Morphological, Cytological, and Molecular-Based Genetic Stability Analysis of In Vitro-Propagated Plants from Newly Induced Aneuploids in Caladium
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Aneuploids are valuable materials of genetic diversity for genetic analysis and improvement in diverse plant species, which can be propagated mainly via in vitro culture methods. However, somaclonal variation is common in tissue culture-derived plants including euploid caladium. In the present study, the genetic stability of in vitro-propagated plants from the leaf cultures of two types of caladium (Caladium × hortulanum Birdsey) aneuploids obtained previously was analyzed morphologically, cytologically, and molecularly. Out of the randomly selected 23 and 8 plants regenerated from the diploid aneuploid SVT9 (2n = 2x − 2 = 28) and the tetraploid aneuploid SVT14 (2n = 4x − 6 = 54), respectively, 5 plants from the SVT9 and 3 plants from the SVT14 exhibited morphological differences from their corresponding parent. Stomatal analysis indicated that both the SVT9-derived variants and the SVT14-originated plants showed significant differences in stomatal guard cell length and width. In addition, the variants from the SVT14 were observed to have rounder and thicker leaves with larger stomatal guard cells and significantly reduced stomatal density compared with the regenerants of the SVT9. Amongst the established plants from the SVT9, two morphological variants containing 3.14–3.58% less mean fluorescence intensity (MFI) lost one chromosome, and four variants containing 4.55–11.02% more MFI gained one or two chromosomes. As for the plants regenerated from the SVT14, one variant with significantly higher MFI gained two chromosomes and three plants having significantly lower MFI resulted in losing four chromosomes. Three, out of the twelve, simple sequence repeat (SSR) markers identified DNA band profile changes in four variants from the SVT9, whereas no polymorphism was detected among the SVT14 and its regenerants. These results indicated that a relatively high frequency of somaclonal variation occurred in the in vitro-propagated plants from caladium aneuploids, especially for the tetraploid aneuploid caladium. Newly produced aneuploid plants are highly valuable germplasm for future genetic improvement and research in caladium.Keywords:
Somaclonal variation
The technology of plant tissue culture is recognized worldwide as a creative, promising and feasible option for propagation and genetic manipulation of plant, particularly those with special demands. The most popular application of tissue culture method is in the genetic transformation system. As a tool of genetic engineering of plant, plant regeneration using tissue culture system is taken a crucial part. Another important use of tissue culture system is propagation of plant, which is an alternative method to vegetative plant multiplication. While using conventional propagation methods, one cutting produces one plant. In sexual propagation, one seed produces one plant. In contrast, one explant (a piece of stem, leaf, bud, root, anther, etc.) can produce an infinity number of plants using tissue culture system within a relatively short period. Somaclonal variation can occur when plant regeneration and multiplication involves tissue culture, particularly when there is a callus phase. Today, somaclonal variation tends to be viewed as a source of undesirable variants that need to be screened out of breeding programmes that involve plant regeneration.
Somaclonal variation
Callus
Plant tissue culture
Plant propagation
Plant tissue
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Contents Summary 1018 I. Introduction 1018 II. Guard cell photosynthesis 1019 III. Guard cell central metabolism 1022 IV. Guard cell starch metabolism differs from that of mesophyll cells and plays a key role in stomatal movement 1025 V. Connectors between mesophyll and guard cells 1026 VI. Challenges and perspectives in understanding and modelling guard cell metabolism 1029 Acknowledgements 1030 References 1030 Summary Stomata are leaf epidermal structures consisting of two guard cells surrounding a pore. Changes in the aperture of this pore regulate plant water‐use efficiency, defined as gain of C by photosynthesis per leaf water transpired. Stomatal aperture is actively regulated by reversible changes in guard cell osmolyte content. Despite the fact that guard cells can photosynthesize on their own, the accumulation of mesophyll‐derived metabolites can seemingly act as signals which contribute to the regulation of stomatal movement. It has been shown that malate can act as a signalling molecule and a counter‐ion of potassium, a well‐established osmolyte that accumulates in the vacuole of guard cells during stomatal opening. By contrast, their efflux from guard cells is an important mechanism during stomatal closure. It has been hypothesized that the breakdown of starch, sucrose and lipids is an important mechanism during stomatal opening, which may be related to ATP production through glycolysis and mitochondrial metabolism, and/or accumulation of osmolytes such as sugars and malate. However, experimental evidence supporting this theory is lacking. Here we highlight the particularities of guard cell metabolism and discuss this in the context of the guard cells themselves and their interaction with the mesophyll cells.
Osmolyte
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Abstract The in vitro tissue cultures are, beyond all difficulties, an essential tool in basic research as well as in commercial applications. Numerous works devoted to plant tissue cultures proved how important this part of the plant science is. Despite half a century of research on the issue of obtaining plants in in vitro cultures, many aspects remain unknown. The path associated with the reprogramming of explants in the fully functioning regenerants includes a series of processes that may result in the appearance of morphological, physiological, biochemical or, finally, genetic and epigenetic changes. All these changes occurring at the tissue culture stage and appearing in regenerants as tissue culture-induced variation and then inherited by generative progeny as somaclonal variation may be the result of oxidative stress, which works at the step of explant preparation, and in tissue culture as a result of nutrient components and environmental factors. In this review, we describe the current status of understanding the genetic and epigenetic changes that occur during tissue culture.
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Plant tissue culture
Reprogramming
Plant tissue
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In order to seek a simple and rapid method of determinating the ploidy level of chromosome in watermelon,the chloroplasts number of stomatal guard cell in two diploid cultivars,TS and 0517 its autotetraploid were observered.The result showed that the chloroplast numbers among different ploidy had obvious diversities.The plant was diploid when the number of chloroplast in stomata guard cell was less than 15,otherwise it was tetraploid.The ploidy identification by counting chloroplast number of stoma guard cell was highly reliable,with an average accurate rate of 90.2%,the large the number of chloroplast,the higher the ploidy level showing that measurement of the number of stoma chloroplast in guard cell by universal fluorescence microscope could be considered as a fast and accurate method to determine the diploid or tetraploidy of watermelon in seedling stage.
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A ploidy series including haploid, diploid, triploid, tetraploid and pentaploid plants were identified among mono-embryonic and polyembryonic progeny of male-sterile (ms 1 /ms 1 ) soybeans (Glycine max (L.) Merrill). Ploidy level was positively correlated with guard cell length, number of chloroplasts per guard cell, leaflet width to length ratio and flower size; and negatively correlated with stomatal frequency and number of internodes. The influence of ploidy level was significant for all characteristics examined. All of these characteristics responded linearly with ploidy level, with coefficients of determination ranging from 0.54 to 0.94. Guard cell length and number of chloroplasts per guard cell appeared to be excellent indicators of ploidy level.
Polyembryony
Plant stem
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Somaclonal variation
Callus
Protoplast
Plant tissue culture
Rice plant
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The occurrence of somaclonal (tissue culture-derived) variation in plants regenerated from tissue culture will influence the efficiency with which techniques such as genetic transformation can be used in the development of new barley cultivars. To assess the effect of somaclonal variation on malting quality, 12 families of tissue culture-derived lines from three barley cultivars were analyzed using standard micromalting techniques. Each family was derived from a single regenerated plant that, in turn, was derived from an immature embryo callus culture. Five to six plants from each family were selected in the R 2 generation based on phenotypic similarity to their uncultured parental controls, and advanced to the R 4 and R 5 generations for replicated field tests. The malting quality of the majority of these lines was altered by passage through tissue culture, and most alterations were undesirable. These results suggest that efforts should be made to delineate in vitro (tissue culture) conditions that are less mutagenic to cultured barley cells.
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Plant tissue culture
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As discussed in Chapter 19 (“Genetic Engineering Technologies”), plant cell culture is an important technique for plant genetic improvement. Historically, and as implied throughout this book, plant cell culture has been viewed by most to be a method for rapid cloning. In essence, it was seen as a method of sophisticated asexual propagation, rather than a technique to add new variability to the existing population. For example, it was believed that all plants arising from such tissue culture were exact clones of the parent, such that terms like “calliclone,” “mericlone,” and “protoclone” were used to describe the regenerants from callus, meristems and protoplasts, respectively. Although phenotypic variants were observed among these regenerants, often they were considered artifacts of tissue culture. Such variation was thought to be due to “epigenetic” factors such as exposure to plant growth regulators (PGRs) and prolonged culture time. As more and more species were subjected to tissue culture, however, reports of variation among regenerants increased. In a historically significant review, Larkin and Scowcroft (1981) proposed the more general term “somaclones” for the regenerants coming out of tissue culture, irrespective of the explant used. Variation displayed by such regenerants from tissue culture would then be somaclonal variation. Tissue culture studies in the 1970s and early 1980s started to focus their attention on this type of variation, and it was soon recognized that somaclonal variation exists for almost all the phenotypic characters. To a plant scientist, somaclonal variation is perhaps the best route for studying somatic cell genetics. In contrast to the earlier view of “true to type” regeneration among plants derived from tissue culture, the frequency of genetic variation may actually be quite high. In some species, such as oil palm and banana, variation among tissue culture derived progenies is higher than one would expect to occur in vivo . In perennial crops that are asexually propagated, somaclonal variation offers an excellent opportunity to add new genotypes to the gene pool. In such cases it is important to understand and identify the causal mechanism behind the variations, so that we can effectively control them to our advantage.
Somaclonal variation
Callus
Plant tissue culture
Variation (astronomy)
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Somaclonal variation
Inbred strain
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