Subsequent Plant Regeneration Research Articles

Shoot Organogenesis from Tetrastigma hemsleyanum Leaf and Petiole Explants, and Subsequent Plant Regeneration and Acclimatization

Shoot Organogenesis from Tetrastigma hemsleyanum Leaf and Petiole Explants, and Subsequent Plant Regeneration and Acclimatization

An Efficient In Vitro Shoot Organogenesis and Comparative GC-MS Metabolite Profiling of Gaillardia pulchella Foug

Gaillardia pulchella Foug. is a widely studied plant because of its high pharmacological and ornamental value. The leaves of G. pulchella were used for inducing callus and subsequent plant regeneration as it is the primary source of phytocompounds. The purpose of the present investigation was to formulate an in vitro propagation method for Gaillardia by using leaf explants in MS (Murashige and Skoog) medium. The best callus induction was observed on high (2.0 mg/L) α-naphthalene acetic acid (NAA) and a low (0.5 mg/L) 6-benzylaminopurine (BAP) with callus induction frequency of 91.66%. The leaf callus also demonstrated high caulogenesis ability (95.83%), with an average 5.2 shoots/callus mass at 0.5 mg/L BAP and 2.0 mg/L NAA. Indole Acetic acid (IAA) at 1.0 mg/L had the maximum rooting percentage (79.17%) with 12.4 roots per shoot. Rooted plantlets were later transferred to greenhouse conditions, showing a survivability rate of 75–80%. The physiological parameters, i.e., phenolic compounds and the flavonoids’ level, in the DPPH assay were higher in leaves obtained in vitro compared to callus formed from leaves and field-obtained (mother) leaves. Gas chromatography–mass spectrometry (GC–MS) analysis of methanol extracts of leaves (in vivo and in vitro) and leaf callus presented a wide array of compounds. In callus extract, some 34 phytocompounds were identified. Some of them were 3-hydroxy-2,3-dihydromaltol (25.39%), isoamyl acetate (11.63%), palmitic acid (11.55%), 4-methyloxazole (7.54%), and 5-methoxypyrrolidin-2-one (7.49%). Leaves derived in vivo and in vitro had 45 and 28 phytocompounds, respectively, belonging to different classes like lignans, phenols, terpenoids, alkaloids and fatty acids, etc. Those findings demonstrated that the leaf derived callus and the leaves are the potential stable source of several compounds with medicinal importance. The developed protocol may provide an alternative source of compounds without affecting wild flora.

Enhancing salinity tolerance in Salix linearistipularis through advanced plant tissue culture techniques

Salinity is a major environmental challenge that hampers agricultural sustainability by affecting vast expanses of arable land globally, including significant portions of the Songnen Plain, which hosts substantial areas of saline-sodic soil detrimental to most plant life. This study focuses on Salix linearistipularis, a naturally salt-tolerant woody plant endemic to the region, to explore its potential in ecological restoration and soil improvement. Through extensive laboratory experiments, we employed various plant tissue culture methods to ascertain the most effective techniques for regenerating Salix linearistipularis, thereby facilitating its use in combating soil salinization. The research utilized several culture media and hormonal treatments to determine optimal conditions for seed germination, callus induction, and subsequent plant regeneration. Key findings indicate that specific combinations of growth hormones significantly enhance the rate of callus formation and adventitious bud differentiation, essential for the plants survival and growth in saline conditions. Furthermore, the study demonstrates the potential for these methods to produce genetically stable plants capable of thriving in hostile environments. The findings provide a valuable foundation for future applications in ecological restoration projects aimed at mitigating soil salinity issues.

Melatonin supplementation enhances browning suppression and improves transformation efficiency and regeneration of transgenic rough lemon plants (Citrus × jambhiri).

Enzymatic browning poses a significant challenge that limits in vitro propagation and genetic transformation of plant tissues. This research focuses on investigating how adding antioxidant substances can suppress browning, leading to improved efficiency in transforming plant tissues using Agrobacterium and subsequent plant regeneration from rough lemon (Citrus × jambhiri). When epicotyl segments of rough lemon were exposed to Agrobacterium, they displayed excessive browning and tissue decay. This was notably different from the 'Hamlin' explants, which did not exhibit the same issue. The regeneration process failed completely in rough lemon explants, and they accumulated high levels of total phenolic compounds (TPC) and polyphenol oxidase (PPO), which contribute to browning. To overcome these challenges, several antioxidant and osmoprotectant compounds, including lipoic acid, melatonin, glycine betaine, and proline were added to the tissue culture medium to reduce the oxidation of phenolic compounds and mitigate browning. Treating epicotyl segments with 100 or 200 μM melatonin led to a significant reduction in browning and phenolic compound accumulation. This resulted in enhanced shoot regeneration, increased transformation efficiency, and reduced tissue decay. Importantly, melatonin supplementation effectively lowered the levels of TPC and PPO in the cultured explants. Molecular and physiological analyses also confirmed the successful overexpression of the CcNHX1 transcription factor, which plays a key role in imparting tolerance to salinity stress. This study emphasizes the noteworthy impact of supplementing antioxidants in achieving successful genetic transformation and plant regeneration in rough lemon. These findings provide valuable insights for developing strategies to address enzymatic browning and enhance the effectiveness of plant tissue culture and genetic engineering methods with potential applications across diverse plant species.

Thidiazuron Enhanced Somatic Embryogenes is from Callus Lines of Arabica Coffee and Subsequent Plant Regeneration

Thidiazuron Enhanced Somatic Embryogenes is from Callus Lines of Arabica Coffee and Subsequent Plant Regeneration

Phytochemical Composition and Detection of Novel Bioactives in Anther Callus of Catharanthus roseus L.

Catharanthus roseus L. (G.) Don is the most widely studied plant because of its high pharmacological value. In vitro culture uses various plant parts such as leaves, nodes, internodes and roots for inducing callus and subsequent plant regeneration in C. roseus. However, till now, little work has been conducted on anther tissue using plant tissue culture techniques. Therefore, the aim of this work is to establish a protocol for in vitro induction of callus by utilizing anthers as explants in MS (Murashige and Skoog) medium fortified with different concentrations and combinations of PGRs. The best callusing medium contains high α-naphthalene acetic acid (NAA) and low kinetin (Kn) concentrations showing a callusing frequency of 86.6%. SEM-EDX analysis was carried out to compare the elemental distribution on the surfaces of anther and anther-derived calli, and the two were noted to be nearly identical in their elemental composition. Gas chromatography-mass spectrometry (GC-MS) analysis of methanol extracts of anther and anther-derived calli was conducted, which revealed the presence of a wide range of phytocompounds. Some of them are ajmalicine, vindolinine, coronaridine, squalene, pleiocarpamine, stigmasterol, etc. More importantly, about 17 compounds are exclusively present in anther-derived callus (not in anther) of Catharanthus. The ploidy status of anther-derived callus was examined via flow cytometry (FCM), and it was estimated to be 0.76 pg, showing the haploid nature of callus. The present work therefore represents an efficient way to produce high-value medicinal compounds from anther callus in a lesser period of time on a larger scale.

Is the soil seed bank a reliable source for passive restoration of intensive grazed habitats in river riparian areas of western Iran?

Soil seed banks serve as reservoirs of seeds for subsequent regeneration of plants particularly in areas prone to disturbance, such as intensive grazed areas along rivers. The potential of soil seed bank (SSB) for vegetation restoration was assessed in overgrazed riparian areas in Gamasyab river, Iran. Therefore, SSB composition, density and diversity and, its relationship with aboveground vegetation (AGV) were investigated along the river. Fifteen transects were set perpendicular to the river and along each transect, 6 plots (1 m × 1 m) were established, in total 90 plots. In each plot, soil was collected for SSB experiments and AGV was sampled during the growing season. The similarity between SSB and AGV was estimated by the Jaccard similarity index and non-metric dimensional scaling (NMDS). Spatial beta diversity was calculated by the mean Bray-Curtis index for the SSB and vegetation compositions, separately. A total of 136, 108 and 134 species were identified in the upper (0–5 cm) and lower (5–10 cm) soil layers and, AGV, respectively. Annual and perennial grasses were dominant in SSB composition at 0–5 cm depth while the frequency of perennial grasses was highest in SSB at deeper depth and vegetation composition. The beta diversity of AGV was significantly higher than for SSB. Similarity between SSB and vegetation was low (7.92%–22.40%), nevertheless, most species in AGV were found frequently in the SSB. So, we argued that restoration of degraded vegetation in our riparian could rely on SSB. It is expected that after grazing exclosure, vegetation would restore mostly with perennial grasses through SSB and vegetative growth.

<i>In vitro</i> regeneration of grape

Considering the global and Russian experience in grape accessions preservation, one of the most reliable ways is the creation of a duplicate in vitro collection. However, in connection with the creation of duplicate grape collections and development of genome editing techniques, there is a need for selecting the most optimal medium composition that will ensure the maximum rate of callus formation and regeneration during the introduction of grape plants into in vitro culture. This will make it possible to obtain the necessary amount of material for further editing and subsequent regeneration of plants with knockout of target genes to improve economically valuable traits. For grapes, this is primarily an increase in resistance to powdery mildew caused by Uncinula necator Burill.The effect of active substances of biological and synthetic origin on the morphogenesis has been studied for many crops, including regenerant plants of the genus Vitis L. However, the genus Vitis is very diverse and heterogeneous in its genetic, physiological and morphological structure, as a result of which the recommended media and components for cultivation under in vitro conditions may not suit every cultivar. Local Russian grape cultivars are better suited to local growing conditions, so efforts should be focused on the development of techniques related to the preservation of local varieties in collections in vitro.Knowledge of genes controlling certain traits, as well as the availability of grape accessions whose genome has been sequenced, contribute to successful in silico analysis for creating editing constructs.

IN VITRO PLANT REGENERATION IN LOCAL AUS RICE THROUGH MATURED SEED CULTURE

An investigation was carried out to decipher the effect of genotypes and concentrations of growth regulators for callus induction and subsequent plant regeneration from mature seeds of rice. Two local rice cultivars viz. Nayanmony and Kalomona and two growth regulators viz. 2, 4-D and NAA were used to evaluate their performance. The seeds were cultured under aseptic condition onto MS basal medium at varying concentrations and combinations of 2, 4-D (1.0, 1.5 and 2.0 mgL- 1) and NAA (0.0, 0.5 and 1.0 mgL-1) for callus induction. For plant regeneration MS medium fortified with 0.5 mgL- 1 NAA and 3.0 mgL-1 BAP was applied. Between the two cultivars Nayanmony showed better responses in callus induction frequency (91.38%), weight of callus (0.90 g)as well as diameter of callus (7.48 mm). Among the combinations 2, 4-D (1.5 mgL-1) alone produced the best callus (95.16%) and the highest weight of callus (1.88 g) but diameter of callus (8.27 mm) was the highest from 2, 4-D 1.0 mgL-1 +0.5 mgL-1 NAA. It was observed that both genotype and growth regulator significantly affected callus induction and plant regeneration. The medium supplemented with 2, 4-D 1.5 mgL-1 + NAA0.0 mgL-1 exhibited better performance in callus induction (99.33%) and weight of callus (1.92 g) in cultivar Nayanmony whereas Kalomona was better for diameter of callus (8.38 mm) at 2, 4-D 1.0 mgL-1 + NAA0.5 mgL-1. The cultivar Nayanmony performed better regarding plant regeneration (88.15%) compared to Kalomona (86.67%). Plantlets derived from the 2, 4-D 1.5 mgL-1+ NAA 0.0 mgL-1 showed the best results for survival. Therefore, it is suggested that application of MS medium supplemented with 2,4-D 1.5 mgL-1 and NAA 0.0 mgL-1 is advantageous to accomplish overall efficiency of callus induction, subsequent plant regeneration and survival from seeds of Nayanmony cultivars. Thus, selection of better responsive cultivar like Nayanmony and MS medium fortified with 1.5 to 2.0 mgL-1 2, 4-D would be beneficial in transformation attempts to improve the crop.

Species richness, diversity, density and spatial distribution of soil seed banks in the riparian woodland along the Thamalakane River of the Okavango Delta, northern Botswana

Soil seed banks serve as reservoirs of seeds for subsequent regeneration of plants. Soil seed banks were investigated along the Thamalakane Riparian Woodlands (hereafter referred to as TRWs) of the Okavango Delta, northern Botswana, from January-July 2015 and January-July 2016. The study aimed at examining species richness and diversity, determining densities, assessing the spatial distribution of seeds in the soil and comparing the similarity in species composition between the standing vegetation and soil seed bank flora. The vegetation was sampled in 71 plots (20 × 50 m) and soil samples were collected from 568 subplots. Agglomerative hierarchical cluster analysis was used to determine soil seed bank communities. Indicator species analysis was used to calculate indicator values for species in each community of germinated seeds and across different soil layers. Multi-response permutation procedures (MRPP) were used to compare similarity in soil seed bank composition. Bray-Curtis ordination was used to infer spatial relationships of soil layers in terms of soil seed bank composition. A total of 109 plant species were identified in the litter and top 9 cm soil layers with a mean density of 2101 seeds m−2. Herbs, grasses, sedges and woody plants were represented by 68, 19, 9 and 13 species, respectively, in 30 families and 87 genera. The overall total diversity and evenness of the soil seed bank in the TRWs was 3.25 and 0.69, respectively. Four plant communities were identified from the soil seed bank, namely Setaria verticilata-Amaranthus hybridus, Acanthospermum hispidum-Setaria sagittifolia, Digitaria eriantha-Eclipta prostrata and Cyperus longus-Fimbristylis dichotoma. Bray-Curtis ordination showed that there was overlap between these communities in terms of seed bank composition. However, MRPP analysis showed that there was significant (P < 0.05) separation between germinated soil seed bank communities. The overall horizontal distribution of seeds varied among sampling quadrats while the vertical distribution of seeds exhibited the highest densities occurring in the upper 3 cm of the soil and gradually decreasing densities with increasing depth. Relatively high densities also occurred in the litter layer. There were large differences in depth distribution between species, suggesting differences in seed longevity. The similarity in species composition between the soil seed flora and standing vegetation was low (27%). The results demonstrated that many species in TRWs store quantities of seeds in the soil. The fact that most of the woody species do not accumulate more seeds in the soil suggests that their regeneration from seeds would be unlikely if mature individuals disappeared (die or are harvested). Because of its diverse seed banks, the herbaceous flora would have a better chance of re-establishing in the event of anthropogenic or natural disturbances. Therefore, the future of the TRW woody flora seems to depend on the successful conservation of the standing vegetation.

Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes

Abstract. Carsono N, Juwendah E, Liberty, Sari S, Damayanti F, Rachmadi M. 2021. Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes. Biodiversitas 22: 2555-2560. The development of callus in the course of transgenic rice avoids the somaclonal variants. To obtain a high number of normal phenotypes and a low number of somaclonal variants requires an appropriate 2,4-D concentration. In this study, we obtained the best callus induction time and a high number of green plant regeneration for three responsive rice genotypes on different 2,4-D concentrations in NB5 medium. The mature seeds of rice embryos were used as explants. A completely randomized factorial design was applied with four levels of 2,4-D concentrations (0, 1, 3, and 5 ppm), two levels of induction time (one and two weeks), and three rice genotypes (cv. Fatmawati, Nipponbare, and Kitaake). The study revealed that there was no interaction effect among genotype, 2,4-D concentration, and callus induction time. Three rice genotypes performed best in callus proliferation and regeneration. One-week callus induction time showed higher callus growth capacity (CGC) as compared to two-week callus induction time. Shoot regeneration capacity (SRC) was independently affected by genotype as well as by callus induction time. The interaction effect between 2,4-D concentration and callus induction time was observed on plant regeneration capacity (PRC). Without the addition of 2,4-D and 1 ppm of 2,4-D, the green plant regeneration capacity (GRC) was comparatively higher. Addition of 2,4-D showed a significant effect, especially at the plant regeneration stage. We found that one-week callus induction was the best treatment for callus proliferation and plant regeneration. We recommend the use of one-week callus induction and 1 ppm of 2,4-D for rice callus proliferation (sub-culture) and subsequent plant regeneration.

Cryopreservation of apple shoot tips by vitrification and subsequent plant regeneration

Cryopreservation of apple shoot tips by vitrification and subsequent plant regeneration

In vitro Callus Induction under NaCl Salt Stress and Subsequent Plant Regeneration in Oats (Avena sativa L.)

An effort was made to identify the in vitro responsiveness for callus induction in NaCl (salt stress) and subsequent plant regeneration of oats (Avena sativa L.). Callus induction was tested in different concentrations (0, 5, 10, 15 and 20 mg/l) of NaCl salt added in MS. The experiment was laid out in CRD considering 2,4-D growth regulator and NaCl salt as factors. The best response in callus induction was observed on MS containing 3 mg/l 2,4- D and 5 mg/l NaCl where the frequency of callus induction was 68.57%, callus weight was 0.36 g and callus diameter was 7.63 mm. During regeneration, the maximum frequency of regeneration (41.67%), regenerated shoots per callus (14.80), shoot length (12.14 cm), root number (10.90) and root length (3.76 cm) was observed from the callus derived from MS containing 3 mg/l 2,4-D and 5 mg/l NaCl. Plantlets that were regenerated from callus induced in MS with 3 mg/l 2,4-D and 5 mg/l NaCl showed higher rate of ex vitro survivability. Therefore, MS with 3 mg/l 2,4-D and 5 mg/l NaCl found better for in vitro callus induction and subsequent plant regeneration from mature seeds of oat.&#x0D; Plant Tissue Cult. &amp; Biotech. 30(2): 253-266, 2020 (December)

Effective callus induction and plant regeneration in callus and protoplast cultures of Nigella damascena L.

In this study we report the development of effective in vitro systems for a medicinal plant Nigella damascena L. comprising: (1) callus induction, (2) somatic embryogenesis in callus cultures with subsequent plant regeneration, and (3) isolation and regeneration of callus-derived protoplasts. Callus development was achieved on 83–100% of hypocotyl and cotyledon explants, whereby Murashige and Skoog medium (MS) supplemented with 3 mg L−1 6-benzylaminopurine and 0.5 mg L−1α-naphthaleneacetic acid (NAA; BN medium) was more advantageous than MS with kinetin and NAA (KN medium). Histological observations of calli revealed the presence of embryogenic zones from which somatic embryos developed on the hormone-free medium. Plant regeneration was observed on 76–95% of calli. A high capacity to form somatic embryos and regeneration was maintained in long-lasting cultures, i.e. even in 2 year old callus.The obtained callus was also a good source tissue for protoplast isolation. By applying a mixture of cellulase and pectolyase, the acceptable yield of viable protoplasts was achieved, especially from hypocotyl-derived callus maintained on BN medium. Protoplasts embedded in an alginate matrix and cultured in modified Kao and Michayluk media re-constructed their cell wall and re-entered mitotic divisions. About 30% of small cell aggregates formed microcalli, which, after the release from alginate, proliferated continuously on KN and BN media, irrespective of the tissue variant used as the protoplast source. Somatic embryo formation and plant regeneration were successful on hormone-free media. An effective plant regeneration system of N. damascena protoplast cultures has been developed and is being reported for the first time.

Application of cryobanking for Platycodon grandiflorum in vitro axillary buds using cryo-plate methods

The application of the V cryo-plate method using either PVS2 or PVS3 and the D cryo-plate method to Platycodon grandiflorum in vitro axillary buds was investigated. Precultured buds (1 mm) were attached to cryo-plates using calcium alginate gel. Then, osmoprotection treatment (2 M glycerol and 1 M sucrose) was performed by immersing the cryo-plates for 30 min at 25°C. The procedures for each cryogenic method were as follows: in the V cryo-plate method, the buds on cryo-plates were exposed to PVS2 for 40 min (highest regrowth 71.1%) or PVS3 for 50 min (highest regrowth 82.2%). In the D cryo-plate method, the buds on the cryo-plates were dehydrated under the air current of a laminar air flow cabinet for 60 min (highest regrowth 80.0%) at 25°C. Then, the cryo-plates were plunged directly into liquid nitrogen. After cryopreservation, buds on the cryo-plates were rewarmed and unloaded by immersion in 1 M sucrose solution for 15 min at 25°C for subsequent plant regeneration. The average regrowth seen on the V cryo-plates exposed to PVS3 and in the D cryo-plate method was higher than that of the V cryo-plates exposed to PVS2. The regrowth after cryopreservation in the V cryo-plate method with PVS2 was not stable. Thus, the V cryo-plate method with PVS3 and the D cryo-plate method are considered to be practical cryopreservation methods for P. grandiflorum germplasm.

Phytotoxic Effect of Herbicides on Various Camelina [Camelina sativa (L.) Crantz] Genotypes and Plant Chlorophyll Fluorescence

Camelina is an oil plant classified as a minor crop. The small acreage is the main cause of the small amount of plant protection products that are registered for use on camelina plantations. This contributes to difficulties in the protection of this plant. In the conducted experiment, the genetic similarity of genotypes of camelina was compared. The effect of selected herbicides (propaquizafop at rate 70 g a.i. ha−1, quizalofop-p-ethyl at rate 50 g a.i. ha−1, clopyralid at rate 90 g a.i. ha−1, and picloram at rate 24 g a.i. ha−1 applied in the three-four-leaves growth stage of camelina) on six individual genotypes of the plant and plant chlorophyll fluorescence after the use of these substances was also determined. The Przybrodzka variety showed the lowest level of damage in the assessment carried out 42 days after herbicide application and the damages of plants after quizalofop-p-ethyl and propaquizafop was completely gone. The variety Przybrodzka had the lowest genetic similarity to all analyzed genotypes. In other cases, genetic similarity of analyzed genotypes could not be linked to herbicide-related damage. Picloram contributed to the greatest damage to test plants and had the greatest impact on the operation of photosystem II (PSII). However, the level of plant chlorophyll fluorescence parameter values indicates small PSII damage for all substances and the possibility of subsequent plant regeneration. The results of the presented research indicate that it is worth referring to several plant varieties in phytotoxicity studies of herbicides towards arable crops.

Somatic Embryogenesis and Plant Regeneration from Cotyledon and Hypocotyl Explants of Fagopyrum esculentum Moench lpls Mutant

Buckwheat (Fagopyrum esculentum, Family Polygonaceae) is an annual pseudo-cereal crop with healing benefits. However, the genetic improvement of common buckwheat has achieved only limited success, mainly due to buckwheat’s dimorphic flowers and heteromorphic self-incompatibility. Here, we develop a useful protocol for indirect somatic embryogenesis and subsequent plant regeneration from hypocotyl explants of F. esculentum. Firstly, the initial calli of hypocotyl explants were induced on Murashige and Skoog (MS) basal medium containing 2.0 mgL−1 2,4-D and 1.5 mgL−1 6-BA for 30 days culture, and then the yellowish white friable embryogenic calli were developed when the initial calli were transferred to fresh MS basal medium supplemented with 1.0 mgL−1 6-BA and 0.5 mgL−1 thidiazuron (TDZ)two to three times subculture at 40–60 days intervals. Subsequently, the somatic embryos were able to germinate from embryogenic callus sub-cultured on MS basal medium containing 1.0 mgL−1 6-BA and 0.5 mgL−1 TDZ with 15% potato puree for 20 days subculture. Finally, maximum mean percentage (75.75%) of somatic embryo-derived plants were obtained when the mature somatic embryos were transferred to MS basal medium without growth regulators for 40 days culture. Our result provides a useful protocol for plant regeneration and SE from hypocotyl explants of F. esculentum.

Targeted genome modifcation in protoplasts of a highly regenerable Siberian barley cultivar using RNA-guided Cas9 endonuclease

The modifcation of crop genomes employing functional components of the microbial CRISPR/Cas immune system is a rapidly developing area of applied research. Site-directed plant genome modifcation by this technology involves the construction of Cas endonuclease- and guide-RNA-encoding vectors, delivery of the plasmid DNA into plant cells, processing of the chosen genomic target site by the corresponding gene products and regeneration of plants from modifed cells. The utilization of this technology in local breeding programs is mainly limited by the typically strong genotype dependence of gene transfer andin vitroregeneration procedures, which holds particularly true in cereals. In the present study, an evaluation ofin vitroregeneration efciency of immature embryos of ten Siberian barley cultivars revealed that only one of these is on a par with the experimental standard cultivar Golden Promise. This cultivar, namely cv. Aley, was consequently chosen for further experiments on site-directed mutagenesis in leaf mesophyll protoplasts. Two genes controlling hulledvsnaked (Nud) and two-rowedvssix-rowed barley (Vrs1) were used as targets to be modifed via polyethyleneglycol-mediated cellular uptake of guide-RNA/Cas9-encoding plasmid DNA. Deep-sequencing of amplicons obtained from protoplast genomic DNA revealed that 6 to 22 percent of the target sites were mutated. The detected modifcations comprised deletions in all three target sites and of various sizes, whereas insertions were observed in only one of the target genes (Vrs1) and were confned to the size of 1 nucleotide. This study demonstrates the possibility of site-directed genome modifcation in Siberian barley. Further steps in technology advancement will require the development of protocols with reduced genotype dependence in terms of both the gene transfer to totipotent cells and the subsequent plant regeneration originating from such cells.

In vitro callus induction and indirect organogenesis of Mentha piperita (L.) - an aromatic medicinal plant

The effect of different concentrations and combinations of plant growth regulators were tested for in vitro callus induction and indirect plant regeneration of Mentha piperita L. from young leaves and internodal explants. Greenish, organogenic and nodular nature of callus was achieved only from leaf disc explant when cultured on MS medium containing 1.5 mg/l NAA+ 0.2 mg/l BAP and then subsequent plant regeneration was obtained on MS medium supplemented with 2.0 mg/l BAP+0.5mg/l NAA from leaf derived callus. The highest frequency of shoot regeneration (90%) with the maximum number of shoots (15.1±0.11) was achieved in this medium. In vitro raised microshoots showed 90% response with maximum number of roots (16.3±0.23) on 1.0 mg/l IBA after 11-12 days. Then in vitro rooted plantlets were move to polybags bearing soil and vermiculite mixture in 1:1 ratio for hardening and hardened plantlets showed 95% survivability on field conditions.

Agrobacterium rhizogenes-mediated transformation of a dioecious plant model Silene latifolia

Silene latifolia serves as a model species to study dioecy, the evolution of sex chromosomes, dosage compensation and sex-determination systems in plants. Currently, no protocol for genetic transformation is available for this species, mainly because S. latifolia is considered recalcitrant to in vitro regeneration and infection with Agrobacterium tumefaciens. Using cytokinins and their synthetic derivatives, we markedly improved the efficiency of regeneration. Several agrobacterial strains were tested for their ability to deliver DNA into S. latifolia tissues leading to transient and stable expression of the GUS reporter. The use of Agrobacterium rhizogenes strains resulted in the highest transformation efficiency (up to 4.7% of stable transformants) in hairy root cultures. Phenotypic and genotypic analyses of the T1 generation suggested that the majority of transformation events contain a small number of independent T-DNA insertions and the transgenes are transmitted to the progeny in a Mendelian pattern of inheritance. In short, we report an efficient and reproducible protocol for leaf disc transformation and subsequent plant regeneration in S. latifolia, based on the unique combination of infection with A. rhizogenes and plant regeneration from hairy root cultures using synthetic cytokinins. A protocol for the transient transformation of S.latifolia protoplasts was also developed and applied to demonstrate the possibility of targeted mutagenesis of the sex linked gene SlAP3 by TALENs and CRISPR/Cas9.