In Vitro Propagation Plum Plants. Agricultural Biotechnology. Laboratory Experiment. Macro.

In-Vitro Propagation Plum Plants. Agricultural Biotechnology.

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Environmental Condition, Methods, Types and Application

Meaning of Plant Tissue Culture:

Plant tissue culture is the technique of maintaining and growing plant cells, tissues or organs especially on artificial medium in suitable containers under controlled environmental conditions.

The part which is cultured is called explant, i.e., any part of a plant taken out and grown in a test tube, under sterile conditions in special nutrient media. This capacity to generate a whole plant from any cell/explant is called cellular toti-potency. In fact, the whole plant can be regenerated from any plant part (referred to as explant) or cells. Gottlieb Haberlandt first initiated tissue culture technique in 1902.

Hormones used in Plant Tissue Culture:

  1. Auxins neoline (Indole-3-acetic acid, Indole-3-butyric acid, Potassium Salt— Naph­thalene acetic acid 2, 4-Dichlorophenoxyacetic acid p-Chloro-phenoxy acetic acid)

  2. Cytokinins (6-Benzylaminopurine, 6-Dimethylallylaminopurine (2ip), Kinetin)

  3. Gibberellins (Gibberellic Acid)

  4. Abscisic Acid (ABA) (Abscisic Acid)

  5. Polyamines (Putrescine, Spermidine)

Environmental Conditions:

There are three important aspects in vitro (outside the living organism and in an artificial environment) culture namely:

(i) nutrient medium,

(ii) aseptic conditions and

(iii) aeration of the tissue

  1. Nutrient Medium:

The composition of plant tissue culture medium can vary depending upon the type of plant tissues or cell that are used for culture. A typical (generalized) nutrient consists of inorganic salts (both micro and macro ele­ments), a carbon source (usually sucrose), vitamins (e.g., nicotonic acid, thiamine, pyridoxine and myoinositol), amino acids (e.g., arginine) and growth regulators (e.g., auxins like 2,4-D or 2,4-dichlorophenoxyacetic acid and cytokinins such as BAP = benzlaminopurine and gibberellins). Other compounds like casein hydrolysate, coconut milk, malt extract, yeast extract, tomato juice, etc. may be added for specific purposes.

Plant hormones play impor­tant role in growth and differentiation of cultured cells and tissues. An optimum pH (usually 5.7) is also very important. The most extensively used nutrient medium is MS medium which was developed by Murashige and Skoog in 1962. Usually a gelling agent agar (a polysac­charide obtained from a red algae Gelidium amansi) is added to the liquid medium for its solidification.

  1. Aseptic Conditions (Sterilization):

Nutrient medium contains ample sugar which increases growth of microorganisms such as bacteria and fungi. These microbes compete with growing tissue and finally kill it. It is essential to maintain aseptic conditions of tissue culture. Thus sterilization means complete destruction or killing of microorganisms so that complete aseptic conditions are created for in vitro culturing.

  1. Aeration of the Tissue:

Proper aeration of the cultured tissue is also an important aspect of culture technique. It is achieved by occasionally stirring the medium by stirrer or by automatic shaker.

Plant Material—the Explant:

Any part of a plant taken out and grown in test tube under sterile conditions in special nutrient media is called explant.

 

Methods of Plant Tissue Culture:

Plant tissue culture includes two major methods:

(A) Type of in vitro growth-callus and suspension cultures.

(B) Type of explant— single cell culture, shoot and root cultures, somatic embryo culture, meristem culture, anther culture and haploid production, protoplast culture and somatic hybridisation, embryo culture, ovule culture, ovary culture, etc.

Types of Plant Tissue Culture:

Callus and Suspension Cultures:

In callus culture, cell division in explant forms a callus. Callus is irregular unorganised and undifferentiated mass of actively dividing cells. Darkness and solid medium gelled by agar stimulates callus formation. The medium ordinarily contains the auxin, 2,4-D, (2, 4- dichlorophenoxy acetic acid) and often a cytokinin like BAP (Benzyl aminopurine). Both are growth regulators. This stimulates cell divison in explant. Callus is obtained within 2-3 weeks.

A suspension culture consists of single cells and small groups of cells suspended in a liquid medium. Usually, the medium contains the auxin 2,4-D. Suspension cultures must be constantly agitated at 100-250 rpm (revolutions per minute). Suspension cultures grow much faster than callus culture.

Sub culturing:

If tissue cultures are kept in the same culture vessel, they die in due course of time. Therefore, cells/tissues are regularly transferred into new culture vessels containing fresh media. This process is called sub culturing. It is important to note that during subculture; only a part of the culture from a vessel is transferred into the new culture vessel.

The callus and suspension cultures may be used to achieve cell biomass production, regeneration of plantlets, and production of transgenic plants and isolation of protoplasts.

 

 

Single Cell Culture (Cell Cloning):

As stated earlier, cells derived from a single cell through mitosis constitute a clone and the process of obtaining clones is called cloning (asexual progeny of a single individual make up a clone). There are two popular techniques for single cell culture.

  1. Bergmann’s Plating Technique:

This is widely used technique. The cells are sus­pended in a liquid medium at a cell density that is twice the desired density in the plate. Sterilized agar (Ca 1%) medium is kept malted in a water bath at 35°C. Equal volumes of the liquid and agar media are mixed and spread in Ca 1 mm thick layer in a petridish. The cells remain embedded in the soft agar medium which is observable under a microscope. When large colonies develop they are isolated and cultured separately.

  1. Filter Paper Raft Nurse Tissue Technique:

Single cells are placed on small pieces (8×8 mm) of filter paper, which are placed on top of callus cultures several days in advance. This allows the filter papers to be wetted by the callus tissues. The single cells placed on the filter paper derive their nutrition from the callus. The cells divide and form macroscopic colonies on the filters. The colonies are isolated and cultured.

Shoot and Root Cultures:

Shoot culture is promoted by a cytokinin like BAR However; root culture is promoted by an auxin like NAA (naphthalene acetic acid). The shoot and root cultures are generally controlled by auxin-cytokinin balance. Usually, an excess of auxin promotes root culture, whereas that of cytokinin promotes shoot culture. Roots culture from the lower end of these shoots to give complete plantlets.

Somatic Embryo Culture:

A somatic embryo develops from a somatic cell. The pattern of development of a somatic embryo is comparable to that of a zygotic embryo. Somatic embryo culture is induced by a high concentration of an auxin, such as 2,4-D. These embryos develop into mature embryos. Mature somatic embryos or embryoids germinate to give complete plantlets.

Establishment in the Field:

The plantlets are removed from culture vessels and estab­lished in the field. This transfer is done by specific procedures called hardening. During hardening, plantlets are kept under reduced light and high humidity. Hardening procedures make the plantlets capable of tolerating the relatively harsher environments outside the culture vessels.

Endosperm Culture:

Tissue culture methods are also used for culturing endosperm. It is unique because it supplies nutrition to the developing embryo. It is also triploid in its chromosome constitution. Triploid plants are used for the production of seedless fruits (e.g., apple, banana etc.). The technique of endosperm culture involves the following:

(i) The immature seeds are dissected under aseptic condition. Endosperms along with embryos, are excised. Sometimes, mature seeds can also be used.

(ii) The excised endosperms are cultured on a suitable medium and embryos are removed after initial growth.

(iii) The initial callus phase is developed.

(iv) The shoots and roots may develop and complete triploid plants are formed for further use.

Meristem Culture:

Meristem is a localized group of cells, which are actively dividing and undifferentiated but ultimately giving rise to permanent tissue. Although the plant is infected with a virus, yet the meristem is free of virus. Therefore, meristem can be removed and grown in vitro to obtain virus free plants. Cultivation of axillary or apical shoot meristems is called meristem culture. The apical or axillary meristems are gener­ally free from virus. Meristem culture involves the development of an already existing shoot meristem and subsequently, the regeneration of adventitious roots from the developed shoots.

It usually does not involve the regeneration of a new shoot meristem. The explants commonly used in meristem culture are shoot tips and nodal segments. These explants are cultured on a medium containing a cyto­kinin (generally BAP). The plantlets thus obtained are subjected to hardening and, ultimately, established in the fiddi Meristem culture is carried out in Potato, Banana, Cardamom, Orchids (protocorm stage), Sugar­cane, Strawberry, Sweet Potato, etc. It is used in (i) Production of virus-free plants like potato, sugarcane, banana and apple, (ii) Germplasm conservation, (iii) Production of transgenic plants, (iv) Rapid clonal multiplication.

Anther Culture and Haploid Production:

An individual/cell having the chromosome number found in the gametes of the species is called haploid. Formation of haploid is called haploid production. Thus haploid individu­als arise from the gametes. A haploid has only one copy of each chromosome. Haploids are sterile and of no direct value.

When the chromosome number of a haploid plant is doubled, the plants of normal chromosome number for particular species are obtained. These plants are homozygous and are produced in 2-3 years. The chromosome number of these haploid plants is doubled by using colchicine to obtain homozygous plants.

In nature, haploid plants originate from unfertilized egg cells, but in laboratory, they can be produced from both male and female gametes. Anther is the part of the flower of Angiosperms producing pollen (microspores), borne at the end of the stamens and usually consisting of four sporangia. When anthers of some plants are cultured on a suitable medium to produce haploid plants, it is called anther culture.

The technique was developed by Guha and Maheshwari (1964) who cultured mature anthers of Datura innoxia. It is highly useful for the improvement of many crop plants. It is also useful for immediate expression of mutations and quick formation of purelines. This technique was first used in India to produce haploids of Datura. In many plants, haploids are also produced by culturing unfertilized ovaries/ovules. Sometimes, pollen grains are separated from anthers and cultured on suitable medium.

Embryo Culture:

Culturing young embryos on a nutrient medium is called embryo culture. Young embryos are obtained from the developing seeds. The embryos complete their development on the medium and grow into seedlings. In general, older embryos are more easily cultured in vitro than young embryos.

Embryo culture is useful as follows:

(i) Orchid seeds do not have any form of stored food. Embryos of such seeds can be cultured to obtain seedlings and maximum seedling formation can be achieved. Embryo culture in orchids can be applied for rapid clonal propagation.

(ii) In certain species, inhibitors present in the endosperm or seed coat make the seed dormant. Such embryos can escape dormancy by culturing on a suitable medium.

(iii) In certain hybrid seeds developed after interspecific crosses, the endosperm degen­erates at an early stage and the young embryo is left with no food, consequently it also dies. Such young embryos can be excised from the seeds and cultured on the nutritive medium. Getting nutrition, they develop into seedlings which can be transplanted in the field.

(iv) A popular example includes hybridization of barley and wheat with Hordeum bulbosum leading to the production of haploid barley and haploid wheat respectively. Haploid wheat plants have also been successfully obtained through culture of hybrid embryos from wheat x maize crosses.

Ovule Culture:

Ovule culture technique is utilized for raising hybrids which normally fail to develop due to the abortion of the embryos at an early stage. Ovules can easily be excised from the ovary and cultured on the basal medium. The loss of a hybrid embryo due to premature abscission of fruits may be prevented by ovule culture. In some cases, addition of fruit/vegetable juice increase the initial growth.

Ovary culture:

Ovary culture technique has also been successfully employed to raise interspecific hybrids between sexually incompatible species, Brassica campestris and B. oleracea. Ovaries are excised from the flowers and cultured at the zygote or two-celled proembryo stage for obtaining normal development on culture medium.

Sometimes coconut milk when used as a supplement to the medium promote formation of fruits that are larger than those formed in vivo (within the living organism). In Anethum, addition of kinetin in the medium caused polyembryony which gave rise to multiple shoots.

Micro propagation:

Micropropagation is the tissue culture technique used for rapid vegetative multiplication of ornamental plants and fruit trees by using small sized explants. Because of minute size of the propagules in the culture, the propagation technique is named as mircopropagation. This method of tissue culture produces several plants. Each of these plants will be genetically identical to the original plant from where they were grown.

The genetically identical plants developed from any part of a plant by tissue culture/micropropagation are called somaclones. The members of a single somaclone have the same genotype. This micropropagation is also known as somaclonal propagation. It is the only process adopted by Indian plant biotechnologists in different industries mainly for the commercial production of ornamental plants like lily, orchids, Euca lyptus, Cinchona, Blueberry, etc. and fruit trees like tomato, apple, banana, grapes, potato, citrus oil palm, etc.

There are four defined steps in micro propagation method. These are:

(i) Initiation of culture from an explant like shoot tip on a suitable nutrient medium.

(ii) Shoot formation multiple shoots formation from the cultured explant.

(iii) Rooting of shoots rooting of in vitro developed shoots.

(iv) Transplantation the hardening of tissue culture raised plants and subsequent trans­plantation to the field.

Advantages of Micro propagation:

These are as follows:

  1. It helps in rapid multiplication of plants.
  2. A large number of plantlets are obtained within a short period and from a small space.
  3. Plants are obtained throughout the year under controlled conditions, independent of seasons.
  4. Sterile plants or plants which cannot maintain their characters by sexual reproduction are multiplied by this method.
  5. It is an easy, safe and economical method for plant propagation.
  6. In case of ornamentals, tissue culture plants give better growth, more flowers and less fall-out.
  7. Genetically similar plants (somaclones) are formed by this method. Therefore, de­sirable characters (genetope) and desired sex of superior variety are kept constant for many generations.
  8. The rare plant and endangered species are multiplied by this method and such plants are saved.

Regeneration of Plantlets:

  1. Preparation of Suitable Nutrient Medium:

Suitable nutrient medium as per objec­tive of culture is prepared and transferred into suitable containers.

  1. Selection of Explants:

Selection of explants such as shoot tip should be done.

  1. Sterilisation of Explants:

Surface sterilization of the explants by disinfectants and then washing the explants with sterile distilled water is essential.

  1. Inoculation:

Inoculation (transfer) of the explants into the suitable nutrient medium (which is sterilized by filter-sterilized to avoid microbial contamination) in culture vessels under sterile conditions is done.

  1. Incubation:

Growing the culture in the growth chamber or plant tissue culture room, having the appropriate physical condition (i.e., artificial light; 16 hours of photoperiod), temperature (-26°C) and relative humidity (50-60%) is required.

  1. Regeneration:

Regeneration of plants from cultured plant tissues is carried out.

  1. Hardening:

Hardening is gradual exposure of plantlets to an environmental condi­tions.

  1. Plantlet Transfer:

After hardening plantlets transferred to the green house or field conditions following acclimatization (hardening) of regenerated plants.

Protoplast Culture and Somatic Hybridisation:

When a hybrid is produced by fusion of somatic cells of two varieties or species, it is known as somatic hybrid. The process of producing somatic hybrids is called somatic hybridisation. First, the cell wall of the plant cells is removed by digestion with a combination of pectinase and cellulase. The plant cells without cell wall are called protoplasts.

The protoplasts of the two plants are brought together and made to fuse in a solution of polyethylene glycol (PEG) or sodium nitrate. The fusion of protoplasts with the help of chemicals is called chemo-fusion. Fusion of protoplasts with the help of high voltage pulse is known as electro-fusion. The fusion of protoplasts not only involves the fusion of their cytoplasm but also their nuclei. The fused protoplasts are allowed to grow on culture medium. Soon they develop their own walls when they are called somatic hybrid cells.

The hybrid cells give rise to callus. Callus later differentiates into new plant which is somatic hybrid between two plants. Somatic hybrids in plants were first obtained between two species of Tobacco (Nicotiana glauca and N. langsdorfit) by Carlson et al in 1972. Successful somatic hybrids have also been got from different species of Brassica, Petunia, and Solanum.

Pomato is somatic hybrid between Potato and Tomato that belong to two different genera and Bomato is somatic hybrid between Brinjal and Tomato. Somatic hybrids are also produced between rice and carrot. The hybrid plant bears both fruits and tubers of the two parents.

(a) Protoplast technology has opened up avenues for development of hybrids of even asexually reproducing plants.

(b) There is a distinct possibility of development of new crop plants, e.g., Pomato.

(c) Somatic hybrids may be used for the production of useful allopolyploids (Individu­als produced by interspecific polyploidy).

(d) Genetic manipulations can be carried out more rapidly when plant cells are in protoplast state. New genes can be introduced (e.g., male sterility, herbicide resistance). Mutations will be easier.

If we conclude, plant tissue culture is a broad term used to define different types of in vitro plant culture. It may be recog­nized in the following types. Each type can result in a whole plant. (1) Callus cul­ture — culture of differentiated tissue from an explant that dedifferentiates. (2) Cell culture — culture of cells or cell aggre­gates (small clumps of cells) in liquid medium. (3) Protoplast culture — culture of plant cells with their cell walls removed. (4) Embryo culture — culture of isolated embryos. (5) Seed culture — culture of seeds to generate plants. (6) Organ cul­ture — culture of isolated plant organs such as anthers, roots, buds and shoots.

Artificial Seeds:

There are many plants which neither have seeds nor produce a small quantity of seeds. To overcome this problem the concept of artificial seeds has become popular, where somatic embryos are en­capsulated in a suitable matrix composed of sodium alginate, along with substances like mycorrhizae, herbicides, fungicides and insecticides. The technique involved in the production of artificial seeds is based on cellular totipotency and somatic embryogenesis.

An artificial seed is a bead of gel containing a somatic embryo (or shoot bud) and the nutrients, growth regulators, antibiotic, etc. needed for the development of a complete plantlet. Artificial seeds may be produced using one of the following two ways: desiccated systems and hydrated systems. In the desiccated systems the somatic embryos (SEs) are first hardened to withstand desiccation and then are encapsulated.

In the hydrated systems, the beads become hardened as calcium alginate is formed, after about 20-30 minutes the artificial seeds are removed, washed with water and used for planting. Hydrated artificial seeds become dry rapidly in the open air. Therefore, hydrated artificial seeds have to be planted soon after they are produced.

In India, this technique of synthetic seeds is being done for sandalwood and mulberry at BARC (Bhaba Atomic Research Centre), Mumbai.

Advantages (i) They can be directly sown in the soil like natural seeds, (ii) They can be stored upto a year without loss of viability, (iii) They are easy to handle, and useful as units of delivery.

The only disadvantage of artificial seeds is the high cost of their production.

Practical Applications of Plant Tissue Culture:

The use of plant cells to generate useful products and/or services constitutes plant biotechnology. In plant biotechnology, the useful product is a plantlet. The plantlets are used for the following purposes.

  1. Rapid Clonal Propagation:

A clone is a group of individuals or cells derived from a single parent individual or cell through asexual reproduction. All the cells in callus or suspension culture are derived from a single explant by mitotic division. Therefore, all plantlets regenerated from a callus/suspension culture generally have the same genotype and constitute a clone. These plantlets are used for rapid clonal propagation. This is done in oil palm.

 

  1. Somaclonal Variation:

Genetic variation present among plant cells of a culture is called somaclonal variation. The term somaclonal variation is also used for the genetic variation present in plants regenerated from a single culture. This variation has been used to develop several useful varieties.

  1. Transgenic Plants:

A gene that is transferred into an organism by genetic engineer­ing is known as transgene. An organism that contains and expresses a transgene is called transgenic organism. The transgenes can be introduced into individual plant cells. The plantlets can be regenerated from these cells. These plantlets give rise to the highly valuable transgenic plants.

  1. Induction and Selection of Mutations:

Mutagens are added to single cell liquid cultures for induction of mutations. The cells are washed and transferred to solid culture for raising mutant plants. Useful mutants are selected for further breeding. Tolerance to stress like pollutants, toxins, salts, drought, flooding, etc. can also be obtained by providing them in culture medium in increasing dosage. The surviving healthy cells are taken to solid medium for raising resistant plants.

  1. Resistance to Weedicides:

It is similar to induction of mutations. Weedicides are added to culture initially in very small concentrations. Dosage is increased in subsequent cultures till the desired level of resistance is obtained. The resistant cells are then regenerated to form plantlets and plants.