WO2020171786A1 - A new nutrient media and method ensuring the multiplication of anatolian orchids - Google Patents
A new nutrient media and method ensuring the multiplication of anatolian orchids Download PDFInfo
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- WO2020171786A1 WO2020171786A1 PCT/TR2019/051098 TR2019051098W WO2020171786A1 WO 2020171786 A1 WO2020171786 A1 WO 2020171786A1 TR 2019051098 W TR2019051098 W TR 2019051098W WO 2020171786 A1 WO2020171786 A1 WO 2020171786A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/20—Growth substrates; Culture media; Apparatus or methods therefor based on or containing natural organic material
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
Definitions
- This invention about agricultural sciences, biotechnology, tissue culture and nutrient media, is on a new nutrient media and method enabling the multiplication of especially tuber plants with tissue culture and ensuring the multiplication of anatolian orchids.
- Anatolian orchid of the Orchidaceae family is a tuber, rooted and herbaceous plant. It’s often used in ice cream making due to the glucomannan contained in its’ tubers. Anatolian orchid, boosting energy, reliefing chest, increasing mind power and significantly supporting the body in many other aspects, also provides important opportunities for entrepreneurs with the tubers it has. It’s determined that germination of anatolian orchid happens in spring, in the first days of summer, in August and early winter. Tubers and leaves grow from the germinated seed after many years. Average shortest time is 2-4 years. It generally reproduces with seed in areas it spreads but because some orchid species having more than one tuber and this tuber seperating there can rarely be seen vegetative orchid aggregates.
- tubers the raw materials of salep, a traditional drink in our country and important ones in food industry especially for their use in ice cream making, are obtained only by harvesting the land orchids in the nature so they’re rapidly decreasing in number and many of the species becomne endangered.
- the invention is a method to keep germinating and growth of orchid seeds containing a plant embryo which doesn’t have any nutrient source and it’s described as facilitating an isolated fungus growth by way of taking a culture from a mature orchid plant and feding it in a nutrient agar environment containing a soluble carbonhydrate and suitable fort he growth of both the fungus and the orchid.
- the method subject to invention is the plant germination inhibitor by fungal pigment without any environmental contamination hazard that dowsn’t have any hazardous impact on the seeded product and with quite a low dosage there can be achieved high effectivity.
- the solution subject to invention contains 165 g NH4NO3, 190 g KNO3, 17 g KH2PO4, 620 mg H3BO3, 2.23 g MnS0 4 .4H 2 0, 860 mg ZnS0 4 .4H 2 0, 83 mg Kl, 25 mg Na 2 Mo 4 ⁇ 2H 2 0, 2.5 mg CuS0 4 .5H 2 0 and 2.5 mg COC1 2 .6H 2 0 in 1.5 liter distilled water.
- the invention also contains sucrose, agar and potato.
- This invention is a new nutrient media and method that ensures multiplication of anatolian orchids and its’ characteristic is being a lower cost, easily implemented new technology that ensures the seed to germinate and become a tuber seedling without adding any plant growth regulator and vitamin to the nutrient media and enables faster and better root and tuber growth compared to other nutrient medias.
- the invention ensures higher germination, protochorm formation, plant growth and tuber formation rate than other cultures (KC (Knudson C, 1921), MS (Murashige and Skoog, 1962).... etc.) used in in vitro multiplication of various land anatolian orchids and enables obtaining tuber seedlings from seed in up to 6-10 months shorter times.
- the method subject to invention is designed for in vitro germination in order to eliminate the problems generally faced in germination in tissue culturing studies of land orchids. In vitro germination studies are being carried out this way in various land orchid species.
- the biggest advantage of the nutrient media in the invention is its’ convenience to be implemented in several anatolian orchids and its’ outstanding feature of germination, protochorm formation rate and shorter respond to plant growth compared to other nutrient medias.
- root and tuber growth in tissue culture in this nutrient media are happening faster and better than other nutrient medias.
- the nutrient media in the method subject to invention is lower in cost and easier to implement than other nutrient medias. In addition it enables the seed to germinate and become a tuber seedling without adding any growth regulator and vitamin to the nutrient media.
- FIG. 1 Schematic view of the method subject to invention.
- FIG. 1 Schematic view of the method subject to invention.
- FIG. 3 Schematic view of the method subject to invention.
- the invention is about a new nutrition media and method ensuring the multiplication of anatolian orchids and its’ characteristic is being consisted of following steps; drying the dry and browned capsules containing seeds of anatolian orchid plant species in a penumbra place for a week; obtaining the seeds by shivering and then screening the cracked capsules with a 60-mesh screen (10); after passing through blender and adding 15 g/1 sucrose, 1.5 g/1 activated carbon, 50 ml/1 freshly squeezed pineapple juice and 100 g/1 fresh potato into 120 mg/1 NH4NO3 , 75 mg/1 MgS0 4 .7H 2 0, 80 mg/1 KH2PO4 , 85 mg/1 Ca(N0 3 ) 2 .4H 2 0, 30 mg/1 FeSCri.dthO chemicals, maintaining the pH value at 5.7 with 1 N NaOH or 1 N HO, adding 6.5 g/1 agar, sterilizing in autoclave at 121°C under 1.2 atmosphere pressure for 15 minutes and obtaining the nutrition media (20); after packaging the obtained
- the invention has the process step of preparing nutrition media that enables asymbiotic (without mycorhizal fungus) germination from anatolian orchid seeds (20).
- the invention has the process step of, besides the seeds of anatolian orchids differ for each species, taking individuals beginning to form regenerated shoots from vivid white color protochorms after 1-2 months, to a last sub culture in 350 ml vials containing 65 ml prepared nutrient media in order for plantings to grow to a size enough to be transferred to outer environments (100).
- the invention has the process step of taking plantings to a last sub culture to ensure optimum growth of anatolian orchid plant according to its’ size and in order for them to grow to a size enough to be transferred to outer environments within 45-60 days (100).
- the invention has the process step of decreasing the number of anatolian orchid seedlings in the culture plate in every process of taking to a sub culture, in order to ensure they get back to the size before they’re transferred to outer environments.
- Basic process steps of the method subject to invention are; obtaining the seeds (10), preparing the nutrient media (20), keeping the seeds in commercial bleach solution (30), rinsing the seeds with sterile pure water (40), obtaining sterilized seeds (50), seeding the seeds into the nutrient media (60), incubating the anatolian orchid seeds (70), taking values on germination, protochorm and plant growth (80), taking them to sub culture in new nutrient media (90), taking plantings to a last sub culture in order for them to grow enough to be transferred to outer environments (100), washing the roots under tap water (110), transferring to viols (120), keeping in acclimatization greenhouses (130), maintaining the greenhouse interior humidity between % 80-90 (140), maintaining the temperature at 21-22 °C (150), ensuring 16/8 (lightness/darkness) photoperiod in days with inadequate daylight (160), reducing the relative humidity (170) and finishing the transferring process (180).
- the content of the nutrient media of the method subject to invention is consisted of taking specific amount of the chemicals given above in the table.
- the preperation of the nutrient media (20) is made by weighing 15 g/1 sucrose, 6.5 g/1 agar, 1.5 g/1 activated carbon, 50 ml freshly squeezed pineapple juice and 100 g fresh potato, passing them through blender and adding them together.
- pH Prior to adding agar to nutrient medias pH is maintained to 5.7 with 1 N NaOH or 1 N HCI.
- nutrient medias are sterilized under autoclave at 1.2 atmosphere pressure, 121°C for 15 minutes.
- Obtaining the sterilized seeds (50) with the method subject to invention is ensured by using‘Packaging Technique’. Because anatolian orchid seeds are very small and in dust-like form, they’re packed as 2 mg inside roughing filter papers. To prevent these packets to open they’re tightly tied with white thread and packaging is finished. In many surface sterilization studies on anatolian orchid seeds, it’s determined that germination rate is low and contamination rate is increasing in 5 and 10 minute applications, 20 minute is the optimum duration and 30 minute applications are decreasing germination. (See Palaz, 2012; Ellialtioglu, 2011; Ozdener, 1994).
- Protocorm rate (%) Protocorm forming total seed count / Total seed count x 100 Plant Growth (3rd Phase): Phase where growth cone completes its’ development and first leaflets and roots begin to grow.
- Plant growth rate (%) Total seed count turning into plant / Total seed count x 100
- protocorm forms out after seeds are germinated in in vitro culture.
- protocorms By taking obtained protocorms to a sub culture in a new nutrient media (90) it’s ensured that they turn into plants.
- plantings are taken to a last sub culture (100) to maket hem grow to a size enough to be transferred to outer environments. For those individuals that forms out roots from plantings to grow bigger before transferring to outer environments, they’re transferred to a new culture plate and healthy tuber plants are being grown.
- Fungus turns starch and samelike compounds created with the decomposition of the organic humus in its’ environment, to water soluble sugar and provides the energy the young orchid plant needs for germination.
- This symbiotic relation is repressed and taken under control by orchid’s cells after germination and orchid plant no longer needs fungus after gaining the autotrophic feeding ability (Ingold and Hudson 1993). Because the rooted and tuber plantings we obtained in in vitro culture environment (gained the autotrophic feeding ability) will be used during acclimatization, they did’t in need of a mycorhizal relation.
- the next step is outer environment acclimatization studies of healthy, rooted tuber ones of those that turned into plants after germinated and formed protocorm in in vitro culture. Rooted plantings are pulled out of their nutrient medias and their roots are washed under tap water (110) and it’s ensured that roots are cleaned of the nutrient media containing agar. After root washing process plantings are transferred into viols containing garden soil, turf and cocopeat at 1:1:1 ratios and placed inside acclimatization mini greenhouses within the greenhouse. A high relative humidity is ensured with the current multiplying within the acclimatization greenhouses and with a humidity meter the greenhouse interior humidity is continously checked and maintained between % 80-90.
- the greenhouse interior temperature is set at 21-22 °C and the required 16/8 (lightess/darkness) photoperiod is provided by the lamps with 3000 lux light intensity located on top of the mini greenhouses in days with inadequate daylight.
- About 10-12 days after maturizing the sliding doors of the mini greenhouses are opened and multiplying processes are reduced it’s ensured for relative humidity to come down first to % 70 and then to % 50.
- At the end of the third week all of the viols are taken out of acclimatization greenhouses and plantings are placed on the plates in the greenhouse. There’s subsurface irrigation method used in plates and by continuing caring and fertilization processes the transferring process is finalized.
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
This invention about agricultural sciences, biotechnology, tissue culture and nutrient media, and a new nutrient media and method enabling the multiplication of especially tuber plants with tissue culture and ensuring the multiplication of anatolian orchids and its' characteristic is being consisted of process steps of drying the dry and browned capsules containing seeds of anatolian orchid plant species in a penumbra place for a week; obtaining the seeds by shivering and then screening the cracked capsules with a 60-mesh screen (10); after passing through blender and adding 15 g/l sucrose, 1.5 g/l activated carbon, 50 ml/l freshly squeezed pineapple juice and 100 g/l fresh potato into 120 mg/l NH4NO3, 75 mg/l MgS04.7H20, 80 mg/l KH2PO4, 85 mg/l Ca(N03)2.4H20, 30 mg/l FeS04.4H20 chemicals, maintaining the pH value at 5.7 with 1 N NaOH or 1 N HCI, adding 6.5 g/l agar, sterilizing in autoclave at 121°C under 1.2 atmosphere pressure for 15 minutes and obtaining the nutrition media (20); after packaging the obtained seeds as 2 mg in roughing filter paper, keeping them in %25 commercial bleach solution with 1-2 drops of tween 20 in it in order to maintain the superficial sterilization of packed seeds (30); rinsing the seeds 3 times each for 5 minutes with sterile pure water (40); taking the seeds to pre-drying in petris within sterile cabin with sterile roughing filter papers placed inside petrises and obtaining sterilized seeds (50); opening the packages containing sterilized seeds with sterile lance and clamp and seeding the seeds in the packages into the nutrition media (60); taking the anatolian orchid seeds into in vitro culture and incubating them under 40 pE/m^s1 fluorescent white light in 18/6 (lightness/darkness) photoperiod and in a climate room at 22±2 °C (70); taking values on germination, protochorm and plant growth in seeds of anatolian orchid species (80); taking obtained protochorms to a new sub culture with same nutrition media in order for them to turn into plants (90); in order to ensure root and tuber formation in obtained protochorms turned into plants and minimizing plant deaths during transferring to outer environments, taking the plantings to a last sub culture for them to grow enough to be transferred to outer environments (100); taking out the rooted plantings of their nutrition media and washing their roots under tap water in order to clean them of the nutrition media with agar (110); transferring the plantings of which roots are washed to viols containing garden soil, turf and cocopeat at 1:1:1 ratios (120); keeping the orchid seedlings in acclimatization greenhouses where being multiplied in order to maintain their relative humidity (130); maintaining the interior humidity of the greenhouse between % 80-90 (140); maintaining the inside temperature in the greenhouse as 21-22 °C (150); maintaining 16/8 (lightness/darkness) photoperiod in days with inadequate sunlight (160); about 10-12 days after the maturity of orchid seedlings reducing the multiplying process and reducing the relative humidity first to %70 at then to %50 (170); taking out all viols of acclimatization greenhouses at the end of the third week and finishing the transferring process (180).
Description
A NEW NUTRIENT MEDIA AND METHOD ENSURING THE
MUUTIPUICATION OF ANATOUIAN ORCHIDS
Technological Area:
This invention about agricultural sciences, biotechnology, tissue culture and nutrient media, is on a new nutrient media and method enabling the multiplication of especially tuber plants with tissue culture and ensuring the multiplication of anatolian orchids.
Current Situation of The Technique:
Anatolian orchid of the Orchidaceae family, is a tuber, rooted and herbaceous plant. It’s often used in ice cream making due to the glucomannan contained in its’ tubers. Anatolian orchid, boosting energy, reliefing chest, increasing mind power and significantly supporting the body in many other aspects, also provides important opportunities for entrepreneurs with the tubers it has. It’s determined that germination of anatolian orchid happens in spring, in the first days of summer, in August and early winter. Tubers and leaves grow from the germinated seed after many years. Average shortest time is 2-4 years. It generally reproduces with seed in areas it spreads but because some orchid species having more than one tuber and this tuber seperating there can rarely be seen vegetative orchid aggregates. Its’ seeds are very small and in dust-like form. Embryos quickly loose their viabilities in seeds without endosperms and only less than %5 of them can germinate under natural environment. In order for a mature plant to grow up one must wait for a long time like 2-16 years after germination. Tubers of the plant create one single baby tuber every year and as a new tuber grows the old one will be used as a food source so it creases and dissolves. Seeds of this plant are very small and because they don’t have any nutrient tissues they must involve in a
symbiosis with a soil fungus in order to live. Therefore it doesn’t have a normal seed planting and plant growth. Altough there are seedling growths under laboratory conditions by germinating anatolian orchids with fungus (symbiotic) this process takes too long and it becomes too hard to maintain all conditions (temperature, humidity, light etc.) at optimum due to in vitro during this. In addition it takes too long for plants grown from the seed obtained with the help of fungus, to become suitable to be pulled up.
Today, tubers, the raw materials of salep, a traditional drink in our country and important ones in food industry especially for their use in ice cream making, are obtained only by harvesting the land orchids in the nature so they’re rapidly decreasing in number and many of the species becomne endangered.
In the application issue numbered TR201619161“Reproduction of The Salep Orchid From The Seed in Soil” has been described. The characteristics of this invention is being achieved by following steps: making packets ready by putting small amounts of Serapias vomeracea subsp. laxiflora between fine-porous mesh clothes, preparing soil mixture, sterilizing the prepared soil mixture by autoclaving in autoclave resistant oven bags for an hour twice every 24 hours to kill various insect larvas and eggs, transferring the sterilized soil in equal amount to pots, transferring the seed packets into soil and grafting fungi (a kingdom of livings and parasol mushrooms including eucaryotic livings which can be both single cell and multi cell) into the soil.
In the application issue numbered GB 19840032453 “Germination and Continous Growth of Orchid Seeds” has been described. The invention is a method to keep germinating and growth of orchid seeds containing a plant embryo which doesn’t have any nutrient source and it’s described as facilitating an isolated fungus growth by way of taking a culture from a mature orchid plant and feding it in a nutrient agar environment containing a soluble carbonhydrate and suitable fort he growth of both the fungus and the orchid.
In the application issue numbered W02005JP05901“Plant Germination Inhibitor and Its’ Use” has been described. The method subject to invention is the plant germination
inhibitor by fungal pigment without any environmental contamination hazard that dowsn’t have any hazardous impact on the seeded product and with quite a low dosage there can be achieved high effectivity. The solution subject to invention contains 165 g NH4NO3, 190 g KNO3, 17 g KH2PO4, 620 mg H3BO3, 2.23 g MnS04.4H20, 860 mg ZnS04.4H20, 83 mg Kl, 25 mg Na2Mo4 · 2H20, 2.5 mg CuS04.5H20 and 2.5 mg COC12.6H20 in 1.5 liter distilled water. In addition the invention also contains sucrose, agar and potato.
In the methods of orchid cultivation mentioned above the fungus isolation, fungus nutrietn media and symbiotic use (with fungus) of the mycorhizal fungus has been determined and it causes cost increase. Besides not enough productivity is achieved with these methods and they take long to germinate.
In conclusion there’s a need for a lower cost, easily implemented new technology that ensures the seed to germinate and become a tuber seedling without adding any plant growth regulator and vitamin to the nutrient media and enables faster and better root and tuber growth compared to other nutrient medias.
The Invention Definition:
This invention is a new nutrient media and method that ensures multiplication of anatolian orchids and its’ characteristic is being a lower cost, easily implemented new technology that ensures the seed to germinate and become a tuber seedling without adding any plant growth regulator and vitamin to the nutrient media and enables faster and better root and tuber growth compared to other nutrient medias.
The invention ensures higher germination, protochorm formation, plant growth and tuber formation rate than other cultures (KC (Knudson C, 1921), MS (Murashige and Skoog, 1962).... etc.) used in in vitro multiplication of various land anatolian orchids and enables obtaining tuber seedlings from seed in up to 6-10 months shorter times.
The method subject to invention is designed for in vitro germination in order to eliminate the problems generally faced in germination in tissue culturing studies of land orchids. In vitro germination studies are being carried out this way in various land orchid species.
The biggest advantage of the nutrient media in the invention is its’ convenience to be implemented in several anatolian orchids and its’ outstanding feature of germination, protochorm formation rate and shorter respond to plant growth compared to other nutrient medias. In addition root and tuber growth in tissue culture in this nutrient media are happening faster and better than other nutrient medias.
In order to realize all purposes mentioned above and those that will arise of the detailed description below, the invention has been developed recently in 2013 and tested in several different orchid species until 2018 and taken its’ last form. The nutrient media in the method subject to invention is lower in cost and easier to implement than other nutrient medias. In addition it enables the seed to germinate and become a tuber seedling without adding any growth regulator and vitamin to the nutrient media.
All adavantages of the method subject to invention will be more clearly understood by means of the scheme given below and the detailed description written referring this scheme so fort his reason assessment should be made considering this scheme and detailed description.
Description of Figures:
The invention will be described by referring the figures given in annex, this way the characteristics of the invention will be understood and assessed more clearly but this is not intended to limit the invention with these specific arrangements. Vice versa it’s intended to cover all alternatives, changes and equivalents of the invention which may be included in the area the invention is defined in by the claims in the annex. It must be understood that shown details are shown only to describe the preferred arrangements of the current invention and to provide the most utilisable and clearer definition of both the
figuring of methods and rules and conceptual characteristics of the invention. In these figures;
Figure 1 Schematic view of the method subject to invention.
Figure 2 Schematic view of the method subject to invention.
Figure 3 Schematic view of the method subject to invention.
Figures that will help out to understand this invention are numbered as specified in the annexed figure and given below by their names.
Description of References:
10. Obtaining seeds
20. Preparing nutrient media
30. Keeping seeds in bleach solution
40. Rinsing seeds with sterile pure water
50. Obtaining sterilized seeds
60. Seeding seeds into nutrient media
70. Incubating anatolian orchid seeds
80. Getting values on germination, protochorm and plant growth
90. Taking to sub culture in new nutrient media
100. Taking plantings to a last sub culture to make them grow to sizes enough to be transferred to outer environments
110. Washing roots under tap water
120. Transferring to viols
130. Storing in acclimatization greenhouses
140. Keeping greenhouse interior humidity between % 80-90
150. Maintaining temperature at 21-22 °C
160. Ensuring 16/8 (lightness/darkness) photoperiod in days with low sunlight 170. Decreasing relative humidity
180. Finishing transferring process
Description of Invention:
The invention is about a new nutrition media and method ensuring the multiplication of anatolian orchids and its’ characteristic is being consisted of following steps; drying the dry and browned capsules containing seeds of anatolian orchid plant species in a penumbra place for a week; obtaining the seeds by shivering and then screening the cracked capsules with a 60-mesh screen (10); after passing through blender and adding 15 g/1 sucrose, 1.5 g/1 activated carbon, 50 ml/1 freshly squeezed pineapple juice and 100 g/1 fresh potato into 120 mg/1 NH4NO3, 75 mg/1 MgS04.7H20, 80 mg/1 KH2PO4, 85 mg/1 Ca(N03)2.4H20, 30 mg/1 FeSCri.dthO chemicals, maintaining the pH value at 5.7 with 1 N NaOH or 1 N HO, adding 6.5 g/1 agar, sterilizing in autoclave at 121°C under 1.2 atmosphere pressure for 15 minutes and obtaining the nutrition media (20); after packaging the obtained seeds as 2 mg in roughing filter paper, keeping them in %25 commercial bleach solution with 1-2 drops of tween 20 in it in order to maintain the superficial sterilization of packed seeds (30); rinsing the seeds 3 times each for 5 minutes with sterile pure water (40); taking the seeds to pre-drying in petris with sterile roughing filter papers placed inside within sterile cabin and obtaining sterilized seeds (50); opening the packages containing sterilized seeds with sterile lance and clamp and seeding the seeds in the packages into the nutrition media (60); taking the anatolian orchid seeds into in vitro culture and incubating them under 40 pE/rrr/s1 fluorescent white light in 18/6 (lightness/darkness) photoperiod and in a climate room at 22±2 °C (70); taking values on germination, protochorm and plant growth in seeds of anatolian orchid species (80); taking obtained protochorms to a sub culture in the new nutrition media (90); in order to ensure root and tuber formation in obtained protochorms turned into plants and minimizing plant deaths during transferring to outer environments, taking the plantings to a last sub culture for them to grow enough to be transferred to outer environments (100); taking out the rooted plantings of their nutrition media and washing their roots under tap water in order to clean them of the nutrition media with agar (110); transferring the plantings of which roots are washed to viols containing garden soil, turf and cocopeat at 1:1:1 ratios (120); keeping the orchid seedlings in acclimatization greenhouses where being multiplied in order to maintain their relative humidity (130); maintaining the interior humidity of the greenhouse between % 80-90
(140); maintaining the inside temperature in the greenhouse as 21-22 °C (150); maintaining 16/8 (lightness/darkness) photoperiod in days with inadequate sunlight (160); about 10-12 days after the maturity of orchid seedlings reducing the multiplying process and reducing the relative humidity to be first %70 at then %50 (170); taking out all viols of acclimatization greenhouses at the end of the third week and finishing the transferring process (180).
The invention has the process step of preparing nutrition media that enables asymbiotic (without mycorhizal fungus) germination from anatolian orchid seeds (20).
The invention has the process step of taking values on germination, protochorm and plant growth (80) by calculating these as follows; Germination rate (%) = Total germinated seed count / Total seed count x 100, Protochorm rate (%) = Protocorm forming total seed count / Total seed count x 100 and Plant growth rate (%) = Total seed count turning into plant / Total seed count x 100.
The invention has the process step of, besides the seeds of anatolian orchids differ for each species, taking individuals beginning to form regenerated shoots from vivid white color protochorms after 1-2 months, to a last sub culture in 350 ml vials containing 65 ml prepared nutrient media in order for plantings to grow to a size enough to be transferred to outer environments (100).
The invention has the process step of taking plantings to a last sub culture to ensure optimum growth of anatolian orchid plant according to its’ size and in order for them to grow to a size enough to be transferred to outer environments within 45-60 days (100).
The invention has the process step of decreasing the number of anatolian orchid seedlings in the culture plate in every process of taking to a sub culture, in order to ensure they get back to the size before they’re transferred to outer environments.
Detailed Description of Invention:
Basic process steps of the method subject to invention are; obtaining the seeds (10), preparing the nutrient media (20), keeping the seeds in commercial bleach solution (30), rinsing the seeds with sterile pure water (40), obtaining sterilized seeds (50), seeding the seeds into the nutrient media (60), incubating the anatolian orchid seeds (70), taking values on germination, protochorm and plant growth (80), taking them to sub culture in new nutrient media (90), taking plantings to a last sub culture in order for them to grow enough to be transferred to outer environments (100), washing the roots under tap water (110), transferring to viols (120), keeping in acclimatization greenhouses (130), maintaining the greenhouse interior humidity between % 80-90 (140), maintaining the temperature at 21-22 °C (150), ensuring 16/8 (lightness/darkness) photoperiod in days with inadequate daylight (160), reducing the relative humidity (170) and finishing the transferring process (180).
With the method subject to invention, dry and browned capsules containing seeds of anatolian orchid plant species in nature, are dried in a penumbra place for about a week and then cracked capsules are well-shivered and screened with 60-mesh screen. Following this, obtained seeds are transferred into brown vials. Prepared vials are tagged and kept at +4 °C in refrigerator until the time they’ll be used, preferably at least for a month. This way it’s ensured seeds are obtained (10).
After obtaining seeds (10) vials, petris, clamp, lancet used in tissue cultivating studies and water filled vials used in surface sterilization, are sterilized under autoclave at 121°C for 20 minutes. Prior to autoclave process clamps and lancets are wrapped in aluminium folio and put in refrigerator bags, mouth of vials are strapped with autoclave tape and petris are placed inside autoclave bags. The transferring room where the sterile cabin is in and herbal materials are taken to cultures, are regularly wiped with bleach (NaOCl). In addition UV (ultraviolet) lamp is turned on in cabin before and after the study to fully sterilize the cabin. When considered necessary, the sterilized cabin interior will be wiped with bleach. Clamps and lancets used in the sterile cabin are kept
inside the sterilizer device within the cabin and used repeatedly by spraying with alcohol.
The content of the nutrient media of the method subject to invention is consisted of taking specific amount of the chemicals given above in the table. The preperation of the nutrient media (20) is made by weighing 15 g/1 sucrose, 6.5 g/1 agar, 1.5 g/1 activated carbon, 50 ml freshly squeezed pineapple juice and 100 g fresh potato, passing them through blender and adding them together. Prior to adding agar to nutrient medias pH is maintained to 5.7 with 1 N NaOH or 1 N HCI. After adding agar and dissolving it, nutrient medias are sterilized under autoclave at 1.2 atmosphere pressure, 121°C for 15 minutes.
Obtaining the sterilized seeds (50) with the method subject to invention, is ensured by using‘Packaging Technique’. Because anatolian orchid seeds are very small and in dust-like form, they’re packed as 2 mg inside roughing filter papers. To prevent these packets to open they’re tightly tied with white thread and packaging is finished. In many surface sterilization studies on anatolian orchid seeds, it’s determined that germination rate is low and contamination rate is increasing in 5 and 10 minute applications, 20 minute is the optimum duration and 30 minute applications are decreasing germination.
(See Palaz, 2012; Ellialtioglu, 2011; Ozdener, 1994). In guide of these studies, in order to ensure the superficial sterlization of the packed seeds, seeds are kept in %25 commercial bleach solution with 1-2 drops of tween dripped in (30), rinsed 3 times each for 5 minutes with sterile pure water at the end of this duration (40) and taken top re drying in petris with sterile roughing filter paper placed in, inside sterile cabin. By this measn it’s ensured to obtain sterilized seeds (50). Later after removing the raw water of seed containing packets, they’re transferred to new sterilized petri dishes and dried well enough by keeping petris half-open for 6-8 hours inside the sterile cabin. After making sure that seeds inside the packets are dried well enough, with the help of clamp and lancet priorly sterilized under autoclave at 121 °C for 20 minutes, seeds are seeded into nutrient media within the sterile cabin (60).
In the method subject to invention, after taking into in vitro culture and incubating the anatolian orchid seeds (70) in climate chamber at 22±2 °C and in 18/6 (lightness/darkness) photoperiod at 40 pE/rrr/s1 fluorescent white light, each petri is photographed under stereo microscope at the end of every month to determine the growth situation of the seeds seeded into nutrient media and by using a specific software and photographs in the computer, values on germination, protochorm and plant growth of anatolian orchid seeds are taken (80) and observed values are determined as % using the formulas below.
Germination (1st Phase): Noted as the phase where the embryo grows and boll swells. Germination rate (%) = Total germinated seed count / total seed count x 100
Protochorm (2nd Phase): Noted as the phase where embryo totally gets out of the boll, swells and becomes a white sphere and growth cone begins to develop.
Protocorm rate (%) = Protocorm forming total seed count / Total seed count x 100 Plant Growth (3rd Phase): Phase where growth cone completes its’ development and first leaflets and roots begin to grow.
Plant growth rate (%) = Total seed count turning into plant / Total seed count x 100
In the method subject to invention protocorm forms out after seeds are germinated in in vitro culture. By taking obtained protocorms to a sub culture in a new nutrient media
(90) it’s ensured that they turn into plants. In order to ensure root and tuber growth in those that turned into plants and minimize plant deaths while transferring to outer environments, plantings are taken to a last sub culture (100) to maket hem grow to a size enough to be transferred to outer environments. For those individuals that forms out roots from plantings to grow bigger before transferring to outer environments, they’re transferred to a new culture plate and healthy tuber plants are being grown.
Orchids with no endosperms in their seeds, need tat symbiotic relation continued with fungus during germination phase. Fungus turns starch and samelike compounds created with the decomposition of the organic humus in its’ environment, to water soluble sugar and provides the energy the young orchid plant needs for germination. This symbiotic relation is repressed and taken under control by orchid’s cells after germination and orchid plant no longer needs fungus after gaining the autotrophic feeding ability (Ingold and Hudson 1993). Because the rooted and tuber plantings we obtained in in vitro culture environment (gained the autotrophic feeding ability) will be used during acclimatization, they weren’t in need of a mycorhizal relation.
In the method subject to invention, the next step is outer environment acclimatization studies of healthy, rooted tuber ones of those that turned into plants after germinated and formed protocorm in in vitro culture. Rooted plantings are pulled out of their nutrient medias and their roots are washed under tap water (110) and it’s ensured that roots are cleaned of the nutrient media containing agar. After root washing process plantings are transferred into viols containing garden soil, turf and cocopeat at 1:1:1 ratios and placed inside acclimatization mini greenhouses within the greenhouse. A high relative humidity is ensured with the current multiplying within the acclimatization greenhouses and with a humidity meter the greenhouse interior humidity is continously checked and maintained between % 80-90. In addition the greenhouse interior temperature is set at 21-22 °C and the required 16/8 (lightess/darkness) photoperiod is provided by the lamps with 3000 lux light intensity located on top of the mini greenhouses in days with inadequate daylight. About 10-12 days after maturizing the sliding doors of the mini greenhouses are opened and multiplying processes are reduced it’s ensured for relative humidity to come down first to % 70 and then to % 50. At the
end of the third week all of the viols are taken out of acclimatization greenhouses and plantings are placed on the plates in the greenhouse. There’s subsurface irrigation method used in plates and by continuing caring and fertilization processes the transferring process is finalized.
Claims
1- The invention is about a new nutrient media and method ensuring the multiplication of anatolian orchids and its’ characteristic is being consisted of the following steps; drying the dry and browned capsules containing seeds of anatolian orchid plant species in a penumbra place for a week and obtaining the seeds by shivering and then screening the cracked capsules with a 60-mesh screen (10),
after passing through blender and adding 15 g/1 sucrose, 1.5 g/1 activated carbon, 50 ml/1 freshly squeezed pineapple juice and 100 g/1 fresh potato into 120 mg/1 NH4NO3, 75 mg/1 MgS04.7H20, 80 mg/1 KH2PO4, 85 mg/1 Ca(N03)2.4H20, 30 mg/1 FeS04.4H20 chemicals, maintaining the pH value at 5.7 with 1 N NaOH or 1 N HCI, adding 6.5 g/1 agar, sterilizing in autoclave at 121°C under 1.2 atmosphere pressure for 15 minutes and obtaining the nutrition media (20), after packaging the obtained seeds as 2 mg in roughing filter paper, keeping them in %25 commercial bleach solution with 1-2 drops of tween 20 in it in order to maintain the superficial sterilization of packed seeds (30),
rinsing the seeds 3 times each for 5 minutes with sterile pure water (40), taking the seeds to pre-drying in petris within sterile cabin with sterile roughing filter papers placed inside petrises and obtaining sterilized seeds (50)
opening the packages containing sterilized seeds with sterile lance and clamp and seeding the seeds in the packages into the nutrition media (60),
taking the anatolian orchid seeds into in vitro culture and incubating them under 40 pE/rrr/s1 fluorescent white light in 18/6 (lightness/darkness) photoperiod and in a climate room at 22±2 °C (70),
taking values on germination, protochorm and plant growth in seeds of anatolian orchid species (80),
taking obtained protochorms to a sub culture in the new nutrition media (90), in order to ensure root and tuber formation in obtained protochorms turned into plants and minimizing plant deaths during transferring to outer environments, taking the plantings to a last sub culture for them to grow enough to be transferred to outer environments (100),
taking out the rooted plantings of their nutrition media and washing their roots under tap water in order to clean them of the nutrition media with agar (110), transferring the plantings of which roots are washed to viols containing garden soil, turf and cocopeat at 1:1:1 ratios (120),
keeping the orchid seedlings in acclimatization greenhouses where being multiplied in order to maintain their relative humidity (130),
maintaining the interior humidity of the greenhouse between % 80-90 (140), maintaining the inside temperature in the greenhouse as 21-22 °C (150), maintaining 16/8 (lightness/darkness) photoperiod in days with inadequate sunlight (160),
about 10-12 days after the maturity of orchid seedlings reducing the multiplying process and reducing the relative humidity first to %70 at then to %50 (170), taking out all viols of acclimatization greenhouses at the end of the third week and finishing the transferring process (180).
2- What is mentioned in Claim 1 is a new nutrient media and method ensuring the multiplication of anatolian orchids and its’ characteristic is preparing nutrition media that enables asymbiotic (without mycorhizal fungus) germination from anatolian orchid seeds (20).
3- What is mentioned in Claim 1 is a new nutrient media and method ensuring the multiplication of anatolian orchids and its’ characteristic is taking values on germination, protochorm and plant growth (80) by calculating these as follows; Germination rate (%) = Total germinated seed count / Total seed count x 100, Protochorm rate (%) = Protocorm forming total seed count / Total seed count x 100 and Plant growth rate (%) = Total seed count turning into plant / Total seed count x 100.
4- What is mentioned in Claim 1 is a new nutrient media and method ensuring the multiplication of anatolian orchids and its’ characteristic is besides the seeds of anatolian orchids differ for each species, taking individuals beginning to form regenerated shoots from vivid white color protochorms after 1-2 months, to a last sub
culture in 350 ml vials containing 65 ml prepared nutrient media in order for plantings to grow to a size enough to be transferred to outer environments (100).
5- What is mentioned in Claim 1 is a new nutrient media and method ensuring the multiplication of anatolian orchids and its’ characteristic is taking plantings to a last sub culture to ensure optimum growth of anatolian orchid plant according to its’ size and in order for them to grow to a size enough to be transferred to outer environments within 45-60 days (100). 6- What is mentioned in Claim 1 is a new nutrient media and method ensuring the multiplication of anatolian orchids and its’ characteristic is decreasing the number of anatolian orchid seedlings in the culture plate in every process of taking them to a sub culture for enough growing to be transferred to outer environments (100), in order to ensure they get back to the size before they’re transferred to outer environments.
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| TR2019/02498A TR201902498A2 (en) | 2019-02-19 | 2019-02-19 | A NEW BREEDING ENVIRONMENT AND METHOD THAT ENABLES THE REPRODUCTION OF SALEP ORCHIDS |
| TR2019/02498 | 2019-02-19 |
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| WO2020171786A1 true WO2020171786A1 (en) | 2020-08-27 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112655561A (en) * | 2021-01-14 | 2021-04-16 | 上饶师范学院 | Method for in vitro preservation of red bud taro test-tube plantlet |
| CN113025503A (en) * | 2021-04-27 | 2021-06-25 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Culture medium for culturing, preserving and rejuvenating edible fungus strains as well as preparation method and application thereof |
| CN113079927A (en) * | 2021-04-13 | 2021-07-09 | 永德沃森农业开发有限公司 | Large-scale planting method of domesticated bitter fruits |
| CN114190277A (en) * | 2021-12-07 | 2022-03-18 | 中国科学院昆明植物研究所 | Method for promoting blooming and fructification of large root orchid test tube |
| CN114455710A (en) * | 2022-01-28 | 2022-05-10 | 湖北大学 | Plant screening and community constructing method suitable for urban landscape water body restoration |
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| US5656482A (en) * | 1988-03-17 | 1997-08-12 | University Of Delaware | Rapid germination of orchid seeds from immature capsules |
| CN101352117A (en) * | 2008-08-15 | 2009-01-28 | 中国科学院植物研究所 | Culture medium of germination of orchid seed |
| CN107056414A (en) * | 2017-01-23 | 2017-08-18 | 中国林业科学研究院林业研究所 | A kind of broad spectrum activity hot bandwidth aseptic seeding culture medium two |
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| US5656482A (en) * | 1988-03-17 | 1997-08-12 | University Of Delaware | Rapid germination of orchid seeds from immature capsules |
| CN101352117A (en) * | 2008-08-15 | 2009-01-28 | 中国科学院植物研究所 | Culture medium of germination of orchid seed |
| CN107056414A (en) * | 2017-01-23 | 2017-08-18 | 中国林业科学研究院林业研究所 | A kind of broad spectrum activity hot bandwidth aseptic seeding culture medium two |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112655561A (en) * | 2021-01-14 | 2021-04-16 | 上饶师范学院 | Method for in vitro preservation of red bud taro test-tube plantlet |
| CN113079927A (en) * | 2021-04-13 | 2021-07-09 | 永德沃森农业开发有限公司 | Large-scale planting method of domesticated bitter fruits |
| CN113025503A (en) * | 2021-04-27 | 2021-06-25 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Culture medium for culturing, preserving and rejuvenating edible fungus strains as well as preparation method and application thereof |
| CN114190277A (en) * | 2021-12-07 | 2022-03-18 | 中国科学院昆明植物研究所 | Method for promoting blooming and fructification of large root orchid test tube |
| CN114455710A (en) * | 2022-01-28 | 2022-05-10 | 湖北大学 | Plant screening and community constructing method suitable for urban landscape water body restoration |
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| TR201902498A2 (en) | 2019-03-21 |
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