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Cryopreservation, a widely utilized technique for the long-term preservation of in vitro cultures, effectively arrests metabolic processes, obviating the need for frequent subcultures and mitigating the risk of somaclonal variation. In this study, we applied cryopreservation methods to intact rice (Oryza sativa L.) calli to determine the optimal age for cryopreservation, investigating the timelines for greening and shoot initiation in R0 plants. Results revealed that three-month-old calli exhibited the highest regeneration percentage, with greening observed within twelve days and shoot initiation within fifteen days. Using 3% mannitol in the callus culture medium provided osmotic stress, aiding in the formation of compact calli masses suitable for regeneration. Vitrification with cryoprotectants (DMSO, PEG, and glucose) and gradual dehydration improved cell survival. Thawing and post-thaw damage were minimized using rapid thawing, fast cryoprotectant removal, and gradual rehydration. We assessed the phenotypic variations in R1 and R2 generation and genotypic fidelity of regenerants in R1. Phenotypic variations from seed-derived plants were observed in seed characters both in vitrified and cryopreserved calli-derived plants. However, these variations were unstable and disappeared in the R2. SSR markers were used to detect genetic variations in R1, with results showing a 3.6% change in vitrified calli-derived plants and an 8.61% change in cryopreservation-derived plants, likely due to reversible DNA methylation or SNPs in non-coding region. Our study confirms the feasibility of cryopreservation for rice calli, ensuring high regeneration rates and minimal long-term genetic variations.Key message: Three-month-old rice calli maintained in callus proliferation medium with 3% mannitol and vitrified in a combination of cryoprotectants ensure high regeneration rates post-cryopreservation, maintaining phenotypic stability and minimal genetic variation. [ABSTRACT FROM AUTHOR]

• Multiple shoots were successfully regenerated from shoot tip explants of Tecoma stans L., under the influence of meta-topolin (mT). • in vitro raised leaf tissues revealed higher phytochemical diversity and significant increase in antidiabetic alkaloid. • No genetic variation was observed among micropropagated plants with the explant donor plant (mother plant) via RAPD marker assessment. Tecoma stans (Bignoneaceae) is a valuable medicinal shrub having commercial and industrial importance for its bioactive phyto-constituents. In the current study, multiple shoots were successfully regenerated from shoot tip explants of Tecoma stans L., under the influence of meta -topolin (m T). On evaluation of the morphogenic effect of its various doses either singly or in combination with different auxins, MS medium fortified with 7.50 μM m T and 0.50 μM NAA yielded maximum shoots (11.8 ± 0.37) per explant with an average length (5. 08 ± 0.28 cm), after 8 weeks. For studying the effect of subculturing passages, the cultures were grown on to optimized medium and an enhanced shoot multiplication upto 35 shoots per explant were observed at 8th subculture passage. For ex vitro rooting, healthy microshoots were excised and pulse treated for 3 h with higher doses (100 - 500 μM) of auxins IAA, IBA or NAA separately before their transplantation to Soilrite. Of the tested treatments, best rooting was obtained in 300 μM IBA treated microshoots, after 4 wk of plantation in Soilrite. Further, on studying the effects of secondary metabolite production, in vitro raised leaf tissues revealed higher phytochemical diversity and significant increase in antidiabetic alkaloid (Tecomine) from 0.36% control plant to 1.68% in in vitro raised plants. Inspite of different phytochemical profiles, no genetic variation was observed among micropropagated plants with the explant donor plant (mother plant) via RAPD marker assessment. The established micropropagation system appears to be practicable for large scale production of T. stans progenies with higher number of biochemical compounds. [ABSTRACT FROM AUTHOR]

Crapemyrtle (Lagerstroemia sp.) is the top-selling flowering tree in the U.S. However, threats from arthropod pests, including the recently emerged crapemyrtle bark scale (CMBS; Acanthococcus lagerstroemiae), severely jeopardize the aesthetic and production attributes of crapemyrtle. A tropical species, L. speciosa (L.) Pers. (“Queen’s Crapemyrtle”) exhibits partial resistance to CMBS and other pests, but conventional breeding to incorporate the characteristics of L. speciosa into existing hybrids remains challenging. Recognizing the potential of tissue culture in facilitating molecular breeding, but also the possibility of undesirable somaclonal variations from in-vitro organogenesis, we utilized leaf explants of L. speciosa to develop a callus-induced regeneration protocol and assessed genetic fidelity of regenerated plantlets using inter-simple sequence repeat (ISSR) markers. Using woody plant medium (WPM) supplemented with 0.2 mg/L 2,4-D and 1.0 mg/L 6-BA achieved 97.9% callus induction. Shifting the growth regulators to 10.0 mg/L 6-BA and 0.5 mg/L NAA resulted in 32.4% of callus explants differentiating into adventitious buds. Finally, nodal segment proliferation (94.6%) and new shoot growth was maximized by using WPM supplemented with 1.0 mg/L 6-BA and 0.02 mg/L NAA. Explants rooted 100% using half-strength WPM supplemented with 0.2 mg/L IBA, and acclimatization survival was 98.3%. The ISSR primer analysis revealed 98.7% monomorphic markers, confirming the genetic integrity of the regenerated plantlets. We describe a reliable callus-induced regeneration system for L. speciosa, which will facilitate future molecular breeding and biotechnology to enhance cold hardiness, pest resistance, and other desired traits in this important genus.Key message: This research developed a callus-induced crapemyrtle regeneration protocol and verified plantlet genetic fidelity, laying the groundwork for advancing molecular breeding to optimize horticultural traits in this important landscape plant. [ABSTRACT FROM AUTHOR]

The efficiency and method of regeneration in in vitro culture conditions depend primarily on the plant growth regulators (PGRs) used. Even growth regulators belonging to one group may have different effects, stimulating the process of direct or indirect organogenesis, thus possibly disturbing the genetic stability among regenerants. The main aim of this study was to identify the genetic stability of Scutellaria baicalensis regenerates obtained by in vitro culture method using start codon targeted (ScoT) markers. S. baicalensis nodal explants were regenerated on MS medium supplemented with kinetin (KIN) at concentrations of 0.25, 0.5, 1.0, and 2.0 mg × dm−3 or benzylaminopurine (BAP)—0.25, 0.5, 1.0, and 2.0 mg × dm−3. The effects of the number of propagated shoots, length, number of nodes, and fresh mass of regenerants were assessed. Moreover, the genetic stability of the regenerants was analyzed using start codon targeted (SCoT) markers. Direct shoot organogenesis was observed on an MS medium containing kinetin, while indirect shoot induction occurred on an MS medium supplemented with BAP. The highest average number of shoots (3.6) was achieved for the MS + KIN medium at a concentration of 0.25 and 5.8 for the MS + BAP 1.0 medium. The average length and average number of nodes were the highest on the MS + BAP 0.25 medium (50.0 and 6.0, respectively), while the lowest values of these features were observed on the MS + KIN 2.0 medium (40.3 and 4.9, respectively). A total of 111 amplified bands were exhibited by SCoT primers. Three of the analyzed primers revealed four unique genotype-specific markers. The average percentage of polymorphism obtained was 36.7%. The analysis of genetic similarity revealed a high level of genetic similarity between the donor plant and regenerants obtained on MS "0" (medium without the addition of phytohormones). A slightly lower value of genetic similarity was observed for regenerants obtained by direct organogenesis (MS + KIN medium at all concentrations). Indirect shoot organogenesis observed on the MS + BAP medium (all concentrations) resulted in the highest differentiation, both in relation to the donor plant and MS "0" regenerants. The results of our work indicate that, in the case of S. baicalensis, the maintenance of genetic stability depends primarily on the presence of the cytokinin type in the medium. [ABSTRACT FROM AUTHOR]

In vitro micro rhizome technology is a highly effective approach in combating seed-borne diseases and ensuring the production of healthy and high-quality planting material in ginger (Zingiber officinale Rosc.). To gauge the efficiency of micro rhizome production and their viability ex vitro, an experiment was conducted on several ginger varieties viz., IISR Varada, IISR Mahima, IISR Rejatha, and Karthika, at ICAR- Indian Institute of Spices Research, Kozhikode, Kerala, India. This experiment adhered to established protocols and standardized procedures. All four varieties exhibited varying rates of micro rhizome production after 180 days of culture. Among these, IISR Varada demonstrated the highest mean weight of cultured plant mass (96.0 ± 4.41 g), followed by Karthika (91.4 ± 5.72 g), IISR Rejatha (78.45 ± 5.59 g), and IISR Mahima (72.4 ± 3.56 g). IISR Rejatha exhibited the maximum number of micro rhizomes per bottle (11.35 ± 0.81) compared to IISR Mahima (10.8 ± 0.54), Karthika (9.8 ± 0.58), and IISR Varada (9.0 ± 0.63). The highest total weight of micro rhizome and mean weight of a single micro rhizome per bottle were recorded in IISR Varada (32.6 ± 1.92 g and 3.9 ± 0.29 g, respectively), followed by Karthika (27.1 ± 1.19 g and 2.9 ± 0.16 g, respectively), IISR Rejatha (27.0 ± 1.79 g and 2.5 ± 0.18 g, respectively) and IISR Mahima (24.5 ± 1.10 g and 2.4 ± 0.16 g, respectively). Besides, IISR Varada, followed by Karthika, emerged as the most promising varieties for micro rhizome production in terms of their multiplication rate. The evaluation extended to the first and second-generation progenies of micro rhizomes from IISR Varada. Results indicated the successful establishment of first-generation micro rhizomes in grow bags and second-generation micro rhizomes in the field, employing both direct planting and transplanting methods. Assessment of quality parameters revealed that the second-generation (V2) transplanted plants of micro rhizomes of IISR Varada exhibited the highest essential oil content 0.78%. The total phenolic content was highest in second-generation (V2) rhizomes directly planted in soil (23 mg GAE/g), whereas the first-generation micro rhizomes raised in grow bags registered the highest total flavonoid content (TFC) of 1.39 mg QE/g. Moreover, the genetic fidelity test conducted on the first and second generations (V1 and V2, respectively) of micro rhizome-derived plants, using molecular markers, exhibited a monomorphic banding pattern similar to that of the mother plant, confirming their genetic stability.Key message: In vitro micro rhizomes of ginger have the potential to combat pathogens transmitted through seed materials, while simultaneously preserving clonal fidelity and ensuring high quality standards. [ABSTRACT FROM AUTHOR]

Bretschneidera sinensis is a monotypic species of rare and tertiary relic trees mainly distributed in China. B. sinensis is a potentially valuable horticultural plant, which has significant ornamental and research value, and is a crucial tool for the study of phylogeography. The artificial cultivation of B. sinensis is of great scientific value and practical significance. In this study, we developed a direct organogenesis process of B. sinensis using mature zygotic embryos as initial materials. The highest sterile germination induction (54.5%) from the mature zygotic embryo was obtained in a Murashige and Skoog (MS) medium with 2.0 mg·L-1 6-benzylaminopurine (6-BA) and 0.2 mg·L-1 a-naphthaleneacetic acid (NAA). The highest percentage of shoot regeneration (90.37%) was attained using 1.0 mg·L-1 6-BA and 0.01 mg·L-1 NAA in the MS medium. The Woody Plant Medium (WPM) had the greatest adventitious shoot elongation rate of 93.33%. The most optimized rooting rate was 88.89% in a half-strength MS medium containing 2.0 mg·L-1 indole-3-butyric acid (IBA) and 1.0 mg·L-1 NAA. The genetic fidelity of in vitro regenerated plantlets was assessed using inter-simple sequence repeats and random amplified polymorphic DNA molecular markers, confirming the genetic uniformity and stability of regenerated B. sinensis plantlets. Our research presents an effective in vitro propagation system for B. sinensis, laying the groundwork for its germplasm conservation and large-scale production while maintaining high genetic integrity. [ABSTRACT FROM AUTHOR]

Establishing vegetative propagation techniques to promote the rejuvenation/reinvigoration of genotypes is essential for the rescue of adult trees of the Eucalyptus genus used in species and provenance tests. The aim of this study was to evaluate the induction of epicormic buds and shoots in pruned branches and the in vitro establishment, multiplication, genetic fidelity, and elongation of three 44-year-old Eucalyptus pilularis selected plants. M1, M2 and M3 represent selected plants. M1 produced the highest number of epicormic buds, number of shoots, and with the lowest rate of tissue oxidation and non-reactive explants. In the establishment stage, M3 exhibited the lowest contamination percentage, number of shoots, and shoot length. The emission of shoots occurred only for M3, and it was the unique genotype subjected to the other stages (i.e. multiplication, elongation, and rooting). In the multiplication stage, the highest vigour and shoot length values of were found in the 15th subculture. Phenolic oxidation had its highest value in the 12th subculture, decreasing from the 13th. The highest value for number of shoots was found in the 11th subculture. No polymorphism was observed in the selected plant (M3) and the clonal plants obtained in the 15th subculture. For shoots elongation, the use of culture medium containing 0.10 mg L-1 BAP and 1.00 mg L-1 NAA provided the lowest means for oxidation, and the highest for vigour, number, and length of shoots. The emission of adventitious roots was observed, demonstrating that, through micropropagation, it was possible to induce the competence to root the material, even at an advanced ontogenetic age. [ABSTRACT FROM AUTHOR]

Cryopreservation is an alternate approach for the long-term preservation of vegetatively propagated crops like sugarcane. This technique is used to preserve plant cells in vitro, enabling long-term storage of plant materials without alteration or modification of their natural state. The goal of this study was to develop a simple and efficient cryopreservation method for the long-term storage of Saccharum species. The cryopreservation techniques were optimized for three distinct explants of Saccharum species, which include the apical meristem, meristem-derived axillary buds, and dormant nodal buds. Various low-temperature cryopreservation strategies were used in this study, viz., two-step freezing, desiccation followed by direct immersion, vitrification, and encapsulation. The regeneration of material after cryogenic treatment at different time intervals exhibited germination in meristem-derived axillary buds and dormant nodal buds. Meristem culture was undertaken to obtain disease-free meristem-derived shoots for cryopreservation. Under encapsulation followed by dehydration, meristem-derived axillary buds of IND 19–2045 and Co 86032 regenerated after cryo treatments at various time intervals from 15 min to 70 h. The genetic fidelity of Co 86032 was confirmed using SRAP markers. Dormant nodal buds of four clones, namely IND 03–1310, IND 03–1294, IND 03–1295, and IND 03–1296, responded well after cryo treatments at different time intervals ranging from 15 min to 70 h under the desiccation–direct immersion method and the encapsulation–dehydration method. [ABSTRACT FROM AUTHOR]

Purple flesh sweet potato var. 'Bhu Krishna' is a rich source of vitamins, minerals, and several important secondary metabolites, including anthocyanin. The tuber of this variety contains a high level of anthocyanin (85-90 mg/100 g). This plant is propagated through stem cutting, which is time-consuming and diseases are transmitted to the next generation. Therefore, there is an urgent need to develop a micropropagation system for the production of huge number of healthy plants to fulfil the demand of farmers as well as industrial utilization. The present study reports a reproducible micropropagation system for purple flesh sweet potato var. 'Bhu Krishna' using mature nodal explants on Murashige and Skoog's (1962) medium fortified with various plant growth regulators (PGRs). Maximum shoots regeneration was observed on MS medium fortified with 2.0 mg/l meta-Topolin. In vitro nodal segments were excised from established shoot cultures and inoculated on the above said optimum medium to produce more shoots (ca. 1300 shoots). In vitro regenerated shoots were successfully rooted on half-strength MS medium augmented with 0.5 mg/l Indole-3-butyric acid. The micropropagated plants were acclimatized on soil, coco peat, and sand (1:1:1) and about 95% of plants survived. Finally, these plants were transferred to the field. The genetic unifomity and phytochemical analysis of micropropagated plants vis-à-vis mother plant were demonstrated using Inter Simple Sequence Repeat markers and spectrophotometric method, respectively. The therapeutic activity in terms of anticancer activity of tubers of micropropagated plants with the mother plant was also tested. For the first time we are reporting a detailed micropropagation system for purple flesh sweet potato var. 'Bhu Krishna' for its mass propagation to fulfil the demand of farmers as well as industries. [Display omitted] • Micropropagation protocol was developed for anthocyanin-rich Sweet potato first time. • Multiple shoot proliferation from nodal segment on MS + 2.0 mg L−1 mT. • Rooting of in vitro regenerated shoot on ½ MS + IBA 0.5 mg L−1. • Genetic fidelity was analyzed by molecular marker (ISSR). • Phytochemical and anticancer activity were evaluated both the mother vis-à-vis micropropagated plant tuber. [ABSTRACT FROM AUTHOR]

Papaya is an important crop with a short juvenile phase among the fruit crops. Direct and indirect regeneration protocols were developed for papaya cultivar Red Lady 786, by using apical, nodal, petiole, leaf and root segments. For direct regeneration, plant growth regulators (PGRs) i.e. BAP, Zeatin and NAA and indirect regeneration BAP, Kinetin, TDZ and NAA were investigated. The study showed that apical and nodal segments can undergo direct regeneration with distinct shoot differentiation without the callus phase, resulting in a high success rate of shoot development (65–88%). The indirect method involves the initiation of callus tissues, followed by morphogenesis of somatic embryogenesis significantly influenced by PGRs. A high success rate of somatic embryogenesis (ranging from 75–85%) was observed in cultures derived from multiple explants. The presence of somatic embryogenesis at specific differential stages, namely globular, heart, torpedo and cotyledonary had confirmed through stereo-microscopic analysis. The result significantly indicated that the supplementation of MS media with TDZ (0.3 mg/l), BAP (1.0 mg/l), NAA (0.10 mg/l), and 30 mg/l sucrose as the most efficient medium for somatic embryogenesis. Somatic embryos were sub-cultured on BAP, Kinetin and NAA resulted in a high frequency (75–80%) of complete plantlet regeneration except for root segment explant derived somatic embryos. The in-vitro regenerated plants via direct and indirect regeneration were successfully acclimatized in greenhouse with 88.89% survival rate. The genetic fidelity was checked by using 22 RAPD and 18 ISSR markers. This developed protocol may be utilized for commercial production of ‘Red Lady 786’ papaya plantlets. [ABSTRACT FROM AUTHOR]

Camelina (Camelina sativa L. Crantz) is an oil and medicinal crop in the Brassicaceae family that possesses many good agronomic qualities, such as stress resistance, strong adaptation, and low water and fertilizer inputs. The objectives of the present study were to optimize somatic embryogenesis and evaluate the genetic fidelity of regenerated Camelina plants using RAPD markers and flow cytometry. The experiment was performed in a factorial form using a completely randomized design with two factors and three replications. The studied factors included two explants of hypocotyls and cotyledons and four different combinations of PGRs composed of NAA, BAP, 2,4-D, and Kin. The observation of many somatic embryonic developmental stages occurring simultaneously on an embryogenic callus, including the globular, heart-shaped, torpedo-shaped, and cotyledonary phases, suggested that camelina embryogenesis accurately reflects an unsynchronized process. The cotyledon explants cultured on MS + 0.3 mg L− 1 NAA + 0.7 mg L− 1 BAP and 1.0 mg L− 1 Kin showed the highest rates of somatic embryogenesis and plant regeneration. Using RAPD markers, genetic fidelity in mother plants and regenerated plants was assessed for the first time in camelina. Sixty-four well-resolved bands were amplified using nine primers, ranging from four to eleven bands, with an average of 7.11 bands per primer. Molecular investigation revealed that there was no genetic variation in either the mother plants or the regenerated plants. The study of regenerated and mother plants using flow cytometry revealed monomorphic patterns, confirming the stability of the ploidy level across plant regeneration. These findings are helpful for breeding and genetic engineering of camelina. Key message: Camelina somatic embryogenesis is an unsynchronized process. This is the first study on the genetic fidelity of regenerated camelina plants using RAPD markers and flow cytometry. [ABSTRACT FROM AUTHOR]

Crinum brachynema is a bulbous plant belonging to the family Amaryllidaceae which is restricted to Western India. Due to its high rarity and low distribution range, it has been classified as "critically endangered". Establishing an efficient and unprecedented somatic embryogenesis protocol is necessary for its conservation and large-scale propagation. In this study, regeneration was achieved through somatic embryogenesis using bulb explants on MS media supplemented with various concentrations of 2,4-D alone and in combination with N6-benzyl-adenine. Different advanced phases with maturation of somatic embryo were obtained on MS medium with different ratios of picloram and thidiazuron (TDZ). The highest number of somatic embryos (50.33 ± 1.52) was obtained after eight weeks in the medium supplemented picloram (2.0 mg L−1) in combination with TDZ (0.5 mg L−1). MS medium with reduced concentration of salts in combination with GA3 (1.0 mg L−1) was used for somatic embryo germination. The maximum embryo germination frequency (82.22) was recorded on half-strength MS medium fortified with 1 mg L−1 GA3. The genetic true-to-typeness of regenerated plants was confirmed by ISSR, SCoT and RAPD primers based molecular analyses. This confirmed their genetic homogeneity compared to the mother plant and it also demonstrated the reliability of our somatic embryogenesis system for C. brachynema. The protocol developed may facilitate efforts in reintroduction, restoration, and ex situ conservation of C. brachynema in its natural habitat and its potential commercial utilization. Key message: We provided the first report on somatic embryogenesis system in C. brachynema. SEM indicated the morphogenesis and several molecular markers revealed genetic homogeneity of the regenerated plants. [ABSTRACT FROM AUTHOR]

A micropropagation protocol via direct shoot organogenesis from leaf explants of six commercial varieties of Saintpaulia ionantha Wendl was established in this study. The shoot induction was successfully achieved on Murashige and Skoog (MS) media supplemented with 0.2 mg L-1 indole-3-acetic acid (IAA) and 0.5 mg L-1 benzylaminopurine (BA). In proliferation stage, the effects of two combinations of PGRs (V1-0.2 mg L-1 IAA + 0.2 mg L-1 BA and V2-0.2 mg L-1 NAA +1 mg L-1 BA) on shoot number and length were examined for each genotype. The results suggest that PGR's combinations significantly influenced shoot proliferation in all analysed variety and among the treatments 0.2 mg L-1 NAA in combination with 1 mg L-1 BA was the most effective for in vitro shoot multiplication. The in vitro rooting percentage was 86.86-96.66% and was varieties-dependent. In vitro-raised plants showed a very high rate of survival (82-94 %). The genetic fidelity between the selected vitro-plants and mother plants were confirmed by CDDP markers. [ABSTRACT FROM AUTHOR]

Adenium obesum is a popular ornamental plant propagated through seeds. But its hybrids are mostly infertile or need assisted pollination for seed production. In the present investigation, an efficient and reliable indirect regeneration protocol for infertile adenium hybrid was developed from leaf explants. Surface sterilization using mercuric chloride 0.2 percent recorded the lowest incidence of contamination and highest survival percentage. Callus from shoot tip showed the lowest number of days for shoot regeneration with 13.2 days in Halfstrength MS medium containing 3 mg L-1 NAA and 3mg L-1 GA3. Maximum shoot length of 2.40 cm was recorded in Half-strength MS medium + 3 mg L-1 NAA+ 3mg L-1 GA3 two weeks after sub culturing. Half-strength MS+2mg L-1 IBA recorded root initiation in 16.16 days. The rooted plantlets were successfully hardened and acclimatized with a survival rate of 92 %. The genetic fidelity of regenerated plantlets was assessed using ten primers and the in vitro cultured plants did not show polymorphism in ISSR analysis. This in vitro propagation protocol could be effectively used for the large-scale propagation of non-seed setting hybrids of adenium. [ABSTRACT FROM AUTHOR]

Rhodiola imbricata is a rare and endangered plant of the Trans-Himalayan region having important medicinal properties. It holds immense therapeutic value against a wide range of diseases and health problems including hypoxia, cancer, stress, anxiety, fatigue, and gastrointestinal problems. The plant which is normally propagated through seeds suffers from drawbacks such as limited seed availability, low seed viability, and germination, limited geographical distribution, slow growth, and slow accumulation of secondary metabolites. Owing to the growing demand for this plant, a novel, highly efficient liquid culture system using meta-Topolin (mT) was developed for its rapid multiplication and continuous production of important bioactive compounds. A comparative analysis was conducted to evaluate the response of shoot multiplication, rooting, and secondary metabolite content in the solid and liquid culture media. In vitro seedlings were inoculated on Murashige and Skoog’s (MS) (1962) medium supplemented with different concentrations of cytokinins. Among the tested cytokinins, the maximum number of shoots were observed in 20 mL of liquid MS medium supplemented with mT (5.0 µM). While Indole-3-butyric acid (IBA) (10.0 µM) exhibited the highest rooting response (95%). Mass propagation of microshoots was achieved using a specialized box, resulting in an improved survival rate of 85% during the subsequent hardening process. The secondary metabolite content, including rosavin, salidroside, tyrosol, total polyphenolic content (TPC), and antioxidant properties were estimated for shoots grown in both agar-gelled solid and liquid culture media. Overall, liquid MS medium supplemented with mT (5.0 µM) was found to be the optimum medium for secondary metabolites production in comparison to solid medium. Further, the genetic and phytochemical stability of the prolong culture of this plant under in vitro conditions were confirmed. This system facilitates large scale production of in vitro plants as well as secondary metabolites throughout the year, which is crucial for various industrial applications.Key message: Rhodiola imbricata is an important, rare and endangered medicinal plant. A novel and efficient protocol for improved shoot proliferation and secondary metabolite production was developed using meta-Topolin and liquid culture. [ABSTRACT FROM AUTHOR]

The prized Red banana, selected for superior qualities, demands strong genetic uniformity for successful clonal propagation and preservation. Ensuring this uniformity early in the growth of in vitro Red banana plants is essential, as gene mutations and chromosome rearrangements during tissue culture can jeopardize both cloning and germplasm conservation. In this situation, molecular markers play a pivotal role in confirming genetic stability. Thus the study aims to discover a marker that identifies tissue-cultured Red bananas from their virescent variants during initial sub-culturing. A marker linked to anthocyanin has been identified which effectively differentiated Red bananas from virescent variants and it was further validated in various banana cultivars, ornamental Musa species and their interspecific hybrids. The PCR-based marker showed remarkable specificity, discerning Red bananas from virescent variants during tissue culture. It also distinguished green and red offspring, cutting time and resource costs, and shortening the banana breeding cycle. [ABSTRACT FROM AUTHOR]

Solena amplexicaulis (Lam.) Gandhi is a creeping cucurbit with high medicinal values in terms of its therapeutic uses. Traditionally, this plant species is used as a folklore medicine for the treatment of various disorders like spermatorrhea, urinary retention, jaundice, and cardiovascular disorders. Owing to high medicinal importance, it is overexploited, which has resulted in decrease in its population size in the wild. Furthermore, its natural propagation through root tubers and seeds is limited. In view of this constraint, the present study of in vitro micropropagation was chosen as an alternative for its propagation and conservation. Murashige and Skoog (MS) media amended with plant growth regulators like 6-benzylaminopurine (BAP) and thidiazuron (TDZ) independently and with a combination of indole-3-acetic acid (IAA) were tested for in vitro shoot induction by using nodal explants. The highest number of shoots (32.84±0.02) per explant was achieved on Murashige and Skoog (MS) medium with 1.0 mg L-1 TDZ and 1.5 mg L-1 IAA. The induced shoots were elongated with a maximum shoot length of 10.25±0.01 cm on MS medium augmented with 2.0 mg L-1 gibberellin (GA3) and 1.5 mg L-1 TDZ. Rooting of in vitro raised shoots was attained on half-strength MS medium fortified with 1.0 mg L-1 indole-3-butyric acid (IBA) and 2.0% sucrose. The hardening of resultant complete plantlets was carried out in plastic cups by using a 2:1 ratio of sterile black soil and sand; initially, they were kept in greenhouse conditions and subsequently shifted to natural field conditions with 78% of survival rate. The genetic fidelity of tissue-cultured plants was confirmed by ISSR (inter simple sequence repeats) and SCoT (starting codon targeted) primer-based analysis. [ABSTRACT FROM AUTHOR]

Artemisia vulgaris is a medicinally important essential oil–yielding plant belonging to Asteraceae, used as a traditional remedy against chronic diseases. The essential oil of this plant has been used as an insect repellent and antimicrobial agent. The growing concern of antibiotic resistance has forced to find an alternative way to control multi-drug-resistant urinary tract–infecting bacterial pathogens by using the essential oil and leaf extract of A. vulgaris for the first time. The antibacterial activity showed that the essential oil (in situ, ex vitro) and leaf crude extract (ex vitro) are highly effective on all human urinary tract–infecting pathogens. In contrast, ex vitro plant-mediated essential oil is most significant with an 18.07 ± 0.17-mm inhibition zone against Klebsiella pneumoniae. The ex vitro plants are also superior to the in situ plants for obtaining greater extract and essential oil content (950 µl), over a time period of 12 wk. To obtain the ex vitro plants, meta-topolin is used for in vitro regeneration of A. vulgaris for the first time, showing the highest mean shoot number (98.11 ± 0.31) regeneration after 42 d, which is more significant in comparison with other studies conducted till date. The cytogenetic stability of the regenerated plantlets has been checked using start codon targeted polymorphism and cytological studies, conferring the homogeneity among regenerants along with in situ plant. Furthermore, the essential oil of the ex vitro plants was analysed through gas-chromatography mass spectroscopy, which detected a few compounds with bio-significance from A. vulgaris, including 2-carene, 2-(4-nitrophenyl) acetamide, β-guaiene, α-acorenol, and 10,12-Tricosadiyonic. [ABSTRACT FROM AUTHOR]

• Biotechnological strategies and phytochemical profiling of medicinally important Saussurea spp. are discussed. • Medium-term and long-term conservation strategies are reviewed. • Plant cell and hairy root culture proved a non-destructive way of metabolites production. • Genetic transformation serves as an effective approach for engineering metabolic pathways. Saussurea DC. is a well-recognized genus for its commercially important secondary metabolites with its maximum diversity in the Himalayas and East Asia. The rising demand for versatile metabolites (costunolide, syringin, hispidulin, jaceosidin, rutin) poses a threat to the natural habitat of Saussurea spp. As a result, plant cell and tissue culture technology have attained cutting-edge progress for its conservation and secondary metabolites production. To date, various protocols of in vitro micropropagation, cell culture, somatic embryogenesis, and cryopreservation have been studied extensively for the conservation and specialized metabolite production in the genus Saussurea. Furthermore, to enhance in vitro biosynthesis of these metabolites, the application of elicitors has also played a significant role. The evolution of protocols that have been achieved during the past years for the augmentation of secondary metabolites is envisaged to design species-specific elicitation strategies in this genus. Also, molecular cues unpinning the genes involved in secondary metabolite biosynthesis have been studied in this genus. Therefore, the present review summarizes the up-to-date information on biotechnological prospects such as plant, cell, tissue, and organ culture, cryopreservation, and secondary metabolites production in Saussurea spp. This comprehensive review provides a better understanding of biotechnological tools implemented for conservation and specialized metabolite production to support its sustainability. [ABSTRACT FROM AUTHOR]

A reliable and reproducible embryogenic cell suspension culture system established successfully for regeneration of plants using pro-embryogenic cell mass derived from secondary somatic embryos as explants. A semi-solid medium was employed, which consisted of Murashige and Skoog (MS) basal salts and vitamins supplemented with 4 mg l− 1 2,4-dichlorophenoxyacetic acid (2,4-D), 1 mg l− 1 biotin, 1 mg l− 1 indole acetic acid (IAA), 1 mg l− 1 naphthalene acetic acid (NAA), 25 mg l− 1 ascorbic acid, 3% (w/v) sucrose and 2 g l− 1 phytagel. The genetic integrity of the plants regenerated from the suspension cells was confirmed through various techniques, such as flow cytometry, simple-sequence repeats (SSR) markers and inter simple sequence repeats (ISSR) markers. The field planting displayed no phenotypic deviations and no adverse effects on the vegetative and yield characteristics in banana cv. Sabri, a rapidly declining landrace in Tripura, India due to severe Fusarium wilt incidence. Based on these insights, we propose research priorities and complementary strategies aimed at the efficient conservation and rejuvenation of such valuable landraces. Key Message: The secondary somatic embryogenesis technique has demonstrated efficiency in establishing an embryogenic cell suspension culture system, offering great promise for producing genetically identical plants to rejuvenate banana cv. Sabri, a declining landrace. [ABSTRACT FROM AUTHOR]

Somaclonal variation in micropropagated plants improves crops. Genetic transformation research relies on micropropagated plant genetic integrity. Somaclonal variation frequently occurs in micropropagation, prompting an investigation of plant genetic stability. The high number of shoot buds was seen when Decalepis hamiltonii shooting tip explants were inoculated with reversed polarity in Murashige and Skoog (MS) medium supplemented with 0.5 mg L-1 benzylaminopurine (BAP). The shoot buds increased in length on an MS medium supplemented with 0.5 mg L-1 Benzylaminopurine (BAP) and 0.1 mg L-1 indole-3-acetic acid (IAA). The lengthened shoots were established in Murashige and Skoog (MS) medium supplemented with 1 mg L-1 Indole-3-butyric Acid (IBA). Well-rooted plants underwent greenhouse hardening. Micropropagated and seedpropagated plants have similar physical features. Regenerated plant genetic stability was assessed using ten primers in a Random Amplified Polymorphic DNA (RAPD) assay. Eighty-five bands were identified. Monomorphic features and original-type clones were found in field-grown plants and bands. The regeneration approach might help study D. hamiltonii micropropagation. [ABSTRACT FROM AUTHOR]

An important, endemic, and imperilled tropical medicinal plant called Tabernaemontana alternifolia is successfully micropropagated in this article. On Murashige and Skoog's media that had thidiazuron (TDZ; 2.5, 5.0, 7.5, and 10 µM) supplied, high-frequency indirect shoot regeneration was induced from the callus produced from internode explants. After 12 weeks of culture, 10 µM TDZ produced the greatest number of shoots among the different TDZ concentrations examined (15.33 shoots). From the original callus, up to three subcultures could regenerate shoots. On full and half-strength MS media containing indole-3-butyric acid (IBA) after 6 weeks of culture, rooting of the shoots was seen. Rooting of shoots was facilitated by a half-strength MS medium supplemented with 5 µM IBA. After successfully being transplanted into a potting mixture including vermiculite and perlite and being raised there for 4 weeks, the plants were moved to one containing soil, sand, and farmyard manure and kept in the greenhouse. Inter simple sequence repeat (ISSR) and directed amplification of minisatellite DNA (DAMD) primer assays were used to assess the genetic stability of Tabernaemontana alternifolia micropropagated plants. When randomly chosen regenerated plants were compared to donor plants, a homogenous amplification profile was found, supporting the clonal fidelity of micropropagated plants. Morphometric analysis of regenerated plants was comparable with that of mother plants suggesting the genetic fidelity of regenerated plants. [ABSTRACT FROM AUTHOR]

The efficiency and method of regeneration in in vitro culture conditions depend primarily on the plant growth regulators (PGRs) used. Even growth regulators belonging to one group may have different effects, stimulating the process of direct or indirect organogenesis, thus possibly disturbing the genetic stability among regenerants. The main aim of this study was to identify the genetic stability of Scutellaria baicalensis regenerates obtained by in vitro culture method using start codon targeted (ScoT) markers. S. baicalensis nodal explants were regenerated on MS medium supplemented with kinetin (KIN) at concentrations of 0.25, 0.5, 1.0, and 2.0 mg × dm−3 or benzylaminopurine (BAP)—0.25, 0.5, 1.0, and 2.0 mg × dm−3. The effects of the number of propagated shoots, length, number of nodes, and fresh mass of regenerants were assessed. Moreover, the genetic stability of the regenerants was analyzed using start codon targeted (SCoT) markers. Direct shoot organogenesis was observed on an MS medium containing kinetin, while indirect shoot induction occurred on an MS medium supplemented with BAP. The highest average number of shoots (3.6) was achieved for the MS + KIN medium at a concentration of 0.25 and 5.8 for the MS + BAP 1.0 medium. The average length and average number of nodes were the highest on the MS + BAP 0.25 medium (50.0 and 6.0, respectively), while the lowest values of these features were observed on the MS + KIN 2.0 medium (40.3 and 4.9, respectively). A total of 111 amplified bands were exhibited by SCoT primers. Three of the analyzed primers revealed four unique genotype-specific markers. The average percentage of polymorphism obtained was 36.7%. The analysis of genetic similarity revealed a high level of genetic similarity between the donor plant and regenerants obtained on MS “0” (medium without the addition of phytohormones). A slightly lower value of genetic similarity was observed for regenerants obtained by direct organogenesis (MS + KIN medium at all concentrations). Indirect shoot organogenesis observed on the MS + BAP medium (all concentrations) resulted in the highest differentiation, both in relation to the donor plant and MS “0” regenerants. The results of our work indicate that, in the case of S. baicalensis, the maintenance of genetic stability depends primarily on the presence of the cytokinin type in the medium.

Rubber tree is economically important rubber producing plant of Thailand. At present, a rubber tree plantation is susceptible to white root disease. Therefore, the use of rootstock from early introduce clone that proved to be resistant to white root disease could help sustain growing of rubber tree. Thus, the objectives of this research were to study the effects of plant growth regulators and different explants on somatic embryo (SE) induction of this rubber clone and assessment genetic stability. The results revealed that mix flower explant cultured on MS medium supplemented with 2.0 mg/L 6-benzyladenine (BA) and 1.5 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) provided callus subsequent somatic embryo (SE) formation at the highest frequency of 39.84 % and number of cotyledonary embryos (CEs) at 3.25 embryos/callus after 3 passages of subculture in the same culture medium (4 weeks/passage). SEs germinated into embryonic axis at 50 % and further development into shoot at 25 % after transfer to 0.25 mg/L GA3 containing MS medium with the best concentrations of BA and 2,4-D for 4 weeks. The assessment gene stability by RAPD and SSR markers showed no variation between mother plant and in vitro plantlets. In this work, a novel explant source - the floral section of the rubber tree - was used for the first time in order to design an effective technique for in vitro somatic embryogenesis. [ABSTRACT FROM AUTHOR]

Teucrium (Teucrium polium L.) is one of the valuable medicinal plants, but its poor seed germination is one of the principal problems of its extensive cultivation. Somatic embryogenesis is crucial for medicinal plant genetic improvement, germplasm preservation, and in vitro mass propagation. Synthetic seed production is a suitable alternative for natural seed production in biotechnology and plant development. The objectives of the present study were to optimize somatic embryogenesis, produce synthetic seeds, and evaluate the genetic fidelity of the regenerated T. polium. Results suggested that MS medium supplemented with 1.0 mg L−1 kinetin (Kin) alone and without auxin demonstrated the maximum embryogenesis and regeneration rates in leaf and petiole explants. The simultaneous occurrence of several somatic embryonic developmental stages, such as the globular, heart-shaped, torpedo-shaped, and cotyledonary stages, on an embryogenic callus clearly suggested that T. polium embryogenesis accurately captures an asynchronous process. The petiole and leaf explants cultured on MS medium with 1.0 mg L−1 Kin showed the highest rates of plant regeneration with spontaneously root development. Compared to somatic embryos, synthetic seeds with meristem sources lacked callogenesis. At 25 °C for 4 wk and at 4 °C for 3 wk, the meristem source caused the highest percentage of synthetic seeds to germinate (96.66%), while − 18 °C had a negative impact on the germination of the synthetic seeds. The results suggested that there was no genetic variation identified in the ISSR analysis on in vitro–generated T. polium plants. [ABSTRACT FROM AUTHOR]

• First report on application of meta -Topolin for augmented in vitro mass propagation of stevia. • meta -Topolin outperformed the other cytokinins during large-scale shoot proliferation. • Growth pattern assessed via micromorphological and leaf gas exchange measurement. • 100% genetic fidelity during flow cytometry and CDDP, DAMD, ISSR and SCoT markers analyses. • 2.4-folds higher rebaudioside-A production from acclimatized plantlets compared to mother plant. Present report ascertains the efficacy of meta -Topolin (m T) for improved in vitro mass propagation of stevia (Stevia rebaudiana Bert.) with high-frequency shoot initiation, multiplication, and proliferation over other cytokinins. Shoot tips were inoculated on Murashige and Skoog (MS) medium fortified with 6-benzyladenine, kinetin, m T, thidiazuron, and zeatin individually, at different concentrations ranged from 0.5—1.5 mg L−1. Among various combinations, MS medium + 1.5 mg L−1 m T developed the maximum number (15.33) and length (57.33 mm) of shoots with a greater number of leaves (38.67) after 4 weeks of culture. Elongated shoots were then transferred for root induction on MS medium fortified with a indole-3-butyric acid (IBA), indole-3-acetic acid, and α-naphthalene acetic acid individually, at concentrations ranged from 0.5—1.5 mg L−1. MS medium + 1 mg L−1 IBA was found as the superior rooting medium with the maximum number (10.67) and length (19.00 mm) of roots after next 4 weeks of culture. Rooted plantlets were acclimatized with 95% survival rate in sand followed by sand-soil (1:1; w w −1) mixture in next 8 weeks. Proper growth and development of acclimatized plantlets without any abnormalities was confirmed by micromorphological assessment (at cellular level), plant pigment analyses as well as leaf gas exchange measurement. Stability of ploidy level of the acclimatized plantlets was confirmed by flow cytometry. Furthermore, genetic fidelity of the acclimatized plantlets with their mother plant was confirmed by various molecular markers such as primers from directed amplification of minisatellite-region DNA, conserved DNA-derived polymorphism, inter simple sequence repeats, and start codon targeted polymorphism markers. High-performance liquid chromatography analysis indicated that acclimatized plantlets produced significantly higher (2.4-folds) rebaudioside-A (96.50 mg g−1 dry weight) in comparison to mother plant (40.20 mg g−1 dry weight). The current report, for first time, accounts the m T-mediated large-scale in vitro propagation of stevia ensuring genetic fidelity and simultaneously ameliorating the rebaudioside-A production. [ABSTRACT FROM AUTHOR]

Manglietiastrum sinicum Y.W. Law is a critically endangered species with great ornamental and commercial value, which urgently requires protection. We tested different combinations of basal media and plant growth regulators to determine (i) the optimal conditions for bud induction and proliferation of explants and (ii) optimal rooting conditions. RAPD- and ISSR-PCR were used to assess the genetic fidelity of regenerated plantlets. Murashige and Skoog medium (MS) supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.05 mg/L indole-3-butyric acid (IBA) is the optimal medium for bud induction (100% induction). MSM medium (a special basal medium for M. sinicum) was more suitable for the efficient proliferation and rooting of M. sinicum. Maximum bud proliferation rate (446.20%) was obtained on MSM, with 0.4 mg/L BA, 0.5 mg/L kinetin, and 0.06 mg/L IBA, while maximum root induction rate (88.89%) was obtained on MSM supplemented with 0.4 mg/L 1-naphthylacetic acid and 1.0 mg/L IBA with a 7-day initial darkness treatment. The rooted plantlets were transferred to a substrate containing peat soil, perlite, coconut chaff, and bark (volume ratio 2:1:1:1), with a resulting survival rate of 92.2%. RAPD and ISSR markers confirmed the genetic uniformity and stability of regenerated plants. [ABSTRACT FROM AUTHOR]

Determination of the somaclonal variation of in vitro-propagated plants is crucial to determine the appropriate micropropagation protocol and growth regulators for commercial scale multiplication. In this research, nine multiplication media (MM) augmented with different concentrations of 6-benzyl adenine (BA), Kinetin (Kin), and Thidiazuron (TDZ), Three rooting media (RM) supplemented with three levels of α-naphthalene acetic acid (NAA) and three types of soil mixtures (v/v); Coco peat/Vermiculite/Sand (CVS), Peat moss/Perlite/Sand (PPS) and Peat moss/Perlite (PP) were used in the micropropagation protocol of daylily plants. MM2 showed the maximum shoot length and the number of leaves, while MM9 showed the maximum number of shoots. The RM1 showed the maximum root length and the number of roots. During acclimatization, CVS, PPS, and PP soil mixture showed similar performance except the CVS mixture showed lower performance regarding plant height and diameter. The genetic fidelity of micropropagated plants was evaluated using Start Codon Targeted (SCoT) Markers. Six SCoT primers amplified 51 scorable bands with an approximate range from 146 bp to 1598 bp size. Thirty one out of 51 loci were presented in the mother plants. 40 loci were polymorphic, 11 were monomorphic and 7 were unique. The amplification patterns of the micropropagated plants demonstrated genetic integrity to the mother plant ranging from 84.32 to 47.06 and somaclonal variations ranging from 52.94 with 5 mg/l BA pathway to 15.68 with 1mg/l TDZ pathway, thus demonstrating that the homogeneity and the variation of the micropropagated plants affected by the type and the quantity of the plant growth regulator used during multiplication subcultures. This research can be successfully used for other ornamental and medicinal plants' bulk multiplication, germplasm conservation, and future genetic improvement. [ABSTRACT FROM AUTHOR]

Ardisia crenata var. bicolor is a multi-purpose plant and has important ornamental and medicinal properties. Conventional methods of propagating the species from seeds and cuttings have low efficiency because of the recalcitrant properties of seeds and low survival rate of high-quality cuttings. This work aims to study the in vitro regeneration protocol for direct organogenesis from nodal segments of A. crenata var. bicolor on Murashige and Skoog (MS) medium, with different combinations and concentrations of plant growth regulators (PGRs). The treatments used for the establishment and proliferation of shoots included MS medium supplemented with different concentrations of Benzyl-aminopurine (BAP) and indole-3-butyric acid (IBA). For rooting, IBA was used in combination with naphthaleneacetic acid (NAA) in full- and half-strength MS media. Maximum shoot establishment (76.67%) and the highest shoot length (6.6 cm) were observed on MS medium with 1.0 mg·L−1 BAP with 0.5 mg·L−1 IBA, while BAP at 1.0 mg·L−1 with 0.25 mg·L−1 IBA obtained the highest shoot proliferation (4.5 ± 1.53). The best rooting response (83.33%) was achieved on half-strength MS including 1.0 mg·L−1 IBA with 0.25 mg·L−1 NAA, and the maximum survival rate of 84.4% was observed after acclimatization under 75% shading. To define their genetic stability, using eleven primers of ISSR markers to assess the genetic stability of the unstable leaf color samples compared with their mother plant, the ISSR markers demonstrated a level of genetic polymorphism in plantlets, but without other morphological variations. This indicates the genetic resemblance to the mother plant and the reliability of this protocol for the efficient micropropagation of A. crenata var. bicolor.

Abstract Background Codonopsis pilosula (Franch.) Nannf. is a medicinal plant traditionally used in China, Korea, and Japan to treat many diseases including poor gastrointestinal function, low immunity, gastric ulcers, and chronic gastritis. The increasing therapeutic and preventive use of C. pilosula has subsequently led to depletion of the natural populations of this species thus necessitating propagation of this important medicinal plant. Here, we developed an efficient and effective in vitro propagation protocol for C. pilosula using apical shoot segments. We tested various plant tissue culture media for the growth of C. pilosula and evaluated the effects of plant growth regulators on the shoot proliferation and rooting of regenerated C. pilosula plants. Furthermore, the tissues (roots and shoots) of maternal and in vitro-regenerated C. pilosula plants were subjected to Fourier-transform near-infrared (FT-NIR) spectrometry, Gas chromatography-mass spectrometry (GC–MS), and their total flavonoids, phenolics, and antioxidant capacity were determined and compared. Results Full-strength Murashige and Skoog (MS) medium augmented with vitamins and benzylaminopurine (1.5 mg·L−1) regenerated the highest shoot number (12 ± 0.46) per explant. MS medium augmented with indole-3-acetic acid (1.0 mg·L−1) produced the highest root number (9 ± 0.89) and maximum root length (20.88 ± 1.48 mm) from regenerated C. pilosula shoots. The survival rate of in vitro-regenerated C. pilosula plants was 94.00% after acclimatization. The maternal and in vitro-regenerated C. pilosula plant tissues showed similar FT-NIR spectra, total phenolics, total flavonoids, phytochemical composition, and antioxidant activity. Randomly amplified polymorphic DNA (RAPD) test confirmed the genetic fidelity of regenerated C. pilosula plants. Conclusions The proposed in vitro propagation protocol may be useful for the rapid mass multiplication and production of high quality C. pilosula as well as for germplasm preservation to ensure sustainable supply amidst the ever-increasing demand.

Indian tea germplasm is known for its diversity, which not only assists the development of superior planting material for tea breeding but also functions as a genetic resource for other tea-producing countries. In this study, the aim was to develop an improved cryo-protocol for long-term conservation of tea germplasm. Shoot tips (approximately 2 mm) of Camellia sinensis were cryopreserved using vitrification and V cryo-plate techniques. The recovery rate of cryopreserved shoot tips obtained using vitrification was 33.2% at 90 min of Plant Vitrification Solution 2 (PVS2) exposure. To improve recovery rates and tolerance to cryo-injury, shoot tip donor plants were preconditioned with cold hardening, high sucrose, abscisic acid (ABA), and proline prior to cryopreservation. The vitrification technique resulted in an increase of regrowth of shoot tips to 60.5% after preconditioning of shoot tip donor plants for 30 d at 25 °C with 10 mg L−1 ABA and 3 mg L−1 proline. Regrowth further increased to 75.6% with the V cryo-plate technique using shoot tip donor plants preconditioned with the above concentrations of ABA and proline. After cryopreservation, shoot tips were recovered on regrowth medium that developed into shoot cultures in 5 wk. Random amplified polymorphic DNA (RAPD) analysis was performed to test genetic fidelity after cryopreservation and results showed 100% monomorphism among all the regenerants and 10-mo-old in vitro mother cultures. Results showed that the preconditioning strategies along with V cryo-plate technique influenced and improved the cryo-recovery rate of shoot tips of tea and provided a robust protocol for tea cryopreservation. [ABSTRACT FROM AUTHOR]

• In vitro multiplication of sweet potato using meta-topolin is reported for the first time. • An improved and reproducible micropropagation protocol for sweet potato was developed. • The multiplication rate and rooting efficiency of sweet potato has been significantly enhanced. • In vitro propagated plants showed 100% genetic similarity with mother plants. An improved in vitro propagation protocol by use of meta-Topolin (m T) hormone was developed in sweet potato (Ipomoea batatas (L.) Lam) using nodal explants derived from 12 weeks old in vitro plantlets of different sweet potato accessions. This is the first study that analyzes the effect of meta-Topolin (m T) hormone on sweet potato micropropagation. Enhanced in vitro multiplication was achieved on Murashige and Skoog (MS) medium supplemented with 0.54 µM 1-Naphthaleneacetic acid (NAA), 0.09 µM Gibberellic acid (GA 3) and varying concentrations of m T. A hundred percent shoot induction was obtained on m T concentrations ranging from 3.11 – 16.58 µM (except 4.15 µM). The best multiplication response with maximum number of shoots (7.26 ± 0.17) and axillary nodes (65.25 ± 1.81) was achieved on MS medium containing 8.29 µM m T with an average shoot length of 2.79 ± 0.27 cm. Shoots multiplied on m T containing medium gave 100% rooting with the best response (9.75 ± 1.21 roots per explant) recorded on MS medium supplemented with 0.49 µM Indole-3-butyric acid (IBA). Profuse rooting with multiple secondary roots was observed on this medium. Rooted plantlets were successfully acclimatized and hardened with a survival percentage of 90.48 ± 0.38%. The effect of different concentrations of m T (low, best and high) on the genetic integrity of regenerated plantlets was tested using 48 ISSR primers. All the successful primers (32) yield monomorphic bands in mother as well as tested plants, thereby confirming the uniformity and genetic integrity of regenerated sweet potato plants. The optimized protocol may be used for large-scale production of true-to-type quality planting material of sweet potato. [ABSTRACT FROM AUTHOR]

Evaluation of genetic uniformity is important in a perennial plant like coffee propagated through somatic embryogenesis. In this study, we compared the genetic homogeneity of somatic embryo-derived plants and the seed-derived plants obtained from a single mother plant of robusta coffee cultivar by employing Sequence Related Amplified Polymorphism (SRAP) and Start Codon Targeted (SCoT) molecular markers and sequencing four functional genes. The SRAP and SCoT molecular markers revealed 96% genetic similarity between somatic embryo-derived and seed-derived plants with the mother plant. The sequencing analysis has shown the minimum sequence variability in the chloroplast maturase K gene and the highest sequence variability in the zinc finger protein gene compared to NAC25 and UDP-glycosyltransferase genes. The presence of SNP and indels created polymorphism in gene sequences. The percent frequency of SNP varied from 0.0 to 0.45 in matK, 0.36 to 1.40 in NAC25, 0.81 to 2.63 in the UDP-glycosyltransferase gene and 3.30 to 8.70 in the zinc finger protein gene. In the case of NAC25 and UDP-glycosyltransferase genes, the seed-derived plants have shown a higher frequency of SNP compared to somatic embryo-derived plants. However, in the case of zinc finger protein gene, tissue culture-derived plants accumulated a two-fold higher frequency of SNP compared to the seed-derived plants. The sequence characterization of the four genes has revealed the presence of a higher frequency of non-synonymous SNPs compared to synonymous SNPs. This is the first report on the comparative genetic uniformity analysis of somatic embryo-derived and seed-derived plants in robusta coffee cultivar with significant practical implications. Key message: Molecular marker based genetic homogeneity assessment of somatic embryo and seed-derived plants of Coffea canephora revealed 96% similarity with the mother plant. The SE derived plants exhibited higher sequence variability in zinc finger protein gene. [ABSTRACT FROM AUTHOR]

Ficus religiosa LQuery. has several ornamental, medicinal, and economical applications. The in vivo propagation of this species has shown various limitations. Due to this reason, the present study efforts on genetically uniform artificial seed production from in vitro developed shoot tips of this species. The in vivo shoot tips were cultivated on Murashige and Skoog (MS) media containing different growth regulators. The maximum shoot response (93.67%) and the longest shoot length (3.85 cm) were exhibited with 0.5 mg L−1 6-furfuryl-amino purine (Kn), 0.2 mg L−1 benzyladenine (BA) and 0.1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) in combination. A treatment of 3% sodium alginate and 75 mM calcium chloride having a polymerization time of 15 min was exhibited to be superior for artificial seed production of these in vitro grown shoot tips. Artificial seed-derived micro shoots yielded the highest root response (94.44%) and roots per shoot (4.61) with 0.5 mg L−1 indole-3-butyric acid (IBA) and 0.1 mg L−1 BA in combination on ½-strength MS media. In comparison to 4 °C-kept artificial seeds, 24 °C-stored artificial seeds had superior germination potential across all storage times. The soil:organic manure (1:1) generated 90% of plantlet survival after 28 days of primary hardening than other mixtures tested. The secondary hardening displayed 92% of plant survival after 60 days. The banding patterns of ISSR analysis between the mother plant and hardened plants were discovered to be monomorphic. This methodology provides a promising and affordable approach to the large-scale plant production of this significant species. [ABSTRACT FROM AUTHOR]

The species belonging to the Rhododendron genus are well-known for their colorful corolla. Molecular marker systems have the potential to elucidate genetic diversity as well as to assess genetic fidelity in rhododendrons. In the present study, the reverse transcription domains of long terminal repeat retrotransposons were cloned from rhododendrons and used to develop an inter-retrotransposon amplified polymorphism (IRAP) marker system. Subsequently, 198 polymorphic loci were generated from the IRAP and inter-simple sequence repeat (ISSR) markers, of which 119 were derived from the IRAP markers. It was shown that in rhododendrons, IRAP markers were superior to the ISSRs in some polymorphic parameters, such as the average number of polymorphic loci (14.88 versus 13.17). The combination of the IRAP and ISSR systems was more discriminative for detecting 46 rhododendron accessions than each of the systems on their own. Furthermore, IRAP markers demonstrated more efficiency in genetic fidelity detection of in-vitro-grown R. bailiense Y.P.Ma, C.Q.Zhang and D.F.Chamb, an endangered species recently recorded in Guizhzhou Province, China. The available evidence revealed the distinct properties of IRAP and ISSR markers in the rhododendron-associated applications, and highlighted the availability of highly informative ISSR and IRAP markers in the evaluation of genetic diversity and genetic fidelity of rhododendrons, which may facilitate preservation and genetic breeding of rhododendron plants. [ABSTRACT FROM AUTHOR]

Ixora coccinea is one of the important ornamental plants, commercially propagated by stem cuttings. This study aims to determine the effects of different media on micropropagation and rooting and to investigate the genetic fidelity of regenerated plants using the Inter Simple Sequence Repeat (ISSR) marker. For this purpose, Murashige and Skoog (MS), and Woody Plant Medium (WPM) media were used for micropropagation and rooting experiments. All media were supplemented by 5.5g /l gelling agent (Agar) for micropropagation, MS media with different level of sucrose (15,30 ,45 and 60 g/l), 2.0 mg/l BAP with different concentrations of IBA (0.0, 0.1, 0.25 and 0.5 mg/l), for rooting 10mg /l NAA used. In vitro rooted plantlets were transferred to plastic pots containing; peat moss with vermiculite (1:1; 2:1, and 1:2 v/v) for acclimatization to ex vitro conditions. The ISSR markers were used for the assessment of genetic fidelity between the mother plant and acclimatized plantlets ISSR marker used. Total DNA was extracted from leaves of the acclimated plant by the cetyltrimethyl ammonium bromide method (CTAB), and the data obtained were analyzed and used to generate a similarity matrix using Jaccard’s similarity coefficient. The best result in terms of regeneration of shoots was obtained from 30 g/l sucrose on a full MS medium. MS media during the incubation period gave the best results in terms of vegetative growth parameters compared to WPM. The estimation of the genetic similarity coefficient based on the ISSR band indicated that acclimatized plantlets derived from the 6th subculture were 100% similar to the mother plant. Keywords: growth regulator; acclimatization; in vitro; genetic fidelity; DNA

Abstract Background Artocarpus heterophyllus is an important tropical agroforestry species that bears multiple applications. However, the population of this species is reduced due to various anthropogenic activities. For this reason, in vitro approach is needed to propagate or conserve this species as in vivo propagation methods face various obstacles. In this respect, the present investigation was undertaken to produce genetically stable jackfruit trees through in vitro technology. In vivo grew shoot tips were harvested on Murashige and Skoog (MS) medium containing several plant growth regulators to achieve this. Results The 6-benzylaminopurine (BAP) at the concentration of 1.5 mg L-1, indole-3-butyric acid (IBA) at 0.5 mg L-1, and α-naphthaleneacetic acid (NAA) at 0.1 mg L-1 in combination on MS media yielded superior shoot response (94.44%), longest shoot length (4.02 cm), and the maximum number of shoots per explant (4.78). They were further multiplied by repeated subculturing on the same media composition and the third subculture resulted in a maximum number of shoots (5.92) with the largest shoot length (5.85 cm). Among the different media screened for rooting, the ¼ MS media yielded 94.44% rooting response, the longest root length (3.78 cm), and the maximum number of roots per shoot (8.44) with 0.1 mg L-1 NAA, 0.5 mg L-1 IBA and 0.1 mg L-1 BAP in combination. Primary hardening showed 88.89% of plant survival under greenhouse conditions after 4 weeks of incubation having a sterilized mixture of garden soil and vermiculite mixture (1:1, w/w). It increased to 90.60% after the secondary hardening process in a vermiculite-soil mixture (2:1; w/w). No polymorphism was detected on random amplification of polymorphic DNA (RAPD) profiling between the mother plant and hardened plants, indicating high genetic stability among the clones. Conclusions This is the first report of the genetic fidelity study of in vitro grown regenerants of A. heterophyllus. This study established a micropropagation protocol for genetically uniform in vitro regeneration of this species to supply plant resources to various industries or conservation of elite germplasm.

The improved sugarcane varieties grown in and around the world are majorly evolved through interspecific hybridization of Saccharum species. Only few promising hybrids have been developed from allied genera such as Erianthus and Sclerostachya. Several other genera, including Phragmites, Vetivera, Narenga and Neyurida, deserve further research. Successful exploitation of these allied genera resources require careful characterization, evaluation and conservation using different methods including tissue culture techniques. Among them, Phragmites australis is used to remediate waste water and remove heavy metals. In this study, meristem tip culture was standardized for five clones of Phragmites to evaluate their response and growth. Among the five clones of Phragmites studied, two clones (IND 89-711 and IND04-1326) showed better regeneration and growth. The in vitro derived plants were compared with the mother plants using four SRAP markers to check their genetic fidelity. Monomorphic bands were obtained in both the in-vitro and mother plants. No polymorphism was detected, thus proving the genetic uniformity of the micropropgated plants.

Panax ginseng Meyer grows in east Russia and Asia. There is a high demand for this crop due to its medicinal properties. However, its low reproductive efficiency has been a hindrance to the crop's widespread use. This study aims to establish an efficient regeneration and acclimatization system for the crop. The type of basal media and strength were evaluated for their effects on somatic embryogenesis, germination, and regeneration. The highest rate of somatic embryogenesis was achieved for the basal media MS, N6, and GD, with the optimal nitrogen content (≥35 mM) and NH4+/NO3− ratio (1:2 or 1:4). The full-strength MS medium was the best one for somatic embryo induction. However, the diluted MS medium had a more positive effect on embryo maturation. Additionally, the basal media affected shooting, rooting, and plantlet formation. The germination medium containing 1/2 MS facilitated good shoot development; however, the medium with 1/2 SH yielded outstanding root development. In vitro-grown roots were successfully transferred to soil, and they exhibited a high survival rate (86.3%). Finally, the ISSR marker analysis demonstrated that the regenerated plants were not different from the control. The obtained results provide valuable information for a more efficient micropropagation of various P. ginseng cultivars. [ABSTRACT FROM AUTHOR]

An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in vitro-developed shoots (tips) were treated individually with 0.1%, 0.25% and 0.5% (w/v) colchicine solutions for 4, 6, 8, and 12 h. The colchicine-treated shoot tips were then inoculated on Murashige and Skoog (MS) medium fortified with 1.5 mg/l meta-Topolin for multiple shoot proliferation and later transferred into 1.5 mg/l indole-3-acetic acid-fortified MS medium for rooting of shoots. The ploidy levels of the colchicine-treated and regenerated plantlets along with the non-treated ones were confirmed via flow cytometry analysis and metaphasic chromosome count. The highest frequency of tetraploid plantlets (50%) were obtained when shoot tips were treated with 0.1% colchicine for 4 h. Morphological observations revealed that induced tetraploid plantlets exhibited delayed fresh shoot initiation, fewer but longer shoots, as well as fewer but broader leaves. Likewise, the study of stomata revealed that in comparison to their diploid counterparts, the tetraploid plantlets exhibited less frequent yet significantly larger stomata, and higher number of chloroplasts. The tetraploids were recorded with significantly higher chlorophyll, carotenoid, and anthocyanin content during the photosynthetic pigment analyses. During ex vitro acclimatization and field growth, the tetraploid plants exhibited delayed proliferation but with higher vigor and thickened broad leaves. The genetic uniformity among the diploid and the tetraploid plants was confirmed using conserved DNA-derived polymorphism (CDDP), directed amplification of minisatellite-region DNA (DAMD), inter simple sequence repeats (ISSR), and start codon targeted (SCoT) polymorphism marker systems. The tetraploids developed in the present study would be of immense importance for the genetic improvement of gerbera as far as its ornamental values are concerned. [ABSTRACT FROM AUTHOR]

Background: Codonopsis pilosula (Franch.) Nannf. is a medicinal plant traditionally used in China, Korea, and Japan to treat many diseases including poor gastrointestinal function, low immunity, gastric ulcers, and chronic gastritis. The increasing therapeutic and preventive use of C. pilosula has subsequently led to depletion of the natural populations of this species thus necessitating propagation of this important medicinal plant. Here, we developed an efficient and effective in vitro propagation protocol for C. pilosula using apical shoot segments. We tested various plant tissue culture media for the growth of C. pilosula and evaluated the effects of plant growth regulators on the shoot proliferation and rooting of regenerated C. pilosula plants. Furthermore, the tissues (roots and shoots) of maternal and in vitro-regenerated C. pilosula plants were subjected to Fourier-transform near-infrared (FT-NIR) spectrometry, Gas chromatography-mass spectrometry (GC–MS), and their total flavonoids, phenolics, and antioxidant capacity were determined and compared. Results: Full-strength Murashige and Skoog (MS) medium augmented with vitamins and benzylaminopurine (1.5 mg·L−1) regenerated the highest shoot number (12 ± 0.46) per explant. MS medium augmented with indole-3-acetic acid (1.0 mg·L−1) produced the highest root number (9 ± 0.89) and maximum root length (20.88 ± 1.48 mm) from regenerated C. pilosula shoots. The survival rate of in vitro-regenerated C. pilosula plants was 94.00% after acclimatization. The maternal and in vitro-regenerated C. pilosula plant tissues showed similar FT-NIR spectra, total phenolics, total flavonoids, phytochemical composition, and antioxidant activity. Randomly amplified polymorphic DNA (RAPD) test confirmed the genetic fidelity of regenerated C. pilosula plants. Conclusions: The proposed in vitro propagation protocol may be useful for the rapid mass multiplication and production of high quality C. pilosula as well as for germplasm preservation to ensure sustainable supply amidst the ever-increasing demand. [ABSTRACT FROM AUTHOR]