Highest Regeneration Frequency Research Articles

Efficient Regeneration of Transgenic Rice from Embryogenic Callus via Agrobacterium-Mediated Transformation: A Case Study Using GFP and Apple MdFT1 Genes

Genetic transformation is a critical tool for gene manipulation and functional analyses in plants, enabling the exploration of key phenotypes and agronomic traits at the genetic level. While dicotyledonous plants offer various tissues for in vitro culture and transformation, monocotyledonous plants, such as rice, have limited options. This study presents an efficient method for genetically transforming rice (Oryza sativa L.) using seed-derived embryogenic calli as explants. Two target genes were utilized to assess regeneration efficiency: green fluorescent protein (eGFP) and the apple FLOWERING LOCUS T (FT)-like gene (MdFT1). Antisense MdFT1 was cloned into a vector controlled by the rice α-amylase 3D (Ramy3D) promoter, while eGFP was fused to Cas9 under the Ubi promoter. These vectors were introduced separately into rice embryogenic calli from two Korean cultivars using Agrobacterium-mediated transformation. Transgenic seedlings were successfully regenerated via hygromycin selection using an in vitro cultivation system. PCR confirmed stable transgene integration in the transgenic calli and their progeny. Fluorescence microscopy revealed eGFP expression, and antisense MdFT1-expressing lines exhibited notable phenotypic changes, including variations in plant height and grain quality. High transformation efficiency and regeneration frequency were achieved for both tested cultivars. This study demonstrated the effective use of seed-derived embryogenic calli for rice transformation, offering a promising approach for developing transgenic plants in monocot species.

Development of regenerated plants of interspecific wheat hybrids using immature embryos as explants

Nowadays particular attention in winter wheat breeding is paid to the introduction of biotechnological methods. The culture of immature embryos in vitro is an alternative way to produce hybrids that often do not develop fully in vivo. The purpose of the current study was to estimate the ability of immature embryos of interspecific winter wheat hybrids for callus induction, morphogenesis, and plant regeneration on artificial nutrient media, as well as to select optimal conditions for obtaining full-fledged plants with all the botanical characteristics of the species. The research material included 5 hybrid combinations of interspecific crosses between durum (Triticum durum DESF.) and common (Triticum aestivum L.) winter wheat. The material was collected during one field season (2022). Immature embryos were used as explants. Culture media were according to Murashige-Skoog (MS), and they differed in the content of hormones and organic substances. There has been found that the hybrid combinations were characterized by a variety of morphogenic responses. The manifestation of callusogenesis was not high enough and averaged 22–32 % depending on the nutrient medium. Rhizogenesis averaged from 1–6.7 %, and ended with the formation of roots. The process of formation of embryoid- and hemmorizogenesis was best demonstrated by the hybrid combination No.1, and on two media. All processes of morphogenesis excepting ‘rhizogenesis’ varied greatly among hybrid combinations. The largest number of regenerated plants was obtained from hybrid combination No.1 (61 pcs., 54.5 %). After vernalization and acclimatization, 38 % of the plants survived from the number of planted embryos. The largest number of heads (79 pcs.) was formed according to hybrid combination No.1, on average 1.46 heads per plant. However, not all heads were fertile. The results of the correlation analysis made it possible to identify indicators that closely correlated with the amount of plant regeneration, namely, the high yield of embryoid- and hemmorizogenic structures ensured a high frequency of plant regeneration (r = 0.769).

Leaf Culture Technique For High-frequency Regeneration of Non-toxic Variants of Jatropha curcas Plantlets

Leaf Culture Technique For High-frequency Regeneration of Non-toxic Variants of Jatropha curcas Plantlets

Colchicine induced polyploidy from thallus segments of Kappaphycus alvarezii (Doty) for genetic improvement

Regeneration of polyploidy from young thallus segments of Kappaphycus alvarezii was optimized for genetic improvement. Kappaphycus thallus segment cultured on sterile sea water supplemented with various combinations of Indole acetic acid, Kinetin and Acardian Marine Plant Extract Powder revealed differential response on callus proliferation and development of new thallus. Presence of Acardian Marine Plant Extract Powder (3 mg/l) in combination with Indole acetic acid and Kinetin (0.01 mg/l each) had induced the longest emerging thallus. Exposure of thallus to colchicine at 0.01% with above combination was optimal to induce high frequency regeneration of polyploidy mostly from the meristematic cells. Anatomical study of colchicine induced polyploidy revealed larger cortical cells with irregular thickening of epidermal layer. Phase contrast and Scanning Electron Microscopic study revealed increase in cell size in cortical region with significantly larger number of spherical shaped carrageenan globules in colchicine induced polyploidy than normal thallus. Single cells isolated using enzymatic treatments from colchicine induced polyploidy, shown chromosome number with a ploidy status of 4n ≈ 40. Whereas in normal thallus, only half the number of chromosomes (2n ≈ 20) were observed. Polyploidy were successfully acclimatized gradually using raft method for further evaluation. This is the first report reveals the induction and regeneration of polyploidy in Kappaphycus. The possible application of this finding in genetic improvement of Kappaphycus is discussed.

Morphogenesis features of <i>Hedysarum alpinum</i> L. in <i>in vitro</i> culture

The plants of Hedysarum alpinum L. in the North are characterized by a long pregenerative period, the laboriousness of seed propagation, low field germination of seeds, rare self-sowing, and the impossibility of vegetative propagation. In connection with this, it seemed relevant to introduce this rare species (included in the Red Book of the Komi Republic) into aseptic culture to preserve and maintain the gene pool, as well as to further develop the technology of its cultivation to obtain medicinal raw materials. The morphogenesis was studied, and the peculiarities of the regeneration of adventitious shoots of Hedysarum alpinum in in vitro culture were revealed. During three subcultivations, the influence of two cytokinin growth regulators, 6-benzylaminopurine (BAP) and kinetin (KIN), at concentrations from 0,5 to 1,0 mg/l, on the regeneration potential of different explants of Hedysarum alpinum in in vitro culture was determined on the WPM environment. A high frequency of regeneration of shoots from single-node segments and cotyledon nodes was obtained 79% and 65%, respectively. In the second and third passages, there was an increase in the regenerative activity of the cotyledon node explants and an increase in the multiplication factor to 5 shoots per explant. It was shown that relatively high concentrations (1,0 mg/l) of BAP and CIN increase the number of shoots per explant but inhibit their growth and development. For the reproduction stage proper, the medium containing BAP 0,5 + CIN 0,5 + IAA 0,1 mg/l is optimal. It was shown that the initiation of rhizogenesis actively took place on the WPM medium supplemented with IAA 0,5 and IBA 0,5 mg/l, on which the proportion of rhizogene explants was 77%. The maximum values for the length and number of roots were obtained on the medium with IAA 1,0 mg/l and with the combined use of two auxins (IAA with IBA at 0,5 mg/l). The regenerated plants were transplanted into non-sterile conditions, and their survival rate under ex vitro conditions was 50%.

Computing artificial neural network and genetic algorithm for the feature optimization of basal salts and cytokinin-auxin for in vitro organogenesis of royal purple (Cotinus coggygria Scop)

This study presents the in vitro regneration protocol for Royal purple [(Cotinus coggygria Scop. (syn.: Rhus cotinus L.)] from nodal segment explants followed by optimizing the input variable combinations with the aid of PyTorch ANN and Genetic Algorithm (GA). The Murashige and Skoog (MS) culture medium yielded relatively higher regeneration frequency (91.52 %) and shoot count (1.96) as compared to woody plant medium (WPM), which yielded 84.58 % regeneration and shoot count (1.61) per explant. The supplementation of plant growth regulators (PGRs) + MS medium yielded 80.0–100.0 % shoot regeneration and 1.48–3.25 shoot counts compared to 60.0–100.0 % shoot regeneration and 1.00–2.37 shoots from the combination of PGRs + WPM. In order to predict the shoot count and regeneration with the aid of a mathematical model, the machine learning algorithms of Multilayer Perceptron (MLP), Support Vector Regression (SVR), Extreme Gradient Boosting (XGB), and Random Forest (RF) models were utilized. The highest R2 values for both output variables were acquired using MLP model in PyTorch platform. The R2 scores for regeneration and shoot counting were recorded as 0.69 and 0.71 respectively. NSGA-II algorithm revealed the 1.25 mg/L BAP (6-Benzylaminopurine), 0.02 mg/L NAA (Naphthalene acetic acid), and 0.03 mg/L IBA (Indole butyric acid) in WPM medium as an optimum combination for 100 % regeneration. On the other hand, the algorithm suggested multiple combination in MS medium for maximum shoot counting.

In Vitro Regeneration of Aromatic Rice (Oryza Sativa L. Var. Doairgura)

Rice (Oryza sativa L.) is the world’s most important cereal crop. The Doairgura variety (O. sativa L.) is a low-yielding, high-quality aromatic rice-producing variety in Bangladesh. This study was undertaken to develop an in vitro regeneration protocol through in vitro callus induction. MS media supplemented with different concentrations of 2, 4- D (0.5, 1.0, 2.0 and 3.0 mg /l) were used for callus induction from the mature dehusked rice seeds. The best results for callus formation was obtained on MS media supplemented with 2.0 mg/l 2, 4-D. Shoot regeneration from in vitro cultured callus was tested with different combinations (BAP, NAA, Kn mg/l) of hormone containing MS media. The highest regeneration frequencies (100%) as well as the highest average shoot per culture (15.3) were recorded on the MS media supplemented with 2.0 mg/l BAP and 1.0 mg/l NAA. For root induction, hormone free MS media showed better results. After hardening, the plantlets were transferred to soil. This protocol is relatively simple and reproducible. Bangladesh J. Bot. 51(4): 677-682, 2022 (December)

Callus induction and regeneration in high-altitude Himalayan rice genotype SR4 via seed explant

SR4 genotype of rice is high altitude Himalayan rice prone to various abiotic stresses such as cold stress and therefore gives a poor yield. An efficient protocol for callusing and regeneration via direct and indirect means was established using mature seeds as an explant which can be utilized for molecular studies for genetic advancement of Himalayan rice genotype SR4 through transformation. Highest frequency (96.6%) of callus induction was obtained on MS media 3.0 mg/L 2, 4-D. While maximum regeneration frequency (100%), number of shoots with maximum length 9.14 ± 0.204 (cm) from callus was recovered from MS media amended with 5.0 mg/L BAP in combination with 0.5 mg/L NAA with highest number of shoots having an average shoot length 9.14 ± 0.204 (cm) after four weeks of culture. Direct multiple shoot regeneration from seed explants was obtained using various concentrations of TDZ and BAP with highest regeneration frequency was observed on MS media fortified with 6 mg/L of TDZ with maximum number of shoots. The shoots developed roots on MS media supplemented with 0.6 mg/L IBA.

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.

Supplementation of Urea as the Nitrate Source of MS Medium and High-frequency Regeneration of Plantlets of Musa paradisiaca cv. Malnad Rasbale

Background: A consistent practice for mass multiplication of banana cv. Malnad Rasbale has been accomplished, which is an endemic cultivar of Western Ghats of Karnataka, India. Methods: Decrease in microbial contagion have been managed by dealing with outward sterilization agents ethanol, sodium hypochlorite, mercuric chloride solution, antibiotic solutions containing cefotaxime and gentamicin. MS media augmented with 350 mg/l urea as the nitrogen source by substituting KNO3 and NH4NO3 which is commonly used in terroristic activities. Organogenic media was supplemented with a range of 2-6 mg/l BAP and 0.3-2.7 mg/l TDZ. Result: The addition of urea substituting KNO3 and NH4NO3 gave the eminent grades in overall initiation, shoot multiplication and development and also in the rooting phase. Supplementation for shoot development with BAP (4 mg/l), TDZ (1.8 mg/l) and with 350 mg/l urea showed enthusiastic progress in shoot elongation with specific concentrations.Plants established under in vitro circumstances were reassigned to the greenhouse and after the progress, they were promoted to field conditions.

Comparative studies on regeneration of different tissues of Bacopa monnieri (L.) Wettst in vitro and analysis of genetic homogeneity by random DNA markers (RDMs)

The natural populations of Bacopa monnieri L., is severely affected by increased indiscriminate collection and overexploitation. There is a need to establish a micropropagation method that should perform effectively for commercial propagation and conservation. In this study, various physiological elements such as medium strength, carbon source (1% to 4%), concentration of agar (0.6%–0.9%), medium PH (5.4–6.0) was optimized for the high frequency regeneration of B. monnieri. Among different cytokinins tested, N6-benzyladenine (BA) was found to be better cytokinin over kinetin for shoot multiplication. The shoot cluster, when subcultured to its same medium, the multiplication rate of the shoot increase significantly by the end of the fifth subculture. The highest number of rooted shoots (10.75) was obtained with half-strength MS medium supplemented with 1.0 mg/l Indole-3-butyric acid (IBA). Among the experiments tested on different soil mixtures, the experience of higher survival of plants in sand, soil and compost (2:2:1) showed a condition survival rate of 87.7% in field conditions. Randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker analysis indicated a high level of uniformity between regenerated plants and together with the mother plant. This study provides important information about selection of suitable explants, micropropagation medium and strategies to improve mass multiplication of B. monnieri which offers a scope for commercial propagation.

Modern bioengineering approaches to creating resistance to the Plum pox virus in stone fruit crops

In recent years, Plum pox virus (PPV) has become a major threat to the cultivation of stone fruit plants. This virus has caused enormous economic damage and caused a significant decrease in production areas in eastern Europe and the Mediterranean. Plum pox virus has spread around the world and is classified by plant quarantine services as the most dangerous pathogen for apricots, plums and peaches Unfortunately, at present, science cannot offer any ways to treat viral plant diseases, and the destruction of infected trees remains the only way to contain the spread of viruses. Given the seriousness of the disease, the difficulty of controlling its spread, the lack of disease-resistant existing varieties, the need to create commercial varieties with increased resistance to this pathogen is obvious. Modern methods of genetic engineering make it possible to significantly accelerate the processes of creating highly productive plum varieties with increased or complete resistance to viruses, unattainable by traditional breeding methods. However, most of the work on the modification of the genomes of stone fruit crops was carried out using juvenile material of zygotic origin, which has a higher morphogenetic potential compared to varieties. The use of modern bioengineering techniques in the selection of stone fruit plants is hampered by the lack of reliable techniques that can provide a sufficiently high frequency of regeneration of shoots from somatic tissues. These and other reasons call for the development of an effective genotype-independent system for regenerating and modifying the genomes of commercial plum varieties. Given the rapid development of plant genome editing techniques, targeted mutation of host genes involved in replication and widespread PPV in infected tissues may be a promising approach for engineering resistance to viruses that exclude the introduction of foreign sequences into the plum genome.

High throughput anther culture response in an upland rice cross ‘Khandagiri x Dular’

Aim: The experiment was conducted to maximize anther culture response in an upland indica rice cross amenable for drought tolerance breeding. Methodology: The cold pre-treated anthers of a cross Khandagiri (drought sensitive) x Dular (drought tolerant) were cultured on N6, SK3, MS and CIM media at varying concentration of hormones (2,4-D, Kn and NAA) to assess callusing response. Embryogenic calli were placed on regeneration medium (RM) with varying concentration of Kn, BAP and NAA for plant regeneration. The plantlets were acclimatized in half-strength MS basal liquid medium for one week before transferring to pot mixture (peat moss: perlite 2:1, v/v) for plant establishment in glasshouse under partial shade. Finally, the plantlets were checked for doubled haploid status by cytological study of root tips. Results: Anther culture response was found to be media and genotype-specific. F1-progenies responded better than either of the parents. A modified MS callus induction medium (CIM) with 1.5mg/l 2,4-D + 0.5mg/l Kn resulted impressively higher callusing response (30.2%) with nodular calli than SK3>MS>N6. High frequency (12.8%) of albino-free green plant regeneration with well developed rooting was achieved in RM medium (a modified MS) containing 2mg/l BAP and 0.5mg/l NAA. A total 129 doubled haploid (DH) plants (each with 12 pairs of chromosomes) were recovered which maintained normal growth, set seeds and resulted in true breeding DH lines. Interpretation: The high throughput regeneration system is amenable for doubled haploid production from indica crosses. Besides, the present doubled haploid stock can serve as an ideal mapping population and as such targeted for marker aided selection for early development of drought tolerant rice varieties.

English

Sorghum is a tropical grass used for various purposes including food, animal feed, and biofuels source, but its in vitro propagation still found an obstacle due to tissue browning. This study aims to propagate and regenerate callus in the Indonesian sorghum hybrid, named Numbu cultivar from seed and meristematic leaf whorl explants. The combined effect of plant growth regulators (PGRs), namely 2,4-D and proline as well as NAA (naphthalene acetic acid) and BAP (6-benzyl amino purine) was investigated on embryogenic callus induction and regeneration, respectively. The results showed that callus induction was significantly faster in the seed explants compared to leaf whorl. The optimal medium used to obtain a large induction in the seed explants was MS salt containing 3 mg L-1 2,4-D and 5000 mg L-1 proline, which gave a 68% yield. Furthermore, the best medium used to obtain a high plant regeneration frequency was the MS medium containing 0.1 mg L-1 NAA and 3 mg L-1 BAP, which regenerated 78% of the callus into plantlet. The addition of 560 mg L-1 proline to the combination medium increased doubled the regeneration into the planlet. Based on the results, PGRs combinations are required to improve embryogenic callus induction and regeneration into planlet in sorghum. © 2022 Friends Science Publishers

Cryo-Technologies for Ex Situ Conservation of Rosa Germplasm.

In this study, we compare two rapid cryopreservation (−196 °C) procedures, droplet-vitrification and encapsulation-dehydration for rose (Rosa × hybrida L., cultivars ‘Ioana’, ‘Mariana’, ‘Vulcan’). Significant factors for cryopreservation, such as sucrose concentration during osmoprotection, treatment duration with plant vitrification solution 2 (PVS2) in droplet-vitrification, duration of air desiccation and moisture content of alginate beads in encapsulation-dehydration, were investigated. In addition, the morphogenetic response to in vitro culture and to liquid nitrogen storage and the content in photosynthetic pigments have been assessed. The in vitro cultures were initiated from plant material originating from field collection. The highest regeneration frequencies were obtained for cv. ‘Vulcan’ in both of the cryopreservation procedures tested, 72% in droplet-vitrification and 65% following encapsulation-dehydration. The morphogenetic response (multiplication index and height of shoots) to liquid nitrogen storage was direct multiple shoot formation per initial shoot tip for all genotypes. The content in chlorophyll a and b was statistically comparable in plant material resulting from cryopreserved and non-cryopreserved shoot tips in all cultivars. The findings expand the information on Rosa‘s response to in vitro culture conditions and cryopreservation, providing protocols with a high regeneration capacity for the storage of genotypes with high ornamental value.

Optimization of Agrobacterium-mediated transformation procedure for an indica rice variety-Khang Dan 18

In addition to traditional methods, advanced biotechnologies, especially CRISPR/Cas9-mediated genome editing, have emerged as effective tools for improving important agronomic traits in rice. However, the critical step for utilizing these systems is to develope an effective system for rice transformation and regeneration. This study was performed to establish procedures for Agrobacterium-mediated transformation and regeneration of rice cultivar Khang Dan 18 (KD18) – a popular indica variety in the North of Vietnam. The tissue cultures procedure with optimized medium compositions showed high frequencies of callus induction and shoot regeneration, at 94.7% and 45.3% respectively. We found that 30 mg/L of hygromycin was an effective concentration for transgenic KD18 selection. Light-yellow friable calli were used as the starting material for transformation mediated by A. tumefaciens strain AGL1 harbouring pHUE411 vector containing gus intron and hygromycin resistance gene (hptII). Important factors related to transformation procedure had been optimized in this study. The high transformation frequency (12.8%) was achieved by using the optimized procedure for KD18 cultivar. In which, bacterial density (OD600), infection time and co-cultivation period were performed at 0.1, 20 minutes and 3 days, respectively. PCR analysis using specific primers and the histological GUS staining demonstrated the presence of hptII and gus genes in transgenic rice lines. This result provides a potential protocol to transfer genes of interest into KD18 as well as other indica rice cultivars.

Expression analysis of OsSERK, OsLEC1 and OsWOX4 genes in rice (Oryza sativa L.) callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species, including rice. This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon1 (LEC1) and WUSCHEL-Related Homeobox4 (WOX4) and also a helpful model for embryo development and clones and transformations. Here, we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice (pigmented and non-pigmented) using modifified N6 media supplemented with Kinetin (2.0 mg/L) and NAA (1.0 mg/L). Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties, rice is still unexplored, especially during somatic embryo development. Moreover, for the formation of callus induction from immature embryos, 2,4-D (2.0 mg/L, 3.0 mg/L) was used. This study analysed the gene expression of OsSERK, OsWOX4 and OsLEC1 genes through RT-PCR analysis. Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media. This study found that rice varieties of pigmented rice (MS Pendek and Gogoniti II) and non-pigmented rice (Pandan Ungu) showed high regeneration frequency, showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14. However, the contrast with Genjah nganjuk may be effective because of other regulatory genes. RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties, which correlate with the percentage of plant regeneration, but not for Gogoniti II. In conclusion, the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.

Efficient Plant Regeneration via Indirect Organogenesis in Carnation (Dianthus caryophyllus semperflorens flore pleno) Cultivars

Abstract Callus induction and plant regeneration are important steps of in vitro plant breeding of ornamental plants. In this study, the effects of different combinations of plant growth regulators (PGRs), promoters, and minerals on callus induction and plant regeneration in different carnation cultivars were studied in a completely randomized design with three replications. For callus induction, 16 different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and casein hydrolysate (CH) were studied using in vitro leaf explants. The Murashige and Skoog (MS) medium supplemented with 0.2 mg·dm−3 of 2,4-D and 200 mg·dm−3 of CH showed the highest frequency of callus induction. Among the cultivars, ‘Noblesse’ showed the highest rate of callus induction (91.67%). Regarding regeneration, BA, NAA, silver nitrate (AgNO3), and adenine hemisulfate (As) were used in ten different combinations. The ‘Cameron’, ‘Tabasco’, and ‘Noblesse’ cultivars with 95.24% regeneration percentage showed the highest rate of plant regeneration. Generally, in most cultivars, the highest regeneration rate and shoot number per explant were found in the MS medium supplemented with 3 mg·dm−3 of BA, 0.6 mg·dm−3 of NAA, 5 mg·dm−3 of AgNO3, and 40 mg·dm−3 of As. According to the results, the highest regeneration frequency was obtained when 40 mg·dm−3 of As was added to the medium. Finally, the flow cytometry analysis indicated that there were no significant differences between in vitro regenerated and control plants in terms of DNA ratios.

Methylglyoxal-glyoxalase system as a possible selection module for raising marker-safe plants in rice

Methylglyoxal (MG) is ubiquitously produced in all living organisms as a byproduct of glycolysis, higher levels of which are cytotoxic, leading to oxidative stress and apoptosis in the living systems. Though its generation is spontaneous but its detoxification involves glyoxalase pathway genes. Based on this understanding, the present study describes the possible role of MG as a novel non-antibiotic-based selection agent in rice. Further, by metabolizing MG, the glyoxalase pathway genes viz. glyoxalase I (GLYI) and glyoxalase II (GLYII), may serve as selection markers. Therefore, herein, transgenic rice harboring GLYI-GLYII genes (as selection markers) were developed and the effect of MG as a selection agent was assessed. The 3mM MG concentration was observed as optimum for the selection of transformed calli, allowing efficient callus induction and proliferation along with high regeneration frequency (55 ± 2%) of the transgenic calli. Since the transformed calli exhibited constitutively higher activity of GLYI and GLYII enzymes compared to the wild type calli, the rise in MG levels was restricted even upon exogenous addition of MG during the selection process, resulting in efficient selection of the transformed calli. Therefore, MG-based selection method is a useful and efficient system for selection of transformed plants without significantly compromising the transformation efficiency. Further, this MG-based selection system is bio-safe and can pave way towards better public acceptance of transgenic plants.

Cytokinin-Based Tissue Cultures for Stable Medicinal Plant Production: Regeneration and Phytochemical Profiling of Salvia bulleyana Shoots.

Salvia bulleyana is a rare Chinese medicinal plant that due to the presence of polyphenols lowers the risk of some chronic diseases especially those related to the cardiovascular system. The present study examines the organogenic competence of various combinations and concentrations of plant growth regulators to develop an efficient protocol for in vitro regeneration of S. bulleyana via leaf explants, maintaining the high production of active constituents. The purpose of the study was also to assess the possibilities of using a cytokinin-based regeneration to effectively produce therapeutic compounds. The adventitious shoot formation was observed through direct organogenesis on media with purine derivatives (meta-topolin, mT and benzylaminopurine, BAP), and through indirect organogenesis on media with urea derivatives (tidiazuron, TDZ and forchlorfenuron, CPPU). The highest regeneration frequency (95%) with 5.2 shoots per explant was obtained on leaves cultured on Murashige and Skoog (MS) medium containing 0.1 mg/L naphthalene-1-acetic acid (NAA) and 2 mg/L BAP. Following inter simple sequence repeat (ISSR) marker-based profiling, the obtained organogenic shoot lines revealed a similar banding pattern to the mother line, with total variability of 4.2–13.7%, indicating high level of genetic stability. The similar genetic profile of the studied lines translated into similar growth parameters. Moreover, HPLC analysis revealed no qualitative differences in the profile of bioactive metabolites; also, the total polyphenol content was similar for different lines, with the exception of the shoots obtained in the presence of CPPU that produced higher level of bioactive compounds. This is the first report of an effective and rapid in vitro organogenesis protocol for S. bulleyana, which can be efficiently employed for obtaining stable cultures rich in bioactive metabolites.