini et al., 1996; D’egidio et al., 1998). The main drawbacks of this crop are related to the harvest time of the mature tubers (generally in December) when the heavy rains make it difficult to use harvest machines in the waterlogged fields. Since Jerusalem artichokes also store temporary amounts of fructans in the stalk, some authors (Incoll and Neales, 1970; Maijer and Mathijssen, 1991) have suggested using it as stalk crop, that can be harvested (using a conventional forage harvester) at the beginning of flowering, i.e. August–September in Europe. The stalks become the raw material for sugar extraction, thus transforming it into a multi-year crop, avoiding the sowing of tubers in the following year (Caserta and Cervigni, 1991; Voltolina, 1994; D’egidio et al., 1998). Paolini et al. (1996), following several experiments carried out in Mediterranean areas, have suggested harvesting the “integral” crop, not just tubers, but stalks and tubers at the same time around the beginning of flowering, when a notable yield in tubers has been recorded, presumably owing to environmental conditions. Both the above strategies have been tested in order to gather information on the yield potential that can be obtained using new crop and management techniques that could open up novel perspectives for Mediterranean farmers. At present, new ideotypes, suitable for the above techniques, are completely scarce and in fact, just clones and “commercial varieties” selected for tuber production are utilised. Although several experiments have been carried out to investigate the existing variability of agronomic characteristics of Jerusalem artichoke accessions in different environ-

ments (Kiehn and Chubey, 1993; Zubr and Pedersen, 1993; Toxopeus et al., 1994; Ben Chekroun et al., 1996; Paolini et al., 1996), the only information available to breeders, is strictly related to increasing the tuber yield (Soja and Haunold, 1991; Soja et al., 1993; Maijer and Mathijssen, 1996). The main objectives when developing new genotypes, suitable for stalk yield or early “integral” crop harvesting, are the maximisation of inulin and total sugar yield in the stem (with the production of large amounts of biomass), and clones with early tuberisation and tuber filling. Among the methods for rapid determination and easy selection indices required for breeding, many authors (Kosaric et al., 1984; Lambers, 1987; Soja and Haunold, 1991) report elevated carbon exchange as a suitable parameter related to stem biomass production and Van Waes et al. (1998) found the refractive index (Brix value) of stalk and tuber juice positively related to the inulin content in chicory roots. Moreover, the inulin degree of polymerisation (DP), depending on Jerusalem artichoke cultivar and date of harvest (Chabbert et al., 1985, 1993), can assume very interesting perspectives in inulin application; in fact inulin with a high DP (>20) could be utilised for diagnostic use, while smaller inulin with DP 6–10, could be utilised in improving consistency of cakes and other bakery products (Vokov et al., 1993). For the above reasons, the aims of this work were to develop new Jerusalem artichoke clones and evaluate the variability in sugar and dry matter yield potential of tuber, stalk and “integral crop”. In addition, useful information was obtained to aid the possibility of using the studied traits as selection criteria.

2. Materials and methods 2.1. Genotypes utilised Six clones of Jerusalem artichoke, five new clones (coded as 17, 22, 56, 69 and 70) and the cultivar “Violette de Rennes”, as control, were used. Their main characteristics are reported in Table 2. 2.2. Experimental site and design adopted Two experiments were carried out, one in 1999 and the other in 2000, at the experimental farm of the

M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40 Table 1 Pre-planting chemical and physical characteristics of the soil for experimentation Soil taxonomy

Udifluvents-Eutrochrepts

pH in water Organic matter (%) Total N (‰) Assimilable P (mg/kg) Exchangeable K (mg/kg) Carbonates (CaCO3 , %) Sand (2 > ∅ < 0.05 mm, %) Silt (0.02 > ∅ < 0.05 mm, %) Clay (∅ < 0.02 mm, %)

7.8 2.9 1.9 41 200 3 40 43 17

Experiment was carried out at the experimental farm of University of Udine, at S. Osvaldo (UD), northern Italy.

University of Udine, at S. Osvaldo (UD) in northern Italy, (latitude 45◦ 2 N, altitude 60 m) on a loamy-sandy, shallow soil (about 50 cm). The main physical and chemical soil characteristics are reported in Table 1. In both years, plots were ploughed 40 cm deep and harrowed twice slightly. The previous crop grown on the plot was sunflower. At the end of winter, before each sowing, 150 kg N/ha, 100 kg P2 O5 /ha and 100 kg K2 O/ha were applied to the soil. No supplemental nitrogen dressing was considered. Data on daily minimum and maximum temperatures and rainfall, obtained in the same site, are reported in Fig. 1. Irrigation maintained the soil under unlimited water supply during the entire crop cycle. Weeds were removed by hand, after plants emerged and no pesticides were applied. The first year, because of the limited number of tubers available for carrying out row plots, the experiment was performed by planting, on 25 March, about 100 g of tubers at a depth of 10 cm in hill plots, with a distance of 1 m × 1 m between them, in order to avoid mixing tubers of adjacent genotypes. The plants emerged on 2 April. The experiment had a completely randomised block design, with two replications (hill plot). Half of each plot (0.5 m2 ) was used to determine the production of the stalks and tubers at the first harvest time (30 September), while the other half was used for the determination of final tubers yield (30 November). Two guard rows of Violette de Rennes around the experimental plants were also provided.

27

In 2000, in the second experiment, on 16 March, 60–70 g of tubers, were planted by hand every 40 cm, with a distance of 0.70 m between rows and at a depth of 10 cm, giving a sowing density of 3.6 plants/m2 . The plants emerged on 30 March. The experimental scheme was a factorial experiment with 12 treatments (6 clones × 2 harvest times) in a randomised block design with four replications. The experimental unit was of 4 rows, 3 m long and 1 m apart (12 m2 of area). The first harvest (for stalks and tubers) and the second (for final tuber yield) were carried out on 4 October and 3 December, respectively. An area of about 3 m2 , corresponding to the two central rows of each experimental unit, was harvested by hand each time. 2.3. Characteristics studied, data acquisition and chemical analysis During the crop cycle the following parameters were determined: • Flowering time, as days from emergence to beginning of flowering, corresponding to the R5.5 stage (Schneiter and Miller, 1981); • Leaf photosynthesis (␮M CO2 m−2 s−1 ) was carried out on fully developed upper leaves on 1, 15 and 30 September 1999 and on upper, middle and lower leaves of the stem, on 16 August 2000, utilising an open portable gas exchange instrument (LICOR 6400 IRGA, Lincoln, Nebraska, USA). Measurements were carried out from noon to 3 p.m. on three different plants per plot, at 1700 PPFD (␮E m−2 s−1 ), with artificial light; • “Refractometrically measured” solids (Brix), was performed by a digital refractometer “model Palette 101” (ATAGO, Tokyo, Japan). The refractometer, that was used extensively in sugar beet programme breeding as a cheap method to estimate the total sugar content, could be used in Jerusalem artichokes also, which, however, apart from free glucose, contains fructose, sucrose and other sugars with different chain lengths (polyfructans) that can contribute in a different way to the refractive index (Frese and Damborth, 1987). The measurements were carried out on tissue juice (few millilitres) of small fresh samples of tubers and aboveground parts, crushed by a portable hydraulic press; the samples were collected and analysed on 15, 30 September and 18

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M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

1999 40

250

Rainfall (mm)

20 150 10 100 0 50

0

Temperature ( C)

30

200

-10

Mar

Apr

May

Jun

Aug

Jul

Sep

Oct

Nov

-20

2000 250

40

Rainfall (mm)

20 150 10 100 0 50

Temperature ( C)

30

200

-10

0

-20 Mar

Apr

May

Jun

Rainfall

Jul

Aug

min temp

Sep

Oct

Nov

max temp

Fig. 1. Ten-day rainfall and minimum and maximum temperatures recorded at the experiment site during 1999 and 2000.

October 1999, and on 6 July, 2 and10 August, 12 and 30 September and 4 December 2000. The amount of soluble components in the Jerusalem artichoke juice was expressed as the Brix-value: this gives the total sugar concentration in (w/v) percentage of a solution with the same refractive index.

The times of measurement are reported in the text and figures as days of the year (doy). During the first harvest time, for each plot, the total weight of separately hand-collected fresh tubers and aboveground biomass, divided into stalks and leaves, were determined. Samples of about 2 kg

M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

of tubers were weighed before and after washing, in order to determine the dirt tare and then used in calculating the corrected total tuber yield. In 2000, tubers, stalks and leaves were mashed in a cutter and two samples, each 10 g, were stored at −20 ◦ C for sugar analysis. In addition to this, about 500 g each of tubers, stalks and leaves were dried at 105 ◦ C for 3 days to determine the dry matter content. At the second harvest time, the aboveground biomass, with the leaves partially or completely lost, was not considered. In 2000, free and total sugar contents were analysed using the following procedure: 1. To extract fructans (inulin), 1 g of sample was mixed with 10 ml of distilled water at 85 ◦ C for 3 h, with mild shaking. The solution was cooled to room temperature, adjusted to 10 ml, homogenised and filtered through a 45 ␮m membrane filter. The free sugars (sucrose, glucose and fructose) were analysed by the HPLC method, using a Bio-Rad Aminex HPX-87C column (Bio-rad Laboratories, Milan, Italy) run at 85 ◦ C with degassed HPLC-grade water at a flow rate of 0.6 ml min−1 and an injection volume of 20 ␮l. Carbohydrates were detected by a Waters 410 refractive index detector (Millipore Corporation, Milford, USA). 2. To determine the total fructose and glucose content, 1 g of fresh sample was hydrolysed with hydrochloric acid (0.2 M) at 85 ◦ C for 2 h, and then neutralised with sodium hydroxide. After centrifugation for 10 min at 1400 rpm, the sample was filtered and adjusted to 10 ml and the fructose and glucose released by hydrolysis were determined by the HPLC methodology. Harvest index (HI) was calculated as tuber dry weight divided by the total biomass (leaf shedding included) dry weight. Total glucose and fructose per hectare were calculated by multiplying storage fresh matter yield by the total glucose and fructose content, respectively. The inulin content was obtained by: I = (F + G) − (fg + ff + fs ) where F and G are the total content of fructose and glucose, respectively and fg , ff and fs the content of free glucose, free fructose and glucose and fructose released from sucrose (amounts/1.9) (Van Loo et al., 1995), respectively.

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The fructose/glucose ratio, strictly related to the average inulin chain length, (Van Waes et al., 1998), in the text is reported as the average DP. All measured and derived data were subjected to analysis of variance. Because clone 69 was not harvested in 1999 and due to the differences in sowing density for the measurement times between 1999 and 2000, each year was analysed separately. Cultivars and measurement time effects were considered fixed and analysed as a factorial design each year. When F-ratios were significant, least significant difference values (LSD, P < 0.05) were calculated and used to compare means.

3. Results and discussions All clones came from a large collection of H. tuberosus L., which starting from 1996, Udine University had obtained by making several crosses between “commercial varieties” (Violette de Rennes, Bianca, C146), some local clones, wild types (coded as FVG-CA, FVG-FO and FVG-UD) and sources around the world (Baldini et al., 2000). In more detail, Violette de Rennes and clone 17 were characterised and selected for tuber production, clone 22 and 56 for their ability to produce stolons instead of “normal sized” tubers, supposing stalks to be the main “sink” for sugars and finally clone 69 was chosen chiefly for its ability to produce a high amount of aboveground biomass, and clone 70 for both large biomass and “normal sized” tuber yield (Table 2). The ANOVA results, for each year, are reported in Tables 3 and 4. For all the charactersitics determined in Tables 3 and 4, the effects “clones”, “days of the year” (when measured) and their interaction were significant (P < 0.01), with the exception of “Brix of the leaves” and the “aboveground biomass yield” during 1999, which were not significant and relatively less significant (P < 0.05), respectively. This indicated that the clones analysed had different response patterns at different times of the year. In both years, all genotypes significantly increased their tuber dry matter yield at the final harvest time with respect to the first (at flowering time), with the exception of clones 22 and 56 in the second year, confirming their poor ability to produce tubers as sugar accumulating organs. Clone 17 showed the highest

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M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

Table 2 Main characteristics of the clones analysed Clones

Mean tuber dry weight (g)

Tuber colour

Tuber yield potential

Emergence–flowering (days)

Mean plant height (cm)

Violette de Rennes Clone 17 Clone 22 Clone 56 Clone 69 Clone 70

7.7 10.8 2.2 2.7 3.3 8.1

White–red White–red Red Red White White

Good Good Poor Poor Medium–poor Good

271 273 266 274 273 272

267 253 257 271 260 242

Values obtained in the second year of the experiment (2000).

Table 3 Analysis of variance with the respective significance level for some characteristics observed in the first year of experimentation (1999) Source Clones Doy Clones × doy

Tuber yield (t/ha)

Aboveground mass yield (t/ha)

HI

Brix leaf (%)

Brix stalk (%)

Brix tuber (%)

Photosynthesis (␮M CO2 m−2 s−1 )

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗

∗∗

n.s.

∗∗

∗∗

∗∗

n.s.: not significant; doy: day of the year of the measurement times. ∗ Significant at P = 0.05. ∗∗ Significant at P = 0.01.

tuber yield potential (8.3 and 17.4 t/ha in 1999 and 14.0 and 17.3 t/ha in 2000, in the first and last harvest, respectively) (Fig. 2). These values were significantly higher than that obtained by the control, Violette de Rennes (12.9 t/ha), which confirmed its tuber yield potential as obtained in previous experiment carried out in the same environment and with the same plant density (Paolini et al., 1996). The yield of dry matter of aboveground biomass is very important at the time of the first harvest, when it represents a temporary storage organ and the crop can be utilised as a stalk and multiyear crop (Caserta and Cervigni, 1991). The highest stalk yield was shown by clone 70 in both years (25.0 and 22.9 t/ha in 1999

and 2000, respectively) and by clone 69 (23.1 t/ha) in the second year (Fig. 2). To evaluate the suitability of a clone for a stalk crop, it is not enough to know the aboveground biomass alone; in fact, if the ratio of leaves/stalks is too high, it could affect the above possibility. The clones utilised in the second year of experimentation (when the leaves were considered), showed a similar ratio ranging between 0.35 and 0.43 with the exception of clone 22, with a value of 0.65 (data not shown), that could be explained by the strong reduction in the aboveground dry matter at the time of the final harvest (Fig. 2). The potentiality of clones to be used as an “integral crop” (harvest at flowering time of both tubers

Table 4 Analysis of variance with the respective significance level for some characteristics observed in the second year of experimentation (2000) Source

Clones Doy Clones × doy

Tuber yield (t/ha)

Aboveground mass yield (t/ha)

HI

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

n.s.: not significant; doy: day of the year of the measurement times. ∗∗ Significant at P = 0.01.

Brix leaf (%)

Brix stalk (%)

Brix tuber (%)

SPAD

n.s.

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

∗∗

M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

31

1999 50

Harvest for stalks (30/09)

Harvest for tubers (30/11)

Yield of dry matter (t/ha)

45 40 35 30 25 20 15 10 5 0 V.R.

17

22

56

70

V.R.

17

22

56

70

clones

2000 50

Harvest for stalks (04/10)

Harvest for tubers (03/12)

Yield of dry matter (t /ha)

45 40 35 30 25 20 15 10 5 0 V.R.

17

22

56

69

70

V.R.

17

22

56

69

70

clones tubers

stalks

total

Fig. 2. Yield of dry matter of the plant organs in correspondence to the two harvest times considered: the first, suitable for stalk and the second for tubers yield. “Total” in the legend, is tubers + stalks in 1999, while it includes also the leaves in 2000. Bars represent the standard errors.

and stalks) is indicated by the total biomass yield at the time of the first harvest (at flowering). Among the clones studied, clone 69, in the second year (42.2 t/ha) and clone 70 in both years (30.4 and 37.3 t/ha, in 1999 and 2000, respectively) showed the highest values (Fig. 2).

Although the two experiments cannot be compared because of differences in number of clones and time of measurement, it is possible to observe certain consistency in the ranking of clones during the two years. In particular, clones 70 and 22 showed the highest and the lowest stalk yield, respectively, in both years

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M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

and the highest yield for tubers at final harvest was obtained by the same clones (17, 70 and Violette de Rennes) in 1999 and 2000. The dry matter content (%) of tubers did not change significantly with respect to the years, times of harvest and clones, ranging between 0.22 and 0.26; in contrast, the dry matter of the stalks increased significantly in all clones, from flowering (0.23–0.26) to the end of the crop cycle (0.34–0.41). The highest final harvest indexes were obtained by clone Violette de Rennes (0.64 and 0.65, for the first and second years, respectively) and 17 (0.65 and 0.78 in first and second year, respectively) (Fig. 3), confirming the ability of these two clones to produce tubers (Table 2). Moreover, the same clones demonstrated an early and very efficient translocation and transport of sugars from the stems to tubers, as seen also in the harvest index values, during the first harvest time in both years (Fig. 3). The exceptionally high value given by clone 22 at the first harvest time in 1999 is mainly due to the very limited aboveground yield, as reported in Fig. 2. The harvest index values in the second experiment, with a plant density (3.6 plants/m2 ) comparable to a standard field crop, were similar to those of other experiments carried out in different locations and with different genotypes (Conde et al., 1991; Schittenhelm, 1999). During the final harvest time, the range of the refractometer indexes (Brix values) of the tubers, considering both years, was 15.8–24, similar of that obtained by Zubr and Pedersen (1993) using different clones under different environmental conditions (18.4–22.3). During the first year, in particular, the Brix values of the tuber increased significantly at the final harvest (30 November) in only two clones (22 and 70); however, the same value did not significantly change from the middle of September (before flowering) in the other clones. At the final harvest, the clones most suitable for tuber yield, such as 17, 70 and Violette de Rennes showed the lowest Brix value in tubers (Fig. 4). The latter two clones showed the highest Brix values in the stalks at flowering (30 September) time indicating a great variability in this character. In 2000, the Brix values was analysed from 1 July, a longer period than 1999 (151 and 76 days, respectively). In this year, all clones studied showed a significant increase in Brix values corresponding to 230

doy (middle of August) in both stalks and tubers (but even in leaves of a few clones) (Fig. 4). During the final harvest time (336th day of the year), all clones displayed a significant Brix value increase in the tubers, with clones 22 and 69 reaching the maximum values. However, clone 17 and Violette de Rennes, showed values similar to that found at the start of experimentation (Fig. 4). Violette de Rennes reached the maximum Brix values in the stalks earlier than the other clones (end of July) exhibiting a different time course for this character (Fig. 4). The rate of photosynthesis (Pmax ) during 1999 and 2000, is reported in Fig. 5. In the first year of measurement, (middle of September, 258 doy), the clones reached their maximum Pmax and among these, clones 69 and 17 showed the highest (30 ␮M CO2 m−2 s−1 ) and the lowest (11.3 ␮M CO2 m−2 s−1 ) values, respectively (Fig. 5a). At the next measurement period, during flowering (stalk harvest time), all clones, except one, showed a decreasing trend in activity (mainly clone 70), but clone 17 had a significantly increased value. These values are slightly lower than those found by Soja and Haunold (1991) in a similar experiment, because they measured 14-week- old plants in contrast to the 23-week-old plants used for this experiment and a decrease in photosynthesis in ageing leaves was common in all plants. In 2000, Pmax activity was measured once, that is in the middle of August (230 doy) and three different leaf positions (upper, medium and basal) were considered. As a mean of the leaves, the highest activity was shown by clone 56 (14.2 ␮M CO2 m−2 s−1 ) and the lowest by clone 69 (5.4 ␮M CO2 m−2 s−1 ). The upper leaf displayed a lower photosynthetic activity than the middle and basal leaves in all, except clone 17 (Fig. 5b). Apparently this result is in contrast with that obtained by Soja and Haunold (1991), but in this year, the measurement was carried out 28 days earlier than the first measurement in 1999, when the plants had not yet reached their final stem length, and upper leaves were still developing. The fructose content, expressed as percentage of fresh weight, showed higher mean values in the tubers than in the stalks in all genotypes, except the control and with higher values in the tubers of the first harvest than in the final one, with the exception of clone 17

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1999 1,0 0,9

Harvest index

0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 V.R.

17

22

56

70

clones Harvest for stalks (04/10) Harvest for tubers (03/12)

2000 1,0 0,9

Harvest index

0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 V.R.

17

22

56

69

70

clones Harvest for stalks (30/09) Harvest for tubers (30/11) Fig. 3. Harvest index in correspondence to the two harvest times considered. Bars represent the standard errors.

(Fig. 6). Among clones, Violette de Rennes had the highest value (16.6%) for fructose content in the stalks, while clone 69 gave the highest in the tubers (23.3 and 22.9% in the first and last harvest, respectively).

With the exception of clone 17 at first harvest time, the glucose content, like that of fructose, was significantly higher in the tubers than in the stalks (Fig. 6). The glucose content in tubers at the last harvest

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M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40 Leaves - 1999

Leaves - 2000

30

30

Brix values

25

25

20

20

15

15

10

10

5

5

0

0 180

258

273

291

334

L.S.D. for P < 0.05 200

220

240

Brix values

Stalks - 1999

280

300

320

340

Stalks - 2000

30

30

25

25

20

20

15

15

10

10

5

L.S.D. for P < 0.05

0

5

L.S.D. for P < 0.05

0

258

273

291

334

180

200

220

Tubers - 1999

Brix values

260

240

260

280

300

320

340

Tubers - 2000

30

30

25

25

20

20

15

15

10

10

5

L.S.D. for P < 0.05

0 258

273

291

334

5 0 180

L.S.D. for P < 0.05

200

220

240

days of the year V.R.

17

22

56

69

260

280

300

320

340

days of the year

70

V.R.

17

22

56

69

70

Fig. 4. The effect of the clones × measurement time interaction on the Brix values of the storage organs, with least significant difference for 0.05 probability level (LSD). Arrows represent the flowering time.

significantly increased with respect to the first harvest. The content of this sugar, if compared to fructose content, was very low, ranging between 0.92 (clone 22 in the stalk) and 3.73% (clone 69 in tubers at the last harvest).

The above result influenced the fructose/glucose ratio, like DP and was highest during the flowering time in both stalks and tubers ranging between 7.5 and 11.2, while, at the final harvest in tubers, it significantly decreased to values ranging from 4.8 to 6.7 (Fig. 7a).

M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

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Fig. 5. (a) The effect of clones × measurement time interaction on leaf photosynthesis of fully developed upper leaves in 1999. Least significant difference for 0.05 probability level (LSD). Arrow represents the flowering time; (b) photosynthesis of three leaf at three different stalk positions, measured on 16 August (229th day of the year) in 2000. Bars represent the standard errors.

The inulin content (as a percentage of fresh weight) resembled the behaviour of the fructose content, ranging between 7.7 and 25.4 (Fig. 7a) and generally, the inulin content in the stalk was lower than that in tubers.

The relationship between inulin content (percentage fresh weight) and DP are depicted in Fig. 7a. Region I with a high inulin content and low DP, contains the tubers of all clones at final harvest, plus the tubers at stalk harvest of clone 70; Region

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M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

Fig. 6. Total glucose and fructose content (percentage on fresh matter) of stalks, tubers at stalk harvest time and tubers at final harvest. Bars represent the standard errors.

II with a low inulin content and low DP, where fortunately just clone 70 falls, with stalks and tubers at final harvest; Region III is characterised by a high DP but low inulin content and includes stalks of all clones (with the exception of clone 70) and the tubers at stalk harvest of clone 17; and finally Region IV with the highest inulin content and

DP, i.e. the most desirable, contains the tubers at stalk harvest of clones 22, 56, 69 and Violette de Rennes. Maijer and Mathijssen (1996) obtained similar results in chicory with a depolymerisation and shorter fructose polymers after the beginning of October, where decreasing the incident radiation in

M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

37

2000 35 I

IV

Inulin content (%)

30 69

25 20

V.R.

15

17

56

69 22

56

70

V.R.

17

10

17

69

70

5

22

V.R.

70 56 22 III

II

0 2

4

6

8

10

12

14

DP

(a)

stalks

tubers at stalk harvest

tubers at final harvest

Inulin yield (t/ha)

2000 20 18 16 14 12 10 8 6 4 2 0

V.R.

I

IV

69 17 V.R.

17

56

70

69

22

70

69 56

22

56

70 17

V.R.

22

II

2

III

4

6

8

101

21

4

DP

(b)

stalk

tubers + stalk

tubers at final harvest

Fig. 7. (a) Relationship between DP (average degree of polymerisation of the inulin chain) and inulin content (percentage on fresh matter) of the storage organs and (b) between DP and inulin yield (t/ha) and of the six clones considered. Lines, inside the graph, represent the averages of inulin content, inulin yield and DP. Clone codes are reported close to the symbols.

late autumn, caused a decrease in assimilation and reduced sucrose supply to the tubers. This latter sugar is fundamental to enhancing the polymerising activity and inhibiting depolymerisation (Edelman and Jefford, 1968). Analogous to this,Fig. 7b, shows the relationship between inulin yield (t/ha) obtained by the different harvest methods and DP. The early harvest, at flowering time, determined the highest DP values (Regions IV and III). In particular, the “integral utilisation” of

the crop (tubers + stalks) and Violette de Rennes with the clones in Region IV, assured the highest inulin yield (17.9 t/ha) and a very high DP (10.2). The same clones obtained the highest DP (11.0) and inulin yield for the stalk. The classical harvest of tubers at the end of the crop cycle, showed the lowest DP (between 4.8 and 6.7) values (Regions I and II), while the inulin yield was very close to the average in all clones, with the exception of 17 (13.7 t/ha).

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M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

Fig. 8. Total sugars and inulin yield (t/ha) of the storage organs at the two harvest times considered. Bars represent the standard errors.

4. Conclusions The yield of total sugars (fructose+glucose) and inulin per unit area (t/ha) were very similar. Considering the crop as a raw material for the industrial production of total sugars, the maximum inulin yields were 10.4 and 8.0 t/ha, respectively, utilising clone 69 and

harvesting just the stalks at flowering time (from the end of September to 1 October) (Fig. 8). The advantage is that the yield in the following years will be obtained simply by re-growing shoots (polyannual crop), thus avoiding tuber sowing and the harvesting technique is simplified. On the other hand, the choice of a polyannual crop restricts the farmer, especially now

M. Baldini et al. / Industrial Crops and Products 19 (2004) 25–40

that the agricultural policy is continuously changing and in evolution. The conventional harvesting of tubers at the end of the crop cycle (generally at the beginning of December in this environment), gave a very satisfactory sugar yield using clone 17 (13.3 and 13.7 t/ha fructose and inulin, respectively), which is significantly higher than that obtained by Violette de Rennes (10.4 and 10.7 t/ha, sugars and inulin, respectively) (Fig. 8). The sugar amount produced by tubers harvested at the end of the cycle is higher than that obtained from the stalks of all genotypes studied, even if, from an economic point of view, the sugar yield in the following years without sowing costs, is considered at least partially. Finally, there is the possibility of utilising the integral crop, harvesting the stalks and tubers at the beginning of flowering. This harvest method, with the exception of clones 17 and 70, significantly increased the yield. In particular, total sugars and inulin yield obtained by Violette de Rennes (18.6 and 17.9 t/ha, respectively) and clone 69 (18.6 and 15.9 t/ha, respectively) reached very high levels compared to the other methods (Fig. 8). This solution, providing both a high yield potential and the agronomic advantage of freeing the soil soon, could give very good results, especially if a suitable genotype is utilised. This ideotype must have a good stalk yield potential, high sugar content in the stalk and the capacity to develop tubers early on. It is suggested that breeders exploit the existing genetic variability among clones regarding sensitiveness to photoperiod stimuli to induce early tuber formation, as the carbohydrate storage pool capacity in the stalk is strictly related to delay or even prevent tuber induction (Soja and Dersch, 1993). The “refractometric index” (Brix) and leaf photosynthesis showed a very high variability among the genotypes tested, demonstrating their exploitability in breeding programmes. In particular, the preliminary data using the refractometer index, a very quick and easy method to use, indicated a significant, positive relationship with the inulin content in the tubers (0.90∗∗ and 0.85∗∗ , n = 6, utilising the averages of the clones) in the first and second harvest, respectively (data not shown), confirming the results obtained by Van Waes et al. (1998) for chicory root. On the contrary, no relationship was found with the stalk inulin content.

39

Acknowledgements This research is supported by the MURST national project Crops for inulin production: modelling, environmental effects and cropping strategies, co-ordinator Prof. Francesco Danuso.

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