WO2016079758A2

WO2016079758A2 - Aquatic- in vitro-ex vitro terrarium

Info

Publication number: WO2016079758A2
Application number: PCT/IN2015/000431
Authority: WO
Inventors: Priya Khetarpal
Original assignee: Priya Khetarpal
Priority date: 2014-11-19
Filing date: 2015-11-19
Publication date: 2016-05-26
Also published as: WO2016079758A3; WO2016079758A9

Abstract

The product invented belongs to Plant tissue culture field and find its application for aesthetic purpose. The prior art exists as bottle garden/terrarium, but it has be maintained. These days the fast pace of life does not allow people to be in touch with plants. Being in touch with nature is very essential for overall well- being of the human race. So, here, the designed products find their place at various sections of the society such as offices, hotels, restaurants, hospitals, old age homes, residential homes, educational institutes and rehabilitation centers, without taking pain to maintain them. The products can be a replacement for cut flowers and bouquets which do not last forever. Incorporating various decorative elements enhance the aesthetics of the product, thus making a positive impression on the observer. Each product designed has different appearance and thus there is a lot of scope for changing and its personalization. The products thus designed have a lot of scope for commercialization in plant tissue culture industry.

Description

ble to the Description-

Field of the Invention And Use of the Invention-

This invention relates to the field of Plant biotechnology. Using the technique of Plant tissue culture. In vitro grown plants are utilized to make a product for aesthetic purpose.

Variegated ornamental In vitro plants are inoculated in semi-solid colored medium in a glassware containing various decorative elements such as sand ,stones, shells, gravel, jelly balls and sculptures( sometimes incorporating themes) in a way that the finished product becomes of commercial value for its aesthetics. The extension of In vitro terrarium was founded in preparing Ex vitro terrariums and in Aquatic terrarium where

combination of all the elements described above was used. However, plants used were either ornamental or succulents tissue cultured ex vitro in Ex vilro terrarium and aquatic plants in Aquatic terrarium.

I his technique can be appl ied on a large scale in Plant 1 issue Culture Indusir; to commercialize such decorative pieces.

Prior art And Problem to be solved-

Prior art exists as Terrariums, a. k. a., Bottle gardens. Making bottle gardens does not necessarily need tissue cultured plants.

Bottle gardens are majorly prepared using following materials- Garden soil/ Cocopeat; small sized plants; sculptures (highly optional) and choice of vessels utilized varies greatly from used/wasteful plastic bottles to fancy and/or elegant glassware.

Terrariums are used as a substitute for houses which cannot accommodate gardens. The product invented here is inspired by the motive behind making of terrariums; however, this product has much more aesthetic value due to v ariously combined elements in an appropriate way . Unlike terrariums, this product adds various colors together (sometimes theme- based) and thus becomes very vibrant.

I have been able to design a product that is the extended version of In vitro terrarium also and has been described as Ex vitro terrarium and Aquatic terrarium.

This product overcomes various problems-

The In vitro terrarium invented does not needs to be maintained or taken care of , since it is prepared aseptically ,it has to be kept sealed. Preparing bottle gardens needs to be maintained from time to time (watering; removing dead leaves and keeping in proper sunlight or shade).

Using certain plants in bottle garden might cause problem to those allergic to them, but. since In vitro terrarium has to be kept sealed strictly so no allergic response will occur.

These products find their place at various sections of our society, such as offices, hotels, restaurants, hospitals, old age homes, residential homes, educational institutes, rehabilitation centers, etc. for keeping people in touch with nature and enjoy its aesthetics without the need of maintaining it. Since, terrariums have to be maintained, no one will take the pain of keeping them at work places.

These products have a high aesthetic value, therefore, finds its application in various sections of the society thus keeping people in touch with nature at their desk, unlike terrariums which will normally be restricted to residential homes.

Bottle gardens are not available commercially (to the best of my knowledge), however, these products can be commercially exploited because they have superior aesthetics than the original counterparts. These products can replace various conventionally used items for decoration, which have various problems associated with them, namely -

^■ Fresh cut flowers: have to be changed from time to time.

^■ Bouquets: serve only one time purpose just like fresh cut flowers.

^■ Terrariums: have to maintain with water, sunlight, removing dead leaves and replacing plants sometimes.

^■ Potted plants: Indoor plants in pots have to be taken care of just as

terrariums and do not provide the same aesthetics as these products.

^■ Gift articles: These products can be a great gift to people who love to be in touch with plants, it will keep people in touch with nature at their desks! !

^■ Decoration pieces: These products make a great decoration pieces to enjoy their aesthetics, growth of plants without the need for maintaining them. It finds its applications in various sections of the society.

Objectives-

• Fhe principle objective of this product is to provide a substitute to bottle garden, for which special care has to be taken.

• In vitro plants are utilized in a way by incorporating various decorative elements, to create a decoration piece.

• To provide a

option for those who like to stay in touch with plants, but don't have time to maintain it.

• To provide a substitute to potted plants in a more decorative and attractive way.

• To provide a substitute to cut flowers and bouquets, that must be long lasting in their comparison.

• To spread the importance of plants and their association in different sections of the society.

• To let people enjoy the aesthetics of In vitro terrarium and its extended version of products, i .e.. Ex vitro terrarium and Aquatic terrarium.

• Can serve as Gift article: These products can be a great gift to people who love to be in touch plants.

Summar -

The product developed is basically an application of Plant Biotechnology for aesthetic purpose. It applies the tissue cultured plants to make a product that does not needs to be maintained, however, keep people in touch with nature. The inspiration of this product came from terrariums but has a wide range of applications. It can be used to replace terrariums. cut flowers, bouquets, vases, potted plants. It can also be presented as a gift article. Incorporating various elements such as colored medium, stones, sand, shells, gravels, jelly balls, sculptures (,in combination with various themes sometimes), variegated ornamental plants (succulents sometimes also are used) and different sizes & shapes of elegant glassware makes it much more aesthetically pleasing than the ordinary terrariums themselves. This product can be prepared in a plant tissue culture industry on a large scale for commercialization. It finds its application in various sections of the society such as offices, educational institutes, hotels, restaurants, residential homes, old age homes, hospitals, rehabil itation centers not only for decorative purpose, but also keeping people in touch with nature at their desk.

Detailed Description-

A product has been invented using Plant Tissue Culture, a technique of Plant

Biotechnology. The product thus prepared is of aesthetic value and can be placed as a decorative item anywhere namely, offices, educational institutes, hotels, restaurants, residential homes, old age homes, hospitals, rehabilitation centers.

The product named In vitro terrarium is inspired from the basic concept of bottle garden. The idea behind In vitro terrarium was extended to be applied for other newl invented products namely Ex vitro terrarium and Aquatic terrarium.

The product does not need to be maintained unlike its existing counterpart known as Terrarium or Bottle garden.

The product can be placed anywhere indoors, away from the direct sunlight, as a replacement for potted plants, cut flowers, bouquets. It can also serve as a replacement for natural gardens in residential homes w here spac is a constraint.

Unlike bottle gardens. In vitro terrarium is prepared aseptically in laboratory conditions and should be kept closed for its maintenance. People suffering from allergies of certain plants won^'t come in contact with plants directly, therefore won't create any problem.

This product can be prepared in a plant tissue culture industry on a large scale for commercialization. The counterpart of these products have not been commercialized (to the best of my knowledge), however, these products can be commercialized. Especially In vitro terrarium and Ex vitro terrarium, since some new elements are to be added and rest all the facilities in a Plant tissue culture Industry can be exploited.

Ex vitro terrariums specifically, can find their place in restaurants; to be general, in places where cut flowers are required in large amounts and have to be replaced daily In vitro terrariums find their place almost everywhere, i.e., wherever nature-loving people are there.

Aquatic terrariums, on the other hand, have a very soothing effect on the observer's mind due to sight of water and plants, which is combined with the sculptures depicting aquatic life. Other themes can also be applied in aquatic terrariums.

In place of garden soil and/or coco peat that are used for making bottle gardens, the binding medium is semi-solid agarose based medium in In vitro terrarium; sand/ stones in Ex vitro terrarium and sand in aquatic terrarium.

In In vitro terrarium, semi-solid medium provides support for plants as well as all the necessary nutrients for their growth. To add some vibrancy of colors, edible synthetic food colors have been utilized to give different colors in the product and to enhance the aesthetics by providing contrast via elements namely sand, stones, gravel, shells, jelly balls, sculptures (sometimes theme based), variegated ornamental In vitro plantlets and elegant glassware. In some of the products a basic theme has been incorporated to give a background. T his is achieved with the help of a set of sculptures. The themes included are forest; village; beach theme; fairy garden, etc., these will be discussed further in detail.

Another product- Extended versions of In vitro terrarium:

The materials stones, sand, shells, gravel, jelly balls are utilized to prepare soil-less ( also without coco peat) products, here named as Ex vitro terrariums and Aquatic terrarium.

Ex vitro terrariums: can be artificial or natural. Artificial Ex vitro terrariums uses sand/stones/jelly balls/gravel/shells/sculptures in combination with artificial trees and plants/flowers [as depicted in Figure 18] . Natural Ex vitro terrariums uses ornamental tissue cultured plants or succulents with sand/stones/jelly balls/gravel/shells [as depicted in Figure 15, 16, 17 & 20-23]. Agar medium (semi-solid without any additional nutrients; colored/without color) in combination with colored stones solely is used to give variation in the appearance of Ex vitro terrarium.

Aquatic terrariums: uses aquatic plants along with sand/stones/jelly

balls/gravel/shells. Coco peat can also be used just to provide a layer for enhancing its appeal and it does not serve any other purpose. In other words, coco peat is not used as binding material or as a substitute to garden soil in this aquatic terrarium. The binding material is sand, in place of garden soil (referring to the prior art).

Materials used (Brief Explanation)-

- Facilities and Materials needed for In vitro Terrarium-

Plant tissue culture laboratory setup as follows -

1. Preparation room:

Equipments-Autoclave; pH meter; Hot air oven; Micropipette; Stove/Hot plate (for melting agar); Weighing balance; Measuring cylinders.

Autoclave sterilized distilled water.

Chemicals- Sucrose; Agar (High quality); Antibiotics (Rifampicin and Amphotericin B); 1 N NaOH; 1 M HC1;

Chemicals for preparing MS basal medium: Stock solutions A, B, C & D as described in the given Table 1 .

Murashige, T. and Skoog F ( 1962) medium formulation [MS]-

Table 1.

,O 2016/079758

Ιί. I 166

Organic salts: H₃ B0₃ 1 ,240 6.2 Minor nutrients MnS0₄.4H₂0 4,460 22.3 (200X) ZnS0₄.7H₂0 1 ,720 8.6

Na₂ Mo0₄.2H₂0 50 0.025

CuS0₄.5H₂0 0.025

CoCl₂.6H₂0 0.025

C. FeS0₄.7H₂0 5,570 27.85

Iron supple- Na₂.EDTA.2H₂0 7.450 37!85 ments(200X)

D. Inositol 20,000 100

Organic salts: Nicotinic acid 100 O.f Vitamins and Pyridoxine HC1 100 0.5 Amino acids Thiamine HC1 20 f 0.1

Glycine 400 2.0

2. Inoculation room [Aseptically maintained (fumigated)] with equipments/ instruments chemicals:

Laminar Air flow cabinet (Horizontal); Glass bead sterilizer; Autoclave sterilized forceps and scalpel; Sterilized blades; Autoclave sterilized glass Petri plates; 70% alcohol (prepared essentially with autociaved distilled water); Autoclave sterilized absorbeni cotton; Quick fix (to be kept under UV in laminar for 1 hour prior to use) (its surface to be wiped with 70% alcohol prior to opening it).

3. Growth room:

Culture conditions should be maintained under 35 μΜ m^0"2 s^"2 illuminations provided by cool-white fluorescence lamps and temperature at 25 ± 2°C and 50-60% humidity. After keeping the products under observation for 2 weeks, if no contamination occurs, they are sold.

->Other necessary material (common for all products)-

1. Colored medium (For In vitro and Ex Vitro terrarium ):

One function of semi-solid medium is to provide support for the plants and sometimes provide nutrition for their growth, in case of In vitro terrarium.

Apart from these functions, edible food color has been added for providing color to the medium, to enhance its aesthetics.

Colors added are either in contrast of the sand/ stones/shells/jelly balls and/or sculptures, or based upon the theme.

These food colors are non-toxic to plants since they are edible.

Colors utilized are: Raspberry red; Rose pink; Sunset orange; Lemon yellow; Forest green; Sea green; Light blue; Dark blue; Violet and

Brown.

Following colors are obtained from grocery store: In a dried form- Forest green; Lemon yellow and Sunset orange.

In a liquid form- Raspberry red; Rose pink; Blue and Violet. Remaining colors are made by mixing two colors-

• Dark and Light shades of blue are made by manipulating the quantity of pure liquid color.

• Sea green color is made by mixing working solutions of Blue and Green.

• Brown color is prepared by mixing working solutions of Red and Green.

• White color considered is the colorless medium. Stones:

Various stones that are used: White; Multicolored; Double-shaded (Green and Yellow; Red and Orange; etc.); Individual colored (such as Magenta; Blue; Red etc.)

For Example:

• White stones contrast with virtually all the colors.

• Orange and Red double-shaded stones go with Red/Yellow/Orange and White colored medium.

• These stones are used to provide contrasting colors to the product in In vitro terrarium and as a binding material in Ex vitro terrarium; also to provide color in aquatic terrarium.

Stones can be acquired from a local market aquarium shop. Sand:

Sand provides a natural look to the product. It suits well with all the colors of the medium. Here, white colored sand has been utilized, however, for experimenting with the appearance; different colored sand can be utilized as well.

Sand is acquired from local aquarium shop. Shells:

Shells are more often used in a theme based product such as aquatic life/beach, etc.

Shells therefore have been utilized with blue shades of colored medium in the product.

They can be utilized in combination with various colored medium to provide a unique appearance. Generally, bigger sized shells will be used in bigger vessels and small sized shells will be utilized in smaller vessels for In vitro, Ex vitro and aquatic terrarium.

Shells can be collected either directly from the sea-shore or acquired from a person who sells them.

Gravel:

Gravel gives a very different look to the overall appearance of the product, whether; it is In vitro, Ex vitro and aquatic terrarium.

Gravel, whether, it is big or small sized; it is easily utilized in this product.

Gravel matches well with all colors of the medium and can be utilized with or without sand and stones.

I had acquired them from a local aquarium shop.

Jelly balls:

These are the commercially available jelly balls of unknown composition; probably of expandable synthetic polymer.

They swell up when placed in water for 4 hours or more. I prefer to keep them in autoclave sterilized distilled water.

These come in various colors, individual or multi colored. I have preferred to use the multi-color ones but the individual colored ones can also be used.

These jelly balls have been utilized for In vitro and Ex vitro terrarium. In In vitro terrarium, it is used to provide contrast to the colored medium [as depicted in Figure 3 & 4]. In Ex vitro terrarium, it is used as a binding medium in place of semi-solid medium [Figure 15].

Generally, sturdy ornamental plants e.g., money plant or succulents survive well in this type of Ex vitro terrariums. Sculptures:

Sculptures are incorporated in the product to give a catchy appearance, with or without a basic theme.

Sculptures made of porcelain are used because of its advantages; they are autoclavable and non-toxic to plants. Porcelain sculptures provide very elegant look to the product. Sculptures made of hard plastic which is autoclavable can also be utilized, when porcelain ones are not available.

Sculptures mould the overall aesthetics of the product, as various themes are based upon the combinations of sculptures selected to be placed in the vessel.

For Example: to depict a village theme, the available sculptures were utilized: cottage, rural woman, well and animals (cow/monkey/squirrels) [as depicted in Figure. 14].

The scope of utilizing sculptures to provide different looks is very expandable as depicted in Table 2.

Further it can be illustrated with the help of another example:

• Exotic location such as Leaning tower of Pisa, Italy; Statue of

Liberty, U.S.A; The Taj Mahal, India.

• Cartoon characters such as Noddy; Tweety [Figure 10 & 17], can be utilized to create variations, and thus give a different appearance to the product.

• Artificial palm trees had been used to create The Beach Theme.

Sculptures are acquired from a local gift shop. Glassware:

All glassware for In vitro terrarium should be well-fitted with caps/lids to provide aseptic conditions inside the vessel. Depending upon the type of glassware used, the product can get an elegant look. Glassware has to be essentially autoclavable for In vitro terrarium, so should be of borosilicate quality.

Vessels of different shapes have been used and the shape cannot be restricted to one type, as one of the main aspects of this product is different shape glassware.

Mainly symmetric shapes such as Round; Oval and Cylindrical have been utilized; however, asymmetrical ones can also be utilized.

A. Fish bowls (For preparing In vitro terrarium)-

• 10 inch and 12 inch vessels were utilized to provide a basic theme as they can accommodate a number of sculptures. [Figure 12, 13 & 14].

Themes such as Village; City; Fairy garden; Beach; Fish pond etc. have been prepared. [Figure 12, 13 & 14].

• 8 inch vessels were prepared to give a funky look to the

product by using sculptures of cartoon characters [Figure 10] or exotic locations around the globe and in combination with contrasting colored medium.

B. Big glasses-

For preparing In vitro terrarium: 8 inch big glasses were utilized to give a funky appearance by combining unusual elements such as pink colored medium with a cow's sculptures or a cartoon characters' sculpture [Figure 1].

For preparing Ex vitro terrarium: Using jelly balls as a binding medium, sturdy ornamental shoots were planted in the Ex vitro terrarium [Figure 17].

C. Small glasses- Glasses of 350 mL capacity were utilized to create elegant and funky products both for In vitro terrarium [as depicted in Figure 5, 6 & 7] and Ex vitro terrarium [as depicted in Figure. 20-23].

D. Conical flasks-

Borosil conical flasks ranging from 100 ml to 1 liter are utilized for making In vitro terrarium. Sand/stones/jelly balls/ gravel/sculpture(s) along with contrasting colored medium, the products thus prepared get a very unique appearance [Figure 1 to 4].

E. Test tubes-

Along with stones/sand/gravel/jelly balls/shells, colored medium is used to prepare huge variations in the test tubes. The test tubes used are flat-bottomed and borosilicate [Figure 8].

F. Vases-

Exclusively used for preparing Ex vitro terrarium, with sand/ stones/ jelly balls/shells/gravel as a medium [as in Figure 15 & 16].

This product can replace the need for fresh cut flowers in

restaurants/residential homes/hotels/hospitals.

G. Irregular/other shape vessels- Big cylindrical shaped vessel was used to prepare aquatic terrarium [as depicted in Figure 19. a). & b).]. Cylindrical shape vessel was also used for preparing In vitro terrarium [as depicted in Figure. 9]. Themes (mainly used in bigger vessels):

An element such as a basic theme has been incorporated in this product, so that the elegance of the product increases. Incorporation of a combination of sculptures whether made of porcelain or hard plastic have been utilized to create a variety of themes.

The name of the theme and the sculptures utilized has been discussed in the following table. Color of medium should also match with the theme to make it more natural. In other words, color of the medium should make sense in the basic theme of the product and not stand out of context.

Table 2.

with the

color of

sculpture

Exotic The statue of liberty; The Color

locations leaning tower of Pisa; The Taj should

Mahal. contrast

Any one exotic/historically with the

important location can be used color of

in the form of sculpture. sculpture

Marriage Sculpture depicting bride and Brown

theme groom.

Funky Animal sculpture, for e.g., cow/ Color Figure 5 to 7 theme squirrel/ should &11.

monkey/tortoise/deer/penguin. contrast

etc. with the

color of

sculpture

This idea has a vast scope of changes by varying the name of the theme and incorporating different set of sculptures. The basic idea remains the same, i.e., using different set of sculptures in the product to create a basic theme/ background to increase the aesthetics. This idea has been used mainly in In vitro terrarium; same can be applied to Ex vitro terrarium [as depicted in figure 17, 18 & 21] and Aquatic terrarium [as depicted in Figure 19] as well. Antibiotics( Exclusively used only in In vitro terrarium):

Use of antibiotics is necessary to avoid any chances of contamination. Specifically in bigger vessels, a combination of antibiotics is essential. The combination constitutes one bactericide and one fungicide.

The individual effect of the antibiotic should be non- toxic to plants. I have selected 'Rifampicin'- the Bactericide and 'Amphotericin B'- the Fungicide.

The combination of antibiotics, thus, should:

• Not affect the growth of plants.

• Stop the growth of contaminants, particularly bacteria and fungi.

• Not cause toxicity to the plants.

• Not cause any change in color of the medium and therefore, overall appearance of the product.

The final concentration of antibiotics used in the medium is: Rifamipicin (1 mg/1) and Amphotericin B (250 μg l). . Plants:

• Ornamental plants- Plants valued for their foliage and flowers are called Ornamental plants.

Plants with variegated foliage are utilized for this product. Tissue cultured ornamental plants are very sturdy and healthy ; and therefore, grow easily. For both, In vitro and Ex vitro terrariums plants that were utilized are as follows- Syngonium (various varieties); Money plant; Ornamental pineapple. • Succulents-

The product allows scope for utilizing different types of plants, out of which succulents are very favorable. Succulents are considered good luck plants except for the ones bearing thorns.

They do not require much care. They easily survive under tough conditions. Plants such as aloe vera; snake plant; etc. have their scope in Ex vitro terrarium products.

• Aquatic plants- Plants such as aquatic banana and others have been utilized to make the aquatic terrarium [as shown in Figure 19.]. And this product gives freshness because it has water which gives soothing effect to the observers.

Preparation of materials-

1. Colored medium :

Media preparation for In vitro terrarium-

MS basal medium is prepared by adding following:

• Stock solutions:

> A= 50 mL

> B= 5 mL

> C= 5 mL

> D= 5 mL

• Sucrose= 30 grams

• Agar= 8 grams (0.8%)

• Autoclaved distilled water

The calculation given above is for preparing 1 liter of semi-solid MS basal medium. a) . The Stock solutions are measured with the help of a graduated measuring cylinder in their respective quantities.

b) . Sucrose is added in the stainless steel bowl along with the stock

solutions and mixed gently.

c) . After sucrose is completely dissolved, the desired volume of media is made up by adding autoclaved distilled water.

d) . With the help of a pH meter, the pH of the media is checked. It should be adjusted to 5.8 to 6, by adding 1 N NaOH (for basicity) or 1 M HC1 (for acidity).

e) . Addition of antibiotics is now done- Rifampicin (lmg/1) and

Amphotericin B (250 μg/l). For eg. If 1 liter of media is prepared, 1 mL of Rifamipicin from lmg/ml of stock solution and 250 μΐ of Amphotericin B from lmg/ml of stock solution is added, with the help of a micropipette.

f) . The media solution is boiled on a stove/ hot plate.

g) . Gradually the agar powder is added and stirred continuously to avoid formation of clumps.

h) . After taking it off the heat, color solution (working solution) is added immediately in the prescribed amount.

i) . Poured the media in the vessel and placed it on a flat surface,

j). Allowed it to cool. Media preparation for Ex vitro terrarium- a) . Measured 1 liter of distilled water with the help of graduated measuring

cylinder.

b) . Poured it into a stainless steel bowl.

c) . Brought it to boil.

d) . Added 8 grams (0.8%) of agar gradually with continuous stirring.

e) . Added the desired color solution in the prescribed amount with gentle stirring. f) . Poured it into the desired vessel before it gets cool.

g) . After pouring, placed the vessel on a flat surface and allowed it to cool.

■- Preparation of antibiotics (needed only for In vitro terrarium)-

10 mg/10 ml (1 mg/ml) of stock solution for each antibiotic is prepared.

^■ Opened the capsule/vial carefully in an autoclave sterilized Petri plate.

^■ Weighed 10 mg (0.010 grams) of the antibiotic powder on a weighing balance.

^■ Dissolved this powder in 10 ml of autoclaved distilled water.

^■ Keep this solution in a vial

^■ The antibiotic solution is refrigerated after adding in media for further use.

a) . Rifampicin:

The concentration to be added from this stock solution in the medium is lmg/1.

b) . Amphotericin B:

The concentration to be added from this stock solution in the medium is 250 μg/l.

Special note- Both these antibiotics are added before boiling the medium on the stove, but, after adjusting the pH of the medium. Preparation of working solutions of Colors-

->For preparing working solution from liquid food colors (concentrated solution):

a) . Took 1 ml of the concentrated color solution from the bottle into a

100 ml borosil flask.

b) . Added 9 ml of autoclaved distilled water in the flask. c) . Stirred gently.

d) . This working solution is added in 1 liter of boiling hot media prepared above, for a standard dark shade of the color.

Liquid colors used are- Blue; Raspberry red; Rose pink and

Violet.

Special note- For preparing light shade of blue, 2.5 to 5 ml/1 concentration of working solution was added.

->For preparing working solution from dry food color (powdered form): a) . Weighed 500 mg of dry color using a weighing balance.

b) . Added 10 ml of autoclaved distilled water in a 100 ml borosil flask. c) . Added the powder color in it and stirred well.

d) . This 10 ml of working solution is used for preparing 1 liter of media, for a dark shade of the color.

Dry colors used are- Green; Sunset orange and Lemon yellow. Special note- The amount of this working solution is decreased if the desired shade has to be light.

-> For preparing other colors that are not readymade available: a) . Brown-

Added 1 ml of green working solution prepared above and 3 ml of red working solution, in a separate borosil flask.

This 4 ml of Brown colored working solution is added in 1 liter of boiling hot media to make a brown colored medium.

b) . Sea green-

Added 1 ml of green colored working solution and 3 ml of blue colored working solution, in a separate borosil flask.

Used 3 ml of this Sea green colored working solution in 1 liter of boiling hot media to make sea green colored medium. c). White- Colorless medium is considered as white. The agar gives the medium a white appearance.

> After pouring of the colored media in the desired vessel, for preparing In vitro terrarium, the media is autoclaved, since it has to be aseptically maintained.

^'r Autoclaved the closed vessel with the lid/cap tightly placed above the vessel at 15 psi for 15-20 minutes.

After autoclaving, the vessels are immediately transferred in the inoculation room.

Amount of medium added in the vessels is given in the following table:

2. Stones/Sand/Shells/Gravel:

> After acquiring the Stones/Sand/Shells/Gravel from the local market, all of them are washed separately under tap water. Thorough rinsing is done.

For In vitro terrarium: All of these are put in separate autoclavable containers with tight lids. They are autoclaved at 15 psi for 15- 20 minutes. This step is additional and is done to avoid any chances of contamination in the product. elly balls:

1 packet of the jelly balls is placed in a 400 ml container and half filled with autoclaved distilled water.

After approximately 4 hours, the balls swell up and occupy whole the container.

For In vitro terrarium: All of these are put in separate autoclavable containers with tight lids or put directly in the medium at the time of autoclaving.

They are autoclaved at 15 psi for 15- 20 minutes.

This step is additional and is done to avoid any chances of contamination in the product. Sculptures:

Sculptures are rinsed with tap water and kept aside for drying.

Sculptures to be used for In vitro terrarium are rinsed thoroughly under tap water. Then, they are rinsed with 70% alcohol.

Placed in separate containers with lids tightly closed and then autoclaved at 15 psi for 15 to 20 minutes. Glassware:

All the glassware has to be rinsed thoroughly with tap water and dried (in hot air oven). For preparing In vitro terrarium, the glassware has to be autoclaved at 15 psi for 15 to 20 minutes after drying. 6. Plants: For preparing- In vitro terrarium:

In vitro plants are acquired from a tissue culture laboratory. These plants should be kept under observation in the growth room for at least 2 weeks, to check for any contamination, before inoculating them under the laminar cabinet in sterile conditions.

> Ex vitro terrarium:

Use of tissue cultured grown plants are preferred over nursery plants. Tissue cultured plants are more healthy and sturdy, especially in case of ornamental plants.

Aquatic terrarium:

Aquatic plants are acquired from a local aquarium shop. Plants appearing healthy and young are acquired.

Special note-

• Dried and older leaves of each plantlet should be removed before placing any type of plant in the product.

• Young plants are preferred.

• Roots should be excised and shoots with intact meristem be placed in the medium (in all products).

7. Instruments- Instruments needed for inoculation are (for all products): Scalpel; Blade and Forceps.

> Directions for their use under In vitro conditions are- Scalpel and Forceps are wrapped separately in aluminum foil and kept for autoclaving at 15 psi for 15-20 minutes. Fresh sterile blades are used. > For In vitro terrarium: Absorbent cotton is placed in bottle and autoclaved. Quick fix glue is required for sealing the lid/cap of the vessel.

70 % alcohol is prepared using autoclaved distilled water and poured in autoclaved container. reparation of laminar cabinet (only for In vitro terrarium):

Wiped the surface of laminar cabinet with 70 % alcohol.

> Placed all the containers to be inoculated on the left side of working bench.

> Placed all the instruments along with sterilized cotton and 70 % alcohol on the right side of the bench.

Wiped a packet of quick fix glue with the 70 % alcohol cotton swab and place it inside the cabinet.

Bottles containing autoclaved sculptures are kept at the right side. Switched ON the glass bead sterilizer.

Turned ON the UV light for 1 hour.

> After one hour, turned OFF the UV lights.

The cabinet is ready for inoculation.

Shifted the bottles containing In vitro plants, from the growth room to the inoculation room.

Turned ON the light and Air Flow of the cabinet.

Method/ Procedure of Performing (Best known)-

> In vitro terrarium:

Combinations used for creating various appearances- Test Tubes (Figure 8.):

Table 4.

White(colorless

c). Double )

Raspberry red

shaded:

Light White(colorless and )

Dark Lemon yellow 05 blue Sea green

Orang White(colorless e and )

Red Red

Lemon yellow

Sunset orange 05

Brown

Green White(colorless and )

Yello Lemon yellow 03 w Green

Sand 2. White Raspberry red

Rose pink

Dark blue

Light blue

Brown

Lemon yellow

Green

Sunset orange

Sea green Violet 11

White

Gravel a). Big sized Raspberry red

Rose pink

Dark blue

Light blue

Brown

Lemon yellow

Green

Sunset orange

Sea green

Violet 11

White(colorless

)

b). Small sized Raspberry red

Rose pink

Dark blue

Light blue

Brown

Lemon yellow

Green

Sunset orange

Sea green 1 1

Violet

White(colorless )

Shells Miscellaneous Raspberry red

size Rose pink

Dark blue

Light blue

Brown

Lemon yellow Green

Sunset orange Sea green

Violet 11 White

(colorless)

Jelly balls 5. Miscellaneous Raspberry red

color

Rose pink

Dark blue

Light blue

Brown

Lemon yellow Green

Sunset orange Sea green

Violet 1 1

White

(colorless)

Total

number of 95 combination

s made =

For other glassware:

The combinations depend upon number of different size and shape of vessel* number of colored medium (i.e., 11)* Use of gravel/sand/stones/jelly balls/shells, individually or in combination; with or without sculptures. In other words, number of possible combinations for preparing different appearance of the product can be 'η'.

1. After the laminar cabinet is ready, wiped the working bench with 70% alcohol using sterilized cotton swab.

2. Wiped hands and arms with 70% alcohol.

3. Wiped the bottles containing In vitro plants with 70% alcohol before opening.

4. Opened and taken out one at a time plantlet with the help of a sterilized forceps.

5. Placed the plantlet on the sterilized Petri plate and cut its roots by using sterilized blade fixed on a sterilized scalpel.

6. Opened the vessel to be inoculated with the left hand. With the right hand, the plantlet was holded with the forceps and placed in the medium.

7. The vessel was immediately recapped.

8. Repeated the steps again from Step 4, if more plantlets are to be inoculated in the vessel or sealed the cap immediately with the help of quick fix glue.

9. Kept the vessel in growth room under observation for at least two weeks, to check for contamination. Various combinations used as explained with reference to the figures:

Table 5.

Jelly balls on surface of medium; 2 shoots of Syngoni m and 2 shoots of Sedum.

8. 9 200 ml Light blue colored medium; Shells; 2

Cylindrical shoots of Syngonium.

glass(medium

poured 50 ml)

9. 10 8 inch fish bowl Orange colored medium; Small sized

gravel at the bottom; 1 noddy sculpture and 1 tweety sculpture; number of plantlets depend upon the space available, here,- 10 Syngonium plantlets weTe inoculated.

10. 1 1 8 inch big 1^st: Pink colored medium; Gravel at the glasses(2) bottom; 1 cow sculpture; 4- White and green variegated shoots + 4- Red and green variegated shoots of Syngonium. 2^nd: Light-green colored mediunT ; White stones at the bottom; 1 deer sculpture; 3-White and green variegated shoots + 3- Red and green variegated shoots of Syngonium.

1 1. 12 10 inch fish bowl Forest-green colored medium; 1 red parrot sculpture; 8- Sedum shoots + 2- Red and green variegated shoots of Syngonium.

12. 13 10 inch fish bowl Dark-blue colored medium; 1 Big yellow fish sculpture; 2 small tortoise sculpture; 1 medium duck sculpture; Multi-colored jelly balls on the surface; 3 - Red and 1 green variegated Syngonium shoots + 10

White and green variegated Syngonium shoots.

13. 14 10 inch fish bowl Brown colored medium; 1 deer, 1

cottage, 1 well and 1 rural woman figurine sculptures ;Multi-colored jelly balls on the surface; White stones at the bottom ;3 - Red and green variegated Syngonium shoots + 15 White and green variegated Syngonium shoots.

*for knowing the amount of medium used in each vessel, refer to Table 3.

A Light-green colored medium was prepared by adding half (5 ml of 10 ml working solution) the original amount of color needed for preparing Forest green color. r- Ex vitro terra rium:

• Prepared the desired combination of glassware with the desired elements such as sand and stones/ gravel and stones/jelly balls and stones/only stones/agar medium with stones; with or without sculptures [as depicted in Figure 15 to 18 & 20 to 23].

• Selected the tissue cultured plants and cut off their roots, taking care not to damage their meristem.

• Placed the shoot(s) in the vessel.

• Kept it under observation for one week.

• Product is ready for sale. Description for Figure 8. From left to right

Ex vitro terrarium depicted in the figures are explained as follows:

Table 6.

• Artificial Ex vitro terrarium: Plant/flower/shoots are artificial; with the combination of other elements. Aquatic terrarium:

Glassware for aquatic terrarium should necessarily be elegant and preferably cylindrical in shape [depicted in Figure 19]. Other shapes can well be used.

• Aquatic terrarium requires layering, so we start with a preparing a layer of coco peat (optional).

• Another layer of big sized gravel.

• Next layer should be of sand (our main binding medium in place of soil). Sand will hold the plants in place.

• With the help of a big forceps, place the aquatic plants inside the vessel. They should be properly distributed.

• Drinking water should be immediately poured in the vessel, till the height of the tallest shoot.

• Place the desired set of sculptures.

• I utilized two fish sculptures, one tortoise sculpture and multi-colored stones to complete the product.

• Lid should be opened from time to time, to provide aeration to the plants.

• Installation of an air pump is essential for its longevity. Aquatic terrarium depicted in the figures is explained as follows:

Table 7.

vessel with sand thick layer as binding medium; fancy lid 10 aquatic plant shoots ; 2 big fish sculptures + 1 big tortoise sculpture;

3 liters drinking water.

Claims

I Claim-

1. The combination of two or more of these elements: Sand/Stones/ Gravel/ Jelly balls (synthetic, water-absorbing)/ Shells Sculptures/ Semi- solid medium (with or without color) for decoration purpose with plantlets in a glass vessel is the basic idea for this invention.

2. This invention varies from the prior art as the indicated invention has one of the

following basic medium: colored semi-solid medium (nutritional, in case of In-vitro terrarium and non-nutritional, in case of Ex- vitro terrarium); sand; stones; gravel; jelly- balls; shells (for Ex-vitro and Aquatic terrarium).

3. In-vitro terrarium includes the use of autoclavable and exotic glassware with air-tight lids. For decoration, one or more of these elements: sculpture(s) (with or without basic theme); sand/ stones/ gravel/ jelly balls/ shells with semi-solid nutritional medium (with or without color) are included along with In-vitro plantlets.

4. In-vitro terrarium will include exotic glassware of varied shapes and sizes. The inventor claims the various shapes of the test tubes including: Rectangle; Square, Circle; Oval; Triangle; Pentagon and Hexagon which may necessarily be flat bottomed.

5. Ex-vitro terrarium includes the use of exotic glassware for decoration purpose, by

incorporating ornamental or succulent plants and two or more of these materials:

sculpture(s); sand/stones/gravel/ jelly balls/shells with non-nutritional agar medium (with or without color).

6. Aquatic terrarium includes the use of exotic glassware for decorating purpose,

incorporating two or more of these materials: sculpture(s) (with or without a basic theme); sand; stones; gravel; jelly balls; shells with aquatic plants.

7. The use of themes by combining various set of sculptures with use of one or more of these: Sand/ Stones/ Gravel/ Jelly balls/ Shells/ Semi- solid medium (with or without color) in glassware to make Aquatic- Ex vitro- In vitro terrarium.