Meristematic Dome Research Articles

Multilayered regulation of developmentally programmed pre-anthesis tip degeneration of the barley inflorescence.

Leaf and floral tissue degeneration are common features in plants. In cereal crops such as barley (Hordeum vulgare L.), pre-anthesis tip degeneration (PTD) starts with growth arrest of the inflorescence meristem dome, which is followed basipetally by the degeneration of floral primordia and the central axis. Due to its quantitative nature and environmental sensitivity, inflorescence PTD constitutes a complex, multilayered trait affecting final grain number. This trait appears to be highly predictable and heritable under standardized growth conditions, consistent with a developmentally programmed mechanism. To elucidate the molecular underpinnings of inflorescence PTD, we combined metabolomic, transcriptomic, and genetic approaches to show that barley inflorescence PTD is accompanied by sugar depletion, amino acid degradation, and abscisic acid responses involving transcriptional regulators of senescence, defense, and light signaling. Based on transcriptome analyses, we identified GRASSY TILLERS1 (HvGT1), encoding an HD-ZIP transcription factor, as an important modulator of inflorescence PTD. A gene-edited knockout mutant of HvGT1 delayed PTD and increased differentiated apical spikelets and final spikelet number, suggesting a possible strategy to increase grain number in cereals. We propose a molecular framework that leads to barley PTD, the manipulation of which may increase yield potential in barley and other related cereals.

Meta-Topolin enhanced direct shoot organogenesis and regeneration from leaf explants of Coleus forskohlii (Willd.) Briq

A novel and effective direct shoot regeneration technique from stomatal complexes was developed for a highly sought after medicinal plant Coleus forskohlii. Meta-Topolin was more efficient than 6-benzylaminopurine (BAP) in promoting direct organogenesis from leaf surface. Highest direct organogenesis was achieved from in vitro leaf explants on Murashige and Skoog (MS) medium augmented with meta-Topolin at the concentration of 2.0 mg/L. The regenerated leaves were fixed at weekly intervals and sectioned longitudinally to evaluate the anatomical developments. The results of light microscopic analysis suggested that the in vitro developed stomatal complexes showed new altered cell divisions, after a week of incubation, and the rapid periclinal divisions resulted in the formation of a meristematic dome. Distinct regions of meristematic activity were visible in the second week characterized by promeristems with small and densely stained parenchymatous cells. Promeristems lead to the development of a distinct tunica layer, which grew into shoot-like protuberances converting into adventitious shoots after 4 weeks of incubation. A clear three phases of shoot organogenesis viz. induction (7 days), initiation and organization (14 days), and shoot formation (28 days onwards) were identified. The shoots were proliferated (21.8 shoots/ single leaf explant) and elongated (5.0 cm length) using half-MS medium fortified with 0.5 mg/L mT and indole-3 acetic acid (IAA) at 0.1 mg/L. Notable rhizogenesis of the elongated shoots was achieved when cultured on half-MS medium containing indole-3 butyric acid (IBA) at the concentration of 1.5 mg/L. About 92.0% of the plantlets survived upon field transfer. Start Codon Target (SCoT) markers were employed to examine the regenerated plants’ genetic uniformity with the mother plant, similar banding patterns confirming the genetic uniformity. Hence, the developed protocol of direct in vitro regeneration of C. forskohlii using mT can be used for the commercial production.

Elimination of rose viruses using microshoot tip tissue culture

Virus elimination therapy for roses is very challenging. Foundation Plant Services at the University of California, Davis provides virus-tested rose propagation material to the rose nursery industry in the United States. Before 1999, heat treatment of potted plants was used to eliminate viruses. Success was varied and often the plant died during the treatment or was still virus-infected at the end of treatment period. In 2007 we reported on successful elimination of Apple mosaic virus (ApMV) and Prunus necrotic ringspot virus (PNRSV) using microshoot tip culture for six cultivars. However, survival was low and was cultivar dependent. In the period 2013 to 2016, 35 rose cultivars were subjected to microshoot tip tissue culture therapy. Microshoot tips measuring <0.5 to 0.7 mm, consisting of the meristem dome plus several pairs of leaf primordia, were excised from actively growing vegetative shoots and cultured in aseptic conditions. Different media, growth factors and light intensity were trialed. To date the most promising medium is: Murashige and Skoog (MS) salts and vitamins with 3% sucrose, 2.5 µmol 6-benzyladeninepurine (BA) and 0.4 µmol indole-3-acetic acid (IAA). Rooting was ex vitro by transplanting shoots that were approximately 5 cm long into a sealed container of saturated perlite to keep humidity high. One or more plants survived from 20 cultivars of the 35 that were treated (56%). Overall percent survival averaged 3% and ranged from 0 to 22%, depending on cultivar. Preliminary testing by ELISA shows that ApMV was eliminated; plants were still infected with PNRSV. Further testing using qPCR and other methods and trials are ongoing.

In vitro establishment of shoot meristems of Ilex paraguariensis and identification of endophytic bacteria

Yerba mate (Ilex paraguariensis A. St.-Hil.) is a species of great economic, social and environmental importance for the southern regions of Brazil, Uruguay and Argentina. Currently the most diverse products are obtained from mate leaves, including mate tea. The objective of this study was to establish shoot meristem cultures (meristematic dome and a few primordia) of elite clones and identify the endophytic bacteria present in the explants. We tested the effect of clones (F1, F2, A03 and A07), culture media (MS, 1/2MS, 1/4MS and WPM), cytokinins (kinetin, BA and 2iP), activated charcoal (1, 2 and 3 g L−1), and disinfecting agent (sodium hypochlorite and mercuric chloride) on in vitro establishment. F1 and F2 clones were the most responsive for shoot meristem in vitro culture. WPM medium supplemented with 8.8 µM 2iP, 0.2 µM NAA and 3 g L−1 activated charcoal was the most suitable for the in vitro establishment of the F1 clone. No phytotoxic effect of the disinfectant was observed and some meristems sprouted. The isolated endophytic bacterium was identified for the first time in yerba mate as Agrobacterium larrymoorei. To conclude, we were able to establish in vitro culture of yerba mate using meristems as explants but the tissues were not free of endophytic microorganisms which could interfere with explant response.

A Specific Gibberellin 20-Oxidase Dictates the Flowering-Runnering Decision in Diploid Strawberry

Asexual and sexual reproduction occur jointly in many angiosperms. Stolons (elongated stems) are used for asexual reproduction in the crop species potato (Solanum tuberosum) and strawberry (Fragaria spp), where they produce tubers and clonal plants, respectively. In strawberry, stolon production is essential for vegetative propagation at the expense of fruit yield, but the underlying molecular mechanisms are unknown. Here, we show that the stolon deficiency trait of the runnerless (r) natural mutant in woodland diploid strawberry (Fragaria vesca) is due to a deletion in the active site of a gibberellin20-oxidase (GA20ox) gene, which is expressed primarily in the axillary meristem dome and primordia and in developing stolons. This mutation, which is found in all r mutants, goes back more than three centuries. When FveGA20ox4 is mutated, axillary meristems remain dormant or produce secondary shoots terminated by inflorescences, thus increasing the number of inflorescences in the plant. The application of bioactive gibberellin (GA) restored the runnering phenotype in the r mutant, indicating that GA biosynthesis in the axillary meristem is essential for inducing stolon differentiation. The possibility of regulating the runnering-flowering decision in strawberry via FveGA20ox4 provides a path for improving productivity in strawberry by controlling the trade-off between sexual reproduction and vegetative propagation.

Analysis of Surface Growth in the Conifer Shoot Apical Meristem

Premise of research. The structure and organization of the conifer shoot apical meristem was investigated using confocal microscopy of whole-mount shoot apical meristems. This article presents detailed surface and internal patterns that provide an informative and comparative model of meristem growth that is able to account for the diversity and variation of conifer meristem form.Methodology. Confocal microscopy; light microscopy.Pivotal results. On the surface of the meristem, two zones with distinct cell division patterns were recognized. The first is the summit zone that covers the apex of the meristem dome, and the second is the peripheral zone that covers the flanks of the meristem. In the summit zone, successive anticlinal cell divisions are perpendicular to each other and produce isotropic growth. In the peripheral zone, surface cells divide with repetitive transverse divisions to form long axial chains that create packets with anisotropic growth. Only cells in the summit zone divide periclinally to contribute daughter cells to the underlying tissue. Those meristems with a small number of cells in the summit zone have a correspondingly small number of periclinal divisions of apical initial cells. Those meristems with larger summit zones and many wedge-shaped packets have a larger number of apical initials dividing periclinally.Conclusions. Conifer meristems have consistent cell division patterns that define the summit and peripheral zones. Apical initials are transient, and their number varies between meristems, depending on the size of the summit zone.

Morphological Characterization of Buds Being Donors of Apex for Molecular Confirmation of Differentiation of Meristem into Permanent Tissues of Stone Fruit Trees (Prunus sp.)

Molecular confirmation of the process of differentiation of meristematic tissue of plants is primarily based on histomorphological characterization of the tissues carrying these processes. For perennial plants – fruit trees, the knowledge of these processes is important to define runtime type and pomotechnical treatments but also to identify the genes responsible for determination of the apices into a generative phase of differentiation. Which apex extraction techniques will be applied depends on the structure of buds, i.e. whether an apex differentiates only into generative elements – pure flower buds, or the differentiation goes into two directions: differentiation of leaf primordium with axillar meristematic dome and differentiation of generative elements of flowers in the peak or lateral zone of an apex – mixed buds. The fruit trees of the genus Prunus have purely flower buds and for this purpose the whole apex is taken, between protection – cover leaves, on lateral positions of all collateral buds at shoot nodes. The moment of initiation of determination and the dynamics of differentiation of generative buds are different at different type of shoots on a tree. It is therefore necessary to know the shoots growth cessation time which is in correlation with apex determination. Fruit trees of the genus Prunus are ending the growth of shoots by rejection of shoot peaks, and the cast away time depends on the type, length and the position of shoots.

Efficient elimination of virus complex from garlic (Allium sativum L.) by cryotherapy of shoot tips

Vegetative propagation of plants, such as garlic (Allium sativum L.), is known to facilitate the transmission of several virus species throughout the plant cycles. This process favors the onset of complex diseases by accumulation of different species in the same plant, resulting in decreased productivity and production quality. Studies have reported the use of cryotherapy of shoot tips, or meristematic clusters, as an efficient tool for obtaining virus-free plants. This study aimed to evaluate the ability of cryotherapy to eradicate virus complex in garlic plants. Bulbils naturally infected with Onion Yellow Dwarf Virus (OYDV), Leek Yellow Strip Virus (LYSV) and Garlic Common Latent Virus (GCLV) were employed as explants for different virus-cleaning treatments tested. Dot-ELISA and RT-PCR analysis were used to demonstrate the presence/absence of virus complex, and histological analysis was also performed to confirm these results. Five days after cryotherapy, structural analysis revealed that cooling had caused cell damage, as indicated by the increased vacuolization of cells after cryotherapy, as well as slight plasmolysis after thermotherapy. Immunolocalization analysis indicated the subcellular distribution of OYDV in garlic shoot tips in association with the development of plasmodesmata, while no OYDV was detected in the first cell layers of the meristematic dome. Cryotherapy successfully removed virus complex, resulting in virus-free plants with enhanced efficiency, compared to conventional meristem culture-based techniques. Moreover, the synergistic effects of cryotherapy and thermotherapy resulted in a 40 % survival rate of shoot tips and the regeneration of 90, 100 and 80 % OYDV-, LYSV- and GCLV-free plants, respectively.

Histocytological analysis of yam (Dioscorea alata) shoot tips cryopreserved by encapsulation–dehydration.

In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation-dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1-3) and seven to nine (L7-9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1-3 and L7-9 cells. Nuclear protein content increased, particularly in L1 and L1-3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7-9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1-3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1-3 and L7-9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.

Elimination and detection of viruses in meristem-derived plantlets of sweetpotato as a low-cost option toward commercialization.

Viral diseases affecting sweetpotato are the most devastating and cause up to 98 % yield loss. In this paper, we report, meristem culture, graft transmission and virus indexing for management of viral pathogens in seven elite sweetpotato cultivars. Plantlets were developed in vitro from the apical meristematic dome with one to two leaf primordia. Mericlones were grafted on virus-sensitive indicator plant Ipomoea setosa and no viral disease symptoms were seen on I. setosa leaves in most cases. This indicates that no viruses translocated from meristem-derived scions to the virus-sensitive root stock. On the other hand, most of the non-tested traditional planting material induced distinct disease symptoms upon grafting, which revealed the presence of one or more viruses in it. About 85 % of mericlones recovered from 0.3–0.5 mm size meristem were tested as virus free, whereas it is difficult to culture meristems smaller than 0.3 mm due to dissection damage and too small a size. Virus-tested mericlones were further micropropagated and transferred to the field. Only few plants were found to be diseased in the R1 field trial. Root yield in the R2 generation was increased significantly when compared with non-tested control plants. During field exposure, only a low percentage of healthy plants were found infected with viruses when managed in a net house. This implies that viral vectors were present during the growing season and reinfection could be effectively reduced by net house management. We concluded that this low-cost technique of producing virus-tested planting material would significantly boost the yield through efficient removal of yield-reducing pathogens.

The transcription factor BELLRINGER modulates phyllotaxis by regulating the expression of a pectin methylesterase in Arabidopsis

Plant leaves and flowers are positioned along the stem in a regular pattern. This pattern, which is referred to as phyllotaxis, is generated through the precise emergence of lateral organs and is controlled by gradients of the plant hormone auxin. This pattern is actively maintained during stem growth through controlled cell proliferation and elongation. The formation of new organs is known to depend on changes in cell wall chemistry, in particular the demethylesterification of homogalacturonans, one of the main pectic components. Here we report a dual function for the homeodomain transcription factor BELLRINGER (BLR) in the establishment and maintenance of the phyllotactic pattern in Arabidopsis. BLR is required for the establishment of normal phyllotaxis through the exclusion of pectin methylesterase PME5 expression from the meristem dome and for the maintenance of phyllotaxis through the activation of PME5 in the elongating stem. These results provide new insights into the role of pectin demethylesterification in organ initiation and cell elongation and identify an important component of the regulation mechanism involved.

ATTEMPTS TO ERADICATE TWO PELARGONIUM VIRUSES (PFBV AND PLPV) BY MERISTEM CULTURE AND SHOOT-TIP CRYOTHERAPY

Attempts to eradicate the Pelargonium flower break virus (PFBV) and Pelargonium line pattern virus (PLPV) by meristem culture and apex droplet-vitrification cryopreservation was carried out using 5 different cultivars. A simple meristem culture did not permit to eliminate PFBV and only 15% of Pelargonium x hortorum 'Stellar Artic' plants regenerated from meristems was PLPV-ELISA-negative. Plants regenerated from cryopreserved apices were tested by DAS-ELISA after a 3-month growing period. Viruses were not detected in 25 and 50% of the tested plants for PFBV and PLPV respectively. Immunolocalisations were carried out for virus localisation in apices from greenhouse plants (control) and vitroplants regenerated after meristem culture or cryopreservation. Immunolocalisations realised on control explants excised from DAS-ELISA positive plants showed that PFBV and PLPV were present in the apices, even in the meristematic dome. However, viral particles were more numerous in the cells of the basal zone than in the more meristematic ones. Immunolocalisations realised on apices from the DAS-ELISA negative cryoregenerated plants showed the viruses were still present. Our results firstly demonstrated that PFBV and PLPV are even present inside meristematic cells and secondly that cryopreservation could decrease their amount in Pelargonium plants but without eliminating them totally. More knowledge on virus behaviour during cryopreservation processes could optimize the management of genetic resources using this conservation method.

The mechanics behind plant development

Morphogenesis in living organisms relies on the integration of both biochemical and mechanical signals. During the last decade, attention has been mainly focused on the role of biochemical signals in patterning and morphogenesis, leaving the contribution of mechanics largely unexplored. Fortunately, the development of new tools and approaches has made it possible to re-examine these processes. In plants, shape is defined by two local variables: growth rate and growth direction. At the level of the cell, these variables depend on both the cell wall and turgor pressure. Multidisciplinary approaches have been used to understand how these cellular processes are integrated in the growing tissues. These include quantitative live imaging to measure growth rate and direction in tissues with cellular resolution. In parallel, stress patterns have been artificially modified and their impact on strain and cell behavior been analysed. Importantly, computational models based on analogies with continuum mechanics systems have been useful in interpreting the results. In this review, we will discuss these issues focusing on the shoot apical meristem, a population of stem cells that is responsible for the initiation of the aerial organs of the plant.

Pathogen‐tested material of grapevine varieties and rootstocks

Pathogen‐tested material of grapevine varieties and rootstocks

Vitrification–cryopreservation, an efficient method for eliminating Candidatus Liberobacter asiaticus, the citrus Huanglongbing pathogen, from in vitro adult shoot tips

Huanglongbing disease (HLB), caused by Candidatus Liberobacter asiaticus, constitutes a most serious problem for the Chinese citrus industry. In this work, the use of vitrification-cryopreservation for eliminating Ca. L. asiaticus from naturally infected plants of several citrus species was investigated. Proliferating meristems were produced in vitro and excised tissue clumps were cryopreserved through vitrification using a plant vitrification solution 2. The health status of regenerated in vitro plants was checked by nested PCR. The putative HLB bacterial-free materials were subsequently re-tested after greenhouse acclimatization. Up to 98.1% of the plants obtained by cryopreservation were free from HLB bacterium, as compared with a sanitation rate of 25.3% yielded by conventional meristem tip culture. Light and electron microscopy observations of the meristem tips showed that the majority of the meristematic cells were injured either during the freezing/thawing step or during the osmotic dehydration step with plant vitrification solution 2. Only small areas of the meristematic dome survived the cryopreservation process, thereby increasing the probability of regenerating cells free of Ca. L. asiaticus. Large cells with big vacuoles and high water content, which are more likely to be infected by Ca. L. asiaticus, apparently cannot survive freezing in liquid nitrogen (LN). By contrast, small cells with dense cytoplasm located in the top layers of the meristem are more likely to escape invasion by Ca. L. asiaticus and can survive freezing in LN.

Relationship between starch accumulation and organ development at the different growth stages of callus in Kihada (Phellodendron amurense Rupr.)

Microscopic observations of Kihada (Phellodendron amurense) callus revealed a strong correlation between starch accumulation and organ development. The starch accumulation started before any observable organ development and was prominent in cells which ultimately gave rise to organ primordia. A large amount of starch accumulation was observed in the three- or four-celled proembryo-like structure (PLS) organization of callus cultured on MS media supplemented with BAP plus 2,4-D. Two-celled PLS showed low performance for accumulation of starch. No starch accumulation was observed in globular and heart-shaped embryo-like structure ELS organization. During shoot primordium development, a decrease in the starch content of the cultured tissues was found, indicating the utilization of the glucan in the organogenic process. Starch granules disappeared with the development of the meristematic dome and leaf primordium and also started to disappear gradually with the development of cell walls and the tracheary elements.

Reversion of inflorescence development in Euphorbia millii and its application to large-scale micropropagation in an air-lift bioreactor

SummaryAn efficient, simple micropropagation protocol was developed for Euphorbia millii Des Moulines using inflorescence explants and an automated, low cost, air-lift bioreactor system. Inflorescence meristems of the main axis usually reverted to vegetative meristems (a meristematic dome with leaf primordia) 1 week after inoculation on MS medium supplemented with benzyladenine (BA) and indole-3-butyric acid (IBA). Of the various plant growth regulator combinations tested, MS medium with 1 mg l–1 BA and 0.3 mg l–1 IBA proved to be most effective. On this medium the percentage reversion was maximal, and the number of explants producing multiple shoots was the highest. The highest percentage of inflorescence reversion and the highest number of multiple shoots induced per explant were observed when inflorescences from the first stage of development were used. The lowest response was observed on third stage inflorescences. Vegetative shoots obtained from reverted explants of inflorescences were used as inocula for multiplication studies. Comparisons between solid and bioreactor cultures (i.e., continuous immersion with or without a net, or temporary immersion in liquid media using ebb and flood) revealed that shoot multiplication and growth were more efficient using a continuous immersion bioreactor (with a net). In vitro regenerated shoots were cultured hydroponically for 6 weeks; 100% of plants rooted and were acclimatised successfully in growing media containing peat moss:vermiculite:perlite (1:1:1). RAPD DNA marker analysis of micropropagated plants and their mother plant confirmed the genetic uniformity of the regenerants.

Elimination of chrysanthemum stunt viroid from an infected chrysanthemum cultivar by shoot regeneration from a leaf primordium-free shoot apical meristem dome attached to a root tip.

In this research we eliminated chrysanthemum stunt viroid (CSVd) from a highly infected chrysanthemum cultivar using a newly established method. 'Piato' is one of the most difficult cultivars in which to obtain CSVd-free plants by conventional methods. Leaf primordium-free shoot apical meristems (LP-free SAMs) of 'Piato' plants were dissected and attached to CSVd-free chrysanthemum or cabbage root tips. As shown by nested-PCR, CSVd was not detected in some shoots regenerated on both types of root tip. The production rates of CSVd-free plants using chrysanthemum and cabbage root tips were 14% and 3%, respectively. Regeneration of plants from LP-free SAMs of chrysanthemum plants by attaching these SAMs to root tips is an efficient method of generating CSVd-free chrysanthemum plants.

Rescue of shoot apical meristems of chrysanthemum by culturing on root tips.

A new method to regenerate plants from leaf primordia-free shoot apical meristem domes (LP-free SAMs) was developed by establishing the meristem dome on the cut surface of root tips. Ten days after culture, the viable rate of LP-free SAMs of chrysanthemum 'Piato' attached to chrysanthemum root tips was >40%. Shoot regeneration was not observed from LP-free SAMs without the root tips. When LP-free SAMs of chrysanthemum were transferred to root tips of either petunia, cabbage, or carnation, the highest shoot regeneration rate was observed with cabbage root tips. Microscopic observation documented that the LP-free SAM temporarily adhered to the cut surface of the root tip of cabbage.

Separation of genetic functions controlling organ identity in flowers.

Comparative studies on the ABC model of floral development have revealed extensive conservation of B and C class genes, but have failed to identify similar conservation for A class genes. Using a reverse genetic approach, we show that the previous inability to obtain Antirrhinum mutants corresponding to the A class gene AP2 of Arabidopsis reflects greater genetic redundancy in Antirrhinum . Antirrhinum has two genes corresponding to AP2, termed LIP1 and LIP2, both of which need to be inactivated to give a mutant phenotype. Analysis of interactions between LIP and class B/C genes shows that unlike AP2 in Arabidopsis, LIP genes are not required for repression of C in outer whorls of the flower. However, like AP2, LIP genes play a role in sepal, petal and ovule development, although some of their detailed effects are different, reflecting the diverse morphologies of Antirrhinum and Arabidopsis flowers. The dual functions for which AP2 is required in Arabidopsis are therefore separate in Antirrhinum, showing that the genetic basis of some aspects of organ identity have undergone major evolutionary change.