Haberlandt’s Dogmatic Dream and Its First Realisation

As vividly described in the review by Vasil (2008), the alleged friendship between the botanist Matthias Jakob Schleiden (1804–1881) and the animal physiologist Theodor Schwann (1810–1882) stimulated, among others, the formation of the “cell theory”. Schleiden (1838) was the first to formulate the hypothesis that all plant or animal structures are composed of cells (or their derivatives) that preserve the complete functional potential of the organism.

Cell Biology in Plant Propagation and Breeding

Self-repair of individual somatic cells is an almost universal property of multicellular organisms, both plants and animals. This ability is necessary to allow continuous replacement of cells lost through senescence or damaged by wounding. In both lower animals and most plants, the regeneration process can lead to the formation of new organs. In plants particularly, various regeneration strategies have culminated in mechanisms of vegetative propagation that either complement or even entirely substitute sexual propagation.

From Neˇmec and Haberlandt to Plant Molecular Biology

The high regenerative capacity of plants is a crucial feature of their life strategy. It is an essential part of the mechanisms that both allow these sessile organisms to repair injury caused by pathogens, herbivores and abiotic factors and to undergo rapid vegetative reproduction, so allowing them to dominate in particular environmental niches. Furthermore, various forms of natural regeneration contribute to techniques that are widely used in plant propagation and plant breeding. The biological nature of plant regeneration has been studied since the very beginnings of plant physiology as a science. Research on regeneration of intact plants in vivo was conducted by Bohumil Ne ˇmec, and early studies of in vitro regeneration in plant tissue cultures were carried out by Gottlieb Haberlandt. At this stage, however, suggestions that somatic plant cells possessed a regeneration “totipotency” were in practice often not acknowledged. Nevertheless, real experiments demonstrated that the regenerative ability of particular cells and tissues is clearly determined by the specific interplay of both genetic (or epigenetic) and physiological factors. This makes some systems “nonresponsive” to the standard regeneration procedures.

This regenerative recalcitrancy hampers both the routine vegetative propagation of various plant species and the construction of genetically modified crops. This chapter addresses the basic historical background of studies on plant regeneration and discusses both the results and ideas acquired by means of classical anatomical and morphological studies in the light of our current state of information obtained using modern molecular techniques. The present knowledge of plant regeneration is also viewed in the light of studies of structure and function of the “stem cell niches” of multicellular organisms, examining their role in the ontogenesis of intact plants and in the processes of embryogenesis and organogenesis in vitro. With reference to other chapters in this book, the role of genetics for the realisation of these processes as well as the role of various regulatory factors, of both exogenous and endogenous nature – especially phytohormones – is also examined. The importance to classify regenerative processes unambiguously using exact terminology (in the context of the allied field of regenerative medicine) as a prerequisite for the formation and validation of appropriate working hypotheses is discussed. Finally, this chapter summarises the main problems of current research on regenerative processes in plants and outlines possible directions for solving problems of recalcitrant materials in the context of their use for application.

Potentials and Prospects of Cryopreservation of Plant Cell, Tissue and Organ Culture and Establishment of 'Germplasm Bank'

Due to gradual disappearance of economic and rare plant species the necessity for storage of genetic resources of plant realm in general and agricultural plants in particular was realized by the biologists (Bajaj and Reinert, 1977; Wilkins and Dodds, 1983). The conventional methods of storage fail to prevent from losses caused by (i) attack of pathogens and pests, (ii) climatic disorders, (iii) natural disorders, and (iv) political and economic causes. However, the conventional methods could not save the viability of short lived seeds of economic plants, for example, oil palm (Elaeis guineensis)i rubber (Hevea brasiliensis), Citrus sp. and Coffes sp. (Dodds, and Roberts, 1985).

These materials are stored at low temperature, due to which growth-rate of cells retards; consequently biological activities are conserved for long time.

Cryobiology deals with the study of metabolic activities and their responses in plant materials (and animal ceils) stored at low temperature (- 196°C) by using liquid nitrogen in the presence of cryoprotectants. Dodds and Roberts (1985) have discussed 3 principal methods used for growth suppression in plant tissue culture (i) the alteration of physiological conditions of culture i.e. temperature or gas composition within the vessel; (ii) changing the composition of basal medium e.g. using sub or supra-optimal concentrations of nutrients (some factors essential for normal growth may be either omitted or employed at a reduced level) and (iii) supplementing the medium with growth retardants (e.g. abscisic acid) or osmocegulatory compounds (e.g. mannital, sorbital, etc.).

Storage at reduced temperature has been very affective for tissue culture of most of the plant species such as potato, cassava (Manihot esculentum), pea (Pisum sativum), chickpea (Cicer arietinum), rice (Oryza sativa), wheat (Triticum vulgare), coconut (Cocos nucifera), oil palm (E. guineensis) strawberry (Fragaria vesca) and sugarcane (Saccharum officinarum) (Bajaj, 1987).

Content

⇒ Difficulties in cryopreservation

⇒ Methods for cryopreservation

⇒ Plant cell bank

⇒ Pollen bank

⇒ Achievements through cryopreservation

Difficulties in Cryopreservation
A number of reviews available during the last two decades illustrate the significant progress made in this field and also the outline of the existing problem (Witherr, 1980; Henshaw, 1982). The difficulties are (i) high specific feature of plant cells, such as their large size, strong vacuolization and abundance of water, (ii) cell damage during freezing and subsequent thawing caused by ice crystals formed inside the cells and by cell dehydration, and (iii) gradual formation of large crystals of more than 0.1mm whose facets rupture many cell membranes (Shimada and Ashahina, 1975). However, in the presence of cryoprotectants (the chemicals decreasing cryodestructJ0h) and reduced temperature, free water has enough time to leave the cells. Therefore, it can freeze on the crystal surface in the solution (Samygin, 1974). This results in marked dehydration and protoplast shrinkage (Muzur, 1977). Excessive time and degree of plasmolysis are the reasons of cell destruction during slow freezing, since they cause irreversible contraction ofthe plasmalemma (Wiest and Steponkus, 1978).

Methods of Cryopreservation
The freezing-storage-thawing cycle is an external procedure consisting of the following basic stages:

(i) Selection of Materials. For selecting the plant materials a number of factors are taken into account ; the important ones are, nature and density of cells in the vials/ampules to be cryopreserved ; because the cryoability of the cell cultures depends on these. Young meristematic, highly cytoplasmic and small cells which are non-vacuolated and thin walled and in small aggregates, are good materials to be selected for this purpose. Cell density in vials or amples should be high, as it shows prolonged survival at high cell density.

(ii) Addition of Cryoprotectors. Cryoprotectors are the chemicals which decrease cryodestruction. These are sugars, glycols, sugar alcohols, alcohols, polyvinylpyrrollidone, polyethylene glycol (PEG), polyethylene oxide (PEO), dextrans, hydroxystarch, glycerine, sucrose, and some amino acids (e.g. proline). Bajaj (1987) has advised to use a mixture of two or three cryoprotectants at low concentrations rather than a single cryoprotectant at a high concentration as it could be toxic. During treatment, the cultures should be maintained in ice to avoid deleterious effects.

(iii) Freezing. Freezing should be done in such a way that it does not cause intracellular freezing and crystal formation, as it is possible in sudden freezing. To avoid this problem, regulated rate of cooling or pre-freezing is done. Moreover, freezers have also been developed which allow the uniform temperature decrease at a desired rate, commonly not less than 1°C per minute (Popov, 1985), In 1987, the Institute of Cryobiology and Cryomedicine of the Ukrainian Academy of Sciences (erstwhile U.S.S.R.) devised the programme freezer which envisaged lower rate (0.5°C per minute) of temperature decrease.

(iv) Storage in Liquid Nitrogen. If the cells are not stored at sufficiently low temperature, an additional injury to the cultures may be caused. The storage temperature should be such that it stops all metabolic activity and prevents biochemical injury (Bajaj 1987). Prolonged storage of frozen materials is possible only when the temperature is lower than -130°C. This can be simply achieved with the help of liquid nitrogen, which keeps the temperature -196°C. Popove (1988) stored the cultures of carrot cells for about 5 years by doing so.

(v) Thawing. Thawing is the process of releasing the vials containing cultures from the frozen state to elevate the temperature between 35 and 40°C. It should be done quickly but without overheating. As soon as the last ice crystals disappear, the vials are tranferred into a water bath at 0°C (Popov, 1985).

(vi) Washing and Reculturing. Washing of plant materials is done to remove the toxic cryoprotectants. When low toxic or non-toxic cryoprotectants are used, the cultures should not be washed, but simply recultured. Washing becomes necessary only when cryoprotectants have toxic effects on cells. Washing follows the following procedure : dilution, resuspension, centrifugation and removal of cells. It is, however, possible that some cells die due to storage stress and the most stable ones survive. Therefore, determination of cell viability by culturing them on growth medium is essential.

vii) Regeneration of Plantlets. The viable cells are cultured on non-specific growth media to regenerate into plantlets. Bajaj (1987) has given an extensive list of works on cryopreservation of cells, tissue, and organ culture of various plants e.g. potato, cassava, sugarcane, soybean, groundnut, carrot, cotton, citrus, coconut, etc.

Plant Cell Bank / Germplasm Bank / Cell Crygbank

Cryopreservation of genetic stock i.e. germplasm (or vegetatively propagated crops, recalcitrant producing plants, rare plant species, medicinal, horticultural and forest plants, and VAM fungi) is a novel approach for their conservation in liquid nitrogen on a long term basis. To achieve this goal, a plant cell bank ( = germplasm bank and cell cryobank) has been suggested by Bajaj (1977 a), Bajaj and Reinert (1977) and Popov (1985). Suggestions have also been made that germplasm bank should be attached to some of the International Research Institutes (e.g. IRRI) that would hold responsibility for the storage, maintenance, distribution (at national and international level), and exchange of these disease free germplasm of the important plants. Fig. 10.1. shows the potential and prospects of cryopreservation of plant cell, tissue and organ and establishment of germplasm bank.

Facilities for storage of genetic stock of plants can be developed in large sized cylinders (30-50 liters capacity) where liquid nitrogen does not require refilling for 6-8 months (Bajaj, 1987). Potentials and prospects of cryopreservation of plant cell, tissue and organ culture and establishment of .'Germplasm Bank' (after Bajaj, 1977a).

Fig. 10.1. Potentials and prospects of cryopreservation of plant cell, tissue and organ culture and establishment of .'Germplasm Bank' (after Bajaj, 1977a). Thus, germplasm bank is such a device where facilities of cryopreservation of genetic resources of a variety of plants are available and on demand, the germplasm can be supplied nationally and internationally.


Pollen Bank
Besides germplasm bank, the storage of pollen grains in liquid nitrogen and establishment of pollen bank have also been suggested to retain their viability for various lengths of time. The freeze storage of pollen would enable (i) hybridization between plants with flowers at different times, (ii) growth at different places, (iii) reducing the dissemination of diseases by pollination vectors, and (iv) maintenance of germplasm and enhancement of longevity (Bajaj, 1987).

Achievement made through Cryopreservation
Various forms of plant materials viz. cell suspensions clones, callii, tissues, somatic embryos, root/shoot tips propagules (tubers) pollen grains, etc. have been preserved in liquid nitrogen for prolonged time and tested for their survival and regeneration potential.

No doubt, in most of the cases, the cells/tissues, organs regenerated into plants. Bajaj (1987) has described a number of plant species that have been successfully cryopreserved. Some of the observations made are as below :

(i) Cryopreservation of cell lines : For example, cell suspensions (soybean, tobacco, dhatura, carrot) and somatic hybrid protoplasts (rice x pea, wheat x pea).

(ii) Cryopreservation of pollen and pollen embryos : For example, fruit crops, trees, mustard, carrot, peanut, etc.

(iii) Cryopreservation of excised meristems : For example, potato, sugarcane, chickpea, peanut, etc.

(iv) Cryopreservation of germplasm of vegetatively propagated crops: potato, sugarcane, etc.

(v) Cryopreservation of recalcitrant seeds and embryos: Large sized seeds that are shortlived and abortive, such as oil palm, coconut, walnut, mango and cocoa.

In Vitro Micropropagation and Clonal Multiplication of Crops in Africa

Among the agrobiotechnology tools, in vitro micropropagation of plant tissues or organs, followed by clonal multiplication of the in vitro plants, ranks first in the propagation of a wide range of herbaceous and tree crop species.

In the case of the banana tree, the example of Kenya is very illustrative of the benefits provided by agrobiotechnology. Unlike large parts of Latin America and other banana exporting countries, small farmers, mostly women, are the main producers in Kenya. They grow bananas for home consumption and the national market. It is the most popular fruit in Kenya, and cooking varieties are also an important staple food. Yet, the average banana yield in Kenya—14 tons per ha—is less than one-third of the crop potential under favourable conditions of the humid tropics. The main problem is the infestation of banana stock with weevils, nematodes and fungi, which cause severe diseases, such as Panama disease and black sigatoka. The resulting yield losses make banana a relatively expensive item for consumers. Producers also suffer reduced cash earnings, and the crop potential to contribute to the food security of rural households is undercut. A biotechnology project for the benefit of small-scale banana producers was facilitated by the International Service for Acquisition of Agri-Biotech Applications (ISAAA) and hosted by the Kenya Agricultural Research Institute (KARI) with funding from the Rockefeller Foundation and the International Development Research Centre, Ottawa (Wambugu, 2001). This model project included tissue culture technology for banana propagation and was awarded the 2000 Medal Prize Award by the Global Development Network—an initiative of the World Bank and the Japanese Government. The project benefited from a private–public partnership that demonstrated the feasibility of North–South technology transfer and the ability of resource-poor farmers to have access to research and technology innovations ensuing from appropriate linkages among partners. The project also benefited from a micro-credit programme that allowed small-scale farmers to buy superior pest- and pathogen-free planting materials.

The potential impact of bananas derived from tissue culture was analysed on three types of farms: small, medium and large (although even large-scale farmers have a mean banana area of only about 2 ha). Large farms increased average yields by 93% and medium-scale farmers gained 132%. For small holders, however, the increase was 150%. One farmer (Esther Gachugu) made up to $300 in one-day sale—more than she could earn in a year from a traditional banana orchard (Wambugu and Kiome, 2001). Other farmers built new houses, installed water tanks or sent their children to school. This success story shows the benefits African farmers can draw from horticultural crop biotechnology.

In addition to the direct impacts of the project, biotechnology distribution channels were established in order to facilitate the development of future innovations. For instance, as the international availability of transgenic banana varieties with resistance to major biotic stresses is expected by 2009, the project opens up avenues for the quick introduction of these and other promising biotechnologies for resource-poor farmers (Qaim, 1999).

In Uganda, substantial investments in research on banana and plantain have been made in recent years. This has culminated in the development of a biotechnology project in which the Government of Uganda provides the largest funding. The US Agency for International Development (USAID), the Rockefeller Foundation and Directorate General for International Cooperation (DGIC, Belgium) also allocate resources. The hub of the project is the National Agricultural Research Organization (NARO) together with Makerere University. Important partners include the Katholieke Universiteit Leuven (KULeuven), International Institute of Tropical Agriculture (IITA, Ibadan), French Agricultural Research Institute for Overseas Development (CIRAD), University of Pretoria and International Plant Genetic Resources Institute (IPGRI), which through its International Network for the Improvement of Bananas and Plantains (INIBAP) coordinates the project (Ortiz et al., 2002). The project aims at creating a biotechnological centre in Uganda and using genetic transformation for enhancing the resistance of the local East African highland bananas to the wide range of pests and diseases currently affecting the crop. The IITA provides technical backstopping for gene mapping of banana weevil resistance through an associated project, funded by a grant given to IPGRI/INIBAP by the Rockefeller Foundation.

In Morocco, with a production capacity of more than 1 million banana in vitro plants per annum, the company Domaines Agricoles, based near the city of Meknès, can meet the national needs for banana plants. These in vitro plants are raised in the nurseries located near Rabat and in the Massa area (Agadir region), which are the main banana-producing regions (Sasson, 2000).

Shoot-tip micrografting, together with thermotherapy, is the technology selected to clean citrus varieties from their viral, mycoplasmic and bacterial pathogens. Cleansed plant material can be obtained in 3 to 6 months instead of 10 to 15 years using conventional technologies such as nucellar selection or mass selection through indexing. Thus, since 1994, Morocco's Domaines Agricoles Unit of Plant Control has been able to clean about 20 commercially important citrus varieties, and produce certified and well-performing plant material (Sasson, 2000).

In air-conditioned greenhouses, one can experimentally control almost all viral diseases which affect citrus plants, i.e. about 30. These facilities enable the company to play an important role at both national and regional level for controlling the tristeza viral disease, a major threat to citrus cultivation. They also serve as quarantine facilities for introduced citrus species or varieties, in full cooperation with the Ministry of Agriculture Services of Plant Protection. The company took the initiative of undertaking a programme for the biological control of a citrus borer, Phylacnistis citrella, which originated from South-East Asia and invaded all citrus-growing Mediterranean countries in less than 3 years, causing heavy losses. Two natural enemies of the insect pest were introduced from Florida and Australia, Ageniaspis citricola and Semielacker petiolatus; hundreds of thousands of Ageniaspis citricola were produced and disseminated throughout the citrus-growing regions of Morocco. Algeria, Egypt and Spain also benefited from the Moroccan experience concerning the breeding of the useful insects. Another example of biological control is that against Aonidiella auranti, one of the oldest and major pests of citrus plants; chemical control is expensive, rather inefficient and can harm the exports of fruits because of the pesticide residues remaining on the fruit surface. A pilot unit was set up at the Domaines Agricoles to produce Aphitis milinus insects and to disseminate them in order to control the Californian lice (Aonidiella auranti) (Sasson, 2000).

The date palm is part of the landscape and a key element of land-use planning in large areas of African countries. It is also found beyond the eastern boundaries of the North African region, in the Near and Middle East, and has been introduced in several sub-Saharan countries, such as Namibia. It is the typical multipurpose tree crop of the oases from Morocco to Egypt, not only to supply dates (for local consumption and export), leaves and trunks as building materials, but more so to provide shade for barley and alfalfa cultivation under irrigation, and animal husbandry (sheep, goats and camels). Except for the case of plantations managed for exporting dates, e.g. in Algeria, Tunisia and Egypt, most date palm groves belong to families of resource-poor farmers and are the pivot of horticulture-type farming. Maintaining this tree crop is, therefore, a crucial socioeconomic issue for the development of marginal areas where date palm is growing; it is a way of controlling rural exodus and of mitigating rural poverty.

Although the date palm varieties grown throughout North Africa are quite sturdy, a fungal disease caused by soil-inhabiting Fusarium oxysporum subsp. albedinis, locally named as bayoud (meaning whitening, because white streaks appear on the leaves of the diseased tree), is causing havoc among the palm groves, particularly in Morocco. In this country, tens of millions of trees have been killed since the beginning of the 20th century. There is no effective chemical remedy for eradicating the fungus whose filaments penetrate through the roots and multiply in the vascular bundles, finally choking the tree (tracheomycosis). In addition to Morocco, which is severely affected, Algeria is also affected by this disease, although slightly less. The fungus may spread to Tunisia and even farther (in the Middle East, especially in the Gulf area, where the main pest of the date palm is the red weevil and the bayoud disease is unknown).

The threat is, therefore, very serious, and the whole ecosystem and way of life is being threatened, at least in the Moroccan oases. Fortunately, the Moroccan scientists of the National Institute for Agricultural Research (INRA) have been identifying, selecting and gathering many bayoud-tolerant date palm varieties, the clonal multiplication of which could be a viable solution. These scientists, with bilateral and multilateral assistance, have succeeded, many years ago, in micropropagating the date palm, starting from the caulinary meristems of offshoots (the tree produces a few offshoots over its life-span), and leading to uniform in vitro plants (through organogenesis). A private corporation, working in collaboration with INRA, is producing around 250 000 date palm in vitro plants per annum. This figure is far from meeting national needs, which are estimated as several millions per year if the medium-term objective is to replace the dead trees by tolerant varieties, and rehabilitate the oases and their specific agriculture (a National Plan for the Rehabilitation of Palm Groves was initiated in 1978).

The only agrobiotechnological tool available for such a purpose is that of somatic embryogenesis, which has been successfully used for several crop species, including the oil-palm, coconut and other tree species. Starting from leaf or inflorescence explants, using liquid instead of semi-solid medium, the production figures are of an additional order of magnitude compared to the organogenesis process. A difficulty relates to the inadvertent production of somaclonal variants, which may have abnormal inflorescences. Recent research overcomes this problem, and in fact several research teams in Europe have been able to produce large populations of normal date palm plants in vitro. Time has, therefore, come for the Moroccan relevant institutions to make the appropriate decisions in order to meet the huge needs of oasis farmers and to contribute to solving a very important socioeconomic problem.

Morocco has cooperated with Mali to introduce tissue culture-derived bayoud-free date palms into the north-eastern region of this country (Menaka), within the framework of an FAO project. Similar cooperation has been established with Libia (Sasson, 2000).

In Egypt, at El-Menoufia University, Sadat City, research is being carried out on the production of plants in vitro. A number of scientists belonging to this university have been trained in Germany and in the USA. The Genetic Engineering and Biotechnology Research Institute of this University succeeded in cloning date palm (Phoenix dactylifera cv. Zaghloul) through somatic embryogenesis and organogenesis. Successful regeneration of plantlets from the shoot-tip and leaf primordia derived from adult plants were reported. The Egyptian researchers are of the opinion that this method holds good chances for achieving mass production of true-to-type plants from adult date palm since the callus stage is avoided (Sasson, 2000).

Moving to somatic embryogenesis for multiplying bayoud-tolerant varieties does not preclude the pursuance of basic research on the molecular basis of the host–parasite relationship as well as on the genome of these varieties in order to identify a resistance gene(s) and stimulate plant-defence mechanisms. Any breakthrough achieved in date palm propagation, physiology and genetics will have a great impact on the socioeconomic development of the whole North Africa region and beyond.

In the Republic of South Africa, the Vegetable and Ornamental Plant Institute of the Agricultural Research Council (ARC-Roodeplaat) has developed tissue-culture protocols during the last 25 years for many vegetable crops—including root and tuber crops such as cassava and the research-neglected Livingstone potato (Plectranthus esculentus). Meristem culture and thermotherapy are used routinely for eliminating viruses in potato, sweet potato, cassava, garlic and indigenous ornamentals. The ARC-Roodeplaat provides all the virus-free material of sweet potato in South Africa. Its in vitro gene bank contains cultivars and breeding materials of potato, sweet potato, cassava and the ornamental Lachenalia spp. The Institute also carries out genetic transformation and molecular marker-aided selection (Ortiz, 2002b).

Introduction to tissue culture callus

In 1902, G. Haberlandt noticed that the plant cells can be grown in synthetic media. The discovery by Haberlandt that the plant cells have the capacity to grow in a nutrient medium in presence of sufficient light made an impact in plant propagation and crop improvement. This has become possible with the development of techniques to regenerate whole plants from the tissue cultured cells.

The ability of a plant cell to give rise to a whole plant is called totipotency. In this method, instead of taking cuttings from a plant, a cell (or few cells) or a tissue is taken from a plant and cultured in suitable containers in which nutrient medium is present. Through these methods new plants can be obtained from single cells or a clump of cells or a bud or other organ of a plant. The portion of the plant that is taken from the desired plant is referred as Explant. By this method, thousands of plants can be produced from a single desirable parent inone generation. These techniques require maintenance of sterile conditions in the medium. The explant need to be sterilised before it is introduced into the medium. All the elements that are required for the growth of the plant are provided in the medium. In addition, the media will contain one or more growth regulators as supplements, depending on the purpose for which the explant is introduced in to the medium. The medium is kept free from bacteria and fungi. Cells or tissues are grown n culture under aseptic conditions.

Tissue Culture Callus Description

Culture techniques are now available for cells, tissues or organs from the plant. In the tissue culture, the explant often divides to give rise to an unorganised mass of tissue called Callus. Te callus or the explant as such, will undergo differentiation into shoots; roots or embryo like structures (Embryoids). This differentiation is dependent on the concentration and combination of the plant growth substances like auxins, kinetin, gibberellin etc., in the medium. Some times numerous independent shoots (multiple shoots) differentiate from the explant or callus.

Plants raised through tissue culture are routinely used in agriculture, horticulture and forestry. When we use a diploid explant, we get a diploid plant like in any of the vegetative propagation methods. Similarly, if we want to develop a haploid plant, it is possible to obtain it through tissue culture. For this, we need to select a haploid cell in the plant. The best choice of a haploid cell is pollen grain . Development of haploid plants through tissue culture was discovered by Indian Scientists Shipra Guha and Satish Maheswari.

The Components of Plant Tissue Culture Media I : Macro- and Micro-Nutrients

INORGANIC MEDIUM COMPONENTS

Plant tissues and organs are grown in vitro on artificial media, which supply the nutrients necessary for growth. The success of plant tissue culture as a means of plant propagation is greatly influenced by the nature of the culture medium used. For healthy and vigorous growth, intact plants need to take up from the soil:

• relatively large amounts of some inorganic
elements (the so-called major plant nutrients): ions of nitrogen (N), potassium (K), calcium (Ca),
phosphorus (P), magnesium (Mg) and sulphur (S);
and,
• small quantities of other elements (minor plant nutrients or trace elements): iron (Fe), nickel (Ni), chlorine (Cl), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum (Mo).

According to Epstein (1971), an element can be considered to be essential for plant growth if:
1. a plant fails to complete its life cycle without it;
2. its action is specific and cannot be replaced completely by any other element;
3. its effect on the organism is direct, not indirect on the environment;
4. it is a constituent of a molecule that is known to be essential.

The elements listed above are - together with carbon (C), oxygen (O) and hydrogen (H) - the 17
essential elements. Certain others, such as cobalt (Co), aluminium (Al), sodium (Na) and iodine (I), are essential or beneficial for some species but their widespread essentiality has still to be established.

History of Tissue Culture Techniques

History of Tissue Culture Techniques - The in vitro techniques were developed initially to demonstrate the totipotency of plant cells predicted by Haberlandt in 1902. Totipotency is the ability of a plant cell to perform all the functions of development, which are characteristic of zygote, i.e., ability to develop into a complete plant. In 1902, Haberlandt reported culture of isolated single palisade cells from leaves in Knop's salt solution enriched with sucrose.
The cells remained alive for up to 1 month, increased in size, accumulated starch but failed to divide. Efforts to demonstrate totipotency led to the development of techniques for cultivation of plant cells under defined conditions.

This was made possible by the brilliant contributions from RJ. Gautheret in France and P.R. White in U.S.A. during the third and the fourth decades of 20th century. Most of the modern tissue culture media derive from the work of Skoog and coworkers during 1950s and 1960s.

The first embryo culture, although crude, was done by Hanning in 1904; he cultured nearly mature embryos of certain crucifers and grew them to maturity. The technique was utilised by Laibach in 1925 to recover hybrid progeny from an interspecific cross in Linum. Subsequently, contributions from several workers led to the refinement of this technigue.
Haploid plants from pollen grains were first produced by Maheshwari and Guha in 1964 by culturing anthers of Datura. This marked the beginning of anther culture or pollen culture for the production of haploid plants.

The technique was further developed by many workers, more notably by JP. Nitch, C. Nitch and coworkers. These workers showed that isolated microspores of tobacco produce complete plants.
Plant protoplasts are naked cells from which cell wall has been removed. In 1960, Cocking produced large quantities of protoplasts by using cell wall degrading enzymes.

The techniques of protoplast production have now been considerably refined. It is now possible to regenerate whole plants from protoplasts and also to fuse protoplasts of different plant species. In 1972, Carlson and coworkers produced the first somatic hybrid plant by fusing the protoplasts of Nicotiana glauca and N. langsdorfii. Since then many divergent somatic hybrids have been produced.

A successful establishment of callus cultures depended on the discovery during mid-thirties of IAA (idole-3-acetic acid), the endogenous auxin, and of the role of B vitamins in plant growth and in root cultures.

The first continuously growing callus cultures were established from cambium tissue in 1939 independently by Gautheret, White and Nobecourt. The subsequent discovery of kinetin by Miller and coworkers in 1955 enabled the initiation of callus cultures from differentiated tissues. Shoot bud differentiation from tobacco pith tissues cultured in vitro was reported by Skoog in 1944, and in 1957 Skoog and Miller proposed that root-shoot differentiation in this system was regulated by auxin-cytokinin ratio.

The first plant from a mature plant cell was regenerated by Braun in 1959. Development of somatic embryos was first reported in 1958- 1959 from carrot tissues independently by Reinert and Steward.

Thus within a brief period, the tissue culture techniques have made a great progress. From the sole objective of demonstrating the totipotency of differentiated plant -cells, the technique now finds application in both basic and applied researches in a number of-fields of enquiry.

source: http://www.molecular-plant-biotechnology.info

Haberlandt ( The father of plant tissue culture )

The father of plant tissue culture is considered to be the German Botanist HABERLANDT who conceived the concept of cell culture in 1902.

"There has been, so far as I know, up to present, no planned attempt to cultivate the vegetative cells of higher plants in suitable nutrients. Yet the results of such attempts should cast many interesting sidelights on the peculiarities and capacities which the cell, as an elementary organism, possesses: they should make possible conclusions as to the interrelations and reciprocal influences to which the cell is subjected within the multicellular organism. Without permitting myself to pose further questions, I believe, in conclusion, that I am not making to bold a prediction if I point to the possibility that, in this way, one could successfully cultivate artificial embryos from vegetative cells".

Haberlandt, 1902.

HABERLANDT, when he embarked upon his attempt to culture plant cells was the first to consider culturing cells aseptically in a nutrient solution.
HABERLANDT did not realise that because photosynthetic cells are relatively differentiated their meristematic potential is not expressed easily and he did not know that this would require stimulating substances ie. plant growth regulators which were unknown at the time. Thus he chose to work with pallisade cells, pith cells, stamen hairs and stomatal guard cells. HABERLANDT cultured these cells in a simple organically enriched medium containing glucose under aseptic conditions and was totally unsucessful in all cases. His cells did not divide but were maintained in a living state for several weeks.

HABERLANDT failed to recognise that the meristematic cells of the plant body are basically heterotrophic and he did not know that the dedifferentiation of a cell into a meristematic state requires the presence of plant growth regulators.

source: http://users.ugent.be/~pdebergh/his/his2az1.htm