Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Culture – maintenance – or preservation techniques – per se
Utility Patent
1998-08-04
2001-01-02
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Culture, maintenance, or preservation techniques, per se
C435S420000, C435S430100, C047S05810R
Utility Patent
active
06168952
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for the floral induction of orchid plants grown aseptically using in vitro system. It can be used for the application of biotechnological purposes and for orchid hybrid breeding.
2. Description of Related Art
Orchids are well-known to have long-time juvenile stage after seed germination and after in vitro plant regeneration. It takes from about couple of years up to 10-13 years to get flowering orchid plant from the seed. Due to various variability in orchids living forms and specific biological properties of different species, hybrids, varieties and cultivars, there is no uniform methodology and even approach to make the juvenile period short and to induce orchid flowering with commercially employed ones. Various treatments were applied traditionally by farmers and researchers using the treatments of long/short day regimens, chilling, high temperature, phytohormone, retardants or specifically designed chemicals etc. to orchid plants. Nevertheless those approaches mentioned above are partially effective for mature orchid plants. In vitro techniques was proved to be applicable for controlling blooming process in higher plants, at least, at certain cases.
Several reports to produce precocious flowering in in vitro system have been published. One of the most efficient methods inducing in vitro orchid flowering was described by Wang et al (1993). This method was applied to
Dendrobium candidum
and includes the following steps:
(A) Protocorm, shoots (without roots) and plantlets (with roots) were obtained from 4-5 months old surface sterilized mature capsules;
(B) These explants mentioned in (A) are transferred to the medium containing abscisic acid (ABA) for two months;
(C) Pre-induced explants were cultured in the medium containing 6-benzyladenine to induce flower bud formation for about 5 months.
In the second method (Duan and Yazawa 1995), three main steps were employed with:
(A) Adventitious shoots formed from nodal sections of floral stalks of Phalaenopsis;
(B) Propagation and growth of floral stalk derived from shoots on Hyponex medium;
(C) Induction of floral bud formation on Vacin-Went medium containing 6-benzyladenine using 9 month old Phalaenopsis shoots.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for inducing in vitro early flowering orchids regenerated from the specific vegetative form named rhizome or protocorm like bodies (PLB). This is achieved by 3-4 month old orchid plants regenerated from rhizome and PLB with root excision. Those explants with root excision were cultured on the medium containing low nitrogen content, enhanced phosphorus content and 6-benzyladenine.
Further scope of the applicability of the present invention will become apparent from the detailed description provided below.
DETAILED DESCRIPTION OF THE INVENTION
The following detailed description is provided to help in practicing the present invention. As employed below, the phrase “low nitrogen” preferably denotes that the total nitrogen content is between about 2 mM and 8 mM, and the most preferable one is about 3 mM.
“High phosphorus” denotes that the total phosphorus content is between about 3 mM and 9 mM, and the most preferable one is 6.25 mM.
“Root excision” denotes that plant roots are cut off about 95% to 100% of roots, and the most preferable ones are cut off 100% of roots.
Step (1) Orchid rhizome and PLB are cultured on Murashige and Skoog (MS) medium (1962) supplied with auxins, at 25-28° C., under an illumination of 4,000-8,000 lux, for 14-18 hours a day. The medium can be MS medium, containing 4% (w/v) of sucrose, pH 5.7. This medium is hereinafter referred to as “plant growing medium”. Linsmayer and Skoog's medium (Linsmayer and Skoog 1965), Eriksson's medium (Eriksson 1965), and other standard plant tissue culture and orchid media can also be employed in place of “MS medium”. Of sugars as carbon source in the medium, glucose, fructose, maltose etc, can be employed in place of sucrose. The sugar concentration can be in the range from 3-6%, preferably 4%, and the pH can be in the range from 4-8, preferably 5.2-6.5. The number of explants can be 1-50, preferably 20-30 per liter. The volume of vessel may be 0.3-1.0 liters, and the volume of medium per 1 liter vessel can be 0.1-0.4 liters, preferably 0.25-0.3 liters.
Step (2) Then the resulted explants are transferred to MS medium supplemented with cytokinin or cytokinin and auxin with high cytokinin/auxin ratio to regenerate plants. The young plants regenerate from the rhizome 90-150 days after transfer to the medium.
Step (3) The resulted about 3 to 4 months old plants undergo root excision and are transferred to the fresh medium containing low nitrogen, high phosphate and cytokinin. Of cytokinins in the medium, 6-benzylaminopurine, kinetin, zeatin, thidiazuron or other routinely applied cytokinins can be employed, preferably 6-benzylaminopurine. The concentration of cytokinin can be in the range from 10-50 &mgr;M, preferably 35-45 &mgr;M. Photoperiod and temperature regimen should be the same as in the first step. The explants are planted in the range from 1 to 5, preferably 2 per 1 liter vessel. In the step mentioned above, orchid plants eventually change vegetative growth to reproductive development displaying producing flower stalks with flower buds.
Step (4) In 1.5-3 months from the beginning of the previous step, the resulted induced plants should be transferred to the medium containing about 20-40 mM, preferably 30 mM of total nitrogen, high phosphate, and cytokinin, preferably, 6benzyladenine. The concentration of cytokinin can be in the range 0-40 &mgr;M, preferably 22 &mgr;M.
Plants cultured on the medium mentioned above develop flowers and can posses fruit onset with self pollination within about 30 to 90 days after the combined treatment. The resulted plants can be used for the purposes of orchid hybrid breeding and for the development of commercial product using flowering orchid plants in tissue culture vessel.
Differences Between the Present Invention and Prior Art Methods
A characteristic feature of the present invention is that aseptically regenerated and cultured orchid plants undergoing root excision are efficiently induced to flowering in vitro when being transferred on medium containing low nitrogen, high phosphate and cytokinin. Fully developed flowers and fruit onset can be reached when pre-induced plants are cultured on medium containing higher nitrogen content and lower cytokinin concentration.
In the first method described, the sterilized mature capsules were used as a source of protocorm, shoots and plantlets for the further induction of in vitro flowering. Thus, the idea to use specific vegetative form (PLB or rhizome) already cultured in vitro for a long time as a source for rapidly and numerous regenerated plants is out of consideration. The same point can be equally applied to the second method described.
Neither the first nor the second method did not employ root excision. Instead, they operated with immature rootless plants exposing them to appropriate treatments. Alternatively, both methods were not able to induce flowering when orchids had fully developed root.
In the first method, the idea to reduce total nitrogen content was not conducted. Although the second method employed low nitrogen content, the lowest concentration was mentioned at the level of 4.5 mM. However, in our experiments, the highest efficiency of orchid flowering induction was achieved at 3.0 mM of total nitrogen (33% lower than that in the first method described).
In both analogous methods described, no orchid species regenerated from rhizome was induced to flowering in vitro. Also, no fruit onset with self pollination has been reported. In order to produce flowers in orchids in vitro, both methods took over 12 months.
The method of present invention allows to overcome drawbacks mentioned above. Although the method of the present invention includes the combined treatments of conventional
Kostenyuk Igor
Oh Boung-Jun
Kenyon & Kenyon
Korea Kumho Petrochemical Co. Ltd.
Lankford , Jr. Leon B.
LandOfFree
Method for producing flowering orchids in vitro does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for producing flowering orchids in vitro, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for producing flowering orchids in vitro will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2547394