Direct Shoot Regeneration Research Articles

A Novel Solid Media-Free In-Planta Soybean (Glycine max. (L) Merr.) Transformation Approach

Soybean’s lengthy protocols for transgenic plant production are a bottleneck in the transgenic breeding of this crop. Explants cultured on a medium for an extended duration exhibit unanticipated modifications. Stress-induced somaclonal variations and in vitro contaminations also cause substantial losses of transgenic plants. This effect could potentially be mitigated by direct shoot regeneration without solid media or in-planta transformation. The current study focused primarily on developing a rapid and effective media-free in-planta transformation technique for three soybean genotypes (Wm82) and our newly developed two hybrids, designated as ZX-16 and ZX-3. The whole procedure for a transgenic plant takes the same time as a stable grown seedling. Multiple axillary shoots were regenerated on stable-grown soybean seedlings without the ectopic expression of developmental regulatory genes. An approximate amount of 200 µL medium with a growth regulator was employed for shoot organogenesis and growth. The maximal shoot regeneration percentages in the Wm82 and ZX-3 genotypes were 87.1% and 84.5%, respectively. The stable transformation ranged from 3% to 8.0%, with an average of 5.5%. This approach seems to be the opposite of the hairy root transformation method, which allowed transgenic shoots to be regenerated on normal roots. Further improvement regarding an increase in the transformation efficiency and of this technique for a broad range of soybean genotypes and other dicot species would be extremely beneficial in achieving increased stable transformation.

Plantlet regeneration of Cucumis melo L. Glamour cv. using different types of cytokinin and explants

Plantlets or clones’ regeneration in Cucurbitaceae species can be performed in vitro by applying the tissue culture techniques. Hypocotyls, cotyledons, leaves, cotyledonary nodes and petioles can be used as the explants or starting materials to initiate the regeneration under laboratory condition. BAP (Benzyl aminopurine) and TDZ (Thidiazuron) were among the well-known cytokinin used in tissue culture as plant hormones to regulate the plant growth in vitro. The effectiveness of BAP and TDZ was investigated in this study to determine the effect of different types of cytokinin hormone on C. melo explants since both hormones might show differences in their activity towards C. melo explants. This study found that BAP was superior at all used concentrations compared to TDZ during most of the growing stage of the explants (cotyledons and hypocotyls). Based on this finding, the experiment was repeated by using different types of explants which were nodal explants, petioles and young shoots along with cotyledons and hypocotyls, by using BAP that showed excellent plantlet regeneration. Cotyledon and nodal explants successfully regenerated plantlets with excellent height and significant number of shoots compared to the rest of the explants. The plantlets from cotyledons and nodal explants were regenerated via direct regeneration without any formation of callus. The direct shoot regeneration was the preferred method for mass production via tissue culture because the chances of somaclonal variation is low. Contrarily, the hypocotyls, petioles and shoot tips mostly developed callus and only several treatments formed very small plantlets.

Direct shoot regeneration from cotyledonary node of Uraria picta (Jacq.) Desv. ex DC., an important plant of dashmula drugs, and assessment of genetic fidelity, metabolic profiling, and anti-diabetic activity

Uraria picta root is used in the polyherbal product ‘Dashmula’. Its exploitation for formulation preparation has depleted its availability, leading to medicine adulteration. Direct shoot regeneration from the cotyledonary node holds promise as a source of raw material. This study aimed to develop a regeneration protocol for U. picta and validate its genetic and metabolic fidelity. The seeds of U. picta showed low germination rates, prolonged dormancy, and poor viability. Root exploitation in the wild poses a threat to its availability in nature. Seedling’s derived cotyledonary nodes cultured on B5 medium supplemented with BAP (0.5–3 mg L−1), Kinetin (0.5–3 mg L−1), Thidiazuron (0.01–1 mg L−1), and meta-Topolin (0.1–4 mg L1). To address hyperhydricity in regenerated shoots, cotyledonary nodes were cultured on high-agar concentration media. Microshoots were exposed to IBA solution (50–800 mg L−1) pulse treatment for rooting. Tissue-cultured plants genetic fidelity was assessed using ISSR and SCoT markers, while metabolic fidelity was studied with HRMS. The chlorophyll content, antioxidant, and antidiabetic activity of micropropagated plants were evaluated. The highest shoot regeneration frequency, with a maximum of 6.57±0.278 shoots per explant, was achieved using 2 mg L−1meta-Topolin. The shoots were elongated, had expanded leaves, and were hyperhydrated. BAP (2 mg L−1) induced a maximum of 9.83±0.333 shoot buds per explant. BAP caused explant browning, profuse callus formation, dwarfing, and hyperhydric shoots. Hyperhydricity was alleviated with a higher agar concentration (1 %). IBA (400 mg L−1) induced a maximum of 2.18±0.090 roots per shoot and a root length of 9.23±0.033 cm. Tissue-cultured and mother plants exhibited clonal fidelity, similar metabolite and chlorophyll content, strong antioxidant activity, and equal efficacy for inhibiting α-amylase and α-glucosidase. This method can propagate elite clones of U. picta and offer its improvement via genetic transformation.

ESTABLISHMENT OF AN EFFICIENT MICROPROPAGATION PROTOCOLS FOR THREE KENAF (HIBISCUS CANNABINUS L.) CULTIVARS

This study was aimed to establish an efficient micropropagation protocol for three popular kenaf (Hibiscus cannabinus L.) cultivars (V36, HF992 and KB6) using different types of plant growth regulators (PGR’s) on Murashige and Skoog medium (MS). The shoot tip, cotyledon and hypocotyl isolated from 2 weeks old seedlings were used as explants. MS medium fortified with different types and concentrations of cytokinins and auxin were evaluated for establishment callus formation, shoot multiplication and rooting. Among different PGR’s used, 5.0 mg/l 2iP + 0.1 mg/l NAA was the best concentration for direct shoot regeneration from cotyledon with shooting percentage of 55% in HF992 cultivar. In multiple shoot induction experiment, 1.0 mg/l TDZ + 0.1 mg/l NAA mg/l concentration has induced the highest (10.4) shoots/explant. Moreover, indirect shoot regeneration was established from callus which was induced from cotyledon, maximum callus induction percentage (88.75%) was recorded on 0.5 mg/l TDZ and 1.0 mg/l NAA in V36 variety. While, for shoot elongation and rooting, MS media half strength supplemented with 1.0 mg/l kinetin + 1.0 mg/l IBA was found to be an optimum concentration.

A novel approach for direct shoot regeneration, anatomical characterization, and withanolides content in micropropagated plants of Withania somnifera (L.) Dunal—an important medicinal plant

A novel approach for direct shoot regeneration, anatomical characterization, and withanolides content in micropropagated plants of Withania somnifera (L.) Dunal—an important medicinal plant

Exogenous Cytokinin Induces Callus and Protocorm-Like-Bodies Formation in In Vitro Root Tips of Vanilla planifolia Andrews.

Vanilla is a popular flavouring essence derived from the pods of vanilla orchid plants. Due to the high demand for vanilla flavour, high yielding vanilla plantlets are necessary for establishing vanilla plantations. Clonal micropropagation is a viable technique for the mass production of high yielding vanilla plantlets. This study reports an efficient regeneration protocol by using cytokinin as the sole plant growth regulator to regenerate plantlets from the root tips of a commercial vanilla orchid species, Vanilla planifolia. Most studies to date have reported using seeds and nodes as starting explants for in vitro micropropagation of vanilla orchids. So far, regeneration from roots has not been very successful. Previous studies favoured the use of auxins only or high auxin to cytokinin ratios to induce callus, and sole cytokinins were used for direct shoot regeneration. However, it was sporadically observed in plantlets regeneration of V. planifolia that multiple shoots were regenerated from the tips of intact aerial roots submerged in media. This study therefore investigated the regeneration of excised vanilla root tips through the application of most commonly used auxins (1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid) and cytokinins (6-benzylaminopurine and thidiazuron). High auxin presence is known to promote callusing in in vitro plants. However, in this study, auxin treatment inhibits callusing in root tips. While cytokinin treatments, even at low levels, has promoted high rate of callusing. These callus cells regenerate into protocorm-like-body (PLB) shoots when cytokinin levels are increased to 0.5 mg/mL 6-benzylaminopurine (BAP) under light conditions. The findings of the study have the potential of providing large quantity of high yielding vanilla plantlets through clonal micropropagation.

Optimization of an Efficient Direct Shoot Induction System and Histological Evaluation of Shoot Formation in Cucurbita maxima Duch

Improving plant regeneration ability and shortening regeneration time can promote the development of genetic transformation breeding technology for horticultural crops. We optimized several culture conditions, including explant type, mother plant genotype, and medium, to improve shoot formation in winter squash (Cucurbita maxima Duch.). Histological analysis of the occurrence of shoots was also carried out. The results indicate that cotyledon was the most suitable explant for inducing the shoot regeneration of winter squash. We found that ‘Jin-li’ had a shorter shoot induction time and a higher average number of shoots. The highest induction rate of 95.23% among the five lines. The average shoot induction rate of five lines was the highest (84.85%) on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 6-benzylaminopurine (6-BA) and 0.2 mg/L indole-3-acetic acid (IAA). We also found that there was an interaction between genotypes and induction media, and their interaction had a greater impact on the shoot induction rate than individual effects. Histological observation revealed that the induced shoots of winter squash cotyledons originated from subepidermal cells. We also found that the optimal medium for de novo root regeneration was 1/2 MS. We acclimatized and cultivated regenerated plants and harvested their fruits, which maintained the characteristics of mother plants. These findings lay an important foundation for further research on direct shoot regeneration and accelerate its application in winter squash genetic transformation.

Establishment of an Effective in vitro Regeneration System for Sugar Beet (Beta vulgaris L.)

Plant regeneration and dedifferentiation are known to be difficult for sugar beet (Beta vulgaris L.) due to its recalcitrant nature. This leads to low transformation efficiency and thus successful application of plant molecular techniques is limited in sugar beet for its genetic enhancement. A prolific regeneration method has been established by modulating several plant growth regulators on the in vitro regeneration of Beta vulgaris var. V6 KWS Serenada. Several types of explants excised from young seedlings of this variety were used for both direct and indirect regeneration of shoots. The highest response towards direct shoot formation and callus induction were obtained from cotyledonary nodes and hypocotyls, respectively. Explants were cultured on MS medium supplemented with different concentrations of BAP, GA3 and 2, 4 -D for callus induction as well as formation of shoots. Hypocotyls responded well for callus induction on MS medium containing 0.1 mg/l BAP + 2.0 mg/l 2, 4-D, while cotyledonary nodes exhibited the highest responses towards shoot formation on MS medium containing 1.0 mg/l BAP and 1.5 mg/l GA3. MS medium containing 2.0 mg/l IBA produced the highest number of roots per shoot. The in vitro raised rooted plantlets were successfully transferred to soil for acclimatization. Plant Tissue Cult. & Biotech. 33(2): 181-188, 2023 (December)

Direct Shoot Regeneration of Plumbago zeylanica Linn. through Tissue Culture Technology

ABSTRACT: Chitrak is scientifically known as Plumbago zeylanica Linn. belongs to Plumbaginaceae. Root shows medicinally valuable drug in pharmacological enterprise as the humanity is certainly prone to numerous contagious ailments due to ecological imbalance triggered by the current technology development. One of its remarkable phytoconstituents is plumbagin, a naphthoquinone molecule used to treat skin conditions, tumours, and tenacious chronic rheumatoid arthritis. The objective was to standardize the procedure of Plumbago zeylanica direct-organogenesis micropropagation. The solution contained various concentrations and combinations of auxin and cytokinins, and the nodal explant was used for quick micropropagation. The maximum multiple shoots (18.24 ± 0.51) per explant with shoot length 4. 2cm ± 0.20 cm was produced as the MS medium supplemented with B5 vitamins together with 2mg/l 6- Benzyleaminopurine (BAP) and 1mg/l Indole-3- acetic acid (IAA). The regenerated shoots were rooted in half strength MS medium with B5 vitamins that contained indole-3-butyric acid (IBA). The highest rooting frequency (100%) was seen in the 1.5 mg/l IBA-containing rooting media. The extreme roots were 9.2±0.14 per explant. The average root length was 4.2± 0.10 cm. The in vitro rooted Shoots were then transplanted in the field showing a 100 percentage survival rate.

In Vitro Regeneration, Conservation, and Field Evaluation of a Medicinal Plant– Greater Burdock (Arctium Lappa L.)

A suitable micropropagation protocol and ex vitro acclimation method have been developed from in vitro grown seedling explants through cotyledonary node and leaf explants in consideration of the vegetable and medicinal properties of Greater Burdock. MS medium with 0.5-4.0 μM BAP showed highest percentage of axillary shoot regeneration from the cotyledonary nodal explants. Direct shoot regeneration was achieved by culturing 1.0 cm2 sections of about 25 days old leaves of in vitro grown shoot on MS medium enriched with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. Within six weeks of incubation on medium enriched with 4.0 M BAP and 2.0 M IBA or NAA, the leaf explants also developed callus from the cut margins. The greatest number of adventitious shoots could then be formed from the leaf-derived callus within 10 weeks of culture on the same media mix. More than 20 shoots were formed per callus clump at the third subculture, which had the highest rate of shoot multiplication. A. lappa's shoot and callus were both preserved at 5 ºC in MS medium with 4.0 μM Kn and 2.0 μM IBA, as well as 4.0 μM BAP and 2.0 μM IBA, respectively. The in vitro proliferated and elongated shoots were separated from callus clump for rooting. A root-induction MS medium with 6.0 μM IBA or NAA was used to cultivate the microshoots individually. All of the cultured microshoots generated 2-16 roots within 4 weeks of being moved to the rooting medium. Regenerated plantlets were transferred to vermiculite and successfully established in an ex vivo environment with a 98% survival rate. J. Bio-Sci. 31(1): 1-15, 2023

Conservation in Tissue Culture of Malacocarpus crithmifolius (Retz.) Fisch. C.A.Mey. - Relict Species from Mangyshlak

Malacocarpus crithmifolius is an endangered rare relict with small size populations grown in a limited area. The purpose of this study was to initiate in vitro and micro replication of Malacocarpus crithmifolius. The vegetative and reproductive explants and Murashige & Skoog medium (MS) supplemented with different concentrations and combinations of cytokinin and auxins were used. The response reaction of different explants and the plant growth regulators' effects on shooting frequency, the number of shoots, callusogenesis, and rooting percent were investigated. Culturing generative explants on the MS medium containing 6-Benzylaminopurine (BAP) combined with indole-3-acetic acid (IAA) or indole-butyric acid (IBA) led to direct shoot regeneration. The bud mass appeared in the leaf axil under inflorescences after one week of cultivation on the initiated medium. The replication was reached by micro-cutting of induced additional shoots and 3-fold passaging them on the fresh mediums. The average multiplication result at MS with BAP in combination with 1-Naphthaleneacetic acid (NAA) or IAA composed 5.85 after three months of cultivation. In conclusion, the applicable explant and phytohormonal composition of nutrient medium for initiation and micropropagation of M. crithmifolius were developed.

Highly efficient microtuber formation, direct shoot regeneration, and root induction in Cyclamen persicum Mill. from in vitro seedling-derived tuber and leaf segments

Highly efficient microtuber formation, direct shoot regeneration, and root induction in Cyclamen persicum Mill. from in vitro seedling-derived tuber and leaf segments

Direct and indirect shoot regeneration from leaf explants of Centratherum punctatum Cass., A wild ornamental plant

Centratherum punctatum Cass. is an important wild ornamental medicinal plant belonging to Asteraceae family. It has anti-inflammatory, anti-cancerous, anti-hypersensitive, anti-bacterial and anti-fungal properties and is commonly used in hair care preparations. In this study we describe a protocol for high frequency direct and indirect shoot regeneration from leaf explants. In vitro derived leaves were cultured on MS medium supplemented with different concentrations of TDZ, ZN or BAP (0.1–2.0 mg/L) alone or in combination with NAA (0.1–0.7 mg/L) for shoot regeneration and varying concentrations of 2, 4-D or NAA (0.2–2.0 mg/L) for callus induction. Optimum direct shoot regeneration response was obtained from in vitro derived leaves in MS medium supplemented with 0.5 mg/L TDZ (61.4 ± 3.90% response; 27.4 ± 1.99 shoots per explant; mean shoot length of 4.8 ± 0.81 cm). Out of the 7, 14, 21 and 28-day-old leaves, 14-day-old leaves resulted in maximum shoot induction indicating the influence of explant age. Lamina with petiole exhibited a significant increase in shoot regeneration compared to that without petiole. Pretreating the explants with 1.0 mg/L TDZ for 24 h prior to culture produced the highest response of 92 ± 1.84% with a mean number of 38.7 ± 1.24 shoots per explant. 2, 4-D was found favourable for callus induction than NAA for callus induction from leaf explants of C. punctatum. The 21-day-old leaf showed the optimum callus induction. The optimum shoot regeneration from callus was obtained in 1.5 mg/L TDZ (84.2 ± 4.09% response with 58.2 ± 2.46 shoots per explant). Well-developed shoots with 3.0–4.0 cm length were rooted on half strength MS medium containing 0.5 mg/L IBA. The rooted shoots were transplanted to soil and acclimatized with 96.8 ± 3.26% success. Analysis of clonal fidelity of the micropropagated plants through SCoT marker analysis revealed the genetic similarity of mother plant and in vitro derived plants.

Control of media browning during micropropagation and assessment of biochemical and clonal fidelity of in vitro-derived and mother plants in Thottea siliquosa (Lamk.) Ding Hou., an important ethnomedicinal shrub

BackgroundThottea siliquosa (Lamk.) Ding Hou., an important medicinal shrub, is widely used in both ayurvedic and indigenous systems of medicine. Root being the most useful part, the plant is constantly uprooted and thus puts pressure on the natural population. Until date, no micropropagation study is available in this plant. The objective of the study is to develop an efficient in vitro propagation protocol and assessment of clonal fidelity of T. siliquosa. ResultsMedia browning was a serious issue during micropropagation, and the addition of 40.0 mg/L ascorbic acid reduced the media browning. For direct shoot regeneration, the optimum response (92% frequency with 20.9 shoots per explant) was obtained when 7-day-old cotyledons were cultured on WPM supplemented with 1.0 mg/L thidiazuron and 0.25 mg/L α-naphthalene acetic acid. The cultures were transferred to WPM augmented with 0.4 mg/L thidiazuron for shoot elongation and growth. On this medium, 100% of cultures responded with a mean number of 27.6 shoots. For callus induction, MS medium with 1.0 mg/L 2,4-dichlorophenoxyacetic acid and 0.5 mg/L N6-benzylaminopurin was used. Shoot organogenesis was initiated on the same medium, and calli with minute shoots were transferred to MS medium fortified with 0.5 mg/L N6-benzylaminopurin and 0.25 mg/L α-naphthalene acetic acid for highest shoot regeneration (100% cultures responded with a mean number of 26.5 shoots per explant). Maximum rooting frequency (82%) and number (20.8) were obtained on half-strength MS medium with 1.0 mg/L indole-3-butyric acid. The rooted plants were acclimatized and transferred to the field. The HPTLC and SCoT analysis revealed the phytochemical and clonal similarity between the in vitro propagated plants and mother plant. ConclusionsIn this study, it is confirmed that cotyledon is an excellent explant for direct and indirect shoot organogenesis in T. siliquosa. For direct shoot induction WPM and indirect organogenesis, MS medium was found to give better response. The true-to-type nature of in vitro-derived plants were confirmed by phytochemical and SCoT analysis. The protocol described here could be used for the large-scale propagation of elite clones of T. siliquosa.

Transverse thin cell layer (tTCL) technology: a promising tool for micropropagation of Centratherum punctatum Cass.

The present study describes an efficient protocol for the micropropagation of Centratherum punctatum Cass. through transverse thin cell layers (tTCLs). The effect of plant growth regulators (PGRs), thickness of tTCLs, and source of tTCLs were evaluated. The tTCLs of varying thickness (0.5 to 5.0 mm) were excised from 10-d-old leaf and 45-d-old node and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of PGRs (BAP, TDZ, KN; 0.1 to 2.0 mg L−1), alone or in combination with NAA (0.1 to 1.0 mg L−1) for shoot induction. The leaf and node tTCLs responded with direct shoot regeneration. A significant effect of thickness or width on shoot induction was observed. For leaf, 1.0-mm-wide and for node 2.0-mm-thick tTCLs showed a maximum response. MS medium supplemented with 1.5 mg L−1 TDZ and 1.0 mg L−1 BAP in combination with 0.2 mg L−1 NAA was found to be optimum for shoot induction from tTCLs of leaf (96% response with 41.5 shoots per explant) and node (87% response with 11.9 shoots per explant), respectively. Rhizogenesis was obtained when micropropagated shoots were transferred to half strength MS medium supplemented with various concentrations (0.25 to 2.0 mg L−1) of IBA. The rooted plantlets were eventually acclimatized and transferred to soil. The clonal fidelity assessment by SCoT markers of the mother plant and micropropagated plants revealed their genetic similarity. Chlorophyll content estimation indicated lower levels in in vitro plants, which increased to a comparable level to that of field-grown plants after 3 mo of acclimatization, showing absence of stress during acclimatization. The procedure described here is a promising tool for micropropagation of C. punctatum as it produces high-frequency healthy shoots from a minimum explant source.

Meta-Topolin enhanced direct shoot organogenesis and regeneration from leaf explants of Coleus forskohlii (Willd.) Briq

A novel and effective direct shoot regeneration technique from stomatal complexes was developed for a highly sought after medicinal plant Coleus forskohlii. Meta-Topolin was more efficient than 6-benzylaminopurine (BAP) in promoting direct organogenesis from leaf surface. Highest direct organogenesis was achieved from in vitro leaf explants on Murashige and Skoog (MS) medium augmented with meta-Topolin at the concentration of 2.0 mg/L. The regenerated leaves were fixed at weekly intervals and sectioned longitudinally to evaluate the anatomical developments. The results of light microscopic analysis suggested that the in vitro developed stomatal complexes showed new altered cell divisions, after a week of incubation, and the rapid periclinal divisions resulted in the formation of a meristematic dome. Distinct regions of meristematic activity were visible in the second week characterized by promeristems with small and densely stained parenchymatous cells. Promeristems lead to the development of a distinct tunica layer, which grew into shoot-like protuberances converting into adventitious shoots after 4 weeks of incubation. A clear three phases of shoot organogenesis viz. induction (7 days), initiation and organization (14 days), and shoot formation (28 days onwards) were identified. The shoots were proliferated (21.8 shoots/ single leaf explant) and elongated (5.0 cm length) using half-MS medium fortified with 0.5 mg/L mT and indole-3 acetic acid (IAA) at 0.1 mg/L. Notable rhizogenesis of the elongated shoots was achieved when cultured on half-MS medium containing indole-3 butyric acid (IBA) at the concentration of 1.5 mg/L. About 92.0% of the plantlets survived upon field transfer. Start Codon Target (SCoT) markers were employed to examine the regenerated plants’ genetic uniformity with the mother plant, similar banding patterns confirming the genetic uniformity. Hence, the developed protocol of direct in vitro regeneration of C. forskohlii using mT can be used for the commercial production.

High‑frequency plant regeneration and genetic fidelity assessment of regenerants by molecular and biochemical markers in Woodland Sage (Salvia nemorosa L.)

Salvia nemorosa L. is a valuable medicinal and ornamental plant from Lamiaceae . A high-frequency protocol for in vitro regeneration from S. nemorosa leaf explants was developed based on our results. Various plant growth regulators were evaluated for their effects on direct and indirect organogenesis , callogenesis, shoot multiplication, and rooting of explants at different concentrations. The maximum percentage of direct shoot regeneration (97.5%) and the number of shoots per explant (8.6) were obtained on MS medium supplement with 8 µM TDZ plus 8 µM NAA . The result revealed that media amended by 16 µM 2, 4-D with 8 or 16 µM BA was the best treatment for callus induction. Media containing the combination of 8 µM TDZ plus 2 µM NAA provided the highest rate of shoot induction (84%) and the highest number of shoots (5.4) from calli. Regenerated shoots were multiplied efficiently with an average of 7.4 shoots and 32.4 leaves per explant on media containing 12 µM TDZ. In the present study, TDZ and BA treatments led to the highest proliferation rate; however, adenine sulfate was preferred for proliferation because it did not produce hyperhydric plants. The genetic fidelity of 19 plantlets (one donor plant, nine direct and nine indirect regenerants) was appraised by 10 ISSR markers. The molecular analysis revealed that direct organogenesis produced true-to-type plantlets and could be employed for clonal multiplication of Woodland Sage. Indirect regenerants showed polymorphic bands (12.5–33.33%), confirming the somaclonal variation occurrence during indirect organogenesis. Also, HPLC analysis of regenerants showed that two indirect regenerants accumulated higher rosmarinic acid content than mother plants, while three of them accumulated lower rosmarinic acid content. As part of this study, molecular and biochemical markers have been employed to investigate somaclonal variation in in vitro regeneration of the Salvia genus for the first time, which could be utilized to develop reliable methods for multiplying elite lines and achieving high-yield lines. • A high-frequency protocol for S. nemorosa in vitro regeneration was developed. • TDZ and NAA combination produced plantlets with high genetic and biochemical fidelity. • Adenine Sulphate prevents the Hyperhydricity in regenerated plantlets. • The indirect regeneration affected PAL activity, rosmarinic acid, and genetic content.

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.

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.

Influence of Plant Growth Regulators on Glandular Trichome Density and Steviol Glycosides Accumulation in Stevia rebaudiana.

With the advent of modern lifestyles, diabetes-related comorbidities attributed the importance of low-caloric natural sweetener plants such as Stevia rebaudiana. This plant is the viable source of steviol glycosides (SGs) and other economically important secondary metabolites. Glandular trichomes (GTs) play the role as a reservoir for all secondary products present in the plant species. Therefore, the present study was carried out to evaluate the influence of different plant growth regulators (PGRs) on GT density and its impact on the SG content. The direct shoot regeneration system was developed on Murashige and Skoog (MS) + benzyl aminopurine (BAP) (1.0 mg/L) + naphthaleneacetic acid (NAA) (0.5 mg/L), and MS + BAP (1.5 mg/L) + NAA (0.5 mg/L) from nodal and leaf explants, respectively. Among the combination of PGRs used, MS medium fortified with BAP (1.0 mg/L) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0.5 mg/L) played a significant role in increasing the GT density on leaf and stem tissues of S. rebaudiana. Furthermore, high-performance thin-layer chromatography and gas chromatography–mass spectrophotometry data confirmed a notable rise in SGs and other valuable secondary metabolites. Thus, the protocol developed can be used for the propagation of stevia with an improved metabolic profile at a large scale.