HAPLOID PLANT PRODUCTION

 Haploid plants are the plants that have half the number of chromosomes as the diploid fertile sporophytes. Naturally, haploid plants are produced by parthenogenesis.

Haploid plants are produced using androgenesis or Gynogenesis. Androgenesis uses pollen grain for the production of the haploid plant, gynogenesis use ovary or ovule for the generation of the haploid plant.

ANDROGENESIS ( Guha and Maheshwari 1964)

Androgenesis is done through the anther culture or pollen culture.

 ANTHER CULTURE

Anther culture requires the culture of Anther at the uninucleate stage of pollen/microspore development. For Anther culture, flower buds are taken from healthy, young plants.

STERILIZATION

 These flower Buds are surface sterilized using hypochlorite solution. Flower buds are cut under aseptic conditions to isolate the Anther.

Flower buds can be sterilized by flaming, dip the flower in 100% ethanol, and pass it through the flame.

Isolating Anthers from flower buds

 The stage appropriate for the Anther culture is the uninucleate stage of pollen grain development. For this anther from a flower bud is crushed and stained with acetocarmine to observe the exact stage of Pollens development. 

Androgenesis- pollen culture, Anther culture

 These anthers are inoculated in a simple nutrient medium. Sometimes, however, growth hormones are essential for androgenesis. Anther cultures are maintained in alternating periods of light from 12 to 18 hours at 28ºC and darkness from 12 to 6 hours at 22ºC.

 After 3 to 8 weeks anthers burst open due to growing Pollen callus or Pollen embryo. The pollen embryo may germinate in a medium to form plantlets. These plantlets are haploid for their chromosome number.

 Heterogeneous plantlets are produced by the Anther culture, as different Pollens/microspores are genetically different, so they produce genetically different plants.

 Further, the Anther shows asynchronous Pollen development, because pollens are in different stages of development in the Anther. A homogenous population of haploid plants is produced using Pollen's cultures ( Kyo and Harada 1990).

POLLEN CULTURE

  Pollen culture involves the following steps are followed

 Preparation of explant,

Isolation of pollen grain,

 Inoculation of the pollens/ microspores,

 Formation of Haploid plants, and

 Diploidization of haploid plants.

 Preparation of Explant

Flower buds are taken from healthy and young plants. Flower buds perform better in nutrient medium culture. These flower buds are sterilized using chemicals. 

Flower buds

These buds are cut under aseptic conditions to separate Anther from the flower buds. The anthers are homogenized in a B5 medium that contains 13% sucrose with a glass homogenizer.

 The medium is filtered through a nylon mesh of 42μm pore size. This removes large debris from the solution. The filtrate is centrifuged at 1000 rpm for 3 minutes, the supernatant is discarded and the palette is suspended in a B5-13 medium. The suspended palette is loaded on a Percoll gradient centrifuge tube, that has 24% 32%, and 40% gradient Percoll solution. It is again centrifuged at 1000 rpm for 5 minutes.

CLOCKWISE FROM LEFT-(A) Flower bud,( B)

homogenization, (C) filtration,( D) Centrifugation, (E,F,G) gradient centrifugation, 

(H) inoculation of pollens, (J) pollen embryos, (K) plantlet,

 The supernatant is discarded and Pollens are suspended in a B5 medium. The density of pollen grain is adjusted to 20000- 50000 pollens ml^-1 in the suspension B5 medium. The pollens are incubated in dark at 32ºC for 4-5 days and then at 25ºC.

 Pollen embryos are then transferred to a hormone-free medium in a flask. Finally, the mature embryos are transferred to a B5 medium that contains 2% sucrose. 

This will result in the formation of haploid plants, that are sterile. Fertile plants are produced by the diploidization of the Pollen embryo.

 Pollen embryos are formed using four different Pathways depending upon the stage of the microscope. These Pathways also depend upon the species.

Pathway I

 Equal division occurs in the pollen grain of brassica. Both of these cells participate in pollen embryo formation.

Pathway II

 In pathway II the generative cell is formed by first mitotic division which does not participate formation of the pollen embryo. This pathway is taken up by tobacco and Capsicum vegetative cell divide equally to produce pollen grain embryo.

Pathway III

 In this pathway generative cells actively participate in the formation of pollen embryo.

Pathway IV

 In pathway IV, vegetative and generative cells are formed by first mitotic division these are unequal cells, but both of these cells participate in the formation of pollen embryos. This pathway is taken up by Datura pollen grains.

Pollen embryos generation pathways

 Pollen embryo can be transferred to a nutrient medium to form a callus and the callus is used to generate haploid plantlet, or pollen embryo is allowed to develop into different stages of embryogenesis such as globular,  torpedo, and heart shape. The embryos ultimately result in the formation of plants.

GYNOGENESIS

 Gynogenesis was first used by( San and Noeum 1976) gynogenetic haploid plantlets arise from unfertilized egg cells as observed by Huang et al., 1982)

 Young flower ovaries or ovule has been used as explant for the production of gynogenetic haploids. Ovule attached to placentae perform well in gynogenesis treatment

The diploidization haploid plants

 Haploid plants are sterile. During gamete formation, normal meiosis cannot occur.  To obtain the fertile homozygous diploid plant, the haploid plant chromosomes are duplicated. For this three methods are used, first is spontaneous duplication in this process self-duplication of chromosomes occurs by itself, its rate, however, is low.

Diploidization

Colchicine

 0.4% solution of colchicines is used to diploidized the haploid plants. Young plantlets obtained from pollen culture are dipped in 0.4% filter sterilized colchicine solution for 96 hours and then transferred to culture medium this will produce diploid plants.

 Lanolin paste is applied on the Axis of the upper leaves of the main axis. This results in the formation of auxiliary buds that are diploid and fertile ( Lichter et al., 1988).

 Application of haploid plant production

 Production of homozygous line.

 Detection of recessive mutation.

 Shortening of the bleeding cycle.

Study of gametoclonal variation.

 Mutagenesis study, that is the result of a recessive mutation in a plant's genetic transformation. The pollen of some plants acts as an effective system for gene transfer and production of Supermale.

GENERATION OF VIRUS FREE PLANT

 

The vegetatively propagated plants are systematically infected by pathogens. This reduces the yield of plants. The yield increases up to 300% if Virus-free stocks are developed ( Murashige 1980). Viruses are not transported through the seeds, but genetic variations are carried when plants are propagated through seeds. To produce plant clones vegetative propagation is used.  The virus-free plants are produced through plant tissue culture by generating a plant through the vegetative part of the plant. It has been observed that apical meristems are usually free from pathogens ( Quack 1977, Wang and Hu 1980).

Pathogen free plant culture or generation of virus-free plant: Meristem culture

The older tissues are always infected by viruses and other pathogens. The presence of viruses/pathogens increases as we go down or away from the Meristem. It has been proposed that viruses/pathogens are transported through the vascular system so they are also called systemic pathogens. As the vascular system is not developed in the Meristem so there is a reduced chance of the presence of virus in meristematic tissue. Cell to cell transport of viruses occurs through plasmodesmata which are very slow. The presence of a high concentration of auxin inactivates the virus in meristematic tissue. So, meristem culture is used to produce pathogen-free/virus-free plants.

The treatment of viruses

 The light and high-temperature treatment of tissues reduce or inactivate viruses present in the plant tissue (Baker 1962). High-temperature treatment is given to plants using hot water or hot air. Heat treatment has been found effective against isometric and thread-like viruses and diseases that are caused by mycoplasmas.

PRODUCTION OF VIRUS-FREE/ PATHOGEN FREE PLANTS

EXPLANT

Explant of virus-free plant generation is apical Meristems, the shoot tip Meristems that measures 100 μm in diameter to 250 μm in length. The explant is taken with 2-3 primordial leaves, that constitute shoot Apex.

STERILIZATION OF EXPLANT

The explant is surface sterilized by dipping into 70% ethanol and sterilizing with 0.1% sodium hypochlorite for 10 minutes. Fleming after dipping into 95% ethanol has been used for garlic. 

CUTTING

The explant is cut under a microscope using forceps and a needle. The leaf primordia are removed, topical Meristem is cut using a separate sterile needle.  cultured into the medium under aseptic conditions. 

Shoot tip meristem with leaf primordia

INOCULATION

After cutting, the explant is inoculated in an MS medium.

It is cultured for shooting and rooting to obtain plantlets.

The plantlets are mapped for the presence of pathogens.

If the plantlets are found to be pathogen-free these are then used for production stocks of pathogen-free plants.

Clockwise from the above; Explant, Sterilization of explant, Cutting, Inoculation, shooting, rooting, hardening

FACTORS AFFECTING THE GENERATION OF VIRUS-FREE PLANT

 Factors affecting Virus-free plant culture medium Morey, White, and MS media are widely used for tissue culture. Potassium and nitrogen in tissue culture are balanced. MS medium has been found good for Meristem culture as healthy shoots are produced in this medium (Kartha, 1975; Quak, 1977; Wang and Hu, 1980). 

The sugar concentration for meristem culture is 2-4 percent. 

The length of 240 μm the explant develops into a callus.

 BAP and NAA have been shown to be essential to raise full plants from excised Meristem.

The size of the explant 

A complete plantlet is produced from an explant of 200 μm, but an explant smaller than this size will result in the formation of callus. If two or three leaf primordia remain intact, this ensures natural auxin supply to the growing explants ( Smith and Murashige 1970).

LIGHT AND TEMPERATURE

 Light incubation results in the production of plants ( Huth and Bode). Heat and temperature treatment Meristem culture improves the efficiency and effectivity of virus-free plant tissue culture. 

The diurnal low temperature and high-temperature cycle improve the generation of pathogen-free plants in the plant that is sensitive to high temperatures where 40-degree Celsius is given for 16 hours and 22-degree Celsius is given for 8 hours. This eliminated CMV from Nicotiana rustica( Walkey, 1978).

 Maintenance of stock virus-free plant

Stock is maintained in sterile soil in greenhouse or insect Proof cages. Multiple subcultures are used to maintain the stock of virus-free plants.

SINGLE CELL CULTURE

Single cells are cultured to produce clones of plants, to study genetic modification as well to avoid the chimeric effect of tissue culture. Isolated protoplasts and single cells obtained from plant tissues are cultured using different techniques. The single-cell culture is difficult as plant cells require some metabolic products that are synthesized by cells present in close vicinity. 

Single-cell culture uses

 Single cells isolated from plant tissue are cultured to obtain clones of a single cell.

 These single cells cultures are used for crop improvement programs,

 for the generation of plant clones, 

for the generation of genetically modified plants.

 These are used in industry for the production of single-cell protein and other such uses. 

 Different techniques are used to successfully culture single cells.

There are different techniques are used to culture single cells

 Cell plating technique

 Micro chamber technique

 Nurse callus culture technique

 Micro droplet technique and

 Paper raft nurse culture technique

Cell plating technique

Single-cell isolated from loose callus or explants plated in solid medium. For this technique, the single cells are adjusted for their concentration. The concentration can be from 1000 cells to 10000 cells per ml of medium. 

Single cells are present in the suspension medium, this medium is diluted for the required concentration of 100 cells/ml. 

15 ml of the single-cell suspension medium is added to an equal amount of agar medium which is cool down to 35 degrees Celsius to 45 degrees Celsius and single cells are added into this medium. 

The medium is mixed gently to make the suspension into an agar medium. The medium is allowed to solidify.

 This culture is incubated for 16 hours at 25 degrees Celsius under white light. Cells in the Agar medium start to divide and form small colonies of the cells.

 The cells can form a small callus, the callus can be isolated and cultured into a rooting or shooting medium to obtain plantlets.

 Micro chamber technique

 A microchamber is prepared under aseptic conditions. For this technique, two drops of mineral oil are placed on the slide.

 A microchamber is created on this slide between the two drops of mineral oil. In this microchamber is placed a suspension medium, that carries a single cell.

 The two drops of oil are then covered with cover glasses these are called raisers, and a third glass is placed above these two glasses.

 The oil prevents water loss but allows gas exchange. This microchamber is then placed inside the Petri plate and incubated. 

A single cell present in the suspension medium divide and forms the callus of single cells is used to produce plantlets under suitable conditions.

 Nurse culture technique

 Under this technique, the actively dividing callus is irradiated to suspend cells in the dividing phase. This callus is producing phytohormone and metabolites that are required for the division and metabolism of single cells. The single cells are plated on this carpet of irradiated cells. The single-cell will divide and form colonies. Which will later develop into a Callus and this callus can be used for the development of plantlets.

Nurse culture technique

Micro drop technique

Under the microchamber technique, special microchambers are prepared on slides. For this silicon is used. Silicon creates small wells on the Petri plates. The wells are prepared of silicone, and a suspension medium is placed between the small wells, single cells are cultured in these prepared microwells.

Kao and Gleba and Hoffmann developed this method in the late 1970s. They used special Cuprak dishes that had two chambers of different sizes. The inner chamber had microwells. Single cells/ protoplasts are cultured in these wells. Outer chambers are filled with sterilized distilled water, to maintain humidity. It is covered by a disc and sealed with parafilm.

Micro drop method 

This method was developed by Koop and Schweiger (1985). In this method, the protoplast or single cells are cultured in a drop of 1μl culture medium. this micro drop is developed by following steps.

1μl micro drops of 2M sucrose solution are dispensed on a cover glass.

This cover glass is overlaid with silica. Allow drying.

Rinse with water and sterilize under UV.

Dispense mineral oil 1microliter in these microchambers.

Dispense culture medium under the micro drop of mineral oil.

Keep the cover glass in a large chamber that is filled with sterile distill water. It will maintain humidity.

A whole plant can be obtained from single-cell culture, using this method.

 

 

Nurse raft culture technique

 The single cells are cultured on a raft of filter paper. Actively dividing callus is separated from the single cells using filter paper.

 The filter paper is dipped into a nutrient medium. 

On the filter paper, single cells are placed.

 The filter paper raft is placed on the callus. 

The callus will provide metabolites required for the single cells to divide and develop into the colony. 

The colony formed by single-cell on filter paper is used to form plantlets.

PROTOPLAST ISOLATION AND CULTURE

 

Protoplast isolation

 Plant cells have a cell wall around their protoplast. The cell wall is composed of cellulose and pectin. This makes the fusion of plant cells difficult in vitro. It is also difficult to transfer genes to intact plant cells. So, to overcome this barrier plant cell is isolated from the cell wall. The isolation of protoplast is done through the mechanical method or enzymatic method.

 Mechanical method

The mechanical method involves cutting the plasmolyzed cell. Thus releasing the viable protoplast. This method is highly inefficient as the yield of the protoplast is very low. Only vacuolated cells can be isolated through a mechanical method. Klecker used this method in 1892.

Enzymatic method

 In 1960 Cocking demonstrated enzymatic isolation of protoplast. He used cellulase enzyme to isolate the protoplast.

 The successful isolation of protoplast requires cellulase and macerozyme. The commercial preparation for protoplast isolation was made by Takebe et al in 1968. 

Sequential method( Takebe et al)

The enzymatic isolation of protoplast can be done through the sequential method and through the simultaneous method. In the sequential method cellulase and Macerozyme are used sequentially. The macerozyme dissolves pectin of the middle lamina, which frees the plant cells from each other.  The cellulase dissolves the cellulose cell wall to free the protoplasts.

 Simultaneous method( Power and Cocking)

 However, protoplast can be separated using both of these enzymes together. This method is called the simultaneous method of protoplast isolation. The protoplast can be isolated from nonlignified cells mesophyll cells. Sometimes cellulase, pectinase, and hemicellulase are also used for enzymatic isolation of protoplast.

 

Explants, Enzyme solutions A and B,  isolated protoplasts   

 Callus and non-embryonic suspension culture, female gametes can be used for isolation of protoplast.

Factors affecting protoplast isolation

 Different factors affect the isolation of protoplasts. The viability is one of the most important concerns of protoplast isolation. So, some modification to enzymatic treatment has been used. These modifications include pretreatment for protoplast isolation, choice of source, and use of osmoticum for keeping the protoplast viable during enzymatic isolation. The isolated protoplasts are studied for their viability, regeneration of cell walls. These are also important factors in protoplast isolation and culture.  However, the protoplast culture is similar to the single-cell culture.

Choice of source material

 The Source of material for protoplast isolation can be mesophyll cells, as they are loosely arranged. They can be isolated from active callus. Protoplasts are difficult to be isolated from seeds of cereals. From cultured material, protoplasts are isolated from the cells that are in the log phase of division.

 Pre-treatment for protoplast isolation

Mechanical treatment is done of tissue for protoplast isolation when protoplasts are isolated from mesophyll cells of the leaf. The source material is treated under aseptic conditions.  It is sterilized and then washed with water. It is peeled to remove the epidermal tissue. Gentle brushing and cutting the leaf into small pieces also increase yield, as it increases surface area for enzyme action.

Peeled and cut explant is incubated in an enzyme solution and osmoticum
( 0.5%macerozyme, 2% cellulase, 13% sorbitol/ mannitol at pH 5.4

Enzymes for protoplast isolation

 Common enzyme treatments are cellulase, pectinase, and hemicellulase. The commercial preparation of enzymes is available, such as Onizuka cellulase SS, Onizuka macerozyme SS. Pectolyase Y 23 is a highly powerful macerozyme in combination with cellulose, it releases protoplast from mesophyll cells of pea (Nagata and Ishii) 1979). Sometimes enzymatic treatment required cellulase, pectinase as well as hemicellulase.

Protoplast isolation steps- 1 explant sterilization, 2 peelings, 3,4 cutting and

brushing, 5 pretreatments, 6,7 enzyme treatments, 8,9 purification of protoplasts, 10 culture of protoplasts.

pH and temperature for enzymatic isolation

 pH for enzyme activation is adjusted as 4.7-6 and the temperature for good enzyme action is 40 degrees Celsius to 50 degrees Celsius but for protoplast viability that temperature is adjusted as 25 degrees Celsius to 30 degrees Celsius. The incubation period is 30 minutes for an enzyme treatment.

 

 

Osmoticum

 The isolated protoplasts are very fragile, to protect them osmoticum stabilizers are used in enzymes solution. This solution is slightly hypertonic. Sorbitol and mannitol are used as osmoticum for enzymatic isolation of protoplasts. The CaCl₂ also improves protoplast isolation.

 Ionic osmoticum is also used for enzymatic isolation of protoplast. Here KCl and MgSO4 are used as osmoticum.

Purification of protoplasts

Once protoplasts are isolated with an enzyme the next step is to purify protoplast from the osmoticum. For this, the large debris is squeezed and removed by hand. Then the remaining solution is filtered through a nylon sieve or metal sieve. The protoplasts are then present in the solution and these are then isolated through centrifugation. The solution is centrifuged at 100 x g for 10 minutes. This will result in the formation of the palate, the palate has protoplasts.

 The palate is dissolved in the washing solution and centrifuged at 50 x g for 5 minutes. Protoplasts are then isolated through differential centrifugation. These are loaded on a sucrose pad( 21%) above the enzymatic solution. They form a clear layer at the junction of the solutions.  Protoplasts are then taken by pipette and then washed again 2-3 times to obtain pure protoplasts. Sucrose and sorbitol are also used to form gradients for protoplast purification.

 Viability test

 Freshly isolated protoplasts are tested for their viability. this can be done by many methods. The viability of isolated protoplast can be established by observing cytoplasmic streaming of protoplast under the microscope.

Isolated protoplasts

 Measuring oxygen uptake by the protoplasts using oxygen electrodes.

 Through exclusion staining by Evans blue dye is done to exclude dead protoplasts.  Fluorescein diacetate is used to stain viable protoplasts.

Protoplast culture(1)

 Viable protoplasts are cultured like the single-cell culture they can be cultured into a suspension medium to obtain callus or they can be plated in a nutrient agar medium through Bergman's technique of cell plating. Protoplast can also be cultured on agar beads. These techniques are given in single-cell culture methods in detail.

Significance of protoplast isolation

Isolated protoplasts are used to form cybrids and somatic hybrids.

These are used for physical gene transfer experiments.

Protoplast fusion can be done for sexually incompatible organisms.

Protoplasts are used to study the effect of drugs.

These are used for physiological studies.

Micropropagation: Organogenesis

 Organogenesis

 It is the process in plant tissue culture where adventitious shoots, roots, or other organs are developed. 

Organogenesis has two steps the caulogenesis and rhizogenesis.  Caulogenesis is the formation of the shoot. Rhizogenesis is the formation of the root.

ORGANOGENESIS OVERVIEW

 For plant tissue culture, partially differentiated or poorly differentiated tissues are used. These tissues when grown under suitable conditions develop into the root or shoot.

 Organogenesis involves two cellular processes, dedifferentiation, and Redifferentiation.

 Dedifferentiation is a reversal of differentiation that is partially differentiated or fully differentiated cells become meristematic lose their specialty and now become totipotent. That can divide multiple times and can produce all types of plant tissues. 

Redifferentiation is the differentiation process that is regained by the dedifferentiated cells to undergo organogenesis.

The organogenesis can be direct that is the cultured tissue may directly develop into the shoot bud and which is done subjected to rooting.

 Organogenesis can be indirect where culture tissues undergo the formation of callus and the callus is subcultured to form shooting and his shoot and culture to for root formation.

 Organogenesis can be divided into three steps, callus formation, shoot formation, and root formation. 

 Explants for organogenesis can be any issue from roots, stems, buds, or leaves. The most common tissue used is mesophyll cells of the leaf.

 For this leaves are taken from the plant. These leaves are sterilized using hypochlorous acid or HgCl2, then washed with autoclaved tissue grade distill water. The explant is cut under aseptic conditions into small pieces. These pieces are then inoculated into a culture medium. The cut pieces of leaves start showing the development of callus in 2-3 weeks. This callus can be used for the formation of more calluses or it may be subjected to organogenesis. The callus is cut into pieces and subcultured into a shooting medium. In shooting medium callus develop vascular tissue and result in the formation of buds, which result in shoot formation. 

Rooting requires the transfer of shoots in the rooting medium.

Shooting and rooting medium(1)

 The formation of shoot requires the presence of cytokinin, auxin, and carbohydrates. The hormones act in combination or they may be produced by the tissue itself.

 Where external supply is required NAA and kinetin have shown good results for the development of shoot. Where a high ratio of kinetin and auxin determines the formation of the shoot. A 1.5 mg per liter kinetin in addition to 0.15 or 0.3 mg per liter NAA induces shooting.

 The reduced amount of cytokinin to that of auxin results in the formation of callus.

Shoots are cut and inoculated to form roots in the rooting medium. The rooting medium consists of different types of auxin and cytokinin. A 1.0 mg/ L IBA or 1.0 mg/L NAA induces the production of rooting in incubated shoots. 

When complete organogenesis occurs the plantlet is produced, which has roots and shoots. The plantlets are further inoculated multiple times to achieve a perfect length of plantlet.  These plantlets are then allowed to harden. For hardening, they are cultured in soil under aseptic conditions. If the plant survives in the soil they are then cultured in poly farms for further hardening, if they survive there are also, they are cultured in the field.

 

Factors affecting organogenesis

 Organogenesis is affected by internal phytohormones present in cultured tissue.

 If the plant has the internal level of auxin and cytokinin and is appropriate for the formation of bud it will form bud without an external supply of these hormones.

 Again the level of cytokinin to auxin determines the formation of organ whether the tissue will develop into to shoot or root.

The presence of vascular tissue in cultured explant also favors the formation of the shoot in cultured tissue.

 The presence of 2,4-D along with a low level of cytokinin induces callus formation in the cultured tissue.

 High intensity of light increases the formation of callus.

 Regular light and dark periods induce the formation of the shoot in culture tissue.

 Ethylene blocks the formation of shooting in cultured tissue.

Kinetin along with NAA found to be more appropriate for induction of shooting in the cultured plants.

Material and methods

Organogenesis involves following steps,

Preparation of instruments

Preparation of medium

Preparation of explants

Inoculation of explants

Inoculation for shooting

Inoculation for rooting

Hardening

Preparation of instruments

Instruments required for plant tissue culture are scissors, blades, blade handles. These are autoclaved.

Glasswares i.e. flasks, Petri plates, tissue papers, beakers, pipette, and pipette tips, test tubes are washed and packed in aluminum foils and autoclaved, along with tissue grade distilled water.

Preparation of medium

Prepare stocks of MS medium.

Use powdered MS medium.

Weigh 12 g of MS medium.

Dissolve it in 800 ml of tissue-grade water.

Add sucrose to it.

Dissolve gelling agent (agar) in 200 ml of water by heating.

Mix the two solutions and maintain the pH at 5.4 to 5.8.

Autoclave the medium in a flask plugged with a cotton plug.

Pour the medium in culture vessels under aseptic conditions.

Allow it to cool.

Preparation of explants

Take the explants. It must be taken from healthy, disease-free, and actively growing plants.

EXPLANTS

Wash with water and mild detergent.

 Sterilize it with chemicals. Antibiotics are also used for sterilization.

Wash with sterilized water.

Cut into small pieces.

Inoculation of explants

CUTTING OF EXPLANT

Prepared plant tissue is inoculated under aseptic conditions in a culture medium. A callus will develop at the cut ends of the explants in 2-3 weeks.

INOCULATION OF EXPLANT

Inoculation for shooting

SHOOTING

The callus is cut into pieces and inoculated in a shooting medium. Buds will appear in a week. The shoot will develop within two weeks. It is subcultured to separate it from the callus and obtain an appropriate height of shoot.

Inoculation for rooting

ROOTING

Shoots are inoculated to rooting medium for rooting.

In this way, a plantlet is obtained.

This plantlet is culture to obtain a full-length plant. These are then subjected to hardening.

 

Hardening

PLANTING IN POTS

 Plantlets are cultured in pots and subjected to hardening.

Where these plantlets are cultured in greenhouses and hardened for temperature, pH, light intensity, etc.

Hardened plants are then cultured into fields.

PRECAUTIONS

Aseptic conditions should be maintained during cutting, inoculation, media preparation, and pouring.

Laminar airflow should be used as the working platform.

It should be sterilized using UV radiation before working.

UV should be switched off before you start working in LAF and the fan should be switched on while working.

Ethanol should be used to sterilize hands and instruments while culturing.

Surgical blades are used for cutting.

Metal instruments should be sterilized by flaming.

Keep a record of your culture experiments.

After inoculation, close the culture vessels tightly and place them in the culture room.

 

 

 

 

 

PLANT TISSUE CULTURE MEDIA

 

CULTURE MEDIA

 A large number of preparations have been used for plant tissue culture in the laboratory. These preparations are called culture media. The plant requires simple inorganic molecules for its growth. Most of the molecules that are required for plant growth are synthesized by the plant from the simple inorganic molecule absorbed from the soil. As we grow plants in the laboratory these simple inorganic molecules are provided to the plant by culture media. 

The culture media are classified as simple media and complex media. The simple media have all the nutrients required for plants that are measured and calibrated for better growth of plants in the laboratory.

 In complex media, we do not know the exact quantity of nutrients present in the media. They may contain tomato extract, potato extract, coconut water, and other such components. The exact quantity and quality of molecules present in them are unknown.

 Simple media have inorganic salt, sugar, vitamin, and hormone added in a known quantity. Now many preparations are available in powder form that can be used to prepare culture media. 

The different components of culture media

The culture media have inorganic molecules, organic nutrients, vitamins, carbon sources, and hormones.  12 inorganic molecules that are essential for plant growth. These nutrients are grouped as macronutrients and micronutrients. 

Macronutrients are the nutrients that are required in large quantities. Nitrogen, carbon, phosphorus, potassium, calcium, are the main macronutrients.

 Micronutrients are required in small amounts, but they are also essential for the growth of plants.  The micronutrients are usually required for many biochemical reactions. The micronutrients used in culture media are copper, ferric, zinc, magnesium, Boron, Cobalt, and Nickel.

 Organic nutrients

 The organic nutrients that are required for plant growth in culture media are vitamins, amino acids, and growth hormones. The plants can synthesize vitamins that are required for their growth by themselves, but animals require some of the vitamins to be supplemented in their diet. Plant when cultured in the laboratory requires vitamin to be added to their culture media.

VITAMINS

 Vitamins used in culture media are thymine, pyridoxine, Myo-inositol, pantothenic acid, Vitamin C, D, and E. Myoinositol is a natural constituent of plant and pyridoxine is the crucial factor for the functioning of the cell membrane. This also acts as a secondary messenger. Vitamin E is an antioxidant and vitamin C prevents the blackening of tissues in culture.

 AMINO ACIDS

Amino acids are synthesized by plants under normal conditions. Amino acids are provided directly to the plant tissues in a culture medium, and plants absorb and utilize them. Amino acids in nutrient media act as the source of Nitrogen to the plant. Cysteine is included in culture media as an antioxidant, and it controls the oxidation of phenolics and prevents the blackening of tissue.

CARBON SOURCE

 Sucrose, glucose, and Fructose are used as a carbon source in plant tissue culture. Monocots usually require dextrose for their growth in culture media. However, dicots better perform in presence of sucrose. When sucrose is autoclaved, it is naturally broken down into glucose and fructose, which, ensures the availability of carbon sources to the plant. Further, sucrose is required for the differentiation of cells into xylem and phloem. So, for the development of vascular tissue in plant tissue culture sucrose is needed.

 GROWTH HORMONES

 Auxin, cytokinin, gibberellins, ethylene, and abscisic acid are required for the normal growth and development of plants. These growth hormones are supplemented into culture media.

 Auxin

It is required for growth of internode, apical dominance, abscission, and rooting in tissue culture. IAA and indole 3 butyric acid, naphthalene acetic acid, 2,4-D, IBA are widely used for rooting, in interaction with cytokinin. For shoot proliferation 2,4D and 2,4,5-T are effective. Callus growth occurs in presence of 2,4-D. Auxins are usually dissolved in ethanol or in dilute NaOH.

 Cytokinin

 It is concerned with cell division, modification of apical dominance, and shoot differentiation. The most common cytokinin are benzyl amino purine, iso- pentenyl-adenine, furfuryl amino purine. Cytokinin is dissolved in HCl or in NaOH.

Gibberellins

  There are 20 known Gibberellins. GA3 is most commonly used in culture mediums.

 Ethylene

 Tissue produces ethylene on its own and its production increases under stress conditions. Ethylene is also produced in the medium due to heat, oxidation, or sunlight. Ethylene induces somatic embryogenesis in Maze ( Vain et al., 1989).

 Abscisic acid is required for the normal growth and development of somatic embryos.

 GELLING AGENT

 the culture media can be used as liquid broth which is usually used during single-cell culture. but for the formation of plantlets, semi-solid culture media is used. For this gelling agents are used such as Agar, agrose, gelrite.

 Agar

Agar is produced from Gelidium amansii.  It is a natural polymer that is used in 0.8 to 1 percent of the concentration.

 Agarose 

 Agarose consists of β-D(1-3) galactose 3,6-anhydrous α-L(1-4) galactopyranose, linked into a polymer chain of 60-120 monosaccharides unit with Sulphur side group. Agarose has a high cost. It is used only when the high-strength gel is required. Agarose is used in the concentration of 0.4 %.

 Gelrite

Gelrite or phytagel, gellan gum, a linear polysaccharide produced from the bacteria Pseudomonas rhamnose and cellobiose molecules. It consists of potassium, sodium, calcium, and magnesium. But it is free from organic impurities. However, it may form clumping so, when prepared, it should be continuously added by stirring.

 pH

 The pH of the medium is usually adjusted between 5.0 to 6 before sterilization.

In general pH of higher than 6.0 give a fairly hard medium, and pH 5.0 does not allow satisfactory gelling.  The pH of the medium usually changes at different stages of preparation. So, pH is adjusted after adding the gelling agent. pH also changes when we culture plants in the medium. As a ratio of NH4⁺ and NO3^- Ion determines the pH of medium change.  pH influences the availability of different iron in the medium.

DIFFERENT COMPONENTS OF CULTURE MEDIA AND THEIR FUNCTION

NUTRIENT	SOURCE	CONSTITUENT OF (ROLE)	FUNCTION
NITROGEN	NO3, NH4	Amino acid, proteins, hormone, and chlorophyll	Growth of plant
Phosphorus	PO4	DNA, RNA, ATP, etc	Normal growth, plant become sickly in absence of phosphorus
Potassium	KCl, KH2PO4		Cell division, protein synthesis, chlorophyll synthesis
Sulfur	Na2NO4	Protein and amino acid
Calcium	CaCl2	Membrane and cell wall	Promote callose formation thereby inhibiting cell extension, regulating hormone function
Magnesium	MgSO4.7H2O	Component of chlorophyll and co-factor of many reactions, low pH inhibits Mg absorption.	Normal chlorophyll and normal metabolic reactions
Manganese, Zinc, Iron	MnSO4, ZnSO4, Fe-EDTA,	Cofactor, chlorophyll, Mn and Fe interfere with the absorption of each other.	Deficiency of result in chlorosis reduced lignifications
Boron	H3BO3		Shoot tip necrosis
Vitamin, vitamin C, D, E, and B		Metabolism, reduce stress, prevent tissue blackening	Thymine Biosynthesis of amino acid, vitamin E acts as an antioxidant, riboflavin inhibit callus formation
Hormone Auxin Cytokinin Gibberellin Abscisic acid and ethylene	IAA, 2,4-D 2-ip, GA3		Growth and differentiation
Carbon source Sucrose Glucose and fructose			Xylem and phloem differentiation

PREPARATION OF MEDIUM

Here the preparation of medium is given for plant tissue culture.

Dissolve a given amount of prepared powder of media in 80% quantity of tissue-grade water.

Add vitamin and hormone from the stock solution.

Add sucrose.

Add gelling agent in the remaining 20% of water.

Heat the solution to obtain a clear solution or till the gelling agent dissolve completely.

Mix the two solutions to obtain the final quantity of medium.

Maintain the pH to 5.0 to 6.0.

Autoclave the medium.

Pour the medium into the culture vessels.

Allow it to cool.

Culture plant tissue in it, under aseptic condition.