PUCHI regulates very long chain fatty acid biosynthesis during lateral root and callus formation.

Lateral root organogenesis plays an essential role in elaborating plant root system architecture. In Arabidopsis, the AP2 family transcription factor PUCHI controls cell proliferation in lateral root primordia. To identify potential targets of PUCHI, we analyzed a time course transcriptomic dataset of lateral root formation. We report that multiple genes coding for very long chain fatty acid (VLCFA) biosynthesis enzymes are induced during lateral root development in a PUCHI-dependent manner. Significantly, several mutants perturbed in VLCFA biosynthesis show similar lateral root developmental defects as puchi-1 Moreover, puchi-1 roots display the same disorganized callus formation phenotype as VLCFA biosynthesis-deficient mutants when grown on auxin-rich callus-inducing medium. Lipidomic profiling of puchi-1 roots revealed reduced VLCFA content compared with WT. We conclude that PUCHI-regulated VLCFA biosynthesis is part of a pathway controlling cell proliferation during lateral root and callus formation.

Mass propagation through direct and indirect organogenesis in three species of genus Zephyranthes and ploidy assessment of regenerants through flow cytometry.

Genus Zephyranthes consists of economically important plant species due to their high ornamental value and presence of valuable bioactive compounds. However, this genus propagates by asexual division only which gives slow propagation rate. Plant tissue culture has the potential to provide efficient techniques for rapid multiplication and genetic improvement of the genus. In this work, a dual in vitro regeneration system through callus mediated shoot regeneration and direct shoot regeneration in species Zephyranthes candida, Zephyranthes grandiflora and Zephyranthes citrina was investigated. Bulb, leaf and root explants were cultured on Murashige and Skoog (MS) medium amended with different plant growth regulators (PGR's) viz. 2,4-dichlorophenoxyacetic acid (2,4-D), 1-Naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), N-phenyl-N'-1,2,3 -thiadiazol-5-ylurea (TDZ), 6-Furfuryl- aminopurine (KIN) alone or in combinations for callus induction and regeneration. Only bulb explants showed callus induction and regeneration response on different PGR combinations with a varied response in callus induction percentage, callus color and callus texture. Creamish compact callus (CC) was induced on 2 mg L[Formula: see text] 2,4-D, brown friable callus (BF) on 2 mg L[Formula: see text] NAA + 1 mg L[Formula: see text] BAP and green friable callus (GF) callus on 1 mg L[Formula: see text] KIN + 3 mg L[Formula: see text] NAA. The maximum shoot multiplication from different callus types (indirect organogenesis) was achieved on 2 mg L[Formula: see text] BAP alone without combinations. Bulb explants of Z. grandiflora induced maximum callus induction percentage (86.4%) and shoot regeneration percentage (83.5%) with the maximum 08 shoots per 150 mg callus mass. The induction and regeneration response was followed in the order of Z. grandiflora > Z. candida > Z. citrina. Similarly, maximum direct organogenesis from bulb explants was obtained in Z. grandiflora (93.3%) followed by Z. candida (91.5%) and Z. citrina (90.4%) on 3 mg L[Formula: see text] TDZ amended MS media. Adventitious root induction was achieved on 2 mg L[Formula: see text] IBA with a maximum of 8 roots per shoot. The in vitro raised plantlets were successfully acclimatized in the field with 85% survival efficiency. The genome size (2C DNA content) of the field-grown plants and in vitro regenerated plants, evaluated through flow cytometry technique, were similar and showed no ploidy changes. An efficient mass propagation protocol was established for obtaining plants with unaltered genome size in the three species of Zephyranthes.

Global Transcriptomic Analysis Reveals Differentially Expressed Genes Involved in Embryogenic Callus Induction in Drumstick (Moringa oleifera Lam.).

The plant embryogenic callus (EC) is an irregular embryogenic cell mass with strong regenerative ability that can be used for propagation and genetic transformation. However, difficulties with EC induction have hindered the breeding of drumstick, a tree with diverse potential commercial uses. In this study, three drumstick EC cDNA libraries were sequenced using an Illumina NovaSeq 6000 system. A total of 7191 differentially expressed genes (DEGs) for embryogenic callus development were identified, of which 2325 were mapped to the KEGG database, with the categories of plant hormone signal transduction and Plant-pathogen interaction being well-represented. The results obtained suggest that auxin and cytokinin metabolism and several embryogenesis-labeled genes are involved in embryogenic callus induction. Additionally, 589 transcription factors from 20 different families were differentially expressed during EC formation. The differential expression of 16 unigenes related to auxin signaling pathways was validated experimentally by quantitative real time PCR (qRT-PCR) using samples representing three sequential developmental stages of drumstick EC, supporting their apparent involvement in drumstick EC formation. Our study provides valuable information about the molecular mechanism of EC formation and has revealed new genes involved in this process.

Cartilage to bone transformation during fracture healing is coordinated by the invading vasculature and induction of the core pluripotency genes.

Fractures heal predominantly through the process of endochondral ossification. The classic model of endochondral ossification holds that chondrocytes mature to hypertrophy, undergo apoptosis and new bone forms by invading osteoprogenitors. However, recent data demonstrate that chondrocytes transdifferentiate to osteoblasts in the growth plate and during regeneration, yet the mechanism(s) regulating this process remain unknown. Here, we show a spatially-dependent phenotypic overlap between hypertrophic chondrocytes and osteoblasts at the chondro-osseous border in the fracture callus, in a region we define as the transition zone (TZ). Hypertrophic chondrocytes in the TZ activate expression of the pluripotency factors [Sox2, Oct4 (Pou5f1), Nanog], and conditional knock-out of Sox2 during fracture healing results in reduction of the fracture callus and a delay in conversion of cartilage to bone. The signal(s) triggering expression of the pluripotency genes are unknown, but we demonstrate that endothelial cell conditioned medium upregulates these genes in ex vivo fracture cultures, supporting histological evidence that transdifferentiation occurs adjacent to the vasculature. Elucidating the cellular and molecular mechanisms underlying fracture repair is important for understanding why some fractures fail to heal and for developing novel therapeutic interventions.

Production of ascorbic acid, total protein, callus and root in vitro of non-heading Chinese cabbage by tissue culture.

The objective of the present work was the selection of cultivar, suitable medium and explant type for callus, root production, ascorbic acid, total ascorbic acid, dehydroascorbic and total protein of non-heading Chinese cabbage in two cultivars 'Caixin' and 'Suzhouqing'. We compared 10 types of MS media supplemented with 0.0, 1.0, 2.0 and 3.0 mg/l TDZ; 0.0, 0.25, 0.50 and 1.0 mg/l NAA and 0.0, 5.0, 7.5 and 9.0 mg/l AgNO3 and 5 kinds of explants as embryo, leaf, root, cotyledon and hypocotyl. Maximum frequency of callus fresh weight was recorded with hypocotyl explant, which were cultured on MS + 2.0 mg/l TDZ + 1.0 mg/l NAA + 9.0 mg/l AgNO3 in 'Suzhouqing', optimum callus dry weight was obtained on the same media. The highest result for root fresh and dry weight recorded with 'Caixin' with MS + 3.0 mg/l TDZ + 1.0 mg/l NAA + 9.0 mg/l AgNO3 when we used embryo as explant. The highest ascorbic acid content was found with callus cultured on MS + 1.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3, when used leaf explant in 'Caixin' or root in 'Suzhouqing', and there were no significant difference between them. While the highest value of total AsA content was registered with callus cultured on MS + 2.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3 extracted from cotyledon in 'Caixin'. The highest content of DHA was registered with MS + 2.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3 with cotyledon in 'Caixin'. Also, in 'Caixin' MS + 3.0 mg/l TDZ + 0.25 mg/l NAA + 5.0 mg/l AgNO3 recorded the highest value of total protein content with embryo explant.

Establishment of an Agrobacterium mediated transformation protocol for the detection of cytokinin in the heterophyllous plant Hygrophila difformis (Acanthaceae).

KEY MESSAGE: This is the first report of a highly efficient Agrobacterium tumefaciens-mediated transformation protocol for Acanthaceae and its utilization in revealing important roles of cytokinin in regulating heterophylly in Hygrophila difformis. Plants show amazing morphological differences in leaf form in response to changes in the surrounding environment, which is a phenomenon called heterophylly. Previous studies have shown that the aquatic plant Hygrophila difformis (Acanthaceae) is an ideal model for heterophylly study. However, low efficiency and poor reproducibility of genetic transformation restricted H. difformis as a model plant. In this study, we reported successful induction of callus, shoots and the establishment of an efficient stable transformation protocol as mediated by Agrobacterium tumefaciens LBA4404. We found that the highest callus induction efficiency was achieved with 1 mg/L 1-Naphthaleneacetic acid (NAA) and 2 mg/L 6-benzyladenine (6-BA), that efficient shoot induction required 0.1 mg/L NAA and 0.1 mg/L 6-BA and that high transformation efficiency required 100 µM acetosyringone. Due to the importance of phytohormones in the regulation of heterophylly and the inadequate knowledge about the function of cytokinin (CK) in this process, we analyzed the function of CK in the regulation of heterophylly by exogenous CK application and endogenous CK detection. By using our newly developed transformation system to detect CK signals, contents and distribution in H. difformis, we revealed an important role of CK in environmental mediated heterophylly.

Impact of the Sensory and Sympathetic Nervous System on Fracture Healing in Ovariectomized Mice.

The peripheral nervous system modulates bone repair under physiological and pathophysiological conditions. Previously, we reported an essential role for sensory neuropeptide substance P (SP) and sympathetic nerve fibers (SNF) for proper fracture healing and bone structure in a murine tibial fracture model. A similar distortion of bone microarchitecture has been described for mice lacking the sensory neuropeptide α-calcitonin gene-related peptide (α-CGRP). Here, we hypothesize that loss of SP, α-CGRP, and SNF modulates inflammatory and pain-related processes and also affects bone regeneration during fracture healing under postmenopausal conditions. Intramedullary fixed femoral fractures were set to 28 days after bilateral ovariectomy (OVX) in female wild type (WT), SP-, α-CGRP-deficient, and sympathectomized (SYX) mice. Locomotion, paw withdrawal threshold, fracture callus maturation and numbers of TRAP-, CD4-, CD8-, F4/80-, iNos-, and Arg1-positive cells within the callus were analyzed. Nightly locomotion was reduced in unfractured SP-deficient and SYX mice after fracture. Resistance to pressure was increased for the fractured leg in SP-deficient mice during the later stages of fracture healing, but was decreased in α-CGRP-deficient mice. Hypertrophic cartilage area was increased nine days after fracture in SP-deficient mice. Bony callus maturation was delayed in SYX mice during the later healing stages. In addition, the number of CD 4-positive cells was reduced after five days and the number of CD 8-positive cells was additionally reduced after 21 days in SYX mice. The number of Arg1-positive M2 macrophages was higher in α-CGRP-deficient mice five days after fracture. The alkaline phosphatase level was increased in SYX mice 16 days after fracture. Absence of α-CGRP appears to promote M2 macrophage polarization and reduces the pain threshold, but has no effect on callus tissue maturation. Absence of SP reduces locomotion, increases the pain-threshold, and accelerates hypertrophic callus tissue remodeling. Destruction of SNF reduces locomotion after fracture and influences bony callus tissue remodeling during the later stages of fracture repair, whereas pain-related processes are not affected.

Modulation of callus growth and secondary metabolites in different Thymus species and Zataria multiflora micropropagated under ZnO nanoparticles stress.

Thymus species are aromatic plants with diverse applications in food industries and medicine. This study was conducted to evaluate the potential effect of ZnO nanoparticles (NPs) on callus proliferation and thymol and carvacrol production in three Thymus species, that is, T. vulgaris, T. daenensis, and T. kotschyanus, and Zataria multiflora. For this purpose, callus induction was performed on Murashige and Skoog (MS) medium containing different plant growth regulators (PGRs). After optimization of callus growth, the effects of different concentrations of ZnO NPs (100 and 150 mg L-1 ) were investigated. MS containing 2 mg L-1 of 2, 4-dichlorophenoxy acetic acid (2,4-D) and 1 mg L-1 of kinetin (Kin) revealed significantly highest fresh weight (0.18 g) of callus in T. kotschyanus. Callus growth rate (0.079 mm day-1 ) was found highest in T. vulgaris under similar conditions. Moreover, highest callus induction (92.50%) was achieved by T. kotschyanus in MS containing 2.5 mg L-1 of 2,4-D. Regarding the highest content of thymol (22.8 mg L-1 ) and carvacrol (0.68 mg L-1 ) evaluated by high-performance liquid chromatography, best results were achieved under 150 mg L-1 of ZnO NPs in T. kotschyanus and T. daenesis, respectively. This is simple and cost-effective method to be applied on industrial level for production of enhanced secondary metabolites content.

A Cytotoxic and Anti-inflammatory Campesterol Derivative from Genetically Transformed Hairy Roots of Lopezia racemosa Cav. (Onagraceae).

The genetically transformed hairy root line LRT 7.31 obtained by infecting leaf explants of Lopezia racemosa Cav with the Agrobacterium rhizogenes strain ATCC15834/pTDT, was evaluated to identify the anti-inflammatory and cytotoxic compounds reported previously for the wild plant. After several subcultures of the LRT 7.31 line, the bio-guided fractionation of the dichloromethane-methanol (1:1) extract obtained from dry biomass afforded a fraction that showed important in vivo anti-inflammatory, and in vitro cytotoxic activities. Chemical separation of the active fraction allowed us to identify the triterpenes ursolic (1) and oleanolic (2) acids, and (23R)-2α,3ß,23,28-tetrahydroxy-14,15-dehydrocampesterol (3) as the anti-inflammatory principles of the active fraction. A new molecule 3 was characterized by spectroscopic analysis of its tetraacetate derivative 3a. This compound was not described in previous reports of callus cultures, in vitro germinated seedlings and wild plant extracts of whole L. racemosa plants. The anti-inflammatory and cytotoxic activities displayed by the fraction are associated to the presence of compounds 1-3. The present study reports the obtaining of the transformed hairy roots, the bioguided isolation of the new molecule 3, and its structure characterization.

Delayed Fracture Healing in Diabetics with Distal Radius Fractures.

PURPOSE OF THE STUDY: Diabetics may have an increased fracture risk, depending on disease duration, quality of metabolic adjustment and extent of comorbidities, and on an increased tendency to fall. The aim of this retrospective one-centre study consisted in detecting differences in fracture healing between patients with and without diabetes mellitus. Data of patients with the most common fracture among older patients were analyzed. MATERIAL AND METHODS: Classification of distal radius fractures was established according to the AO classification. Inital assessment and follow-up were made by conventional X-rays with radiological default settings. To evaluate fracture healing, formation of callus and sclerotic border, assessment of the fracture gap, and evidence of consolidation signs were used. RESULTS: The authors demonstrated that fracture morphology does not influence fracture healing regarding time span, neither concerning consolidation signs nor in fracture gap behaviour. However, tendency for bone remodeling is around 70% lower in investigated diabetics than in non-diabetics, while probability for a successful fracture consolidation is 60% lower. CONCLUSIONS: To corroborate the authors hypothesis of delayed fracture healing in patients with diabetes mellitus, prospective studies incorporating influencing factors like duration of metabolic disease, quality of diabetes control, medical diabetes treatment, comorbidities and secondary diseases, like chronic nephropathy and osteoporosis, have to be carried out.

Optimization of a biolistic transformation system for transfer of antifreeze gene KN2 and the bar herbicide resistance gene in common wheat.

We studied the effects of different media for callus induction and differentiation, and pre-culture period of immature wheat embryo culture on biolistic transformation efficiency for including antifreeze gene KN2 and bar conferring resistance to the herbicide bialaphos. The percentage of plantlets generated from induction and differentiation media without Cu2+ was lower than those cultured on differentiation media with Cu2+ (71.15%) or induction media with Cu2+ (68.45%) and both induction and differentiation media with Cu2+ (52.17%). The combinations of Nor medium for callus induction and Cu2+ medium for regeneration, and Cu2+ medium for induction and R medium for regeneration were superior for biolistic transformation. The calli induced on Cu2+ medium and pre-cultured for 4 d before biolistic transformation, and cultured on R medium after biolistic transformation produced the highest percentage (65%) of transgenic plantlets with the KN2 gene. Overall, about 50% plantlets regenerated from calli pre-cultured 4d before bombardment carried the KN2 gene; 44.7% of the plantlets carried the bar gene, which was higher than for any other treatment, followed by pre-culture 1d with 31.43% transformation rate for the KN2 gene and 20% transformation rate for the bar gene.

Determination of suitable microspore stage and callus induction from anthers of kenaf (Hibiscus cannabinus L.).

Kenaf (Hibiscus cannabinus L.) is one of the important species of Hibiscus cultivated for fiber. Availability of homozygous parent lines is prerequisite to the use of the heterosis effect reproducible in hybrid breeding. The production of haploid plants by anther culture followed by chromosome doubling can be achieved in short period compared with inbred lines by conventional method that requires self pollination of parent material. In this research, the effects of the microspore developmental stage, time of flower collection, various pretreatments, different combinations of hormones, and culture condition on anther culture of KB6 variety of Kenaf were studied. Young flower buds with immature anthers at the appropriate stage of microspore development were sterilized and the anthers were carefully dissected from the flower buds and subjected to various pretreatments and different combinations of hormones like NAA, 2,4-D, Kinetin, BAP, and TDZ to induce callus. The best microspore development stage of the flower buds was about 6-8 mm long collected 1-2 weeks after flower initiation. At that stage, the microspores were at the uninucleate stage which was suitable for culture. The best callus induction frequency was 90% in the optimized semisolid MS medium fortified with 3.0 mg/L BAP + 3.0 mg/L NAA.

Macroscopic and microscopic process of long bone fracture healing.

The growth of each type of callus (cortical, medullary and periosteal) depends on the mechanical condition of fracture fixation (elastic fixation and instability or rigid immobilization), the type of treatment (non-operative, close or open surgical procedure, intra-medullary nailing, external fixation, plate...) and the high or poor quality of soft tissue and the specific characteristics of the local vascularisation.

The efficacy of allogeneic mesenchymal precursor cells for the repair of an ovine tibial segmental defect.

INTRODUCTION: Synthetic void-fillers offer an alternative to autograft or allograft bone in the repair of segmental defects. However, the reparative process is delayed as only osteoconductive elements are present. The inclusion of pluripotential cells may resolve this limitation, and the use of allogeneic tissue provides the opportunity for an off-the-shelf remedy. The current study evaluated the utilisation of mesenchymal precursor cells (MPC) for the repair of an ovine critical-size tibial segmental defect. METHODS: Twenty-four, mature female sheep underwent surgery for the creation of a 3 cm tibial diaphyseal defect. In one group of 12 sheep the scaffold was used alone, and in the second group the scaffold was seeded with MPC. The defect was stabilised using a locking intramedullary nail and allowed to heal over a nine-month-period. Outcome assessments of healing included radiology of callus formation, computed tomography, assessment of new-bone volume, mechanical attributes, and histological evaluation of linear bone apposition rate and tissue response. RESULTS: The MPC-treated group displayed a significantly greater level of callus formation and rate of bone apposition in the defect. DISCUSSION: The incorporation of allogeneic MPC to a synthetic void filler stimulated early repair of critical-size diaphyseal segmental defects and holds potential as an off-the-shelf therapy for augmenting bone regeneration.

Recent advances in the use of serological bone formation markers to monitor callus development and fracture healing.

The failure of an osseous fracture to heal, or the development of a nonunion, is common; however, current diagnostic measures lack the capability of early and reliable detection of such events. Analyses of radiographic imaging and clinical examination, in combination, remain the gold standard for diagnosis; however, these methods are not reliable for early detection. Delayed diagnosis of a nonunion is costly from both the patient and treatment standpoints. In response, repeated efforts have been made to identify bone metabolic markers as diagnostic or prognostic tools for monitoring bone healing. Thus far, the evidence regarding a correlation between the kinetics of most bone metabolic markers and nonunion is very limited. With the aim of classifying the role of biological pathways of bone metabolism and of understanding bone conditions in the development of osteoporosis, advances have been made in our knowledge of the molecular basis of bone remodeling, fracture healing, and its failure. Procollagen type I amino-terminal propeptide has been shown to be a reliable bone formation marker in osteoporosis therapy and its kinetics during fracture healing has been recently described. In this article, we suggest that procollagen type I amino-terminal propeptide presents a good opportunity for early detection of nonunion. We also review the role and potential of serum PINP, as well as other markers, as indications of fracture healing.

Oral rehabilitation of a maxillectomy defect using bone transport distraction and dental implants.

Bone transport distraction is a reliable procedure in various maxillofacial bony defect reconstruction techniques. It is minimally invasive and it eliminates donor site morbidity. We introduce a new surgical technique for maxillary backward bone transport distraction reconstruction performed in a 77-year-old woman with a posterior partial maxillary defect. Transport distraction was successful for posterior maxillary alveolar bony regeneration, which helped close an oroantral fistula. One month after the distraction device was removed, 3 dental implants were placed in the reconstructed alveolus, followed by successful oral functional rehabilitation by use of an implant-anchored prosthesis. Two and a half years have passed since the patient's dental implant-based prosthesis was activated, and the functional occlusal reconstruction by use of bone transport distraction and dental implants after repair of the maxillectomy defect has proven to be effective with patient satisfaction.

CTRP3 Regulates Endochondral Ossification and Bone Remodeling During Fracture Healing.

C1q/TNF-related protein 3 (CTRP3) is a cytokine known to regulate a variety of metabolic processes. Though previously undescribed in the context of bone regeneration, high throughput gene expression experiments in mice identified CTRP3 as one of the most highly upregulated genes in fracture callus tissue. Hypothesizing a positive regulatory role for CTRP3 in bone regeneration, we phenotyped skeletal development and fracture healing in CTRP3 knockout (KO) and CTRP3 overexpressing transgenic (TG) mice relative to wild-type (WT) control animals. CTRP3 KO mice experienced delayed endochondral fracture healing, resulting in abnormal mineral distribution, the presence of periosteal marrow compartments, and a nonunion-like state. Decreased osteoclast number was also observed in CTRP3 KO mice, whereas CTRP3 TG mice underwent accelerated callus remodeling. Gene expression profiling revealed a broad impact on osteoblast/osteoclast lineage commitment and metabolism, including arrested progression toward mature skeletal lineages in the KO group. A single systemic injection of CTRP3 protein at the time of fracture was insufficient to phenocopy the chronic TG healing response in WT mice. By associating CTRP3 levels with fracture healing progression, these data identify a novel protein family with potential therapeutic and diagnostic value. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:00-19966, 2020.

Callus growth kinetics and accumulation of secondary metabolites of Bletilla striata Rchb.f. using a callus suspension culture.

Bletilla striata is an endangered traditional Chinese medicinal plant with multiple uses and a slow regeneration rate of its germplasm resources. To evaluate the callus growth kinetics and accumulation of secondary metabolites (SMs), a callus suspension culture was proven to be a valuable approach for acquiring high yields of medicinal compounds. An effective callus suspension culture for obtaining B. striata callus growth and its SMs was achieved with the in vitro induction of calluses from B. striata seeds. The callus growth kinetics and accumulation of SMs were analyzed using a mathematical model. The resulting callus growth kinetic model revealed that the growth curves of B. striata suspension-cultured calluses were sigmoidal, indicating changes in the growth of the suspension-cultured calluses. Improved Murashige and Skoog callus growth medium was the most favorable medium for B. striata callus formation, with the highest callus growth occurring during the stationary phase of the cultivation period. Callus growth acceleration started after 7 days and thereafter gradually decreased until day 24 of the cultivation period and reached its highest at day 36 period in both the dry weight and fresh weight analyses. The coelonin concentration peaked during the exponential growth stage and decreased afterward during the stationary stage of the callus suspension culture. The maximum content of coelonin (approximately 0.3323 mg/g callus dry weight) was observed on the 18th day of the cultivation cycle, while dactylorhin A and militarine reached the highest concentrations at day 24, and p-hydroxybenzyl alcohol at day 39. This investigation also laid a foundation for a multimathematical model to better describe the accumulation variation of SMs. The production of SMs showed great specificity during callus growth and development. This research provided a well-organized way to increase the accumulation and production of SMs during the scaled-up biosynthesis of calluses in B. striata callus suspension cultures.

MYB94 and MYB96 additively inhibit callus formation via directly repressing LBD29 expression in Arabidopsis thaliana.

Plant somatic cells can be reprogrammed during in vitro culture. Callus induction is the initial step of a typical plant regeneration system. Recent studies showed that auxin-induced callus formation in multiple organs occurs from the pericycle or pericycle-like cells via a root developmental pathway. However, the molecular control of callus formation is largely unknown. Here, two MYB transcription factors, MYB94 and MYB96, were shown to play negative roles in auxin-induced callus formation in Arabidopsis. MYB94 and MYB96 were expressed in the newly formed callus. myb96, myb94, and myb94 myb96 generated more calli than the WT, with myb94 myb96 producing the most. MYB94 and MYB96 repressed expression of LATERAL ORGAN BOUNDARIES-DOMAIN 29 (LBD29) via directly binding to the gene's promoter. The loss of function of LBD29 partly rescued the callus formation defect of myb94 myb96. Our findings found MYB94 and MYB96 to be important repressors of callus formation and MYB94/96-LBD29 as a new regulatory pathway acting in parallel with ARF7/19-LBDs' pathway to modulate in vitro callus formation.

Defining the genetic components of callus formation: A GWAS approach.

A characteristic feature of plant cells is the ability to form callus from parenchyma cells in response to biotic and abiotic stimuli. Tissue culture propagation of recalcitrant plant species and genetic engineering for desired phenotypes typically depends on efficient in vitro callus generation. Callus formation is under genetic regulation, and consequently, a molecular understanding of this process underlies successful generation for propagation materials and/or introduction of genetic elements in experimental or industrial applications. Herein, we identified 11 genetic loci significantly associated with callus formation in Populus trichocarpa using a genome-wide association study (GWAS) approach. Eight of the 11 significant gene associations were consistent across biological replications, exceeding a chromosome-wide-log10 (p) = 4.46 [p = 3.47E-05] Bonferroni-adjusted significance threshold. These eight genes were used as hub genes in a high-resolution co-expression network analysis to gain insight into the genome-wide basis of callus formation. A network of positively and negatively co-expressed genes, including several transcription factors, was identified. As proof-of-principle, a transient protoplast assay confirmed the negative regulation of a Chloroplast Nucleoid DNA-binding-related gene (Potri.018G014800) by the LEC2 transcription factor. Many of the candidate genes and co-expressed genes were 1) linked to cell division and cell cycling in plants and 2) showed homology to tumor and cancer-related genes in humans. The GWAS approach based on a high-resolution marker set, and the ability to manipulate targets genes in vitro, provided a catalog of high-confidence genes linked to callus formation that can serve as an important resource for successful manipulation of model and non-model plant species, and likewise, suggests a robust method of discovering common homologous functions across organisms.