Final Technical ReportFinal Technical ReportFinal Technical ReportFinal Technical Report
Sustainable Production of PlantSustainable Production of PlantSustainable Production of PlantSustainable Production of Plant----Derived IndigoDerived IndigoDerived IndigoDerived Indigo
Partner 3
Reporting Period: 01.01.2001 � 30.06.2004
Themenblatt-Nr.: 42.12.430
Thüringer Landesanstalt für Landwirtschaft
Thüringer Ministerium für Landwirtschaft, Naturschutz und Umwelt
Langtitel: Sustainable Production of PlantSustainable Production of PlantSustainable Production of PlantSustainable Production of Plant----DerivedDerivedDerivedDerived Kurztitel: SPINDIGO Projekt: Öl-, Energie- und Industriepflanzen Projektleiter: Dr. habil. Armin Vetter Abteilung: Pflanzenproduktion Abteilungsleiter: Dr. habil. Armin Vetter Laufzeit: 01/2001 bis 06/2004 Auftraggeber: Europäische Union Namen der Bearbeiter: Dr. sc. Günter Wurl Dipl. Ing. agr. Andrea Biertümpfel Jena, im August 2004 (Prof. Dr. Gerhard Breitschuh) (Dr. habil. Armin Vetter)
Präsident Projektleiter
ContentContentContentContent Page Summary 3 1 Introduction 5 2 Material and methods 6 3 Results 7 3.1 Development of a modern cultivation scheme of Isatis spp. 7 3.1.1 Generally 7 3.1.2 Sowing time 8 3.1.3 Harvest time and cutting frequency 9 3.1.4 Assessment of various herbicides for use with Isatis spp. 9 3.1.5 Seed production 11 3.1.6 Maintaining of the different strains of woad 12 3.2 Examination of woad to produce indigo 13 3.2.1 Generally 13 3.2.2 Biomass yield, indigo content and indigo yield per area of the different 14
woad strains in 2002 and 2003 3.2.3 Causes for the different indigo yields 16 3.2.3.1 Genetical causes 16 3.2.3.2 Influence of meteorological conditions on the biomass yield, 18
the dye content and the dye yield of woad 3.3 Development of a modern cultivation scheme of Polygonum tinctorium 22 3.3.1 Generally 22 3.3.2 Sowing time 23 3.3.3 Harvest time and cutting frequency for Polygonum tinctorium 25 3.3.4 Optimal N-fertilization of Polygonum 27 3.3.5 Assessment of various herbicides for use with Polygonum tinctorium 28 3.3.6 Seed production 28 3.4 Indigo extraction from Polygonum tinctorium 29 3.4.1 Generally 29 3.4.2 Attempts for production of indigo from Polygonum in Dornburg 31 3.4.3 Possible causes for the different recovery rates 33 3.4.4 Purification of the raw indigo 36 3.4.5 Possibilities to influence the indican content 37 3.5 Recycling of waste products 41 3.6 Analytical determination of indigo and its precursors in Isatis and Polygonum 42 3.6.1 Material and methods 42 3.6.2 Results and discussion 42 3.6.3 Conclusions 43 4 Discussion 44 5 Conclusions 45 6 Exploitation and dissemination 45
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SummarySummarySummarySummary
The objectives of the project were the re-establishment of the cultivation of blue dye deliv-ering plants in Europe and their use for dyeing purposes in an industrial scale. That in-cludes the development of an ecologically and economically sustainable cultivation system, efficient low-cost dye production methods and environmentally friendly textile dyeing up to not dangerous disposal of the residues of the dye winning process.
The task of the Thüringer Landesanstalt für Landwirtschaft (TLL) was to find out, which plant of the three in Europe only cultivated species with precursors of indigo, Isatis tincto-ria, Isatis indigotica and Polygonum tinctorium, is the most suitable for the cultivation un-der Middle-European conditions. From the historical point of view it should be Isatis tinc-toria. Woad had been the only plant species for winning the blue dye in Germany, espe-cially in Thuringia up to the end of the 17th century. In 1990 this plant has been already rein-troduced in the agriculture of Thuringia and there was developed a modern cultivation for it by the TLL.
At the comparative cultivation of the three species in the frame of the project has been shown that the indigo precursor content in woad is very low. Investigations of 1262 single plants gave only values from 0.025 % to 0.975 % of indigo in the dry mass with a mean of 0.27 % of indigo in the dry mass. In the trial of the year 2002 with 12 accessions of Isatis tinctoria and 1 of Isatis indigotica could be detected values between 1 and 2 % of indigo in the dry mass but always with the exception on the material of one of the three cuts. Gener-ally the indigo content of Isatis indigotica seems to be higher than that of Isatis tinctoria. The cold requirement for vernalization of Isatis indigotica is very low, so that under Thur-ingian conditions the most plants bolt after sowing in spring and therefore a successful cultivation is hindered. Apart from the low dye content Isatis tinctoria has a further draw-back. Its biomass yield changes in a great extent (up to 100 %) from year to year. Especially in years with cold and/or dry weather, the formation of biomass in woad is low.
For Middle-European conditions Polygonum tinctorium seems to be the most suitable plant for indigo production. The yield of Polygonum leaves (only the leaves contain the indigo precursor) may be in some years up to 30 % lower , in the most years it is nearly the same as at Isatis tinctoria. The indigo content in the Polygonum leaves is about 3 to 5fold higher than in woad. For example, 92 Polygonum probes gave values from 0.56 to > 2.44 % of indigo in the dry mass (calculated from the determined precursor content) with a mean of 1.40 % of indigo in the dry mass. Furthermore the changes of the biomass yield from year to year are only low. So the indigo yields per area land are always higher to very much higher than those of woad. Unfortunately, Polygonum tinctorium, the classical plant for winning blue dye in Japan, is widely unknown in Europe. Therefore, no cultivation schemes existed for this plant on which could be gone back. The TLL won the first characteristics of Polygonum tinctorium by sowing out some seeds in a greenhouse and than transplanting them out in the field on a very small plot. Also pot experiments were carried out. In the frame of the project it was possible to develop a modern cultivation system. The working out of a short agronomical blueprint on the bases of the results enables the farmers to cul-tivate this crop successfully with standard equipment, currently used by them.
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Although the Polygonum plant is very sensitive to low temperatures below 5 °C the sowing of the crop should be done at the beginning of April. Because of its hard semen shell the duration of germination is very long., and so the seeds germinate not before the middle of May at a frost-free time. Late sowing of the crop can cause an insure emergence because there is not enough water in the soil. 5 kg pure seeds per hectare for sowing by a drill is a reliable sowing strength. The inter row distance should be 30 cm. First, Polygonum plants grow only slowly. Therefore, it is necessary to combat the weeds. If the crop is cultivated in a greater extent, the use of herbicides is absolutely necessary. Because of a high succepti-bility of Polygonum to the most herbicides, only an application of means before emergence is possible. The use of 1.5 kg/ha Afalon (=Linuron) was the best variant.
The same problems exist at woad. The efficiency of different compounds and the tolerance to the crop were determined in extra trials in 2002 and 2003. The best variant in this case was a mixture of Lentagran (=Pyridat) + Butisan (= Metazachlor) + Starane 180 (=Flu-roxipur) in an amount of 1.5 kg + 1.5 l + 0.2 l per hectare applicated at the 4 to 6-leave-stage of the woad plant.
While the optimal harvest time and the cutting frequency for woad was already known from the cultivation practice in Middle Age, this parameters had to be developed in many field trials for Polygonum. The time of the 1st cut is reached, when the rows are closed by the leaves of the plants. The relation of stalks : leaves is then 1.25 : 1 or less. This date changes from year to year under Central European conditions. It is before the end of July, but gener-ally at the beginning of August. Polygonum plants need at least 6 weeks for a full regrowth, and so the stand can be harvested in a 2nd cut at latest at the middle of September.
For a high yield Polygonum tinctorium needs 160 kg plant available N/ha (one gift before sowing, under consideration of mineralic N in the soil in 0 to 60 cm depth). This amount is something lower than at woad (200 to 220 kg N/ha).
The production of seeds in the desired quantities at Polygonum is not always possible, because of the late flowering of the crop. It is better to produce the seeds in more southern and warmer countries. For the seed production 0f woad exists no difficulties.
Some differences exist between woad and Polygonum in regard to an optimal dye winning process of the both plants, determined by the different chemical nature of the dye precur-sors. While the main precursor of woad, isatan-B (= indoxyl-5-ketogluconate) is a very in-stable compound, which can be cleaved already by water, the precursor of Polygonum indi-can (= indoxyl-ß-glucoside) is very stable, which in watery solution can be cleaved by strong acids ore bases or enzymatically. After results from Japan, the Polygonum plant contains ß-
glucosidase, which cleaves the cmpound at the extraction process. Temperatures of ≥ 60 °C inhibited the enzyme completely. So, the extraction machinery for woad, working at 70 °C, is not suitable for Polygonum. An extraction temperature of 40 °C seems to be the best variant for Polygonum to get a moderate extraction time (about 20 hours) and simultane-ously a cleavage of the precursor.
The purity of the won raw indigo is determined to a high degree by the precursor content of the source material. The harvest time, the water status of the soil, the height of N-fertilization and the genotype influence the dye content of the leaves.
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It was possible to select types with a higher dye formation ability and higher indigo yields per area of land after a mutagenic treatment of Polygonum seeds.
Because of the great importance of the quality of the leaves for the further processing for the determination of dye a method must be generated, which allow to work with a greater number of samples in a sufficient precision. This task was solved by co-work of the TLL with the Spindigo partners.
Summarising it can be said, that now exist the prepositions for a successful cultivation of woad and Polygonum in the agricultural practice and that the dye production of apprecia-ble amounts of indigo is possible.
1111 IntroductionIntroductionIntroductionIntroduction
Indigo, the oldest dye for blue dyeing of textiles, has also a great importance for dyeing Jeans well, although there are better blue dyes in regard to dyeing properties and fast-nesses. Up to 1897 indigo were produced exclusively from plants. At this time, alone 8.000 t of pure indigo was produced from the Indigofera-plant in India. This amount corre-sponded with 80 % of the world production (H. SCHMIDT, 1997). In 1897 K. Heumann in the BASF discovered a practicable method for the technical production of the dye. As a result of this indigo naturalis disappeared nearly completely from the market. This had a positive effect in those days. The land used for cultivation of dye plants became free for the cultivation of food for humans and feed for animals.
Now an enormous increase of yield took place by breeding, application of anorganic fertil-izers, weed and disease control and mechanisation of agriculture. So, the full available soil is not needed for the production of foodstuffs. Therefore, the European agriculture looks for other possibilities to use the surplus land. One possibility could be the reintroduction of dye plants into the agricultural production, especially the cultivation of blue dye (= in-digo) delivering plants. The dye stuff of the plant is chemically the same as that of the tech-nical product. So, there haven’t to be developed new methods for dyeing. But which plant is the most suitable for Middle European conditions? Indigofera ssp. as tropical species was ruled out at the beginning. It remain only the three species Isatis tinctoria, Isatis indigotica and Polygonum tinctorium. In fact, Polygonum is like Indigofera of subtropical origin, but it grows very well under European conditions as could be determined in forego-ing attempts. After the literature Polygonum should have a dye precursor content like Indigofera. So, Isatis species together with Polygonum tinctorium were cultivated within the Spindigo-project in the field on experimental plots, to compare their formation of biomass. Also, the three plant species have been examined for their potential to produce indigo. The aims of the investigations were to develop an optimal cultivation scheme, to discover the most suitable species for the production of indigo and to give concrete recommendations for a cultivation in the agricultural practice. In fact, the handling of the harvested material was to investigate.
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2222 Material and methodsMaterial and methodsMaterial and methodsMaterial and methods
A modern cultivation method had to be worked out for Polygonum tinctorium. It contains the following parameters:
- sowing time
- sowing strength
- distance between the rows
- harvesting time, including the cutting frequency
- N-fertilization and amount of the single fertilization gifts
- weed control
In former attempts the plants were sown out in a greenhouse and then planted out into the field. Planting of ten plants per square meter gave always a full yield.
For Isatis species a modern cultivation system was developed by the TLL already earlier. Its main goal was to secure high yields of biomass for the production of woad juice, which can be used for the conservation of wood, paper and stone and also as a mean for fire preven-tion. For theses purposes yearly 10 to 80 ha woad have been cultivated in Thuringia since 1990.
Within the Spindigo-project woad has been examined for its potential to produce indigo. For this purpose 10 to 13 accessions were cultivated on experimental plots in Dornburg after the recommendations for the juice production:
- drilling of 0,5 g (about 250 pure seeds) per square meter at a row distance of 13.5 cm as early as possible in spring
- fertilization before sowing to a level of 120 kg plant available N/ha and further gifts of 50 kg N/ha after the 1st and the 2nd cut with Calciumammoniumnitrate
Obviously more than three cuts per year are not useful under Central European conditions. The beginning of the harvest was around July, 1st. Two further cuts were carried out with a distance from 4 to 6 weeks between each cut.
In the Spindigo-project the former cultivation recommendations were completed by the assessment of various herbicides for use with Isatis species in an extra trial. The same was done with Polygonum.
To get enough seed amounts for the production of woad and Polygonum in the practice the conditions for a sure seed production were determined for all species.
All cultivation attempts took place on 13.5 m² plots with 2 to 4 replications in the trial sta-tion Dornburg. Dornburg has the following geographical, climatic and soil parameters (tab. 1).
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Table 1:Table 1:Table 1:Table 1: Characterisation of the trial station Dornburg Parameter
Height over sea level [m] 250 to 270 Mean annual precipitation [mm] 595,8 Mean annual temperature [°C] 8,8 Geological subsoil Median limestone with loess overlay Soil type Clay to sandier clay Value of soil 46 to 80, mean 65 Climatic area Moderate humid location
The influence of the different agrotechnical measures on the dye precursor content has been determined by chemical methods, developed in co-work with a part of the other Spin-digo-partners.
Finally, a mutagenic treatment of Polygonum seeds with Nitro-methyl-urea was carried out to induce types with a higher dye content than the source strain.
More information on the single attempts can be found at the description of the results.
3333 ResultsResultsResultsResults
3.13.13.13.1 Development of a modern cultivation scheme of Isatis spp.Development of a modern cultivation scheme of Isatis spp.Development of a modern cultivation scheme of Isatis spp.Development of a modern cultivation scheme of Isatis spp.
3.1.1 Generally
Woad (Isatis tinctoria) had been the only plant species for winning of blue dye in Europe in the Middle Age. At the end of the 17th century this plant disappeared from the European agriculture because it had not been competitive with the tropical plant Indigofera spp.. In 1990 this plant has been reintroduced because of the fungistatical compounds of the woad juice which can be used for the conservation of wood, paper and stone and for fire preven-tion. The TLL has developed a modern cultivation system. The results can be summarized as following:
- The plant is sown in spring as early as possible with a seed strength of 5 kg/ha at an interrow distance of 13.5 cm (like cereals).
- N-fertilization of the crop up to 120 kg plant available N (mineralic N in the soil in 0 to 60 cm depth + N-fertilizer) per ha before sowing and 50 kg N/ha as cal-cium ammonium nitrate after the 1st and the 2nd cut.
- Woad can be harvested in three cuts per year and the harvest takes place with a forage harvester.
- The harvest starts about July, 1st. Further cuts follow in a distance of about 5 to 6 weeks. More than three cuts are impossible under Central European condi-tions.
The cultivation practice of woad began with a distance of the rows of 30 to 40 cm. This was also the row distance in the Middle Age. It makes possible a hand or a machine hoeing, resp. for the weed control. The nearer row distance of 13.5 cm has shown an appreciable increase of yield, especially at the phenotypes with an erect growth habit. They are only suitable for loss-poor machine harvest. High and secure yields of leaves are wished for the winning of juice. Within the SPINDIGO project woad has been examined for its potential to
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produce indigo. Some of the earlier experiments have been repeated from this point of view to determine its dye precursor content. At the same time the improvement of the cul-tivation scheme was a goal of the attempts. Especially the assessment of herbicides for use in Isatis spp. was carried out. The weed control in woad is only possible by herbicides. Be-sides attempts were carried out for the security of seed production in a greater extent.
3.1.2 Sowing time
Under Central European conditions the sowing of Isatis is possible in late autumn (later than the middle of October) and in early spring. Sowing in early autumn (Au-gust/September) gives always flowering plants in the next year. Usually the sowing in spring is as early as possible. Moreover sowing in autumn may be advantageous as can be seen in table 2. Table 2:Table 2:Table 2:Table 2: Influence of the sowing time on the yield of different woad strains, trial station Dornburg
Strain Cut Sowing in autumn Sowing in spring GD(Tukey,5%) Yield
(dt dry mass) S Yield
(dt dry mass) s
Thür. Waid
1st 2nd
3rd
8,16 14.25 11.35
0.685 1.940 2.269
9.08 12.45 12.13
1.700 1.846 2.702
2.19 2.65 3.15
�������� 33.7633.7633.7633.76 4.5404.5404.5404.540 33.6733.6733.6733.67 5.0425.0425.0425.042 2.612.612.612.61 BG8 1st
2nd
3rd
9.98 13.75 10.16
1.525 2.806 2.807
9.11 13.39 12.28
2.660 2.804 2.925
2.29 0.21 1.94
������������ 33.8933.8933.8933.89 7.0377.0377.0377.037 34.7834.7834.7834.78 7.8387.8387.8387.838 2.642.642.642.64 740 1st
2nd
3rd
12.00 15.94 12.70
1.612 2.629 1.930
10.53 15.03 13.02
1.913 2.732 2.565
5.24 2.21 4.76
������������ 40.6540.6540.6540.65 5.5.5.5.110110110110 38.5838.5838.5838.58 5.4635.4635.4635.463 10.0910.0910.0910.09 757 1st
2nd
3rd
14.64 20.03 15.83
1.618 2.376 2.835
13.15 19.40 13.20
2.143 4.093 2.673
2.77 5.06 5.52
������������ 50.5050.5050.5050.50 5.1365.1365.1365.136 45.7545.7545.7545.75 7.5797.5797.5797.579 10.5910.5910.5910.59 758 1st
2nd
3rd
12.74 18.46 14.43
2.421 3.799 2.920
12.03 18.57 14.56
1.719 2.961 5.503
3.67 2.93 7.91
������������ 45.6345.6345.6345.63 9.0509.0509.0509.050 45.1645.1645.1645.16 9.8209.8209.8209.820 13.3413.3413.3413.34 769 1st
2nd
3rd
14.07 18.34 15.35
3.215 2.325 2.854
12.96 16.88 11.12
2.985 1.816 2.398
6.04 3.32 3.67
������������ 47.7647.7647.7647.76 8.0868.0868.0868.086 40.9640.9640.9640.96 6.9156.9156.9156.915 12.6912.6912.6912.69
The weed control is not different in autumn and spring sown woad, but the duration of the processing time is longer and so the yield of biomass is higher in the first case. So already in the Middle Age in Thuringia farmers have sown the woad (hulled seeds) partially in win-ter on the snow on the soil by hand. Nowadays husked seeds are drilled. The autumn drilled woad plots often become muddy and must be broken off in spring as it took place in Dornburg in the year 2002. Sowing under cover-crop, which decease in winter may be use-ful. Also the sowing of pure seeds with a fertilizer spreader in a stand of crop like white mustard, peas or garden cress may be useful. The amount of seeds per hectare must be chosen then a bit higher. Despite of the higher risk of an autumn sowing, it is wise to drill
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about 25 % of the whole area in this time at a greater extent of woad cultivation. The Thur-ingian farmer cultivating 20 ha in 2004 for the juice production has sown the whole area in autumn 2003.
Isatis indigotica has only a very low cold requirement for vernalisation and therefore sow-ing is not possible before middle of May.
3.1.3 Harvest time and cutting frequency
Formerly much experimental work was done to determine the optimal cutting time and cutting frequency for woad. Such an attempt was repeated in 2002. The results of the fore-going attempts could be confirmed (tab. 3). Table 3:Table 3:Table 3:Table 3: Yield (dt dry matter per ha) of woad at different cutting regimes (4 replications, 13.5 m² plot size) Variant Weeks Harvest time ����
between harvests
14.06. 28.06. 12.07. 26.07. 09.08. 26.08. 06.09. 21.09. 05.10. 17.10. 01.11.
1 4 3.6 14.5 17.4 7.4 42.942.942.942.9 2 6 3.6 19.1 17.4 6.3 46.446.446.446.4 3 8 3.7 22.2 19.2 45.145.145.145.1 4 4 11.0 14.5 17.8 4.8 47.847.847.847.8 5 6 10.8 19.3 16.2 4.1 50.450.450.450.4 6 8 11.5 23.8 12.2 47.547.547.547.5
Obviously more than 3 cuts per year are not useful under Central European conditions after sowing woad in spring. This result corresponds with the experiences in the Middle Age. Like at the former attempts an earlier beginning of harvesting than about July, 1st and a dis-tance between the further cuts longer than 6 weeks were not satisfactory. Better results only can be got, if the woad is already sown in autumn or winter and not in spring (see above).
3.1.4 Assessment of various herbicides for use with Isatis spp.
The near row distance at the cultivation of woad makes a mechanical weed control impos-sible. Therefore, the use of herbicides (and pesticides?) is necessary, if woad is cultivated in a greater extent. In Thuringia the yearly cultivation of this crop in a size of about 100 ha is planned for the next years, but not for dye winning purposes. The dye may be a by-product at the production of woad juice for the conservation of wood, paper and stones and as a means of fire prevention.
In Germany the application of herbicides is only permitted for plants mentioned in the reg-istration of the herbicide, pesticide etc. (indication) registration by the BBA. For the regis-tration are needed certificates of
a) the efficiency of the compound against combated weeds,
b) the tolerance of the crop, for which the means are intended and
c) the freedom of the harvested material from residues of the means.
The big companies which apply for registration do not show any interest in minor crops with a small cultivation area. Potential users of herbicides, pesticides etc for such minor crops as medical or spice (or dye) plants must look after the permission. Results of two
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years must be available to get the permission of the Biologische Bundesanstalt für Land-wirtschaft (BBA) for using the herbicides in a crop. The TLL has now carried out attempts for two years.
For this purposes extra field trials with woad and Polygonum were carried out in 2002 and 2003. The woad was sown on 08.04.2002, resp. 25.03.2003 with a seed strength of 5 kg/ha in 4 replications (plot size 13.5 m²). It emerged on 30.04.2002, resp. 15.04.2003. The amount of applicated herbicides and the appilcation time can be seen in table 4. The re-sults of the second year were the same ones as in the foregoing year. Table 4:Table 4:Table 4:Table 4: Results of the herbicide trials in woad, Dornburg 2002 and 2003 Variant Application Weeds/m²/
Efficiency of herbi-cides (%)*
Phytotoxicity Yield (1st cut) (dt dm/ha)
Amount Time (kg or l/ha) 2002 2003 2002 2003 2002 2003 2002 2003 UC - - - 48 65 - - 13.6 14.0 Treflan**** (Trifluralin)
2.0 04.04.
25.03. 0 - 99 0 – 96 ** 8 % deceased plants (dp)
10 % of the plants with growth depression
(gd)
0 13.9 13.4
Fusilade Max (Fluazifop)
1.0 17.05. 16.05. 0 Agropy-
ron repens
100
0 winter
barley 100
0 0 13.6 12.2
Lontrel 100 (Clopyralid)
1.2 17.05. 06.05. 0 - 100 5 - 8 0 0 14.6 12.7
Butisan (Metazachlor)
1.5 08.05. 06.05. 0 - 100 0 - 15 0 0 13.4 13.8
Starane 180 (Fluroxipyr)
0.5 08.05. 06.05. 0 - 100 0 - 20 5 % dp 10 % gd
15 % gd 12.8 11.4
Splitting Lenta-gran WP (Pyridat)
1.0 + 1.0 08.05. 21.05.
06.05.16.05.
40 - 100 50– 70 *** 5 % dp 10 % gd
10 % gd 13.7 13.6
Mixture: Lentagran + Buti-san + Starane 180
1.5 + 1.5 + 0.2 08.05. 06.05. 98 - 100 64 - 99 18 % dp 10 % gd
gd 12.0 13.9
GD t, 5 % 1.8 2.0 * last assessment 05.06.2002, 04.06.2003 ** no effect on Thlaspi arvense *** weakness against Polygonum ssp. **** working into the soil before sowing
The main weeds in the two years were rape, Chenopodium alba, Solanum nigrum, Poly-gonum species, Thlaspi arvense and Galium aparine, possibly also cereals. Most of the examined means have a weakness against one ore also more weeds. Only the mixture of Lentagran + Butisan + Starane 180 combats the weeds effectively. It can be recommended without restrictions for the weed control at woad. Its registration as an official means with woad is proposed.
The growth depression after its application could be observed always, but it had no influ-ence on the yield of leaves. The plants didn’t show any growth depression after the first cut.
3.1.5 Seed production
Difficulty at the seed production consists in the very strong connection of the seeds within the husks. At the harvest of woad the seeds are not separated by the reap-threshing. Be-cause of the good flying characteristics of the siliquated seeds the reap-threshing of woad
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is something difficult, but quite possible with each usual reap-thresher. The results, got from experimental plots with a small reap-thresher with two woad strains, are shown in table 5. Table 5:Table 5:Table 5:Table 5: Seed yields of two strains on experimental plots in 2003 (13.5 m², 8 replications) Strain-No. Conditions Seed yield (kg/plot) Unhulled,
uncleaned Hulled,
pure seeds Share of
pure seeds 1 2 sieves, the under sieve for
cereals 2.20 0.57 25.9 %
2 sieves, the under sieve for soybeans
3.50 1.06 30.3 %
1 sieve (the usual over sieve) 3.70 0.99 26.8 % ∅∅∅∅ 7.76 kg/108 m² = 7.19 7.76 kg/108 m² = 7.19 7.76 kg/108 m² = 7.19 7.76 kg/108 m² = 7.19
dt/hadt/hadt/hadt/ha
2 1 sieve (the usual over sieve) 2.88 0.77 26.7 % Harvesting by hand 4.26 1.04 24.4 %
It can be seen that threshing with sieves with big holes gives good results, but with a sieve like used at cereals the loss is high and the harvested material is not cleaner, neither. The sieves should be adjusted so steep as possible. Threshing of woad without a sieve is not possible because of the high share of stalks and leaves, which then go into the harvested good. The speed of the reap-thresher must be low and also the wind. The loss of seeds at harvesting is then about 25 % and lies in order of other oil crops, for example rape.
Woad must be threshed namely, when the husks are blue-black and the leaves are still green. Waiting for a full ripeness of the plants causes a very strong decrease of seed yield because of the high seed shattering of the woad plant. The harvested material has a low dry substance content of only 65 %.
The harvested good must be dried in a drying room immediately after the threshing. The dried material consists of about 80 % siliquated woad seeds, the rest are stems and leaves. The share of pure seeds in the siliquated material is about 25 % of the weight. Their win-ning is possible by a clover huller. After they are freed from the valvaes, their further clean-ing takes place by wind sifting.
The relative high expenditures are needed to get a material which can be sown without complications. The use of siliquated seeds for sowing causes a quick to stopping up of the drill.
From plots in the practice (5 ha) were gotten 300 kg pure seeds per hectare, from the small experimental plots even much more (719 kg per hectare, see tab. 5).
For the seed production of woad the production plots can be used in the second year on the one hand. On the other hand it is possible to sow the woad at the end of August like winter rape. In August sown woad flowers surely in the next year. The flowering of the au-tumn sown plots is much uniformer than in the production plots in the second year of cul-tivation. In the last case a considerable number of plants is still green whereas another part is overripe. Also not bolted plants could be determined. All this is not the case at the Au-gust sown woad.
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In all there are no problems at the production of seeds at woad. The multiplication factor is
high (1 : ≥ 60), that means, it is possible to sow 60 ha woad with the low harvest of 300 kg from 1 ha and at a sowing strength of 5 kg pure seeds per hectare.
Similar should be the seed production at Isatis indigotica. The only difference to Isatis tinc-toria is the low cold requirement of Isatis indigotica and therefore the cultivation of this crop in the field for dye winning is very difficult.
In 2003, two accessions of Isatis indigotica were sown in Mitcherlich pots (5 seeds/pot) in early spring. All plants began to flower and a great amount of seeds could be harvested. Because of the cold weather in May all plants of the Isatis indigotica strain in the field trials with woad became bolting and flowered.
3.1.6 Maintaining of the different strains of woad
Seed stocks from the in WP 3 enumberated woad accessions were got during more than 10 years by isolation of about 100 plants of the production plots in the 2nd year shortly before flowering. Always two stalks of two different plants were isolated under one bag. Only plants which correspond with the strain character were isolated. The non isolated plants of the plot were cut. After ripeness the isolated stalks were harvested, threshed with a clover huller and cleaned by an exhauster.
In 2003 a great extent of plants was killed by frost in the last December days and again at the beginning of March, but the reaction of the single strains was very different (tab. 6). Table 6:Table 6:Table 6:Table 6: Assessment of the single woad strains after the winter 2002/2003 (3 replications, 13.5 m² per
plot) Strain-No. Accession Number of plants before winter* Assessment after winter
(notes 1 – 9)** Per plot Per square meter
Standard Thüringer Waid 171 13 2.3 2 Bordeaux 684 51 3.0 3 Montreal 507 38 4.0 4 Lausanne 621 46 4.7 5 Bordeaux 573 38 3.0 6 Chateau de Magrin 489 36 3.0 7 Frankfurt a. M. 567 42 3.7 8 Heidelberg 360 27 5.0 9 Kiel 621 46 5.7 10 Jena 498 37 4.0 11 Bristol 174 13 1.0 12 Isatis indigotica 285 21 2.3 13 Pisa 408 30 2.0
* after counting out of 1 m² of each plot ** assessment notes: 1 <= 3 plants/plot, 9 full stock of plants
It can be seen that the accession “Bristol “ was the most susceptible strain against frost, but also the local accession “Thüringer Waid” showed a high damage by the cold. Only at the accessions “Frankfurt” and “Kiel” a high winter hardiness could be observed. They gave the foreseen number of plants for isolation.
For the multiplication in a greater extent (to produce more kilograms) the harvested seeds of the single strains were drilled on plots from 40 – 100 m² at a distance of more than 300
13
m from each other in the field at the end of August like winter rape. In each year of the pro-ject 2 to 4 strains were multiplicated this way, in 2002/2003 two strains, 2003/2004 four strains. No damage by the frost could be observed on this plots.
3.23.23.23.2 Examination of woad to produce indigoExamination of woad to produce indigoExamination of woad to produce indigoExamination of woad to produce indigo
3.2.1 Generally
In former attempts, carried out with four woad strains over two years, the indigo yield cor-responded highly with the dry matter yield of the strains, as can be seen in figure 1.
Indigo yield (kg/ha)
302520151050
60
50
40
30
20
10 R-Qu. = 0,8781
Figure 1:Figure 1:Figure 1:Figure 1: Correlation between the dry mass yield and the indigo yield of woad, Dornburg 1995 and 1996
Each of the four strains were cultivated with two sowing quantities (3 resp. 5 kg pure seeds/ha) and two row distances (13.5 resp. 30 cm). Therefore, it seems to be more suc-cessful to develop strains with a high and possibly constant yielding ability in regard to high biomass. We hoped to get a high indigo yield at the same time this way, so that the same strains can be used for juice winning and also if necessary for the production of in-digo. To identify high and constant yielders, 12 resp. 13 strains (accessions) were cultivated over three years (2001 to 2003). Already in the first year there was the yield difference up to more than 100 % between the single strains (tab. 7).
14
Table 7:Table 7:Table 7:Table 7: Yields of different woad strains, Dornburg 2001 (field trial, 13.5 m² plot size, 3 replications) Strain origin Yield (dt dry mass/ha) Range
1st cut 2nd cut 3rd cut ���� 1 (Thüringer Waid) Thuringia 11.1 8.3 7.7 27.127.127.127.1 10
2 3 4 5 6 7 8 9 10 11
Bordeaux Montreal Lausanne Bordeaux 2 Château de Magrin Frankfurt a. Main Heidelberg Kiel Jena Bristol
14.3 11.8 9.2 13.1 14.5 16.6 11.7 13.4 14.2 14.8
14.8 10.8 9.8
12.4 11.4 13.6 9.4 11.3 12.6 13.7
12.1 11.3 10.1 11.6 10.5 10.9 8.0
10.7 11.1
10.0
41.241.241.241.2 34.034.034.034.0 20.220.220.220.2 37.137.137.137.1 36.336.336.336.3 41.041.041.041.0 29.129.129.129.1 35.535.535.535.5 37.937.937.937.9 38.538.538.538.5
1 8
11 5 6 2 9 7 4 2
12 (I. indigotica) Bristol 6.2 6.4 6.8 19.519.519.519.5 12 GD t, 5 % 3.6 3.0 2.7 8.48.48.48.4
Unfortunately, the values of the determination of the indigo content in 2001 were not uti-lizable, so that nothing can be said of the indigo yields of the strains this year.
3.2.2 Biomass yield, indigo content and indigo yield per area of the different woad strains 2002 and 2003
The biomass yield, the indigo content and the calculated indigo yield per hectare of the single strains were very different in the two years. So the biomass yields of the 13 examined woad strains in 2003 were in the mean only 61.3 % of the yields of 2002 with variation from 40 % to 78 % between the single strains. The hierarchy in regard to the biomass yields is still the same as in 2003 and corresponds also far-reaching with the year 2001 (tab. 8). Table 8Table 8Table 8Table 8 Dry mass yield (dt/ha) of woad strains in comparison to the standard ‚Thüringer Waid‘
Dornburg 2002 and 2003 Strain Accession 2002 2003 % % % %
1st cut
2nd cut
3rd cut
�������� RangeRangeRangeRange 1st cut
2nd cut
3rd cut
�������� RangeRangeRangeRange to 2002to 2002to 2002to 2002
Stan-dard
Thüringer Waid 14.6 14.5 14.4 43.543.543.543.5 8888 7.3 12.0 7.9 27.227.227.227.2 9999 50.850.850.850.8
2 Bordeaux 13.4 21.1 20.2 54.754.754.754.7 3333 11.2 16.2 9.3 36.736.736.736.7 2222 67.167.167.167.1 3 Montreal 18.3 17.6 15.6 51.551.551.551.5 5555 10.7 13.4 9.1 33.233.233.233.2 4444 64.564.564.564.5 4 Lausanne 5.6 18.3 16.9 40.840.840.840.8 10101010 6.7 10.4 6.7 23232323.8.8.8.8 10101010 58.358.358.358.3 5 Bordeaux 16.2 22.4 20.5 59.159.159.159.1 2222 9.0 15.1 8.9 33.033.033.033.0 5555 55.855.855.855.8 6 Chateau de
Magrin 11.3 17.4 15.3 44.044.044.044.0 7777 9.4 13.6 8.0 31.031.031.031.0 8888 70.570.570.570.5
7 Frankfurt a. M. 12.5 15.3 17.6 45.445.445.445.4 6666 9.2 16.3 9.7 35.235.235.235.2 3333 77.877.877.877.8 8 Heidelberg 7.0 18.0 17.7 42.742.742.742.7 9999 8.7 16.0 7.4 32.132.132.132.1 6666 75.275.275.275.2 9 Kiel 18.4 22.0 22.3 62.762.762.762.7 1111 6.5 15.8 9.2 31.531.531.531.5 7777 50.250.250.250.2 10 Jena 14.6 18.6 18.6 51.851.851.851.8 4444 9.8 17.0 11.6 38.438.438.438.4 1111 74.174.174.174.1 11 Bristol 14.5 12.5 7.8 34.834.834.834.8 12121212 4.0 9.8 5.2 19.019.019.019.0 11111111 54.654.654.654.6 12 Isatis indigotica 14.1 11.4 11.0 36.536.536.536.5 11111111 5.8 7.4 4.4 17.617.617.617.6 12121212 48.248.248.248.2
GD t. 5% 4.4 3.7 4.1 8.98.98.98.9 3.0 3.1 2.1 7.37.37.37.3 Mean 47.6 = 47.6 = 47.6 = 47.6 =
100 %100 %100 %100 % 29.229.229.229.2 61.361.361.361.3
The values for the calculated indigo yields in 2003 are still lower than the biomass yields. They reach in the mean only 36.3 % of the mean in 2002 (tab. 9).
15
Table 9:Table 9:Table 9:Table 9: Indigo yield (kg/ha) of woad strains in comparison to the standard ‚Thüringer Waid‘ Dornburg 2002 and 2003 Strain. Accession 2002 2003 % to% to% to% to
1st cut
2nd cut
3rd cut
���� RangeRangeRangeRange 1st cut
2nd cut
3rd cut
���� RangeRangeRangeRange 2002200220022002
Stan-dard
Thüringer Waid 25.4 3.1 15.1 43.643.643.643.6 5555 1.4 18.7 2.4 22.522.522.522.5 1111 51.651.651.651.6
2 Bordeaux 8.9 5.2 7.4 21.521.521.521.5 12121212 1.0 6.8 2.2 10.010.010.010.0 9999 46.546.546.546.5 3 Montreal 23.7 8.4 17.6 49.749.749.749.7 4444 3.8 8.3 1.8 13.913.913.913.9 7777 28.028.028.028.0 4 Lausanne 7.9 2.3 2.8 13.013.013.013.0 13131313 0.6 7.0 2.2 9.89.89.89.8 10101010 75.475.475.475.4 5 Bordeaux 17.8 5.9 12.9 36.636.636.636.6 8888 2.0 7.4 3.4 12.812.812.812.8 8888 35.035.035.035.0 6 Chateau de
Magrin 15.1 12.4 7.4 34.934.934.934.9 9999 1.6 4.9 3.0 9.59.59.59.5 11111111 27.227.227.227.2
7 Frankfurt a. M. 27.7 5.2 4.4 37.337.337.337.3 6666 4.7 6.2 4.4 15.315.315.315.3 5555 41.041.041.041.0 8 Heidelberg 4.0 12.1 6.8 22.922.922.922.9 11111111 0.7 2.9 3.6 7.27.27.27.2 13131313 31.431.431.431.4 9 Kiel 16.2 29.8 17.7 63.763.763.763.7 2222 3.8 13.7 3.9 21.421.421.421.4 2222 34.034.034.034.0
10 Jena 26.4 25.7 12.7 64.864.864.864.8 1111 4.2 6.0 4.1 14.314.314.314.3 6666 22.122.122.122.1 11 Bristol 16.8 7.6 5.3 29.729.729.729.7 10101010 1.3 4.3 3.4 9.09.09.09.0 12121212 30.330.330.330.3 12 Isatis indi-
gotica 26.1 12.4 14.6 53.153.153.153.1 3333 3.0 8.7 8.7 20.420.420.420.4 3333 38.438.438.438.4
13 Pisa 21.4 7.1 8.4 36.936.936.936.9 7777 0.8 16.3 0.9 18.018.018.018.0 4444 48.848.848.848.8 GD t. 5% 10.7 9.4 9.3 18.718.718.718.7 1.8 6.6 2.1 6.86.86.86.8 Mean 39.1 = 39.1 = 39.1 = 39.1 =
100 %100 %100 %100 % 14.214.214.214.2 36.336.336.336.3
Obviously, the low values of 2003 in comparison to those of 2002 may be the result of the weather conditions during the vegetation period in Dornburg. They were too warm and too dry compared with the long standing mean (fig. 2).
April May June July August September0
20
40
60
80
100
0
5
10
15
20
25Rainfall, long standing mean (y1)Rainfall 2003 (y1)Temperture, long standing mean (y2)Temperature 2003 (y2)
Figure 2Figure 2Figure 2Figure 2: Rainfall and temperature in 2003 in comparison with the long standing mean in Dornburg
The climatic water balance became negative from the middle of June up to the end of September (fig. 3).
16
March April May June July August September-150
-100
-50
0
50
100
150
200
Figure 3:Figure 3:Figure 3:Figure 3: Climatic water balance in 2003 in Dornburg
So, the missing water seems to be the most important factor, which restricted the forma-tion of the biomass and the indigo yield. On the contrary, 2002 was a very useful year. This can to be seen not only at the good values of the biomass, but also at the height of the in-digo content in the woad leaves (tab. 9). Table 9:Table 9:Table 9:Table 9: Indigo content (% i. dry mass) of woad strains in comparison with the “Thüringer Waid” Dornburg 2002 and 2003
Strain. 2002 2003 Accession 1st cut 2nd cut 3rd cut 1st cut 2nd cut 3rd cut
Standard Thüringer Waid 1.68 0.21 1.07 0.17 1.20 0.29 2 Bordeaux 0.66 0.24 0.36 0.08 0.46 0.24 3 Montreal 1.26 0.49 1.12 0.34 0.56 0.20 4 Lausanne 1.37 0.13 0.16 0.09 0.48 0.33 5 Bordeaux 1.14 0.23 0.62 0.22 0.52 0.38 6 Chateau de Magrin 1.30 0.72 0.49 0.16 0.35 0.37 7 Frankfurt a. M. 2.23 0.34 0.25 0.51 0.40 0.45 8 Heidelberg 0.59 0.67 0.38 0.08 0.21 0.50 9 Kiel 0.87 1.36 0.76 0.53 0.92 0.42 10 Jena 1.82 1.39 0.69 0.46 0.41 0.36 11 Bristol 1.14 0.60 0.68 0.28 0.25 0.67 12 Isatis indigotica 1.80 1.11 1.34 0.44 0.55 1.98 13 Pisa 1.45 0.41 0.45 0.32 1.15 0.14
GD t. 5% 0.64 0.52 0.52 0.20 0.42 0.48
With maximal 2.23 % in the dry mass at the 1st cut of the accessions Frankfurt could be determined a value, which was reached never before (s. later). That the determined high indigo contents was exact, has been shown at the attempts of the indigo production with the machinery of partner 6. From 120 kg of fresh woad leaves could be got 150 g raw indigo with a purity of 9 % (analysed by TLL) resp. 10 % (analysed by Critical Processes). The dry substance of the fresh leaves was 13 %, so that the 120 kg fresh mass are equivalent to 15.6 kg dry mass. The indigo content of the waod leaves was then 0.87 % resp. 0.96 %, pro-
17
vided all indigo precursors were extracted and changed into indigo. This corresponds very well with the values determined at the leaves.
3.2.3 Causes for the different indigo yields
3.2.3.1 Genetical causes
The high biomass yields, connected with high indigo yields in 2002 on the one hand and the low biomass yields and the low indigo yields in 2003 at the same time on the other hand imply that there is a narrow relationship between the two characters and therefore the breeding on high biomass yield may be successful. But the calculation of the correlation for the single strains shows that there isn’t such a relationship (Fig. 4).
Dry mass yield (dt/ha)
7060504030
70
60
50
40
30
20
10 R-Qu. = 0,1334
2002
Dry mass yield (dt/ha)
40302010
24
22
20
18
16
14
12
10
8
6 R-Qu. = 0,0424
2003
I. Indigotica
Jena Kiel
Montreal
Thüringer
Thüringer
KielI. Indigotica
Pisa
Figure 4:Figure 4:Figure 4:Figure 4: Relationship between the biomass and the indigo yield in the years 2002 and 2003
18
In 2002 only two of the six strains with the highest biomass yields (> 50 dt dry mass/ha) gave also extraordinary high indigo yields/ha (calculated from the dye content and the biomass yield). The other four showed only low or middle dye yields. In 2003 again no rela-tionship between the heights of the indigo yield and the biomass yield of the single strains is existing. From the high biomass yielders (> 30 dt dry mass/ha) four showed also indigo yields over the mean.
There is every indication that the formation (and stability and accumulation) of the indigo precursors in the single strains is genetically determined. So in all years “Thüringer Waid”, a land race (population) of woad from Thuringia, gave only low biomass yields with an ac-ceptable dye content, whereas the best strain from France always has shown brilliant bio-mass yields with always low dye contents. The dye yield of Chinese woad is also high de-spite of the low yield of biomass.
From Isatis indigotica is known that its precursor content is somewhat higher than that of Isatis tinctoria (HILL, 1992). Because of its high tendency to bolting, Isatis indigotica is not suitable for cultivation under Central European conditions. Sowing of Chinese woad a bit later than Isatis tinctoria can not overcome this drawback. Only a few days of cold weather are enough to satisfy its vernalization requirement. Isatis indigotica may be a useful cross-ing partner for the breeding of better woad strains. Isatis indigotica and Isatis tinctoria can be crossed with each other without difficulties.
3.2.3.2 Influence of meteorological conditions on the biomass yield, the dye content and the dye yield of woad
To find out, which meteorological data may be of influence on the enumerated characters, the dry mass yield and the indigo yield of three woad strains are represented graphically (fig. 5). They show a wide variation from year to year.
Thür. Waid Jena France0
5
10
15
20
25
30
35
40
45
50
55
60
653d cut2nd cut1th cut
Dry mass yield
1995
1995
1995
1996
1996
1996
2001
20012001
2003
20022002
2002
20032003
Thür. Waid Jena France0
5
10
15
20
25
30
35
40
45
50
55
60
65
703d cut2nd cut1th cut
Indigo yield
199519951995
1996
1996
1996
2002
2002
20022003
2003
2003
Figure 5:Figure 5:Figure 5:Figure 5: Dry mass yield and indigo yield of three strains of woad in the years 1995, 1996, 2001, 2002 and
2003
19
The belonging to meteorological values as rainfall, temperature and radiation is listed in table 10. Table 10:Table 10:Table 10:Table 10: Comparison of rainfall, temperatures and radiation during the vegetation periods in 1995, 1996, 2001, 2002
and 2003 Month Rainfall (mm) ∅ Temperature (°C) Radiation (kW/m²)
Decade 1995 1996 2001 2002 2003 1995 1996 2001 2002 2003 1995 1996 2001 2002 2003
May 1 2 3
4.6 26.7 53.4
16.3 55.7 22.2
30.0 14.9 20.1
35.1 17.1 10.1
12.622.65.9
11.9 8.2 14.2
7.8 10.713.5
13.0 14.2 14.9
14.418.3 18.2
15.0 11.8 16.1
127.2
101.6
31.6 35.2 39.1
31.2 45.844.3
43.3 42.251.6
June 1 2 3
58.5 14.1 13.0
3.2 2.4 12.0
25.0 23.6 18.2
35.7 44.4 2.2
14.926.96.0
11.2 13.4 14.5
17.6 14.912.7
11.5 13.8 16.8
19.1 22.3 21.6
20.818.2 18.1
208.7
137.4
35.2 44.3 51.8
44.041.655.1
59.5 55.3 58.1
July 1 2 3
24.0 15.6 46.7
73.4 9.5
49.6
45.4 43.9 74.5
4.5 51.5 1.4
16.016.045.6
19.220.419.6
14.1 14.8 16.8
18.2 16.220.6
21.1 20.121.4
16.3 20.320.2
155.2
127.4
48.4 40.5 61.8
41.426.947.8
38.2 57.947.1
August 1 2 3
39.3 29.4 24.6
7.6 32.9 32.9
14.2 8.0 7.9
33.2 37.7 72.3
0.0 5.4
20.4
19.419.215.6
17.4 17.5 17.6
18.020.418.4
20.721.1 22.2
23.7 20.916.8
126.7
123.0
36.1 39.5 40.6
34.5 35.3 41.5
56.046.639.7
Septem-ber
1 2 3
19.1 15.9 12.8
30.0 9.2 17.4
30.1 19.6 13.1
15.4 9.4 25.8
12.7 44.717.2
13.5 13.7 10.5
12.69.9 10.2
13.0 11.1 11.6
17.8 14.911.4
13.4 14.1 12.5
?
?
20.2 19.5 18.2
35.9 28.821.9
28.5 28.227.8
October 1 14.0 7.0 23.4 - - 9.9 14.1 14.0 10101010.9.9.9.9 - ? ? 20.2 19.6 - Up to 1st cut
���� 209.9209.9209.9209.9
���� 200.7200.7200.7200.7
���� 221.1221.1221.1221.1
���� 142,4142,4142,4142,4
���� 47.847.847.847.8
���� 14.1 14.1 14.1 14.1 ���� 13.3 13.3 13.3 13.3 ���� 14.7 14.7 14.7 14.7 ���� 18.5 18.5 18.5 18.5 ���� 16.7 16.7 16.7 16.7 ���� 326.1326.1326.1326.1
���� 300.0300.0300.0300.0
���� 310.0310.0310.0310.0
From 1st to 2nd cut
���� 115.4115.4115.4115.4
���� 123.0123.0123.0123.0
���� 34.634.634.634.6
���� 59.659.659.659.6
���� 77.677.677.677.6
���� 19.4 19.4 19.4 19.4 ���� 17.2 17.2 17.2 17.2 ���� 19.4 19.4 19.4 19.4 ���� 21.0 21.0 21.0 21.0 ���� 19.0 19.0 19.0 19.0 ���� 137.4137.4137.4137.4
���� 171.2171.2171.2171.2
���� 143.2143.2143.2143.2
From 2nd to 3rd cut
���� 86.486.486.486.4
���� 63.663.663.663.6
���� 94.194.194.194.1
���� 168,0168,0168,0168,0
���� 38.538.538.538.5
���� 12.6 12.6 12.6 12.6 ���� 12.9 12.9 12.9 12.9 ���� 12.4 12.4 12.4 12.4 ���� 19.3 19.3 19.3 19.3 ���� 18.6 18.6 18.6 18.6 ���� 118.7118.7118.7118.7
���� 147.3147.3147.3147.3
���� 170.7170.7170.7170.7
From emerging to 3rd cut
411.7411.7411.7411.7 387.3387.3387.3387.3 349.8349.8349.8349.8 370.0370.0370.0370.0 266.9266.9266.9266.9
Only in 2002 was reached a high yield of leaves with a very high precursor content. This seems to be a result of the high temperatures, esp. in the first vegetation period, and suffi-cient (not optimal) precipitation. The temperature seems to play an important role not only at the forming of the biomass at woad, but also at the formation of precursor content. Woad as a plant of Mediterranean origin needs above all warmth for its development. This can be seen by the comparison of the values of the crop and the meteorological data of the years 2002 and 1996. Despite of a good distribution of the precipitations and nearly the same height of the global radiation as in 2002, 1996 was up to now the worst year because of the low temperatures during the full vegetation period.
If the dye content of 2002 and 2003 is compared, only the values of the first cut of the year 2002 are appreciably higher than in 2003. This may be the result of an about 2.5 °C higher temperature in the mean of the first vegetation period.
A wide influence on the height of dye precursors is ascribed by the partners from Pisa and Bristol. It is the intensity of lightening (total global radiation – PAR) of the crop. High radia-tion should increase this character. This is not always the case, how results of 2003 have shown. Besides of an approximately by 25 % higher total global radiation during the main vegetation period (tab. 3) the mean of dye content in the woad leaves is at all three cuts of the harvest 2003 lower than that of 2002.
To study the influence of the radiation on the precursor content in 2001 and 2003 attempts were carried out with different row distances. Starting from the assumption, that the expo-
20
sure of the woad leaves to the sunlight is better at wider row distance than at nearer row distance there was expected a higher precursor content in the first case.
In a preattempt three woad strains were drilled with two different row distances (13.5 cm resp. 30 cm). The results has been confirming the assumption. At the nearer row distance (13.5 cm) the values of indigo content were always lower than at the greater row distance (tab. 11). Table 11:Table 11:Table 11:Table 11: Indigo content (g/kg dry matter) in the mean of two replications of 3 woad strains in a field
experiment with a different row distance Strain 1st cut 2nd cut 3rd cut
30 cm 15 cm 30 cm 15 cm 30 cm 15 cm 1 4.2 4.1 5.0 3.6 4.7 4.1 2 4.4 4.3 4.4 3.2 4.7 5.0 3 3.4 2.2 2.9 2.0 4.1 2.6
In 2003 10 strains (3 replications, 13.5 m² plot size) were drilled once at an interrow dis-tance of 13.5 cm (like cereals) and on the other hand at a row distance of 30 cm. The results can be seen in figures 6, 7 and 8.
Row distance (cm)13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30
0
5
10
15
20
25
30
35
40
3rd cut 2nd cut 1st cut
Dry mass yield (dt/ha)St. 1 St. 2 St. 3 St. 4 St. 5 St. 6 St. 7 St. 8 St. 9 St. 10
Figure 6:Figure 6:Figure 6:Figure 6: Influence of the row distance on the dry mass yield of woad strains, Dornburg 2003
21
Row distance (cm)13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30
0
0,2
0,4
0,6
0,8
1
1,2
1,41st cut 2nd cut 3rd cut
Indigo content (% of dry mass)
St. 1 St. 2 St. 3 St. 4 St. 5 St. 6 St. 7 St. 8 St. 9 St. 10
Figure 7:Figure 7:Figure 7:Figure 7: Influence of the row distance on the indigo content of woad strains, Dornburg 2003
Row distance (cm)13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30 13,5 30
0
5
10
15
20
25
3rd cut 2nd cut 1st cut
Indigo yield (kg/ha)
St. 1 St. 2 St. 3 St. 4 St. 5 St. 6 St. 7 St. 8 St. 9 St. 10
Figure 8:Figure 8:Figure 8:Figure 8: Influence of the row distance on the indigo yield of woad strains, Dornburg 2003
As in many attempts before the biomass yield is a bit higher at the nearer row distance, especially at the types with a postrate growth habit by a completer harvest. Contrary to the assumption the dye content and dye yield at the wider row distance is lower than at near row distance.
An interpretation of the causes of these results is difficult. The evaporation and the transpi-ration of the leaves may be higher at the wider row distance and by this the minimum fac-
22
tor “water” got still a greater negative influence on the formation of dye precursors in the plant. But the results show that light is in no account the only factor, which determines the height of dye precursor content in the leaves. In the year 2003 the low water content should have been the restricting factor. Woad gives only high dye yields, if the temperatures and the radiation are high and the water status of the soil is optimal. One of these factors is in the minimum in the most years in the Central European region. Obviously we must expect a crop failure in every 4th year.
Maximal 22.5 kg (theoretical) indigo per hectare is too low for an economical indigo pro-duction. The cultivation of Isatis ssp. should be much better in Spain or Italy because of the warmer and more uniform climate.
3.33.33.33.3 Development of a modern cultivation scheme of Polygonum tinctoriumDevelopment of a modern cultivation scheme of Polygonum tinctoriumDevelopment of a modern cultivation scheme of Polygonum tinctoriumDevelopment of a modern cultivation scheme of Polygonum tinctorium
3.3.1 Generally
Polygonum tinctorium was the classical plant to deliver indigo. In Japan it has been culti-vated in a small extent untill nowadays for the production of blue dye for dyeing of tradi-tional clothes (MÜLLEROTT, 1992). In Europe this plant is widely unknown and therefore, no cultivation scheme exists for it on which could be gone back. A first cultivation of some Polygonum plants in the field has shown that it grows constantly from its germination in the middle of May up to the autumn. At first temperatures nearly 0 °C in Septem-ber/October the plant dies immediately. The dye of the leaves then changes from green to blue. The only dye precursor of Polygonum is indican = indoxyle-ß-D-glucoside. After early statements in the literature the indigo content reaches up to 5 % in the dry matter (v. WIESE, 1928). This could be confirmed by a reexamination of the Polygonum plants in Dornburg.
First trials showed that the drilling of 5 kg seeds per hectare brought the same yield as the planted stands.
In the frame of the project a modern cultivation system was developed, which enables the farmers to cultivate Polygonum without risk. For the determination of the optimal sowing time, the crop was sown from the beginning to the end of April. The harvest of the plants also took place at different data with different cutting frequency (2 or 3 times). Special at-tention was given the N-fertilization of the plant. In preattempts in Mitscherlich-pots has been shown that the height of the indigo precursor content depends in a great degree from this factor. Finally the possibilities of an effective weed control, the production of seeds and the extraction of indigo precursors out of the leaves and the production of indigo were investigated in many attempts.
3.3.2 Sowing time
The attempts to determine the optimal sowing time were carried out over the years 2001 to 2003 with two accessions on 13.5 m² plots in 4 replications. The planned sowing times were April the 10th, 20th and 30th. In 2001, the planned earliest variant was impossible be-cause of the very wet soil, so that only two sowing times were possible in this year. Table 12 shows the results.
23
Table 12:Table 12:Table 12:Table 12: Influence of the sowing time on dry mass yield, indican content and indigo yield of two acces-sions of Polygonum tinctorium, Dornburg 2001 to 2003
Sow-ing time
Acces-sion Cut
Yield of leaves (dt dry mass/ha) Leave:stalk-relation
Indican content (% dry mass)
Indigo yield (kg/ha)
2001 2002 2003 2001 2002 2003 2001 2002 2003 2001 2002 2003
10.04. Dorn-burg
1 2 -
14.7 24.0
9.9 19.4 -
1 : 0.901 : 1.12
1 : 0.791 : 0.84 -
6.696.23
5.60 8.23 -
49.175.1
28.379.8
���� 38.738.738.738.7 29.329.329.329.3 124.2124.2124.2124.2 108.1108.1108.1108.1
Bristol 1 2 -
15.9 26.8
12.320.9 -
1 : 1.221 : 1.21
1 : 0.861 : 078 -
6.344.81
5.20 9.05 -
50.864.2
31.794.9
���� 42.742.742.742.7 33.233.233.233.2 115.0115.0115.0115.0 126.6126.6126.6126.6
20.04. Dorn-burg
1 2
9.1 14.1
9.8 18.6
16.615.4
1 : 0.971: 0.63
1 : 0.981 : 0.99
1 : 1.031 : 0.62
2.533.88
6.895.81
6.84 9.91
12.1 27.8
33.353.0
56.776.3
���� 23.123.123.123.1 28.428.428.428.4 32.032.032.032.0 39.839.839.839.8 86.386.386.386.3 133.0133.0133.0133.0
Bristol 1 2
9.3 10.4
10.1 17.0
21.016.4
1 : 1.331 :
0.66 1 : 0.961 : 0.95
1 : 1.041 : 0.57
2.193.36
6.783.83
6.44 9.26
9.4 18.4
34.031.8
67.475.9
���� 19.719.719.719.7 27.127.127.127.1 37.437.437.437.4 27.927.927.927.9 65.865.865.865.8 143.3143.3143.3143.3
30.04. Dorn-burg
1 2
10.9 11.2
14.5 23.1
15.612.4
1 : 0.811 : 0.74
1 : 1.081 : 0.86
1 : 1.371 : 0.50
2.174.52
6.413.93
3.63 4.49
11.8 25.5
46.245.3
28.027.7
���� 22.122.122.122.1 37.637.637.637.6 28.028.028.028.0 37.237.237.237.2 91.591.591.591.5 55.755.755.755.7
Bristol 1 2
17.9 16.1
18.1 24.8
18.813.9
1 : 0.971 :
0.80 1 : 1.651 : 1.17
1 : 1.381 : 0.47
2.404.20
6.753.84
2.38 5.75
21.0 34.1
62.447.4
22.240.3
���� 34.034.034.034.0 42.942.942.942.9 32.732.732.732.7 55.155.155.155.1 109.8109.8109.8109.8 62.562.562.562.5
GD t. 5% 1 2
4.4 4.8
4.0 5.3
4.2 3.1
0.860.67
0.881.15
1.72 2.19
7.0 11.3
15.417.5
18.225.5
The values of 2001 are very different from those of 2002 and 2003. While the accession Dornburg gave the same leave and calculated indigo yields at the both sowing times, the yield of the accession Bristol was twice as high at the last sowing time. This late sowing seems to be advantageous for the yield because of the high susceptibility of Polygonum plants to cold.
The yield of leaves at the three sowing times with the exception of the variant “20.04.” is not significantly different in 2002 and 2003. It was the follow of an error in the trial that in 2002 the sowing time “middle of April” gave by 35 % (accession Dornburg), resp. by 58 % (accession Bristol) lower yields than the two others. The applicated herbicide was washed into the deeper soil layers by an intensive rainfall shortly after the application. This way a great part of the germinating Polygonum seeds was killed together with the weeds. The low number of plants has been lowered the yield of leaves, too.
On principle, the results of the last two years are corresponding very well. Also the yields of the calculated indigo amounts have the same tendency in the two years. They are always in spite of same leave yields lower at the last sowing time than at the first:
2002: 35 % (accession Dornburg), resp. 5 % (accession Bristol)
2003: about 50 % for both accessions.
This is the follow of a lower dye precursor content in the Polygonum leaves of the 2nd cut of the last sowing time. Because of the longer time between sowing and harvest, the devel-opment of the roots may have been better and so the uptake of N was more intensive after the 1st cut.
24
The second sowing time (middle of April) gave the highest dye yields in 2003, but the dif-ferences to the first sowing time are not significant.
The conclusion of the results is, that the sowing should be done as early as possible in the spring under Central European conditions, that means, when the soil is dry enough for drilling and warm enough for a quick germination of the crop. Beside Polygonum has a hard semen shell and therefore a long duration of germination (> 3 weeks). So the emer-gence of crop takes place usually only in May. In most years May has no days below 0 °C in Central Europe. Sowing up to the middle of May is possible as could be shown in 2002, but then the dye yield is always lower than at earlier sowing dates.
3.3.3 Harvest time and cutting frequency for Polygonum tinctorium
Additionally to the sowing time the harvest time plays a great role with regard to the dye content in the harvested plant material and to the yield of biomatter per area. The time for the first cut is reached when the rows are closed by the Polygonum plants. The formation of new leaves is then far-reaching concluded and the further increase of biomass concerns in the first line the stalks, so that the relation leaves:stalks becomes higher (tab. 13).
Table 13:Table 13:Table 13:Table 13: Influence of the harvest time and cutting frequency on the yield and the dye content of Polygonum tinctorium Var. Cut Harvest time Yield of leaves
(dt dry mass/ha) Leave:stalk-relation Indican content
(% of dry mass) Indigo yield d (kg/ha)
2001 2002 2003 2001 2002 2003 2001 2002 2003 2001 2002 2003 2001 2002 2003
1 1 2
08.08. 08.10.
08.07. 18.09.
22.07. 26.09.
9.3 19.3
10.4 22.6
14.8 21.1
1 : 1.091 : 0.68
1 : 0.751 : 1.01
1 : 1.051 : 0.97
2.491.78
1.17 4.94
6.54 8.94
11.5 16.9
6.3 55.6
47.994.4
�������� 28.628.628.628.6 33.033.033.033.0 35.935.935.935.9 28.428.428.428.4 61.961.961.961.9 142.3142.3142.3142.32 1
2 15.08. 11.10.
15.07. 18.09.
29.07. 26.09.
10.2 10.8
11.4 25.1
35.9 16.9
1 : 1.021 : 0.56
1 : 1.111 : 1.24
1 : 0.821 : 0.87
2.51 2.30
3.91 4.35
4.69 9.91
12.8 12.6
22.554.3
82.084.2
�������� 21.121.121.121.1 36.536.536.536.5 52.52.52.52.8888 25.425.425.425.4 76.876.876.876.8 166.2166.2166.2166.23 1
2 22.08. 16.10.
22.07. 18.09.
05.08. 26.09.
11.2 13.0
14.8 19.2
22.015.5
1 : 1.041 : 0.54
1 : 1.261 : 1.27
1 : 1.311 : 0.90
3.62 2.19
4.67 4.80
7.08 5.03
19.7 14.8
34.545.8
77.438.9
�������� 24.224.224.224.2 34.034.034.034.0 37.537.537.537.5 34.534.534.534.5 80.380.380.380.3 111116.316.316.316.34 1
2 29.08. 16.10.
29.07. 18.09.
13.08. 26.09.
12.4 10.6
17.1 17.0
27.4 10.7
1 : 1.311 : 0.38
1 : 1.221 : 1.00
1 : 1.571 : 0.57
2.75 2.01
5.34 4.00
5.82 5.48
16.9 10.8
46.136.6
79.229.6
�������� 23.023.023.023.0 34.134.134.134.1 38.138.138.138.1 27.727.727.727.7 82.782.782.782.7 108.8108.8108.8108.8GD
t. 5% 1
2 1.6
4.2 3.5 4.7
9.8 3.9
0.780.52
1.74 0.85
1.15 2.40
4.4 4.9
17.013.6
17.530.0
Because only the leaves contain the dye precursor, from this point of view the harvest should be done as early as possible. The higher share of stalks in the harvested material of later harvests only needs more space at the transport and also the extraction and higher amounts of extractant. But it seems that the formation of dye precursors reaches its climax in the full expanded leaves. Therefore, the 1st cut should be done not too early.
This date changes from year to year. Under Central European conditions it lies very rarely before the end of July, but generally at the beginning of August. For a full regrowth and a full formation of precursor content Polygonum plants need at least 6 weeks, as has already be shown in a preattempt in 2000 (tab. 14).
25
Table 14:Table 14:Table 14:Table 14: Influence of the harvest regime on yield of dry leaves, dye content and the dye yield in Polygonum tictorium, Dornburg 2000 (field trial with 13.5 m²/plot, 4 replications)
Var. Cut Harvest time Yield of leaves (dt dry mass/ha)
Indican content (% of dry mass)
Indigo yield (kg/ha)
1 1 2 3
19.07. 31.08. 04.10.
13,7 15,7 10,0
2,97 3,49 2,48
18,0 24,8 10,6
�������� 39,439,439,439,4 53,453,453,453,4 2 1
2 19.07. 04.08.
13,7 28,5
2,97 2,12
18,0 27,0
�������� 42,342,342,342,3 45,045,045,045,0 3 1
2 3
26.07. 03.09. 04.10
18,8 13,4 10,0
2,80 4,57 1,97
23,2 27,4 8,8
�������� 42,242,242,242,2 59,459,459,459,4 4 1
2 26.07 04.10.
18,8 27,2
2,80 2,52
23,2 30,2
�������� 46,046,046,046,0 53,453,453,453,4 5 1
2 02.08. 13.09.
16,1 13,5
3,14 3,46
22,0 20,2
�������� 29,529,529,529,5 42,242,242,242,2 6 1
2 02.08. 04.10.
16,1 24,8
3,14 2,72
22,0 30,2
�������� 40,940,940,940,9 52,252,252,252,2 7 1
2 09.08. 15.09.
19,5 13,1
3,23 1,44
28,0 8,4
�������� 32,632,632,632,6 36,436,436,436,4 8 1
2 09.08. 04.10.
19,5 21,6
3,23 2,77
28,0 26,7
�������� 41,141,141,141,1 54,754,754,754,7 GD t, 5% 1
2 3
2,8 6,4 2,0
0,66 1,19 0,47
5,3 9,6 4,4
Despite nearly the same yield of leaves, the calculated indigo yield is only half at the har-vest interval of 5 weeks (09.08. to 15.09.) in contrary to the harvest interval of 6 weeks (02.08. to 13.09.). Therefore, more than two cuts are not useful under Central European conditions.
In fact, in 2000 the highest yield of dye was got after threefold cutting (11.1 % more than in the comparable variant with two cuts, not significant). But if the additional costs for one cut more will be covered by the small morease of dye yield, seems to be doubtful. The last cut in 2000 was done on October, the 17th. Most years have one day with temperature be-low 0 °C already at the end of September under Central European conditions and the plants die then immediately and they are then useless for dye winning. To be on the safe side, the last harvest should be in the middle of September.
The first cut should not be done after long-lasting dryness. In 2002 this was the case and the indican content was very low. In 2003, on July the 17th, the precipitation was 15.6 mm, which evidently increased the yield of biomass and the indican content. The proposal is, not to harvest the crop at great dryness of soil, but to wait for rain or to irrigate a few days before the harvest.
26
3.3.4 Optimal N-fertilization of Polygonum
For getting Polygonum plants with a high content of indigo precursors a high N-fertilization is needed. This could be shown in a first experiment with Polygonum in Mitscherlich pots (tab. 15).
Table 15:Table 15:Table 15:Table 15: Influence of different N-fertilization on dry mass yield and indigo yield in Mitscherlich pots (4 replications/variant)
N-fertilization (g N/pot)
Dry mass yield (g/pot)
Indigo content (% in the dry mass)
Indigo yield (g/pot)
0.5 1.0 1.5
135.6 130.1 129.0
1.29 1.35 1.90
1.77 1.75 2.48
The increase of the N-gift per pot has a slight negative effect on the dry mass yield, but the dye content and also the indigo yield per pot increase. The results do not say, how high the N-gift must be to get the highest indigo yield in each year in the field. For this purpose many field trials were carried out, at which the single plots were fertilized with different amounts of N and the yield of leaves, their dye content and the calculated indigo yield of these plots were compared with the corresponding values of an unfertilized plot. As the best variant in Dornburg has been shown the fertilization of the soil with up to 160 kg/ha plant available N (N from fertilizer and mineralic N in the soil at 0 to 60 cm depth) before sowing the crop. A splitting of the N-gift (the greater amount before sowing and the rest after the 1st cut) is not useful. A reexamination of the findings in 2002 and 2003 confirmed the former results (tab. 16).
Table 16:Table 16:Table 16:Table 16: Influence of the N-fertilization on dry mass yield, indican content and indigo yield of Polygonum tinctorium, VS Dornburg 2001 to 2003
N-fertilization (kg/ha)
cut Yield of leaves (dt dry mass/ha)
Leaf:stalk-relation Indican content(% of dry mass)
Indigo yield (kg/ha)
1.1) + 2.2) gift � 2002 2003 2002 2003 2002 2003 2002 2003 % to 2002 without 0 1. 22.7 18.8 1 : 1.00 1 : 1.14 2.48 5.40 28.0 50.6
2. 17.8 14.6 1 : 0.73 1 : 0.60 3.82 5.80 34.0 42.5 ���� 40.540.540.540.5 33.433.433.433.4 62.062.062.062.0 93.193.193.193.1 150.2150.2150.2150.2
140 140 1. 22.0 18.0 1 : 0.82 1 : 1.13 2.73 6.66 30.6 60.0 2. 21.4 14.1 1 : 0.69 1: 0.53 4.25 5.31 45.5 37.3 ���� 43.443.443.443.4 32.132.132.132.1 76.176.176.176.1 97.397.397.397.3 127.9127.9127.9127.9
140 + 40 180 1. 22.5 18.7 1 : 0.88 1 : 1.09 3.16 6.42 35.7 60.3 2. 22.4 15.4 1 : 0.59 1 : 0.50 3.97 6.48 45.1 50.7 ���� 44.944.944.944.9 34.134.134.134.1 80.880.880.880.8 111.0111.0111.0111.0 137.4137.4137.4137.4
160 160 1. 26.3 17.9 1 : 0.84 1 : 1.01 2.80 6.39 36.9 57.6 2. 22.5 14.9 1 : 0.60 1 : 0.53 4.52 6.04 50.8 45.0 ���� 48.848.848.848.8 32.832.832.832.8 87.787.787.787.7 102.6102.6102.6102.6 117.0117.0117.0117.0
160 + 20 180 1. 20.0 18.1 1 : 0.89 1 : 1.01 3.13 6.21 31.9 56.2 2. 22.2 15.4 1 : 0.57 1 : 0.54 4.48 6.34 49.3 48.7 ���� 42.242.242.242.2 33.533.533.533.5 81.281.281.281.2 104.9104.9104.9104.9 129.2129.2129.2129.2 GD t. 5% 1. 3.3 2.8 0.64 0.59 9.1 10.8
2. 3.4 1.7 0.63 1.01 10.6 10.5 MeanMeanMeanMean 44.044.044.044.0
= 100 = 100 = 100 = 100 %%%%
33.2 33.2 33.2 33.2 = 75.5 %= 75.5 %= 75.5 %= 75.5 %
77.677.677.677.6 101.8101.8101.8101.8 132.3132.3132.3132.3
1) N-fertilization + N content of the soil (0 - 60 cm) 2) N-gift after the 1st cut
27
It is to be seen that the dye precursor content increases stronger than the yield of leaves. Again, a N-supply of 160 kg available N/ha has shown the best effect. Heightening the gift on 180 kg/ha gives no better indigo yields.
3.3.5 Assessment of various herbicides for use with Polygonum tinctorium
Similarly as at Isatis, the farmer needs a sure herbicide for Polygonum if he cultivates this crop in a greater extent. Polygonum is relatively fast-growing. Therefore a single application of herbicides in an early stage of the plant should be enough. In later stages the plant is able to depress germinating weeds.
The Polygonum plant is very susceptible against the most herbicides. Only Compete (Fluoroglycofen) has not damaged the crop, but this mean has a weekness against the usual weeds in Polygonum. Therefore, for an effective weed control only means are suit-able, which can be applicated before the emergence of Polygonum. To combat weeds in a later stage of development, only mechanical measures are a possibility. They are only pos-
sible at an interrow distance ≥ 30 cm. At Polygonum Afalon gave the best results of the three tested herbicides, as can be seen in table 17. All the herbicides were applicated shortly after sowing the Polygonum. The as-sessment took place on the 23.05.2002 and the 23.06.2003 respectively. Afalon has not only the best efficiency against the weeds but also the lowest phytotoxicity.
Table 17: Table 17: Table 17: Table 17: Results of the herbicide trials in Polygonum, Dornburg 2002 and 2003 Variant Applicated
amount (l or kg/ha)
Weeds/m²/Efficiency (%)
Phytotoxicity
2002 2003 2002 UC 0 n. d. 100 - Bandur (Aclonifen)
2.0 0 – 100 0 – 100 50 % of the plants with growth depression 20 % deceased plants, clear delay of emer-gence
Basta (Glufosinat-NH4)
3.0 0 – 92.5 25 - 77 20 % of the plants with growth depression 4 % deceased plants
Afalon (Linuron)
1.5 80 - 100 81 - 96 10 % of the plants with growth depression light delay of emergence
The application of Patoran FL (Metobrumuron) resp. Roundup (Glyphosat) was not carried out in this year. Both herbicides gave good results, but their application can be problem-atic. In 2002 the application of Patoran FL shortly before a strong rainfall gave heavy dam-ages of the Polygonum plants. For Roundup, the right time of application is difficult to de-termine. Too early application meets not all weeds, too late application may damage the crop.
3.3.6 Seed production
The seed production at Polygonum tinctorium is unsafe under Central European condi-tions. Polygonum is a strict short-day-plant. Therefore, its flowering does not begin earlier than in the middle of August in Thuringia, mostly even still later. Polygonum achieves the full ripeness in no year on the field. The always green plants must be cut off shortly before the first frost. After leaving them to the far-reaching drying the harvest is to thresh with a
28
reap-thresher. So, the yield got by this method was about 5 g/m² in 2002. This is a pleasing yield, but not very high. In years with wet autumn, the necessary dryness for threshing can’t be reached this way. Therefore, the multiplication of seeds should be done on locations with a short day or a long dry and warm autumn. Spain or Italy seem to be very favourable for the multiplication of Polygonum seeds.
The seed yield of Polygonum is very high under favourable conditions, as can be seen in table 18.
Table 18:Table 18:Table 18:Table 18: Production of Polygonum seeds in China and India Country Geographical coordinates
of the location Duration of vegetation Yield (pur seeds)
India 16 °N, 76 °W Begin of June – middle of September 130 kg/1000 m² = 13 dt/ha China 35 °N, 115 °W Begin of May – middle of November 95 kg/1200 m² = 7,9 dt/ha
Although there did not exist any experiences with the cultivation of Polygonum tinctorium, in both locations the seed production was very satisfactory. Especially the results of the Indian location confirms, how extremely the beginning of flowering and accordingly the duration of vegetation period of Polygonum depends on the day length. With 90 – 100 days from sowing to ripeness the vegetation period in India is only half as long as in China (about 180 days). Small amounts can also be produced very successfully in a greenhouse (tab. 19).
Table 19:Table 19:Table 19:Table 19: Seed yield of Polygonum tinctorium in Mitscherlich pots at 3 different N-levels in two years (4 pots/variant)
N-fertilization (g N/pot) Seed yield (g/pot) 1st year 2nd year
1.0 1.5 2.0
52.7 63.1 58.7
27.7 31.4 29.0
� 58.2 29.4 In fact the yield varies in the two years by more than 100 %, but the low yields in the 2nd year are still pleasing high.
Polygonum seeds quickly lose their ability to germinate. After storage of one year at room temperature their germination ability is at best only half of the original one. Therefore they must be stored at low temperatures, for example in a refrigerator or in a deep-freeze.
3.43.43.43.4 Indigo extraction from Polygonum tinctoriumIndigo extraction from Polygonum tinctoriumIndigo extraction from Polygonum tinctoriumIndigo extraction from Polygonum tinctorium
3.4.1 Generally
Polygonum tinctorium seems to be the only suitable plant for the winning of blue dye un-der Central European conditions. Its dye content in the leaves, calculated on the basis of determined dye precursor content, is three- to fivefold higher compared with woad as fig-ure 9 shows.
29
Indigo content (% dry mass)0,025 0,125 0,225 0,325 0,425 0,525 0,625 0,725 0,825 0,925
0
50
100
150
200Isatis (n = 1262)Isatis (n = 1262)Isatis (n = 1262)Isatis (n = 1262)
Indigo content (% dry mass)0,56 0,78 0,99 1,22 1,44 1,66 1,89 2,11 > 2,44
0
5
10
15
20
25Polygonum (n = 92)Polygonum (n = 92)Polygonum (n = 92)Polygonum (n = 92)
Mittel: 1,40 +/- 0,49 %
Figure 9:Figure 9:Figure 9:Figure 9: Variability of the indigo content in Isatis and Poygonum
If also in 2002 a twofold higher indigo content could be determined for some woad strains as it is shown in figure 1, in the most years the dye content lies between 0.3 and 0.4 % of the dry mass.
Indigo is an artefact of the secondary metabolism: it is not found as a native compound in the plant. In Polygonum its precursor is indican (indoxyl-ß-D-glucoside). This is a very sta-
30
ble compound in contrary to the main precursor of woad, isatan-B (indoxyl-5-ketogluconate). On the one hand, this may be advantageous because of the more safe handling, but on the other hand it is disadvantageous, as at the attempt with the machin-ery, developed from partner 6 for the production of indigo from woad leaves has shown. No indigo from Polygonum could be produced this way. Obviously, nearly the whole amount seems to have been extracted by the 70 °C hot water (the working temperature of the machine). In fact considerable amounts of indican could be detected in the extractant, but no indigo could be precipitated after alkalisation and aeration. The content of indican in the watery extract was exactly so high as at the first determination immediately after the extraction. Obviously, the pH-value was too low for a hydrolisation of indican. On the other hand we got always appreciable amounts of indigo if we extracted the Polygonum plants at surrounding temperatures of 14 to 40 °C. This corresponds with the experiences of the producers of Indigo from Indigofera spp. in the 19th century, which extracted the plants in great basins with water at the surrounding temperature (v. WIESE, 1928). Indigofera con-tains the same indigo precursor as Polygonum.
In Japan, the blue dye from Polygonum tinctorium was used (and is used) as “Sukomo”. For its production the harvested plants were dried in the sunlight, followed by a separation of the leaves from the stalks. The leaves were then fermentated at about 60 °C in a more month process. The result is a plant paste with a high indigo content, ready to be used by the dyer (MÜLLEROTT, 1994). However, this method is now unacceptable for modern dyers, because it relies on “dirty” processing, it is inefficient and produces a putrid odeur.
3.4.2 Attempts for the production of indigo from Polygonum in Dornburg
For a watery extraction only fresh harvested good is suitable. Storage of Polygonum like of woad in frozen state is impossible (tab. 20).
Table 20Table 20Table 20Table 20:::: Influence of storage conditions on the Indican content of Polygonum Probe Conditions of storage before extraction Indican content
(mg/g dry mass) 1/1 1/2 1/3 1/4
extracted immediately after harvest 1 d cooled at -20 °C 1 month at -20 °C 2 h at room temperature
17.69 0.84 0.40 10.09
2/1 2/2 2/3 2/4 2/5 2/6
extracted immediately after harvest 1 d cooled at -20 °C 0.5 h at room temperature 1 h at room temperature 2 h at room temperature 3 h at room temperature
15.54 9.67 14.48 15.24 12.22 13.21
Proceeding from the observation that at the extraction of Polygonum plants in a water bucket in the lab for 24 h in a second extraction of the same material further 20 % of indigo could be won, we have tried a continuous countercurrent extraction in a greater extent.
The installation consisted of 3 vats of 400 l which contain the extractant (water). The Poly-gonum (about 70 kg of fresh mass) was put into the first vat and left there for 24 h. After-wards it was lifted out by a crane and put into a second vat with fresh water. The first vat was loaded with new harvested material. 24 h later the procedure was repeated once more:
31
the Polygonum of the second container was taken to the third one while that of the first container was put into the second. Another 24 h later the material of the third vat was taken out, dropped carefully and than composted. The water of the first vat was brought to pH = 11 by adding NaOH, CaO, Na2CO3, NH3...., and aerated for 4 h. The formed indigo was let settle down, the water pumped up, the slurry was filtrated and then the indigo dried.
It became clear quickly that the indican in the extracted leaves was always very high (about the half) after 24 h extraction. Only after 48 h extraction, the indican content in the ex-tracted leaves was near 0. Fresh harvested good, incubated in the solution of the first ex-traction, contained only small amounts of indican (tab. 21).
Table 21Table 21Table 21Table 21:::: Indican and indigo content in Polygonum leaves after different extraction Variant Indican content
(mg/g fresh mass) Indigo content
(mg/g fresh mass) Fresh plant material 24 h in fresh water (1st filling of vat ) 5.28 0.27 Fresh plant material 24 h in the solution (2nd filling of vat) 0.07 0.50 Fresh plant material 24 h in the solution (3rd filling of vat) 0.06 0.44 Fresh plant material 48 h in fresh water (1st filling of vat) 0 0.26 Fresh plant material 48 h in the solution(2nd filling of vat) 0.11 0.41 Fresh plant material (cut) 48 h in fresh water (1st filling of vat) 0 0.10
This may be the follow of the quicker going numb of the leaves in the light acid solution. Therefore, the continuous process was changed in a discontinuous.
At the moment, in the TLL in Dornburg the following method for the production of appre-
ciable amounts of indigo (about 10 kg per year) is used:
- Incubating of Polygonum plants (about 70 kg), included in a perforated steel container, in water containing vats (300 l).
- After different times the container is lifted out by a crane and loaded with fresh Polygonum. This procedure is repeated once more.
- After threefold incubation of fresh plant material the watery solution is
brought on a pH ≥ 9.0 and then the indigo is precipitated by aeration
with air, filtered, dried and grinded.
We have produced about 1 dt raw indigo from 1998 up to now. The evaluation of 92 at-tempts has shown, that the variation of the dye yields was very high from attempt to at-tempt and altogether very low.
A yield higher than 60 % of the theory was very rare, but in some cases it was reached (fig. 10).
32
Recovery rate (%)5 15 25 35 45 55 65 > 70
0
5
10
15
20
25
30
35
Mean:26.3 +/- 19.8 %
Figure 10:Figure 10:Figure 10:Figure 10: Recovery rate of indigo (theory vs. yield), n = 92
3.4.3 Possible causes for the different recovery rates
To get safe and high recovery rates (≥ 70 %) the process of the extraction was investigated more exactly in the last years.
Causes for the variability of the indigo yield may be the threefold dipping of Polygonum into the same extraction solution and the change of indoxyl into non-indigoid compounds because of the long extraction time. But this is not the case, as can be seen in table 22.
Table 22:Table 22:Table 22:Table 22: Yield of raw indigo after 1 to 3fold incubation of fresh Polygonum plants in the same extractant Incubated amount Incubation duration Yield of raw indigo
1 x 10 kg 48 h 37 g = 1 2 x 10 kg 96 h 67 g = 1.81 3 x 10 kg 144 h 102 g = 2.76
The raw indigo amounts, got after single, two- and threefold incubation of each 10 kg fresh mass, were nearly equivalent to the expected ration of 1 : 2: 3. A spontaneous formation of indigo could not be observed. Therefore, the cause for the varieing yields must be the dif-ferent dissolution of the indican from the leaves, although no appreciable amounts of indi-can or indigo could be determined in the extracted material after 48 h.
The operation was done in a greenhouse with very fluctuating temperatures (from 14 to 40 °C) during the extraction. Especially in the nights the water temperatures decreased quickly. If the temperatures were very low during the full time, the extraction have been incomplete.
33
In order to study the influence of the temperature during the extraction and to determine the optimal extraction time at different temperatures extractions were carried out at three constant temperatures: 20, 40 and 60 °C. During the extraction time the indican content of the extraction solution was monitored by taking samples and measuring indican using HPLC.
Surprisingly the results of these investigations showed a totally different behaviour of the three temperatures (tab. 23). Table 23:Table 23:Table 23:Table 23: Indican content in the extraction solvent (mg/l) after extraction at different temperatures
Duration of extraction (h) Temperature of extraction (°C) 20 40 60
0.5 - - 14.7 1.0 - - 39.9 1.5 - - 65.4 1.75 0.0 - - 2.0 - 3.8 82.3 2.5 - - 95.7 3.0 - - 108.9 4.0 0.0 7.7 125.4 5.0 - - 136.2 6.0 0.0 15.3 143.1 7.0 - - 147.1 8.0 0.0 13.5 150.6
24.0 1.1 12.3 138.5 26.0 1.2 - - 27.0 - 1.3 - 28.0 1.3 - - 30.0 1.3 0.0 - 48.0 1.6 0.0 - 52.0 2.2 - - 72.0 3.3 - -
At 20 °C the indican content of the extracts increased very slowly up to low contents. This contents were very far from the contents at higher temperatures and also very far from theoretical values derived from the indican content in the leaf material.
At increasing temperatures (up to 40 °C) the relation between indican content in the solu-tion and time changed completely. Corresponding to the theory the values increased to a maximum followed by a decrease to nearly zero.
In case of the 60 °C extraction the indican concentration increased to a concentration in the range of the theoretical yield from the leaves. This concentration remained stable for more than 30 hours (tab. 24).
34
Table 24:Table 24:Table 24:Table 24: Indican extraction in dependence from the temperatureTrial-number Temperature (°C) Extraktion duration (h) Indican in the solvent (% of theory
1 20 72 0.2 2 20 72 0.9 12 40 8 2.7 13 40 48 53.6 14 43 6 19.2 15 48 8 13.1 5 40 6 0.9 6 40 6 56 7 40 6 0.3 8 40 6 0.3 3 40 4 0.8 9 40 6 9.2 4 40 4 0.4 10 40 6 1.4 11 40 6 0.9 16 60 3 45.1 17 60 3 21.7 25252525 60606060 8888 93939393 26262626 60606060 8888 98989898 27 57 24 49 28 60 27 62.7 18 60 3 23.6 19 60 3 25.1 24 60 6 70.6 20 60 3 24.7 21 60 3 14.9 22 60 3 20.4
Obviously the kinetics of the extraction process from Polygonum is strongly influenced by
enzyme(s). A ß-glucosidase from Polygonum leaves, which catalyses preferentially the
hydrolysis of indican, has been characterised first by MINAMI et al. (1996). Their findings
have been confirmed by MAUGARD et al. (2002) and by ANGELINI et al. (2003).
At 20 °C the dissolution seems to be very slow compared with the following enzymatic
processes. This ratio between both processes becomes more similar at 40 °C resulting in a
maximum in the concentration-time curves. Very interesting is the behaviour at 60 °C
where only a dissolution of indican out of the leaves takes place without a following hy-
drolisation forming other indoxyl compounds because the native-ß-glucosidase was com-
pletely inhibited. So it becomes clear that with the machinery for indigo production no dye
could be gotten from Polygonum, because the extraction temperature is 70 °C.
From the results follow at our opinion, that Polygonum should be extracted at 40 °C. Be-
cause of the very much shorter extraction time, an extraction at 60 °C may be better, but
indican is a very stable compound, which can be cleaved only with strong acids or bases or
enzymatically.
The application of 40 °C hot water for the extraction of Polygonum was done in Thuringia in a cooperative of land women in 2003. From an area of 300 m² Polygonum they got 4 kg raw indigo with a purity of 29 %. This yield corresponds with 46 kg pure indigo per hectare. It should be an excellent result.
35
3.4.4 Purification of the raw indigo
The won indigo is more or less contaminated by anorganic compounds. The impurities come from soil and dust particles on the plants, but also from the anorganic constituents of the plant itself or out of the extractant “water”. They could be to reduced by different simple operations (tab. 25).
Table 25:Table 25:Table 25:Table 25: Indigotin content of raw indigo after different pretreatment resp. aftertreatment Treatment Indigotin content No treatment 17 % Washing of the leaves 33 % Washing of the leaves, washing of the raw material, with acetic acid 58 % No pretreatment, washing of the raw material, with acetic acid 27 %
Obviously a great amount of impurities comes from the water, which is very hard in Dorn-burg. A possibility to hinder that is the use of softened water, but this operation makes the product more expensive.
Without a pre-treatment the indigotin content is always low (fig. 11).
Indigo (%)2,5 7,5 12,5 17,5 22,5 27,5 32,5
0
5
10
15
20
25
30
35
Mean: 14.9 +/- 7.3 %
Figure 11:Figure 11:Figure 11:Figure 11: Indigo content in the raw indigo (n = 92)
Washing the plants with water before the extraction should be enough for purposes of tex-tile dyeing. A further cleaning is necessary, if the indigo shall be applicated for special pur-poses, f. e. as print dye.
If the raw material has a high indican content, a high indigotin content in the raw indigo will be guaranteed from the start. For example, from the theoretical considerations follows that the indigotin content of raw indigo can be doubled, if plants with 2 % indican content
36
were extracted with 1 % indican content. For this purposes it is important to know, which factors influence the indican content in the Polygonum leaves.
3.4.5 Possibilities to influence the indican content
Agrotechnical measures
Among this fall the N-fertilization and the sowing and the harvest date. About these factors has been reported in detail. Here should be still remarked that a determination of the indi-can content before harvest may be very useful. Therefore, a close cooperation between the farmer and the laboratory is very important.
Meteorological factors
In publications from England (STOKER et al, 1998), but also from Italy (ANGELINI et al, 2004) is described that the amount of the dye precursors seems to be positively affected by the light intensity.
Investigations to this question were carried out also in Dornburg.
For this purpose the determination of the indican content in the leaves of Polygonum tinc-torium was carried out for over about 3 weeks. In 2001 the samples were taken from sev-eral single plants (up to 50) in a time distance of two to five days. In 2002 and 2003 the leaves were harvested daily (2 samples/day). The determined indican values were com-pared not only with the total global radiation, but also with the daily temperatures and the precipitations to find out if there are correlations between the precursor values and the meteorological parameters (fig. 12 - 14).
date25.7. 27.7. 29.7. 31.7. 2.8. 4.8. 6.8. 8.8. 10.8. 12.8. 14.8. 16.8. 18.8. 20.8. 22.8.
0
0,1
0,2
0,3
0,4
0,5
0,6
0
5
10
15
20
25
30
Precipitation (mm) (y2) Temperature (middle °C) (y2)Indican content (% FM) (y1) Radiation (kW/m²) (y2)
Figure 12:Figure 12:Figure 12:Figure 12: Influence of precipitation, radiation and temperature on the indican content of Polygonum, Dorn-burg 2001
37
date5.7. 7.7. 9.7. 11.7. 13.7. 15.7. 17.7. 19.7. 21.7. 23.7. 25.7.
0
0,2
0,4
0,6
0,8
1
1,2
1,4
0
5
10
15
20
25
30
Precipitation (mm) (y2) Temperature (middle °C) (y2)Indican content (% FM) (y1) Radiation (kW/m²) (y2)
Figure 13:Figure 13:Figure 13:Figure 13: Influence of precipitation, radiation and temperature on the indican content of Polygonum, Dorn-burg 2002
Datum20.7. 22.7. 24.7. 26.7. 28.7. 30.7. 1.8. 3.8. 5.8. 8.8. 10.8. 12.8. 14.8.
0
0,5
1
1,5
2
0
5
10
15
20
25
30
35
Precipitation (mm) (y2) Temperature (middle °C) (y2)Indican content (% FM) (y1) Radiation (y2)
Figure 14:Figure 14:Figure 14:Figure 14: Influence of precipitation, radiation and temperature on the indican content of Polygonum, Dorn-burg 2003
38
There could not be determined clear correlations between the indican content in the leaves and the climatic data. The interpretation of the results of each single year is difficult, but the comparison of the summarized values of each year shows, that the amount of the pre-cipitation during the trial run has obviously the greatest influence on the indican content (tab. 26).
Table 26:Table 26:Table 26:Table 26: Comparison of the mean of the daily temperature, the precipitations and the total global radiation and the mean of indican content during the trial run of the years 2001, 2002 and 2003 (precipita-tions + 10 days before the attempt begun)
Character 2001 2002 2003
∅ Temperature (°C) 19.2 17.5 23.0
� Rainfall (mm) 28.3 57.7 62.8
� Global radiation (kW/m²) ∅ Global radiation per day
85.5 3.72
65.2 3.62
129.4 5.62
∅ Indican content (% of fresh mass) 0.37 0,91 1.36
This fact can be realized not only at the comparison of the mean values of 2001 with those of 2002 and 2003, it can also be seen at the curves of indican content in each year. After a rainfall they begin to increase with a delay of a few days. This fact seems to be an indirect effect of water on the indican formation in the plant.
The following explanation for the observed effect is conceivable:
The N-availability in the soil and its uptake by the plants is better because of the better wa-ter status of the soil. As former pot experiments have shown (see above) the dye content increases always with an increase of N much more than the formation of the biomass. The same effect is to be observed in field experiments, esp. in the year 2003.
The mean yield of leaves over all fertilization-variants was 25 % lower than in the year 2002. Besides of this fact the calculated dye yields per hectare were about 30 % higher. This may be the result of harvesting shortly after rainfall. A positive influence of the very high global radiation in 2003 can’t be excluded. For practical use follows from the results, that the first cut should not be done after long-lasting dryness. In 2002 this was the case and the indi-can content was very low. In 2003, on July the 17th, the precipitation was 15.6 mm, which evidently increased the yield of biomass and the indican content. The proposal is, not to harvest the crop at great dryness of soil, but to wait for rain or to irrigate a few days before the harvest.
Genotypical factors
There are small differences between the Polygonum tinctorium lines available in Europe in regard to biomass yield and dye content so far as they are white flowering (ANGELINI et al, 2004). The also existing pink flowering genotypes show always an appreciable lower dye content, so that they reach only half or one third of the dye yields per area of the white ones (BIERTÜMPFEL, VETTER, 1999). The white flowering accessions of Polygonum tinctorium, placed in the TLL at disposal only show a small variation in regard to the dye content be-tween the individual plants. For the widening of the variability seeds of one accesion were treated with a watery buffered solution (m/15 KH2PO4 = pH 4.5 – 4.75) of the mutagenic
39
agents NaN3 or nitroso-methyl-urea (NMH) for 6 hours and each with 4 different concen-trations.
For the further selection the M0 was sown into the field, each concentration on a separate plot. At ripeness only the 2 plots with the highest concentration (10 mM NaN3 and 6 mM NMH resp.) were harvested. Only the variant treated with 6 mM NMH was used for the subsequent sowing and selection. In 2000 the indican content was determined from 231 and in 2001 from 303 single plants of the M1 resp. the M2-mutaion bulk. The frequency dis-tribution of this character of the year 2001 is shown in figure 15.
Indican (% i. d. FM)
70
60
50
40
30
20
10
0
N = 302,00
Deviation = 0.16mean = 0.37
Figure 15:Figure 15:Figure 15:Figure 15: Frequency distribution of the indican content of Polygonum M2-plants, Dornburg 2001
The most types are negative, but a small amount shows also a positive deviation. In 2000 25 and in 2001 30 plants with a high indican content were selected for cultivation in 2001 resp. 2002 (2 replications, 5.25 m² plot size). As expected only a few “mutants” confirmed their high indican content. The high dye content of these types could be determined not only at the first cut, but also at the second cut, although their superiority was not so plain as at the first cut.
In 2003 a trial was set up in the field (4 replications, 5.25 m² plot size) with the 20 best forms. Five of them have shown an indigo yield of more than 15 % (up to 30 %) in com-parison with the original accession (Tab. 27). Their superiority in regard to their high dye content and dye yield shall be confirmed further in the next years. They should be the start-ing material for better breeding stocks.
40
Table 27:Table 27:Table 27:Table 27: Indican content and Indigo yield of mutants of Polygonum tinctorium 2002 and 2003 Field-no.. Indican content
(% dry mass) Indigo yield
(kg/ha) 2001 2002 2003 2002 2003 (% fresh mass) 1st cut 2nd cut 1st cut 2nd cut 1st cut 2nd cut ���� 1st cut 2nd cut ����
1 (Standard) - 3,96 5,02 3,59 2,88 36,9 55,3 92,292,292,292,2 39,9 35,4 75,375,375,375,36 1,34 5,53 7,35 4,28 2,68 38,3 94,9 132,7132,7132,7132,7 44,0 32,7 76,776,776,776,78 1,33 4,11 7,82 4,09 2,86 33,9 77,1 111,0111,0111,0111,0 33,4 28,2 61,661,661,661,69 0,59 4,61 5,34 3,77 2,15 40,0 62,7 102,7102,7102,7102,7 35,5 24,0 59,559,559,559,512 0,64 5,58 6,55 2,75 2,95 43,8 85,4 129,2129,2129,2129,2 22,6 27,6 50,250,250,250,213 0,54 4,47 7,03 3,81 2,35 50,5 87,6 138,1138,1138,1138,1 42,3 22,6 64,964,964,964,914 0,55 5,89 6,47 2,55 2,32 52,4 75,6 128,0128,0128,0128,0 35,2 26,0 61,261,261,261,215 0,58 6,32 6,48 5,19 2,59 41,2 75,2 116,4116,4116,4116,4 36,9 21,1 58,058,058,058,016 0,55 6,12 6,52 4,20 2,84 47,6 77,5 125,1125,1125,1125,1 44,8 34,6 79,479,479,479,417 0,62 5,05 6,36 3,60 2,65 47,1 64,3 111,4111,4111,4111,4 32,3 23,2 55,555,555,555,520 0,59 7,33 7,22 3,45 1,94 53,1 77,2 130,4130,4130,4130,4 44,0 19,1 63,163,163,163,127 0,68 6,45 5,98 3,66 2,68 63,6 59,4 122,9122,9122,9122,9 59,9 27,6 87,487,487,487,431 0,90 7,09 7,21 3,42 2,39 51,6 82,1 133,7133,7133,7133,7 63,3 28,0 91,391,391,391,333 0,91 6,09 4,78 3,97 2,71 50,3 64,5 114,8114,8114,8114,8 43,7 28,7 72,472,472,472,434 0,81 4,46 7,20 4,70 3,74 36,0 83,7 119,9119,9119,9119,9 51,5 40,2 91,791,791,791,736 0,87 1,13 8,42 3,73 2,96 8,1 111,8 119,9119,9119,9119,9 46,8 36,1 82,982,982,982,941 - 5,60 7,09 3,43 2,73 47,0 70,7 117,8117,8117,8117,8 53,5 32,2 85,785,785,785,743 - 5,63 5,35 3,31 3,45 45,2 79,7 124,9124,9124,9124,9 31,1 31,6 62,762,762,762,748 - 7,88 8,78 4,69 4,11 71,8 96,1 167,9167,9167,9167,9 54,1 43,9 98,098,098,098,051 - 6,80 6,46 2,50 2,99 82,3 73,9 156,3156,3156,3156,3 26,8 32,1 58,958,958,958,9 4,784,784,784,78 5,655,655,655,65 3,733,733,733,73 0,650,650,650,65 40,740,740,740,7 63,463,463,463,4 104,1104,1104,1104,1 42,1 9,49,49,49,4 26,026,026,026,0
GD t, 5 % 2,11 2,62 1,10 21,0 29,8 27,9 20,0
The processing of material should not only gives a “cleaner” product. For the whole econ-omy of the dye production from the plant material the level of the dye content in the leaves of the Polygonum plants plays an important role. If it is possible with simple extraction procedures to get nearly the total indigo amounts, grown in the field, the price difference between natural and synthetic indigo should be substantially lower as at the moment. The chances of the application of natural indigo for dyeing should be better then.
3.53.53.53.5 Recycling of waste productsRecycling of waste productsRecycling of waste productsRecycling of waste products
The waste water of the indigo extraction can be used like a thin liquor for fertilisation of plants (tab. 28). Table 28:Table 28:Table 28:Table 28: Comparison of the nutrient content of waste water from the extraction and thin liquor1)
N (%) P (%) K (%) pH-value
Waste water from the extraction 0.67 0.007 0.49 8.1
Thin liquor 0.40 0.02 0.40 > 7 1) KERSCHBERGER et al. (1997)
The plant residues can be composted. Composted Polygonum plants were investigated for heavy metals and organic pollutants. The determined values are considerably lower than values demanded from the legislator (tab. 29).
41
Table 20:Table 20:Table 20:Table 20: Heavy metals and organic pollutants in the compost from Polygonum plants after six month Parameter Content of Polygonum leaves
(mg/kg dry mass) Border values
(mg/kg dry mass) Cu 16 800 Cd 0.25 10 Cr 188 900 Pb 16 900 Ni 8 200 Zn 101 2500 Hg 0.08 8
AOX 150 500 PCB 0.005 0.2
During the composting a great number of earthworms always settled the substrate. This speaks for the environmental safety of the Polygonum wastes.
3.63.63.63.6 Analytical determination of indigo and its precursors in Isatis and PolygonumAnalytical determination of indigo and its precursors in Isatis and PolygonumAnalytical determination of indigo and its precursors in Isatis and PolygonumAnalytical determination of indigo and its precursors in Isatis and Polygonum
3.6.1 Material and methods
There are several opportunities for the determination of indigo-related compounds in plants. Reversed phase HPLC using detection by means of DAD and/or ELSD is the basic technology. The analytical base-line separation has to be optimised in dependence of the used solvents and columns in each laboratory.
For the purpose of comparison independent methods like spectrophotometry and HPLC with derivatization have to be used.
3.6.2 Results and discussion
Details of all described procedures and measurements are described in the reports for 2001 and 2002 and as a summary in:
Quantitative Analysis of Indigo and Indigo-precursors in Leaves of Isatis spp. and Poly-gonum tinctorium by KG Gilbert, H G Maule, B. Rudolph, H. Vandenburg, E. Sales, S. Tozzi and DT Cooke, published
Sampling and storage
During the first two reporting periods of the project a lot of work has been done from part-ners 2, 3 and 8 in order to establish standardised methods for the characterization of Isatis and Polygonum. That means the quantitative determination of the precursors as well as of total indigo content of the plants.
One of the most important difficulties is the instability of the indigo precursors in har-vested plant material. In case of Isatis the main precursor Isatan-B degrades very rapidly during storage and after the extraction in the solvent. Indican as dominating compound in Polygonum is very sensitive against enzymatic degradation in cutted or frozen leaves.
As a consequence it is necessary to analyse the plant material very quickly after the harvest avoiding storage. Only deep-freezing could be a compromise for Isatis but not in case of Polygonum.
42
Extraction
In order to achieve an extraction rate of 100 % of the total content of the precursors with-out any kind of degradation polar and “enzyme-blocking” solvents are necessary. Due to this reasons methanol or mixtures of methanol and water ore water with additives are pre-ferred. Formic acid or acetonitril are suitable additives, but methanol seems to be the best solvent for analytical extractions.
Recovery tests with indican showed a sufficient stability of this precursor against heat treat-ment at the temperature of boiling methanol. The behaviour of Isatan-B from Isatis during several extraction procedures is not well known because of this compound is not available as pure substance.
The use of methanol seems to be safer for the stability of the extraction procedure in prac-tical analyses but the number of extracted compounds is much larger than in the case of water. This can cause some difficulties for laboratories using HPLC with ELSD detection.
Methods of determination
In the case of experienced laboratories it should be not very difficult to establish a stable, base-line separation for the indigo precursors, especially for extracts from Polygonum. A standard RP 18-column is sufficient for a gradient separation with water and methanol as eluent mixture. UV-detection at 290 nm coupled with peak identification by means of Di-ode-Array-Detection gives stable results. The most important problem is the identification and quantification of the not commercially available precursor Isatan-B from Isatis. There are several opportunities to overcome this problem with indirect quantification after com-parison with indican. The derivatisation with isatin and following determination of the reac-tion product indirubin gives safe results which are comparable with independent methods like High Performance Thin Layer Chromatography (HPTLC).
Spectrophotometric determination of indigo formed in the extracts after alkaline hydrolisa-tion and oxidation by aeration is a suitable alternative with simple analytical technologies but only for Isatis. The indigo is removed from the extract by phase partition into ethylace-tate and afterwards quantified spectrophotometrically. It has been taken care because of the low solubility of indigo in ethylacetate.
3.6.3 Conclusions
A robust and efficient analytical method suitable for daily work is absolutely necessary in order to receive stable, reproducible and correct results. Therefore it is very important to adapt the method to the kind and number of plants which have to be investigated but also to the capacity and the time-table of the laboratory. Harvested crops have to be analysed as soon as possible at the same day or the following after storage in a fridge. In case of Isatis deep freezing is possible with the uncertainty of partial degradation.
However a quick collaboration between farmer and laboratory is very important! For the reason of efficiency of the laboratory this could be a limitation fact for the number of sam-ples as well as for the date of the harvest. Simplified methods like spectrophometry can be used for field tests and semi quantitative investigations with the risk of losses in precision and accuracy.
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4444 DiscussionDiscussionDiscussionDiscussion
Among all problems in regard to the reintroduction of natural indigo for dyeing purposes the question of cultivation can be easily solved. This can be seen on the example of the woad, which has been cultivated successfully in the agricultural practice of Thuringia since 1990 after an agronomic blueprint worked out by the TLL and further improved in the frame of the Spindigo-project. Now an agronomic blueprint also exists for a second blue delivering plant, Polygonum tinctorium. It seems to be the only suitable plant for dye win-ning purposes with an indigo content of up to 5 % in the dry mass under Middle European conditions. The blue dyeing plant Isatis tinctoria has a too low content of dye precursors as could be shown at the investigation of 1262 woad samples. It gave indigo values from 0.125 % to 0.95 % with a mean of 0.3 % in the dry mass. The values correspond with information of VOLTOLINA and VALERIANA (1996) from Italia, which found in their woad attempts indigo contents of 0.4 to 0.44 % in the dry woad leaves. Even if in 2002 some strains of woad showed a dye content between 1 % and 2 % in the dry leaves and the calculated dye yield was up to 65 kg indigo per hectare, Polygonum tinctorium gave calculated yields up to 124 kg per hectare at the same time, because of the higher dye content in ist leaves. Gener-ally, in the most years the difference in the calculated indigo yield of Polygonum tinctorium and Isatis tinctoria is much higher.
The expenditure to produce these amounts is lower at Polygonum as at Isatis. Woad needs N-gifts of 200 to 220 kg/ha, Polygonum only 160 kg/ha. The first must be cut three times, the last only twice. With this the costs for the production of 1 kg indigo is somewhat lower in the case of using Polygonum instead of woad. Of course the price of dye plays an impor-tant role for opening a market for indigo naturalis.
Substantially more difficult as the working out of modern cultivation methods is the further processing of the harvested material to indigo. It needs special equipment, which is not available in an agricultural enterprise. The machinery, developed by partner 6 for woad, can not be used for Polygonum. The cleavage of indican into indoxyl is incompletely inhibited at 70°C, the extraction temperature of woad, by the inactivation of the plant-own enzyme. The extracted indican is very stable in the extraction solution and the formation of indigo is impossible. It can be only cleaved by strong acids or bases or enzymatically. Therefore, Polygonum should be extracted at a moderate temperature (35 °C to 40 °C). By this way, the dissolution of indican doesn’t take a too long time (about 20 h) and its cleavage into indigo yielding compounds takes place at the same time. Morefold dipping of fresh Poly-
gonum into the same extractant (≤ 3fold) seems to be useful to minimize the amount of waste water. The won raw dye contains only about 20 % indigotin in the mean. It can be increased up to 30 – 40 % by washing the leaves before the extraction with water. This should be enough for purposes of textile dyeing.
All in all, the technical and financial expenditures for the production of indigo from Poly-gonum are not very high.
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5555 ConclusionsConclusionsConclusionsConclusions
The cultivation of the blue delivering plants Isatis tinctoria and Polygonum tinctorium makes no problems. The at the moment available seeds in Thuringia make it possible to cultivate about 10.000 ha woad in the next years. The agricultural enterprises, which are able to cultivate rape successfully, should be able to grow woad or Polygonum with suc-cess, too. In Thuringia nearly all enterprises cultivate rape.
The question who produces indigo from woad or – better – Polygonum leaves is not solved to the same extent. A first attempt to install a plant at the “Verein Ländliche Kerne”, where people who had difficulties to find employment, among other cultivate dye plants and dye clothes, which they then sell, was a flop. They are unable to grow dye plants in a greater extent. For a successful introduction of indigo naturalis on the market the producer needs
an amount of ≥ 100 kg dye stuff, how it was shown in many inquiries to the TLL: Only if there is the possibility for traders or small enterprises to buy and to get the indigo just in time, they will be regular customers. The best variant seems to be the cultivation and the processing in the same agricultural enterprise. Only in this case the transport ways are short. The waste water of the dye winning can be used as a thin liquor to fertilize the own fields and the plant residues as an organic manure. So the price may be lower as in the case that the producer of the dye must sell the waste products. Mostly, only the farmer has also the location for the intermediate storage.
The TLL is anxious to win at least one farmer for the cultivation of ≥ 1 ha Polygonum for the production of indigo in the next year. The chances to reach this aim seem to be good. Naturally, this is a risky venture, but it is hopeless to waite for a greater order of dye. The resonance from the side of the big dyeing industry is absolutely absent.
6666 DisseminationDisseminationDisseminationDissemination
The TLL makes an intense effort since 10 years to reintroduce natural dyes for the dyeing of textiles, leather goods, paper, etc. For this purposes we have taken many initiatives to propagate the cultivation of dye plants and the application of their dyes, especially woad and Polygonum, at many scientific and popular meetings. Just in May 2004 took place a meeting in Dornburg on natural dyes, organized by the TLL and the Fachagentur Nach-wachsende Rohstoffe for interested parties. The number of scientific and popular publica-tions is very high, alone in the last year we have had 8 publications on woad and Poly-gonum. For the most important dye plants the TLL has worked out and distributed cultiva-tion recommendations to interested parties. So the cultivation of weld, madder and woad took place resp. takes place on some hectares.
At the moment, in Thuringia are cultivated 30 ha woad according to recommendations of the TLL, which were improved in the project “SPINDIGO”, but the woad will not be used for the production of the blue dye.
Despite all our efforts the breakthrough of natural dyes suffers. The dyeing with natural dyes is still a matter for fancy of housewifes as at the begining of this and all our other pro-jects.
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To change this situation the TLL has planned for the next year the installation of a plant for the production of indigo from Polygonum in a 100 kg measure. For this purpose we have discussed with some farmers, if they are willing and able to win indigo after our experi-ences. On the other hand the TLL has started an inquiry, which amounts of indigo are needed for dyeing purposes of small plants.
Our aim is at least to begin with the cultivation of some hectares of Polygonum and possi-bly to open a market. The chances to reach this aim seem to be good.
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