Shoot Primordia Research Articles

Transcriptome analysis of apical meristem enriched bud samples for size dependent flowering commitment in Crocus sativus reveal role of sugar and auxin signalling.

Cultivation of Crocus sativus (saffron) faces challenges due to inconsistent flowering patterns and variations in yield. Flowering takes place in a graded way with smaller corms unable to produce flowers. Enhancing the productivity requires a comprehensive understanding of the underlying genetic mechanisms that govern this size-based flowering initiation and commitment. Therefore, samples enriched with non-flowering and flowering apical buds from small (< 6g) and large (> 14g) corms were sequenced. Apical bud enriched samples from small and large corms were collected immediately after dormancy break in July. RNA sequencing was performed using Illumina Novaseq 6000 to access the gene expression profiles associated with size dependent flowering. De novo transcriptome assembly and analysis using flowering committed buds from large corms at post-dormancy and their comparison with vegetative shoot primordia from small corms pointed out the major role of starch and sucrose metabolism, Auxin and ABA hormonal regulation. Many genes with known dual responses in flowering development and circadian rhythm like Flowering locus T and Cryptochrome 1 along with a transcript showing homology with small auxin upregulated RNA (SAUR) exhibited induced expression in flowering buds. Thorough prediction of Crocus sativus non-coding RNA repertoire has been carried out for the first time. Enolase was found to be acting as a major hub with protein-protein interaction analysis using Arabidopsis counterparts. Transcripts belong to key pathways including phenylpropanoid biosynthesis, hormone signaling and carbon metabolism were found significantly modulated. KEGG assessment and protein-protein interaction analysis confirm the expression data. Findings unravel the genetic determinants driving the size dependent flowering in Crocus sativus.

Optimizing callus induction and indirect organogenesis in non-dormant corm explants of Gloriosa superba (L.) via media priming

IntroductionGloriosa superba L. is an endangered ornamental plant of significant medicinal, commercial, and cultural value. This study establishes an efficient protocol for rapid in vitro propagation of this plant species through callus-mediated organogenesis using non-dormant corm explants.MethodsPlant tissue culture techniques were employed to facilitate the in vitro regeneration process of Gloriosa superba L. This encompassed various stages, including the acquisition of plant material, surface sterilization, formulation of growth media, and the execution of callogenesis, shooting, rooting, and acclimatization experiments.Results and DiscussionInvestigation into the impact of plant growth regulators on callogenesis unveiled diverse callus morphologies, dependent on regulator type and concentration, with the NAA and KN combination emerging as the most effective for callus induction. Specifically, the callus induction medium supplemented with 1.5 mg L−1 NAA, 0.5 mg L−1 KN, and 10 mg L−1 casein hydrolysate (CH) achieved remarkable results, boasting an 81.25% callus induction rate and a substantial callus biomass fresh weight of 333 mg. Furthermore, the combination of BAP and NAA facilitated optimal shoot primordia induction and shooting in callus derived from non-dormant corm explants. The shoot induction medium, enriched with 2.0 mg L−1 BAP, 0.5 mg L−1 NAA, 5 mg L−1 CH, and 20% (v/v) coconut water (CW), resulted in an impressive 83.33% shoot primordia formation rate and an average of 6.86 shoot primordia per callus. For root induction, the medium supplemented with 1.0 mg L−1 IBA displayed a high root induction rate of 81.25% and robust rooting (+++). Successful acclimatization of in vitro plantlets in controlled environments and shade net houses culminated in thriving growth, and upon transplantation into a garden soil blend (garden soil : sand : vermiculite, 2:1:1, v/v), with direct sunlight exposure, the plantlets demonstrated commendable development and form, boasting a 69% survival rate at the seven-week mark. These findings offer a robust foundation for scalable and sustainable propagation strategies, ensuring the conservation and utilization of this valuable species.

Harnessing spatial transcriptomics for advancing plant regeneration research

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.

Морфометрические критерии оценки качества контейнерных сеянцев хвойных пород

The quality of planted forest crops depends significantly on the qualities of the planting stock. Numerous studies have been carried out to find clear seedling quality criteria and work out methods for their quantitative assessment to optimize the cultivation regime. Indicators of planting stock that are available for measurement under nursery conditions are needed to predict the success of forest crop establishment. This paper provides a review of the studies that applied different morphological criteria to evaluate coniferous planting stock quality and different determination methods. The suitability of planting stock quality indicators is determined by the effectiveness of predicting the outplanting survival rate (%) and growth. The key morphometric attributes of planting stock are the linear dimensions and biomass of whole seedlings and their individual organs: root collar diameter (RCD), seedling height (SH), dry mass of needles (NDM), stem (SBDM), roots (RDM), shoot/aboveground part of the seedling (SDM), seedling’s total dry matter (TDM), number of needles (NN). Effective indicators of seedling quality are morphometric indicators of roots: total length (TRL), volume (TRV), dry mass (RDM), number of first- (FOLR N), second- (SOLR N), and third-order (TOLR N) lateral roots, root surface area (TRS). TRL is a fairly reliable indicator of absorbent surface area. To minimize errors in the use of one or two traits, integrated seedling quality indices based on two or more indices have been developed. Widely used indices include: SQ = SH(cm)/RCD(mm); SDM/RDM ratio; Dickson quality index DQI = TDM/ ((SH / RCD) + (SDM / RDM)). RCD was the most suitable parameter to indicate seedling quality for many species due to its higher correlation level with the Dickson quality index. The SH index proved to be an effective indicator for analysis only when used together with RCD. When studying the formation of frost resistance, a number of morphological parameters are used: initiation of needle primordia, size of primordial shoots, cell volume, mitotic index (MI) of terminal primordia. Despite advances in testing planting stock quality and predicting field performance, not a single test is universally applicable for all woody plant species and environmental conditions. According to the target seedling concept, the application of morphometric criteria for assessing seedling quality in forest nurseries is a prerequisite for predicting outplanting success and maximizing the predictability of the outcomes of highproductivity forest crop planting. For citation: Robonen E.V., Chernobrovkina N.P., Egorova A.V., Zaitseva M.I., Nelaeva K.G. Morphometric Criteria for Assessing the Containerized Conifers Seedlings Quality. Lesnoy Zhurnal = Russian Forestry Journal, 2023, no. 5, pp. 42–57. (In Russ.). https://doi.org/10.37482/0536-1036-2023-5-42-57

Spatial transcriptomics reveals light-induced chlorenchyma cells involved in promoting shoot regeneration in tomato callus

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.

Plant Growth Hormones and Micro-Tuberization in Breaking the Seed Dormancy of Bunium persicum (Boiss.) Fedts.

Bunium persicum is a valuable medicinal plant with limited production but high market demand. It thrives predominantly in high-altitude regions. The main challenges hindering its widespread cultivation are seed dormancy and a lengthy seed-to-seed cycle, making its large-scale cultivation difficult. Six genotypes of Bunium persicum were collected from different altitudes to evaluate its germination behavior and seed dormancy. The study was conducted during 2020-23 and comprised three experiments (viz., seed germination under an open field, controlled conditions, and micro-tuberization). Under open field conditions, germination percent was genotype dependent, and the highest germination percentage, root length, and shoot length were recorded in Shalimar Kalazeera-1. Germination behavior assessment of the Bunium persicum revealed that treatment T9 (GA3 (25 ppm) + TDZ (9 µM/L)) is effective in breaking the dormancy of Bunium persicum as well as in obtaining a higher germination percent for early development of the tubers. Similarly, with regard to the effect of temperature and moisture conditions, stratification under moist chilling conditions showed effectiveness in breaking seed dormancy as the germination percentage in stratified seeds was at par with the most efficient growth hormone. With regard to the in vitro micro-propagation, direct regeneration showed multiple shoot primordia at the base of the tubers without intervening callus phase from the MS medium supplemented with BA (22.2 µM) and NAA (13.95 µM) 4 weeks after sub-culturing. Similarly, medium supplemented with JA (8.0 mg/L) and BA (22.2 µM) produced well-organized somatic embryos with shiny surfaces, which appeared at the swelled basal portion of apical stems. Further, the combination of JA (6.0 mg/L) and BA (22.2 M) was effective in developing the micro-tubers and also enhanced the weight and length of Bunium persicum micro-tubers.

Histological and molecular insights in to in vitro regeneration pattern of Xanthosoma sagittifolium

A study on the effect of various phytohormonal combinations on in vitro propagation of Cocoyam [Xanthosoma sagittifolium (L.) Schott] was conducted to develop an improved and efficient in vitro regeneration protocol for its mass multiplication. Histological analysis to understand the in vitro regeneration pattern and genetic fidelity assessment of regenerated plants were also carried out. Single shoots excised from in vitro established cultures of X. sagittifolium were used as explants. Among the 32 different phytohormonal combinations tested, indirect organogenesis with intervening callus phase was observed on majority of the media combinations. Meristematic clump formation was optimally achieved on all the tested media combinations with maximum 43.54 ± 0.51 shoot primordia on MS medium containing 0.2 mg/L BAP + 0.1 mg/L NAA followed by 36.44 ± 0.76 shoot primordia on MS medium having 2.5 mg/L TDZ. Micro-morphological analysis of different morphogenetic structures revealed that the regeneration of cocoyam is well executed via meristematic nodules, shoot primordia formation that may evolve in to proper shoots. Adventitious shoots (> 2 cm) were successfully (100.00 ± 0.00%) rooted on the half-strength MS medium containing IBA (0.05–1.0 mg/L) and IAA (0.05–0.5 mg/L). The number of roots ranged from 0.78 ± 0.31 on the control half-strength MS medium to 13.94 ± 0.46 on half-strength MS supplemented with 1.0 mg/L IBA. Considering somaclonal variations as a potential restriction to in vitro multiplication of plants, genetic stability was assessed using 40 ISSR primers. The PCR amplification profiles obtained from all the tested propagules (calli, meristematic clumps, regenerated plantlets) were similar to the mother plants indicating the homogeneity of the individuals raised through the regeneration protocol reported here.

Leaf-based induction of protocorm-like bodies, their encapsulation, storage and post-storage germination with genetic fidelity in Mokara Sayan × Ascocenda Wangsa gold

• First report on direct induction of PLBs Mokara Sayan × Ascocenda Wangsa gold. • Earliest and maximum PLB induction occurred on MS medium fortified with 3 mg L –1 TDZ. • Synthetic seeds were developed encapsulating PLBs with 3 % Na-alginate and 75 mM CaCl 2 . • Better storage of synthetic seeds up to 180 days was attained at 25°C in comparison to 4°C. • RAPD marker-based analysis ensured genetic integrity of synthetic seed-derived plantlets. An innovative and accelerated in vitro mass propagation protocol via direct induction of protocorm-like bodies (PLBs) from leaf sections of orchid hybrid Mokara Sayan × Ascocenda Wangsa gold was developed for the first time. Initially, leaf sections were transferred into Murashige and Skoog (MS) medium supplemented with two cytokinin sources namely, thidiazuron (TDZ) and zeatin with variable concentrations. The earliest (∼8 days) induction of PLBs with maximum number (34 PLBs cm –2 leaf section) and induction frequency (82.8 %) were recorded in MS medium fortified with 3 mg L –1 TDZ. The same medium formulation resulted in the highest growth rate (∼93.7 mg day –1 ) with maximum diameter of PLBs (3.4 mm) when compared with other treatments. Observations on proliferating PLBs under variable pressure scanning electron microscopy illustrated their distinctive growth phases wherein proliferating PLBs exhibited globular shape, typical epidermal texture with a rough surface that steadily became more wrinkled, consisting of basal absorbing hair-bodies and the apical zone forming shoot primordia. Stereo microscopic observations revealed gradual maturation of PLBs, initiation of leaf and shoot primordia. PLBs were encapsulated with calcium-alginate for short-term storage and germplasm exchange. Lower concentrations of sodium alginate (1 and 2 %) produced synthetic seeds that were soft to grip and extremely irregular in shape, whereas higher concentration (4 %) resulted in stiffness of synthetic seeds and reduced germination frequency. The most suitable gel matrix to form uniform spherical beads was optimized at 3 % sodium alginate and 75 mM calcium chloride. Synthetic seeds were effectively stored at 25 °C for up to 180 days. The germination efficiency of synthetic seeds gradually declined after storage at both 4 °C and 25 °C, with synthetic seeds stored at 25 °C being more tolerant than those stored at 4 °C. Even after 180 days of storage, the synthetic seeds that were stored at 25 °C exhibited 71.2 % germination frequency and ∼14 days to germination. Synthetic seeds stored at 4 °C deteriorated rapidly and degenerated completely within 180 days. Random amplified polymorphic DNA marker-based analysis carried out with randomly selected synthetic seed-derived plantlets showed 100 % monomorphism with a total 512 bands of molecular size ranging between 130—2100 bp, ensuring post-storage genetic fidelity.

Histological and in vitro seed culture studies on Biancaea sappan (L.) Tod. (Caesalpiniaceae)

Establishment of a tissue culture protocol for clonal regeneration is an essential prerequisite for the potential and economically important plant species Biancaea sappan (L.) Tod. (syn. Caesalpinia sappan L.; Caesalpiniaceae). This plant is a valuable dye-yielding legume shrub native to Indomalayan and is used in India by the local people of Kerala and Tamil Nadu States for its biological potential. The present study evaluated seed development both in vivo and in vitro through histological analysis. The mature seed was used as explants and cultured on modified Murashige and Skoog (MS) medium supplemented with 2, 4-D, BAP, GA3 and IBA. The cultures showed direct regeneration of shoots from nodular structures in the shoot apical meristem (SAM) region. During in vitro regeneration, the callus cells exhibited ballooning of cells, and the morphogenetic seed developed parenchymatous, polygonal cells filled with polyphenols. Histological studies indicated that in vitro plant regeneration involved the organogenic pathway that leads to de novo shoot primordia development.

Plant regeneration from root segments of Anthurium andraeanum and assessment of genetic fidelity of in vitro regenerates

A highly efficient regeneration system of Anthurium andraeanum Linden was established using root segments as explants of four genotypes, Arizona, SWD, OYH, and Mississippi. The root explants were firstly incubated on callus induction media containing NH4NO3 level of 206 mg L−1 or 825 mg L−1, and the calluses were formed from both ends of the root segments after 1 mo of culture. Root-derived calluses were then transferred to shoot regeneration media with NH4NO3 level of 412 mg L−1 or 1650 mg L−1, and numbers of shoot were successfully produced from the surface of calluses after 2 mo of culture. Medium containing 206 mg L−1 NH4NO3 induced more callus compared to 825 mg L−1. In contrast, the number of shoots per callus was the same between the above two shoot regeneration media indicating that NH4NO3 level had little effect on shoot regeneration. Long-termin vitro maintenance of the callus-bearing shoot initials was possible without compromising shoot regeneration and subsequent rooting. Histological analysis showed that early cell division was initiated from the inner cortical parenchyma cells near the vascular tissue and from the vascular parenchyma cells adjacent to the xylem vessel. The primary callus mainly comprised inner parenchyma cells with scattered procambial-like cells and xylem elements and two different meristematic zones beneath epidermal layers. Transfer of callus to the regenerative medium caused meristemoid formation and subsequent shoot primordia production. Calcium oxalate crystals and tannin-like deposits were visible on the outer callus layer or within the cotyledon-like structure of shoot primordium throughout callus development. Starch grains were abundant in root explants, but disappeared in the profusely growing primary callus, and were mobilized in the shoot primordium, indicating energy requirements of these two developmental stages. Flow cytometry analysis demonstrated that ploidy levels were maintained during in vitro cultures, including long-term regenerated plants. The inter-simple sequence repeat analysis indicated genomic DNA stability of Arizona and OYH but occasional genetic variation in primary regenerates of SWD and Mississippi.

Enhanced in vitro Shoot Regeneration and Biochemical Properties of Stevia rebaudiana using Chitosan

Stevia rebaudiana is a herbaceous perennial plant with great global demand due to its beneficial steviol glycosides(SGs) content. Current conventional breeding technique is unable to cater the need for more S. rebaudiana planting materials. Therefore, an improved in vitro shoot regeneration protocol was developed for S. rebaudiana by using chitosan. The highest fresh weight of plant (0.586 ± 0.176 g/explant), dry weight of plant (0.056 ± 0.02 g/explant) and plant height (4.94 ± 1.17 cm/explant) with maximum number of leaves (25.33 ± 6.95 /explant) were observed on explants grown in optimun treatment of MS basal medium supplemented with 1.0 mg/L of 6-benzyaminopurine (BAP) and 60 mg/L of low molecular weight (MW) chitosan after 4 weeks of culture. Scanning electron microscopy (SEM) analysis showed that new shoot primordia and shoot bud formation can be seen as early as day 3 and 5 of cultures on optimun treatment. Further biochemical assays showed that total phenolic acid content, total protein content and total hydrolyzed sugar content were recorded higher in explants cultured in optimun treatment as compared to control media. In contrast, total chlorophyll content and total flavonoids content were reduced in optimum treatment. Meanwhile, no significant difference in antioxidant activity was observed. All cultures from the optimal treatment were successfully regenerated, acclimatized and grew well with 100% survival rate. Taken together, an enhanced and efficient shoot regeneration protocol of S. rebaudiana was successfully developed which will be useful for rapid and large-scale micropropagation in future.

Exogenous toluene gas removal improvement in recombinant rabbit cytochrome P450 2E1 (CYP2E1)-transgenic Ardisia pusilla DC

The cytochrome P450 2E1 (CYP2E1) enzyme encoded by CYP2E1 plays an important role in the metabolism of organic compounds in mammalian liver cells. In this study, the plasmid pCAMBIA2300 harboring the rabbit CYP2E1 was transferred into Agrobacterium tumefaciens; this was then transferred into internodes of Ardisia pusilla. Shoot primordia, whose appearance resembles that of the in vitro protocorm-like body of orchids, regenerated from 121 of 332 internodes that were cultured in selection medium supplemented with 5 mg·L−1 kanamycin. PCR analysis confirmed that 25 of 89 putative transgenic plants (28.0%) stably harbored the CYP2E1 and NPTII genes, and Southern blot analysis revealed the presence of one to three copies of CYP2E1 within the genome of the 25 transgenic plants. The mRNA expression of the transgene was confirmed in 12 of 25 CYP2E1-transgenic plants by Northern blot analysis. Western blot analysis indicated the presence of a 53-kDa recombinant rabbit CYP2E1 protein in the 12 CYP2E1-transgenic A. pusilla plants. The aniline 4-hydroxylase activity of rabbit CYP2E1 in nine CYP2E1-transgenic A. pusilla plants was higher than that in non-transgenic A. pusilla plants. There was a statistically significant increase (5.1–6.1-fold) in the toluene removal ability of the transgenic line CYP2E1-2–2 compared to that observed in wild-type Ardisia plants at different times after exogenous toluene exposure.

Enhancement of In Vitro Production of Volatile Organic Compounds by Shoot Differentiation in Artemisia spicigera.

Callus initiation, shoot formation and plant regeneration were established for Artemisia spicigera, a traditional medicinal plant growing in Armenia, Middle-Anatolia and Iran, and producing valuable volatile organic compounds (VOCs) that are mostly represented by monoterpenoids. Optimal callus initiation and shoot production were obtained by culture of hypocotyl and cotyledon explants on MS medium comprising 0.5 mg L−1 naphthalene acetic acid (NAA) and 0.5 mg L−1 6-benzyladenine (BA). Consequently, the shoots were transferred onto the MS media supplemented with 1 mg L−1 of indole-3-butyric acid (IBA) or 1 mg L−1 of NAA. Both types of auxin induced root formation on the shoots and the resulting plantlets were successfully grown in pots. The production of VOCs in callus tissues and regenerated plantlets was studied by gas chromatography–mass spectrometry (GC-MS) analysis. Although the potential of undifferentiated callus to produce VOCs was very low, an increased content of bioactive volatile components was observed at the beginning of shoot primordia differentiation. Intriguingly, the volatiles obtained from in vitro plantlets showed quantitative and qualitative variation depending on the type of auxins used for the rooting process. The acquired quantities based on total ion current (TIC) showed that the regenerated plantlets using 1 mg L−1 NAA produced higher amounts of oxygenated monoterpenes such as camphor (30.29%), cis-thujone (7.07%), and 1,8-cineole (6.71%) and sesquiterpene derivatives, namely germacrene D (8.75%), bicyclogermacrene (4.0%) and spathulenol (1.49%) compared with the intact plant. According to these findings, in vitro generation of volatile organic compounds in A. spicigera depends on the developmental stages of tissues and may enhance with the formation of shoot primordia and regeneration of plantlets.

Shoot Regeneration Is Not a Single Cell Event.

Shoot regeneration is a key tool of modern plant biotechnology. While many researchers use this process empirically, very little is known about the early molecular genetic factors and signaling events that lead to shoot regeneration. Using tobacco as a model system, we found that the inductive events required for shoot regeneration occur in the first 4–5 days following incubation on regeneration medium. Leaf segments placed on regeneration medium did not produce shoots if removed from the medium before four days indicating this time frame is crucial for the induction of shoot regeneration. Leaf segments placed on regeneration medium for longer than five days maintain the capacity to produce shoots when removed from the regeneration medium. Analysis of gene expression during the early days of incubation on regeneration medium revealed many changes occurring with no single expression pattern evident among major gene families previously implicated in developmental processes. For example, expression of Knotted gene family members increased during the induction period, whereas transcription factors from the Wuschel gene family were unaltered during shoot induction. Expression levels of genes involved in cell cycle regulation increased steadily on regeneration medium while expression of NAC genes varied. No obvious possible candidate genes or developmental processes could be identified as a target for the early events (first few days) in the induction of shoot regeneration. On the other hand, observations during the early stages of regeneration pointed out that regeneration does not occur from a single cell but a group of cells. We observed that while cell division starts just as leaf segments are placed on regeneration medium, only a group of cells could become shoot primordia. Still, these primordia are not identifiable during the first days.

Control of oxidative stress by a combination of PBU, BAP and DMTU enhances adventitious shoot formation in Eucalyptus urophylla

The application of molecular breeding of eucalyptus is restricted by the difficulty of regeneration. One limitation of eucalyptus regeneration could be the production of excessive reactive oxygen species (ROS) during in vitro culture that greatly affects the shoot regeneration. NADPH oxidases, encoded by the respiratory burst oxidase homologue (rboh), and ascorbate peroxidases (APX) play an important role in the ROS metabolism. The study investigated the combined effect and preliminary mechanism on eucalyptus regeneration of N-phenyl-N′-[6-(2-chlorobenzothiazol)-yl] urea (PBU), 6-benzylaminopurine (BAP), and dimethylthiourea (DMTU) by inoculating Eucalyptus urophylla hypocotyls on SPCa medium supplemented with 0.57 µM indole-3-acetic acid (IAA) and various ratios of PBU and BAP. In the presence of 4.56 µM PBU alone, the rboh transcription levels were relatively low and the APX transcription levels were relatively high in the 2-week-old calli. However, the highest frequency of organogenic calli was induced by the combination of 3.99 µM PBU, 0.57 µM BAP and 0.57 µM IAA. DMTU, a ROS scavenger, was added in 10, 20, or 30 µM increments to the SPCa medium together with 3.99 µM PBU and 0.57 µM BAP to promote callus differentiation. The expression levels of all nine APX genes increased significantly in the calli following treatment with DMTU. The calli treated with 20 µM DMTU showed the highest APX transcriptional levels, which was consistent with the highest APX activity, and formed the most organogenic calli. During the subsequent process of adventitious shoot formation, the transcription levels of rboh1 showed a descending pattern. However, the transcription levels of rboh4, rboh5, and rboh6 showed an ascending pattern and peaked in the calli with visible shoot primordia. Specifically, the expression of rboh5 and rboh6 was 203.2 times and 573.9 times higher than that in the 2-week-old calli, respectively. Additionally, the calli with visible shoot primordia showed the highest H2O2 content. The results implied that control of oxidative stress by a combination of PBU, BAP, and DMTU enhances adventitious shoot formation in E. urophylla by affecting the expression of the rboh and APX genes and regulating ROS metabolism. The results would be helpful for an efficient regeneration protocol for eucalyptus. Cooperation of PBU, BAP, and DMTU enhances adventitious shoot formation in Eucalyptus urophylla by affecting the expression of rboh and APX genes.

Cryopreservation of Multiple Shoot Primordia of Endangered Plant Nymphoides indica and Regeneration of Plants from Cryopreserved Tissues

Cryopreservation of Multiple Shoot Primordia of Endangered Plant Nymphoides indica and Regeneration of Plants from Cryopreserved Tissues

The microvine, a model to study the effect of temperature on grapevine latent bud development and fruitfulness

Aim: The success of inflorescence primordia initiation and differentiation within latent buds (i.e. bud fruitfulness) is a critical issue for grapevine yield sustainability under climate change. The aim of the present study was to track the timing and rate of inflorescence development in latent buds along the cane and to quantify their responses to elevated day/night (D/N) temperatures.Methods and Results: The experiments were conducted under controlled conditions, using the microvine model, which is suitable for establishment in small areas. Two imagery methods for analyzing bud anatomy were assessed: light microscopy and x-ray microtomography. Light microscopy was laborious, but it was the most accurate method for investigating organogenesis in the primordial shoot of the latent bud. In plants grown in a greenhouse (D/N, 25°C/15°C), the number of phytomer primordia in latent buds increased linearly from the apical to the basal buds on the cane. A maximum of six phytomers and two inflorescence primordia were observed beneath the 20th bud position that is, slightly fewer than usually reported with macrovines. The first and second inflorescences started to differentiate at the 14th and 18th bud position, respectively. Temperature increases in the growth chamber (D/N, 20–30°C/15–25°C) only slightly changed the final number of preformed phytomers and the probability of inflorescence primordia differentiation per bud. However, elevated temperature sharply accelerated and thereby shortened development of the latent bud primordial shoot, resulting in differentiation of the first inflorescence primordia straight from the fifth bud position. Based on the spatiotemporal conversion of bud position into thermal time, the first inflorescence started to differentiate 332 growing degree days (°Cd) (or 41 days) after bud emergence at D/N 20°C/15°C, and only 98°Cd (or 5 days) after bud emergence at D/N 35°C/25°C. Finally, the number of preformed phytomers was shown to correlate with primary bud length and cane diameter, independent of temperature. These easily measured variables may be used as indicators of bud developmental stage and potential bud fruitfulness in further studies using the microvine.Conclusions: The microvine appears to be suitable for parameterizing a developmental model of grapevine latent buds under controlled environmental conditions and when evaluating the response to elevated D/N temperatures.Significance and impact of the study: The precise description of the timing and rate of differentiation of phytomers and inflorescences opens new perspectives for understanding the molecular processes underlying the response of bud fruitfulness to environmental constraints.

Expression pattern of CUC3 ortholog in Zeylanidium tailichenoides (Podostemaceae) infers organization of a unique distichous shoot in Podostemoideae.

Shoots of the aquatic eudicot family, Podostemaceae, exhibit unusual organogenesis with mixed leaf and stem identities. New shoots arise at the base of the older shoot with shoot apical meristem (SAM) identity but the entire SAM differentiates into a "leaf" as it develops in the Podostemoideae subfamily. The "leaves" are tightly arranged in a zigzag manner to form an apparent distichous shoot as a whole. Although previous studies have suggested that Podostemoideae shoots have evolved by modifying the ancestral sympodial branching system in the basal Tristichoideae subfamily, this evolutionary scenario requires elucidation at the molecular level. To confirm that the shoots arise as axillary shoots, in the present study, we examined gene expression patterns in plumular shoots of Zeylanidium tailichenoides using CUP-SHAPED COTYLEDON 3 (CUC3) and SHOOT MERISTEMLESS (STM) orthologs, which are involved in the determination of axils and meristem formation in model plants. Expression of the CUC3 ortholog was detected at the adaxial base of cotyledons and parental shoots where the new shoots are initiated, while STM ortholog was expressed at the initiation site and in the young shoot primordia throughout early shoot development. The results demonstrate that each Z. tailichenoides shoot arises as an axillary bud in a manner similar to axillary meristem formation in model plants involving CUC3 and STM genes. Considering that each of the two cotyledons produces an axillary bud that in turn continues to form its own axillary bud independently, the apparent distichous shoot in Z.tailichenoides is not a single shoot, but a composite of two sympodially branched shoots.

Rhythm of Seasonal Development and Minor Life Cycle of Prunella vulgaris L. (Lamiaceae) in Khakasia

Seasonal rhythms of plant development are related to plant adaptation to surrounding ecological coenotic and climatic conditions. To discover the patterns of seasonal development of plants in different phytocoenoses, it is essential to observe individual shoots and the entire course of shoot formation. So far the data on the rhythm of seasonal development of Prunella vulgaris L., a plant of the circumboreal region, have been available for the European part of Russia only. We examined the rhythm of seasonal development and minor life cycle of Prunella vulgaris L., the long-rhizome life form, in Siberia (Khakasia) in 2012-2013. Observations were carried out in a forest meadow every 5-7 days in the spring-summer-autumn period and 1-2 times a month in the winter. The development of dicyclic meso-rosetted and winter monocyclic semi-rosetted generative monocarpic shoots was observed using the method by I.G. Serebryakov. It was determined that the differentiation of vegetative and generative spheres in Prunella vulgaris L. shoots in Siberia occurs in early spring within the year of flowering. The species forms spring and autumn leaf generations and phenologically can be described as a summer-winter green plant with a long growing season. The period of shoot development from its initiation to dying the above-ground parts lasts 26 and 14 months in dicyclic meso-rosetted shoots and winter monocyclic semi-rosetted shoots, respectively. In both cases the development of the primordial shoot inside the bud lasts 8 months while the duration of shoot development after emerging from the bud varies. The latter phase lasts 18 months in dicyclic meso-rosetted shoots and only 6 months in winter monocyclic semi-rosetted shoots. Start times and duration of phenological phases (budding, flowering, fruiting and dissemination) are related to the weather conditions in the habitats including fluctuations in air temperature, precipitation patterns and types

Augmenting a competent in vitro organogenesis etiquette from leaf base of country mallow, Abutilon indicum L. sweet: An ethno-botanically valuable medicinal plant

Abstract An easy, efficient and highly reproducible regeneration system was established through organogenesis from leaf base mediated callus of an ethnobotanical hairy shrub Abutilon indicum L. sweet, which is documented to possess pharmaceutically important phytochemicals. Among various explants, leaf bases produced significant callus induction (89%) with 140 mg fresh weight on Murashige and Skoog (MS) medium supplemented with 11.31 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D). Combinations of 2, 4-D and kinetin (KIN) increased the biomass (191 mg) of embryogenic calli. Regeneration medium with a combination of 8.88 μM of 6-amino benzyl purine (BAP) and 8.06 μM of naphthalene acetic acid in MS basal media significantly influenced the initiation of shoot primordium with 91% of regeneration. Proficiently regenerated shoot apical meristems (SAMs) produced higher frequency of multiple shoot induction on 4.10 μM of zeatin. The fully regenerated mature shootlets produced high root biomass (791.67 ± 48.05 mg) on half - strength MS with 4.54 μM indole-3-butyric acid (IBA) with an excellent mat root system for efficient acclimatization. In vitro established plantlets were successfully acclimatized under ex vitro conditions with high frequency. Presence of homologous DNA banding pattern with Single Primer Amplification Reaction markers confirmed the genetic similarity. FTIR and GC-MS spectrum showed similar metabolic profiles between wild and in vitro regenerated plantlets. Thus the protocol is reliable for in vitro regeneration through leaf base derived callus which could pave a way to the large scale production of secondary metabolite through biotechnological approaches.