Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant.

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an ever-increasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg/L BAP, 0.3 mg/L GA3, and 0.3 mg/L ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity. https://doi.org/10.54680/fr24210110412.

Dissecting the Roles of the Cytokinin Signaling Network: The Case of De Novo Shoot Apical Meristem Formation.

Numerous biotechnological applications require a fast and efficient clonal propagation of whole plants under controlled laboratory conditions. For most plant species, the de novo regeneration of shoots from the cuttings of various plant organs can be obtained on nutrient media supplemented with plant hormones, auxin and cytokinin. While auxin is needed during the early stages of the process that include the establishment of pluripotent primordia and the subsequent acquisition of organogenic competence, cytokinin-supplemented media are required to induce these primordia to differentiate into developing shoots. The perception of cytokinin through the receptor ARABIDOPSIS HISTIDINE KINASE4 (AHK4) is crucial for the activation of the two main regulators of the establishment and maintenance of shoot apical meristems (SAMs): SHOOTMERISTEMLESS (STM) and the WUSCHEL-CLAVATA3 (WUS-CLV3) regulatory circuit. In this review, we summarize the current knowledge of the roles of the cytokinin signaling cascade in the perception and transduction of signals that are crucial for the de novo establishment of SAMs and lead to the desired biotechnological output-adventitious shoot multiplication. We highlight the functional differences between individual members of the multigene families involved in cytokinin signal transduction, and demonstrate how complex genetic regulation can be achieved through functional specialization of individual gene family members.

The Shoot Apical Meristem: An Evolutionary Molding of Higher Plants.

The shoot apical meristem (SAM) gives rise to the aerial structure of plants by producing lateral organs and other meristems. The SAM is responsible for plant developmental patterns, thus determining plant morphology and, consequently, many agronomic traits such as the number and size of fruits and flowers and kernel yield. Our current understanding of SAM morphology and regulation is based on studies conducted mainly on some angiosperms, including economically important crops such as maize (Zea mays) and rice (Oryza sativa), and the model species Arabidopsis (Arabidopsis thaliana). However, studies in other plant species from the gymnosperms are scant, making difficult comparative analyses that help us understand SAM regulation in diverse plant species. This limitation prevents deciphering the mechanisms by which evolution gave rise to the multiple plant structures within the plant kingdom and determines the conserved mechanisms involved in SAM maintenance and operation. This review aims to integrate and analyze the current knowledge of SAM evolution by combining the morphological and molecular information recently reported from the plant kingdom.

Tipping the balance: The dynamics of stem cell maintenance and stress responses in plant meristems.

Plant meristems contain pools of dividing stem cells that produce new organs for plant growth and development. Environmental factors, including biotic and abiotic stresses and nutrient availability, affect meristem activity and thus the architecture of roots and shoots; understanding how meristems react to changing environmental conditions will shed light on how plants optimize nutrient acquisition and acclimate to different environmental conditions. This review highlights recent exciting advances in this field, mainly in Arabidopsis. We discuss the signaling pathways, genetic regulators, and molecular mechanisms involved in the response of plant meristems to environmental and nutrient cues, and compare the similarities and differences of stress responses between the shoot and root apical meristems.

Illuminating the path to shoot meristem regeneration: Molecular insights into reprogramming cells into stem cells.

Plant cells possess the ability to dedifferentiate and reprogram into stem cell-like populations, enabling the regeneration of new organs. However, the maintenance of stem cells relies on specialized microenvironments composed of distinct cell populations with specific functions. Consequently, the regeneration process necessitates the orchestrated regulation of multiple pathways across diverse cellular populations. One crucial pathway involves the transcription factor WUSCHEL HOMEOBOX 5 (WOX5), which plays a pivotal role in reprogramming cells into stem cells and promoting their conversion into shoot meristems through WUSCHEL (WUS). Additionally, cell and tissue mechanics, including cell wall modifications and mechanical stress, critically contribute to de novo shoot organogenesis by regulating polar auxin transport. Furthermore, light signaling emerges as a key regulator of plant regeneration, directly influencing expression of meristem genes and potentially influencing aforementioned pathways as well.

Quantifying Gene Expression Domains in Plant Shoot Apical Meristems.

The shoot apical meristem is the plant tissue that produces the plant aerial organs such as flowers and leaves. To better understand how the shoot apical meristem develops and adapts to the environment, imaging developing shoot meristems expressing fluorescence reporters through laser confocal microscopy is becoming increasingly important. Yet, there are not many computational pipelines enabling a systematic and high-throughput characterization of the produced microscopy images. This chapter provides a simple method to analyze 3D images obtained through laser scanning microscopy and quantitatively characterize radially or axially symmetric 3D fluorescence domains expressed in a tissue or organ by a reporter. Then, it presents different computational pipelines aiming at performing high-throughput quantitative image analysis of gene expression in plant inflorescence and floral meristems. This methodology has notably enabled the quantitative characterization of how stem cells respond to environmental perturbations in the Arabidopsis thaliana inflorescence meristem and will open new avenues in the use of quantitative analysis of gene expression in shoot apical meristems. Overall, the presented methodology provides a simple framework to analyze quantitatively gene expression domains from 3D confocal images at the tissue and organ level, which can be applied to shoot meristems and other organs and tissues.

ISSR markers endorsed genetically stable plants regeneration of neem (Azadirachta indica A. Juss.) through indirect organogenesis using different explants.

BACKGROUND: Various parts of neem (Azadirachta indica) have high demand in several industries. However, the inadequate supply of sources hampers the commercialization of different neem products. In this scenario, the current research was undertaken to produce genetically stable plants through indirect organogenesis. METHODS AND RESULTS: Several explants like shoot tips, internodal segments, and leaves, were cultivated on MS media with different growth regulators. Maximum callus formation was achieved using 1.5 mg/L NAA, 0.5 mg/L 2,4-D and 0.2 mg/L both for Kn and BAP in combination with shoot tip (93.67%). These calli showed an organogenic potentiality on MS medium having coconut water (15%) without growth regulators. This medium along with 0.5 mg/L Kn and 0.1 mg/L both for BAP and NAA yielded the maximum adventitious shoot production with shoot tip-derived callus (95.24%). These calli further produced the most buds per shoot (6.38) and highest average shoot length (5.46 cm) with 0.5 mg/L both for BAP and Kn and 0.1 mg/L NAA in combination after the fifth subculture. The 1/3 strength of MS media was found to be best along with 0.5 mg/L IBA and 0.1 mg/L Kn in combination to generate maximum root response (92.86%), roots per shoot (5.86) and longest average root length (3.84 cm). The mean plant survival after initial hardening was 83.33% which increased to 89.47% after secondary hardening. The lack of variation in ISSR markers among the regenerated trees is evidence of clonal fidelity between hardened plants. CONCLUSIONS: This protocol will accelerate the propagation of neem for utilization of its sources.

WUSCHEL-RELATED HOMEOBOX 13 suppresses de novo shoot regeneration via cell fate control of pluripotent callus.

Plants can regenerate their bodies via de novo establishment of shoot apical meristems (SAMs) from pluripotent callus. Only a small fraction of callus cells is eventually specified into SAMs but the molecular mechanisms underlying fate specification remain obscure. The expression of WUSCHEL (WUS) is an early hallmark of SAM fate acquisition. Here, we show that a WUS paralog, WUSCHEL-RELATED HOMEOBOX 13 (WOX13), negatively regulates SAM formation from callus in Arabidopsis thaliana. WOX13 promotes non-meristematic cell fate via transcriptional repression of WUS and other SAM regulators and activation of cell wall modifiers. Our Quartz-Seq2-based single cell transcriptome revealed that WOX13 plays key roles in determining cellular identity of callus cell population. We propose that reciprocal inhibition between WUS and WOX13 mediates critical cell fate determination in pluripotent cell population, which has a major impact on regeneration efficiency.

Genome-wide analysis of MdPLATZ genes and their expression during axillary bud outgrowth in apple (Malus domestica Borkh.).

BACKGROUND: Branching is a plastic character that affects plant architecture and spatial structure. The trait is controlled by a variety of plant hormones through coordination with environmental signals. Plant AT-rich sequence and zinc-binding protein (PLATZ) is a transcription factor that plays an important role in plant growth and development. However, systematic research on the role of the PLATZ family in apple branching has not been conducted previously. RESULTS: In this study, a total of 17 PLATZ genes were identified and characterized from the apple genome. The 83 PLATZ proteins from apple, tomato, Arabidopsis, rice, and maize were classified into three groups based on the topological structure of the phylogenetic tree. The phylogenetic relationships, conserved motifs, gene structure, regulatory cis-acting elements, and microRNAs of the MdPLATZ family members were predicted. Expression analysis revealed that MdPLATZ genes exhibited distinct expression patterns in different tissues. The expression patterns of the MdPLATZ genes were systematically investigated in response to treatments that impact apple branching [thidazuron (TDZ) and decapitation]. The expression of MdPLATZ1, 6, 7, 8, 9, 15, and 16 was regulated during axillary bud outgrowth based on RNA-sequencing data obtained from apple axillary buds treated by decapitation or exogenous TDZ application. Quantitative real-time PCR analysis showed that MdPLATZ6 was strongly downregulated in response to the TDZ and decapitation treatments, however, MdPLATZ15 was significantly upregulated in response to TDZ, but exhibited little response to decapitation. Furthermore, the co-expression network showed that PLATZ might be involved in shoot branching by regulating branching-related genes or mediating cytokinin or auxin pathway. CONCLUSION: The results provide valuable information for further functional investigation of MdPLATZ genes in the control of axillary bud outgrowth in apple.

Cell signaling in the shoot apical meristem.

Distinct from animals, plants maintain organogenesis from specialized tissues termed meristems throughout life. In the shoot apex, the shoot apical meristem (SAM) produces all aerial organs, such as leaves, from its periphery. For this, the SAM needs to precisely balance stem cell renewal and differentiation, which is achieved through dynamic zonation of the SAM, and cell signaling within functional domains is key for SAM functions. The WUSCHEL-CLAVATA feedback loop plays a key role in SAM homeostasis, and recent studies have uncovered new components, expanding our understanding of the spatial expression and signaling mechanism. Advances in polar auxin transport and signaling have contributed to knowledge of the multifaceted roles of auxin in the SAM and organogenesis. Finally, single-cell techniques have expanded our understanding of the cellular functions within the shoot apex at single-cell resolution. In this review, we summarize the most up-to-date understanding of cell signaling in the SAM and focus on the multiple levels of regulation of SAM formation and maintenance.

The genetic structure within a single tree is determined by the behavior of the stem cells in the meristem.

Genomic sequencing revealed that somatic mutations cause a genetic differentiation of the cells in a single tree. We studied a mathematical model for stem cell proliferation in the shoot apical meristem (SAM). We evaluated the phylogenetic distance between cells sampled from different portions of a shoot, indicating their genetic difference due to mutations accumulated during shoot elongation. The plant tissue has cell walls that suppress the exchange of location between cells. This leads to the genetic differentiation of cells according to the angle around the shoot and a larger genetic variance among cells in the body. The assumptions are as follows: stem cells in the SAM normally undergo asymmetric cell division, producing successor stem cells and differentiated cells. Occasionally, a stem cell fails to leave its successor stem cell and the vacancy is filled by the duplication of one of the nearest neighbor stem cells. A mathematical analysis revealed the following: the genetic diversity of cells sampled at the same position along the shoot increases with the distance from the base of the shoot. Stem cells hold a larger variation if they are replaced only by the nearest neighbors. The coalescent length between two cells increases not only with the difference in the position along the shoot but also in the angle around the shoot axis. The dynamics of stem cells at the SAM determine the genetic pattern of the entire shoot.

LAZY3 interacts with LAZY2 to regulate tiller angle by modulating shoot gravity perception in rice.

Starch biosynthesis in gravity-sensing tissues of rice shoot determines the magnitude of rice shoot gravitropism and thus tiller angle. However, the molecular mechanism underlying starch biosynthesis in rice gravity-sensing tissues is still unclear. We characterized a novel tiller angle gene LAZY3 (LA3) in rice through map-based cloning. Biochemical, molecular and genetic studies further demonstrated the essential roles of LA3 in gravity perception of rice shoot and tiller angle control. The shoot gravitropism and lateral auxin transport were defective in la3 mutant upon gravistimulation. We showed that LA3 encodes a chloroplast-localized tryptophan-rich protein associated with starch granules via Tryptophan-rich region (TRR) domain. Moreover, LA3 could interact with the starch biosynthesis regulator LA2, determining starch granule formation in shoot gravity-sensing tissues. LA3 and LA2 negatively regulate tiller angle in the same pathway acting upstream of LA1 to mediate asymmetric distribution of auxin. Our study defined LA3 as an indispensable factor of starch biosynthesis in rice gravity-sensing tissues that greatly broadens current understanding in the molecular mechanisms underlying the starch granule formation in gravity-sensing tissues, and provides new insights into the regulatory mechanism of shoot gravitropism and rice tiller angle.

Morphogenesis at the shoot meristem.

Shoot apical meristems are populations of stem cells which initiate the aerial parts of higher plants. Work during the last decades has revealed a complex network of molecular regulators, which control both meristem maintenance and the production of different types of organs. The behavior of this network in time and space is defined by the local interactions between regulators and also involves hormonal regulation. In particular, auxin and cytokinin are intimately implicated in the coordination of gene expression patterns. To control growth patterns at the shoot meristem the individual components of the network influence directions and rates of cell growth. This requires interference with the mechanical properties of the cells. How this complex multiscale process, characterized by multiple feedbacks, is controlled remains largely an open question. Fortunately, genetics, live imaging, computational modelling and a number of other recently developed tools offer interesting albeit challenging perspectives.

Les méristèmes apicaux caulinaires sont des populations de cellules souches qui initient les parties aériennes des plantes supérieures. Les travaux des dernières décennies ont révélé un réseau complexe de régulateurs moléculaires, qui contrôlent à la fois la maintenance des méristèmes et la production de différents types d'organes. Le comportement de ce réseau dans le temps et dans l'espace est défini par les interactions locales entre régulateurs et implique également une régulation hormonale. En particulier, l'auxine et la cytokinine sont intimement impliquées dans la coordination de l'expression génique. Pour contrôler la morphogenèse au niveau du méristème caulinaire, les éléments individuels du réseau influencent les directions et les taux de croissance cellulaire. Cela nécessite une interférence avec les propriétés mécaniques des cellules. Comment ce processus multi-échelle complexe, caractérisé par de multiples rétroactions, est contrôlé reste largement une question ouverte et sa compréhension représente un défi majeur. Heureusement, la génétique, l'imagerie in vivo, la modélisation informatique et un certain nombre d'autres outils récemment développés offrent des perspectives intéressantes.

Genome size and gas chromatography-mass spectrometry (GC-MS) analysis of field-grown and in vitro regenerated Pluchea lanceolata plants.

Pluchea lanceolata is a threatened pharmacologically important plant from the family Asteraceae. It is a source of immunologically active compounds; large-scale propagation may offer compounds with medicinal benefits. Traditional propagation method is ineffective as the seeds are not viable; and root sprout propagation is a slow process and produces less numbers of plants. Plant tissue culture technique is an alternative, efficient method for increasing mass propagation and it also facilitate genetic improvement. The present study investigated a three-way regeneration system in P. lanceolata using indirect shoot regeneration (ISR), direct shoot regeneration (DSR), and somatic embryo mediated regeneration (SER). Aseptic leaf and nodal explants were inoculated on Murashige and Skoog (MS) medium amended with plant growth regulators (PGRs), 2,4-dichlorophenoxy acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP) either singly or in combinations. Compact, yellowish green callus was obtained from leaf explants in 1.0 mg/l BAP (89.10%) added medium; ISR percentage was high, i.e., 69.33% in 2.0 mg/l BAP + 0.5 mg/l NAA enriched MS with 4.02 mean number of shoots per callus mass. Highest DSR frequency (67.15%) with an average of 5.62 shoot numbers per explant was noted in 0.5 mg/l BAP added MS medium. Somatic embryos were produced in 1.0 mg/l NAA fortified medium with 4.1 mean numbers of somatic embryos per culture. On BAP (1.0 mg/l) + 0.5 mg/l gibberellic acid (GA3) amended medium, improved somatic embryo germination frequency (68.14%) was noted showing 12.18 mean numbers of shoots per culture. Histological and scanning electron microscopic (SEM) observation revealed different stages of embryos, confirming somatic embryogenesis in P. lanceolata. Best rooting frequency (83.95%) of in vitro raised shootlets was obtained in 1.0 mg/l IBA supplemented half MS medium with a maximum of 7.83 roots per shoot. The regenerated plantlets were transferred to the field with 87% survival rate. The 2C genome size of ISR, DSR, and SER plants was measured and noted to be 2.24, 2.25, and 2.22 pg respectively, which are similar to field-grown mother plant (2C = 2.26 pg). Oxidative and physiological events suggested upregulation of enzymatic activities in tissue culture regenerated plants compared to mother plants, so were photosynthetic pigments. Implementation of gas chromatography-mass spectrometry (GC-MS) technique on in vivo and in vitro raised plants revealed the presence of diverse phyto-chemicals. The yields of alpha amyrin and lupeol (medicinally important triterpenoids) were quantified using high-performance thin-layer chromatography (HPTLC) method and enhanced level of alpha amyrin (2.129 µg g-1 dry wt) and lupeol (1.232 µg g-1 dry wt) was noted in in vitro grown leaf tissues, suggesting in vitro conditions act as a potential trigger for augmenting secondary metabolite synthesis. The present protocol represents a reliable mass propagation technique in producing true-to-type plants of P. lanceolata, conserving 2C DNA and ploidy successfully without affecting genetic homogeneity.

The role of shoot-derived RNAs transported to plant root in response to abiotic stresses.

A large number of RNA molecules are transported over long-distance between shoots and roots via phloem in higher plants. Mobile RNA signals are important for plants to tackle abiotic stresses. Shoot-derived mobile RNAs can be involved in the response to different developmental or environmental signals in the root. Some environmental conditions such as climate change, water deficit, nutrient deficiency challenge modern agriculture with more expeditious abiotic stress conditions. Root architecture determines the ability of water and nutrient uptake and further abiotic stress tolerance, and shoot tissue also determines the balance between shoot-root relationship in plant growth and adaptations. Thus, it is necessary to understand the roles of shoot-derived RNA signals and their potential function in roots upon abiotic stresses in the model plants (Arabidopsis thaliana and Nicotiana benthamiana) and agricultural crops. In this review, we summarize the so-far discovered shoot-derived mobile RNA transportation to the root under abiotic stress conditions, e.g. drought, cold stress and nutrient deficiencies. Furthermore, we will focus on the biological relevance and the potential roles of these RNAs in root development and stress responses which will be an asset for the future breeding strategies.

Arrest, Senescence and Death of Shoot Apical Stem Cells in Arabidopsis thaliana.

Shoot stem cells act as the source of the aboveground parts of flowering plants. A precise regulatory basis is required to ensure that plant stem cells show the right status during the stages of proliferation, senescence and cell death. Over the past few decades, the genetic circuits controlling stem cell fate, including the regulatory pathways of establishment, maintenance and differentiation, have been largely revealed. However, the morphological changes and molecular mechanisms of the final stages of stem cells, which are represented by senescence and cell death, have been less studied. The senescence and death of shoot stem cells are under the control of a complex series of pathways that integrate multiple internal and external signals. Given the crucial roles of shoot stem cells in influencing plant longevity and crop yields, researchers have attempted to uncover details of stem cell senescence and death. Recent studies indicate that stem cell activity arrest is controlled by the FRUITFULL-APETALA2 pathway and the plant hormones auxin and cytokinin, while the features of senescent and dead shoot apical stem cells have also been described, with dynamic changes in reactive oxygen species implicated in stem cell death. In this review, we highlight the recent breakthroughs that have enriched our understanding of senescence and cell death processes in plant stem cells.

Plant gene silencing signals move from the phloem to influence gene expression in shoot apical meristems.

BACKGROUND: Small RNAs (sRNA) are potent regulators of gene expression that can diffuse short distances between cells and move long distances through plant vasculature. However, the degree to which sRNA silencing signals can move from the phloem to the shoot apical meristem (SAM) remains unclear. RESULTS: Two independent transgenic approaches were used to examine whether phloem sRNA silencing can reach different domains of the SAM and silence SAM-expressed genes. First, the phloem companion-cell specific SUCROSE-PROTON SYMPORTER2 (SUC2) promoter was used to drive expression of an inverted repeat to target the FD gene, an exclusively SAM-localized floral regulator. Second, the SUC2 promoter was used to express an artificial microRNA (aMiR) designed to target a synthetic CLAVATA3 (CLV3) transgene in SAM stem cells. Both phloem silencing signals phenocopied the loss of function of their targets and altered target gene expression suggesting that a phloem-to-SAM silencing communication axis exists, connecting distal regions of the plant to SAM stem cells. CONCLUSIONS: Demonstration of phloem-to-SAM silencing reveals a regulatory link between somatic sRNA expressed in distal regions of the plant and the growing shoot. Since the SAM stem cells ultimately produce the gametes, we discuss the intriguing possibility that phloem-to-SAM sRNA trafficking could allow transient somatic sRNA expression to manifest stable, transgenerational epigenetic changes.

Establishment of an Efficient In Vitro Propagation of Cnidium officinale Makino and Selection of Superior Clones through Flow Cytometric Assessment of DNA Content.

Cnidium officinale is a valuable medicinal plant cultivated in Asia for its rhizomes. This study reports the in vitro regeneration of Cnidium officinale plants and the induction of rhizomes from microshoots. The rhizomatous buds of Cnidium officinale induced multiple shoots on Murashige and Skoog (MS) medium supplemented with 0.5 mg L-1 BA, which led to the regeneration of plants within four weeks of culture. After four weeks of culture, the plants were assessed for fresh weight, the number of leaves, the number of roots, and the length of roots to compare the performance of the different clones. The clones with good growth characteristics were selected with the aid of a flow cytometric analysis of 2C nuclear DNA content. The plants bearing high DNA values showed better growth characteristics. Various factors, namely, sucrose concentration (30, 50, 70, and 90 g L-1), ABA (0, 0.5, 1.0, and 2.0 mg L-1), the synergistic effects of BA (1.0 mg L-1) + NAA (0.5 mg L-1) and BA (1.0 mg L-1) + NAA (0.5 mg L-1) + ABA (1.0 mg L-1) with or without activated charcoal (1 g L-1), and light and dark incubation were tested on rhizome formation from microshoots. The results of the above experiments suggest that MS medium supplemented with 50 g L-1 sucrose, 1.0 mg L-1 ABA, and 1 g L-1 AC is good for the induction of rhizomes from the shoots of Cnidium officinale. Plantlets with rhizomes were successfully transferred to pots, and they showed 100% survival.

De novo stem cell establishment in meristems requires repression of organ boundary cell fate.

Stem cells play important roles in animal and plant biology, as they sustain morphogenesis and tissue replenishment following aging or injury. In plants, stem cells are embedded in multicellular structures called meristems. The formation of new meristems is essential for the plastic expansion of the highly branched shoot and root systems. In particular, axillary meristems (AMs) that produce lateral shoots arise from the division of boundary domain cells at the leaf base. The CUP-SHAPED COTYLEDON (CUC) genes are major determinants of the boundary domain and are required for AM initiation. However, how AMs get structured and how stem cells become established de novo remain elusive. Here, we show that two NGATHA-LIKE (NGAL) transcription factors, DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4)/NGAL3 and SUPPRESSOR OF DA1-1 7 (SOD7)/NGAL2, redundantly repress CUC expression in initiating AMs of Arabidopsis thaliana. Ectopic boundary fate leads to abnormal growth and organization of the AM and prevents de novo stem cell establishment. Floral meristems of the dpa4 sod7 double mutant show a similar delay in de novo stem cell establishment. Altogether, while boundary fate is required for the initiation of AMs, our work reveals how it is later repressed to allow proper meristem establishment and de novo stem cell niche formation.