The origin of plant cell and tissue culture can be found in a treatise published during the mid-18th century, entitled La Physique des Arbes, that describes the formation of callus tissue following the for mation of a ring of cortex from elm trees. Over the next two centuries, the discovery of plant growth hormones, in particular auxins and cytokinins, and detailed analyses on the nutritional requirements of plants, led to the formulation of media that could maintain actively dividing cultures derived from gymnosperms, and both dicotyledon ous and monocotyledonous angiosperms. However, much of the prog ress and technological development in the in vitro propagation of plant cells, tissues, and organs has occurred during the last 25 years. Recently, plant tissue culture techniques have been used as basic tools in the rapidly expanding field of plant biotechnology for the development and clonal propagation of new and/or improved plant varieties. Plant tissue culture is used for the micropropagation of commercially valuable cultivars that include ornamentals, oil palm, Glycyrrhiza, Pyrethrum, pine, Eucalyptus, sugar cane, and potatoes. Cultured plant tissue is also used for the selection of cells and, ul timately, the regeneration of plants that are tolerant to physical stresses such as pathogens, drought, and temperature extremes, and to chemical stress agents such as salinity, herbicides, proteins, and pyrethrins. In addition, new plants have been produced by the fusion of protoplasts prepared from cultured cells of different species in cluding sunflower and french bean, tomato and potato, and various cultivars of Datura. Finally, bacterial vectors and various mechanical methods have been used to introduce foreign genes into cultured plant tissues. Genetic transformation can result in profound changes in the phenotype and/or biochemical profile of the regenerated trans genic plants that are not characteristic of the wild type. An impressive variety of technologies in tissue culture, genetic manipulation, and molecular biology have been developed for nu merous plant species. Many of these techniques, sometimes referred to as plant biotechnology, have been extensively summarized and compiled in a well-balanced collection of easy-to-follow protocols presented in three separate, but complimentary, volumes. Plant Cell, Tissue and Organ Culture consists of 22 chapters (with 86 figures) and 5 appendices. The chapters cover critical aspects of (a) the es sential requirements for the operation of a plant tissue culture lab oratory; (b) the basic procedures of micropropagation, regeneration, and somatic embryogenesis; (c) some specific applications of organ culture systems such as embryo rescue and culture, and anther and microspore culture for haploid and double haploid production; (d) elementary transformation technology; and (e) useful microtechnique and analytical protocols specifically adapted to cultured tissues and cells. The appendices provide a convenient summary of media for mulations and commercial suppliers for the materials described in the text.

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Plant Cell, Tissue and Organ Culture

A method of identifying microorganisms with reduced adaptability to a particular environment, wherein (1) each microorganism is independent by insertional inactivation of a gene with a nucleic acid comprising a unique marker sequence. Mutating to provide a plurality of microorganisms, or clones of said microorganisms, such that each mutation has a different label sequence; (2) providing storage samples of each mutation produced by step (1), respectively, and providing storage nucleic acids each comprising a unique label sequence from each mutation; (3) introducing a plurality of mutations produced by step (1) into the specific environment such that the microorganisms capable of growing in the specific environment grow in the above environment; (4) recovering the microorganisms or selected portions thereof from the environment and separating nucleic acids from the recovered microorganisms; (5) comparing all label sequences in the nucleic acid isolated in step (4) with the unique label sequences of each of the stored mutations as in step (2); and (6) any label isolated in step (4). Screening for individual mutations that also do not have a sequence.

Identification of genes

Compared with animals, plants generally possess a high degree of developmental plasticity and display various types of tissue or organ regeneration. This regenerative capacity can be enhanced by exogenously supplied plant hormones in vitro, wherein the balance between auxin and cytokinin determines the developmental fate of regenerating organs. Accumulating evidence suggests that some forms of plant regeneration involve reprogramming of differentiated somatic cells, whereas others are induced through the activation of relatively undifferentiated cells in somatic tissues. We summarize the current understanding of how plants control various types of regeneration and discuss how developmental and environmental constraints influence these regulatory mechanisms.

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Plant regeneration: cellular origins and molecular mechanisms

The first haploid angiosperm, a dwarf form of cotton with half the normal chromosome complement, was discovered in 1920, and in the ninety years since then such plants have been identified in many other species. They can occur either spontaneously or can be induced by modified pollination methods in vivo, or by in vitro culture of immature male or female gametophytes. Haploids represent an immediate, one-stage route to homozygous diploids and thence to F(1) hybrid production. The commercial exploitation of heterosis in such F(1) hybrids leads to the development of hybrid seed companies and subsequently to the GM revolution in agriculture. This review describes the range of techniques available for the isolation or induction of haploids and discusses their value in a range of areas, from fundamental research on mutant isolation and transformation, through to applied aspects of quantitative genetics and plant breeding. It will also focus on how molecular methods have been used recently to explore some of the underlying aspects of this fascinating developmental phenomenon.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1467-7652.2009.00498.x

Haploids in flowering plants: origins and exploitation.

The WOX genes form a plant-specific subclade of the eukaryotic homeobox transcription factor superfamily, which is characterized by the presence of a conserved DNA-binding homeodomain. The analysis of WOX gene expression and function shows that WOX family members fulfill specialized functions in key developmental processes in plants, such as embryonic patterning, stem-cell maintenance and organ formation. These functions can be related to either promotion of cell division activity and/or prevention of premature cell differentiation. The phylogenetic tree of the plant WOX proteins can be divided into three clades, termed the WUS, intermediate and ancient clade. WOX proteins of the WUS clade appear to some extent able to functionally complement other members. The specific function of individual WOX-family proteins is most probably determined by their spatiotemporal expression pattern and probably also by their interaction with other proteins, which may repress their transcriptional activity. The prototypic WOX-family member WUS has recently been shown to act as a bifunctional transcription factor, functioning as repressor in stem-cell regulation and as activator in floral patterning. Past research has mainly focused on part of the WOX protein family in some model flowering plants, such as Arabidopsis thaliana (thale cress) or Oryza sativa (rice). Future research, including so-far neglected clades and non-flowering plants, is expected to reveal how these master switches of plant differentiation and embryonic patterning evolved and how they fulfill their function.

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The WUS homeobox-containing (WOX) protein family.

An isolated microspore culture provides an excellent system for the study of microspore induction and embryogenesis, provides a platform for an ever-increasing array of molecular studies, and can produce doubled haploid (DH) plants, which are used to accelerate plant-breeding programs. Moreover, isolated microspore cultures have several advantages over anther culture, wherein presence of the anther walls can lead to the development of diploid, somatic calli and plants. Although protocols for isolated microspore culture vary from laboratory to laboratory, the basic steps of growing donor plants, harvesting floral organs, isolating microspores, culturing and inducing microspores, regenerating embryos, and doubling the chromosomes, remain the same. Over the past few years, a large proportion of the research reports on isolated microspore culture have focused on cereal and Brassica species. For some of these species, isolated microspore culture protocols are well established and routinely used in laboratories around the world for developing new varieties, as well as for basic research in areas such as genomics, gene expression, and genetic mapping. Although these species are considered highly responsive to microspore culture, improvements in efficiency are still being made. However, with many species, isolated microspore culture is simply not yet efficient enough at producing DH plants to be cost-effective for breeding programs. There has been a recent resurgence of haploidy research with response being reported in some species once considered recalcitrant. Future research programs aimed at elucidating pathways involved in microspore induction and embryogenesis will be of benefit, as will novel approaches to improve the efficiency of microspore culture for DH production. With many species, anther culture has proven to be more effective than isolated microspore culture, necessitating more research to clarify the contribution of the anther wall to embryogenesis. The development of molecular markers for use in determining the gametic origin of regenerated plants, irrespective of their ploidy, would also be beneficial. In this review, we aim to provide an overview of the basic isolated microspore culture protocol with an emphasis on recent progress in several crop species.

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Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production

Isolated microspores of Brassica napus are developmentally programmed to form gametes; however, microspores can be reprogrammed through stress treatments to undergo appropriate divisions and form embryos. We are interested in the identification and isolation of factors and genes associated with the induction and establishment of embryogenesis in isolated microspores. Standard and normalized cDNA libraries, as well as subtractive cDNA libraries, were constructed from freshly isolated microspores (0 h) and microspores cultured for 3, 5, or 7 d under embryogenesis-inducing conditions. Library comparison tools were used to identify shifts in metabolism across this time course. Detailed expressed sequence tag analyses of 3 and 5 d cultures indicate that most sequences are related to pollen-specific genes. However, semiquantitative and real-time reverse transcription-polymerase chain reaction analyses at the initial stages of embryo induction also reveal expression of embryogenesis-related genes such as BABYBOOM1, LEAFY COTYLEDON1 (LEC1), and LEC2 as early as 2 to 3 d of microspore culture. Sequencing results suggest that embryogenesis is clearly established in a subset of the microspores by 7 d of culture and that this time point is optimal for isolation of embryo-specific expressed sequence tags such as ABSCISIC ACID INSENSITIVE3, ATS1, LEC1, LEC2, and FUSCA3. Following extensive polymerase chain reaction-based expression profiling, 16 genes were identified as unequivocal molecular markers for microspore embryogenesis in B. napus. These molecular marker genes also show expression during zygotic embryogenesis, underscoring the common developmental pathways that function in zygotic and gametic embryogenesis. The quantitative expression values of several of these molecular marker genes are shown to be predictive of embryogenic potential in B. napus cultivars (e.g. ‘Topas’ DH4079, ‘Allons,’ ‘Westar,’ ‘Garrison’).

Transcript Profiling and Identification of Molecular Markers for Early Microspore Embryogenesis in Brassica napus

The availability of a highly efficient and reliable microspore culture protocol for many Brassica species makes this system useful for studying basic and applied research questions. Microspores and microspore-derived embryos are ideal targets for modification by mutagenesis and transformation. Regenerated doubled haploid plants are widely used in breeding programs and in genetic studies. Furthermore, the Brassica microspore culture system allows the identification of genomic regions and genes involved in the microspore embryogenic response, spontaneous diploidization and direct embryo to plant conversion. This review summarizes current achievements and discusses future perspectives.

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Haploids and doubled haploids in Brassica spp. for genetic and genomic research

Isolated microspore culture techniques are being widely used in Brassica breeding programs to generate haploid and doubled haploid plants. A number of factors influence regeneration response in vitro including genotype. In order to assess the effect of genotype on microspore embryogenesis in B. rapa L. var. oleifera, 17 cultivars and breeding lines were evaluated. Embryos developed from all but one genotype when using NLN medium with 17% sucrose, followed by a reduction in sucrose concentration to 10%, 48 h later. The number of embryos /100 buds differed between genotypes, ranging from 0 to 70. Further studies indicated that sucrose concentration and incubation time influenced embryogenesis. Selection studies carried out with an Agriculture and Agri-Food Canada breeding line have resulted in the identification of a highly embryogenic B. rapa line. This line produced thousands of microspore-derived embryos /100 buds and will be useful in mutant selection and gene transfer as well as biochemical and developmental studies.

Evaluation of Brassica rapa L. genotypes for microspore culture response and identification of a highly embryogenic line.

Brassica napus cultivar Westar is non-embryogenic under all standard protocols for induction of microspore embryogenesis; however, the rare embryos produced in Westar microspore cultures, induced with added brassinosteroids, were found to develop into heritably stable embryogenic lines after chromosome doubling. One of the Westar-derived doubled haploid (DH) lines, DH-2, produced up to 30% the number of embryos as the highly embryogenic B. napus line, Topas DH4079. Expression analysis of marker genes for embryogenesis in Westar and the derived DH-2 line, using real-time reverse transcription-PCR, revealed that the timely expression of embryogenesis-related genes such as LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3, and BABY BOOM1, and an accompanying down-regulation of pollen-related transcripts, were associated with commitment to embryo development in Brassica microspores. Microarray comparisons of 7 d cultures of Westar and Westar DH-2, using a B. napus seed-focused cDNA array (10 642 unigenes), identified highly expressed genes related to protein synthesis, translation, and response to stimulus (Gene Ontology) in the embryogenic DH-2 microspore-derived cell cultures. In contrast, transcripts for pollen-expressed genes were predominant in the recalcitrant Westar microspores. Besides being embryogenic, DH-2 plants showed alterations in morphology and architecture as compared with Westar, for example epinastic leaves, non-abscised petals, pale flower colour, and longer lateral branches. Auxin, cytokinin, and abscisic acid (ABA) profiles in young leaves, mature leaves, and inflorescences of Westar and DH-2 revealed no significant differences that could account for the alterations in embryogenic potential or phenotype. Various mechanisms accounting for the increased capacity for embryogenesis in Westar-derived DH lines are considered.

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Alison M. R. Ferrie

Papers

Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production

Transcript Profiling and Identification of Molecular Markers for Early Microspore Embryogenesis in Brassica napus

Haploids and doubled haploids in Brassica spp. for genetic and genomic research

Evaluation of Brassica rapa L. genotypes for microspore culture response and identification of a highly embryogenic line.

Isolation of an embryogenic line from non-embryogenic Brassica napus cv. Westar through microspore embryogenesis