Plant Cell Culture Protocols, Second Edition, edited by Victor M. Loyola-Vargas and Felipe Vázquez-Flota,. Differential Display Methods and. Following in the footsteps of its much-acclaimed first edition, Plant Cell Culture Protocols, Second Edition has been expanded and revised to include the most. Rosa M. Galáz-Ávalos, Sagrario Aguilar-Díaz, Pedro A. Xool-González, Silvia M. Huchín-May, Víctor M. Loyola-Vargas. Pages PDF.

Plant Cell Culture Protocols Pdf

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Currently, facilities for in vitro cell cultures are found in practically every plant biology Included format: EPUB, PDF; ebooks can be used on all reading devices. Readily reproducible and extensively annotated, the methods cover culture ISBN ; Digitally watermarked, DRM-free; Included format: PDF, EPUB In Plant Cell Culture Protocols, Robert Hall and a panel of expert. Morphology of Cells in Culture. .. Protocol for Passaging Suspension Cells. .. Cell culture refers to the removal of cells from an animal or plant and their.

Therefore, there are efforts to utilize the information in efficient ways, preferably using high-throughput strategies, to accelerate our understanding of plant gene function. For example, high-throughput vector construction utilizing a large set of full-length Arabidopsis cDNA clones was reported Ogawa et al. Over the past few decades, research in functional genomics has accelerated with the use of transgenic plants. However, generating robust transgenic plants is time consuming often requiring a year or more and is limiting often prohibitive for high-throughput functional genomics applications.

Cultured plant cells are a useful alternative to whole plants for high-throughput genetic engineering, because transgenic cell lines that are ready to use in experiments can be generated within a few weeks.

Suspension-cultured cell lines have been established from several plant species, and several such lines, e. Nicotiana tabacum BY-2 Nagata et al. The virtually homogeneous nature of cells in these cultures gives rise to reproducible and reliable results; moreover, the cells are maintained and grown under strictly controlled conditions.

Cultured plant cells have been successfully used for genetic analyses e.

Callard et al. However, continuous culturing with periodic refreshment of medium is laborious; continuous culturing also increases the risk of microbial contamination and loss of the culture.

Genetic engineering can make possible a number of improved crop varieties with high yield potential and resistance against pests. Genetic transformation technology relies on the technical aspects of plant tissue culture and molecular biology for: Production of improved crop varieties Production of disease-free plants virus Genetic transformation Production of varieties tolerant to salinity, drought and heat stresses 5.

Germplasm conservation In vitro cell and organ culture offers an alternative source for the conservation of endangered genotypes [ 40 ]. Germplasm conservation worldwide is increasingly becoming an essential activity due to the high rate of disappearance of plant species and the increased need for safeguarding the floristic patrimony of the countries [ 41 ].

Tissue culture protocols can be used for preservation of vegetative tissues when the targets for conservation are clones instead of seeds, to keep the genetic background of a crop and to avoid the loss of the conserved patrimony due to natural disasters, whether biotic or abiotic stress [ 42 ].


Cryopreservation plays a vital role in the long-term in vitro conservation of essential biological material and genetic resources. It involves the storage of in vitro cells or tissues in liquid nitrogen that results incryo-injury on the exposure of tissues tophysical andchemical stresses.

Successful cryopreservation is often ascertained by cell and tissue survival and the ability to re-grow or regenerate into complete plants or form new colonies [ 43 ]. The fidelity of recovered plants can be assessed at phenotypic, histological, cytological, biochemical and molecular levels, although, there are advantages and limitations of the various approaches used to assess genetic stability [ 45 ].

Cryobionomics is a new approach to study genetic stability in the cryopreserved plant materials [ 46 ]. The embryonic tissues can be cryopreserved for future use or for germplasm conservation [ 47 ].

Embryo culture Embryo culture is a type of plant tissue culture that is used to grow embryos from seeds and ovules in a nutrient medium. In embryo culture, the plant develops directly from the embryo or indirectly through the formation of callus and then subsequent formation of shoots and roots.

The technique has been developed to break seed dormancy, test the vitality of seeds, production of rare species and haploid plants [ 59 , ]. It is an effective technique that is employed to shorten the breeding cycle of plants by growing excised embryos and results in the reduction of long dormancy period of seeds. Somatic embryogenesis and plant regeneration has been carried out in embryo cultures of Jucara Palm for rapid cloning and improvement of selected individuals [ 60 ].

In addition, conservation of endangered species can also be attained by practicing embryo culture technique.

Recently a successful protocol has been developed for the in vitro propagation of Khayagrandifoliola by excising embryos from mature seeds [ 61 ]. The plant has a high economic value for timber wood and for medicinal purposes as well.

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This technique has an important application in forestry by offering a mean of propagation of elite individuals where the selection and improvement of natural population is difficult.

Genetic transformation Genetic transformation is the most recent aspect of plant cell and tissue culture that provides the mean of transfer of genes with desirable trait into host plants and recovery of transgenic plants [ 63 ].

The technique has a great potential of genetic improvement of various crop plants by integrating in plant biotechnology and breeding programmes.

It has a promising role for the introduction of agronomically important traits such as increased yield, better quality and enhanced resistance to pests and diseases [ 64 ]. Genetic transformation in plants can be achieved by either vector-mediated indirect gene transfer or vectorless direct gene transfer method [ 65 ].

Among vector dependant gene transfer methods, Agrobacterium-mediated genetic transformation is most widely used for the expression of foreign genes in plant cells. Successful introduction of agronomic traits in plants was achieved by using root explants for the genetic transformation [ 66 ]. Virus-based vectors offers an alternative way of stable and rapid transient protein expression in plant cells thus providing an efficient mean of recombinant protein production on large scale [ 67 ].

Recently successful transgenic plants of Jatropha were obtained by direct DNA delivery to mature seed-derived shoot apices via particle bombardment method [ 68 ].

This technology has an important impact on the reduction of toxic substances in seeds [ 69 ] thus overcoming the obstacle of seed utilization in various industrial sector.

Regeneration of disease or viral resistant plants is now achieved by employing genetic transformation technique.

Researchers succeeded in developing transgenic plants of potato resistant to potato virus Y PVY which is a major threat to potato crop worldwide [ 70 ]. In addition, marker free transgenic plants of Petunia hybrida were produced using multi-auto-transformation MAT vector system. The plants exhibited high level of resistance to Botrytis cinerea,causal agent of gray mold [ 71 ].

Protoplast fusion Somatic hybridization is an important tool of plant breeding and crop improvement by the production of interspecific and intergeneric hybrids. The technique involves the fusion of protoplasts of two different genomes followed by the selection of desired somatic hybrid cells and regeneration of hybrid plants [ 48 ].

A simple and efficient method for the long-term preservation of plant cell suspension cultures

Protoplast fusion provides an efficient mean of gene transfer with desired trait from one species to another and has an increasing impact on crop improvement [ 3 ]. Somatic hybrids were produced by fusion of protoplasts from rice and ditch reed using electrofusion treatment for salt tolerance [ 49 ]. In vitro fusion of protoplast opens a way of developing unique hybrid plants by overcoming the barriers of sexual incompatibility.

The technique has been applicable in horticultural industry to create new hybrids with increased fruit yield and better resistance to diseases. Successful viable hybrid plants were obtained when protoplasts from citrus were fused with other related citrinae species [ 50 ]. The potential of somatic hybridization in important crop plants is best illustrated by the production of intergeneric hybrid plants among the members of Brassicaceae [ 51 ].

To resolvethe problem of loss of chromosomes and decreased regeneration capacity, successful protocol has been established for the production of somatic hybrid plants by using two types of wheat protoplast as recipient and protoplast of Haynaldiavillosa as a fusion donor. It is also employed as an important gene source for wheat improvement [ 52 ]. Figure 1. Schematic representation of production of hybrid plant via protoplast fusion 9.

Haploid production The tissue culture techniques enable to produce homozygous plants in relatively short time period through the protoplast, anther and microspore cultures instead of conventional breeding [ 53 ].

Haploids are sterile plants having single set of chromosomes which are converted into homozygous diploids by spontaneous or induced chromosome doubling. The doubling of chromosomes restores the fertility of plants resulting in production of double haploids with potential to become pure breeding new cultivars [ 54 ].

The term androgenesis refers to the production of haploid plants from young pollen cells without undergoing fertilization.

Sudherson et al. The haploidy technology has now become an integral part of plant breeding programs by speeding up the production of inbred lines [ 56 ] and overcoming the constraints of seed dormancy and embryo non-viability [ 57 ].

Cell culture

The technique has a remarkable use in genetic transformation by the production of haploid plants with induced resistance to various biotic and abiotic stresses. Introduction of genes with desired trait at haploid state followed by chromosome doubling led to the production of double haploids inbred wheat and drought tolerant plantswere attained successfully [ 58 ].

Current and future status of plant tissue culture The past decades of plant cell biotechnology has evolved as a new era in the field of biotechnology, focusing on the production of a large number of secondary plant products. During the second half of the last century the development of genetic engineering and molecular biology techniques allowed the appearance of improved and new agricultural products which have occupied an increasing demand in the productive systems of several countries worldwide [ 31 , 32 , 33 , 34 ].

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Plant Cell Culture Protocols

FAQ Policy. About this book Following in the footsteps of its much-acclaimed first edition, Plant Cell Culture Protocols, Second Edition has been expanded and revised to include the most up-to-date and frequently used techniques for plant cell and tissue culture.

Show all.The micropropagation technology has a vast potential to produce plants of superior quality, isolation of useful variants in well-adapted high yielding genotypes with better disease resistance and stress tolerance capacities [ 3 ].

Tissue culture in pharmaceuticals Plant cell and tissue cultures hold great promise for controlled production of myriad of useful secondary metabolites [ 72 ]. Plant-made vaccines or antibodies plantibodies are especially striking, as plants are free of human diseases, thus reducing screening costs for viruses and bacterial toxins.

Cell suspension culture: Cell suspension culture systems are used now days for large scale culturing of plant cells from which secondary metabolites could be extracted. Generally, the combined application of bactericide and fungicide products is suggested. Thus, the question was to determine whether it was possible to further diminish the consumption of carbohydrates by cells whilst at the same time keeping cell suspensions physiologically safe.

As cultures grow, pieces are typically sliced off and subcultured onto new media to allow for growth or to alter the morphology of the culture.

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