Organogenesis In Tissue Cultures Research Articles

Allele-specific DNA methylation and gene expression during shoot organogenesis in tissue culture of hybrid poplar.

Plant tissue regeneration is critical for genetic transformation and genome editing techniques. During the regeneration process, changes in epigenetic modifications accompany the cell fate transition. However, how allele-specific DNA methylation in two haplotypes contributes to the transcriptional dynamics during regeneration remains elusive. Here we applied an inter-species hybrid poplar (Populus alba × P. glandulosa cv. 84K) as a system to characterize the DNA methylation landscape during de novo shoot organogenesis at allele level. Both direct and indirect shoot organogenesis showed a reduction in genome-wide DNA methylation. At gene level, non-expressed genes were hypermethylated in comparison with expressed genes. Among the genes exhibiting significant correlations between levels of DNA methylation and gene expression, the expression patterns of 75% of genes were negatively correlated with DNA methylation in the CG context, whereas the correlation patterns in the CHH context were the reverse. The allele-biased DNA methylation was consistent during shoot organogenesis, with fewer than one-thousandth of allele-specific methylation regions shifted. Analysis of allele-specific expression revealed that there were only 1909 genes showing phase-dependent allele-biased expression in the regeneration process, among which the allele pairs with greater differences in transcription factor binding sites at promoter regions exhibited greater differences in allele expression. Our results indicated a relatively independent transcriptional regulation in two subgenomes during shoot organogenesis, which was contributed by cis-acting genomic and epigenomic variations.

A novel chemical inhibitor of polar auxin transport promotes shoot regeneration by local enhancement of HD-ZIP III transcription.

De novo shoot organogenesis is a prerequisite for numerous applications in plant research and breeding but is often a limiting factor, for example, in genome editing approaches. Class III homeodomain-leucine zipper (HD-ZIP III) transcription factors have been characterized as crucial regulators of shoot specification, however up-stream components controlling their activity during shoot regeneration are only partially identified. In a chemical genetic screen, we isolated ZIC2, a novel activator of HD-ZIP III activity. Using molecular, physiological and hormone transport analyses in Arabidopsis and sunflower (Helianthus annuus), we examined the molecular mechanism by which the drug promotes HD-ZIP III expression. ZIC2-dependent upregulation of HD-ZIP III transcription promotes shoot regeneration in Arabidopsis and is accompanied by the induction of shoot specifying factors WUS and RAP2.6L and a subset of cytokinin biosynthesis enzymes. ZIC2's effect on HD-ZIP III expression and regeneration is based on its ability to limit polar auxin transport. We further provide evidence that chemical modulation of auxin efflux can enhance de novo shoot formation in the regeneration recalcitrant species sunflower. Activation of HD-ZIP III transcription during shoot regeneration depends on the local distribution of auxin and chemical modulation of auxin transport can be used to overcome poor shoot organogenesis in tissue culture.

Post-Embryonic Lateral Organ Development and Adaxial-Abaxial Polarity Are Regulated by the Combined Effect of ENHANCER OF SHOOT REGENERATION 1 and WUSCHEL in Arabidopsis Shoots.

The development of above-ground lateral organs is initiated at the peripheral zone of the shoot apical meristem (SAM). The coordination of cell fate determination and the maintenance of stem cells are achieved through a complex regulatory network comprised of transcription factors. Two AP2/ERF transcription factor family genes, ESR1/DRN and ESR2/DRNL/SOB/BOL, regulate cotyledon and flower formation and de novo organogenesis in tissue culture. However, their roles in post-embryonic lateral organ development remain elusive. In this study, we analyzed the genetic interactions among SAM-related genes, WUS and STM, two ESR genes, and one of the HD-ZIP III members, REV, whose protein product interacts with ESR1 in planta. We found that esr1 mutations substantially enhanced the wus and stm phenotypes, which bear a striking resemblance to those of the wus rev and stm rev double mutants, respectively. Aberrant adaxial–abaxial polarity is observed in wus esr1 at relatively low penetrance. On the contrary, the esr2 mutation partially suppressed stm phenotypes in the later vegetative phase. Such complex genetic interactions appear to be attributed to the distinct expression pattern of two ESR genes because the ESR1 promoter-driving ESR2 is capable of rescuing phenotypes caused by the esr1 mutation. Our results pose the unique genetic relevance of ESR1 and the SAM-related gene interactions in the development of rosette leaves.

Efficient plant regeneration in three cultivars of Hami melon [Cucumis melo L. ssp. melo convar. ameri (Pang.) Greb] via organogenesis

SummaryHami melon [Cucumis melo L. ssp. melo convar. ameri (Pang.) Greb] is reputed to be the ‘king of melons’, but commercial development of transgenic Hami melon cultivars is limited by low levels of plant regeneration and a high incidence of polyploid regenerants. Regeneration systems for three Hami melon varieties, namely ‘Jin Queen’, ‘Hongmibao’, and ‘Huangzhuixian’, were developed via organogenesis in tissue culture. Leaf explants ranging from 0.125 – 0.500× the full-size of each leaf were prepared in vitro from seedlings of different ages, then cultured on Murashige and Skoog (MS) medium containing various concentrations of 6-benzylaminopurine (BAP; 1.0 – 9.0 mg l–1) to determine optimal conditions for induction of adventitious shoots. Leaves from 8-d old seedlings were cut into four, approximately-equal segments. Plant regeneration was greatest on MS medium containing 3.0 mg l–1 BAP, with a shoot induction frequency of 90% for ‘Hongmibao’. For ‘Jin Queen’ and ‘Huangzhuixian’, the highest frequencies of shoot induction (86% and 88%, respectively) were obtained on MS medium supplemented with 4.5 mg l–1 BAP.Adventitious shoots were then elongated on MS medium containing 0.1 mg l–1 BAP, but rooted successfully on hormone-free MS medium. Approx. 88% of the regenerated plants were diploid, as determined by flow cytometry.

Metabolic evaluation of cellular differentiation of tobacco leaf explants in response to plant growth regulators in tissue cultures using 1H NMR spectroscopy and multivariate analysis

To investigate the metabolic changes that precede visible organogenesis in tissue culture, tobacco leaf explants were cultured on media supplemented with various plant growth regulators (PGRs) and analyzed with proton nuclear magnetic resonance (1H NMR) spectroscopy. Principal component analysis (PCA) of 1H NMR spectral data was unable to differentiate between leaf explants cultured with α-naphthaleneacetic acid and those cultured with 6-benzyladenine after 4 days of culture; however, a difference was evident after 8 days of culture. A hierarchical dendrogram from PCA analysis could be grouping leaf explants cultured with various auxins separately from those treated by various cytokinins. However, leaf explants cultured with thidiazuron (TDZ) were identified as an outlier group; TDZ appeared to produce pleiotropic metabolic effects that differed from those induced by other PGRs. These results show that dedifferentiation can be initiated by either auxins or cytokinins, which is reflected by similar metabolic changes produced by the two distinct PGRs during the initial incubation period. The subsequent redifferentiation differs according to the PGR treatment, which is reflected by differential metabolic changes, depending on the fate of cells in organogenesis. Glutamine and glutamate levels increased approximately twofold in cytokinin-treated leaf explants compared with auxin-treated explants; however, changes in the levels of sugar compounds did not differ between the two treatments, demonstrating auxin regulation of the carbon/nitrogen ratio in favor of rooting. Taken together, our results suggest that 1H-NMR spectroscopy combined with multivariate analysis is a promising means for the metabolic evaluation of plant growth and differentiation.

Micropropagation of Wild Service Tree (Sorbus torminalis [L.] Crantz): The Regulative Role of Different Aromatic Cytokinins During Organogenesis

The influences of three different aromatic cytokinin derivatives [6-benzylaminopurine, meta-topolin, and 6-(3-methoxybenzylamino)purine-9-s-D-ribofuranoside (MeOBAPR)] on in vitro multiplication and rhizogenesis of the wild service tree (Sorbus torminalis [L.] Crantz) were compared. The highest micropropagation rate (24 new shoots per explant after 3 months of cultivation) was achieved on media containing BAP. On the other hand, the best rooting microcuttings were those multiplied on a medium containing MeoBAPR. To compare these results with the levels of endogenous cytokinins in multiplied explants, a newly developed UPLC-ESI(+)-MS/MS method was used to determine levels of 50 cytokinin metabolites in explants cultivated 12 weeks on media supplemented by BAP and of the two other aromatic cytokinin analogs used. Several significant differences among the levels of endogenous cytokinins, extracted from the explants, were found. The concentration of BAP9G, an important metabolite suspected to be responsible for inhibition of rooting and acclimatization problems of newly formed plantlets, was found to be the highest in microcuttings grown on media supplemented with BAP. This agrees well with the results of our rooting experiments; the lowest percentages of rooted plantlets 6 weeks after transferring shoots on rooting medium were present on explants multiplied on BAP. In contrast, BAP was still the most effective for the induction of bud formation on primary explants. Levels of the most active endogenous isoprenoid cytokinins, tZ, tZR, and iPR, as well as O-glucosides were also suppressed in explants grown on BAP compared with those of explants treated with other cytokinin derivatives. This may be the result of a very high BAP uptake into the explants grown on this cytokinin. On the other hand, endogenous concentrations of cis-zeatin derivatives as well as dihydrozeatin derivatives were not affected. Differences in the production of another plant hormone, ethylene, that plays an important role in controlling organogenesis in tissue culture, were also observed among S. torminalis plantlets grown in vitro on media containing different cytokinins tested. The highest ethylene levels were detected in the vessels containing media supplemented with mT. They were two to four times higher compared with the production by the S. torminalis explants cultivated on other media used. Finally, the levels of free IAA were also determined in the explants. S. torminalis plantlets grown on media containing BAP contained the lowest level of auxin, which is again in good agreement with their loss of rooting capacity. The results found in this study about optimal plant hormone concentrations may be used to improve in vitro rooting efficiency of the wild service tree and possibly also of other plant species.

Arabidopsis mutants with increased organ regeneration in tissue culture are more competent to respond to hormonal signals.

Shoots and roots can be regenerated through organogenesis in tissue culture by subjecting plant explants to the appropriate regime of hormone treatments. In an effort to understand the control of shoot organogenesis, we screened for mutants in Arabidopsis thaliana (L.) Heynh. Columbia ecotype for enhanced shoot development at sub-optimal concentrations of cytokinin. Mutants in four different complementation groups were identified, one of which represents a new locus named increased organ regeneration1 (ire1) and another that is allelic to the previously identified pom1/erh2 mutant. Although the mutants were selected for their response to cytokinin, they were neither hypersensitive to, nor were they over-producers of cytokinins. The mutations identified in this study not only promote more robust shoot production in tissue culture, but also enhance green-callus and root formation. We interpret this to mean that, in tissue culture, IRE genes act before organ specification during the time when root explants acquire the competency to respond to organ formation signals. In normal plant development, IRE genes may down-regulate the competency of vegetative tissue to respond to hormonal signals involved in shoot and root organogenesis.

Molecular genetics of auxin and cytokinin

The indeterminate nature of plant development requires that plants continuously regulate cell division and elongation in response to genetic, as well as a wide variety of different environmental signals [53]. Since their discovery, many physiological studies have shown that the plant hormones auxin and cytokinin have fundamental roles in the regulation of cell growth [30, 43]. Both compounds appear to be essential for growth of plant cells in culture. Organogenesis in tissue culture is regulated by altering the concentration of auxin and cytokinin. In the intact plant, auxin and cytokinin are apparently involved in a bewildering array of growth processes. One explanation for this complexity is that most and perhaps all changes in cell division or elongation are mediated by cytokinin or auxin. According to this view, developmental responses to environmental stimuli such as light or temperature are mediated by these hormones.

Mechanisms of the proliferation and differentiation of plant cells in cell culture systems.

Plant cell functions have been investigated in various cell culture systems. In this review, we summarize results obtained from investigations of gene expression during the cell cycle in synchronized cultures of Catharanthus roseus during somatic embryogenesis in suspension cultures of Daucus carota, during organogenesis in tissue cultures of Arabidopsis thaliana and during the transdifferentiation of isolated mesophyll cells to tracheary elements in single-cell cultures of Zinnia elegans.

Shoot organogenesis in tissue culture of Drimys Winteri

Calluses from stem explants of Drimys winteri Forst. (Winteraceae) initiated shoot organogenesis in vitro in hormone-containing media under light exposure. Shoots formed from callus-derived outgrowths and from the superficial callus layers of the explants. Light inhibited callus initiation but promoted growth of established cultures. Root organogenesis did not occur under any of various hormonal conditions. Active synthesis of anthocyanins was observed in morphogenetic tissue.

Organogenesis in tissue culture of Solanum melongena cv. Florida Market

Cell suspension cultures were initiated from pith callus of eggplant, Solanum melongena cv. Florida Market on Murashige and Skoog (MS) medium supplemented with 13.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The effects of mineral salts (MgSO 4, KNO 3, CaCl 2), sucrose and 2,4-D were studied on aggregate formation in suspension cultures. Aggregates from a cell suspension when cultured on an MS agar medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA) and γ,γ-isopentenyl adenine (2iP) differentiated into green nodules, but they failed to develop into organized shoots on the same medium. Upon transfer of these green nodules to MS medium supplemented with 0.1 μM ascorbic acid (autoclaved with the medium) alone or in combination with 49 μM 2iP, developed further into organized leafy shoots. This suggests that some degradation product of ascorbic acid might be responsible for inducing leafy shoots. Based on leaf morphology and chromosome analysis, regenerated plants were identified as tetraploids.

Axillary Meristem Development in Mammillaria elongata DC (Cactaceae)1

Abstract Axillary meristems of Mammillaria elongata DC were studied with a scanning electron microscope using fresh tissue to determine their degree of development and suitability as explants in tissue culture investigations. Meristems were excised at regular intervals along the stem. The axillary meristem reached maximum size about 10 cm and maximum development about 15 cm below the stem tip. Axillary meristems closer to the base of the stem were less developed. In fully developed meristems, leaf primordia were initiated in a spiral acropetal sequence at the margins of the apical dome, resulting in a phyllotaxis of 8 + 13 contact parastichies of tubercles on the developed stem. Enlarged leaf primordia were 1- or 3-lobed and about 75 μm in length. No flower initiation or lateral branch development was observed; however, a corky region, the result of aborted or senesced flowers, was observed in several axils. Variability in development or absence of axillary meristems in several axils make M. elongata axillary meristems unsuitable for critical studies on factors influencing organogenesis in tissue culture.

Callus, Cell Suspensions, and Organogenesis in Tissue Cultures of Purple Nutsedge (Cyperus rotundus)

Callus of Cyperus rotundus L. was initiated from young rhizomes and tubers on SH basal medium with 5 mg/liter NAA. Callus was subcultured on agar or in liquid shake cultures. In liquid cultures both small callus masses and individual suspended cells developed. Roots were produced when callus was transferred to low-auxin or auxin-free media. Roots and occasionally shoots developed after callus was transferred to media with 20% CW. Nurse cultures with tobacco callus did not affect organogenesis. Kinetin, casein hydrolysate, and 2,4-D had little effect on organogenesis. Histology of the callus, cell suspension, roots, and shoots is described. Chromosome number could not be determined for the callus or induced organs.

Organogenesis in tissue cultures of certain asclepiads

Summary Embryo and organ differentiation in stem callus tissues derived from 5 members of the fam. Asclepiadaceae have been investigated. Pergularia minor formed embryoids and plant-lets, shoot buds and roots during the initial stages of callus growth but showed a decline in organogenetic potential with continuous subculture. In Asclepias curassavica , embryoids were differentiated at the senescent stage of the callus but these never grew into plantlets under any cultural variables. Dregea, Ibatia and Cryptostegia sp. formed profuse calli but lacked the ability to initiate organs.