Green Callus Research Articles

Not everything that is green is Gonolobus uniflorus (Apocynaceae, Asclepiadoideae): a new species for Mexico

A new species of Gonolobus restricted to the Mexican states Jalisco and Michoacán and that has usually been confused with G. uniflorus is described. Gonolobus leotzinii is characterized by having sepals 3.48–5.08 mm long, corolla 1.5–1.75 cm in diameter with a whitish green callus on the margin of the lobes, and a gynostegial corona with five cucculate lobes. The new species is generally confused with Gonolobus uniflorus, which is why it has gone unnoticed in herbaria. This work presents a detailed description, data on the species’ habitat, phenology, and conservation status. A distribution map of the new species and an identification key for the Gonolobus species distributed in Jalisco and Michoacán is included.

Comparative Transcriptomic Insights into the Mechanisms Underlying Maize (Zea mays L.) Embryogenic Callus Differentiation

The regeneration of plant somatic cells is a prerequisite for their biological breeding. Identification of key genes controlling embryogenic callus (EC) differentiation and investigation of the genetic mechanism of cell fate determination are important for improving plant variety. In this study, we used the maize inbred line KN5585 and its gene-edited mutants Zmprx19-1, Zmprx19-2 and Zmprx19-3 as plant materials. Three somatic regeneration-related traits, the embryogenic callus induction rate (EIR), green callus rate (GCR) and plantlet regeneration rate (PRR), were identified by tissue culture of immature embryos. Additionally, the ECs at different differentiation stages (0 d, 5 d, 10 d and 15 d) were subjected to RNA-seq, and comparative transcriptome analyses were performed. The results showed that the somatic regeneration traits of the mutants were all highly significantly lower than those of the wild type (p < 0.01). The PRR value of KN5585 was 75.25%, while the highest PRR of the mutants was only 15.08%, indicating that knockdown of ZmPRX19 inhibited EC regeneration. Transcriptome sequencing yielded a total of 200.30 Gb of clean data from 24 libraries, with an average of 6.53 Gb of clean data per library. Mutant and wild-type gene expression data were compared separately at four differentiation stages, and 689 common differentially expressed genes (DEGs) were screened. WGCNA was used to classify these genes into nine modules, which were subsequently subjected to GO and KEGG enrichment analyses. In total, 40, 23, 17 and 5 genes were significantly (q < 0.05) enriched in plant hormone signal transduction, the MAPK signaling pathway-plant, phenylpropanoid biosynthesis and photosynthesis, respectively. Moreover, protein–protein interaction (PPI) network analysis revealed five MAPKKK17_18 hub nodes involved in the MAPK pathway-plant, which may be the key genes controlling plantlet differentiation from ECs. The above results provide a basis for the final elucidation of the molecular mechanism of plant somatic regeneration.

Chemical profiling of bioactive compounds in the methanolic extract of wild leaf and callus of Vitex negundo using gas chromatography-mass spectrometry.

The investigation of plant-based therapeutic agents in medicinal plants has revealed their presence in the extracts and provides the vision to formulate novel techniques for drug therapy. Vitex negundo (V. negundo), a perennial herb belonging to the Varbanaceae family, is extensively used in conventional medication. To determine the existence of therapeutic components in leaf and callus extracts from wild V. negundo plants using gas chromatography-mass spectrometry (GC-MS). In this study, we conducted GC-MS on wild plant leaf extracts and correlated the presence of constituents with those in callus extracts. Various growth regulators such as 6-benzylaminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthylacetic acid (NAA), and di-phenylurea (DPU) were added to plant leaves and in-vitro callus and grown on MS medium. The results clearly indicated that the addition of BAP (2.0 mg/L), 2,4-D (0.2 mg/mL), DPU (2.0 mg/L) and 2,4-D (0.2 mg/mL) in MS medium resulted in rapid callus development. The plant profile of Vitex extracts by GC-MS analysis showed that 24, 10, and 14 bioactive constituents were detected in the methanolic extract of leaf, green callus and the methanolic extract of white loose callus, respectively. Octadecadienoic acid, hexadecanoic acid and methyl ester were the major constituents in the leaf and callus methanolic extract. Octadecadienoic acid was the most common constituent in all samples. The maximum concentration of octadecadienoic acid in leaves, green callus and white loose callus was 21.93%, 47.79% and 40.38%, respectively. These findings demonstrate that the concentration of octadecadienoic acid doubles in-vitro compared to in-vivo. In addition to octadecadienoic acid; butyric acid, benzene, 1-methoxy-4-(1-propenyl), dospan, tridecanedialdehyde, methylcyclohexenylbutanol, chlorpyrifos, n-secondary terpene diester, anflunine and other important active compounds were also detected. All these components were only available in callus formed in-vitro. This study showed that the callus contained additional botanical characteristics compared with wild plants. Due to the presence of numerous bioactive compounds, the medical use of Vitex for various diseases has been accepted and the plant is considered an important source of therapeutics for research and development.

Exploring the Production of Secondary Metabolites from a Halophyte Tetragonia tetragonoides through Callus Culture

Considering Korea’s gradual shift toward an aging society, consumer interest in compounds with physiological benefits, including antioxidant and anticancer effects, has surged. This study explored the potential of Tetragonia tetragonoides (Pall.) Kunze, commonly known as New Zealand Spinach (NZS), a halophyte with reported health benefits, including efficacy in treating gastrointestinal diseases, high blood pressure, diabetes, and obesity. This study also introduced a novel callus culture system for NZS, allowing for the rapid in vitro production of secondary metabolites. Optimal callus induction (100%) and biomass production (0.416 g) were achieved by adding 2.0 mg·L−1 6-BA (6-Benzylaminopurine) and 0.5 mg·L−1 2,4-D (2,4-Dichlorophenoxyacetic acid) among five auxin and cytokinin combinations. Two distinct callus types, TGC [TDZ (Thidiazuron)-supplemented Green Callus] and TNYC [TDZ + NAA (Naphthalene acetic acid)-supplemented Yellow Callus], were identified, each with unique characteristics. The calli showed total phenolic and flavonoid contents comparable to those of NZS leaves grown in the greenhouse. An expression analysis of six genes (CHS, CHI, F3H, F3′H, FLS, and DFR) involved in the kaempferol biosynthesis revealed an enhanced flavonoid biosynthesis-related gene expression in TGC, emphasizing its potential for compound production. GC-MS analysis identified distinct compound profiles in TGC and TNYC, with 2,3-butanediol and succinic acid being the predominant compounds among the nine and forty-four components, respectively. These calli offer a stable supply of functional compounds and present an environmentally sustainable solution. The derived callus culture system is anticipated to contribute to the development of healthy functional foods or pharmaceuticals from halophyte NZS.

The Effect of NAA and BA on In Vitro Callus Induction of Biduri (Calotropis gigantea)

A family of medicinal plants known as biduri (Calotropis gigantea) has a number of useful traits. Alkaloids, glycosides, phenolics, tannins, flavonoids, terpenes, and sterols are just a few of the chemical substances found in biduri. One method used to boost the generation of secondary metabolites is biduri callus induction by tissue culture. The goal of the study was to find the ideal growth regulator concentration for callus biduri induction. The study employed a completely randomized design (CRD), which was carried out three times and was set up in a factorial fashion with two treatment components and 25 treatment combinations. The growth regulator NAA and the growth regulator BA were the first and second factors, respectively. ANOVA was used to examine the data with a 5% level test. The Duncan Multiple Range Test (DMRT) was conducted at a 5% level if there was a significant difference. A compact, green callus texture was generated by applying one ppm NAA and three ppm BA. At the same time, a callus on the biduri could not have been treated without an NAA application. Further research can be done by subculture callus biduri when the callus is still fresh and green to produce secondary metabolites.

Transcriptomic analysis of salt stress induced chlorophyll biosynthesis-related genes in photoheterotrophic Arabidopsis thaliana calli.

In order to investigate the salt stress induced chlorophyll biosynthesis-related genes in photoheterotrophic cultures, we performed RNA-Seq analysis on A. thaliana calli exposed to 100mM NaCl on MS medium containing 0.5mg/L 2,4-D 30days. Four different conditions of samples were sequenced on Illumina HiSeq Platform in total and generated about 4.49Gb per sample. The average genome and gene mapping rates were 93.52% and 90.78%, respectively. According to expression profile analysis, some DEGs demonstrated altered related to chlorophyll pigment metabolism. According to analysis, green callus color of photoheterotrophic calli were mainly connected with the induction of LHCB4.3 light harvesting complex photosystem II (Gene ID:818599), AT1G49975 photosystem I reaction center subunit N (Gene ID: 841421), PAM68 PAM68-like protein (DUF3464) (Gene ID: 2745715) and AT3G63540 thylakoid lumenal protein (Mog1/PsbP/DUF1795-like photosystem II reaction center PsbP family protein)(Gene ID: 7922413) genes. Furthermore, 8 DEGs were randomly selected to validate the transcriptome profiles via qPCR. These results will provide a foundation for further studies aimed at giving photosynthetic properties to in vitro plant cultures.

Effect of different plant growth regulators on callus and adventitious shoots induction, polysaccharides accumulation and antioxidant activity of Rhodiola dumulosa

ObjectiveAs a medicinal plant, the resource of Rhodiola dumulosa is deficient along with the large collection. For the protection and utilization of R. dumulosa, the influence of plant growth regulators (PGRs) on callus induction and adventitious shoots differentiation, polysaccharide production and the antioxidant activity were tested. MethodsInternodes of R. dumulosa were used as explants and cultured on MS mediumplus different plant growth regulators (PGRs). The anti-oxidative activities of polysaccharides were evaluated using radical scavenging assays. ResultsBy response surface plot, 0.85 mg/L N6-benzyladenine (BA), 0.34 mg/L naphthaleneacetic acid (NAA) and 0.33 mg/L 2,4-dicholorophenoxyacetic acid (2,4-D) were the optimal factors for callus induction (90.03%) from internodes explants on MS medium. The fresh weight of green callus increased 47.26 fold, when callus was inoculated on MS + thidiazuron (TDZ) 0.5 mg/L + NAA 2.0 mg/L. Adventitious buds regenerated from callus on the media of MS were fortified with BA 1.0 mg/L plus NAA 0.5 mg/L, and the induction rate was 40.00%. MS plus indole-3-butyric acid (IBA) 1.0 mg/L produced the highest rooting rate with 10 to 15 roots in a length of 2–3 cm per shoot. The content of total polysaccharides in callus developed on MS + TDZ 0.5 mg/L + NAA 2.0 mg/L and MS + BA 1.0 mg/L + NAA 0.5 mg/L was as high as 1.72%−2.15%. At the dose of 0.5 mg/mL polysaccharides extracted from different callus induced on MS + NAA 2.0 mg/L + TDZ 0.5 mg/L or MS + BA 1.0 mg/L + NAA 0.5 mg/L or MS + BA 0.5 mg/L + 2,4-D 0.5 mg/L, the ABTS radical eliminating percentages were 82.78%, 80.18% and 68.59%, respectively, much higher than that of wild plant. ConclusionA rapid micropropagation system for R. dumulosa has been developed. The combination of TDZ and NAA or BA and NAA can increase the yield of the total polysaccharides. The polysaccharides isolated from callus and whole wild plants had stronger free radicals scavenging activities, indicating that polysaccharides from R. dumulosa are the potential pharmaceutical supplements.

Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma.

Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.

Evaluation of the effect of jasmonic acid elicitation on composition of pigments and biological activities in green callus of neem (Azadirachta indica)

This study was carried out with the aim of determining the effects of jasmonic acid (JA) elicitation on the bioactive pigments' biosynthesis and the antioxidant activities in green callus of Azadirachta indica of two different ages (4- and 8-week-old). Plant tissue culture technique was employed to induce the formation of green callus from leaf explants of A. indica on Murashige and Skoog (MS) medium supplemented with 0.6 mg/L thidiazuron (CM) and three different concentrations of JA (2, 4, and 6 mg/L). The methanolic extracts from the green callus were used for determination of total chlorophyll content (TCh), total carotenoid content (TC), total anthocyanin content (TAC), total phenolic content (TPC), and total flavonoid content (TFC) through colorimetric and HPLC analyses. The highest amount of yield was obtained from CM and 2 mg/L JA (2JA) extracts for 4- and 8-week-old samples, respectively. Phytochemical screening revealed the presence of alkaloids, flavonoids, phenols, tannins, and terpenoids in all 4- and 8-week-old samples elicited with 2, 4 and 6 mg/L JA. The highest value for TAC, TCh, TC, TPC, and TFC of 4- and 8-week-old samples were from callus cultured on media supplemented with 6 mg/L JA (6JA) and 4 mg/L JA (4JA), respectively. The lowest IC50 values were found to be 8.29 ± 0.10 mg/mL (6JA) for 4-week-old and 7.73 ± 0.03 mg/mL (4JA) for 8-week-old samples. The highest Ferric Reducing Antioxidant Power (FRAP) values obtained in this study were 90.60 ± 1.55 g/g (6JA), and 74.59 ± 3.91 g/g (4JA), respectively, for 4- and 8-week-old samples. Moreover, Pearson's correlation analysis revealed a significant correlation between TAC, TCh, TC, TPC, and TFC with ABTS and FRAP assays. In addition, PCA analysis revealed that 83.5% of the information (variances) contained in the data were retained by the first two principal components. Overall, these findings suggested that JA supplementation into the culture media significantly increase the chlorophyll, carotenoid, anthocyanin, phenolic and flavonoid contents and JA concentrations at 6 mg/L JA and 4 mg/L JA yielded the highest pigments content in 4- and 8-weeks-old callus, respectively.

Comparative Analysis on the Role of 2,4-Dichlorophenoxyacetic Acid in the Expression of Bioactive Compounds in Callus of Capsicum frutescens

Plant cell culture technology serves as an effective alternative system for in vitro production of bioactive molecules, as it allows for the exploration of valuable compounds under a controlled environment. The present study was conducted to evaluate the effect of plant growth regulator (PGR); 2,4-dichlorophenoxyacetic acid (2,4-D) on the expression of compounds in coloured callus of Capsicum frutescens, a vital spice in various cuisines worldwide. The differential accumulation of compounds in the callus was analysed using liquid chromatography-mass spectrometry (LCMS) and the PGR concentration that resulted in the highest accumulation of the valuable compounds was identified. In this study, calli of various colours (cream, yellow and green) were successfully produced from C. frutescens through plant tissue culture. The increase in 2,4-D concentrations was found to increase callus growth index (GI) and specific growth rate (Sg), where the highest GI (0.5690) and Sg (0.6348 mg/week) were observed in callus produced in media supplemented with 0.5 mg/L 2,4-D. LCMS data analyses showed that 19 compounds were detected in the callus, with 8 compounds (fatty acids and phenolics) were successfully identified, while the remaining 11 compounds were reported as unknowns. Yellow-coloured callus was observed to contain the highest number of compounds (18 compounds), while green callus contained the least (14 compounds). This analysis provides valuable information on the application of biotechnological tools such as plant tissue culture as an alternative for sustainable production of compounds with high bioactivity in Capsicum frutescens.

Genome size and gas chromatography-mass spectrometry (GC-MS) analysis of field-grown and in vitro regenerated Pluchea lanceolata plants.

Pluchea lanceolata is a threatened pharmacologically important plant from the family Asteraceae. It is a source of immunologically active compounds; large-scale propagation may offer compounds with medicinal benefits. Traditional propagation method is ineffective as the seeds are not viable; and root sprout propagation is a slow process and produces less numbers of plants. Plant tissue culture technique is an alternative, efficient method for increasing mass propagation and it also facilitate genetic improvement. The present study investigated a three-way regeneration system in P. lanceolata using indirect shoot regeneration (ISR), direct shoot regeneration (DSR), and somatic embryo mediated regeneration (SER). Aseptic leaf and nodal explants were inoculated on Murashige and Skoog (MS) medium amended with plant growth regulators (PGRs), 2,4-dichlorophenoxy acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), and 6-benzyl amino purine (BAP) either singly or in combinations. Compact, yellowish green callus was obtained from leaf explants in 1.0 mg/l BAP (89.10%) added medium; ISR percentage was high, i.e., 69.33% in 2.0 mg/l BAP + 0.5 mg/l NAA enriched MS with 4.02 mean number of shoots per callus mass. Highest DSR frequency (67.15%) with an average of 5.62 shoot numbers per explant was noted in 0.5 mg/l BAP added MS medium. Somatic embryos were produced in 1.0 mg/l NAA fortified medium with 4.1 mean numbers of somatic embryos per culture. On BAP (1.0 mg/l) + 0.5 mg/l gibberellic acid (GA3) amended medium, improved somatic embryo germination frequency (68.14%) was noted showing 12.18 mean numbers of shoots per culture. Histological and scanning electron microscopic (SEM) observation revealed different stages of embryos, confirming somatic embryogenesis in P. lanceolata. Best rooting frequency (83.95%) of in vitro raised shootlets was obtained in 1.0 mg/l IBA supplemented half MS medium with a maximum of 7.83 roots per shoot. The regenerated plantlets were transferred to the field with 87% survival rate. The 2C genome size of ISR, DSR, and SER plants was measured and noted to be 2.24, 2.25, and 2.22 pg respectively, which are similar to field-grown mother plant (2C = 2.26 pg). Oxidative and physiological events suggested upregulation of enzymatic activities in tissue culture regenerated plants compared to mother plants, so were photosynthetic pigments. Implementation of gas chromatography-mass spectrometry (GC–MS) technique on in vivo and in vitro raised plants revealed the presence of diverse phyto-chemicals. The yields of alpha amyrin and lupeol (medicinally important triterpenoids) were quantified using high-performance thin-layer chromatography (HPTLC) method and enhanced level of alpha amyrin (2.129 µg g−1 dry wt) and lupeol (1.232 µg g−1 dry wt) was noted in in vitro grown leaf tissues, suggesting in vitro conditions act as a potential trigger for augmenting secondary metabolite synthesis. The present protocol represents a reliable mass propagation technique in producing true-to-type plants of P. lanceolata, conserving 2C DNA and ploidy successfully without affecting genetic homogeneity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13353-022-00727-7.

GhWOX11 and GhWOX12 promote cell fate specification during embryogenesis

The acquisition of transgenic materials relies on an efficient genetic transformation system, in which WUSCHEL-related homeobox genes play imperative roles in enhancing somatic embryogenesis potential. In our study, we isolated and characterized the cotton (Gossypium hirsutum L) homologues of Arabidopsis WOX11 and WOX12, namely GhWOX11_At, GhWOX11_Dt, GhWOX12_Dt and GhWOX12_At. Domain analysis indicated that the putative GhWOX11 and GhWOX12 proteins contain a highly conserved DNA-binding HOX domain or ACR domain. We have also demonstrated that callus initiation and adventitious root formation are simultaneously regulated by GhWOX11/12 genes. The asymmetric distribution of auxin leads to the differential expression of GhWOX11 and GhWOX12, which controls the formation of callus and presents a polar distribution at the ends of hypocotyl. Furthermore, overexpression of GhWOX11/12 significantly promotes the formation of callus and the fate transition from callus cell to pluripotent cell in G. hirsutum. On the other hand, expression of the chimeric GhWOX11/12 repressor in cotton callus inhibited embryogenic callus formation. Overexpression of GhWOX11 also promoted the formations of green callus, adventitious roots and adventitious shoots in Arabidopsis, corroborating its conserved functional role in inducing plant somatic embryogenesis and root development. Our results show that GhWOX11 and GhWOX12 genes play an important role in the process of cell fate transition in the early stage of somatic embryogenesis, providing a new perspective for the optimization of plant transformation systems.

In Vitro Micropropagation Studies Cotyledonary Explants Of Trichosanthes Anguina (L)

A method for Invitro Micropropagation Through Cotyledonary Explants of Trichosanthes anguina was exercised from 10-15 days of young stage explants [1, 2]. Developed a protocol for cotyledonary explants cultures, callus induction, and regeneration through cotyledon callus tissue [3, 4]. Multiple shoot formation was promoted by BAP 1.0 mg/l to 3.0 to 5.0 mg/l and higher combination with NAA 3.0 to 4.0 mg/l Kn [5, 6]. Invitro produced small shoots were green callus induced to cotyledonary callus cultures green compact small shoots buds were induced in 16-20 days cultures respectively on Ms. Medium.

QTL Mapping of Somatic Regeneration-Related Traits in Maize

The somatic regeneration of maize depends on its genotypes, so improving its variety with modern biotechnology is severely restricted. Locating the quantitative trait loci (QTLs) associated with somatic regeneration is important for breeding elite inbred lines that undergo genetic transformations. Here, by crossing the high-regeneration inbred line H99 and non-regeneration inbred line Fr993, an F2 population and its F2:3 and F2:4 population families were constructed. Immature embryos from the family populations were subjected to tissue culture in two independent seasons to determine their embryogenic callus induction rates (EIRs), green callus rates (GCRs) and plantlet regeneration rates (PRRs). Genetic linkage maps were constructed for the F2 population to locate somatic regeneration QTLs. The results showed that variation in the EIR, GCR and PRR ranged from 0.00–99.33%, and their broad-sense heritabilities were 0.50, 0.52 and 0.53, respectively. The total genetic distance of the linkage maps constructed by the GenoBaits 10 K chip was 2319.50 cM, and twelve QTLs were associated with somatic regeneration traits, accounting for 3.90–14.06% of the phenotypic variation. Expression analysis revealed six candidate genes screened from the QTLs with distinct responses to induction culture in the parental lines, suggesting that they may impact commitment to somatic cell fate. These results provide a basis for the molecular breeding of maize varieties with high-frequency somatic regeneration.

Efectos de reguladores de crecimiento para inducción de embriones somáticos a partir de diferentes explantes de Echinocactus parryi Engelm, una especie endémica y amenazada

Introduction: The list of threatened species is enhancing and needs to be revised by integrating plant tissue culture tools with conventional techniques that support the appropriate management of these species. Objective: To assess the effects of the growth regulators for the induction of somatic embryos from mature seeds, shoots, and compact green callus of Echinocactus parryi Engelm. and the histological analysis of the embryogenic structures. Materials and methods: A completely randomized design was utilized to evaluate three types of explants (apical, medium, and basal) cultured on basal Murashige & Skoog media (MS) with different growth regulators concentrations (2, 4-D [dichlorophenoxy acetic acid], BAP [6-benzylaminopurine] and kinetin, at four levels: 0.5, 1, 1.5, and 2 mg∙L -1 ). Histological analysis of the embryogenic structures was performed. Results and discussion: The 2, 4-D induced both embryogenic and organogenic callus from seeds and shoot explants. The globular stage did not evolve to their maturity, presumably because of 2, 4-D accumulation. The compact callus explants were the more efficient to induce 19.2 somatic embryos per explant when they were cultured in the medium with 0.5 mg∙L -1 kinetin. However, the latest phases did not germinate, probably due to abnormalities generated by genetic and epigenetic changes in the DNA that can cause abnormal somatic embryos. The histology image demonstrated that the globular and torpedo structures were visible under a microscope showing stained nucleus and numerous starch grains. Conclusions: E. parryi is a species that can produce a high number of embryogenic structures, which represents a great potential to grow massive plants.

COMPARATIVE STUDIES OF PHYTOCHEMICAL AND ANTIOXIDANT ACTIVITY OF IN VIVO PLANT AND IN VITRO CALLUS EXTRACT OF CARDIOSPERMUM HALICACABUM L.

Objective: The present investigation focuses on the use of Cardiospermum halicacabum L. in their phytochemical and biological activities. Methods: In this study, in vivo stem and in vitro callus ethanolic extracts of C. halicacabum were tested for their phytochemical attributes by qualitative method, Fourier transform infrared (FTIR), antioxidant, antibacterial, and bioactive compound properties. The bactericidal activity of the in vivo stem and in vitro callus extract has been evaluated in both Gram+ve and Gram-ve microorganisms using the disk diffusion method. Results: The highest frequency (78%) of well developed, dark green organogenic callus was induced from stem explant on Murashige and Skoog (MS) medium supplemented with 0.7 mg/l 2,4-Dichlorophenoxyacetic acid (2, 4-D) and 0.5 mg/l benzyl adenine (BA). The results of FTIR spectra confirmed the presence of functional groups in wild stem and in vitro callus extract of C. halicacabum with various peaks. The total phenolic content in ethanolic extract of in vivo plant and in vitro callus was 80.46 mg gallic acid equivalent (GAE)/g dry weight and 76.4 mg GAE/g dry weight, respectively. The highest percentage of tannins was measured at 78.03 in wild stem ethanol extracts followed by 75.22 in callus extract. The antioxidant activity of 2,2-diphenyl-2- picrylhydrazyl (DPPH) ethanol extract was found to be 206.54 μg/ml. IC50 values of the stem extracts of C. halicacabum are 306 μg/ml and 286 μg/ml in callus extract, respectively. Antibacterial activity of the ethanol extract was higher for Staphylococcus aureus (S. aureus) with a 17 mm zone of inhibition. Conclusion: The present investigation recommended that the callus ethanolic extract function as a good source of biologically active compounds and natural antioxidants.

Transformation of Kalanchoe pinnata by Agrobacterium tumefaciens with ZsGreen1

Screening tests in which 30 species of succulent plants were inoculated with A136 or C58 strains of Agrobacterium tumefaciens led to selecting genus Kalanchoe for further study of Agrobacterium-mediated transformation. Regeneration studies showed that 4.55 µM Thidiazuron (TDZ) combined with 2.15 µM α-naphtaleneacetic acid (NAA) significantly proliferated callus development from Kalanchoe pinnata leaf explants in Murashige and Skoog (MS) medium. Subsequently, 4.55 µM TDZ significantly enhanced shoot formation from the green and compact callus. NAA (5.37 µM) accelerated root growth leading to establishment of transgenic plants in soil. A gene encoding the fluorescent protein, ZsGreen1, was cloned into a plasmid vector harboring a constitutive figwort mosaic virus (FMV) 35S promoter, a kanamycin resistance gene, and a NOS-terminator. The plasmids were delivered into the genome of K. pinnata by A. tumefaciens strain C58 (ABI). The ZsGreen1-transgenic plants exhibited very strong, green fluorescence from the callus, leaves, stems, roots, and flowers visible through a green emission filter (GFP, 480/510 nm, excitation/emission) under excitation by ultra-violet light. Transcription analysis by sqRT-PCR and qPCR confirmed the stable integration of the ZsGreen1 in the genome of K. pinnata. The FMV 35S promoter conferred uniform expression throughout whole plant organ. ZsGreen1 was proven to be a stable marker for gene expression in genetic transformation of K. pinnata. The ZsGreen1-transgenic Kalanchoe pinnata mediated by Agrobacterium tumefaciens exhibited strong, green fluorescence from the callus, leaves, stems, roots and flowers under the control of the figwort mosaic virus (FMV) 35S promoter.

In vitro GYNOGENESIS OF Jatropha curcas L. var ALJC01

<pre><strong><span lang="EN-US">Background. </span></strong><em><span lang="EN-US">Jatropha curcas</span></em><span lang="EN-US"> L. is a crop whose oil can be converted to biodiesel. However, there is lack of varieties with high yields that limiting commercial production of the crop. An alternative is to obtain <em>in vitro </em>plants from unpollinated female gametophytes is known as gynogenesis which can provide an alternative to </span><span lang="EN-US">produce </span><span lang="EN-US">varieties with increased seed production.<strong> Objective</strong>. The objective was to establish a protocol for obtaining <em>in vitro</em> plants from unfertilized ovules of <em>Jatropha curcas</em> L. ovules. <strong>Methodology</strong>. The ovules were extracted from unfertilized inflorescences pretreated for 0, 1, 3 and 7 days at 2 to 5 ° C. Three combinations of plant growth regulator treatments were applied to culture media for induction of gynogenic calli. Gynogenic calli were cultured on MS basal medium with BAP and IBA for development of green, friable. The </span><span lang="EN-US">gynogenic </span><span lang="EN-US">calli from the development treatment </span><span lang="EN-US">were transferred to experiments to determine a regeneration treatment. </span><span lang="EN-US">The gynogenic plantlets were transferred to different treatments for root development. <strong>Results</strong>. The results show that pretreatment of inflorescences at 2 to 5°C had no significant effect on the number of ovules that formed friable white calli. Induction of gynogenic friable white calli occurred in the dark conditions, on half-strength </span><span lang="EN-US">Murashige and Skoog basal medium (</span><span lang="EN-US">½</span><span lang="EN-US"> MS) </span><span lang="EN-US">supplemented with two combinations of growth regulators: (i) 5.37 µM naphthalene acetic acid (NAA) combined with 6.65 µM 6-benzylaminopurine (BAP) and, (ii)</span><span lang="EN-US">8.05 µM NAA with 22.09 µM BAP. Development of green, friable, gynogenic calli under light–dark conditions was possible under treatment with complete Murashige and Skoog (1962) (MS) basal medium with 6.66 µM BAP and 4.9 µM indole-butyric acid (IBA).</span><span lang="EN-US">Friable green callus formed gynogenic embryos on MS basal medium supplemented with </span><span lang="EN-US">22.09 </span><span lang="EN-US">µM</span><span lang="EN-US">BAP and </span><span lang="EN-US">3.40 </span><span lang="EN-US">µM</span><span lang="EN-US"> paclobutrazol (PBZ), embryos regeneration occurred in photoperiod conditions</span><span lang="EN-US">. </span><span lang="EN-US">Embryos were able to develop and convert to plantlets in the treatment on MS basal medium containing 2.22 µM BAP and 0.28 µM of indole-3-acetic acid (IAA).</span><span lang="EN-US"> Root development of </span><span lang="EN-US">plantlets</span><span lang="EN-US"> occurred in </span><span lang="EN-US">½</span><span lang="EN-US"> MS basal medium with 18.65 µM IBA. <strong>Implications</strong>. A protocol of <em>in vitro</em> gynogenesis in <em>Jatropha curcas</em> would contribute to the improvement of its cultivation, reducing the time required for the generation of pure lines that would allow us to obtain varieties with increased seed production. <strong>Conclusions. </strong>The<strong> </strong></span><span lang="EN-US">work presented here describes a reproducible protocol to produce plants <em>in vitro</em> from unfertilized ovules of <em>Jatropha curcas</em> L. T</span><span lang="EN-US">his methodology will facilitate to obtain homozygotic lines with significant reduction in the time required by conventional methods. </span><strong></strong></pre>

Tomato protoplasts as cell target for ribonucleoprotein (RNP)-mediated multiplexed genome editing

The possibility to produce plants edited in multiple genes by means of DNA-free approaches opens new perspectives for breeding purposes and acceptance of resultant genotypes. In this work, we have explored the polyethylene glycol (PEG)-mediated delivery of ribonucleoproteins (RNPs) in tomato protoplasts using a multiplexing approach (i.e. two genes targeted simultaneously using two sgRNAs per gene) for the first time. We have analysed the editing outcome in fully developed green calli and demonstrated that tomato protoplasts are a valid cell target for RNP-mediated multiplexed genome editing with high efficiency. RNP could be applied with high efficiency in a multiplexing genome editing approach in tomato protoplasts.

In vitro selection of elite clone of Withania somnifera against leaf blight disease caused by Alternaria alternata

In vitro selection of Withania somnifera has been incurred against Alternaria alternata by selection pressure of fungal toxin in an innovative way. Solid green callus was maintained in Murashige and Skoog's medium supplemented with 6-Benzylaminopurine (2 mgL−1). Toxin was partially purified from A. alternata broth culture. Elite clone was generated by gradual intoxication of solid green callus in the toxin containing medium (5–400 mgL−1). Three highest dose of toxin tolerance was selected viz. 50 mgL−1, 100 mgL−1 and 200 mgL−1. Plants were successfully regenerated from those toxin selected callus. Eventually, various defense enzymes and total phenolics were measured in regenerated plants and toxin selected callus. Callus selected from 100 mgL−1 toxin dose and plants obtained from them give the best results among all and showed more than 2–5 fold increase of defense enzyme activities and total phenolics compared to control. Furthermore, those plants when challenge inoculated showed significant reduction (more than 70%) of leaf spot incidence. Augmentation of disease resistance persists for at least two successive generations. Furthermore, the increase in defense molecules also correlated with increased nitric oxide generation (73%). Presence of withaferin A and withanolide A has also been confirmed in the toxin regenerated plants by High-performance thin-layer chromatography (HPTLC) analysis. Over all observations suggests that fungal toxin at a concentration 100 mgL−1 and by habituation technique can be use as a selection method for the production of disease resistant medicinally important crop like W. somnifera without altering its secondary metabolite production.