CRISPR/Cas9-Mediated Genome Editing in Indica Rice (Oryza sativa L. subsp. indica var. CR-5272).

Tissue culture optimization protocols limit indica rice breeding. Such a challenge is vital because emergent techniques still rely on tissue culture methods and could allow the breeding of new varieties with higher production and toleration of adverse environmental effects caused by climate change. Genome editing technology, using CRISPR/Cas9, is a fast and precise method for accelerated plant breeding. It limited its use in indica subspecies because of the recalcitrant response to in vitro culture methods. This chapter describes a protocol for CRISPR/Cas9 editing in indica subspecies, specifically in the CR-5272 variety derived from parental lines IR-822, using Agrobacterium tumefaciens and biolistic transformation.

Insights into plant regeneration: cellular pathways and DNA methylation dynamics.

Plants, known for their immobility, employ various mechanisms against stress and damage. A prominent feature is the formation of callus tissue-a cellular growth phenomenon that remains insufficiently explored, despite its distinctive cellular plasticity compared to vertebrates. Callus formation involves dedifferentiated cells, with a subset attaining pluripotency. Calluses exhibit an extraordinary capacity to reinitiate cellular division and undergo structural transformations, generating de novo shoots and roots, thereby developing into regenerated plants-a testament to the heightened developmental plasticity inherent in plants. In this way, plant regeneration through clonal propagation is a widely employed technique for vegetative reproduction. Thus, exploration of the biological components involved in regaining pluripotency contributes to the foundation upon which methods of somatic plant propagation can be advanced. This review provides an overview of the cellular pathway involved in callus and subsequent de novo shoot formation from already differentiated plant tissue, highlighting key genes critical to this process. In addition, it explores the intricate realm of epigenetic regulatory processes, emphasizing the nuanced dynamics of DNA methylation that contribute to plant regeneration. Finally, we briefly discuss somaclonal variation, examining its relation to DNA methylation, and investigating the heritability of epigenomic changes in crops.

Plant regeneration capacity in seeds of three species of Miconia (Melastomataceae) may be related to endogenous polyamine profiles.

In plant tissue culture, differences in endogenous levels of species-specific plant growth regulators (PGRs) may explain differences in regenerative capacity. In the case of polyamines (PAs), their dynamics and distribution may vary between species, genotypes, tissues, and developmental pathways, such as sexual reproduction and apomixis. In this study, for the first time, we aimed to assess the impact of varying endogenous PAs levels in seeds from distinct reproductive modes in Miconia spp. (Melastomataceae), on their in vitro regenerative capacity. We quantified the free PAs endogenous content in seeds of Miconia australis (obligate apomictic), Miconia hyemalis (facultative apomictic), and Miconia sellowiana (sexual) and evaluated their in vitro regenerative potential in WPM culture medium supplemented with a combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP). The morphogenic responses were characterized by light microscopy and scanning electron microscopy and discussed regarding the endogenous PAs profiles found. Seeds of M. sellowiana presented approximately eight times more putrescine than M. australis, which was associated with a higher percentage of regenerated calluses (76.67%) than M. australis (5.56%). On the other hand, spermine levels were significantly higher in M. australis. Spermine is indicated as an inhibitor of auxin-carrying gene expression, which may have contributed to its lower regenerative capacity under the tested conditions. These findings provide important insights into in vitro morphogenesis mechanisms in Miconia and highlight the significance of endogenous PA levels in plant regeneration. These discoveries can potentially optimize future regeneration protocols in Miconia, a plant group still underexplored in this area.

Multilayered Gel-Spotting Device for In Vitro Reconstruction of Hair Follicle-like Microstructure.

Hair follicles play an important role in hair development. This study aimed to develop a microgel-spotting device to fabricate a multilayered gel bead culture model and to mimic the early development of skin appendages to regenerate hair follicles in vitro. The model consists of an alginate gel layer containing cytokines as the core layer, a collagen gel layer containing mouse embryonic stem cells as the middle layer, and a collagen gel layer containing fetus-derived epidermal cells as the outer layer. A concentration gradient of cytokines is formed, which promotes interactions between epidermal and stem cells. Histological and immunnohistological analyses confirmed the reconstruction of hair follicle structures. As a result, the cell number and gel bead size could be precisely controlled by the developed microgel-spotting device. In the multilayered gel bead, the embryonic and epidermal cells cultured with the cytokine gradient formed cell aggregates with keratinized tissue in the center similar to "native" hair follicle structure. Sweat gland-like luminal tissue and erector pilorum-like structures were also observed around aggregates with concentric structures. In conclusion, the multilayered gel bead culture model demonstrated potential for in vitro hair follicle regeneration. The findings of this study provide insight into the early development of skin appendages.

Optimized Recovery of Cryostored Dormant Buds of Mulberry Germplasm.

A two-step freezing cryoprotocol preceded by desiccation to 15 to 25% moisture content was developed and successfully applied to winter dormant buds of mulberry (different Morus spp.) of a core set comprising 238 accessions studies in our laboratory. The survival and recovery percentage of diverse accessions cryobanked for various periods were tested under in vitro conditions, and several factors were analyzed to determine their role in optimizing the recovery of low-viability accessions. The effect of rates of freezing and thawing (both fast and slow), were tested and recovery compared. Recovery conditions such as dark incubation and rehydration in sterile moist moss grass for different durations after cryopreservation led to a higher survival percentage compared to controls. Two different recovery culture media were compared for their efficiency in survival. On average, the survival under in vitro culture conditions using optimized conditions was high: above 60% in majority of the accessions. Dormant buds showed viability in the range of 25 to 100% with an average of 50.4%. The recovery percentage of winter dormant buds after cryopreservation via slow freezing and slow thawing with rehydration by moist moss grass for 2 h was recorded in the range from 63.3 to 90.9% with an average of 81.05%. Without rehydration, it ranged from 50 to 75% with an average of 60.4%. Regeneration of cryopreserved mulberry germplasm after 6 years of storage indicated no survival loss over different years of storage, and 33-40% of the accessions showed viability above 40%, up to a maximum of 100%. Maximum shoot formation (100%) was obtained from Morus alba. The majority of the accessions were rooted in vitro within 20-25 days of subculture in the auxin rich rooting media, except in wild species M. latifolia and M. laevigata, which took longer (45 to 60 days) for root development. All the rooted plantlets were then transferred to the field and successfully established in a glasshouse.

Establishment of in vitro regeneration system and molecular analysis of early development of somatic callus in Capsicum chinense and Capsicum baccatum.

Regeneration is extremely important to pepper genetic development; however, the molecular mechanisms of how the callus reactivates cell proliferation and promotes cell reprogramming remain elusive in pepper. In the present study, C. baccatum (HNUCB81 and HNUCB226) and C. chinense (HNUCC22 and HNUCC16) were analyzed to reveal callus initiation by in vitro regeneration, histology, and transcriptome. We successfully established an efficient in vitro regeneration system of two cultivars to monitor the callus induction of differential genotypes, and the regenerated plants were obtained. Compared to C. chinense, there was a higher callus induction rate in C. baccatum. The phenotype of C. baccatum changed significantly and formed vascular tissue faster than C. chinense. The KEGG enrichment analysis found that plant hormone transduction and starch and sucrose metabolism pathways were significantly enriched. In addition, we identified that the WOX7 gene was significantly up-regulated in HNUCB81 and HNUCB226 than that in HNUCC22 and HNUCC16, which may be a potential function in callus formation. These results provided a promising strategy to improve the regeneration and transformation of pepper plants.

Micropropagation of Andrographis producta through axillary and adventitious shoot regeneration.

BACKGROUND: Andrographis producta (C. B. Clarke) Gamble is a valuable medicinal plant that yields several therapeutic compounds. In addition, this species is endemic to the Western Ghats regions of South India. Natural populations of Andrographis producta have dwindled due to the overexploitation of this species. The objective of the present study was to develop a reliable In vitro propagation protocol for this plant species. RESULTS: In vitro plant regeneration protocol has been developed in Andrographis producta using nodal and shoot tip explants. The highest axillary shoots (14.60) were regenerated from nodal explants on MS medium amended with 10 µM 2-iP. Similarly, on MS media amended with 5 µM BAP, 17.50 shoots were regenerated from shoot tip explants. Optimal of 27.66 adventitious shoots were regenerated from the cut end of shoot tip explants on MS medium amended with 10 µM 2-iP. Medium amended with 10 µM 2-iP was optimum for regeneration of multiple axillary shoots from nodal explants and for the adventitious shoots regeneration from shoot tip explants. Shoot tips were ideal explants for the micropropagation of A. producta. Qarter sthrength MS media supplemented with 10 µM IBA has resulted in maximum rooting of the shoots. CONCLUSIONS: A reliable In vitro micropropagation method was developed in Andrographis producta through direct organogenesis, and this method is helpful for the multiplication, conservation, and utilization of this plant.

Development of a Transformation System and Locus Identification Pipeline for T-DNA in Chrysanthemum seticuspe, A Model Species for Hexaploid Cultivated Chrysanthemum.

Chrysanthemum is one of the most popular flowers worldwide and has high aesthetic and commercial value. However, the cultivated varieties of chrysanthemum are hexaploid and highly heterozygous, which makes gene editing and gene function research difficult. Gojo-0 is a diploid homozygous line bred from a self-compatible mutant of Chrysanthemum seticuspe and is expected to become a model plant of the genus Chrysanthemum. After assessment of different growth regulator combinations, the optimal concentrations of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (6-BA) in the regeneration system were 1.0 mg·L-1 and 0.2 mg·L-1, respectively. In the genetic transformation system, the selected concentrations of kanamycin, hygromycin and glufosinate-ammonium were 10 mg·L-1, 2.5 mg·L-1 and 0.6 mg·L-1 for bud generation and 12 mg L-1, 1.5 mg·L-1 and 0.5 mg·L-1 for rooting. The transgenic plants were verified by not only PCR detection and GUS staining, but also identification of the T-DNA insertion locus using high-throughput sequencing. Our results lay the foundation for gene functional research on chrysanthemum and will help with the identification of transgenic plants.

Innovation in the Breeding of Common Bean Through a Combined Approach of in vitro Regeneration and Machine Learning Algorithms.

Common bean is considered a recalcitrant crop for in vitro regeneration and needs a repeatable and efficient in vitro regeneration protocol for its improvement through biotechnological approaches. In this study, the establishment of efficient and reproducible in vitro regeneration followed by predicting and optimizing through machine learning (ML) models, such as artificial neural network algorithms, was performed. Mature embryos of common bean were pretreated with 5, 10, and 20 mg/L benzylaminopurine (BAP) for 20 days followed by isolation of plumular apice for in vitro regeneration and cultured on a post-treatment medium containing 0.25, 0.50, 1.0, and 1.50 mg/L BAP for 8 weeks. Plumular apice explants pretreated with 20 mg/L BAP exerted a negative impact and resulted in minimum shoot regeneration frequency and shoot count, but produced longer shoots. All output variables (shoot regeneration frequency, shoot counts, and shoot length) increased significantly with the enhancement of BAP concentration in the post-treatment medium. Interaction of the pretreatment × post-treatment medium revealed the need for a specific combination for inducing a high shoot regeneration frequency. Higher shoot count and shoot length were achieved from the interaction of 5 mg/L BAP × 1.00 mg/L BAP followed by 10 mg/L BAP × 1.50 mg/L BAP and 20 mg/L BAP × 1.50 mg/L BAP. The evaluation of data through ML models revealed that R 2 values ranged from 0.32 to 0.58 (regeneration), 0.01 to 0.22 (shoot counts), and 0.18 to 0.48 (shoot length). On the other hand, the mean squared error values ranged from 0.0596 to 0.0965 for shoot regeneration, 0.0327 to 0.0412 for shoot count, and 0.0258 to 0.0404 for shoot length from all ML models. Among the utilized models, the multilayer perceptron model provided a better prediction and optimization for all output variables, compared to other models. The achieved results can be employed for the prediction and optimization of plant tissue culture protocols used for biotechnological approaches in a breeding program of common beans.

Efficient regeneration of mature castanopsis hystrix from in vitro stem explants.

Castanopsis hystrix is one of the main timber trees grown in China. However, severe shortage of natural seeds and the difficulty of explant regeneration has limited seedling supply. As such, there is a need for research on asexual multiplication of C. hystrix. This study established a rapid propagation technology system for C. hystrix genotypes, including explant treatment, proliferation, and rooting. HZ (a modified MS medium) supplemented with 4.4 µM BA and 0.5 µM IBA was found to be the optimal medium for shoot sprouting. The maximum proliferation coefficient and the number of effective shoots was obtained on HZ medium supplemented with 2.6 µM BA and 1.0 µM IBA, were 3.00 and 5.63, respectively. A rooting rate of 83.33% was achieved using half-strength HZ medium supplemented with 3.2 µM NAA. Adding vitamin C (80 mg⋅l-1) for 7 days in a dark environment reduced the browning rate, while increasing the proliferation rate. Additionally, through cytological observation, we established how and where adventitious roots occur. The survival rate of transplanted plantlets was > 90%. This is the first report of an in vitro regeneration technique that uses stem segments of mature C. hystrix as explants.

Callus-Mediated High-Frequency Plant Regeneration, Phytochemical Profiling, Antioxidant Activity and Genetic Stability in Ruta chalepensis L.

Efficient methods for callus induction and the high-frequency plant regeneration of Ruta chalepensis L. were established, and the phytochemical potential and antioxidant activity of a donor plant, ex-vitro-established micropropagated plants, and callus were also studied. Yellowish-green callus was induced with a frequency of 97.8% from internode shoot segments of the donor plant growing in soil in the botanical garden cultured on Murashige and Skoog (MS) medium containing 10 µM 2,4-D (2,4-dichlorophenoxyacetic acid) and 1 µM BA (6-benzyladenine). Adventitious shoots were regenerated from the yellowish-green callus on MS medium containing 5.0 µM (BA) and 1.0 µM 1-naphthaleneacetic acid (NAA), with a regeneration frequency of 98.4% and a maximum of 54.6 shoots with an average length of 4.5 cm after 8 weeks. The regenerated shoots were rooted in a medium containing 1.0 µM IBA (indole-3-butyric acid) and successfully transferred to ex vitro conditions in pots containing normal garden soil, with a 95% survival rate. The amounts of alkaloids, phenolics, flavonoids, tannins, and antioxidant activity of the ex-vitro-established micropropagated plants were higher than in the donor plant and callus. The highest contents of hesperidin and rutin (93.3 and 55.9 µg/mg, respectively) were found in the ex-vitro-established micropropagated plants compared to those obtained from the donor plant (91.4 and 31.0 µg/mg, respectively) and callus (59.1 and 21.6 µg/mg, respectively). The genetic uniformity of the ex-vitro-established micropropagated plants was appraised by the ISSR markers and compared with the donor plant. This is the first report describing the callus-mediated plant regeneration, as well as the production of phenolic compounds and antioxidant activities in R. chalepensis, which might be a potential alternative technique for the mass propagation and synthesis of bioactive compounds such as hesperidin and rutin.

Overexpressing Arabidopsis thaliana ACBP6 in transgenic rapid-cycling Brassica napus confers cold tolerance.

BACKGROUND: Rapid-cycling Brassica napus (B. napus-RC) has potential as a rapid trait testing system for canola (B. napus) because its life cycle is completed within 2 months while canola usually takes 4 months, and it is susceptible to the same range of diseases and abiotic stress as canola. However, a rapid trait testing system for canola requires the development of an efficient transformation and tissue culture system for B. napus-RC. Furthermore, effectiveness of this system needs to be demonstrated by showing that a particular trait can be rapidly introduced into B. napus-RC plants. RESULTS: An in-vitro regeneration protocol was developed for B. napus-RC using 4-day-old cotyledons as the explant. High regeneration percentages, exceeding 70%, were achieved when 1-naphthaleneacetic acid (0.10 mg/L), 6-benzylaminopurine (1.0 mg/L), gibberellic acid (0.01 mg/L) and the ethylene antagonist silver nitrate (5 mg/L) were included in the regeneration medium. An average transformation efficiency of 16.4% was obtained using Agrobacterium-mediated transformation of B. napus-RC cotyledons using Agrobacterium strain GV3101 harbouring a plasmid with an NPTII (kanamycin-selectable) marker gene and the Arabidopsis thaliana cDNA encoding ACYL-COA-BINDING PROTEIN6 (AtACBP6). Transgenic B. napus-RC overexpressing AtACBP6 displayed better tolerance to freezing/frost than the wild type, with enhanced recovery from cellular membrane damage at both vegetative and flowering stages. AtACBP6-overexpressing B. napus-RC plants also exhibited lower electrolyte leakage and improved recovery following frost treatment, resulting in higher yields than the wild type. Ovules from transgenic AtACBP6 lines were better protected from frost than those of the wild type, while the developing embryos of frost-treated AtACBP6-overexpressing plants showed less freezing injury than the wild type. CONCLUSIONS: This study demonstrates that B. napus-RC can be successfully regenerated and transformed from cotyledon explants and has the potential to be an effective trait testing platform for canola. Additionally, AtACBP6 shows potential for enhancing cold tolerance in canola however, larger scale studies will be required to further confirm this outcome.

Citrus Genetic Transformation: An Overview of the Current Strategies and Insights on the New Emerging Technologies.

Citrus are among the most prevailing fruit crops produced worldwide. The implementation of effective and reliable breeding programs is essential for coping with the increasing demands of satisfactory yield and quality of the fruit as well as to deal with the negative impact of fast-spreading diseases. Conventional methods are time-consuming and of difficult application because of inherent factors of citrus biology, such as their prolonged juvenile period and a complex reproductive stage, sometimes presenting infertility, self-incompatibility, parthenocarpy, or polyembryony. Moreover, certain desirable traits are absent from cultivated or wild citrus genotypes. All these features are challenging for the incorporation of the desirable traits. In this regard, genetic engineering technologies offer a series of alternative approaches that allow overcoming the difficulties of conventional breeding programs. This review gives a detailed overview of the currently used strategies for the development of genetically modified citrus. We describe different aspects regarding genotype varieties used, including elite cultivars or extensively used scions and rootstocks. Furthermore, we discuss technical aspects of citrus genetic transformation procedures via Agrobacterium, regular physical methods, and magnetofection. Finally, we describe the selection of explants considering young and mature tissues, protoplast isolation, etc. We also address current protocols and novel approaches for improving the in vitro regeneration process, which is an important bottleneck for citrus genetic transformation. This review also explores alternative emerging transformation strategies applied to citrus species such as transient and tissue localized transformation. New breeding technologies, including cisgenesis, intragenesis, and genome editing by clustered regularly interspaced short palindromic repeats (CRISPR), are also discussed. Other relevant aspects comprising new promoters and reporter genes, marker-free systems, and strategies for induction of early flowering, are also addressed. We provided a future perspective on the use of current and new technologies in citrus and its potential impact on regulatory processes.

Genetic diversity and volatile oil components variation in Achillea fragrantissima wild accessions and their regenerated genotypes.

BACKGROUND: Wild medicinal plants are suffering natural environmental stresses and habitat destruction. The genetic diversity evaluation of wild accessions and their in vitro raised genotypes using molecular markers, as well as the estimation of substances of pharmaceutical value in wild plants and their regenerated genotypes are convenient approaches to test the genetic fidelity of regenerated plants as a source of substances of pharmaceutical value. In this study, the genetic diversity of 12 accessions of the medicinal plant Achillea fragrantissima, representing five sites in the mountains of South Sinai, Egypt, were estimated by the inter simple sequence repeats (ISSR) fingerprinting and their volatile oil components were identified using gas chromatography-mass spectrometry (GC-MS) analysis. The same accessions were regenerated in vitro and the genetic diversity and volatile oil components of propagated genotypes were determined and compared to their wild parents. RESULTS: Clustering and principal component analyses indicated that the wild accessions and their regenerated genotypes were genetically differentiated, but the regenerated plants are relatively more diverse compared to their wild parents. However, genetic variation between wild accessions is inherited to their in vitro propagated genotypes indicating genotypic differentiation of the examined accessions. The number of volatile oil compounds in the wild A. fragrantissima accessions was 31 compounds while in the in vitro propagated plants only 24 compounds were detected. Four major compounds are common to both wild and regenerated plants; these are artemisia ketone, alpha-thujone, dodecane, and piperitone. CONCLUSIONS: Genome profiling and essential oil components analysis showed variations in A. fragrantissima accessions from different populations. Genetic differences between wild and regenerated genotypes were analyzed and validated with the final conclusion that in vitro conditions elicited higher genetic variation that is associated with reduced amount and diversity in the essential oil components.

In vitro Regeneration of Hyssopus officinalis L. and Plant Genetic Similarity.

Biotechnological methods are an essential component of plant genetic resources investigation that allow both to preserve a rare natural plant and useful selection genotypes, especially essential oil bearing plants used in medicine, perfume, food, etc. In the clonal micropropagation in vitro, the key moment is to retain genetic stability of in vitro material. It is considered that plant regeneration in vitro from meristem or vegetative buds gives identical clones, but the effect of the medium growth regulators on the genetic stability of in vitro plant material is discussed. Therefore, the objective of our work was to evaluate the genetic similarity of ex situ and in vitro plants. Investigation was performed on Hyssopus officinalis L. cv. Nikitskiy Beliy (NBG selection). Regeneration from the shoot single-node segments on the modified Murashige and Skoog culture medium (MS) supplemented with 6-benzylaminopurine (BAP) in concentration 0.3-0.9 mg/L and 0.1 mg/L Indole-3-butyric acid (IBA) was carried out. It was established, that application of 0.5 mg/L BAP was sufficient for the organ formation and development without morphological deviations. Genetic analysis based on RAPD and ISSR-PCR had shown full genetic similarity of the investigated ex situ and in vitro plants.

Organic and inorganic elicitors enhance in vitro regeneration of Rosa canina.

BACKGROUND: Rosa canina is one of the most popular rose species which is widely used as the rootstock for the propagation of rose cultivars. The purpose of the present study is to improve the in vitro propagation efficiency of this valuable plant species using various growth stimulants in a proliferation medium. In this study, in vitro-derived axillary buds of R. canina were inoculated in Vander Salm (VS) medium supplemented with varying levels of organic or inorganic elicitors including casein hydrolysate (200, 400, and 600 mg/l), glutamic acid (2, 4, 8, and 12 mg/l), proline (500, 1000, 1500, and 2000 mg/l), and silver nitrate (25, 50, 75, and 100 mg/l), separately. Benzyl amino purine (BAP) as well as naphthalin acetic acid (NAA) were added to all media at a constant rate to promote shoot proliferation. RESULTS: The results indicated that the supplementation of casein hydrolysate to the VS medium markedly stimulated shoot regeneration by 173% in comparison to control. Shoot proliferation was also positively influenced by glutamic acid at all levels, however, at a lesser extent compared to casein hydrolysate. Silver nitrate at 100 mg/l induced the longest shoots (2.52 ± 0.248 cm) and maximum leaf number (8.90 ± 0.276) among all treatments. Although it did not encourage efficient shoot regeneration, the highest quality shoots with maximum growth vigor were observed in this treatment. CONCLUSION: In this study, the promising role of casein hydrolysate in combination with plant growth regulators has been emphasized for the improved efficiency of R. canina regeneration protocol. Moreover, the addition of silver nitrate to the culture medium seems vital for enhancing the quality of regenerated shoots. The results of this study could be beneficial either for the further pharmaceutical or biochemical investigations of R. canina or commercial purposes for mass propagation of this specimen.

The Roots of Rye (Secale cereale L.) Are Capable of Synthesizing Benzoxazinoids.

According to current opinion, the first step of benzoxazinoids (BXs) synthesis, that is, the conversion of indole-3-glycerol phosphate to indole, occurs exclusively in the photosynthesising parts of plants. However, the results of our previous work and some other studies suggest that this process may also occur in the roots. In this study, we provide evidence that the first step of BXs synthesis does indeed occur in the roots of rye seedlings. We detected ScBx1 transcripts, BX1 enzyme, and six BXs (2-hydroxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one, (2R)-2-O-ß-d-glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one glucoside, 2,4-dihydroxy- 7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside, and 6-methoxy-2-benzoxazolinone) in the roots developed from seeds deprived of the coleoptile at 2 days after sowing (i.e., roots without contact with aerial parts). In roots regenerated in vitro, both ScBx1 transcripts and BX1 enzyme were detected at a low but still measurable levels. Thus, BXs are able to be synthesised in both the roots and above-ground parts of rye plants.

Global 5-methylcytosine and physiological changes are triggers of indirect somatic embryogenesis in Coffea canephora.

Indirect somatic embryogenesis (ISE) establishment for Coffea species started in the 1970s. Since then, intraspecific variations in the morphogenic pathway have been reported, even in the common environmental condition in vitro. Several authors have suggested that these variations are the result of genetic, epigenetic, and/or physiological events, highlighting the need for investigations to know the causes. Along these lines, this study aimed to investigate and describe, for the first time, the global 5-methylcytosine and physiological changes that occur in the cells of the aggregate suspensions of Coffea canephora during proliferation and somatic embryo regeneration steps. The cell proliferation step was characterized by increase in cell mass in all subcultures; relatively low mean values of global 5-methylcytosine (5-mC%), abscisic acid (ABA), and indole-3-acetic acid (IAA); high mean value of 1-aminocyclopropane-1-carboxylic acid (ACC, an ethylene precursor); and increase followed by decrease in spermidine (Spd, a polyamine) level. Therefore, these epigenetic and physiologic aspects promoted the cell proliferation, which is fundamental for ISE. In turn, the somatic embryo regeneration was correlated with global 5-mC% and physiological changes. The competence acquisition, determination, and cell differentiation steps were marked by increases in mean values of 5-mC%, IAA and ABA, and decreases in ACC and Spd, evincing that these changes are the triggers for regeneration and maturation of somatic embryos. Therefore, dynamic and coordinated epigenetic and physiologic changes occur in the cells of the aggregate suspensions during the C. canephora ISE in liquid system.

Hairy root culture technology: applications, constraints and prospect.

Hairy root (HR) culture, a successful biotechnology combining in vitro tissue culture with recombinant DNA machinery, is intended for the genetic improvement of plants. This technology has been put to use since the last three decades for genetic advancement of medicinal and aromatic plants and also to harvest the economical products in the form of secondary metabolites that are significantly important for their ethnobotanical and pharmacological properties. It also provides an efficient way out for the quicker extraction and quantification of the valuable phytochemicals. The current review provides an account of the in vitro HR culture technology and its wide-scale applications in the field of research as well as in pharmaceutical industries. Different facets of HR with respect to the culture establishment, phytochemical production as well as research investigations concerning the areas of gene manipulation, biotransformation of the secondary metabolites, phytoremediation, their industrial utilisations and different problems encountered during the application of this technology have been covered in this appraisal. Eventually, an idea has been provided on HR about the recent trends on the progress of this technology that may open up newer prospects in near future and calls for further research and explorations in this field. KEY POINTS: • Genetic engineering-based HR culture aims towards enhanced secondary metabolite production. • This review explores an insight in the HR technology and its multi-faceted approaches. • Up-to-date ground-breaking research applications and constraints of HR culture are discussed.

Impact of green synthesized WcAgNPs on in-vitro plant regeneration and withanolides production by inducing key biosynthetic genes in Withania coagulans.

KEY MESSAGE: Withania coagulans (L.) Dunal bio-synthesized silver nanoparticles (WcAgNPs) worked as an abiotic elicitor or auto-catalyst that enhanced root regeneration and withanolides production in in-vitro regenerated W. coagulans. Rapid development in the production / consumption of silver nanoparticles (AgNPs) raised serious concern over its effects on the growth of natural plant community. The knowledge related to impact of AgNPs on plant growth and biocompatibility is increasing day by day, but comprehensive mechanism and gaps regarding their impacts on plant health have yet to be addressed. In the present study, we investigated the impact of Withania coagulans biosynthesized AgNPs (WcAgNPs) on in-vitro plant growth and withanolides production. Obtained results showed that the low concentrations of WcAgNPs significantly induced the plant growth by regulating oxidative stress via anti-oxidative defense system. Physiological, morphology and anatomical features also reflected healthy plant growth under low WcAgNPs exposure. While higher concentrations of WcAgNPs have a negative impact on W. coagulans plant growth due to induced lipid peroxidation, ROS accumulation, and root cell death. At lower concentrations, WcAgNPs have shown a positive effect on in-planta withanolides biosynthesis stimulating withanolide A and withaferin A up to 11.15-22.8-fold, respectively. Furthermore, the expression of withanolides biosynthetic genes were also quantified upon WcAgNPs exposure and terpenes biosynthetic genes showed over-expression. Thus, the present study concludes that the lower concentrations of WcAgNPs positively induced plant growth via improved root organogenesis and also have potential to act as an elicitor for withanolides production.