Seed sterility was verified by plating and deep-sequencing of homogenates from sterile seedlings (Supplementary Fig. 13 ). We established seedling growth, harvesting and DNA preparation pipelines as detailed in the specific sections below. We defined the bacterial community within each soil, and the community associated with plant roots across a number of controlled experimental variables: soil type, plant sample fraction, plant age and plant genotype. For plant age, we harvested roots from two developmental stages: at the formation of an inflorescence meristem (yng) and during fruiting when ≥50% of the rosette leaves were senescent (old). The former represents plants at the peak of photosynthetic conversion to carbon, whereas the latter represents a stage well after the source-sink shift has occurred, marking the change in carbon allocation from vegetal to reproductive utilization23 (link). We prepared two microbial sample fractions from each individual plant: a rhizosphere (bacteria contained in the layer of soil covering the outer surface of the root system that could be washed from roots in a buffer/detergent solution), and EC (bacteria from within the plant root system after sonication-based removal of the rhizoplane; Supplementary Fig. 1 ). We also collected control soil samples (soil treated in parallel, but without a plant grown in it).
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Living Beings
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Plant
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Meristem
Meristem
Meristem: The region of active cell division and growth in plants, responsible for the formation of new tissues and organs.
Meristems are located at the tips of roots and shoots, as well as in other regions of the plant, and play a crucial role in the plant's development and expansion.
These specialized tissues contain undifferentiated cells that can give rise to various cell types, allowing the plant to continually grow and respond to changes in its environment.
Understanding the structure and function of meristems is essential for plant biology reseach, as well as for applications in agriculture and horticultre.
Meristems are located at the tips of roots and shoots, as well as in other regions of the plant, and play a crucial role in the plant's development and expansion.
These specialized tissues contain undifferentiated cells that can give rise to various cell types, allowing the plant to continually grow and respond to changes in its environment.
Understanding the structure and function of meristems is essential for plant biology reseach, as well as for applications in agriculture and horticultre.
Most cited protocols related to «Meristem»
Apicoectomy
Bacteria
Buffers
Carbon
Detergents
Genotype
Inflorescence
Meristem
Photosynthesis
Plant Roots
Plants
Reproduction
Rhizosphere
Specimen Collection
Sterility, Reproductive
Mitotic metaphase chromosomes of barley (Hordeum vulgare L., 2n = 2x = 14) cv. Akcent were flow-sorted according to Lysák et al. [33 (link)]. Briefly, barley seedlings were treated subsequently with hydroxyurea and amiprophos-methyl to accumulate meristem root tip cells at metaphase and the synchronized root meristems were fixed by formaldehyde. Chromosome suspensions were prepared by mechanical homogenization of 25 root tips in 1 ml ice-cold LB01 buffer [34 ] and stained by 2 μg/ml DAPI (4',6-diamidino-2-phenylindole). The stained samples were analyzed using a FACSVantage SE flow cytometer and sorter (Becton Dickinson, San José, USA). Batches of 10,000 chromosomes 1H and of 60,000 chromosomes 2H – 7H were sorted into 50 μl deionized water in a PCR tube. Purity in sorted fractions was checked regularly by FISH using a probe for GAA microsatellite as described in Suchánková et al. [35 (link)].
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amiprophos methyl
Buffers
Cells
Chromosomes
Chromosomes, Human, Pair 10
Cold Temperature
DAPI
Fishes
Formaldehyde
Hordeum
Hordeum vulgare
Hydroxyurea
Meristem
Metaphase
Plant Roots
Root Tip
Seedlings
Short Tandem Repeat
Live confocal time-lapse series of developing flower of A. thaliana Col-0 (Figure 2A–F and Figure 2—figure supplement 2 ), shoot apical meristem of tomato (Solanum lycopersicum) DR5 reporter line (Shani et al., 2010 (link)) (Figure 4—figure supplement 3 ) and leaf trichomes of Capsella rubella (Figure 5A ) were acquired using SP8 or SP5 Leica confocal microscopes, as described previously (Kierzkowski et al., 2012 (link); Vlad et al., 2014 (link)). After dissection samples were stained with 0.1% propidium iodide (PI) and grown in vitro on medium (Bayer et al., 2009 (link)). Confocal imaging was performed with a 63× long distance water immersion objective and an argon laser emitting at the wavelength of 488 nm. PI signal was collected at 600–665 nm. In the case of tomato shoot apex, pDR5::3xVENUS-N7 signal was also collected, at 505–545 nm. Distance between stacks was 0.5 μm. Time intervals were 11 hr for tomato and 24 hr for A. thaliana and C. rubella time lapse series.
Mature A. thaliana embryos (Figure 2H ) were fixed and stained as previously described (Bassel et al., 2014 (link)) and imaged using a Zeiss LSM710 confocal microscope with a 25× oil immersion lens. Confocal stacks of microtubule marker line TUA6-GFP (Ueda et al., 1999 (link)) in live Cardamine hirsuta fruits (Figure 3A ) were acquired using a SP2 Leica microscope, with a 40× long working distance water immersion objective and an argon laser emitting at 488 nm. GFP signal was collected at 495–545 nm. The z step between stack slices was 0.2 μm.
The sequential replica method (Williams and Green, 1988 (link)) was used to acquire a stereopair of SEM images from an Arabidospsis leaf surface (Figure 1D ) as described in (Elsner et al., 2012 (link)). Stereoscopic reconstruction (Routier-Kierzkowska and Kwiatkowska, 2008 (link)) was then performed for the stereo pair and converted into a triangular mesh using a custom MorphoGraphX module. All other data presented in this manuscript were acquired for previously published work or available through on-line catalogs.
Mature A. thaliana embryos (
The sequential replica method (Williams and Green, 1988 (link)) was used to acquire a stereopair of SEM images from an Arabidospsis leaf surface (
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Argon Ion Lasers
Capsella
Cardamine
Dietary Supplements
Dissection
Embryo
Fruit
Lens, Crystalline
Meristem
Microscopy
Microscopy, Confocal
Microtubules
Plant Leaves
Propidium Iodide
Reconstructive Surgical Procedures
Replica Techniques
Rubella
Submersion
Trichomes
There are technical drivers of research priorities: the tools we have. As soon as certain aspects of plant functioning become measurable, we start using those tools and assign overarching significance to these measurements, perhaps, because we aim at doing important things, simply because we are important, at least to ourselves. Things we cannot measure or observe become a matter of unimportance. Although we will continue to depend on scientific tools and their availability, the challenge is, not to get trapped in studying what we have tools for, but to go beyond, based on the challenges posed by theory, developing novel approaches that will permit us entering the terrain that remained largely unexplored for methodological reasons.
As was explained above, water shortage and low temperature, and to some extent nutrient shortage, are not primarily affecting plant carbon capture (photosynthesis), but rather affect tissue formation directly. Well known in plant physiology, plant ecologists tend to overlook the great sensitivity of meristematic tissues to low turgor pressure, low temperature, and shortage in key nutrients. These tissues stop building new cells at water potentials, low temperatures, and critically low nutrient supply that still permit reasonably high rates of photosynthetic CO2 uptake. Not surprisingly, the initial response of plants to such tissue-level growth constraints leads to an accumulation of non-structural carbon-metabolites (osmotically inactive ones such as starch and lipids), rather than to carbon-starvation (Körner, 2003 (link)). This discrepancy between awareness and reality roots in the convenient tools and techniques we have to measure photosynthesis and the absence of tools to monitor cell division and cell differentiation in situ, and/or to assess discrepancies between demand and supply of photoassimilates.
Another example for our methods driven priorities is the generally great significance attributed to air conditioning or to climate aspects in general when manipulative experiments are designed (e.g., CO2-enrichment works), although soils exert far greater influences on plant responses to what ever treatment we apply. I invite readers to check the length authors spend on describing atmospheric conditions in their experiments versus the soil conditions. The simple reason is that we can engineer atmospheric conditions, but we have no means to engineer plant–soil or plant–soil–microbe interactions, as decisive these might be. To my knowledge, the only experiment where the response of plants to a high CO2-environment were tested with plants growing in two different soil types (see Spinnler et al., 2002 (link)), revealed two different story lines, just depending on which soil was chosen. The challenge is to arrive at a broad appreciation that soil conditions (e.g., disturbed or undisturbed) are pre-determining experimental results. I join Högberg et al. (2005 ) in their viewing soil microbiota associated with roots as an integral part of plant functioning, to the extent, that they may actually be seen as part of the autotrophic system, rather than belonging to the heterotrophic world.
On a similar avenue, root research was and still is a minor fraction compared to leaf research, although there is no theoretical reason for such a posteriority. Both are equally significant, in fact roots may be more influential with respect to limiting resources. The only reason is methodology. While a leaf can be studied in isolation (e.g., some sensors mounted to it), a root does not function properly without its intact rhizosphere, apart from its poor visibility. We can “bring” the atmosphere into the lab (growth chambers), but we cannot bring a coupled rhizosphere to the lab. Any pot experiment is confounded as soon as plants respond differently to two treatments, because, inevitably, the treatment changes the root-space/plant size relationship. So the challenge here is testing hypothesis on plant responses with plants grown with unconstrained, well-developed soil biota in action. Most commonly, this can only be done in the field.
As was explained above, water shortage and low temperature, and to some extent nutrient shortage, are not primarily affecting plant carbon capture (photosynthesis), but rather affect tissue formation directly. Well known in plant physiology, plant ecologists tend to overlook the great sensitivity of meristematic tissues to low turgor pressure, low temperature, and shortage in key nutrients. These tissues stop building new cells at water potentials, low temperatures, and critically low nutrient supply that still permit reasonably high rates of photosynthetic CO2 uptake. Not surprisingly, the initial response of plants to such tissue-level growth constraints leads to an accumulation of non-structural carbon-metabolites (osmotically inactive ones such as starch and lipids), rather than to carbon-starvation (Körner, 2003 (link)). This discrepancy between awareness and reality roots in the convenient tools and techniques we have to measure photosynthesis and the absence of tools to monitor cell division and cell differentiation in situ, and/or to assess discrepancies between demand and supply of photoassimilates.
Another example for our methods driven priorities is the generally great significance attributed to air conditioning or to climate aspects in general when manipulative experiments are designed (e.g., CO2-enrichment works), although soils exert far greater influences on plant responses to what ever treatment we apply. I invite readers to check the length authors spend on describing atmospheric conditions in their experiments versus the soil conditions. The simple reason is that we can engineer atmospheric conditions, but we have no means to engineer plant–soil or plant–soil–microbe interactions, as decisive these might be. To my knowledge, the only experiment where the response of plants to a high CO2-environment were tested with plants growing in two different soil types (see Spinnler et al., 2002 (link)), revealed two different story lines, just depending on which soil was chosen. The challenge is to arrive at a broad appreciation that soil conditions (e.g., disturbed or undisturbed) are pre-determining experimental results. I join Högberg et al. (2005 ) in their viewing soil microbiota associated with roots as an integral part of plant functioning, to the extent, that they may actually be seen as part of the autotrophic system, rather than belonging to the heterotrophic world.
On a similar avenue, root research was and still is a minor fraction compared to leaf research, although there is no theoretical reason for such a posteriority. Both are equally significant, in fact roots may be more influential with respect to limiting resources. The only reason is methodology. While a leaf can be studied in isolation (e.g., some sensors mounted to it), a root does not function properly without its intact rhizosphere, apart from its poor visibility. We can “bring” the atmosphere into the lab (growth chambers), but we cannot bring a coupled rhizosphere to the lab. Any pot experiment is confounded as soon as plants respond differently to two treatments, because, inevitably, the treatment changes the root-space/plant size relationship. So the challenge here is testing hypothesis on plant responses with plants grown with unconstrained, well-developed soil biota in action. Most commonly, this can only be done in the field.
Atmosphere
Awareness
Biological Community
Carbon
Cells
Climate
Cold Temperature
Differentiations, Cell
Division, Cell
Fixation, Carbon
Heterotrophy
Hypersensitivity
isolation
Lipids
Meristem
Microbial Community
Microbial Interactions
Nutrients
Photosynthesis
Plant Leaves
Plant Roots
Plants
Pressure
Rhizosphere
Starch
Tissues
Vision
Actively growing root-tip meristems were pretreated with 0.05% aqueous solution of colchicine for 4 h at room temperature, fixed in ethanol : acetic acid (3 : 1) for at least 3 h at room temperature, and stored at −20°C until use.
Chromosome counting and basic karyotype analyses were performed using the standard Feulgen staining technique [55 (link)]. Ideograms (Additional file2 : Figure S2) were constructed based on measurements of at least five well-spread metaphase plates per individual (not shown) and measurements were used to calculate Haploid Karyotype Length (HKL). A single ideogram of each species and cytotype is provided, except for cytotypes B7B7 and B6B6 in which structural chromosomal variants were found (Table 2 ). Idiograms were constructed using Autoidiogram software (courtesy of Dr Wolfgang Harand, formerly University of Vienna; for details see [55 (link)]).
Chromosomal spreads for FISH were prepared by enzymatic digestion and squashing, as described earlier [4 (link),16 (link)] with some modifications. Briefly, material was digested with 1% cellulase Onozuka (Serva, Heidelberg, Germany), 1% cytohelicase (Sigma-Aldrich, Vienna, Austria), and 1% pectolyase (Sigma-Aldrich) for 18 min at 37°C. Cover slips were removed at −80°C and preparations air-dried. FISH followed the established protocol [16 (link),56 (link)]. Probes used for FISH were: 35S (18S/25S) rDNA from Arabidopsis thaliana in plasmid pSK+; 5S rRNA genic region from Melampodium montanum in plasmid pGEM-T Easy. Probes were labeled with biotin or digoxygenin (Roche, Vienna, Austria) either directly by PCR (5S rDNA) or using a nick translation kit (35S rDNA; Roche, Vienna, Austria). Digoxygenin was detected with antidigoxygenin antibody conjugated with FITC (5 μg mL-1: Roche, Vienna, Austria) and biotin with ExtrAvidin conjugated with Cy3 (2 μg mL-1: Sigma-Aldrich, Vienna, Austria). Preparations were analyzed with an AxioImager M2 epifluorescent microscope (Carl Zeiss, Vienna, Austria), images captured with a CCD camera, and processed using AxioVision ver. 4.8 (Carl Zeiss, Vienna, Austria) with only those functions that apply equally to the whole image. For rDNA localization, a minimum of 20 well-spread metaphases and prometaphases was analysed for each individual.
Chromosome counting and basic karyotype analyses were performed using the standard Feulgen staining technique [55 (link)]. Ideograms (Additional file
Chromosomal spreads for FISH were prepared by enzymatic digestion and squashing, as described earlier [4 (link),16 (link)] with some modifications. Briefly, material was digested with 1% cellulase Onozuka (Serva, Heidelberg, Germany), 1% cytohelicase (Sigma-Aldrich, Vienna, Austria), and 1% pectolyase (Sigma-Aldrich) for 18 min at 37°C. Cover slips were removed at −80°C and preparations air-dried. FISH followed the established protocol [16 (link),56 (link)]. Probes used for FISH were: 35S (18S/25S) rDNA from Arabidopsis thaliana in plasmid pSK+; 5S rRNA genic region from Melampodium montanum in plasmid pGEM-T Easy. Probes were labeled with biotin or digoxygenin (Roche, Vienna, Austria) either directly by PCR (5S rDNA) or using a nick translation kit (35S rDNA; Roche, Vienna, Austria). Digoxygenin was detected with antidigoxygenin antibody conjugated with FITC (5 μg mL-1: Roche, Vienna, Austria) and biotin with ExtrAvidin conjugated with Cy3 (2 μg mL-1: Sigma-Aldrich, Vienna, Austria). Preparations were analyzed with an AxioImager M2 epifluorescent microscope (Carl Zeiss, Vienna, Austria), images captured with a CCD camera, and processed using AxioVision ver. 4.8 (Carl Zeiss, Vienna, Austria) with only those functions that apply equally to the whole image. For rDNA localization, a minimum of 20 well-spread metaphases and prometaphases was analysed for each individual.
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Acetic Acid
Arabidopsis thalianas
Biotin
Cellulase
Chromosomes
Colchicine
Digestion
DNA, Ribosomal
Enzymes
Ethanol
Fishes
Fluorescein-5-isothiocyanate
Genes
Immunoglobulins
Karyotyping
Meristem
Metaphase
Microscopy
pectin lyase
Plasmids
Prometaphase
prostaglandin M
RNA, Ribosomal, 5S
Root Tip
Staining
Most recents protocols related to «Meristem»
Example 28
As transcription factors, REGULATOR OF AXILLARY MERISTEMS (RAX) play an important role in the formation of branch meristems. In tobacco, there are two RAX genes: RAX1 (SEQ ID NOs: 75 and 76) and RAX2 (SEQ ID NOs: 77 and 78).
RAX1 (SEQ ID NO: 75) and RAX2 (SEQ ID NO: 77) are knocked out in separate tobacco lines. The knockout mutant of RAX1 show the mislocalization of axillary buds in leaf axil (see
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Axilla
AXIN2 protein, human
Genes
Meristem
Nicotiana
Phenotype
Plant Leaves
Transcription Factor
The lateral roots were fixed in formalin–acetic acid–methanol (FAA) according to the method of Livingston et al.62 (link). Blocks were sectioned with a Leica RM2016 rotary microtome at 5 μm. Fast green FCF and safranin O were used to stain the sections. The lignified or corkified cell wall and vessel element will be dyed red and other tissues will be dyed green. Images were captured with a Nikon Eclipse E100. Subsequently, the length of the meristematic zone and the number of meristematic cells were analyzed using ImageJ 1.53.
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Acetic Acid
Blood Vessel
Cell Wall
Fast Green FCF
Formalin
Meristem
Methanol
Microtomy
Plant Roots
safranine T
Stains
Tissues
Since the end of the meristem for each cell file is not the same, the meristem size was determined in every cell file of the epidermis. To do so, the distance from the QC to the first cell in focus in the epidermis was measured as well as the length of all other cells in the file. The end of the meristem was considered as the first elongated cell71 (link). The relative position of each cell was calculated by normalization to the length of the RAM in each cell file. Thus, in this analysis, the cell marking the end of the meristem will be at position 1 and 0 corresponds to the QC. The relative position was calculated according to:
Epidermal T-clones and cells undergoing mitosis were removed from the analysis.
Epidermal T-clones and cells undergoing mitosis were removed from the analysis.
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Cells
Clone Cells
Epidermal Cells
Epidermis
Germ Cells
Meristem
Mitosis
Stack images of the roots (1 µm section) were acquired using a ×40 oil objective with a NIKON A1R + confocal microscope. Tile-scanning (4 × 1 tiles) was used to ensure the imaging of the whole meristem and the elongation zone of the roots. The Z Project tool (Projection type: Sum slices) was used to sum the fluorescence intensity of the pixels corresponding to each nucleus present in the epidermal layer. The background fluorescence was subtracted for each color channel and the fluorescent intensity of chromocenters and nuclei was measured as the integrity density of a determined ROI. In all cases, independent measurements were taken for each color channel. Data acquisition and processing were done using FIJI. The Relative Heterochromatin Fraction (RHF) was calculated using the formula RHF = [Ac × (Ʃ Ic − Ib)] / [An × (In − Ib)]. Ac: the total chromocenters area; Ic: fluorescence intensity of chromocenter; Ib: fluorescence intensity of the background; An: area of the nucleus; In: fluorescence intensity of the nucleus.
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Cell Nucleus
Epidermis
Fluorescence
Heterochromatin
Meristem
Microscopy, Confocal
Plant Roots
Composite M. truncatula plants were obtained following the previously described procedure (Boisson-Dernier et al., 2001 (link)). In brief, transgenic A. rhizogenes (ARqua1) carrying the plasmid of interest were grown in liquid LB medium (5 ml) supplemented with appropriate antibiotic for 24 hr until reaching an OD600 of approximately 0.5–0.7. 300 μl of this culture were then spread on a plate containing solid LB medium with antibiotics and incubated at 28 °C in dark for 48 hr. The root meristem of germinated seedlings was removed with a scalpel and the wounded part was dragged on the A. rhizogenes solid culture before transferring seedlings to plates containing solid Fahräeus medium supplemented with 0.5 mM NH4NO3. Transformed seedlings were grown vertically in a controlled environment chamber at 22 °C in dark for 3 days and for additional 4 days with a 16/8 hr light/dark photoperiod providing shading to the root system. Afterwards, composite plants were transferred to new solid Fahräeus plates (0.5 mM NH4NO3) and grown for 10 days at 24 °C with a 16/8 hr light/dark photoperiod. Transformed roots expressing the fluorescent selection marker were selected using a stereo microscope, untransformed roots were excised with a scalpel, and composite plants were transferred to either solid Fahräeus medium supplemented with 0.1 mM NH4NO3 (live-cell imaging of root hairs) or to pots (complementation assays).
Sinorhizobium meliloti (Sm2011) was grown in liquid TY medium (5 ml) supplemented with appropriate antibiotics for 3 days at 28 °C. 100 μl of this culture was used as inoculum for a fresh culture (5 ml), which was grown for 24 hr at 28 °C. A bacterial pellet was then obtained by centrifugation at 3000 rpm for 10 min, washed once with liquid Fahräeus medium (0.1 mM NH4NO3), and resuspended in the same medium to reach a final OD600=0.01 (inoculation of composite plants in plates, 200 μl per plant) or OD600=0.001 (inoculation in pots, 5 ml per plant).
Sinorhizobium meliloti (Sm2011) was grown in liquid TY medium (5 ml) supplemented with appropriate antibiotics for 3 days at 28 °C. 100 μl of this culture was used as inoculum for a fresh culture (5 ml), which was grown for 24 hr at 28 °C. A bacterial pellet was then obtained by centrifugation at 3000 rpm for 10 min, washed once with liquid Fahräeus medium (0.1 mM NH4NO3), and resuspended in the same medium to reach a final OD600=0.01 (inoculation of composite plants in plates, 200 μl per plant) or OD600=0.001 (inoculation in pots, 5 ml per plant).
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Animals, Transgenic
Antibiotics
Antibiotics, Antitubercular
Bacteria
Biological Assay
Cells
Centrifugation
Environment, Controlled
Hair
Light
Marijuana Abuse
Meristem
Microscopy
Plant Roots
Plants
Plasmids
Seedlings
Sinorhizobium meliloti
Vaccination
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Pectinase is an enzyme used in laboratory applications. It functions to break down pectin, a complex carbohydrate found in plant cell walls.
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The RNeasy Plant Mini Kit is a laboratory equipment designed for the isolation and purification of total RNA from plant tissues and cells. It utilizes a silica-membrane-based technology to efficiently capture and purify RNA molecules, enabling subsequent analysis and downstream applications.
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Onozuka R-10 is a cellulase enzyme preparation derived from the fungus Trichoderma viride. It is commonly used in various biotechnology applications, including plant cell wall degradation and sample preparation for electrophoresis.
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More about "Meristem"
Meristems are the powerhouses of plant growth and development, responsible for the formation of new tissues and organs.
These specialized regions of active cell division and differentiation are found at the tips of roots and shoots, as well as in other areas of the plant.
Meristems contain undifferentiated cells that can give rise to a variety of cell types, allowing the plant to continually adapt and respond to changes in its environment.
Understanding the structure and function of meristems is crucial for plant biology research, as well as for applications in agriculture and horticulture.
Techniques like TRIzol reagent, Pectinase, RNeasy Plant Mini Kit, Onozuka R-10, and microscopy tools like LSM 700, Agilent 2100 Bioanalyzer, LSM 780, and LSM 880 are commonly used to study meristems and their role in plant growth and development.
Meristems are the key to unlocking the secrets of plant life, enabling researchers to optimize cultivation protocols and enhance the reproducibility of their findings.
By leveraging the power of meristems, scientists can develop new and innovative solutions to meet the challenges of a rapidly changing world, from improving crop yields to designing more resilient and sustainable plant-based systems.
These specialized regions of active cell division and differentiation are found at the tips of roots and shoots, as well as in other areas of the plant.
Meristems contain undifferentiated cells that can give rise to a variety of cell types, allowing the plant to continually adapt and respond to changes in its environment.
Understanding the structure and function of meristems is crucial for plant biology research, as well as for applications in agriculture and horticulture.
Techniques like TRIzol reagent, Pectinase, RNeasy Plant Mini Kit, Onozuka R-10, and microscopy tools like LSM 700, Agilent 2100 Bioanalyzer, LSM 780, and LSM 880 are commonly used to study meristems and their role in plant growth and development.
Meristems are the key to unlocking the secrets of plant life, enabling researchers to optimize cultivation protocols and enhance the reproducibility of their findings.
By leveraging the power of meristems, scientists can develop new and innovative solutions to meet the challenges of a rapidly changing world, from improving crop yields to designing more resilient and sustainable plant-based systems.