WO2002037953A1 - Multiplication et fleurissement $i(in vitro) de cultivars de roses - Google Patents

Multiplication et fleurissement $i(in vitro) de cultivars de roses Download PDF

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Publication number
WO2002037953A1
WO2002037953A1 PCT/SG2000/000183 SG0000183W WO0237953A1 WO 2002037953 A1 WO2002037953 A1 WO 2002037953A1 SG 0000183 W SG0000183 W SG 0000183W WO 0237953 A1 WO0237953 A1 WO 0237953A1
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medium
culture medium
concentration
buds
culturing
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PCT/SG2000/000183
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English (en)
Inventor
Yan Hong
Mei Fang Yuan
Guang Yuan Wang
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Institute Of Molecular Agrobiology
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Application filed by Institute Of Molecular Agrobiology filed Critical Institute Of Molecular Agrobiology
Priority to AU2001215676A priority Critical patent/AU2001215676A1/en
Priority to PCT/SG2000/000183 priority patent/WO2002037953A1/fr
Priority to JP2002540555A priority patent/JP2004522423A/ja
Priority to CN00820009.2A priority patent/CN1455640A/zh
Priority to EP00978192A priority patent/EP1333714A1/fr
Priority to TW090127409A priority patent/TWI242405B/zh
Priority to AU2002215298A priority patent/AU2002215298A1/en
Priority to PCT/SG2001/000230 priority patent/WO2002037954A2/fr
Publication of WO2002037953A1 publication Critical patent/WO2002037953A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/002—Culture media for tissue culture
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants

Definitions

  • the present invention relates to the fields of plant husbandry, plant cloning and horticulture. Specifically, this invention relates to the vegetative propagation of a rose plant or tissue culture and in vitro induced flowering of rose plantlets derived from the plant or tissue culture. More particularly, the invention is directed to media formulations and efficient methods for the in vitro micropropagation, flower bud induction and flowering of roses and rose plants.
  • the rose plant belongs to the plant family Rosaceae Juss. This family is very large, with over 100 genera and 2000 herbaceous to woody species of plants as members. Many important food and ornamental plants are in the Rosaceae family, including for example strawberry, apple, almond, cherry, peach and blackberry. The exact species involved in the development of the present-day rose is not known. Most rose species are found in the temperate parts of the Northern Hemisphere, especially from southern China and the far East, to the Himalayas and Bengal into Ethiopia and west to North America from the Arctic Circle to New Mexico. Recurrent or perennial flowering rose plants introduced into Europe from the Far East at the end of the eighteenth century were the results of countless generations of breeding in China, India, and Japan. The rose is one of the world's most popular flowers. Roses are commonly grown as cut flowers, potted plants, or specimen plants in home gardens. There are numerous types of cut roses, including for example long stem standards, short stem standards, small flowered sweethearts and multi flowered bunch roses.
  • micropropagation also known as axillary bud propagation or in vitro asexual propagation, which is the method of propagating plants from axillary buds under sterile aseptic conditions.
  • Micropropagation has the advantage of fast propagation of a desirable plant into many genetically uniform plantlets.
  • Plantlets derived from micropropagation are also free of bacterial, fungal and viral infections and also are free of insect pests.
  • Micropropagation procedures involve preparation of explants from the relevant plant, culture of the explant on a medium supplemented with phytohormones, incubation, and recovery of true to type shoots (or shoots with roots) [Douglas, In: Methods in Molecular Biology, Vol. 6, W. Pollard, J. M. Walker, eds. (1990); George and Sherrington, Exegetics, Ltd. U.K., p.3 (1984); and Brown and Thorpe, In: Cell Culture and Somatic Cell Genetics of Plants, p49-65, 1. K. Vasil, ed. (1986)].
  • Micropropagation provides a potential cost effective means for the mass propagation of certain plants.
  • An important consideration of this technology is the ability to add significant economic value to the plants being propagated.
  • Examination of the relevant literature reveals that present propagation protocols are in general very inefficient at inducing the flowering of plantlets in vitro. Efficiency is dependent on the variety of plant used but is nevertheless low (about 10%) even for the best cultivars.
  • potted rose varieties with sizes ranging from 7.5 cm to 20 cm tall have become very popular due to their compact size and longer shelf life as compared to cut roses. Also, the ability to produce multiple flowers at reasonable cost has further expanded the popularity of potted roses. Breeding for miniature rose hybrids has also been carried out around the world. By way of example, there have been more than 300 miniature rose hybrids registered in the United States alone. The most prominent breeders for commercial miniature rose hybrids are Poulsen Roser ApS in Denmark (with registered trademark PARADE®) and De Ruiter's New Rose International in Netherlands. Miniature pot roses are displayed in gardens, patios or even in homes for decorative effect.
  • a miniature potted rose is somewhat limited for use as an indoor decoration, mainly due to the requirement for soil in the pot. Also, considering a total average height between 15-30 cm (including the pot), even the height of miniature potted plants is still too high for placement on tables, for example. Furthermore, due to the increased urbanization around the world, more and more people spend more quality time indoors in the home or office, for example. Thus there presently exists a demand for decorative rose plants with multiple flowers with long shelf life that are both clean and compact.
  • a method of micropropagation of a rose plant comprising culturing a stem bearing a node in a first culture medium comprising a cytokinin, an auxin, and gibberellin and then in a second culture medium comprising a cytokinin, an auxin, and gibberellin to produce a flowering rose plant capable of being transferred to soil.
  • the present invention is directed to the development of novel compositions and methods useful for the propagation of miniature rose plantlets and the efficient induction of in vitro flowering for the production of miniature roses without transfer to soil and the need for a pot.
  • the invention provides the roses flowered in an enclosed container on media for commercial and decorative purposes.
  • the invention provides methods for the vegetative propagation and multiplication of a rose plant or rose plant tissue culture material.
  • the invention provides a method for the in vitro induced flowering of multiplied plantlets derived from a rose plant or rose plant tissue culture material.
  • the invention provides compositions for the micropropagation and in vitro flowering of multiplied plantlets derived from a rose plant or rose plant tissue culture material.
  • rose shoots from potted plants are cultured on a first culture medium comprising inorganic nutrients, vitamins, a cytokinin, an auxin, and sucrose as a carbon source until buds form from the shoots.
  • Buds are cut from the shoots and transferred to an enclosed vessel containing the first culture medium for about fifty (50) days to produce plantlets and new buds.
  • the newly formed buds are cut and transferred to an enclosed vessel containing the first culture medium and cultured to multiply the plantlet and bud yield.
  • rose buds are excised and cultured again to produce plantlets on the first medium.
  • the plantlets are then transferred to a second culture medium comprising inorganic nutrients, vitamins, sucrose as a carbon source, the phytohormone thidiazuron, the cytokinin kinetin, an auxin and optionally myo-inositol to induce flower buds.
  • a second culture medium comprising inorganic nutrients, vitamins, sucrose as a carbon source, the phytohormone thidiazuron, the cytokinin kinetin, an auxin and optionally myo-inositol to induce flower buds.
  • plantlets are transferred to an elongation media comprising inorganic nutrients, vitamins, sucrose as a carbon source, a cytokinin, an auxin, and myo-inositol.
  • plantlets are then cultured on a culture medium comprising inorganic nutrients, vitamins and ampicillin for in vitro flowering.
  • plantlets are then transferred to a culture medium comprising inorganic nutrients, vitamins, sucrose as a carbon source, the cytokinin Zeatin, and inositol to induce flower buds.
  • plantlets are then transferred directly to medium comprising inorganic nutrients, vitamins and ampicillin for in vitro flowering.
  • Figure 1 depicts a flow chart showing the time required for completion of the various steps involved in the propagation and in vitro flowering of miniature rose cultivars.
  • Figure 2 shows a full view of in vitro rose plantlet flowering in an enclosed vessel.
  • Figure 3 shows a full view of enclosed vessels on shelf with rose plantlets flowering.
  • the invention provides methods for the vegetative propagation and multiplication of a rose plant or rose plant tissue culture material.
  • the invention provides a method for the in vitro induced flowering of multiplied plantlets derived from a rose plant or rose plant tissue culture material.
  • the invention provides compositions for the micropropagation and in vitro flowering of multiplied plantlets derived from a rose plant or rose plant tissue culture material.
  • rose shoots from potted plants are cultured on a first culture medium comprising inorganic nutrients, vitamins, a cytokinin, an auxin, and sucrose as a carbon source until buds form from the shoots.
  • Buds are cut from the shoots and transferred to an enclosed vessel containing the first culture medium for about fifty (50) days to produce plantlets and new buds.
  • the newly formed buds are cut and transferred to an enclosed vessel containing the first culture medium and cultured to multiply the plantlet yield.
  • rose buds are excised and cultured again to produce additional plantlets on the first medium.
  • the plantlets after multiplication of plantlets in a first media comprising a cytokinin, an auxin and sucrose as a carbon source, the plantlets are transferred to a second culture medium comprising inorganic nutrients, vitamins, sucrose as a carbon source, the phytohormone thidiazuron, the cytokinin kinetin, an auxin and myo-inositol to induce flower buds.
  • a second culture medium comprising inorganic nutrients, vitamins, sucrose as a carbon source, the phytohormone thidiazuron, the cytokinin kinetin, an auxin and myo-inositol to induce flower buds.
  • flower bud induction plantlets after flower bud induction plantlets are transferred to an elongation media comprising inorganic nutrients, vitamins, sucrose as a carbon source, a cytokinin, an auxin, and myo-inositol.
  • plantlets are then cultured on a culture medium comprising inorganic nutrients, vitamins and auxin for plant elongation followed by transfer of elongated plantlets to culture medium comprising inorganic nutrients, vitamins and ampicillin for in vitro flowering.
  • plantlets are then transferred to a culture medium comprising inorganic nutrients, vitamins, sucrose as a carbon source, the phytohormone zeatin, an auxin and myo-inositol to induce flower buds. Plantlets are then transferred directly to medium comprising inorganic nutrients, vitamins and ampicillin for in vitro flowering.
  • the rose plant derived from vegetative propagation and multiplication may be a flowering rose that is capable of being stored and displayed on the media of the present invention. Unflowered plantlets can be used in an additional propagation procedure.
  • a culture medium for tissue culture micropropagation of a rose plant is also an embodiment of the present invention and comprises about 1.0 to about 2.0 mg/L benzyladenine, 0.05 to about 0.1 mg/L indole acetic acid or naphthalene acetic acid and about 1.5 % to 2.0 % sucrose as a carbon source.
  • the culture medium contains about 2.0 mg/1 benzyladenine, 0.1 mg/L of an auxin, and 2 % sucrose.
  • the media of the present invention do not contain gibberellic acid.
  • a rose plant, including a flowering rose, produced by the method is also within the scope of the present invention.
  • the rose is preferably Rose, and most preferably a cultivar selected from the group consisting of Orange PARADE®, Fiesta PARADE®, Scarlet PARADE®, Bianca PARADE® and Frosty PARADE®.
  • the present invention is also directed to a rose tissue culture comprising a rose stem bearing a node contained in an enclosed vessel containing the culture medium of the present invention.
  • the rose stem may comprise at least one shoot, the rose tissue culture capable of producing a rose, for example, a flowering rose.
  • a rose explant is also within the scope of the present invention.
  • the rose explant may be selected from a rose stem bearing a node, a rose stem bearing a node and at least one shoot, and a rose shoot.
  • the rose explant may be contained in an enclosed vessel comprising culture medium and may be cultured until a rose plant is produced, a flowering rose plant, for example.
  • the present invention is directed, inter alia, to a flowering rose plant which may remain in culture without feeding or watering for a period of time which permits the plant to continue to be sustained. Once the rose flowers in the enclosed vessel, it typically may be maintained for more than one month.
  • the only limitation on the enclosed vessels contemplated for use in the present invention is that they must be able to maintain without leaking the solid culture medium containing water and nutrients such as minerals, salt and vitamins.
  • the culture medium may also contain one or more dyes which are specially selected to enhance the attractiveness of the in vitro cultured flowering plant growing in the container.
  • a cover and base together comprise the enclosed vessel and the cover and 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 and the interior of the container is optionally 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.
  • Suitable materials include polycarbonate, poly (methyl methacrylate) and glass. Any other material that can withstand high heat (e.g. >160°C) and pressure ( ⁇ atmospheric pressure) with high clarity can also be used.
  • antifogging chemicals which can be used for this purpose are Sicanett®, product of Anfora (SV), Italy, and Siclair® (Nettoyant universal), an anti-statique manufactured by Si-International S.A. France.
  • the culture medium of the present invention may further comprise a nutritive medium.
  • the medium is a modified Murashige and Skoog (MS) basal medium that has been described elsewhere (Murashige et al., Physiol. Plant., Vol. 15, pp. 473-97 (1962).
  • Modified MS medium is MS medium containing Gamborg's B5 vitamins (final concentration of 10 mg L thiamine hydrochloride, 1 mg/L nicotinic acid, 1 mg/L pyridoxine, 100 mg/L myo-inositol), pH 5.8, with 2% sucrose and gelled by 0.30% phytagel. In the preferred embodiment, this is the medium that is supplemented with the various additional phytohormones and other additives as disclosed herein.
  • auxins used in the practice of the preferred embodiments of the invention include indole acetic acid and naphthalene acetic acid.
  • the cytokinin include thidiazuron, kinetin 6-benzyladenine and zeatin.
  • Benzyladenine may be substituted with another natural or synthetic cytokinin selected from the group consisting of 6-benzylaminopurine riboside; 6-( ⁇ - ⁇ -dimethylallylamino)purine; DL-Dihydrozeatin; t-zeatin riboside; zeatin; N-(2-Chloro-4-pyridyl)-N'-phenylurea; N-benzyl-9- (2-tetrahydropyranyl)adenine; kinetin; kinetin riboside; and the like, wherein the cytokinin may be used alone or in combination with one or more other cytokinin.
  • cytokinin selected from the group consisting of 6-benzylaminopurine riboside; 6-( ⁇ - ⁇ -dimethylallylamino)purine; DL-Dihydrozeatin; t-zeatin riboside; zeatin; N-(2-Chloro-4-pyridy
  • Indole acetic acid may be substituted with another natural or synthetic auxin selected from the group consisting of naphthalene acetic acid, indole butyric acid, picloram, dicamba, and the like, usually at a concentration of about 0.03 to about 0.3 mg/1, wherein the auxin may be used alone or in combination with one or more other auxin.
  • the media may be modified such that hormonal constituents are provided for specific varieties of plants.
  • Nutrient levels may be modified in the basal media when inducing flowering.
  • Murashige and Skoog (MS) medium which provides the general nutritional and growth requirements of tissue culture plant cells, may be substituted with other conventional culture or growth medium such are known in the art. It will readily be appreciated by the skilled artisan that the precise amount of each component to be employed will depend upon the type of rose to be cultured. Typically, the compounds are initially employed at the lower levels, and the amounts increased as necessary to achieve the desired effect.
  • the concentration of thidiazuron when added to the composition and method for flower bud induction is between 0.4-0.5 mg/L.
  • the concentration of zeatin is between 0.5 and 1.0 mg/L.
  • the optimal concentration is about 2 mg/L.
  • the optimal concentration is about 0.1 mg/L.
  • naphthalene acetic acid When naphthalene acetic acid is the auxin added to the media of the present invention, it is added at a concentration of less than 2 mg/L. Optimally, the concentration of naphthalene acetic acid is between 0.05 and 0.1 mg/L.
  • indole acetic acid When indole acetic acid is the auxin added to the media of the present invention, it is added at a concentration between 0.05 and 0.1 mg/L. Optimally, the concentration of indole acetic acid is. 0.1 mg/L for multiplication of plantlets. When indole acetic acid is added to the media used for the elongation of plantlets with induced flower buds, the optimal concentration is about 1.0 mg/L.
  • micropropagation conditions will be readily apparent to those skilled in the art and include choosing donor tissues for culture initiation which come from plants of appropriate genotype and physiological and development states.
  • variation of explant source, plant variety, and physical environment in which the cultures are grown are contemplated by the present invention.
  • gas environment, temperature and light conditions may be varied to provide for an improved rose plant contained in an enclosed vessel.
  • the optimal temperature for flower bud induction will be ⁇ 25 °C.
  • Any suitable gelling agent such as and not limited to PHYTAGEL.TM. comprising gellan gum, GEL- RITE.TM. comprising gellan gum; AGARGEL.TM.
  • gellan gum comprising gellan gum; agar such as and not limited to agar types A, E, and M, High Gel Strength, Purified, Bacteriological Flake; AGARGEL.TM. a blend of agar and PHYTAGEL.TM.; agarose, such as and not limited to Type VII; alginic acid; carrageenan; transfergel or hydroxyethylcellulose; and the like.
  • a rose explant is an embodiment of the present invention.
  • the rose explant may be contained in an enclosed vessel comprising the aforementioned culture medium, and may be cultured until a rose plant is produced, a flowering rose plant, for example.
  • the explants may be obtained from a plant, such as and not limited to the genus Rosa, including and not limited to the hybrid rose Rosa hybrida, such as and not limited to the cultivars Orange PARADE®, Fiesta PARADE®, Scarlet PARADE®, Bianca PARADE® and Frosty PARADE® and the like; Rosa damascene, Rosa multiflora, and Rosa gallica, and the like.
  • the tissue culture techniques of the present invention comprise culturing a rose explant in an enclosed vessel such as and not limited to a petri dish, a test tube, a flask, an eppendorf tube, a baby food jar, a canning jar, and any other enclosed container capable of supporting the growth of the rose plant of the present invention in accordance with the methods of the present invention.
  • an enclosed vessel such as and not limited to a petri dish, a test tube, a flask, an eppendorf tube, a baby food jar, a canning jar, and any other enclosed container capable of supporting the growth of the rose plant of the present invention in accordance with the methods of the present invention.
  • a rose including a flowering rose, produced by the methods of the present invention, is also within the scope of the present invention.
  • the rose plant may be contained within the enclosed vessel.
  • Rose tissue culture or plantlet is also within the scope of the present invention.
  • the rose tissue culture is capable of producing a rose, such as a flowering rose.
  • the rose in an enclosed vessel may be used to screen new cultivars for flower color and form.
  • plants may be subjected to mutational treatment during tissue culture of rose callus, explants or axillary bud. Mutated rose plants may then be grown in the culture of the present invention and screened for those with altered flower color, form, disease resistance, growth vigor, cold and heat sensitivity, and any other valuable trait.
  • the present invention also permits performing cross pollination of rose plants in culture. For example, since flowers contain visibly developed male and female organs, such flowers may be pollinated with pollen derived from the same flower or from a plant of any other source. Alternatively, flowers may be propagated in vitro and stored or displayed in a clear, transparent container for decorative purposes.
  • Benzyladenine is a plant hormone of the cytokinin type.
  • Zeatin is a plant hormone of the cytokinin type.
  • Naphthalene acetic acid or 2-naphthylacetic acid is a plant hormone of the auxin type.
  • Indole acetic acid or 3-indoleacetic acid is a plant hormone.
  • cytokinin refers to plant hormones. In low concentrations, these organic substances promote elongation of root cells.
  • auxins refers to plant hormones. In low concentrations, these organic substances promote elongation of plant shoots and control other specific growth effects. Auxins include 3-(3-indolyl)-propionic acid (IP A), abscisic acid (ABA), and 2,4-D, Napthalene acetic acid and GA3.
  • cultivar refers to a commercially valuable, horticulturally derived, variety, as distinguished from a naturally occurring variety.
  • micropropagation refers to in vitro asexual clonal reproduction of plants wherein large numbers of new shoots may be obtained in a short time period from the induced buds of parental plants by culture in a medium containing plant hormones, minerals, vitamins and carbohydrates in the appropriate concentrations.
  • RMS 0 Modified MS media containing full strength Murashige Skoog inorganics nutrients and Gamborg's B5 vitamins (final concentration of 10 mg/L thiamine hydrochloride, lmg/L nicotinic acid, lmg/L pyridoxine, 100 mg/L myo-inositol), pH 5.8, with 2% sucrose and gelled by 0.30% phytagel. At different developmental stages, the medium was supplemented with different phytohormones.
  • RMS RMS 0 supplemented with 2 mg/L 6-benzyladenine and 0.05 mg/L napthalene acetic acid.
  • RMS 2 RMS 0 with 3% sucrose and supplemented with 0.4-0.5 mg/L thidiazuron, 0.05 mg/L napthalene acetic acid, 0.1 mg/L kinetin and 400 mg/L inositol.
  • RMS 3 RMS 0 with 3% sucrose and supplemented with 0.5-1.0 mg/L zeatin, 0.05 mg/L napthalene acetic acid, and 400 mg/L inositol.
  • RMS 4 RMS 0 with 3% sucrose and supplemented with 0.1 mg/L benzyladenine, 1.0 mg/L indole acetic acid and 400 mg/L myo-inositol.
  • RMS 5 RMS 0 with 2% sucrose and supplemented with 2 mg/L benzyladenine and 0.1 mg/L indole acetic acid.
  • young shoots with axillary buds are used as explants.
  • the explant is surface sterilized before use.
  • Many sterilizing techniques are available in the art for the purpose of preparing explant for culture. Such techniques involve dipping the explant in the solution containing at least one sterilizing agent.
  • sterilizing agents include, sodium hypochlorite, calcium hypochlorite, mercuric chloride, ethyl alcohol etc.
  • young shoots of 10-12 cm long from mature plants are stripped of the outer leaves and washed under running water first, and then sterilized with 0.1% HgCl 2 for 5-10 minutes and then washed completely with running water for more than 30 minutes.
  • the shoots are then immersed in 10-15% CloroxR (containing 5.25% sodium hypochlorite) solution for 10-15 minutes. Finally, the shoots are rinsed 4-6 times with sterile distilled water.
  • Young shoots can alternatively be derived from rose plant tissue culture using techniques well known within the art. It is thus contemplated that young shoots obtained from tissue culture may be used in place of the young shoots prepared from mature plants without departing from the scope of the present invention.
  • the explants are placed on solid media and cultured under 16 hr photo periods with a light intensity of 4500-5500 lux (provided by daylight type lights). When the plantlets are exposed to the above lighting conditions, the temperature is maintained at 23°C. Alternatively, when the light source is removed, the plantlets are maintained at 19°C.
  • Whole shoots are cultured on RMS, [RMS 0 supplemented with 2 mg/L 6-Benzyladenine, 0.05 mg/L Naphthalene acetic acid]. The explants turn green gradually and will usually form new buds after 10 to 15 days. The buds are cut from shoots at this point in time in preparation for multiplication of the plantlet.
  • Multiplication of plantlets is the process where whole plant material is obtained from a juvenile or rejuvenated growing plant, typically at a growing point or area of rapidly dividing cells at the tip of a root or shoot, referred to as meristem material.
  • the shoot material is placed in a predefined hormonal and nutritional medium to produce explants which gradually form plantlets having new buds. Individual buds are then removed from such explants and cultured in the same predefined hormonal and nutritional medium as above to produce multiple plantlets from a single whole shoot.
  • Such asexual reproduction of plants from a single parent allows cloning of plant progeny having identical genetic characteristics to those of the parent. Cut buds are transferred to RMS, medium and cultured for about 50 days. Utilizing the above procedures, buds grow into plantlets and new buds form on these plantlets. The newly formed buds are transferred when ready to be cut onto new RMS, medium for propagation and multiplication of plantlets.
  • the multiplied ratio will usually range from 4 to 7 times the original starting number of explants.
  • Flower bud induction is critical for in vitro flowering at commercial scale. There are two alternative approaches to flower bud induction contemplated by the present invention.
  • following multiplication flower buds are induced by culturing the multiplied plantlets on RMS 2 medium in an enclosed vessel.
  • the induction culturing lasts about fifty (50) days.
  • the plantlets are transferred to an enclosed vessel containing RMS 4 for an elongation step which lasts about fifteen (15) to thirty (30) days.
  • elongation plantlets are transferred to MS 0 with 20 mg/L to 50 mg/L ampicillin. Flowers usually open within ten (10) to twenty (20) days.
  • following multiplication flower buds are induced by culturing the multiplied plantlets on RMS 3 medium in an enclosed vessel for a period of about fifty (50) days. Following induction, plantlets are transferred directly to MS 0 with 20 mg/L to 50 mg/L ampicillin. Flowers usually open within fifteen (15) to twenty (20) days.
  • EXAMPLES The present invention is further detailed in the following examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described infra are utilized.
  • This example demonstrates the induction of flower buds on medium augmented with thidiazuron.
  • Young shoots with axillary buds from mature plants were used as explants.
  • Rose explants were placed on solid media and cultured under 16 hr photoperiods with a light intensity of 4500-5500 lux (provided by daylight type lights) and a temperature of 23°C with lighting and 19°C without lighting.
  • Whole shoots were cultured on RMS,.
  • the explants turned green gradually and formed new buds after 10 to 15 days.
  • the buds were cut from shoots.
  • Multiplied plantlets (> two times multiplication) were cultured on RMS 2 to induce floral buds. After flower bud induction, plantlets were transferred onto elongation media for in vitro ' flowering.
  • Plantlets were cultured on the elongation media, RMS 4 for 15-30 days before transferring into MS 0 with ampicillin at concentrations between 20-50 mg/L. Flowers opened within 10-20 days following transfer to MS 0 plus ampicillin. Forty to fifty percent plantlets yielded flowers and more than 80% of the flowers were normal in appearance. Unflowered plantlets could go back to propagation. The shelf life of flowers cultured was more than 1 month at less than 25 °C. EXAMPLE 2
  • This example demonstrates the induction of flower buds on medium augumented with zeatin.
  • Young shoots with axilary buds from mature plants were used as explants.
  • Rose explants were placed on solid media and cultured under 16hr photoperiods with a light intensity of 4500- 5500 lux (provided by daylight typelights) and a temperature of 23°C with lighting and 19°C without lighting.
  • Multiplied plantlets (> two times multiplication) were cultured on RMS 3 .
  • plantlets with flower buds were transferred directly into MS 0 with ampicillin 20-50 mg/L for flowering. Flowers would be opened within 15-20 days. Forty to fifty percent plantlets yielded flowers and more than 80% were normal. Unflowered plantlets could go back to propagation.

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Abstract

La présente invention concerne des compositions et des procédés de micropropagation d'une rose et de fleurissement in vitro de cette rose. De jeunes pousses sont induites pour produire des bourgeons dans une cuve fermée contenant un premier milieu de culture comprenant de la benzyladénine, une auxine et 2 % de sucrose comme source de carbone. Les bourgeons sont excisés et cultivés sur un milieu en vue de leur propagation et de la multiplication des plantules. Les plantules sont ensuite transférés dans un milieu comprenant du thidiazuron, une auxine et du myo-inositol en vue de l'induction de bourgeons de fleurs, puis de la culture sur un milieu comprenant du benzyladénine et une auxine pour l'allongement des plantules et enfin de la culture dans le cinquième milieu sans phytohormone pour le fleurissement. Par ailleurs, après la propagation et la multiplication, les plantules sont transférés dans un milieu à base de zéatine, d'auxine et de myo-inositol en vue de l'induction des bourgeons avant de les cultiver sur un milieu sans phytohormone pour l'allongement et le fleurissement.
PCT/SG2000/000183 2000-11-09 2000-11-09 Multiplication et fleurissement $i(in vitro) de cultivars de roses WO2002037953A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
AU2001215676A AU2001215676A1 (en) 2000-11-09 2000-11-09 Multiplication and in vitro flowering of rose cultivars
PCT/SG2000/000183 WO2002037953A1 (fr) 2000-11-09 2000-11-09 Multiplication et fleurissement $i(in vitro) de cultivars de roses
JP2002540555A JP2004522423A (ja) 2000-11-09 2000-11-09 バラ栽培品種の増殖およびインビトロ開花
CN00820009.2A CN1455640A (zh) 2000-11-09 2000-11-09 蔷薇属栽培品种的繁殖和体外开花
EP00978192A EP1333714A1 (fr) 2000-11-09 2000-11-09 Multiplication et fleurissement $i(in vitro) de cultivars de roses
TW090127409A TWI242405B (en) 2000-11-09 2001-11-05 Multiplication and in vitro flowering of rose cultivars
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1750497A2 (fr) * 2004-05-28 2007-02-14 The University Of Toledo Procede permettant de produire un fleurissement in vitro direct et des semences viables a partir de cotyledons, de radicules et d'explants de feuilles, et plantes produites de cette maniere
CN102662372A (zh) * 2012-05-09 2012-09-12 漯河伊人黑玫瑰有限责任公司 基于b/s架构和物联网的黑玫瑰快速繁殖管理系统及方法
CN102986535A (zh) * 2012-12-14 2013-03-27 西南林业大学 无籽刺梨种苗快速扩繁的方法
CN104115743A (zh) * 2013-04-27 2014-10-29 吉林师范大学 一种微型月季花诱导方法
CN104365482A (zh) * 2014-11-17 2015-02-25 苏州市新巷农艺科技园 一种微型月季组培的繁殖方法
US20170000112A1 (en) * 2013-12-24 2017-01-05 Enhold B.V. Method of preserving cut roses during transportation and storage and shipment kit containing cut roses
CN113207678A (zh) * 2021-05-18 2021-08-06 中农实创(北京)环境工程技术有限公司 一种玫瑰花的培育方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN100355334C (zh) * 2005-12-16 2007-12-19 江苏阳光生态农林开发股份有限公司 狗蔷薇的组织培养和育苗方法
CN100409740C (zh) * 2006-06-29 2008-08-13 北京锦绣大地农业股份有限公司 密闭容器中鸡冠花成花培养基及其诱导开花培养方法
CN103355174A (zh) * 2013-08-05 2013-10-23 黑龙江省农垦科学院 一种抗寒月季低成本高效工厂化育苗方法
CN106489739A (zh) * 2016-11-25 2017-03-15 华南农业大学 一种红花丰花月季试管花的生产方法
CN111084107A (zh) * 2020-02-18 2020-05-01 美尚生态景观股份有限公司 一种麦李腋芽诱导及继代增殖培养方法
CN111374057A (zh) * 2020-04-24 2020-07-07 黑龙江省科学院大庆分院 一种耐寒丰花月季环保高效微繁殖方法
CN115299344A (zh) * 2022-08-17 2022-11-08 河北高泽科技有限公司 一种欧月组织培养繁殖方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996024660A1 (fr) * 1995-02-09 1996-08-15 Novaflora, Inc. Micropropagation de plants de rosiers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996024660A1 (fr) * 1995-02-09 1996-08-15 Novaflora, Inc. Micropropagation de plants de rosiers

Non-Patent Citations (21)

* Cited by examiner, † Cited by third party
Title
BRAGANTIA, vol. 51, no. 2, 1992, pages 111 - 119, ISSN: 0006-8705 *
BRESSAN P H ET AL: "FACTORS AFFECTING IN-VITRO PROPAGATION OF ROSE ROSA-HYBRIDA", JOURNAL OF THE AMERICAN SOCIETY FOR HORTICULTURAL SCIENCE, vol. 107, no. 6, 1982, pages 979 - 990, XP002172738, ISSN: 0003-1062 *
CAMPOS P S ET AL: "MASS PROPAGATION OF THE DWARF ROSE CULTIVAR ROSAMINI", SCIENTIA HORTICULTURAE (AMSTERDAM), vol. 43, no. 3-4, 1990, pages 321 - 330, XP001010422, ISSN: 0304-4238 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1982, KHOSH-KHUI M ET AL: "MICRO PROPAGATION OF NEW-WORLD AND OLD-WORLD ROSE SPECIES", XP002172746, Database accession no. PREV198375057035 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1986, ALEKHNO G D ET AL: "CLONAL MICROPROPAGATION OF ROSES", XP002172745, Database accession no. PREV198783107558 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1989, DOUGLAS G C ET AL: "MICROPROPAGATION OF FLORIBUNDA GROUND COVER AND MINIATURE ROSES", XP002172744, Database accession no. PREV199089025674 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1992, TOMBOLATO ANTONIO FERNANDO CAETANO ET AL: "Micropropagation of rose varieties: In vitro propagation of root stock Rosa Shafter.", XP002172743, Database accession no. PREV199396128986 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1993, SHEVADE A ET AL: "In vitro shoot and floral organogenesis from stamen explants from a Rhododendron PJM group clone.", XP002172742, Database accession no. PREV199497122939 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1995, BARNA K S ET AL: "Effects of thidiazuron of micropropagation of rose.", XP002172741, Database accession no. PREV199598176884 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 1995, ROSU A ET AL: "The development of putative adventitious shoots from a chimeral thornless rose (Rosa multiflora Thunb. ex J. Murr.) in vitro.", XP002172740, Database accession no. PREV199698593724 *
DATABASE BIOSIS [online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; 2000, KANE MICHAEL E: "Micropropagation and in vitro flowering of rose.", XP002172739, Database accession no. PREV200100238318 *
FIZIOLOGIYA I BIOKHIMIYA KUL'TURNYKH RASTENII, vol. 18, no. 5, 1986, pages 489 - 493, ISSN: 0256-1425 *
IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY PLANT, vol. 31, no. 1, 1995, pages 44 - 46, ISSN: 1054-5476 *
JOURNAL OF HORTICULTURAL SCIENCE, vol. 57, no. 3, 1982, pages 315 - 320, ISSN: 0022-1589 *
JOURNAL OF HORTICULTURAL SCIENCE, vol. 70, no. 6, 1995, pages 901 - 907, ISSN: 0022-1589 *
KHAN M A ET AL: "IN-VITRO PROPAGATION OF ROSA-HYBRIDA CULTIVAR QUEEN ELIZABETH", PAKISTAN JOURNAL OF SCIENTIFIC AND INDUSTRIAL RESEARCH, vol. 33, no. 9, 1990, pages 396 - 398, XP001010431, ISSN: 0030-9885 *
MA YAN ET AL: "Propagation of rose species in vitro.", IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY PLANT, vol. 32, no. 2, 1996, pages 103 - 108, XP001010420, ISSN: 1054-5476 *
MARCELIS-VAN ACKER C A M ET AL: "Development of axillary buds of rose in vitro.", SCIENTIA HORTICULTURAE (AMSTERDAM), vol. 63, no. 1-2, 1995, pages 47 - 55, XP001010421, ISSN: 0304-4238 *
PLANT CELL TISSUE AND ORGAN CULTURE, vol. 19, no. 1, 1989, pages 55 - 64, ISSN: 0167-6857 *
PLANT TISSUE CULTURE CONCEPTS AND LABORATORY EXERCISES., 2000, CRC Press LLC 2000 Corporate Blvd., N.W., Boca Raton, FL, 33431, USA, pages 119 - 123, ISBN: 0-8493-2029-1 *
SCIENTIA HORTICULTURAE (AMSTERDAM), vol. 56, no. 2, 1993, pages 163 - 170, ISSN: 0304-4238 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1750497A2 (fr) * 2004-05-28 2007-02-14 The University Of Toledo Procede permettant de produire un fleurissement in vitro direct et des semences viables a partir de cotyledons, de radicules et d'explants de feuilles, et plantes produites de cette maniere
EP1750497A4 (fr) * 2004-05-28 2009-08-19 Univ Toledo Procede permettant de produire un fleurissement in vitro direct et des semences viables a partir de cotyledons, de radicules et d'explants de feuilles, et plantes produites de cette maniere
CN102662372A (zh) * 2012-05-09 2012-09-12 漯河伊人黑玫瑰有限责任公司 基于b/s架构和物联网的黑玫瑰快速繁殖管理系统及方法
CN102986535A (zh) * 2012-12-14 2013-03-27 西南林业大学 无籽刺梨种苗快速扩繁的方法
CN102986535B (zh) * 2012-12-14 2014-03-26 西南林业大学 无籽刺梨种苗快速扩繁的方法
CN104115743A (zh) * 2013-04-27 2014-10-29 吉林师范大学 一种微型月季花诱导方法
US20170000112A1 (en) * 2013-12-24 2017-01-05 Enhold B.V. Method of preserving cut roses during transportation and storage and shipment kit containing cut roses
CN104365482A (zh) * 2014-11-17 2015-02-25 苏州市新巷农艺科技园 一种微型月季组培的繁殖方法
CN113207678A (zh) * 2021-05-18 2021-08-06 中农实创(北京)环境工程技术有限公司 一种玫瑰花的培育方法

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