Cultivation and breeding of Digitalis lanata the Netherlands · Cultivation andbreeding ofDigitalis...

Br Heart 7 1985; 54: 262-8

Cultivation and breeding of Digitalis lanata inthe NetherlandsC MASTENBROEKFrom the Stichting "Kruidentuin te Doornspijk", Elburg, The Netherlands

SUMMARY After the second world war Marshall Aid funds were used to establish a cooperativeorganisation for growing, drying, and selling Digitalis lanata (and other medicinal, aromatic, andculinary herbs) in the Netherlands. The crop is sown in mid April and the fully mechanisedharvest of the leaves takes place from September to late November. The leaves are dried for 10-12hours at 50°C maximum. The aim of breeding trials is to improve leaf production, erect leafattitude, resistance to Septoria leaf spot and to bolting, and a higher dry matter and digoxincontent. 9

Cultivation

It is cheaper for the pharmaceutical industry to ex-tract digoxin from the dried leaves of Digitalis lanatathan to synthesise the drug chemically. D lanata(Fig. la) is called the woolly foxglove because thecalyx of the flower is thickly covered with whiteglistening slightly sticky hairs (Fig. lb). Digitalispurpurea, which is native to north west Europe, wasgrown for the extraction of digitoxin until the medi-cal superiority of digoxin was demonstrated. Di-goxin only occurs in D lanata, which is not native tonorth west Europe; its natural habitat is south eastEurope.

After the second world war the Dutch governmentused some Marshall Aid funds to promote the culti-vation of D lanata (and other medicinal plants andaromatic and culinary herbs). Several cooperativedrying plants were established; three of these are stillactive. They are situated in the north east, centre,and south east of the Netherlands. In 1984 230 hec-tares of D lanata were harvested and dried. Con-tracts with growers are arranged by the threeregional drying factories and a central cooperativeVNK (Verenigde Nederlandse Krudencooperatie,United Dutch Herb Cooperative) organises con-tracts with and delivery to the pharmaceutical indus-try.D lanata is cultivated on peaty, sandy, and clay

soils. The seeds are drilled in mid-April withmachines adapted to sow very small quantities (2 kgper hectare) of tiny seeds. Germination is very slow,and it takes about two months before the seedlingsRequests for reprints to Dr C Mastenbroek, De Terminator 8, 8251AD Dronten, The Netherlands.

develop sizeable leaves. The leaves (Fig. 2) are har-vested from the end of September till the middle ofNovember or even later, depending on weather con-ditions. Harvesting is completely mechanised. Themean yield of fresh leaves is about 30 tonnes perhectare. The freshly cut leaves are immediatelytransported to the drying factories. After weighingthey are coarsely cut, washed to remove soil par-ticles, and dried for 10-12 hours at 50°C until theirmoisture content is 8-10%. The dried product,around 5 tonnes per hectare, is pressed into poly-ethylene bags (35 kg per bag) and exported mainly tothe United Kingdom, the United States of America,Germany, and France.

Breeding

The growers, drying plants, and pharmaceutical in-dustry all have an interest in the improvement of theinherited properties of the species. Their variouspriorities are set out below.

THE GROWERSThe yield of fresh leaves is the basis for payment.

The farmer wants to grow a variety that produces aheavy leaf canopy. The first step in breeding, there-fore, is selection of individual plants with manybroad and long leaves. Until about 10 years ago thematerial cultivated had been obtained through natu-ral accession from a botanical garden and did nothave outstandingly large leaves (Fig. 3a). Individualplants with larger than average leaves were selectedand grown for their seed. In the next generation theplant type appeared to have been somewhat im-proved. Variation was still apparent and this was

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Cultivation and breeding of Digitalis lanata in the Netherlands

Fig. 1 (a) Digitalis lanata in full bloom (photographprovided by Professor F L Meijler) and (b) woollyhairs on flower buds.

again exploited for the selection of better plants.Selection over three consecutive generations hasconsiderably improved the plant type (Figs. 3b andc). It should be borne in mind, however, that in a

well established production field more than 1 000 000plants are competing for room, light, and nour-

ishment, and this competition means that plants ofthe same genetic constitution will have a differentappearance when they are grown in closed stands andas spaced plants in nurseries. The yields of individ-ual plants will also differ considerably according totheir growing conditions; nevertheless there is evi-dence that selection has improved leaf yield by about15%.

The leaf attitude ofthe original accession tended to

be prostrate, and some of the lower leaves remainedattached to the plant after harvesting (Fig. 4). Theleaves left behind can only be used as green manure

and represent a loss in income to the grower. Anupright leaf attitude is therefore an advantage. Selec-tion has produced a plant type with a strikingly erectleaf habit. This improvement is more apparent whenD lanata is grown in nurseries of spaced plants ratherthan in close stands in production fields. Plants witherect leaves will tend to produce a cleaner harvested

product since the more upright the leaves are the lessthe driver of the cutting machine will be tempted tomake a deep cut which harvests soil particles too.

Resistance to diseases makes measures to prevent orto check damaging attacks less necessary or evensuperfluous. Thus when resistant varieties are grownproduction costs for the same yield are lower. Incommercial production only one fungus, Septoriadigitalis (Fig. 5a), produces damage suffficient to re-quire chemical control. In comparative trials thefresh leaf yield of the very susceptible original acces-sion was reduced by as much as 20% between mid-September and the end of October when chemicalcontrol was not used (Fig. 5b). Spraying twice dur-ing the growing season costs about £125 per hectare.Selection of plants which have a high degree of nat-ural resistance to disease has produced a varietywhich does not need to be sprayed with fungicides.

Resistance to bolting is important because it saveslabour. Plants that develop stems with flowers andfruits in the first year are called bolters. Because theleaves contain much more digoxin than other parts ofthe plant, the raw material for extraction should con-sist of leaves only. Growers should undertake to re-move bolters from their crops. This can only be done

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Fig. 2 Mechanised harvest of leaf crop in commercial cultivation.

by hand. Thus they prefer to cultivate a varietywhich does not have a tendency to bolt. Bolting isstimulated by exposure of the young plants to lowtemperatures. This encourages the plants to developflowers. The amount of stimulation required to ini-tiate bolting depends on the plant's natural re-sistance to bolting. Selection for this feature is notdifficult. When seeds are sown in cold frames in thefirst days of April- and the seedlings are transplantedat the beginning of June the percentage of plants thatbolt (Fig. 6) is much higher than that found in fieldcrops and in comparative yield trials which are nor-mally drilled around the middle of April. The exclu-sion from further selection of all bolting plants,including those in which bolting is only incipient,has produced a high level of resistance to bolting inonly a few years.

THE DRYING FACTORIESAn upright leaf attitude produces a cleaner crop.Dry matter content-The cost of drying the leaves

by means of natural gas is high. The mean dry mattercontent of the leaves is about 20%, and the moisture

content has to be brought down from 80% (or moreif the product has been washed) to 8-10%. An in-creased dry matter content reduces the cost of dry-ing. There is genetic variation in dry matter contentbetween individual plants and their seed progenies,but non-genetic variation between individual plantscaused by external conditions may also be large. Thismakes effective selection for this character ratherdifficult. Nevertheless, plants with a higher drymatter content have been selected. It is difficult,however, to combine a high dry matter content witha high yield of fresh leaves and a compromise isnecessary.

THE PHARMACEUTICAL INDUSTRYDigoxin content influences production costs. As longas the price of the raw materials is not related to itsdigoxin content, the pharmaceutical industry willbenefit from an increased digoxin content. A de-crease in the content of compounds that resembledigoxin (63 glycosides have been found in D lanata)would also simplify purification. To select for ahigher digoxin content individual plants are assessed

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Fig. 3 (a) Plants of orzginal accession and(b improved selection. (c) An outstandirngi.ndividual plant.

by paper chromatography. This is not a reliablemethod, but it is quick, simple, and cheap; and thisis important since several hundred plants have to betested in two to three weeks. In recent years samplesof selections in yield trials have been investigated byhigh performance thin layer chromatography (Fig.7). The digoxin content of dried leaves of the varietynow in cultivation is around 25-3-0 parts per thou-sand, which is about 50% higher than the content ofthe original accession.

BREEDING PROCEDUREYear 1-Selection of individual plants in spaced

stands for characteristics described above in a nurs-ery under heavy Septoria pressure. Only about 20out of 10 000 plants are retained for further testing.

Year 2-Seeds are harvested from individual se-

lected plants.Year 3-Seed progenies are tested for relevant

characteristics in comparative yield trials (Fig. 8) intwo replicate plots on clay and sandy soil. About 10of the better progenies are retained.

Year 4-Repeated testing of 10 progenies as inyear three. If one or two perform better than thestandard over the two years, they are retained.

Year 5-Retained selections are sown for seedproduction.

Year 6-Seed production.Year 7-Testing of seed progenies as in years 2

and 3 but on more plots and at different harvestdates. If only one selection performs satisfactorily, itwill be used in commercial production from Year 8onwards, after which further seed multiplication willtake place.A new selection cycle is initiated each year, start-

ing from progenies that are outstanding overall or forparticular characteristics.

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Fig. 4 Recumbent leaves remain after harvesting.

Seed production

D lanata is self-fertile. The pollen is sticky, andselfing can be achieved by moving a small soft brusharound inside the flowers. Flowers which have beentreated in this way must be covered (bagged) to ex-clude bees and thus prevent cross pollination.

In the breeding work that I describe, however, theplants are not bagged. Bees are allowed to cross pol-linate selected plants retained from year 1 for seedproduction in year 2. Plants with particular charac-teristics are grown in isolated plots to prevent crosspollination between groups. Seed production in year6 and in successive years also takes place in isolatedplots and fields.Large numbers of bees are attracted by the glis-

tening droplets of nectar at the bottom of the flower(Fig. 9). When the crop is in bloom bees carry thesticky pollen from plant to plant. This cross polli-nation produces heterogeneous progenies, and prog-eny produced by cross pollination of an outstandingplant is as a rule less outstanding than the mother-

IL__ < _.A......b

Fig. 5 (a) Leafspot disease caused by Septoria digitalis and(b) heavy damage in a susceptible variety.

plant itself. The genetic contamination of out-standing plants can be prevented by raising severalconsecutive generations by selfing, but inbreedingdepression may result. This has not so far been in-vestigated.

Natural pollination by bees certainly brings abouta high degree of inadvertent crossing between out-standing plants in year 2. Some of the heterogeneousplants produced may have new and favourable com-binations of genes. Such outstanding individuals canbe selected in year 1 of each new selection cycle. Thevery best plants may be outstanding simply becausethey are cross bred. If this is true, it follows thatgenetic segregation in the sexual progenies is un-avoidable and that some genetic deterioration willtake place. This may indicate that selection shouldcontinue to maintain the favourable inherited char-acteristics of the variety. Investigations into thisquestion have been started, but it will take severalyears before a conclusion can be drawn.

Seed production may be damaged by the greymould Botrytis cinerea, a widely distributed fungus,

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Fig. 7 High performance thin layer chromatography densi-tograms showing a high peak in 23 digoxin in a selected varietyand in the standard I (TNO, Zeist, The Netherlands).

Fig. 6 Bolting plants flower in the first year.

Fig. 8 Comparative yield trial.

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Fig. 9 Bees bring about cross pollination.

which can attack the inflorescence of D lanata. Insevere cases almost all the seed is lost. The fungus isonly weakly pathogenic, but starting from deadflowers that remain stuck in the hairs on top of theyoung fruits or in the leaf axils, it may invade thestems. Growth of the fungus within the stem (Fig.10) causes all the seed in an attacked inflorescence tobe lost. The attack is favoured by rain, high air hu-

Fig. 10 A healthy stem with fruits producing many seeds(left) and a Botrytis-diseased stem producing no seed (right).

midity, and several days of high temperatures. Dlanata has no natural resistance to this disease, butRonilan (vinchlozoline) which was developed to con-trol the fungus in french beans is very effective in Dlanata if it is sprayed once a week (or twice a weekwhen conditions are moist and warm) during the 6-8week flowering period and once or twice afterflowering.

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