Procedures have been developed for production of miniature flowering plants (also referred to as baby flowering plants or plantlets) by in vitro culture of meristems and/or seeds and/or microcuttings. A list of relevant publications is provided at the end of this section. The publications are identified in this discussion and in the following examples by first author and date of publication. Wang et al., 1988, Duan et al., 1994, and Wang et al., 1993 disclose in vitro production of baby orchids from floral stalk.

Duan et al., 1994 also discloses in vitro production of baby orchids from seeds. Chu et al., 1990 and Chu et al., 1993 disclose in vitro production of baby roses from dormant lateral buds. Chen, 1995 reports in vitro tissue culture of Celosia. Chu et al., 1993 states that the plantlets were cultured in GA 7 vessels. Wang et al., 1993 an Duan et al., 1994 state that the plantlets were cultured in 500 ml and 200 ml

flasks, respectively. The English language abstract accompanying the Wang et al., 1988 publication states that the plantlets"flowered in the flask"and that tissue culture induced miniature orchids"flowering in the test tube." There is no disclosure in these references of miniature flowering plants in a closed container.

Chen Y. N., 1995. List of in vitro flowering plants. Plant Physiology Communication. Vol. 31, No. 4, pp 318-320.

Chu, C. Y. and Knight, S. L., 1990."Effects of micropropagation techniques on growth and development of miniature roses,"Hortscience, Vol.

25, No. 9, p 138.

Chu, C. Y., Knight, and S. L., Smith, M. A. L, 1993.

"Effect of liquid culture on the growth and development of miniature rose (Rosa chinensis Jacq. Minima'),"Plant Cell,"Plant Cell, Tissue and Organ Culture, Vol. 32, No. 3, pp 329-334.

Duan, J. X. and Yazawa, S., 1994."In vitro floral development in X Doriella Tiny (Doritis pulcherrina X Kingiella philippinensis),"Scientia Horticulturae, Vol. 59, pp 253-264.

Wang, G. Y., Xu, Z., Chia, T., Wong P., and Chua, N. H., 1993."In vitro flowering of orchid (Dendrobium candidum),"Biotechnoloay_in Agriculture, pp 373-378.

Wang, X., Zhang, J. Y., Lian, H. K., and Gong, S. F., 1988."Studies on Cybidium ensifolium susin clonal propagation and floral bud differentiation by means of tissue culture,"Acta Horticulturae Sinica, Vol. 15, No. 3, pp 205-208.

Summary of the Invention This invention is a miniature flowering plant produced through in vitro culture and growing inside a closed culture container. The container comprises a dish-like base and a tube-like cover.

The dish-like base holds solid culture medium containing water and nutrients such as minerals, salt and vitamins. In a preferred embodiment, the culture medium also contains dye which is specially selected to enhance the attractiveness of the in vitro cultured flowering plant growing in the container. The tube-like cover and dish- like base are attached so that they do not separate in normal handling and transit and so that moisture is retained inside the container.

The cover, and desirably the entire container, is made of high transparency, high clarity material.

In a preferred embodiment, the interior of the container is coated with chemical to prevent water condensation on the inner surface of the container, giving a clear view of the in vitro flowering plantlet inside.

The shape and size of the container may vary to enhance the aesthetic and commercial value of

the miniature flowering plant. In a preferred configuration the dish-like base is in the form of a flat plate portion which is integral with a perpendicular wall portion. In top plan view the wall forms a closed figure within or corresponding to the perimeter of the plate. The tube-like cover has a lower wall portion which has substantially the same interior configuration and dimensions as the exterior configuration and dimensions of the wall portion of the dish-like base, so that the lower wall portion of the cover fits snugly around the wall portion of the base and the cover and base are held together by friction. The tube-like cover is closed at its top end with an integral top portion which can have any shape, such as a plane, a dome, a cone, a polyhedron such as a pyramid, or an irregular shape.

Description of the Preferred Embodiment Fig. 1A and 1B illustrate a presently preferred design of the container. Fig. 1A shows the dish-like base in longitudinal cross section.

Fig. 1B shows the tube-like cover in longitudinal cross section. The dish-like base is in the form of a cylinder portion 1 which is closed at one end by an integral concentric circular plate portion 2. The dish-like base contains culture medium 5.

The tube-like cover is in the form of a cylinder portion 3 which is closed at one end with by an integral concentric dome portion 4. The inside

and outside diameters of the dome portion 4 at its circumference are equal to those of the cover cylinder portion 3. The inside diameter of the cover cylinder portion 3 is substantially the same as the outside diameter of the base cylinder portion 1, so that the cover cylinder portion 3 fits snugly around the base cylinder portion 1.

Thus, the tube-like cover and dish-like base are held together by friction so that they do not separate in normal handling and transit and so that moisture is retained inside the container.

To prevent dislodging of the base and cover, we apply stick glue, elephant glue or Ulu glue at 3- 4 points around the outside of the cylinder portion 1 before putting the base and cover together.

We incorporate a bioactive antiseptic agent in the glue before it is applied. The bioactive agent is BTI=Micro-guarde sustained release agent, a product of Bernard Technologies, Inc., 919 North Michigan Avenue, Chicago, Illinois, USA.

The sustained release product generates chlorine dioxide, activated by ambient humidity that controls all sorts of microbial contamination/activity within the container. The glue and the bioactive antiseptic agent are mixed and delivered through a glue gun.

Dimensions of the container may vary, depending upon the size and shape of the plantlet.

Typical dimensions for a container having the

design illustrated in Fig. 1A and Fig. 1B are as follows. The outside diameter of base cylinder portion 1 and inside diameter of cover cylinder portion 3 are each 74 mm. The thickness of the cylinder portions 1 and 3 is 3 mm. The thickness of dome portion 4 is 3 mm at its circumference and 4 mm at its apex. The thickness of base plate portion 2 is 4 mm. The diameter of base plate portion 2 is 82mm. Outside height of the cover is 100mm and inside height is 96 mm. Height of the base cylinder portion is 29 mm.

In another embodiment of the same design, the outside diameter of base cylinder portion 1 and inside diameter of cover cylinder portion 3 are each 39 mm. The thickness of the cylinder portions 1 and 3 is 3 mm. The thickness of dome portion 4 is 3 mm at its circumference and 4 mm at its apex. The thickness of base plate portion 2 is 4 mm. The diameter of base plate portion 2 is 49mm. Outside height of the cover is 140mm and inside height is 136 mm. Height of the base cylinder portion is 30 mm.

The tube-like cover and desirably also the dish-like base are made of high transparency, high clarity material. Suitable materials include polycarbonate, poly (methyl methacrylate) and glass. Any other material that can withstand high heat (e. g. >160oC) and pressure (= or > atmospheric pressure) with high clarity can be used.

The interior of the cover is desirably coated with a chemical to prevent water condensation, giving a clear beautiful view of the baby flowering plants growing on colorful medium within the culture container. Examples of antifogging chemicals which can be used for this purpose are Sicanett, product of Anfora (SV), Italy, and Siclair0 (Nettoyant universal), an anti-statique manufactured by SI-International S. A. France.

Antifogging agent is sprayed directly onto inner surface of the container. It is left to dry before attaching the tube-like cover to the dish- like base. Waiting time of about 2 minutes is required. Trimming of roots and planting of budding plantlets can be performed while waiting for dry up of antifogging agent.

The interior of the cover is also desirably coated with an antiseptic agent such as BTI: Micro- guardeTM to aid in control of microbial contamination/activity within the container. We apply BTI: Micro-guardeTM by putting the cover on a piece of cheese cloth saturated with a solution of the antiseptic agent. The anti-microbial activity is activated immediately when BTI: Micro- guardeTM contacts the high humidity level inside the container. Contact time of 1-2 minutes is sufficient. This process is usually timed to coincide with selection of budding plantlets, as described below.

Desirably, dyes can be added to the culture medium to enhance the aesthetic effect of the baby plants. Examples of dyes which can be used are food dyes such as Bush Boake Allen (orange), Pandan Paste (green) and Raspberry Red (red).

Experimental procedures are described below for production of baby orchids, roses and celosia.

The orchids were cultivated in 500 ml flasks. The roses and celosia were cultured in GA 7 vesses.

Baby Orchid An in vitro system for generating baby flowering orchids was established. The Dendrobium sp was used for generating baby flowering orchid.

Two different parts of Dendrobium sp plant were used for in vitro tissue culture. The first culture used the floral stalk as the initial plant material (Wang et al., 1988, Duan et al., 1994, Wang et al., 1993). Floral stalk internodal fragments from a mother plant, each about 3 cm in length with an axial bud, were excised. Two ends of the fragment were sealed with wax. The sealed fragments were surface sterilized with 1% active chloride (Antihol) containing 0.1% of Tween 20, and incubated in this solution for 10 min. with constant shaking, followed by rinsing thoroughly with sterile-distilled water for 3 times. After removing bracteal leaves from the axial buds and the wax seals with forceps and scalpel blade, the fragments were used for in vitro culture. The

second method was to use mature Dendrobium seeds (Duan et al., 1994). Mature seeds of Dendrobium were dissected after sterilization. They were then cultured on the Murashige-Skoog (MS) agar medium supplemented with 0.3 mg/L naphaleneacetic acid (NAA) and 1.5% sucrose (pH 5.4) under diffused light conditions. Protocorm formation was induced 2 months after the first cultivation, and masses of protocorm were obtained in subsequent 2 months. The protocorms obtained from this culture were used for the in vitro flowering culture.

All experiments were carried out in 500 ml flasks with 80 ml culture medium. Ten clumps of protocorms or 10 fragments of the sectioned floral stalk were transferred into each flask. Each treatment had 5-6 replicates. The orchid cultures for in vitro flowering were placed under 16 hours light and 8 hours dark regime. The temperature was set at 25-27°C (light) and 21-23°C (dark), respectively. The frequency of the flower formation were counted in the period of 5 months.

It has been shown that the optimum conditions for in vitro flowering of Dendrobium are: the MS mediium with 2 mM spermidine, 2 mg/L abscisic acid to culture the protocorm for 1 month and then the protocorm tissues were transferred to MS medium containing 2 mg/L benzyladenine (BA) and continuing culture for another 2 months. With this method about 80% of the protocorms were induced flowering in 3 months.

Baby Rose An efficient propagation system for the low cost production of high quality miniature rose plants has been reported (Chu and Knight, 1990). According to this report, the dormant lateral buds of miniature roses were cultured on MS medium containing 8 g/L sucrose and 25 combinations of NAA and BA. Initial explant growth was achieved on a medium containing 0.001-0.01 ppm BA and 30 g/L sucrose. Growth was enhanced after culturing when dormant buds had more parental stem tissue.

Explants from the lowest 2 nodes with the shortest internode length exhibited the poorest growth.

The higher the photosynthetic photon flux (PPF), the faster explants grew and aged. Optimal PPF for initial growth was 20 umol/sec/sq. m.

Subculture microcuttings (at least 1 cm long) grew vigorously 1 month after cuttings were dipped in 1000 ppm indolebutyric acid and placed on a mist bench. Results indicate that micrpropagation of miniature roses has high potential for use in the commercial industry.

Baby Celosia Celosia (Celosia crislata) is an annual herbaceous plant which flowers in relatively short time. There has been a report for in vitro tissue culture of Celosia (Chen, Y. N., 1995). For in vitro culture of Celosia, the seeds were sterilized with 70% ethanol and then rinsed 3

times with sterile water. The seeds were germinated on the MS media. The in vitro tissue culture methods were followed the protocol as of those for in vitro tissue culture of baby flowering rose. Basically, the MS media were supplemented with 1 mg/L NAA, 0.3 mg/L BA and 8 g/L of sucrose. The Celosia seedlings were kept at 25°C with 16 hours light and 8 hours dark regime. Under these conditions, the Celosia plants flowered within 3 months.

The procedures which we used to produce baby flowering Celosia plants in the decorative closed culture container described above are summarized in Table 1.

TABLE 1

Time Frame Stage Seed germination 20 days, temperature 23-25°C I Young seedlings Light intensity = 1500 Lux Medium type: S Stage Microcutting and 20-40 days (depend on II into subculture cultivars) different media Conditions of culture as above Medium type: FS Stage Formatting of Medium type: FK III Floral buds Culture conditions: As above The shelf-life of in-vitro flowering baby plant is approximately 60-80 days (time allow for distribution, display Subculture into and sale) decorative container (2-3 plants/container)

Media types S, FS and FK are media which have been modified and improved to give the best growth rate, formation of floral buds, leaf sizes, colour and vigor of plantlets. We designated code number to the respective formulatlions for identification purposes. The chemical compositions and specific rate tested per medium for the production of in vitro flowering Celosia are shown in Table 2. The volume of media used was 50 ml per container.

TABLE 2

Medium type: FS 1S FK Content Amt (mg/L) Amt (mg/L) Amt (mg/L) 16501650NH4NO31650 KNO3 1900 1900 1900 KH2PO4 170 170 340 (170 x 2) 370370MgSO4.7H2O370 440440CaCl2.2H2O440 22.322.3MnSO4.4H2O22.3 8.68.6ZnSO4.7H2O8.6 H3BO3 6.2 6.2 6. 2 KI 0. 83 0. 83 0.83 NaMoO4. 2H20 0. 25 0. 25 0.25 CuSO,. 5H20 0.025 0.025 0.25 Cocu2. 6H2O 0. 025 0.025 0.25 Na2-EDTA 74.6 (37.3 x 2) 37.3 37.3 FeSO4. 7H20 55.6 (27.8 x 2) 27.8 55.6 Myo-Inositol 100 100 100 Glycine Niacin 0. 5 0. 5 0.5 Pyridoxine 0. 5 0. 5 0.5 Thiamine 0. 4 0. 4 0.4 Sugar 50g 50g 50g Agar 12g 12g 12g For sub-culturing in-vitro plantlets into the decorative container, the following criteria are observed and followed. Selection of suitable plantlets will be based on: a. Uniform height, between about 3-4 cm;

b. Thickness of stem, about 0.2 cm measured at base of the stem; c. Number of floral buds, each plantlet carries one floral bud of size 0.5 cm in diameter; d. Number of leaves, each plantlet carries 3 pairs of leaves.

Plantlets are trimmed to the same height and roots are removed. Three plantlets are planted near the center of the dish-like base, spaced at equidistance with gap allowing for full spread of leaves (i. e., leaves do not overlap). Upon completion, 3 plantlets are set in a cluster, nicely positioned near the center of the dish-like base.

Baby roses and orchids produced as described above can be transferred to the decorative closed culture container as described above in the same manner as baby Celosia plants. Besides orchids, roses and Celosia, other flowering plants which are derived from seed germination or tissue culture can also be induced to flower in vitro, and can be produced in the closed, decorative container in accordance with this invention.

Small cuttings of flowering plants, e. g., Chrysanthemum, can be induced to flower in vitro and produced in the closed, decorative container by controlling variables such as daylength and temperature. In addition, our culture system can be adapted to growing of miniature exotic and

colourful non-flowering plants, e. g., fern, moss, Venus fly traps, variegated foliage plants.

The products of this invention will appeal to a wide range of people, especially to plant lovers as house plants. The baby flowering plants can also serve as excellent teaching aids in schools and colleges as the in vitro culture technique permits assessment of flowering morphology and habit without going through a long growth gestation period usually required for flowering and maturity of ornamental plants. This will also be useful for biology classes studying plant physiology such as mineral requirements, etc.

The beauty of the baby flowering plants in closed, decorative container lies not only on the aesthetic value but also on the sheer simplicity of growing and maintaining the plants. One need not be an experienced plant grower to grow and enjoy the baby flowering plants as they are grown in solid medium enriched with nutrients in an aseptic culture container. Watering and application of fertilizers are also unnecessary as the plants will continue to grow autonomously for weeks. The only requirement is to place them under light and at ambient temperature. The closed, decorative container retains sufficient moisture for sufficient time that the plants can live for several weeks, frequently until they have outgrown their containers. When the plants have overgrown their containers, they can be removed and planted in any commercially available potting mixture. Small, handy, convenience and easy maintenance of the plants fit into the busy urban lifestyle of today.