Raphanus with increased anthocyanin levels

#The present invention relates to plants of the genus Raphanus that contain increased levels of anthocyanins, In particular the invention relates to edible Raphanus sprouts containing increased levels of anthocyanins, as well as to methods for their production.

#There is an increasing consumer demand for edible young vegetable plants, also referred to as sprouts. Sprouts may be produced by germinating seeds in either liquid medium and harvested before the cotyledons appear, as is e.g. done with alfalfa. Alternatively, seeds may be sown onto a water-soaked solid support, such as cellulose, and allowed to germinate and grow in e.g. small disposable containers until or beyond the cotyledons appear. Further growth of the plantlets may be arrested, e.g. by cooling the plantlets, usually before the plants reach a height of about 4 to 15 cm. The plantlets are then ready for consumption. Sprouts of e.g. cress, daikon (a type of radish sprout) and mustard grow this way are very popular and many other vegetables and herbs are nowadays also available in the form of sprouts. The popularity of vegetable sprouts may at least in part be explained by the notion that they are healthy. Sprouts are indeed known to be very rich in vitamins and minerals such as the vitamins C and B1, potassium, calcium, phosphorus, magnesium and iron, while at the same time they are low in calories. Daikon sprouts, i.e. Sprouts of Raphanus sativa longipinatus, are particularly popular in the U.S, and in Japan were they are usually sold as young two-leafed green plants. Unfortunately, daikon sprouts do not contain anthocyanins, a compound that could further contribute to the health promoting effect of these sprouts.

#Anthocyanins are present in many plants of higher order where they are responsible for the red, violet, blue or bluish black colours of flowers and fits. They are heterocyclic 2-phenyl-chromenol multiring systems (see also Formula 1) of varying hydroxylation patterns and varying absorption spectra in the visible light range. The sugar-free aglycon components of anthocyanins are referred to as anthocyanidins. They are obtained easily by hydrolysis of the glycosides contained in common fruits.

#FIG. 1. Spectra of the anthocyanins as extracted from sprout of the Raphanus sativa line V33. The absorbance versus the wavelength is shown for each of the anthocyanins eluting from the HPLC at different times as indicated (see Example 4 for experimental details).

#FIG. 2. Spectra of the anthocyanins as extracted from sprout of the Raphanus sativa line CGN 6924 (starting cultivar). The absorbance versus the wavelength is shown for each of the anthocyanins eluting from the HPLC at different times as indicated (see Example 4 for experimental details).

#A total of 568 Raphanus sativa lines as available from public gene banks were screened for their capability to produce a purple sprout. 14 of those lines were found to produce a sprout with at least some purple colouring. After several generations of self-pollination and selection, most descendants did not produce a sufficiently intense purple colour. Only 2 out of the 14 lines produced a significantly intense purple colour and were selected for further breeding. These two lines, CGN 6924 and CGN 7240 were both obtained from the CGN gene bank at Plant Research International (formerly CPRO-DLO), Wageningen, The Netherlands and originated from the former German Democratic Republic and from China, respectively. Starting from these three lines a high frequency further breeding program was undertaken whereby two generations were grown per year, alternating growth in the Northern and Southern Hemisphere. Both self-pollination and crossing of lines were applied whereby new lines were selected on colour intensity, time of fading of purple colour to green, nicking time, seed production and amount of seeds per flower, height/length of the plant(let)s, uniformity of germination time, absence of green descendants, and size and shape of cotyledons. Generally it was found that the lines with purple colour showed reduced seed production. Per generation at least 200 combinations were screened. Several lines with very intense purple colour and that did not divide out green descendants were thus obtained. One such line, with designation V33 was deposited on Aug. 13, 2001 under the Budapest Treaty at the American Type Culture Collection, Manassas, Va., USA, and was assigned ATCC No. PTA-3630. This line may used as such for the production of sprouts or may be used in breeding programs, optionally in combination with CGN 6924 and CGN 7240, to cross the purple colour into conventional green varieties.

#A method for producing Raphanus (daikon) sprout in the form of “cress” (Sakura Cress®) is described in Dutch Patent No. 192969. Basically, seed of a purple Raphanus line is placed in plastic containers (of e.g. 6×8 cm surface) having a water soaked solid support (“substrate”) such as e.g. a sheet or pad of cellulose or fin peat on the bottom. A suitable substrate is described in Dutch Patent No. 1001570. The seeds are sown at a high density of about 3-6 seeds per cm2. The containers wit the seeds are then allowed to germinate at 15-24° C. at high humidity (preferably a relative humidity of 100%). Germination may suitably be performed in the dark or with light and preferably is carried out in a germination cell or room with controlled temperature and humidity. After germination, which usually takes about 48 hours, the containers with seedlings are placed in a greenhouse where the temperature is kept at 15° C. or higher (preferably not higher than 25° C.) and humidity is preferably kept above 70% (relative humidity). The plantlets are then allowed to grow for another 48-72 hours until they reach a height of 5-14 cm and usually having no more than 2 leaves, i.e. the cotyledons. Additional light may be given to make the plantlets more sturdy. When the plantlets have reached the desired height or number of leaves, growth is arrested by cooling down to 2° C. The plantlets are further kept, stored and transported at this temperature, preferably until consumption.

#Seed of the V33 line is washed and soaked in water and placed in a slowly rotating drum at 15-25° C., under addition of a fine mist of water. This mist is provided every hour or differently according to the growing method and planning of the grower. The seeds slowly tumble in the drum, fast enough not to cling together and slow enough not to break the germs. After 100 hours with a variation of about 48 hours, the sprouts have developed into a red/purple shiny product, which is then washed and ready for packaging. The ratio of seed to harvestable product usually is 1:8 till 1:10, i.e. 1 kg seeds provides 8 to 10 kg of sprouts. Additional lighting may be added during the germination resulting in a more intense purple colour.