Shoot Regeneration Capacity Research Articles

Exploring an economic and highly efficient genetic transformation and genome-editing system for radish through developmental regulators and visible reporter.

Radish (Raphanus sativus L.) is one of the most important root vegetable crops worldwide. However, gene function exploration and germplasm innovation still face tremendous challenges due to its extremely low transformation efficiency. Here, an economic and highly efficient genetic transformation method for radish was explored by Agrobacterium rhizogenes-mediated transformation with the help of combining special developmental regulator (DR) genes and the visual identification reporter. Firstly, the RUBY gene, a betalain biosynthesis system, could result in a visual red-violet color used as a convenient and effective reporter for monitoring transgenic hairy roots screening of radish. However, the hairy roots-to-shoots conversion system of radish still stands as a barrier to the obtainment of whole transgenic plants, although different hormone combinations and various culture conditions were tried. Following, two DR genes including Wuschel2 (Wus2) and isopentenyl transferase (ipt), as well as their combination Wus2-ipt were introduced for the shoot regeneration capacity improvement. The results showed that the transgenic shoots could be directly generated without externally supplying any hormones in the presence of a Wus2-ipt combination. Then, Wus2-ipt along with the RUBY reporter was employed to establish an efficient genetic transformation system of radish. Moreover, this system was applied in generating gene-edited radish plants and the phytoene desaturase (RsPDS) gene was effectively knockout through albino phenotype observation and sequencing analysis. These findings have the potential to be widely applied in genetic transformation and genome-editing genetic improvement of other vegetable species.

Effects of plant hormones and genotypes on anther culture response of safflower (Carthamus tinctorius L.)

Safflower (Carthamus tinctorius L.) belongs to the composite family with various uses, from the stem to the seeds. In vitro flowering and embryo rescue are common techniques for improving safflower hybrid fertility by overcoming breeding constraints. However, anther culture expedites the production of superior types. The aim of the study was to evaluate the effect of plant hormones and genotypes on anther culture responses and to develop a suitable protocol. In both safflower genotypes, callus, shoot, and root formation were investigated using ten factorial hormone treatments. To promote callus induction and shoot regeneration, variable doses of Thidiazuron (TDZ) at 1 mg/l indole butyric acid (IBA) and variable benzylaminopurine (BAP) at 0.5 mg/l NAA were added to the MS medium, respectively. ½ MS media with varying IBA concentrations at a fixed 0.5 mg/l NAA were used for root regeneration. Results showed that Turkan was superior in all aspects of callus induction, and the highest degree of callus formation (46 %) was observed at 0.5 mg/l TDZ. On the other hand, the local safflower performed better for shoot and root regeneration. The highest shoot regeneration capacity (20 %) was shown at 2.0 mg/l BAP, while shoot rooting was reached (21 %) at 1.0 mg/l IBA of culture media. Despite successful in vitro regeneration, acclimated plantlets did not survive in the glasshouse. In this study, we demonstrated the higher capability for both shoot and root regeneration of calli produced from local safflower anthers. More effort is required to improve shoot and root development for both genotypes.

Comparative transcriptome and functional analyses provide insights into the key factors regulating shoot regeneration in highbush blueberry.

Establishing an efficient plant regeneration system is a crucial prerequisite for genetic engineering technology in plants. However, the regeneration rate exhibits considerable variability among genotypes, and the key factors underlying shoot regeneration capacity remain largely elusive. Blueberry leaf explants cultured on a medium rich in cytokinins exhibit direct shoot organogenesis without prominent callus formation, which holds promise for expediting genetic transformation while minimizing somatic mutations during culture. The objective of this study is to unravel the molecular and genetic determinants that govern cultivar-specific shoot regeneration potential in highbush blueberry (Vaccinium corymbosum L.). We conducted comparative transcriptome analysis using two highbush blueberry genotypes: 'Blue Muffin' ('BM') displaying a high regeneration rate (>80%) and 'O'Neal' ('ON') exhibiting a low regeneration rate (<10%). The findings revealed differential expression of numerous auxin-related genes; notably, 'BM' exhibited higher expression of auxin signaling genes compared to 'ON'. Among blueberry orthologs of transcription factors involved in meristem formation in Arabidopsis, expression of VcENHANCER OF SHOOT REGENERATION (VcESR), VcWUSCHEL (VcWUS), and VcCUP-SHAPED COTYLEDON 2.1 were significantly higher in 'BM' relative to 'ON'. Exogenous application of auxin promoted regeneration, as well as VcESR and VcWUS expression, whereas inhibition of auxin biosynthesis yielded the opposite effects. Overexpression of VcESR in 'BM' promoted shoot regeneration under phytohormone-free conditions by activating the expression of cytokinin- and auxin-related genes. These findings provide new insights into the molecular mechanisms underlying blueberry regeneration and have practical implications for enhancing plant regeneration and transformation techniques.

MicroRNA396 negatively regulates shoot regeneration in tomato.

Numerous studies have been dedicated to genetically engineering crops to enhance their yield and quality. One of the key requirements for generating genetically modified plants is the reprogramming of cell fate. However, the efficiency of shoot regeneration during this process is highly dependent on genotypes, and the underlying molecular mechanisms remain poorly understood. Here, we identified microRNA396 (miR396) as a negative regulator of shoot regeneration in tomato. By selecting two genotypes with contrasting shoot regeneration efficiencies and analyzing their transcriptome profiles, we found that miR396 and its target transcripts, which encode GROWTH-REGULATING FACTORs (GRFs), exhibit differential abundance between high- and low-efficiency genotypes. Suppression of miR396 functions significantly improved shoot regeneration rates along with increased expression of GRFs in transformed T0 explants, suggesting that miR396 is a key molecule involved in the determination of regeneration efficiency. Notably, we also showed that co-expression of a miR396 suppressor with the gene-editing tool can be employed to generate gene-edited plants in the genotype with a low capacity for shoot regeneration. Our findings show the critical role of miR396 as a molecular barrier to shoot regeneration in tomato and suggest that regeneration efficiency can be improved by blocking this single microRNA.

Regeneration of roots and shoots as a propagation strategy in Eugenia candolleana DC. (Myrtaceae) seeds

Abstract: Seeds of the genus Eugenia have high regenerative capacity, producing roots and shoots from seeds with reduced cotyledon matter. In addition, seeds of this genus regenerate new roots and shoots when the first roots and shoots are eliminated, and this characteristic is maintained even when the seed reserves are reduced by up to half. The aim of this study was to analyze the limits of new root and shoot regenerative capacity in whole and fractionated Eugenia candolleana seeds at different maturity stages. The regenerative capacity of seeds stored for six months was also evaluated. The seeds were sown; and when the first roots and shoots were produced, they were eliminated, simulating herbivory conditions. The results showed that the seed not only has high regenerative capacity at different maturity stages, but also that it maintains root regeneration when the seed is reduced by half, even after the storage period. Such information may indicate that seeds of the Eugenia genus can await the next crop season by regenerating several times as a way to withstand or tolerate predation. This ability can be understood as a propagation strategy of the species.

WUSCHEL controls genotype-dependent shoot regeneration capacity in potato.

Plant cells can reprogram their fate. The combinatorial actions of auxin and cytokinin dedifferentiate somatic cells to regenerate organs, which can develop into individual plants. As transgenic plants can be generated from genetically modified somatic cells through these processes, cell fate transition is an unavoidable step in crop genetic engineering. However, regeneration capacity closely depends on the genotype, and the molecular events underlying these variances remain elusive. In the present study, we demonstrated that WUSCHEL (WUS) -a homeodomain transcription factor-determines regeneration capacity in different potato (Solanum tuberosum) genotypes. Comparative analysis of shoot regeneration efficiency and expression of genes related to cell fate transition revealed that WUS expression coincided with regeneration rate in different potato genotypes. Moreover, in a high-efficiency genotype, WUS silencing suppressed shoot regeneration. Meanwhile, in a low-efficiency genotype, regeneration could be enhanced through the supplementation of a different type of cytokinin that promoted WUS expression. Computational modeling of cytokinin receptor-ligand interactions suggested that the docking pose of cytokinins mediated by hydrogen bonding with the core residues may be pivotal for WUS expression and shoot regeneration in potatoes. Furthermore, our whole genome sequencing analysis revealed core sequence variations in the WUS promoters that differentiate low- and high-efficiency genotypes. The present study revealed that cytokinin responses, particularly WUS expression, determine shoot regeneration efficiency in different potato genotypes.

Efficient organogenesis and taxifolin production system from mature zygotic embryos and needles in larch

The deciduous conifer larch has been widely distributed around the world, which is a high-quality wood species and is also used to extract industrial raw materials and medicines. In this study, we developed an organogenesis protocol for <italic>Larix olgensis</italic> from both mature zygotic embryos and needles, and analyzed the content of taxifolin in different tissues. The highest callus induction (96.8%) from mature zygotic embryo was got in the Douglas-fir Cotyledon Revised (DCR) medium augmented with 2.0 mg/L 6-Benzylaminopurine (6-BA) and 0.2 mg/L α-Naphthaleneacetic acid (NAA), while from needles the highest callus induction (92.03%) was got in the Murashige and Skoog (MS) medium augmented with 3 mg/L 6-BA and 0.3 mg/L NAA. The best shoot regeneration capacity from zygotic embryo-derived calli (83.3%) was obtained in DCR medium augmented with 1.0 mg/L 6-BA and 0.01 mg/L NAA, and needle-derived calli were 77.3%. The shoots achieved the highest elongation (75.6%) in the DCR medium supplemented with 0.5 mg/L 6-BA, 0.05 mg/L NAA and 2g/L activated charcoal (AC). The rooting rate was 62.8% in DCR medium augmented with 3 mg/L Indole-3-butyric acid (IBA) and 100 mg/L phloroglucinol (PG). The accumulation of the taxifolin in elongation shoots and lignified elongation shoots have greatly improved along with the development process, were 28.6 µg/g, and 53 µg/g respectively. The content of the taxifolin in callus was 1.99-5.26 µg/g, adventitious shoots were 4.8µg/g, and adventitious roots were 2.86 µg/g. To date, we reported an efficient organogenesis and taxifolin production protocol in larch for the first time.

Genome-Wide Identification and Expression Analysis of the Aux/IAA Gene Family of the Drumstick Tree (Moringa oleifera Lam.) Reveals Regulatory Effects on Shoot Regeneration

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.

A group of CLE peptides regulates de novo shoot regeneration in Arabidopsis thaliana.

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.

Transcriptome analysis reveals the effects of strigolactone on shoot regeneration of apple

We have demonstrated that strigolactone inhibitor, Tis108, could be used to improve shoot regeneration of apple, and provided insights into the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation. Lack of an efficient transformation system largely stagnated the application of transgenic and CRISPR technology in apple rootstock. High shoot regeneration ability is an important basis for establishing an effective transformation system. In this study, we first demonstrated the inhibitory effects of strigolactones on the adventitious shoot formation of apple rootstock M26. Next, we successfully verified that strigolactone-biosynthesis inhibitor, Tis108, could be used to improve the shoot regeneration of woody plants. Our results also suggest strigolactone-biosynthesis gene, MdCCD7, can be a target gene for biotechnological improvements of shoot regeneration capacity. Furthermore, we have employed transcriptome analysis to reveal the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation. Differentially expressed genes associated with photosynthesis, secondary growth, and organ development were identified. WGCNA suggests SLs might affect shoot regeneration through interaction with other hormones, especially, auxin, cytokinin, and ethylene. We were able to identify important candidate genes mediating the cross-talk between strigolactone and other hormones during the process of adventitious shoot formation. Overall, our findings not only propose a useful chemical for improving shoot regeneration in practice but also provide insights into the molecular mechanism of strigolactone-mediated inhibition of adventitious shoot formation.

HY5 inhibits in vitro shoot stem cell niches initiation via directly repressing pluripotency and cytokinin pathways.

The efficiency of plant regeneration from explants is influenced by phytohormones and environmental conditions. Light has a particularly marked effect on in vitro shoot regeneration, and some light signaling factors are involved in shoot regeneration, while the underlying molecular mechanism remains elusive. Here, ELONGATED HYPOCOTYL5 (HY5), as the key transcription factor of light signaling, was found to inhibit shoot regeneration under a range of light conditions. The heightened shoot regeneration capacity of the hy5-215 mutant was less marked in the dark than in the light, showing that HY5-mediated inhibition of shoot regeneration is partly light dependent. The co-localization of WUSCHEL (WUS) and CLAVATA3 (CLV3) expressions was found to coincide with the initiation of stem cell niches in root explants during shoot regeneration. HY5 could directly repress CLV3 and WUS expression by binding to their respective promoters. In parallel, HY5 indirectly repressed CLV3 and WUS by binding to the ARABIDOPSIS RESPONSE REGULATOR12 (ARR12) promoter. The resulting dual regulation exerted by HY5 on WUS and CLV3 impeded the initiation of shoot stem cell niches. A HY5-mediated inhibitory pathway was identified that links cytokinin signaling and the pluripotency pathway during shoot regeneration.

Warm Temperature Promotes Shoot Regeneration in Arabidopsis thaliana.

Many plants are able to regenerate upon cutting, and this process can be enhanced in vitro by incubating explants on hormone-supplemented media. While such protocols have been used for decades, little is known about the molecular details of how incubation conditions influence their efficiency. In this study, we find that warm temperature promotes both callus formation and shoot regeneration in Arabidopsis thaliana. We show that such an increase in shoot regenerative capacity at higher temperatures correlates with the enhanced expression of several regeneration-associated genes, such as CUP-SHAPED COTYLEDON 1 (CUC1) encoding a transcription factor involved in shoot meristem formation and YUCCAs (YUCs) encoding auxin biosynthesis enzymes. ChIP-sequencing analyses further reveal that histone variant H2A.Z is enriched on these loci at 17°C, while its occupancy is reduced by an increase in ambient temperature to 27°C. Moreover, we provide genetic evidence to demonstrate that H2A.Z acts as a repressor of de novo shoot organogenesis since H2A.Z-depleted mutants display enhanced shoot regeneration. This study thus uncovers a new chromatin-based mechanism that influences hormone-induced regeneration and additionally highlights incubation temperature as a key parameter for optimizing in vitro tissue culture.

Combination of 6-Benzylaminopurine and Thidiazuron Promotes Highly Efficient Shoot Regeneration from Cotyledonary Node of Mature Peanut (Arachis hypogaea L.) Cultivars

Efficient in vitro plantlet regeneration is an important step to successfully transform genes for the improvement of agronomic traits. A combination of 6-benzylaminopurine (BAP) and thidiazuron (TDZ) plant growth regulators was applied to evaluate shoot regeneration capacity whereas α-naphthalene acetic acid (NAA) combination with 6-benzylaminopurine (BAP), and 2, 4-dichlorophenoxyacetic acid (2, 4-D) with 6-benzylaminopurine were tested to optimize root induction for two peanut cultivars. The result showed combination (BAP with TDZ) was found to be effective in promoting shoot. The highest shoot regeneration frequency (93%) was obtained on a medium supplemented with 4 mg/L BAP and 0.5 mg/L TDZ while an average regeneration frequency (87%) was achieved in a medium containing combinations of 2 mg/L BAP with 1 mg/L TDZ. The shooting rate increased for both cultivars as the concentrations of BAP increased and TDZ decreased. The highest rooting rate (93%) was obtained on a medium supplemented with 3.5 mg/L NAA with 2.5 mg/L BAP for both cultivars. The rooting rate increased as the concentration of auxin to cytokinin ratio increased. The maximum rooting rate (83%) was obtained on MS medium supplemented with 0.3 mg/L 2, 4-D with 0.2 mg/L BAP for the cultivar N3. The result indicated that BAP with NAA was much better than BAP with 2, 4-D in rooting rate. Thus, the protocol developed was genotype independent and effective for peanut tissue culture.

Indirect in vitro Regeneration of the Medicinal Plant, Aspilia africana, and Histological Assessment at Different Developmental Stages.

The medicinal plant, Aspilia africana, has been traditionally used in several African countries to treat many diseases such as tuberculosis, cough, inflammation, malaria, osteoporosis, and diabetes. In this study, we developed a protocol for in vitro propagation of A. africana using indirect shoot organogenesis from leaf and root explants of in vitro-grown seedlings and assessed the tissues at different developmental stages. The highest callus induction (91.9 ± 2.96%) from leaf explants was in the Murashige and Skoog (MS) medium augmented with 1.0 mg/L 6-Benzylaminopurine (BAP) and 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) while from root explants, the highest callus induction (92.6 ± 2.80%) was in the same plant tissue culture medium augmented with 0.5 mg/L BAP and 1.0 mg/L 2,4-D. The best shoot regeneration capacity from leaf-derived calli (i.e., 80.0 ± 6.23% regeneration percentage and 12.0 ± 6.23 shoots per callus) was obtained in medium augmented with 1.0 mg/L BAP and 0.05 mg/L α-Naphthaleneacetic acid (NAA); the best regeneration capacity for root-derived calli (i.e., 86.7 ± 6.24% shoot regeneration percentage and 14.7 ± 1.11 shoots per callus) was obtained in the MS medium augmented with 1.0 mg/L BAP, 0.05 mg/L NAA, and 0.1 mg/L Thidiazuron (TDZ). Regenerated plantlets developed a robust root system in 1/2 MS medium augmented with 0.1 mg/L NAA and had a survival rate of 93.6% at acclimatization. The in vitro regenerated stem tissue was fully differentiated, while the young leaf tissue consisted of largely unorganized and poorly differentiated cells with large intercellular airspaces typical of in vitro leaf tissues. Our study established a protocol for the indirect regeneration of A. africana and offers a basis for its domestication, large-scale multiplication, and germplasm preservation. To the best of our knowledge, this is the first study to develop an indirect regeneration protocol for A. africana and conduct anatomical assessment through the different stages of development from callus to a fully developed plantlet.

Casein Stabilized Metal and Metal Oxide Nanoparticles for the Efficient In Vitro Culturing of Scoparia dulcis L.

Unique physicochemical properties of nanoparticles make them a novel candidate in agriculture and related areas. In the present work, we have studied the effect of casein stabilized metal and metal oxide nanoparticles, viz. AgNPs, AuNPs, and CuONPs, on in vitro propagation of Scoparia dulcis L. We have examined the effect of these nanoparticles on the callus induction, shoot regeneration, and root regeneration capacity. It was observed that Ag, Au, and CuO nanoparticles had interacted with the tissues differently and produced calluses with difference in color, nature, and texture. Nanoparticles also affected shoot regeneration, root regeneration, phenolic compound production, and chlorophyll content. Explants showed a very good response when treated with CuONPs. The formed callus underwent shoot and root regeneration without changing the medium

Methods for vegetative propagation of wild enset (Ensete ventricosum (Welw.) Cheesman) that make genotype conservation possible

The declining trends in crop wild relative genetic resources in many crop centers of origins including Ethiopia require short and long-term conservation strategies. Enset (Ensete ventricosum) is arguably the most important cultivated food security crop of Ethiopia with dwindling wild stocks. The cultivated enset is propagated clonally through adventitious bud sprouting from the corm after the distraction of the apical meristem. Shoot regeneration in the cultivated enset has been induced by humans and has not been observed to occur naturally. The technique of shoot induction has not been extended to the wild enset. To determine whether the capacity for shoot regeneration existed in wild enset and optimize the technique, a series of experiments were conducted. These involved: (i) sucker production from corms of wild enset with and without apical meristem removal; (ii) sprouting capacity of corms ranging 22–49 cm diameter, with removed apical meristem; and (iii) a factorial experiment involving two populations of wild enset (from Shebena and Getiba localities in Sheka zone), two ways of preparing or cutting the corms: tero and tubo, i.e. cutting the pseudostem at the corm junction and cutting it at 25–30 cm height, respectively, and three extents of parting the corm (whole, half, and quarter) using corms with a diameter of 45 ± 2.9 cm. The experiments revealed that wild enset can be successfully propagated vegetatively in the same way as the cultivated enset. It also revealed that the regeneration process involved callus formation and adventurous bud proliferation from corms only after the apical meristem was removed. Corms of different sizes varied in their capacity for regeneration significantly with a linear increase in regeneration frequency with corm size. With a one cm increase in corm diameter, regeneration frequency increased by 3.138 %. The two populations of wild enset showed non-significant differences in regeneration capacity; however, the achieved regeneration was generally analogous to that observed among cultivated enset clones: whole corms resulted in a longer time to emergence and fewer sucker per corm than split corms. Specifically, halved corms emerged significantly (p < 0.05) earlier (71 ± 9 and 75 ± 7 days, for Shebena and Getiba populations, respectively) than whole corms (120 days). Regeneration frequency was higher (75–100%) for split than for whole corms (33–56%). The highest rate of suckering (94 ± 14 per corm) was achieved from quarter corms prepared by cutting the pseudo-stem at the junction. In conclusion, the adventitious bud propagation technique developed by farmers to propagate the cultivated enset can successfully be used for the clonal regeneration of wild enset. We recommend the adoption of this shoot induction to conserve and maintain the rapidly eroding wild enset genetic resources in Ethiopia. In addition, wild enset plants with promising characteristics may be fixed using the method to enrich the gene pool of the cultivated enset.

Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes

Abstract. Carsono N, Juwendah E, Liberty, Sari S, Damayanti F, Rachmadi M. 2021. Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes. Biodiversitas 22: 2555-2560. The development of callus in the course of transgenic rice avoids the somaclonal variants. To obtain a high number of normal phenotypes and a low number of somaclonal variants requires an appropriate 2,4-D concentration. In this study, we obtained the best callus induction time and a high number of green plant regeneration for three responsive rice genotypes on different 2,4-D concentrations in NB5 medium. The mature seeds of rice embryos were used as explants. A completely randomized factorial design was applied with four levels of 2,4-D concentrations (0, 1, 3, and 5 ppm), two levels of induction time (one and two weeks), and three rice genotypes (cv. Fatmawati, Nipponbare, and Kitaake). The study revealed that there was no interaction effect among genotype, 2,4-D concentration, and callus induction time. Three rice genotypes performed best in callus proliferation and regeneration. One-week callus induction time showed higher callus growth capacity (CGC) as compared to two-week callus induction time. Shoot regeneration capacity (SRC) was independently affected by genotype as well as by callus induction time. The interaction effect between 2,4-D concentration and callus induction time was observed on plant regeneration capacity (PRC). Without the addition of 2,4-D and 1 ppm of 2,4-D, the green plant regeneration capacity (GRC) was comparatively higher. Addition of 2,4-D showed a significant effect, especially at the plant regeneration stage. We found that one-week callus induction was the best treatment for callus proliferation and plant regeneration. We recommend the use of one-week callus induction and 1 ppm of 2,4-D for rice callus proliferation (sub-culture) and subsequent plant regeneration.

Dynamic Chromatin State Profiling Reveals Regulatory Principle of Pluripotency During Shoot Regeneration

Genome-editing technologies have advanced in recent years but designing future crops is still limited by current methods of improving shoot regenerative capacity. Shoot regeneration is mediated by the sequential action of two phytohormones, auxin and cytokinin. However, the chromatin regulatory landscapes underlying this dynamic response have not yet been studied. In this study, we jointly profiled chromatin accessibility, histone modifications, and transcriptomes to demonstrate that a high-auxin environment primes shoot regeneration by increasing the accessibility of the gene loci associated with pluripotency and shoot fate determination. Cytokinin signaling not only triggers the commitment of the shoot progenitor at later stages, but allows chromatin to maintain shoot identity genes at the priming stage. Our analysis of transcriptional regulatory dynamics further identifies a catalog of regeneration cis-elements dedicated to cell fate transitions, and uncovers previously unrecognized roles of BES1, MYC, IDD, and PIF transcription factors in shoot regeneration. Our results provide a comprehensive resource for studying somatic cell reprogramming in plants and provides potential targets for improving future shoot regeneration efficiency.

ARF4 regulates shoot regeneration through coordination with ARF5 and IAA12.

ARF4-regulated shoot regeneration through competing with ARF5 for the interaction with IAA12. Plant possess the ability to regenerate shoot meristem and subsequent the whole individual. This process is the foundation for in vitro propagation and genetic engineering and provides a system for studying fundamental biological questions, such as hormonal signaling. Auxin response factor (ARF) family transcription factors are critical components of auxin signaling pathway that regulate the transcription of target genes. To date, the mechanisms underlying the functions of class-B ARFs which act as transcription repressors remains unclear. In this study, we found that ARF4, the transcriptional repressor, was involved in regulating shoot regeneration. ARF4 interacted with auxin/Indole-3-Acetic-Acid12 (IAA12). The expression signals of ARF4 displayed a dynamic pattern similar with those of ARF5 and IAA12 during shoot meristem formation. Enhanced expression of IAA12 compromised the shoot regeneration capacity. Induced expression of ARF4 complemented the regeneration phenotype of IAA12-overexpression but did not rescued the defects in the arf5 mutant, mp-S319. Further analysis revealed that ARF4 competed with ARF5 for the interaction with IAA12. The results indicate that ARF4-regulated shoot regeneration through cooperating with ARF5 and IAA12. Our findings provided new information for deciphering the function of class-B ARFs.

MiR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses.

Root growth is modulated by different factors, including phytohormones, transcription factors, and microRNAs (miRNAs). MicroRNA156 and its targets, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, define an age-dependent pathway that controls several developmental processes, including lateral root emergence. However, it remains unclear whether miR156-regulated SPLs control root meristem activity and root-derived de novo shoot regeneration. Here, we show that MIR156 and SPL genes have opposing expression patterns during the progression of primary root (PR) growth in Arabidopsis, suggesting that age cues may modulate root development. Plants with high miR156 levels display reduced meristem size, resulting in shorter primary root (PRs). Conversely, plants with reduced miR156 levels show higher meristem activity. Importantly, loss of function of SPL10 decreases meristem activity, while SPL10 de-repression increases it. Meristem activity is regulated by SPL10 probably through the reduction of cytokinin responses, via the modulation of type-B ARABIDOPSIS RESPONSE REGULATOR1(ARR1) expression. We also show that SPL10 de-repression in the PRs abolishes de novo shoot regenerative capacity by attenuating cytokinin responses. Our results reveal a cooperative regulation of root meristem activity and root-derived de novo shoot regeneration by integrating age cues with cytokinin responses via miR156-targeted SPL10.