Design and Maintenance of Green House 3(2+1)

Lesson 25 Plant Tissue Culture: History, Terminologies and Laboratory Requirements

25.2 HISTORY

History of plant tissue culture is a record of systematic efforts by botanists to culture excised plant tissues and organs to understand their growth and development under controlled conditions.

25.2.1 Cell Culture

The idea of experimenting with the tissues and organs of plants in isolation under controlled laboratory conditions arose during the latter part of the nineteenth century. German botanist Gottlieb Haberlandt was the first person to culture isolated, fully differentiated cells in 1898. He selected single isolated cells from leaves and grew them on Knop’s (1865) salt solution with sucrose. Haberlandt succeeded in maintaining isolated leaf cells alive for extended periods but the cells failed to divide because the simple nutrient media lacked the necessary plant hormones. Although he could not demonstrate the ability of mature cells to divide, he was certain that in the intact plant body, the growth of a cell simply stops due to a stimulus released by the organism itself, after acquiring the features required to meet the need of the whole organism. Haberlandt’s vision was to achieve continued cell division in explanted tissues on nutrient media; that is, to establish true, potentially perpetual tissue culture. This goal was attained only after the discovery of auxins. Although Haberlandt was unsuccessful in his attempts to culture cells, he foresaw that they could provide an elegant means of studying morphogenesis and the result of such culture experiments should give some interesting insight into the properties and potentialities which the cell as an elementary unit of life possesses.

25.2.2 Organ Culture

In the early part of the 20^th century, efforts in growing excised plant tissues in culture continued with the development of sterile working methods and discovery of the need for vitamin B and auxins for tissue growth. In 1922, Robbins (USA) and Kotte (Germany) reported some success with growing isolated root tips. The first successful experiment to maintain growth and cell division in plant cell culture was conducted by White (1934) who established cultures of isolated tomato roots under aseptic conditions. White’s medium was simple, containing only sucrose, mineral salts and yeast extract, which supplied vitamins. The cultured roots maintained their morphological identity as roots with the same basic anatomy and physiology. This happened only because excised plant organs on nutrient media are capable of synthesizing hormones necessary to maintain cell division. Ball (1946) obtained whole plants from cultured shoot meristem. This heralded the present day method of in vitro vegetative multiplication. Ball is considered the father of so called micro propagation. Morel and Martin cultured shoot meristem of virus-infected plants to raise healthy plants from Dahlia. The cells of the shoot tip of virus infected plants are free of virus or contain a negligible number of virus. Axillary bud proliferation has immense practical applications for large scale clonal propagation of plants of importance in agriculture, horticulture and forestry.

25.2.3 Tissue or Callus Culture

A mass of unorganized protoplasmic (undifferentiated living) cells is known as   ‘callus’ (Figure 25.2.1).

White (1939) cultured tissue of plant tumors (galls) that were produced by a hybrid between Nicotiana glauca and N. langsdorffii on the same medium that was used for tomato roots. Proliferated cell masses from the original explants were divided and subcultured. Gautheret and Nobecourt in 1939 reported unlimited growth of cultures derived from carrot tap root tissue, using indole-3-acetic acid (IAA). The goal at that time was to establish unlimited growth of a culture by repeated subcultures. Much effort was devoted to determine the nutritional requirements for sustained growth. White and Braun (1942) initiated studies on crown gall and tumour formation in plants and Skoog (1944) initiated work on organogenesis in tobacco callus. Although continuously growing cultures could be established in 1939, the objective of Haberlandt to induce cell division in isolated vegetative cells, was not achieved, because the tissues used by them were not meristematic in nature.The most significant event that led to advancement in the field was the discovery of the nutritional properties of coconut milk. Van Overbeek and his coworkers (1941) cultured isolated embryo of Datura on a medium containing coconut milk. The combination of 2, 4-D (2, 4-dichlorophenoxy acetic acid) and coconut milk had a remarkable effect on stimulating growth of cultured carrot and potato tissues [6–8]. In a search for cell division factor, Skoog’s group located such a factor in degraded DNA preparation. It was isolated, identified as 6- furfurylaminopurine, and named it Kinetin. The related analogue, 6-benzylaminopurine, was then synthesized and that too stimulated cell division in cultured tissues. The generic term ‘cytokinin’ was given to this group of 6-substituted amino purine compounds that stimulate cell division in cultured plant tissues. Later Zeatin was discovered as a natural plant hormone. Skoog and Miller advanced the hypothesis that shoot and root initiation in cultured callus can be regulated by specific ratios of auxin and cytokinin. The availability of cytokinins made it possible to induce divisions in cells of mature and differentiated tissues. At this stage, the dream of Haberlandt was realized partially, for he foresaw the possibility of cultivating isolated single cells. Only small pieces of tissue could be grown in cultures. Further progress was made by Muir  who transferred callus of Tagetes erecta and Nicotiana tabacum to liquid medium (culture medium devoid of agar-agar) and agitated the cultures on a shaking machine, to break the tissue into single cells and small cell aggregates. Muir et al. (1954) succeeded in making single cells to divide by placing them individually on separate filter papers resting on top of a well-established callus culture. Callus tissue separated from the cells by thin filter paper, supplied the necessary factor(s) for cell division. Jones (1960) et al. designed a micro-chamber for growing single cells in hanging drops in a conditioned medium (medium in which callus has been grown previously). Using a micro-chamber and replacing the conditioned medium with a fresh medium containing coconut milk, Vasil and Hildbrandt (1965) raised whole plants starting from single cells of tobacco. They transferred single tobacco hybrid cells to a drop of culture medium on a slide, and observed separately under phase contrast microscope and photographed their observations. Cells were observed to divide and form callus which differentiated into roots and leafy shoots. However, they did not prove that the whole plants were the direct product of a single cell, rather than the product of a tissue mass within which somaclonal or other genetic changes might have taken place during growth Finally, Haberlandt’s prediction, that one could successfully cultivate artificial embryos from vegetative cells, was proved by the research of Backs-Husemann and Reinert in Berlin. They mounted isolated single cells on microscope slides and photo graphed repeatedly. Isolated cells divided to form a mass of embryogenic and parenchyma cells which developed into heart shaped and torpedo-shaped embryos with recognizable cotyledons, hypocotyls and radicles. Tuleke (1953) cultured pollen grains of Ginkgo biloba in a medium containing vitamins and amino acids and obtained cell clumps, some of which looked similar to embryos. Yamada et al. reported that culture of Tradescantia reflexa anther produced haploid tissues. Guha and Maheshwari reported that immature pollen grains produced embryos. Colchicine treatment can transform them into diploid fertile plants. Klercker (1892) and Kuster (1909) reported isolation and fusion of protoplasts, respectively. Cocking developed enzymatic method of protoplast isolation. The method involved the enzymatic digestion of cell wall by cellulase and pectinase enzymes extracted from the fungus Myrothecium verrucaria. Cultured protoplasts regenerated new walls, developed colonies and eventually plantlets [16]. Protoplasts are now used for creation of somatic hybrids within and between species and genera. The first hybrid between N. Glauca and N. langsdorffii was produced by Carlson. In 1978, Melchers et al. produced a hybrid between potato and tomato, but the hybrid was sterile. Novel application of protoplast fusion is called cybrid production, where cytoplasm of two species or genera is fused with nuclear genome of only one cell (nuclear–cytoplasmic combination)

25.3 ADVANTAGES OF TISSUE CULTURE

• The biochemical engineer can grow plant cells in liquid culture on a large scale bioreactor.

• The production of dihaploid plants from haploid cultures shortens the time taken to achieve uniform homozygous lines and varieties.

• The crossing of distantly related species by protoplast isolation and somatic fusion increases the possibility for the transfer and expression of novel variation in domestic crops.

• Cell selection-increases the potential number of individuals in a screening program.

• Micro-propagation using meristem and shoot culture techniques allows the production of large number of uniform individuals of species from limited starting material.

• Genetic transformation of cells enables very specific information tobe introduced into single cells which can then be regenerated

25.4 TERMINOLOGIES OF TISSUE CULTURE

• Adventitious- Developing from unusual points of origin, such as shoot or root tissues from callus or embryos, from sources other than zygotes.

• Agar- a polysaccharide powder derived from algae used to get a medium. Agar is generally used at a concentration of 6-12 g/liter.

• Aseptic- Free of microorganisms.

• Aseptic Technique- Procedures used to prevent the introduction of fungi, bacteria, viruses, mycoplasma or other microorganisms into cultures.

• Autoclave- A machine capable of sterilizing wet or dry items with steam under pressure. Pressure cookers are a type of autoclaves.

• Callus- An unorganized, proliferate mass of differentiated plant cells, a wound response.

• Chemically Defined Medium- A nutritive solution for culturing cells in which each component is specifiable and ideally of known chemical structure.

• Clone- Plants produced asexually from a single source plant.

• Clonal Propagation- Asexual reproduction of plants that are considered to be genetically uniform and originated from a single individual or explant and will support the continued cell division of mature cells, leading to the formation of callus.

• Coconut milk- The liquid endosperm of coconut contain the cytokinin zeatin

• Contamination- Being infested with unwanted microorganisms such as bacteria or fungi.

• Culture- plant growing in vitro.

• Detergent- Increasing the efficiency of sterilization.

• Differentiated- Cells that maintain, in culture, all or much of the specialized structure and function typical of the cell type in vivo. Modifications of new cells to form tissues or organs with a specific function.

• Explant- Tissue taken from its original site and transferred to an artificial medium for growth or maintenance.

• Horizontal laminar flow unit- An enclosed work area that has sterile air moving across it. The air moves with uniform velocity along parallel flow lines. Room air is pulled into the unit and forced through a HEPA (High Energy Particulate Air) filter, which removes particles 0.3 μm and larger.

• Hormones- Growth regulators, generally synthetic in occurrence that strongly affects growth (i.e. cytokinins, auxins, and gibberellins).

• Internode- The space between two nodes on a stem

• Media- Plural of medium

• Medium- A nutritive solution, solid or liquid, for culturing cells.

• Micropropagation- In vitro Clonal propagation of plants from shoot tips or nodal explants, usually with an accelerated proliferation of shoots during subcultures.

• Node- A part of the plant stem from which a leaf, shoot or flower originates.

• Pathogen- A disease-causing organism.

• Pathogenic- Capable of causing a disease.

• Petiole- A leaf stalk the portion of the plant that attaches the leaf blade to the node of the stem.

• Plant Tissue Culture- The growth or maintenance of plant cells, tissues, organs or whole plants in vitro.

• Regeneration- In plant cultures, a morphogenetic response to a stimulus that results in the products of organs, embryos, or whole plants.

• Somaclonal Variation- Phenotypic variation, either genetic or epigenetic in origin, displayed among somaclones.

• Somaclones - Plants derived from any form of cell culture involving the use of somatic plant cells.

• Sterile - (A) Without life. (B) Inability of an organism to produce functional gametes. (C) A culture that is free of viable microorganisms.

• Sterile Techniques - The practice of working with cultures in an environment free from microorganisms.

• Subculture - See “Passage”. With plant cultures, this is the process by which the tissue or explant is first subdivide, then transferred into fresh culture medium.

• Tissue Culture - The maintenance or growth of tissue, in vitro, in a way that may allow differentiation and preservation of their function.

• Totipotency- A cell characteristic in which the potential for forming all the cell types in the adult organism are retained.

• Undifferentiated - With plant cells, existing in a state of cell development characterized by isodiametric cell shape, very little or no vacuole, a large nucleus, and exemplified by cells comprising an apical meristem or embryo.

25.5 LABORATORY REQUIREMENTS FOR TISSUE CULTURE

25.5.1 General Organization

Localize each portion of the tissue culture procedure in a specified place in the laboratory. An assembly line arrangement of work areas (such as, media preparation, glassware washing, sterilization, microscopy, and aseptic transfers) facilitates all operations and enhances cleanliness.

25.5.2. Glassware

Use glassware that has only been used for tissue culture and not for other experiments.  Toxic metal ions absorbed on glassware can be especially troublesome. Wash glassware with laboratory detergent, then rinse several times with tap water and, finally, rinse with purified water.

25.5.3 High-purity Water

Use only high-purity water in tissue culture procedures.    Double glass distilled water or deionized water from an ion-exchanger are acceptable. Water should not be stored, but used immediately.  Regular maintenance and monitoring of water purification equipment are necessary. Purified water for tissue culture can also be purchased.

25.5.4 Plant Material

Plants  used  in  tissue  culture  need  to  be  healthy  and  actively  growing. Stressed plants, particularly water-stressed plants, usually do not grow as tissue cultures.   Insect and disease-free greenhouse plants are rendered aseptic more readily, so contamination rate is lower when these plants are used in tissue culture procedures. Seeds that can be easily surface sterilized usually produce contamination-free plants that can be grown under clean greenhouse conditions for later experimental use.

25.5.5 Aseptic Technique

The essence of aseptic technique is the exclusion of invading microorganisms during experimental procedures. If sterile tissues are available, then the exclusion of microorganisms is accomplished by using sterile instruments and culture media concurrently with standard bacteriological transfer procedures to avoid extraneous contamination.Media and apparatus are rendered sterile by autoclaving at 15 lbs/inch2 (121°C) for 15 minutes. The use of disposable sterile plastic ware reduces the need for some autoclaving.  Alternative sterilization techniques such as filter sterilization must be employed for healable substances like cytokinins. Aseptic transfers can be made on the laboratory bench top by using standard bacteriological techniques (i.e., flaming instruments prior to use and flaming the opening of receiving vessels prior to transfer).  Aseptic transfers are more easily performed in a transfer chamber such as a laminar flow hood, which is also preferably equipped with a bunsen burner (Bottino, 1981). If experimental tissues are not aseptic, then surface sterilization procedures specific to the tissues are employed.  Common sterilants are ethyl alcohol and/or chlorox with an added surfactant. Concentration of sterilants and exposure time are determined empirically.

25.6 APPLIED ASPECTS OF PLANT TISSUE CULTURE

Establishment of plant tissue culture techniques has enabled botanists to introduce this method in major areas of plant sciences such as plant breeding, industrial production of natural plant products, conservation of germplasm and genetic engineering.

25.6.1 Plant Breeding

Establishment of cellular totipotency, callus differentiation and vegetative multiplication under in vitro conditions has opened up new dimensions in the applied field of plant sciences. Rapid vegetative propagation or micropropagation of plants of elite characteristics is possible through axillary shoot induction (Figure 1b) and rooting them (Figure 1c) in vitro to raise com plete plantlets. Somatic embryogenesis and organogenesis (callus differentiation) are other methods of micropropagation. Seedlings (Figure 1d) derived from mature seeds can also be used as a source for large-scale multiplication of rare and endangered plant species. Virus-free plants can be raised using apical meristems of virus-infected plants. Homozygous plants can be obtained in a single generation by diploidization of the haploid cells such as pollen grains. Protoplast technology has made it possible to develop somatic hybrids and cybrids of distantly related plant species and genus. Protoplasts are also a suitable material for genetic engineering of plants in a manner similar to gene transfer into bacteria. Cell culture may be an important source of induction and selection of cell variants for production of new varieties of economically important plants

25.6.2 Industrial Production of Natural Plant Products

Plants produce a variety of natural compounds that are used as agricultural chemicals, pharmaceuticals and food additives. Cell culture technique is being used as an efficient system for production of high-value natural plant products at industrial level. In the 1950s and 1960s, great efforts were made by the Pfizer Company to culture plant cells in liquid medium (suspension culture) similar to culture of microbes for production of natural plant products as an alternative to whole plants. Different kinds of bioreactors have been designed for large-scale cultivation of plant cells. Culture of hairy roots produced by transformation with Agrobacterium rhizogenes has been shown to be a more efficient system than cell culture for the production of compounds which are normally synthesized in roots of intact plants. The first tissue culture product to be commercialized by Mitsui Petrochemical Co. of Japan is shikonin, a natural colouring substance, from the cell cultures of Lithospermum erythrorhizon .

REFERENCES:

1. H.R.Dagla, 2012. “Plant Tissue Culture:  Historical Developments and Applied Aspects.” Resonance. PP-759-767.

2. Lorraine Mineo, 1990. “Plant Tissue Culture Technique.” Pages 151-174 in tested studies for Laboratory Teaching, volume 11, Proceedings of the Eleventh Workshop/Conference of the Association for Biology Laboratory Education (ABLE), and 195 pages.