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The phytohormone abscisic acid (ABA) is an important regulator of plant growth, but its potential participation in the process of in vitro shoot regeneration has not to date been reported. Here, we found that ABA appeared to inhibit in vitro shoot regeneration. ABA represses the formation of stem cell niches, thereby reducing the shoot regeneration by localizing the expression of WUSCHEL (WUS). During in vitro shoot regeneration, enrichment of H3K9ac in the specific region of WUS is a necessary event for its activation which could be inhibited by exogenous ABA. These findings reveal the potential function, as well as the possible way of ABA in regulating de novo shoot regeneration in Arabidopsis. [ABSTRACT FROM AUTHOR]

Producing uniform Cannabis sativa (Cannabis) for medicinal/recreational flower production through sexual propagation has been problematic, leading to dominance of clonal propagation from "mother plants" in the cannabinoid industry, which also faces significant limitations. Cannabis tissue culture (TC) methods have been developed to overcome these challenges, but the long-term health and maintenance of Cannabis explants in TC have been largely overlooked in previous studies. The current study focused on the development of an efficient and optimized micropropagation protocol covering the entire process, with a specific focus on the health and performance in the multiplication stage. Multiplication media were formulated hormone-free to avoid longer-term vitrification issues, resulting in single-main-shoot cultures rather than multiple-shoot cultures. This instigated the use of stage II explant types different from the standard shoot tips previously used for multiple shoot cultures. Multiplication media were further improved from the basal salt composition via nitrogen and calcium additives. The optimized protocol was used on eight diverse Cannabis cultivars to test its applicability across various genetic backgrounds. Results indicated that the protocol was effective for conservation purposes across all cultivars and achieved good long-term multiplication rates for some but not all. The outcomes of this study mark a significant stride towards an efficient Cannabis TC methodology ready for more comprehensive industrial applications. [ABSTRACT FROM AUTHOR]

In this study, the effects of various light treatments, growth regulators and culture media (agar-solidified and liquid) on in vitro propagation of Alternanthera reineckii Briq. Were evaluated. In in vitro experiments were used red, blue, and white LEDs alone and various combinations of red and blue LEDs. Different levels of 6-benzylaminopurine (BAP) and Kinetin (KIN), ranging from 0.25 mg/L to 1 mg/L, were introduced into the solid and liquid Murashige and Skoog (MS) basal medium. In solid culture, the highest number of shoots per explant (16.67 shoots/explant) was observed when utilizing a proportion of 1:2 red/blue LEDs in MS medium supplemented with 1 mg/L BAP. On the other hand, in the liquid culture, 15.13 shoots/explant were obtained with 1 mg/L BAP under a 2:1 red/blue LEDs configuration. During the experiments involving KIN, 10.42 shoots/explant were observed in the solid culture conditions with 1 mg/L of KIN under a lighting configuration of 1:2 red/blue LEDs. On the other hand, a total of 10.62 shoots/explant was attained in the liquid MS medium with 0.50 mg/L of KIN under a lighting configuration of 2:1 red/blue LEDs. As a result, LED lights were found to be better than fluorescent lights for multiple propagations of A. reineckii. Moreover, higher shoot numbers and longer shoots were achieved with red and blue LED light combinations. [ABSTRACT FROM AUTHOR]

Article Highlights - Effective induction of PLBs from stem thin layer and shoot regeneration using specific BAP and auxin combinations. - Significant role of potato extract in enhancing shoot regeneration. - Highest shoot regeneration rate achieved with 3 mg/L BAP concentration. Abstract Anubias barteri var. nana Petite, a highly valued ornamental aquatic plant in the Araceae family, faces challenges in vegetative propagation due to its slow multiplication rate. This study established a robust micropropagation protocol for rapid and efficient multiplication of A. barteri var. nana Petite. Explants were subjected to thorough sterilization, and shoot induction and multiplication were optimized using varying concentrations of Benzyl adenine (BA) on Murashige and Skoog (MS) medium. Protocorm-like bodies (PLBs) were induced from transverse thin-layer explants using combinations of BAP and Naphthaleneacetic acid (NAA). PLB multiplication and shoot regeneration were achieved through a sequential culture protocol. In addition, the influence of potato extract on shoot regeneration and the role of Indole-3-butyric Acid (IBA) in root development were explored. This study demonstrated a 100% survival rate of regenerated shoots in aquariums. Ongoing research is focused on further enhancing PLB multiplication. This optimized micropropagation protocol holds promise for the large-scale production of A. barteri var. nana Petite, addressing the limitations of natural propagation.

SUMMARY: Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4‐dichlorophenoxyacetic acid (2,4‐D), since natural auxins, such as indole‐3‐acetic acid (IAA) or 4‐chloroindole‐3‐acetic acid (4‐Cl‐IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4‐D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co‐application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux‐inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4‐D system, where only 50–60% of the embryos converted to seedlings, all SEs induced by transport‐inhibited natural auxins converted to seedlings. Efflux‐inhibited IAA, like 2,4‐D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux‐inhibited 4‐Cl‐IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4‐D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux‐inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants. Significance Statement: It has been a longstanding question why the synthetic auxin 2,4‐dichlorophenoxyacetic acid (2,4‐D) is so effective in inducing plant regeneration. Here we show that the secret lies in its inefficient export from plant cells and that using natural auxins can be as effective, even leading to improved regeneration, when their efflux is transiently inhibited. [ABSTRACT FROM AUTHOR]

Shoot regeneration capacity is essential for prosperous genetic transformation. Previous studies have shown that WUSCHEL-related homeobox (WOX) transcription factor plays a crucial role in callus growth, shoot regeneration, and root development. However, the mechanisms and functions of shoot regeneration related to WOX8 remain unclear. In the current research, the HmWOX8 gene was isolated from Hemerocallis middendorffii by RACE (Rapid-amplification of cDNA ends) technology. Overexpression of HmWOX8 improved callus proliferation and shoot regeneration ability of Arabidopsis and rice, whereas silencing HmWOX8 in H. middendorffii resulted in the inverse correlation. Transcriptome analysis revealed that HmWOX8 enhances the efficiency of callus proliferation and shoot regeneration through two different ways of regulation, including hormone signaling pathways and shoot development-related genes. (I) HmWOX8 regulates crosstalk among different hormone signaling pathways by activating and inhibiting the expression of different genes in these pathways, thus ensuring signal integration for efficient callus proliferation and shoot regeneration. (II) HmWOX8 can upregulate the expression level of shoot developmental genes, including WOX5/7, BBM, AIL5/7, PLT1, PIN6, CUC3, and SCR14/30, to regulate shoot emergence and outgrowth. In addition, Yeast two-hybrid assays and Bimolecular fluorescence complementation assay suggested that HmWOX8 directly interacts with HmCUC2, thereby promoting shoot regeneration. The present research improves the understanding of molecular mechanisms for HmWOX8-mediated regeneration and provides valuable gene resources for breeding programs to promote plant regeneration.

The investigation was carried out to evaluate the influence of the dicamba (3,6-Dichloro-2-methoxybenzoic acid) (DIC) and casein hydrolysate (CH) on the callus growth, shoot multiplication, and some biochemical constituents of date palm cv. Barhee cultured in vitro. Both DIC and CH were required for callus growth and shoots regeneration. The medium supplemented with 4.0 mg l−1 DIC in combination with 1.0 g l−1 CH gave the highest callus weight (287 mg), while the maximum response rate and the number of shoots per jar (86.67% and 15.07 shoots/jar) were found in MS media equipped with 4 mg l−1 DIC and 0.5 mg l−1 CH combination. The total amount of phenolic compounds was significantly reduced to 0.82 and 0.79 mg GAE g–1 in shoots cultured in the medium equipped with 4.0 mg l−1 DIC with 0.5 and 1.0 g l−1 CH, which is reflected in the rate of browning. The results showed that the highest shoots content of endogenous IAA (3.71 and 3.50 μg g−1), were obtained in response to 4 mg l−1 DIC + 1.0 g l−1 CH and 4.0 mg l−1 DIC + 0.5 g l−1 CH, respectively. The macronutrient K, P, Ca, and free amino acids content significantly increased in the in vitro shoots regenerated on the media supplemented with 4.0 mg l−1 DIC + 1.0 g l−1 CH. The genetic stability of this study was confirmed by the DNA-based fingerprinting method RAPD. The RAPD binding patterns indicated no variation among tissue culture-derived plants. The in vitro propagation protocol described herein can be introduced to the production of genetically stable date palm plants.

Song et al. utilized spatial transcriptomics to study the molecular characteristics of various cells – such as shoot primordia and chlorenchyma cells – in tomato callus during shoot regeneration. This research enhances our knowledge of shoot regeneration and demonstrates the potential of spatial transcriptomics in advancing plant biology. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Meyer lemon (Citrus × meyeri) is a hybrid citrus fruit from the Rutaceae family, originating from China. It is well-known for its distinctive appearance and flavor, as well as its healthnourishing nutrients. Micropropagation is an efficient alternative in the multiplication of plant stocks suitable for the commercial scale. The inclusion of organic additives in culture media has been found to provide a cost-effective option as a plant growth stimulant for in vitro plant development. The current study intends to assess the effects of coconut water and banana homogenate in the regeneration of Meyer lemon. In vitro, shoots were treated in half-strength Murashige and Skoog media fortified with 2 mg/L 6-benzylaminopurine with varying concentrations of coconut water and banana homogenate without sucrose. Results revealed that the treatment of 30% coconut water and 40 g/L banana homogenate resulted in the greatest proliferation of new shoots (3.00 ± 0.873 and 1.57 ± 0.297, respectively), whereas treatment of 40% coconut water resulted in the greatest shoot elongation of 0.239 ± 0.026 cm. The current study suggested the incorporation of coconut water and banana homogenate as potential substitutes for carbon sources and growth stimulants in the regeneration of Meyer lemon. [ABSTRACT FROM AUTHOR]

Gene transfer technology has great value in ornamental plants toward the generation of varieties with new ornate characteristics. In the previous studies through the transformation of cyclamen, hygromycin was mainly used as a selective marker. However, there have been some drawbacks associated with hygromycin usage as a selecting agent. Therefore, in the current study, the optimization of kanamycin concentration in the regeneration media has been considered. Subsequently, the plant transformation using three different in vitro explants from three Cyclamen persicum cultivars using three Agrobacterium tumefaciens strains has been examined. Accordingly, the optimal kanamycin concentrations for regeneration from root and leaf explants were determined as 10 mg/L and for microtuber explants as 30 mg/L. The successful gene transformation in the antibiotic-resistant shoots were examined by PCR and UV-equipped microscopes. The gfp reporter gene transfer resulted in the highest efficiency of transformation (60%) to date, from the leaf explants of cv. Pure White inoculated with Agrobacterium tumefaciens strain LBA4404. In contrast, the lowest gene transfer efficiency (25%) was observed in root explants of cv. Dark Violet and cv. Neon Pink inoculated with strains GV3101 and AGL-1, respectively. The results of the current project are expandable to the subsequent investigations of Cyclamen persicum transformation. [ABSTRACT FROM AUTHOR]

Vitrification is one of the most significant issues encountered in plant tissue culture applications. It diminishes the quality of in vitro plants, causing their leaves and stems to appear watery and translucent, and it may impede the success of the acclimatization step. In this respect, this study investigates the impact of sucrose and 6-Benzylaminopurine (BAP) concentrations on shoot regeneration and vitrification development in Aronia melanocarpa, known for its high antioxidant content and health benefits. Initially, the presence of BAP, in combination with varying sucrose concentrations, leads to a substantial increase in shoot number, and the largest number (7 shoots per nodal explant) was observed in the Murashige and Skoog (MS) medium containing 3% sucrose and 5.0 mg/L BAP. Furthermore, sucrose concentration plays a crucial role in shoot growth, with higher concentrations promoting more extensive shoot development. However, when 3% sucrose was combined with higher BAP (from 1.0- to 5.0 mg/L), an increased incidence of vitrification was observed over time. Interestingly, lower sucrose concentrations (1% or 2%) combined with 0.5 mg/L or 2.5 mg/L BAP initially delayed vitrification but eventually led to its occurrence. Microscopic analysis of leaf samples with varying levels of vitrification indicates significant differences in the density of stomata, further confirming the detrimental impact of vitrification on cellular structures and physiological processes. The recovery of vitrified plants was evaluated using different growth media combinations. The absence of BAP in the medium led to higher recovery percentages (min 96%) without necrosis, while the addition of 0.5 mg/L BAP promoted shoot growth but potentially inhibited root development. It has been found that media with 1 mg/L BAP and either 10 g/L or 20 g/L sucrose, as well as media with 30 g/L sucrose and 0.5 mg/L BAP, are the most suitable for efficient shoot regeneration with minimal vitrification risk. However, increasing BAP levels for faster shoot regeneration also raises the risk of vitrification. During acclimatization, vitrified plants exhibited stunted shoot growth, shorter and narrower leaves, reduced root numbers and lengths, and decreased survival rates, particularly under lower humidity conditions. The cultivation period required for the recovery of the crop was determined to be 6 weeks under greenhouse conditions for a sustainable plant propagation.Key message: Optimizing Aronia melanocarpa shoot regeneration: Balance sucrose and BAP for vigorous growth. Beware vitrification! Tailor medium and conditions for successful recovery. [ABSTRACT FROM AUTHOR]

Copper (Cu) is an essential plant micronutrient. This report is the first to assess the effects of high copper sulfate (CuSO4) levels on in vitro callus and shoot formation of cucumber. Four-day-old cotyledon explants from the inbred line Wisconsin 2843 and the commercial cultivars Marketer and Negrito were used. Murashige and Skoog (MS)-derived callus and shoot induction medium containing 0.5 mg L−1 indole-3-acetic acid (IAA) and 2.5 mg L−1 6-benzylaminopurine (BAP) was supplemented with CuSO4 (0.2–5 mg L−1). The response on callus-derived shoots showed that the optimal concentration of CuSO4 was 8- to 200-fold greater than in standard MS medium. Shoot frequency (SF) and shoot number (SN) were assessed, and Marketer > Negrito > Wisconsin 2843, where 1, 0.2 and 5 mg L−1 CuSO4 produced the highest results, respectively. SF and SN increased 6- and 10-fold in Wisconsin 2843 and twice in the other cultivars. All explants formed calluses, and in two of the three cultivars, callus extension was significantly affected by CuSO4 application. SN showed a strong relationship with CuSO4 levels and no association with callus extension. The results show that specific CuSO4 concentrations higher than in standard MS medium increase adventitious cucumber shoot organogenesis.

Invasions of alien species have become a global problem worldwide. Our paper presents the results of the study of the chorology of the invasive species Ambrosia artemisiifolia L. within the Right-Bank Forest-Steppe of Ukraine (RBFSU). We identified 13 new growth areas in the study area. These studies cover the observation and recording of the phenological behaviour of this species during 2020-2021. Although phenological observations are mainly carried out on cultivated plants, the data obtained as a result of the study is an important resource for preparing an invasive species control plan. Based on the indicators of temperature fluctuations, drought tolerance, light tolerance, relation to soil fertility, reproduction and shoot regeneration of A. artemisiifolia, a scoring method was proposed using the coefficient of complex adaptive potential (CCAP). However, our work is a pioneering study aimed at counteracting the spread of alien plants.

Under appropriate culture conditions, plant cells can regenerate new organs or even whole plants. De novo organ regeneration is an excellent biological system, which usually requires additional growth regulators, including auxin and cytokinin. Nitrate is an essential nutrient element for plant vegetative and reproductive development. It has been reported that nitrate is involved in auxin biosynthesis and transport throughout the growth and development of plants. In this study, we demonstrated that the ectopic expression of the MdNLP7 transcription factor in Arabidopsis could regulate the regeneration of root explants. MdNLP7 mainly participated in the regulation of callus formation, starting with pericycle cell division, and mainly affected auxin distribution and accumulation in the regulation process. Moreover, MdNLP7 upregulated the expression of genes related to auxin biosynthesis and transport in the callus formation stage. The results demonstrated that MdNLP7 may play a role in the nitrate-modulated regeneration of root explants. Moreover, the results revealed that nitrate–auxin crosstalk is required for de novo callus initiation and clarified the mechanisms of organogenesis.

Producing uniform Cannabis sativa (Cannabis) for medicinal/recreational flower production through sexual propagation has been problematic, leading to dominance of clonal propagation from “mother plants” in the cannabinoid industry, which also faces significant limitations. Cannabis tissue culture (TC) methods have been developed to overcome these challenges, but the long-term health and maintenance of Cannabis explants in TC have been largely overlooked in previous studies. The current study focused on the development of an efficient and optimized micropropagation protocol covering the entire process, with a specific focus on the health and performance in the multiplication stage. Multiplication media were formulated hormone-free to avoid longer-term vitrification issues, resulting in single-main-shoot cultures rather than multiple-shoot cultures. This instigated the use of stage II explant types different from the standard shoot tips previously used for multiple shoot cultures. Multiplication media were further improved from the basal salt composition via nitrogen and calcium additives. The optimized protocol was used on eight diverse Cannabis cultivars to test its applicability across various genetic backgrounds. Results indicated that the protocol was effective for conservation purposes across all cultivars and achieved good long-term multiplication rates for some but not all. The outcomes of this study mark a significant stride towards an efficient Cannabis TC methodology ready for more comprehensive industrial applications.

Copper (Cu) is an essential plant micronutrient. This report is the first to assess the effects of high copper sulfate (CuSO4) levels on in vitro callus and shoot formation of cucumber. Four-day-old cotyledon explants from the inbred line Wisconsin 2843 and the commercial cultivars Marketer and Negrito were used. Murashige and Skoog (MS)-derived callus and shoot induction medium containing 0.5 mg L-1 indole-3-acetic acid (IAA) and 2.5 mg L-1 6-benzylaminopurine (BAP) was supplemented with CuSO4 (0.2-5 mg L-1). The response on callus-derived shoots showed that the optimal concentration of CuSO4 was 8- to 200-fold greater than in standard MS medium. Shoot frequency (SF) and shoot number (SN) were assessed, and Marketer > Negrito > Wisconsin 2843, where 1, 0.2 and 5 mg L-1 CuSO4 produced the highest results, respectively. SF and SN increased 6- and 10-fold in Wisconsin 2843 and twice in the other cultivars. All explants formed calluses, and in two of the three cultivars, callus extension was significantly affected by CuSO4 application. SN showed a strong relationship with CuSO4 levels and no association with callus extension. The results show that specific CuSO4 concentrations higher than in standard MS medium increase adventitious cucumber shoot organogenesis. [ABSTRACT FROM AUTHOR]

Plant tissue regeneration is a key process for genetic transformation and genome editing. The exploration of regulatory mechanisms in plant regeneration would improve regeneration efficiency. In comparison to some model plants, the genomic heterozygosity is much higher in forest trees, increasing the complexity of transcriptional regulation. Here, we report the allele-specific transcriptional analysis in hybrid poplar 84K (Populus alba × P. tremula var. glandulosa cv. 84K) during the shoot regeneration process. Firstly, 180 regeneration-related genes (REGs) and 2446 REG-homologous genes (REGHs) were identified in hybrid poplar. The expression patterns of REGs exhibited that about half of them were positively correlated between poplar and Arabidopsis at the locus level. The expression levels of REGHs vary among the gene family at different stages during callus and shoot induction. Among the gene clusters with similar expression patterns, the distribution of gene families in poplar and Arabidopsis also exhibits large variations. At the allele level, most of the allele pairs of REGs were positively correlated in expression. The expression patterns of genes in auxin synthesis, transport, and signaling pathways agree with the general patterns. Due to the presence/absence of variations between two subgenomes, two YUC alleles and two IAA alleles are present only in one subgenome, and the expression patterns of the two alleles are greatly different. Our analysis indicates the conservativeness and diversity of transcriptional regulation during shoot regeneration in poplar and Arabidopsis. The complexity in allele expression contributed by heterozygosity suggests the importance of genotyping in the screening of explants for plant regeneration. [ABSTRACT FROM AUTHOR]

The current in vitro plant regeneration of purple passion fruit is mainly based on shoot organogenesis. In particular, thin cell layer (TCL) technology has emerged as a powerful technique for shoot organogenesis from many in vitro explant sources of purple passion fruit. This study further optimized the in vitro plant regeneration from ex vitro explant sources (leaves and internodes) via TCL technology combined with the addition of AgNPs on the culture medium. For leaf TCL explants, the results showed that the optimal shoot induction rate (96.30%) and the highest number of shoots (4.33 shoots/explant) were recorded on MS medium supplemented with 1.0 mg L− 1 BA and 1.5 mg L− 1 AgNPs. For internode TCL explants, different internode positions had a significant effect on shoot induction from tTCL explants. After 60 days of culture, the optimal shoot induction rate (70.37%) was observed in the tTCL explants at the 3rd internode from the shoot tip. This study also revealed that the difference in endogenous hormones at different internode positions was one of the significant factors affecting shoot induction from TCL explants. On the other hand, the results indicated that the shoot regeneration coefficient in lTCL was significantly higher than in tTCL. In addition, somatic embryogenesis was recorded for the first time in internode tTCL explants (22.45%). The addition of 3.0 mg L− 1 AgNPs significantly enhanced the proliferation and maturation of somatic embryos derived from internode tTCL explants. The present study contributes to a significant improvement in the micropropagation efficiency of purple passion fruit. Key message: Efficient shoot regeneration was observed for thin cell layer explants of ex vitro leaves and internodes. Differences in endogenous hormones at different internode locations significantly influenced shoot induction. Somatic embryogenesis was first recorded from internode explants of Passiflora edulis Sims f. edulis and this process was significantly enhanced by silver nanoparticles. [ABSTRACT FROM AUTHOR]

Invasions of alien species have become a global problem worldwide. Our paper presents the results of the study of the chorology of the invasive species Ambrosia artemisiifolia L. within the Right-Bank Forest-Steppe of Ukraine (RBFSU). We identified 13 new growth areas in the study area. These studies cover the observation and recording of the phenological behaviour of this species during 2020-2021. Although phenological observations are mainly carried out on cultivated plants, the data obtained as a result of the study is an important resource for preparing an invasive species control plan. Based on the indicators of temperature fluctuations, drought tolerance, light tolerance, relation to soil fertility, reproduction and shoot regeneration of A. artemisiifolia, a scoring method was proposed using the coefficient of complex adaptive potential (CCAP). However, our work is a pioneering study aimed at counteracting the spread of alien plants. [ABSTRACT FROM AUTHOR]

Wild plant species are of great interest as a source of pharmacologically valuable compounds but a great number of them are endemic and/or endangered ones. Modern plant biotechnology can provide reliable methods for their utilization without disturbing natural populations. In vitro culture methods for Rhodiola species are being intensively developed to include them into various biotechnological programmes. Aim. Development of a protocol for direct Rhodiola rosea L. plant regeneration from leaf explants. Methods. The leaves of R. rosea aseptically growing plants were used as the explants. Several variants of Murashige and Skoog (1962) agar-solidified culture medium supplemented with different combinations of auxins (1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)) and cytokinins (kinetin and 6-benzylaminopurine (BAP)) were estimated as potential regeneration-inducing media. Regeneration frequency was calculated as the percentage of leaves that produced shoots. Results. The use of MS medium supplemented with 2.5 mg/l BAP and 1.0 mg/l 2,4-D allowed inducing shoot formation with 100% frequency. An increase in the 2,4-D content up to 2.5 mg/l and decrease in BAP content to 1.0 mg/l resulted in decreasing of the regeneration frequency to 62.5%. Regeneration frequency was 25% and 62%, respectively, on the media containing 1.0 mg/l kinetin + 2.5 mg/l 2,4-D and 2.5 mg/l kinetin + 1.0 mg/l 2,4-D. Conclusions. R. rosea leaf explants have demonstrated high regeneration capacity with using the studied combinations of plant growth regulators. MS medium supplemented with 2.5 mg/l BAP and 1.0 mg/l 2,4-D allowed inducing shoot regeneration in leaf explants with the frequency of 100%. The frequency of regeneration was lower in the case of substitution of BAP for kinetin. The other types of morphogenesis (formation of adventitious roots and/or callus) were also observed.

Callus is a reprogrammed cell mass involved in plant regeneration and gene transformation in crop engineering. Pluripotent callus cells develop into fertile shoots through shoot regeneration. The molecular basis of the shoot regeneration process in crop callus remains largely elusive. This study pioneers the exploration of the spatial transcriptome of tomato callus during shoot regeneration. The findings reveal the presence of highly heterogeneous cell populations within the callus, including epidermis, vascular tissue, shoot primordia, inner callus, and outgrowth shoots. By characterizing the spatially resolved molecular features of shoot primordia and surrounding cells, specific factors essential for shoot primordia formation are identified. Notably, chlorenchyma cells, enriched in photosynthesis-related processes, play a crucial role in promoting shoot primordia formation and subsequent shoot regeneration. Light is shown to promote shoot regeneration by inducing chlorenchyma cell development and coordinating sugar signaling. These findings significantly advance our understanding of the cellular and molecular aspects of shoot regeneration in tomato callus and demonstrate the immense potential of spatial transcriptomics in plant biology. [ABSTRACT FROM AUTHOR]

Plants, unlike animals, possess a unique developmental plasticity, that allows them to adapt to changing environmental conditions. A fundamental aspect of this plasticity is their ability to undergo postembryonic de novo organogenesis. This requires the presence of regulators that trigger and mediate specific spatiotemporal changes in developmental programs. The phytohormone cytokinin has been known as a principal regulator of plant development for more than six decades. In de novo shoot organogenesis and in vitro shoot regeneration, cytokinins are the prime candidates for the signal that determines shoot identity. Both processes of de novo shoot apical meristem development are accompanied by changes in gene expression, cell fate reprogramming, and the switching-on of the shoot-specific homeodomain regulator, WUSCHEL. Current understanding about the role of cytokinins in the shoot regeneration will be discussed. [ABSTRACT FROM AUTHOR]

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Doubled haploid plants are very important for the development of complete homozygous plants from heterozygous parents in one generation as they possess duplicate copy of haploid chromosome. Haploid production is easily obtained from in vitro anther culture. The present study was undertaken with the objective to develop doubled haploids using anthers for in vitro induction of callus on N6 medium supplemented with various combinations and concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5-2.5 mg/L), Kinetin (0.5-1.0 mg/L) and Naphthalene acetic acid (NAA) (2.0 mg/L) as callus induction medium (CIM). The highest callus induction frequency was obtained when N6 medium fortified with 2,4-D (2.5 mg/L), Kinetin (0.5 mg/L) and NAA (2 mg/L) of 10.07 per cent. The induced callus was sub cultured for shoot regeneration on Murashige and Skoog medium (MS) supplemented with growth regulators: Kinetin and NAA (0.5 mg/L each) in combination with BAP (0.0 - 2.5 mg/L). MS medium supplemented with NAA (0.5 mg/L), Kinetin (0.5 mg/L) and BAP (1.5 mg/L) was most responsive exhibiting regeneration frequency of 28.1 per cent which resulted in maximum regeneration of green plantlets and only 5.21 per cent of albinos. Individual plantlets were separated and immersed in liquid MS medium augmented with NAA (0.5-1.0 mg/L) and BAP (0.5-1.0 mg/L). Maximum rooting was observed in MS medium with NAA (0.5 mg/L) and BAP (1.0 mg/L). The survival rate of in-vitro raised plants was 51.51 per cent. Of these surviving plants, 21 plants were observed to have the sterility percentage above 50 percent and hence can be considered as the doubled haploid plants. Plant DH8 is susceptible and DH20 is heterozygous for gene Xa21. Two plants are susceptible for gene xa13

Mango is the fifth most important fruit crop in the world in terms of production with an increasing demand for high-quality plant material for new plantings. Compared to other fruit tree crops, vegetative propagation in mango is slow and allows only a relatively limited production of plant material. To date, efficient procedures for in vitro establishment and micropropagation are not yet available in mango. This work aims at filling this gap. Germination of mango seeds in vitro, compared with ex vitro conditions, significantly increased the germination rate for the monoembryonic genotype tested ('Irwin'). In vitro germination also increased the number of developed embryos for the two polyembryonic genotypes analysed, 'Ataulfo' and 'Gomera-4'. Regarding the use of shoot tips for in vitro establishment and micropropagation, our results confirmed that this explant is not adequate for this purpose. We report for the first time the use of cotyledonary nodes as initial explants in mango. Axillary shoots were obtained in all the genotypes tested ('Ataulfo', 'Sabre', 'Gomera-4', 'Irwin' and 'Keitt'), although the regeneration rate was highly genotype-dependent. Thidiazuron induced high-frequency regeneration patterns. The best results were observed with 'Keitt'. When 3.0 mg l−1 thidiazuron was added to the medium, a 63.15% regeneration rate was reached and about 4 shoots per regenerating explant were obtained. Subsequently, microshoots excised from the cotyledonary nodes were successfully rooted in vitro and acclimatized to ex vitro conditions. Our results show that the use of cotyledonary nodes is efficient for mango mass propagation and, consequently, represents a qualitative advance for in vitro propagation of this recalcitrant species. [ABSTRACT FROM AUTHOR]

Bupleurum chinense DC is an important medicinal plant with many active ingredients that are used for the treatment of different types of diseases and valued in pharmaceutical markets. In vitro shoot regeneration can efficiently contribute to the improvement of B. chinense. In the present study, we investigated the effects of the explant type and plant growth regulators (PGRs) on embryogenic callus induction and plant regeneration in B. chinense. Our investigation demonstrated that 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) combined with 1 mg/L thidiazuron (TDZ) played a major role in promoting callus induction from leaf, hypocotyl and stem 2 explants, whereas the most effective treatment for stem 1 callus formation was Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-D, 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kin). The highest shoot regeneration rate (57.14%) was obtained from hypocotyl-induced calli in MS medium with 0.5 mg/L Kin after 12 weeks of cultivation. This regeneration protocol can be used in large-scale cultivation and may be useful for future genetic modifications of B. chinense. [ABSTRACT FROM AUTHOR]

Plants, unlike animals, possess a unique developmental plasticity, that allows them to adapt to changing environmental conditions. A fundamental aspect of this plasticity is their ability to undergo postembryonic de novo organogenesis. This requires the presence of regulators that trigger and mediate specific spatiotemporal changes in developmental programs. The phytohormone cytokinin has been known as a principal regulator of plant development for more than six decades. In de novo shoot organogenesis and in vitro shoot regeneration, cytokinins are the prime candidates for the signal that determines shoot identity. Both processes of de novo shoot apical meristem development are accompanied by changes in gene expression, cell fate reprogramming, and the switching-on of the shoot-specific homeodomain regulator, WUSCHEL. Current understanding about the role of cytokinins in the shoot regeneration will be discussed.

Auxin plays a critical role in organogenesis in plants. The classical auxin signaling pathway holds that auxin initiates downstream signal transduction by degrading Aux/IAA transcription repressors that interact with ARF transcription factors. In this study, 23 MoIAA genes were identified in the drumstick tree genome. All MoIAA genes were located within five subfamilies based on phylogenetic evolution analysis; the gene characteristics and promoter cis-elements were also analyzed. The protein interaction network between the MoIAAs with MoARFs was complex. The MoIAA gene family responded positively to NAA treatment, exhibiting different patterns and degrees, notably for MoIAA1, MoIAA7 and MoIAA13. The three genes expressed and functioned in the nucleus; only the intact encoding protein of MoIAA13 exhibited transcriptional activation activity. The shoot regeneration capacity in the 35S::MoIAA13-OE transgenic line was considerably lower than in the wild type. These results establish a foundation for further research on MoIAA gene function and provide useful information for improved tissue culture efficiency and molecular breeding of M. oleifera. [ABSTRACT FROM AUTHOR]

Rhododendron dauricum L., a semi-evergreen shrub growing in the south of Western Siberia, Mongolia, and China, is a valuable genetic resource for breeding of new rhododendron cultivars with winter-hardiness, polymorphism of corolla color, and the terms of flowering. To create efficient systems of micropropagation, floral explants consisting of isolated pistil with pedicel were used. Contrary to the other types of vegetative explant, floral explants have a number of advantages, such as a low level of contamination. While micropropagation protocols based on floral explants have been developed for evergreen rhododendrons, they were found not to initiate the regeneration from floral explants of semi-evergreen R. dauricum. As a result, a new optimized two-step procedure for direct shoot regeneration from R. dauricum floral explants was presented: (i) preculture of floral explants on hormone-free Anderson medium for 4 d; (ii) induction of shoot regeneration under 1.0 µM thidiazuron (TDZ) and 2.5 µM zeatin. The detailed histological analysis of morphogenesis pathways revealed that regeneration from floral explants induced by TDZ occurs along the direct pathway of morphogenesis based on two parallel processes: dedifferentiation of epidermal cells with subsequent acquisition of competence to morphogenesis, and activation and development of already existing meristems. According to the present study, this is the first histological study of floral reversion in semi-evergreen R. dauricum. [ABSTRACT FROM AUTHOR]

Vigna umbellata is an unexplored and underutilized grain legume of high nutritional values. This crop can be improved for several agronomic attributes via genetic engineering. The presented study emphasizes the first-time standardization of parameters affecting regeneration and transformation using shoot apices. Various parameters comprise concentration of growth regulators, age of explants, explant's priming with 6-benzylaminopurine (BAP), genotype effect, modulation of explant's compatibility towards Agrobacterium infection via mechanical wounding, and assessment of the type and concentration of antioxidants (silver nitrate and thiourea). Out of various variables, the 2-d-old apices primed with 15 μM BAP formed an average of 10.4 shoots per explant with 100% regeneration frequency on shoot regeneration medium [SRM2: MSB5V (MS – Murashige and Skoog salts and B5 – Gamborg's vitamins), 2.5 μM BAP and 5.0 mg L−1 silver nitrate]. Scanning electron microscopy revealed morphology of explant's meristem for suitability to Agrobacterium infection. Wounded explants pierced with copper metal particles did not affect the regeneration compared to longitudinal incisions or pierced with a needle. A transformation study was carried out using Agrobacterium tumefaciens (strain EHA105 harboring pCAMBIA2301 vector). The stable expression of uidA gene in tissues of transgenics (T0) and seedlings (T1) evidenced the success of the transformation process. Transgenic plants (T0) were characterized by polymerase chain reaction and Southern hybridization and progeny plants via reverse transcriptase–polymerase chain reaction. The highest transformation efficiency (13%) was achieved via copper metal particles comparative to other methods of wounding in a short span of 6 to 7 wk from initiation of cultures to transplantation. [ABSTRACT FROM AUTHOR]

In this work, axillary shoot regenerations were investigated from nodal explants of Hygrophila polysperma (Roxb.) T. Anderson in MS medium (Murashige and Skoog) including different combinations of Gibberellic Acid (GA3) and Naphthalene Acetic Acid (NAA). Nodal explants of H. polysperma were used in propagation studies. The 100% regeneration frequency was determined in MS plus 0.25 and 0.50 mg/L GA3 + 0.15 mg/L NAA. When the shoot numbers were examined, the best nutrient medium was determined as 0.25 mg/L GA3 + 0.15 mg/L NAA (14.72 shoots/explant). The best result in shoot length was reached in MS plus 0.50 mg/L GA3 + 0.15 mg/L NAA (1.91 cm). Then the longest shoot was determined as 1.79 cm in nutrient media plus 0.25 mg/L GA3 + 0.15 mg/L NAA. Longer shoots were obtained in GA3+NAA nutrient media according to control. When the results were examined, it was determined that the number and length of shoots decreased as the amount of GA3+NAA used in the culture medium increased. Regenerated shoots were rooted in MS media including 0.25 mg/L Indole-3-Acetic Acid and successfully acclimatized to aquarium conditions. As a result, multiple and rapid productions of H. polysperma under tissue culture conditions were achieved. [ABSTRACT FROM AUTHOR]

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Date palm is propagated by the offshoots, the number of which is limited. Therefore, date palms may be produced through the use of tissue culture of shoot tips and axillary shoot meristems. Date palm (Phoenix dactylifera L.) micropropagation still faces many problems, such as reduced growth and development of callus, low multiplication efficiency, and low rooting rate. Here, we established an efficient plant-regeneration system for (Phoenix dactylifera L.) cv. Ashgar by tissue culture. Murashige and Skoog medium containing 6-benzyladenine (BA) (2.0 and 5.0 mg L−1) and a-naphthaleneacetic acid (NAA) or phenylacetic acid (PAA) (0.05–2.00 mg L−1) was used to initiate shoot formation from callus tissues. The maximum number of shoots per jar was produced on a medium containing 5.0 mgL−1 BA and 0.5 mg L−1 PAA, while the medium supplemented with 2.0 mgL−1 BA in combination with 0.05 m L−1 PAA gave the highest callus induction (+++). A decrease in browning percentage was observed in the tissue-cultured media supplied with BA in combination with NAA or PAA compared to those provided with BA alone. In comparison with other treatments, the total amount of phenolic compounds was significantly reduced to 0.45 mg g−1 in buds cultured in the media supplemented with 5.0 mg L−1 BA and 0.5 mg L−1 PAA. The genetic stability of this protocol was confirmed by DNA-based fingerprinting technique RAPD. The RAPD banding patterns showed no variation among the tissue culture-derived plants tested. The in vitro micropropagation protocol reported herein could be served production of genetically stable date palm plants. [ABSTRACT FROM AUTHOR]

Although the division of the pericycle cells initiates both lateral root development and root-derived callus formation, these developmental processes are affected differently in the strigolactone and karrikin/KARRIKIN INSENSITIVE 2 (KAI2) ligand signalling mutant more axillary growth 2 (max2). Whereas max2 produces more lateral roots than the wild type, it is defective in the regeneration of shoots from root explants. We suggest that the decreased shoot regeneration of max2 originates from delayed formation of callus primordium, yielding less callus material to regenerate shoots. Indeed, when incubated on callus-inducing medium, the pericycle cell division was reduced in max2 and the early gene expression varied when compared with the wild type, as determined by a transcriptomics analysis. Furthermore, the expression of the LATERAL ORGAN BOUNDARIES DOMAIN genes and of callus-induction genes was modified in correlation with the max2 phenotype, suggesting a role for MAX2 in the regulation of the interplay between cytokinin, auxin, and light signalling in callus initiation. Additionally, we found that the in vitro shoot regeneration phenotype of max2 might be caused by a defect in KAI2, rather than in DWARF14, signalling. Nevertheless, the shoot regeneration assays revealed that the strigolactone biosynthesis mutants max3 and max4 also play a minor role. [ABSTRACT FROM AUTHOR]

In vitro plant cell and tissue cultures are potent tools to propagating germplasm resources in conserving and managing plant genetic resources. A reliable micropropagation protocol was developed for efficient callus proliferation and direct and indirect shoot regeneration of Meseika (Haplophyllum tuberculatum). With the applied sterilization procedure, immature, unopened H. tuberculatum seed pods can be identified as a potent explant with high viability and low contamination percentage. Multiple shoots were regenerated from leaf and stem explants through direct organogenesis on Murashige and Skoog's (MS) + 3% sucrose medium amended with BAP. Indirect regeneration of several shoots was achieved on 1/2 MS + 1% sucrose media amended with 2 and 4 mg/l BAP. An efficient callus proliferation from both explants can be achieved by supplementing the MS media with NAA and BAP. All the cultures were incubated in a controlled growth chamber under 5/19 h light/dark photoperiod, temperature (25 ± 2 ℃), and 60% relative humidity (RH).10 ISSR (Inter Simple Sequence Repeat) markers were screened to test the genetic fidelity of regenerated H. tuberculatum shoots. Callus development was observed after 15 days and shoot regeneration was occurred after 30 days after callus initiation. 10 ISSR primers produced a total of 39 clear, distinct amplicons. 75, 60, 40, and 16% polymorphism percentages were recorded by the ISSR primer 11, 7, 5, and 4, respectively. The developed micropropagation protocol is appropriate for rapid in-vitro multiplication of H. tuberculatum shoots and callus. [ABSTRACT FROM AUTHOR]

The establishment of an efficient protocol for in vitro growth and regeneration of kohlrabi (Brassica oleracea var. gongylodes) allowed us to closely examine the phytohormone profiles of kohlrabi seedlings at four growth stages (T1–T4), additionally including the effects of cytokinins (CKs)—trans-zeatin (transZ) and thidiazuron (TDZ)—and high sucrose concentrations (6% and 9%). Resulting phytohormone profiles showed complex time-course patterns. At the T2 stage of control kohlrabi plantlets (with two emerged true leaves), levels of endogenous CK free bases and gibberellin GA20 increased, while increases in jasmonic acid (JA), JA-isoleucine (JA-Ile), indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) peaked later, at T3. At the same time, the content of most of the analyzed IAA metabolites decreased. Supplementing growth media with CK induced de novo formation of shoots, while both CK and sucrose treatments caused important changes in most of the phytohormone groups at each developmental stage, compared to control. Principal component analysis (PCA) showed that sucrose treatment, especially at 9%, had a stronger effect on the content of endogenous hormones than CK treatments. Correlation analysis showed that the dynamic balance between the levels of certain bioactive phytohormone forms and some of their metabolites could be lost or reversed at particular growth stages and under certain CK or sucrose treatments, with correlation values changing between strongly positive and strongly negative. Our results indicate that the kohlrabi phytohormonome is a highly dynamic system that changes greatly along the developmental time scale and also during de novo shoot formation, depending on exogenous factors such as the presence of growth regulators and different sucrose concentrations in the growth media, and that it interacts intensively with these factors to facilitate certain responses. [ABSTRACT FROM AUTHOR]

Aeschynanthus pulcher (Blume) G. Don, the "lipstick plant" is a prized ornamental plant with distinctive flowers. Here, we introduce a novel in vitro regeneration method for A. pulcher using leaf explants and an optimized combination of phytohormone plant growth regulators (PGRs). The optimal conditions for shoot regeneration included 1 mg L−1 polyvinyl pyrrolidone (PVP) plus 3 mg L−1 thidiazuron (TDZ), inducing a response rate of 82.4% and a shoot/explant ratio of 38.6. When the Murashige and Skoog (MS) medium contained indole-3-butyric acid (IBA) alone, leaves first differentiated into adventitious roots and then adventitious shoots. Leaves cultured on MS medium containing 1 g L−1 PVP, 3 mg L−1 TDZ, 5 mg L−1 casein, and 0.1 mg L−1 α-naphthaleneacetic acid (NAA) for 30 d exhibited the highest embryogenic callus (EC) induction rate (95.6%). The optimal shoot proliferation coefficient (21.5) was obtained when shoots derived from EC were cultured on the same medium as that used for EC induction for 5 weeks. The most effective medium for rooting of elongated shoots was MS medium containing 1 g L−1 PVP, 5 mg L−1 casein, 3 mg L−1 6-benzyladenine (BA), and 0.1 mg L−1 NAA, and the number of roots reached 18.8. The regenerated plants grown in a greenhouse had 100% survival following one week of hardening. Overall, our effective and efficient propagation method should result in shortened culture periods and reduced production costs, allowing for the future selective breeding and genetic improvement of A. pulcher. [ABSTRACT FROM AUTHOR]

Callus induction and plant regeneration are important steps of in vitro plant breeding of ornamental plants. In this study, the effects of different combinations of plant growth regulators (PGRs), promoters, and minerals on callus induction and plant regeneration in different carnation cultivars were studied in a completely randomized design with three replications. For callus induction, 16 different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and casein hydrolysate (CH) were studied using in vitro leaf explants. The Murashige and Skoog (MS) medium supplemented with 0.2 mg·dm−3 of 2,4-D and 200 mg·dm−3 of CH showed the highest frequency of callus induction. Among the cultivars, ‘Noblesse’ showed the highest rate of callus induction (91.67%). Regarding regeneration, BA, NAA, silver nitrate (AgNO3), and adenine hemisulfate (As) were used in ten different combinations. The ‘Cameron’, ‘Tabasco’, and ‘Noblesse’ cultivars with 95.24% regeneration percentage showed the highest rate of plant regeneration. Generally, in most cultivars, the highest regeneration rate and shoot number per explant were found in the MS medium supplemented with 3 mg·dm−3 of BA, 0.6 mg·dm−3 of NAA, 5 mg·dm−3 of AgNO3, and 40 mg·dm−3 of As. According to the results, the highest regeneration frequency was obtained when 40 mg·dm−3 of As was added to the medium. Finally, the flow cytometry analysis indicated that there were no significant differences between in vitro regenerated and control plants in terms of DNA ratios.

Nodal and petiole explants were employed to study the direct and indirect regeneration from Pelargonium odoratissimum in vitro. Direct shoots regeneration of nodal segments was tried in MS medium containing 1 and 2 mg l-1 BAP and 0.5 mg l-1 IBA. The highest mean shoots number and the greatest shoots per explant number were obtained in the medium containing 2 mg l-1 BAP. Nodal segments were the source of petiole explants and the resulting petioles were cultured in ½ MS medium supplemented by 1, 1.5, 2 and 4.5 mg l-1 BAP enriched with 0.1, 1 and 1.5 mg l-1 NAA. With the petiole explants, the lowest browning percentage, the highest callus induction and also, the top number of shoots per explant were recorded in 2 mg l-1 BAP + 0.1 mg l-1 NAA medium. The medium supplemented with 0.2 mg l-1 NAA exhibited the desired effect on rooting percentage and mean root number and length. The rooted young plants were transferred to the pots containing peat-moss and perlite (1:1) and the acclimatization was successful since, more than 90 % of plants adapted-well in the greenhouse conditions. This in-vitro propagation methodology would be advisable to the plant production systems and to whom wish to produce the clonal homogenous plants for the commercial ideas and for the detailed molecular studies.

Summary: Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR‐RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem‐cell regulators. Yet whether and how stem cell‐regulating CLE peptides are implicated in plant regeneration remains unknown.By CRISPR/Cas9‐induced loss‐of‐function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1–CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana.CLE1–CLE7 are induced by callus‐induction medium and dynamically expressed in pluripotent callus. Exogenously‐applied CLE1–CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1–CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1–CLE7‐mediated shoot regeneration suppression is impaired in loss‐of‐function mutants of callus‐expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1–CLE7‐mediated shoot regeneration signaling. CLE1–CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell‐promoting transcription factor known as a principal regulator of plant regeneration.Our results indicate that functionally‐redundant CLE1–CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot‐regeneration capacity, establishing the mechanistic basis for CLE1–CLE7‐mediated shoot regeneration and a novel role for CLE peptides in hormone‐dependent developmental plasticity. [ABSTRACT FROM AUTHOR]

Reversible protein phosphorylation is a post-translational modification involved in virtually all plant processes, as it mediates protein activity and signal transduction. Here, we probe dynamic protein phosphorylation during de novo shoot organogenesis in Arabidopsis thaliana. We find that application of three kinase inhibitors in various time intervals has different effects on root explants. Short exposures to the putative histidine (His) kinase inhibitor TCSA during the initial days on shoot induction medium (SIM) are detrimental for regeneration in seven natural accessions. Investigation of cytokinin signaling mutants, as well as reporter lines for hormone responses and shoot markers, suggests that TCSA impedes cytokinin signal transduction via AHK3, AHK4, AHP3, and AHP5. A mass spectrometry-based phosphoproteome analysis further reveals profound deregulation of Ser/Thr/Tyr phosphoproteins regulating protein modification, transcription, vesicle trafficking, organ morphogenesis, and cation transport. Among TCSA-responsive factors are prior candidates with a role in shoot apical meristem patterning, such as AGO1, BAM1, PLL5, FIP37, TOP1ALPHA, and RBR1, as well as proteins involved in polar auxin transport (e.g., PIN1) and brassinosteroid signaling (e.g., BIN2). Putative novel regeneration determinants regulated by TCSA include RD2, AT1G52780, PVA11, and AVT1C, while NAIP2, OPS, ARR1, QKY, and aquaporins exhibit differential phospholevels on control SIM. LC-MS/MS data are available via ProteomeXchange with identifier PXD030754. [ABSTRACT FROM AUTHOR]