Basic Technique of Plant Tissue Culture

Plant Tissue Culture

Basic Technique of Plant Tissue Culture

Introduction

Plant tissue culture, the growth of plant cells outside an intact plant, is a technique essential in many areas of the plant sciences. Cultures of individual or groups of plant cells, and whole organs, contribute to understanding both fundamental and applied science.
The application of modern biotechnology techniques like plant tissue culture has proven to be a powerful tool in medicinal plant improvement programs. Scientists hit upon a technique where by not only can these plants be preserved from being lost but are also able to develop a complete plant from a small plant part.
Basically plant tissue culture is the technique of growing plant cells, tissues and organs in an artificial, prepared nutrient medium, static or liquid, under aseptic conditions. These explants divide and gradually develop into an unorganized mass of cells called “callus” and subsequently differentiate to form plant or directly give rise to shoots or embryos.
Plant tissue culture is now the major component of technologies which are applied in plant biotechnology. Advances made in genetic engineering and molecular biology can be made manifest in plant through the application of various techniques developed in the field of plant tissue culture. Many of the crop plants regarded as recalcitrant are now amenable to regeneration in vitro using cultured protoplasts, cells or calli, thus each of them can be used as a tool in plant genetic manipulation programs. Considerable progress has been made with regard to the development of media or techniques as well as in understanding the basic aspects, such as cell culture, cellular totipotency. Development of plant tissue culture is closely linked to improvement of techniques of protoplast, cell, tissue and organ culture followed by the success achieved in regenerating whole plants from culture plant materials.
Knowledge of plant tissue culture has contributed greatly to our understanding of the factors responsible for growth, metabolism, differentiation and morphogenesis of plant cells. Plant tissue culture is presently of great interest to molecular biologists, plant breeders and industrialists. Tissue culture methods have been used as a tool for the propagation of genetically manipulated superior clones and for ex-situ conservation of valuable germplasm. The progress in use of cell or tissue culture in producing pathogen free plant as well as in the synthesis of many important secondary compounds (including pharmaceuticals) has been very significant.
Though a considerable progress has been made in tissue culture of plant species, the method is not widely applicable in its present state for cloning, improvement, somaclonal variation, disease resistance, protoplast culture and genetic engineering of many endangered medicinal plant, therefore basic information generated will be useful on these lines of work for specific and selected cases for developing clones for fodder, medicine and various types of resistance.
Most methods of plant transformation applied to GM crops require that a whole plant is regenerated from isolated plant cells or tissue which have been genetically transformed. This regeneration is conducted in vitro so that the environment and growth medium can be manipulated to ensure a high frequency of regeneration. In addition to a high frequency of regeneration, the regenerable cells must be accessible to gene transfer by whatever technique is chosen. The primary aim is therefore to produce, as easily and as quickly as possible, a large number of regenerable cells that are accessible to gene transfer. The subsequent regeneration step is often the most difficult step in plant transformation studies. However, it is important to remember that a high frequency of regeneration does not necessarily correlate with high transformation efficiency. This chapter will consider some basic issues concerned with plant tissue culture in vitro, particularly as applied to plant transformation. It will also look at the basic culture types used for plant transformation and cover some of the techniques that can be used to regenerate whole transformed plants from transformed cells or tissue. Practically any plant transformation experiment relies at some point on tissue culture. There are some exceptions to this generalization, but the ability to regenerate plants from isolated cells or tissues invitro underpins most plant transformation systems.

Plasticity and Totipotency

Two concepts, plasticity and totipotency, are central to understanding plant cell culture and regeneration. Plants, due to their sessile nature and long life span, have developed a greater ability to endure extreme conditions and predation than have animals. This plasticity allows plants to alter their metabolism, growth and development to best suit their environment. Particularly important aspects of this adaptation, as far as plant tissue culture and regeneration are concerned, are the abilities to initiate cell division from almost any tissue of the plant and to regenerate lost organs or undergo different developmental pathways in response to particular stimuli. When plant cells and tissues are cultured in vitro they generally exhibit a very high degree of plasticity, which allows one type of tissue or organ to be initiated from another type. In this way, whole plants can be subsequently regenerated.
This regeneration of whole organisms depends upon the concept that all plant cells can, given the correct stimuli, express the total genetic potential of the parent plant. This maintenance of genetic potential is called ‘totipotency’. Plant cell culture and regeneration do, in fact, provide the most compelling evidence for totipotency.

Types of Tissue Culture

Cultures are generally initiated from sterile pieces of a whole plant. These pieces are termed ‘explants’, and may consist of pieces of organs, such as leaves or roots, or may be specific cell types, such as pollen or endosperm. Many features of the explant are known to affect the efficiency of culture initiation. Generally, younger, more rapidly growing tissue (or tissue at an early stage of development) is most effective. Several different culture types most commonly used in plant transformation studies will now be examined in more detail.

Callus Culture

Explants, when cultured on the appropriate medium, usually with both an auxin and a cytokinin, can give rise to an unorganised, growing and dividing mass of cells. It is thought that any plant tissue can be used as an explant, if the correct conditions are found. In culture, this proliferation can be maintained more or less indefinitely, provided that the callus is subcultured on to fresh medium periodically. During callus formation there is some degree of dedifferentiation (i.e. the changes that occur during development and specialization are, to some extent, reversed), both in morphology (callus is usually composed of unspecialised parenchyma cells) and metabolism. One major consequence of this dedifferentiation is that most plant cultures lose the ability to photosynthesise. This has important consequences for the culture of callus tissue, as the metabolic profile will probably not match that of the donor plant. Callus culture is often performed in the dark (the lack of photosynthetic capability being no drawback) as light can encourage differentiation of the callus.

Cell-suspension Cultures

Callus cultures, broadly speaking, fall into one of two categories: compact or friable. In compact callus the cells are densely aggregated, whereas in friable callus the cells are only loosely associated with each other and the callus becomes soft and breaks apart easily. Friable callus provides the inoculum to form cell-suspension cultures. Explants from some plant species or particular cell types tend not to form friable callus, making cell-suspension initiation a difficult task. The friability of callus can sometimes be improved by manipulating the medium components or by repeated subculturing. The friability of the callus can also sometimes be improved by culturing it on ‘semi-solid’ medium (medium with a low concentration of gelling agent). When friable callus is placed into a liquid medium (usually the same composition as the solid medium used for the callus culture) and then agitated, single cells and/or small clumps of cells are released into the medium.

Protoplasts Culture

Protoplasts are plant cells with the cell wall removed. Protoplasts are most commonly isolated from either leaf mesophyll cells or cell suspensions, although other sources can be used to advantage. Two general approaches to removing the cell wall (a difficult task without damaging the protoplast) can be taken—mechanical or enzymatic isolation. Mechanical isolation, although possible, often results in low yields, poor quality and poor performance in culture due to substances released from damaged cells.
Enzymatic isolation is usually carried out in a simple salt solution with a high osmoticum, plus the cell wall grading enzymes. It is usual to use a mix of both cellulase and pectinase enzymes, which must be of high quality and purity.
Protoplasts are fragile and easily damaged, and therefore must be cultured carefully. Liquid medium is not agitated and a high osmotic potential is maintained, at least in the initial stages. The liquid medium must be shallow enough to allow aeration in the absence of agitation. Protoplasts can be plated out on to solid medium and callus produced. Whole plants can be regenerated by organogenesis or somatic embryogenesis from this callus. Protoplasts are ideal targets for transformation by a variety of means.

Root Cultures

Root cultures can be established in vitro from explants of the root tip of either primary or lateral roots and can be cultured on fairly simple media. The growth of roots in vitro is potentially unlimited, as roots are indeterminate organs. Although the establishment of root cultures was one of the first achievements of modern plant tissue culture, they are not widely used in plant transformation studies.

Shoot Tip and Meristem Culture

The tips of shoots (which contain the shoot apical meristem) can be cultured in vitro, producing clumps of shoots from either axillary or adventitious buds. This method can be used for clonal propagation. Shoot meristem cultures are potential alternatives to the more commonly used methods for cereal regeneration (see the Case study below) as they are less genotype-dependent and more efficient (seedlings can be used as donor material).

Embryo Culture

Embryos can be used as explants to generate callus cultures or somatic embryos. Both immature and mature embryos can be used as explants. Immature, embryo-derived embryogenic callus is the most popular method of monocot plant regeneration.

Microspore Culture

Haploid tissue can be cultured in vitro by using pollen or anthers as an explant. Pollen contains the male gametophyte, which is termed the ‘microspore’. Both callus and embryos can be produced from pollen. Two main approaches can be taken to produce in vitro cultures from haploid tissue. The first method depends on using the anther as the explant. Anthers (somatic tissue that surrounds and contains the pollen) can be cultured on solid medium (agar should not be used to solidify the medium as it contains inhibitory substances). Pollen-derived embryos are subsequently produced via dehiscence of the mature anthers. The dehiscence of the anther depends both on its isolation at the correct stage and on the correct culture conditions. Anthers can also be cultured in liquid medium, and pollen released from the anthers can be induced to form embryos, although the efficiency of plant regeneration is often very low. Immature pollen can also be extracted from developing anthers and cultured directly, although this is a very time-consuming process.

Plant Regeneration

Having looked at the main types of plant culture that can be established in vitro, we can now look at how whole plants can be regenerated from these cultures. In broad terms, two methods of plant regeneration are widely used in plant transformation studies, i.e. somatic embryogenesis and organogenesis.

Somatic Embryogenesis

In somatic (asexual) embryogenesis, embryo-like structures, which can develop into whole plants in a way analogous to zygotic embryos, are formed from somatic tissues (Figure 2.2). These somatic embryos can be produced either directly or indirectly. In direct somatic embryogenesis, the embryo is formed directly from a cell or small group of cells without the production of an intervening callus. Though common from some tissues (usually reproductive tissues such as the nucleus, styles or pollen), direct somatic embryogenesis is generally rare in comparison with indirect somatic embryogenesis. In indirect somatic embryogenesis, callus is first produced from the explant. Embryos can then be produced from the callus tissue or from a cell suspension produced from that callus.

Organogrnesis

Somatic embryogenesis relies on plant regeneration through a process analogous to zygotic embryo germination. Organogenesis relies on the production of organs, either directly from an explant or from a callus culture. There are three methods of plant regeneration via organogenesis. The first two methods depend on adventitious organs arising either from a callus culture or directly from an explants . Alternatively, axillaries bud formation and growth can also be used to regenerate whole plants from some types of tissue culture.

Benefits of Tissue Culture

Application of Tissue Culture

Over 4 million plants have been dispatched for field plantation from these facilities. The tissue culture-raised plants are presently being evaluated under field conditions. This is being done in tandem with the forest departments of Haryana, Uttar Pradesh, Madhya Pradesh, Bihar, Jammu and Kashmir, and Orissa. For initial screening for phenotypically superior trees, only a few hundred plantlets of the same are raised and tested under various agroclimatic zones. The best clones are then mass multiplied and monitored regularly for their performance. Field data suggest a more than 90% survival rate even in the harsh conditions of Aravalis without the life-saving irrigation.

History of Plant Tissue Culture

This cell is evidently the repository of all the information necessary for its subsequent growth into a multicellular, highly organized, complex but co-ordinated system. This tiny totipotent cell conceals the potential for differentiation. The differentiated somatic cells in a plant carry out specialized activities and appear to have surrendered their totipotency in the bargain. The idea of totipotency of plant cells was put forward by G. Haberlandt, the great German physiologist who in 1902 suggested that “one could successfully cultivate artificial embryos from vegetative cells”. He isolated cells from a number of higher plants and maintained them alive in a viable state in simple nutrient solutions for about 10 days. During this period, cell swelling and wall thickening occurred, but the cells failed to divide. Haberlandt’s attempt to grow vegetative cells in an artificial medium did not succeed due to lack of proper techniques and unfortunate choice of highly specialized materials but it opened up new vistas in morphogenesis.
This cell is evidently the repository of all the information necessary for its subsequent growth into a multicellular, highly organized, complex but co-ordinated system. This tiny totipotent cell conceals the potential for differentiation. The differentiated somatic cells in a plant carry out specialized activities and appear to have surrendered their totipotency in the bargain. The idea of totipotency of plant cells was put forward by G. Haberlandt, the great German physiologist who in 1902 suggested that “one could successfully cultivate artificial embryos from vegetative cells”. He isolated cells from a number of higher plants and maintained them alive in a viable state in simple nutrient solutions for about 10 days. During this period, cell swelling and wall thickening occurred, but the cells failed to divide. Haberlandt’s attempt to grow vegetative cells in an artificial medium did not succeed due to lack of proper techniques and unfortunate choice of highly specialized materials but it opened up new vistas in morphogenesis.

Materials and Methods

Methods Involved

The technique of tissue culture is broadly carried out in following three phases:
    a) Pre- experimental Phase
    b) Experimental Phase
    c) Post- experimental Phase
a) Pre-Experimental Phase
It is the preparatory phase and includes following steps:
1. Sterilization of Room
The maintenance of highly aseptic conditions is the most important factor for a successful tissue cultures laboratory. Thus the room of such laboratory should be first washed with disinfectant followed by wiping with 95% ethyl alcohol or 2% sodium hypochlorite. Commercially available disinfectant like extran, Lysol, zephiran and roccal are effective disinfectant (Razdan, 1993).
The final sterilization of the room should be done either by organs mercury lamp of low pressure or by UV radiations or more recently by ozone generating system, which gives 90-100% disinfection in transfer area between 8 m3 to 280 m3. The disinfected area can be used 8 minutes after sterilization is over (Kumar, 1998).
2. Washing of Glassware
All the glassware's used in tissue culture technique are washed with hot or warm water containing soap detergent (labolene). The soap detergent is removed under running tap water followed by a final washing with doubled distilled water. After washing, the glassware should be subjected to over drying at 700 C.
3. Choice of Media
The selection of medium for achieving successful results is an essential criterion in tissue culture technique. The composition of tissue culture medium may range from simple salt mixture to highly complex mixture of organic of inorganic and organic salts.
Table 1: Composition of Murashige and Skoog Media used for in-vitro culture (Bajaj, 1998)
Constituents (Macronutrients) Murashige and Skoog (MS, 1962) mg/L
MgSO4. 7H2O 370
KH2PO4 170
NaH2PO4.H3O ----
KNO3 1900
NH4NO3 1650
CaCl2.2H2O 440
NH4H2PO4 ----


Constituents (Micronutrients) Murashige and Skoog (MS, 1962) mg/L
Boric acid H3BO3 6.2
MnSO4.4 H2O 22.3
ZnSO4.7 H2O 8.6
Na2MoO4.2 H2O 0.25
CuSO4.5 H2O 0.025
CoCl2.6 H2O 0.025
KI 0.83
FeSO4.7H2O 27.8
Na2EDTA 37.3
Thiamine HCI 0.1
Pyridoxine HCl 0.5
Nicotinic Acid 0.5
Myo-inositol 100
Glycine 2.0
Sucrose 30


4. Preparation of Stock Solutions
The preparation of MS media by this method is based on sour concentrated stock solution, which are prepared and kept in refrigerator.
Table 2: Stock of Solution of MS (1962) Macro Salts (*10).
Constituents Amount (g/o) to be taken for stock solution (*100) Final Vol. of stock solution (ml) Amount to be used in litre (ml)
NH4NO3 16.5
KNO3 19.0
CaCl.2H2O 4.4 1000 100
MgSO4. 7H2O 3.7
KH2PO4 1.7


Table 3: Stock of Solution of MS (1962) Micro Salts (*100).
Constituents Amount (g/l) to be taken for stock solution (*100) Final Vol. of stock solution (ml) Amount to be used in litre (ml)
MnSO4. 4H2O 2230
ZnSO4.4H2O 860
H3BO3 620
KI 83 500 5
Na2MoO4.2H2O 25
CuSO4.5H2O 2.5
COCl2.6H2O 2.5
Table 4: Stock of Solution of MS (1962) Vitamins (*100)
Constituents Amount (g/l) to be taken for stock solution (*100) Final Vol. of stock solution (ml) Amount to be used in litre (ml)
Glycine 200
Nicotinic acid 50
Pyridoxine HCL 50 500 5
Thiamine HCL 10
Table 5: Stock of Solution of MS (1962) Inositol (*50)
Constituents Amount (g/L) to be taken for stock solution (*100) Final Vol. of stock solution (ml) Amount to be used in litre (ml)
Myo-Inositol 5 250 5
Table 6: Stock of Solution of MS (1962) Iron Source (*100)
Constituents Amount (g/L) to be taken for stock solution (*100) Final Vol. of stock solution (ml) Amount to be used in litre (ml)
FeSo4.7H2O 2.78
Na2EDTA.2H2O 3.73 500 5
Stock Solutions of Phytohormones
The concentration of phytohormones in plant tissue culture medium is usually represented in milligrams (mg) parts per million (ppm) or micromoles (um). The procedure for the preparation of stock solution of hormones is given in table.
Table 7: Stock Solutions of Phytohormones
Phytohormones Molecular Weight Required Amount of Stock Solution Amount of Solvent required to be dissolved Amount of Water to be added (ml) Final Volume of Stock Solution Final Concentration (mg/l)
Auxin
2,4D 221.04 10 1 ml (0.1) NaOH 99 10 0.1
IAA 175.18 10 1 ml (0.1) NaOH 99 10 0.1
NAA 186.20 10 1 ml (0.1) NaOH 99 10 0.1
IBA 203.23 10 1 ml (0.1) NaOH 99 10 0.1
NDA 202.30 10 1 ml (0.1) NaOH 99 10 0.1
Cytokinin
BA 225.20 10 HCI (0.1)HCI(0.1N 1ml N) 1 ml 99 10 0.1
Kn 215.21 10 HCI (0.1N) 1 ml 99 10 0.1
Z 219.20 10 HCI (0.1N) 1 ml 99 10 0.1
21 IP 203.30 10 HCI (0.1N) 1 ml 99 10 0.1
Giberllin
GA3 346.36 10 1 ml (0.1 N) NaOH 99 10 0.1
MEDIA PREPARATION
The most widely used medium in plant tissue culture is that of Murashige & Skoog (1962). The following steps are used to make 1 L of MS medium from either commercially prepared media packages available from companies such as the sigma chemical company or from stock solutions.
A) PREPARATION OF MS MEDIUM FROM COMMERCIAL PACKAGES
B) PREPARATION OF MS MEDIUM FROM STOCK SOLUTIONS
The preparation of MS media from basic chemicals is time consuming and convenient but it does have the advantage of providing the flexibility to vary individual components of the medium.
Preparation of 1 L of MS medium from stock solutions.
The preparation of 1 litre MS (1962) medium involves following steps:
STERILIZATION OF NUTRIENT MEDIUM
Sterilization of nutrient media can be performed by autoclaving. The nutrient media are generally sterilized by autoclaving at 121 degree centigrade and 1.06 kg/cm2 (15-20 PSC) for 20 minutes. The minimum time required for sterilization depends upon the volume of the medium in the vessel. Prolonged autoclaving may result in breaking and denaturation of various media ingredients. Therefore it is better to dispense media in small aliquots, whenever possible to prevent denaturation of medium ingredients.
STERILIZATION OF GLASSWARE AND INSTRUMENTS
STERILIZATION OF EXPLANTS
Seeds, axillary and Apical meristem and first three axillary buds from nodal segments, leaves were collected from field grown matured plants. The explants were thoroughly washed with 2-4 drops of surfactant (Tween 20 or any soft liquid soap) for 10 minutes under running tap water. Surfactants reduce the surface tension of material being cleaned there by making the disinfectant solution more effective.
After washing with tap water properly the explants were subjected to chemical sterilization in the hood of laminar airflow with 0.1% w/v for 3-5 minutes. After HgC/l2 treatment the explants were washed 3-4 times with sterilized double distilled water to remove the traces of sterilants (HgCl2). Now the explants are ready for inoculation on required medium.
INOCULATION OF EXPLANTS FOR INITIATION OF CULTURE
The inoculation of sterilized explants is carried out in Laminar airflow cabinet, which prior to operation is sterilized with UV light for 30 minutes followed by 15 minutes flow of motor, to Exhaust out ozone from cabinet formed by UV exposure. The hood of the cabinet s sterilized by wet cotton swabs dipped in 75% ethanol. The instruments are further sterilized by flame with repeated dipping in ethanol. The mouth of the culture vial is opened near the flame of the burner and the explants are placed on the medium with the help of sterilized forceps.
The mouth of the culture vial is immediately re plugged. Date of inoculation, medium code and explants code are labeled on culture vials. These vials are then placed in incubation room under controlled environmental conditions.
INCUBATION
Incubation of culture vials is carried out in incubation room under cool white fluorescent light (200-300 flux) of 16 hours photoperiod and 8 hours dark period at a temperature of 25+30C and relative humidity of 50-60%. The cultures were observed after one week.
MERISTEM CULTURE
MULTIPLICATION
Ones aseptic cultures have been established the objective is to induce shoot multiplication. Lateral buds present on the explants may produce shoots which themselves have further buds along them. Such adventitious buds have more potential than the induction of axillary buds for mass clonal propagation of plants. The main advantages of this direct method of clonal propagation are the formation of high multiples of disease free and genetically identical plants. After one weak of initiating experiment the bud start responding by bud break and initiation of shoot after 6 to 10 days of culture, initiated shoots were separated and transferred to different multiplication medium. Numbers of experiments were carried out to maximize the rapid multiplication of shoots. These include use of high concentration of cytokinins, BAP (0.1- 0.5 mg/l), NAA (0.1- 0.5 mg/l) as compared to induction medium.
ELONGATION
Having obtained shoot multiplication it may be necessary to provide particular conditions for shoot elongation to get shoots long to be handled.
ROOT FORMATION
Once the shoots have been produce the elongated multiple shoots have been separated and the next stage for root induction shoots were transferred into the rooting medium containing auxins as a growth regulators.
PLANTING OUT OR ACCLIMATIZATION
As the shoots were well rooted the plantlets were carefully removed out from the culture medium and were transferred to the soil, and were incubated in the green house in the green house condition includes high humidity, free from pathogens, optimum nutrient supply, low light intensity and supply of sucrose liquid. The plants produced adapted to these conditions exposed to outside environment the plants were shifted to the shade house for further growth.

REFERENCES