Improved orchid culturing method

The invention relates to an improved method for the culturing of orchids, more specifically Phalaenopsis.

The sales of potted orchids has increased steadily over the last 30 years. In 2000, wholesale orchid sales were approximately $100,000,000, of which about 75% was accounted for by Phalaenopsis (Griesbach, R.J., 2002. Development of Phalaenopsis Orchids for the Mass Market, p. 458-465, In: Janick J. and Whipkey A. (eds), Trends in new crops and new uses, ASHS Press, Alexandra, VA). Phalaenopsis, or the moth or butterfly orchid, is a genus of the family of Orchidiaceae, which contains at least 60 different species. However, in the recent past many varieties and hybrids have been produced for commercial purposes, resulting in a large assortment of pot flowers. An important group of these hybrids is formed by plants of the genus Doritaenopsis, which arise from a first cross between a Phalaenopsis plant and a plant of the genus Doritus. Very many subvarieties, created through further crosses with Phalaenopsis or Doritaenopsis are known in the meantime. Comprised in the term "orchids of the genus Phalaenopsis and Doritaenopsis" are thus also all hybrids which have been created by a cross with one of these. All orchids have these five basic features :

- the presence of a column

- the flower is bilaterally symmetrical

- the pollen are glued together into the pollinia, a mass of waxy pollen on filaments. - the seeds are microscopically small, lacking endosperm (food reserves).

- the seeds can, under natural circumstances, only germinate in symbiosis with specialized fungi. Under artificial circumstances, however, germination is possible "in vitro" on sterile substrates or agar in specialized laboratories. Phalaenopsis (as well as Doritaenopsis) shows a monopodial growth habit. An erect growing rhizome produces from the top one or two alternate, thick and fleshy, elleptical leaves a year. The older, basal leaves drop off at the same rate. The plant retains in this way four to five leaves. They have no pseudobulbs. The raceme (flowering stem or stalk, spike or inflorescence) appears from the stem between the leaves. Phalaenopsis typically produces a single raceme, which consists of up to 25 flowers. The species and hybrids of Phalaenopsis that bloom in later winter or early spring require a period of about six weeks of exposure from 25°C-30°C (culture) to 15°C-20°C (induction) to trigger the emergence of the inflorescence (Report "Cultivation Guidelines Phalaenopsis Pot Plant. Anthura B.V. and Bureau IMAC Bleiswijk B.V., http://www.anthura.nl). There is an absolute requirement for the presence of light while plants are exposed to the proper temperatures for spiking (= emergence of the inflorescence). Although the plants are marketed year round, winter is an important selling season. This means that for the plants which have to be sold during winter, the cold induction has to be performed during the summer months. This means that the greenhouses need to be cooled down to at least 20 °C, which increases the costs of growing the plants.

It is already known in the prior art that treatment with hormones, especially cytokinions, can induce growth of shoots (JP9233962 and Nayak, N.R. et al., 1998, In Vitro Cell. Dev. Biol. - Plant 34:185-188) or flowering in orchids, as well in vitro (Kostenyuk, I. et al., 1999, Plant Cell Reports, 19:1-5; Kim, T.-J., et al., 1999, J. Kor. Soc. Hort. Sci. 40:619-622; Duan, J.-X. and Yazawa S., 1995a, Plant Cell, Tissue and Organ Culture, 43:71-74) as in vivo (Yoneda, K. and Momose, H., 1990, Bull. Coll. Agr. & Vet. Med., Nihon Univ.,

47:71-74; and Duan, J.-X. and Yazawa, S., 1995b, Acta Horticulturae 397:103- 110).

It is desirable to generate more inflorescences on the same plant, since this would yield more flowers per plant, and thus would increase its value. Depending on the length and temperature of the cold induction Phal;aenopsis plants normally induce one or two flower stalks, and exceptionally three, and very exceptionally four flower stalks.

In the above-mentioned prior art, the presence of multiple flower stalks is what can be expected according to the normal existing growing practice: little number of plants shows one or two additional flower stalks. Induction of multiple flower stalks by the hormone treatment has only been described with the in vitro treatment of shoots with cytokinin (Duan, J.-X. en Yazawa, S, 1995b, supra), wherein the authors, who concomitantly also describe in vivo experiments in these latter plants, did not report an increase in the number of flower stalks. Further, the art is silent about the effect of a combined hormone treatment and cold induction.

Thus, there is still need for a method to induce multiple raceme formation in orchids.

SUMMARY OF THE INVENTION

The present inventors now have found a method to increase the number of inflorescences in an orchid of the genus Phalaenopsis or Doritaenopsis by administration of a cytokinin to said orchid, before or during the period of exposure of said orchid to a cold period for inducing flowering. Said cytokinin is preferably 6-BAP (6-benzylaminopurine), which can be applied in a concentration in the range of about 2 mg to about 30 mg per plant, preferably about 5 mg to about 26 mg per plant, more preferably about 10 mg to about 24 mg per plant, most preferably about 20 mg per plant. In aqueous spraying solution, this would result in a range of about 100 ppm to about 1500 ppm,

preferably about 250 ppm to about 1300 ppm, more preferably about 500 ppm to about 1200 ppm, most preferably about 600-1000 ppm. A preferred embodiment of the invention is wherein the administration of the cytokinin is done by spraying a solution comprising said cytokinin. Additionally, for better uptake of the cytokinin, the orchid is kept in the dark for a period of about 6 to about 14 hours after spraying. A similar effect, enhanced uptake of cytokinine, can be achieved by increasing the relative humidity of the air in the greenhouse. These measures can be taken separately, dependant on the climatic conditions inside and outside the greenhouse. Also for better uptake the spraying solution can additional comprise a wetting agent, preferably Zipper®, and/or a co-solvent, preferably

DMSO .

A further preferred embodiment comprises the administration of a rooting hormone, preferably NAA to the orchid (uptake of NAA takes place both ath the leaves and at the roots). Administration of the rooting hormone preferably takes place at transfer of the young plants to pots and can be performed to about 4 weeks previous to the cold induction period. A following embodiment of the invention is an orchid of the genus Phalaenopsis or Doritaenopsis, which has more than four inflorescences. Also a group of orchids of the genus Phalaenopsis or Doritaenopsis of one and the same variety of 1000 or more, having an average number of inflorescences of more than 2.9, preferably more than 2.5, more preferably more than 2.1, is included in the scope of the present invention. Also contemplated are the use of a cytokinin to induce the forming of adventitious inflorescences in an orchid and the use of a cyokinin to abolish or diminish the need for a cold period to induce inflorescences.

DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a plant of the species Phalaenopsis Hybrid showing four flower stalks.

Fig. 2 shows results of example 1.

DETAILED DESCRIPTION OF THE INVENTION

Cytokinins have the effect of increasing the mitosis (cell division) of shoot and buds. Some cytokinins increase the size or number of the plant cells and e.g. makes leaves larger. The cell differentiation can be controlled (e.g. for production of calli). Growth of roots is inhibited. Cytokinins are adenine derivatives and kinetin can e.g. be synthesised from heating nucleic acid. Known cytokinins are: 2iP N6-(3-methylbut-2-enyl)adenine, Kinetin (6- furfurylaminop urine), Benzyladenine (N6-benzyladenine), BA or BAP (6- benzylaminopurine), Zeatin, Adenine sulfate (2C5H5N5 ^• H2SO4) and

Thidiazurone (l-phenyl-3-(l,2,3-tiadiazole-5-yl)urea). Cytokinins are heavily used in orchid production during tissue culture, to induce shoot regeneration. Further, it has been demonstrated that cytokinins can be used to induce keiki's in orchids (a keiki is a small plant which grows from one of the nodes along the (flower-bearing) stem). Usually these keiki's are introduced by applying cytokinines (commercially available as 'keiki paste', e.g. KeikiGrow from Plant Hormones Canada) after flowering. However, it has been reported (Wang, Y-T.: Medium, Nutrition and Flower Induction in Potting Blooming Orchids, ASHS-2000 Symposium, http://primera.tamu.edu/orchids/wang.htm) that application of cytokinin (BA or a combination of PBA and GA), whereas this was shown to trigger spiking — by application to the mature pseudobulbs, but not the developing ones - in Dendrobium, was unable to induce off-season spiking in Phalaenopsis. One possible reason for this failure is that the development of the inflorescence in Dendrobium occurs through so-called pseudo-bulbs, which are absent in Phalaenopsis.

The inventors, however, have now found that application of a cytokinin yields the emergence of adventitious inflorescences in orchids, especially orchids with a monopodial growth, preferably orchids that do not have pseudobulbs, and more preferably Phalaenopsis or Doritaenopsis. Upon cytokinin application it appears that sometimes four or more additional flowers stalks appear. These additional spikes, incontrast to so-called secondary flower stalks, are about similar to the original one and can also bear a large amount of flowers (under optimal growing conditions).

The cultivation and production of potted orchids in general and specifically Phalaenopsis, nowadays has taken an enormous leap due to the availability of tissue engineering for large scale production of clones. Since the 1970's several tissue culture protocols specific for Phalaenopsis have been developed. These tissue explants (mostly explants from the flower stalk) are regenerated to plantlets in large facilities, which have capacities of producing millions of plantlets a year. It is also possible to cultivate the orchids from seed, which would yield a pottable plant after about 15 months. The grower obtains these small plantlets and cultivates them further (for at least another 20 weeks) before induction of the florescence is initiated. After about 6 weeks at lower temperature, it will take about another 16 weeks to grow the plant, now bearing a flower stalk, into a sellable product, which is a plant which has just started flowering. The cultivation from plantlet to flowering plant is almost exclusively done in greenhouses because of the requirements of temperature control. Normally the plants are held at a temperature of about 25 ⁰ C to about 30 ⁰ C, preferably about 27 °C. When flowering needs to be induced the temperature is lowered to about 15 °C to about 20 °C, preferably about 18 °C. In summer, in temperate climates, this would approximate the outside temperature, which means that this cold period can be achieved by only terminating the previous heating to 27 ⁰ C, whereupon the temperature in the

greenhouse would drop to the outside temperature. However, since this outside temperature is far from constant and — in hot summers — would easily be above the desired temperature of max 20 °C, many breeders opt for active cooling. It has now appeared possible to minimise the active cooling and still be able to induce the inflorescence by applying cytokinin. This treatment not only minimises the cold induction, but it appeared that more flower stalks are formed than with the normal cold induction. Whereas plants which have been cold induced normally would develop only one flower stalk (or two), treatment with cytokinin normally would give two flower stalks en in many cases more than two.

The number of flower stalks is related principally to the age of the plant (in fact the number of leaves and - defined thereby — the number of eyes from which primary flower stalks can grow). The top three leaves of the plant are too juvenile to be of influence on the formation of flower stalks, but every pair of leaves beneath them can form the basis of one flower stalk. The older the plant, i.e. the more (still or not any more present) leaves, the more possibilities the orchid has for the formation of flower stalks. In principle, thus, more than two (to maximally four) flower stalks could be generated from very old plants, but because this necessitates the use of old plants, it is not en economically cost-effective way to increase the number of flower stalks.

Further, typically plants of the genus Phalaenopsis and Doritaenopsis fit the above description. Som exceptions exist of plants, which standard give a multiplicity of flower stalks and yet should be classified within one of both genera. These are: P. amabilis and P. "Anthura Gold". For the purpose of this application, these are regarded as atypical plants, while th plants which normally would give one or sometimes two flower stalks in conventional culture are indicated as typical plants of the genus Phalaenopsis and Doritaenopsis.

Preferably synthetic cytokinines are used, most preferably BAP because of its low cost: purification of naturally occurring cytokinines is time consuming and expensive. Further it is know that synthetic cytokininies are not toxic in the concentrations used, nor for plants nor for animals, including humans (TAP Report for 6-benzyladenine, January 2004, at http://www.ams.usda.gov/nop/NationalList/6BenzyladenineFinal noCBI.pdf). Synthetic cytokinines according to the present invention are substances that show a cytokinine activity en are not to be found as such in nature, notably in plants (e.g. thidiazuron, benzyladenine and 6-benzylaminop urine). Application of the cytokinin can be performed in any conventional way, but in greenhouses with many plants, it is preferable to apply the cytokinin by spraying with an aqueous solution. The concentration of the cytokinin to be sprayed can vary, but should be in the range of about 100 ppm to about 1500 ppm, preferably about 250 ppm to about 1300ppm, more preferably about 500 ppm to about 1200 ppm, most preferably about 600-1000 ppm.

Spraying is done with an amount of about 100 liters of spraying solution for an amount of approximately 20.000 plants. Thus, each plant will receive about 1/200 liter, or 5 ml of spraying solution. Spraying can be performed using standard spraying equipment, such as 5- 25 Bar pressurepump with single or multiple nozzles as commonly used for application of fungicides and/or pesticides. Such equipment is commercially available. This equipment preferably nebulises the spraying solution, in order that both upper and under surface of the leaves come into contact with the spraying solution.

When recalculating the above mention preferred concentrations in amounts of cytokinine applied to individual plants, it would result in about 2 to about 30 mg per plant, preferably about 5 mg to about 26 mg per plant, more preferably 10 mg to about 24 mg per plant, most preferably about 20 mg per plant.

Next to administration via spraying solution, the cytokinine can also be applied according to other ways known in the art, e.g. through powdercoating, electrospray, through normal techniques for watering (e.g. ebb- and floodtechnique) or through direct application on or next to the plant, e.g. in the form of pastes, patches or strips, or plant sticks with slow release of the cytokinin to the substrate.

To increase the uptake of the cytokinin by the plants additives, such as co-solvents or wetting agents, can be added to the cytokinin solution. One co-solvent which is preferably used in the invention is DMSO, which preferably is added to the spraying solution to a final concentration of 1 liter per 100 liter spraying solution. As wetting agent the commercially available Zipper® (an organically modified trisiloxane, obtainable from Asepta, Delft., The Netherlands) can be used in a final concentration of 10-500 ml per 100 liter spraying solution, preferably 100 ml/liter. Generally all means that enhance the uptake of the cytokinin by the plant can be used. Next to the above discussed wetting agent Zipper®, other compounds that decrease the surface tension of the liquid are equally applicable. Also agents that enhance the penetration of the cytokinin, such as DMSO (Broome, O. C, Zimmerman, R.H., (1976) J. Amer. Soc. Hort. Sci. 101:28-30) can be useful in the present invention.

To further increase uptake of the cytokinin by the plants, the plants can be covered from the light source, directly after spraying, e.g. by a plastic tent. By thus inducing an artificial night environment the stomata of the plants will open, which facilitates uptake. Further, because of the low light level, evaporation of the spraying solution will minimalise, which means that the sprayed solution will stay in contact with plants for a longer time. Further, in the case of a tent, ventilation will be reduced, which decreases evaporation and increases the relative humidity of the air, which also enhances uptake. Preferably, covering of the plants only lasts for about 4-48 hours, since orchids need light to grow. After removal of the cover, plants are allowed

to continue to grow at the low temperature conditions for another three to seven weeks, preferably about five weeks. After that, the temperature regimen should be returned to the high temperature, i.e. preferably between about 18 ⁰ C and 25 ⁰ C, to develop the plants to marketable early flowering plants for another 8-23 weeks. If desired, plants can be 'harvested' already earlier and transported or marketed.

Administration of the cytokinin thus is preferably done during or at the end of the cold induction period. Most preferred is administration at the start or just before the period. However, it is also possible to have the administration done plenty of time before (maximally 10 weeks before, but preferably 1, 2 or 3 weeks before) or shortly after (maximally 5 weeks after) the cold induction period.

Growing conditions for the plants throughout the whole greenhouse stay are the same, except for the above discussed variations in light and temperature. Preferably this means that the plants are potted in pots with a diameter of 10-20 cm, with a growing medium of bark, coconut, Styrofoam, sphagnum moss and turf. Preferably they are stored on table-high racks, which facilitates handling. Light intensity on the surface of the pot is 3000- 15000 lux (see Schapendonk, A.H.C.M., "Belichting Phalaenopsis" , PT-Project 12170, productverslag, Productschap Tuinbouw). Normal daylight is maintained, but preferably in wintertime a lights on:lights off regime of 14:10 hours is used. Watering is preferably automated and is taken care of by a sprinkler system or any other spraying system, an ebb and flow system, via gutters running along the racks, or by submerging the pots in a water basin Nutrients and/or fertiliser are added to the water on a regular basis (EC 0,5 — 2, NPK 20-20-20). See also ook "Cultivation Guidelines Phalaenopsis Pot Plants, supra.

A preferred embodiment of the invention is the additional application of hormones which promote root growth, such as commercially available rooting hormone products. To ensure optimal uptake of water and

nutrients, a well-developed root system is essential, especially when more than one inflorescences need to be supported. Therefore, it is preferred to add rooting hormone substances to the water and nutrients to induce extra root growth to be able to support the extra inflorescences. Preferably, the rooting hormone is given when transplanting the pottable plants until 4 weeks prior to cooling for induction.

EXAMPLES

Example 1

In June 2004 three groups of Phalaenopsis plants have been induced 6 months after potting. For testing 100 plants were treated and 100 non- treated controls were used per variety. Both groups of plants were cultured as described above. Induction of spiking started at week 28 by spraying a solution of lOOOppm 6-BAP (BAP-10, Plantwise, Louisville, Kentucky USA), 0.1% Zipper, 1%DMSO] to the treated plants, while the controls did not receive this treatment. The results in Table 1 show the number of inflorescences per plant for the different testgroups.

Table 1

From this experiemnt it appears that groups of orchids of at least 5 plants per variety, or alternative 10, 20, 30, 40, 50, 100 or 1000 plants, can be produced with a method as described above, which have on average more than 2 inflorescences. To discriminate such a group from a random group of five or more control plants of one variety, it can be stated that the group of treated plants has at least an average of 2.9 or more, preferably 2.5 or more and more preferably 2.1 or more flowering stalks. These racemes can support flowering flowers or not.