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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]

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]

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]

• 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]

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]

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system. [ABSTRACT FROM AUTHOR]

In vitro plant propagation techniques play an important role in modern agriculture and horticulture, offering efficient methods for the accelerated multiplication of high-value plant species. Thin cell layer (TCL) culture technique has emerged as a promising approach for large-scale in vitro plantlets production. This study explores the potential of TCL culture as a cutting-edge strategy to enhance the efficiency of multiple shoots formation in Kaempferia parviflora. An attempt was also made to look into the synergistic effects of cytokinin and auxin [6-benzyladenine (BA) - indole-3-acetic acid (IAA)] during micropropagation. Shoot tip explants (from rhizome sprouts) were cultured onto Murashige and Skoog (MS) medium enriched with BA (2.0–8.0 mg/L) and IAA (0.2–0.8 mg/L) in combination where the highest shoot number (7.3 shoots/explant) and length (5.9 cm) was obtained in 8.0 mg/L BA and 0.8 mg/L IAA. Subsequently, the transverse thin cell layer (tTCL) culture system was successfully utilized whereby the shoot tip explants were individually sliced from tips at the meristem and cultured onto MS medium supplemented with BA (2.0–8.0 mg/L) and IAA (0.2–0.8 mg/L) in combination. The regenerated tTCLs exhibited the highest shoot multiplication (9.3 shoots/tTCL; 28 shoots/shoot tip) and length (5.3 cm) in 4.0 mg/L BA + 0.8 mg/L IAA, thus significantly increased number of plantlets were obtained from tTCL culture (953.0 plantlets) compared to shoot tip culture (527.7 plantlets) after six sub-culture cycles. The tTCL regenerants were successfully acclimatized (with 90% survival) and displayed active growth. Genetic fidelity assessment via inter-simple sequence repeats, conserved DNA-derived polymorphism, directed amplification of minisatellite-region DNA, and start codon targeted polymorphism marker systems suggested that a very low level (5.8%; 148 out of 2572 scorable bands) of polymorphism was detected whereas the ploidy level remained unchanged, confirmed by flow cytometry analysis. The implementation of tTCL culture, coupled with a controlled sub-culture regimen, holds significant potential for the efficient commercial-scale production of K. parviflora. • First report on utilization of transverse thin cell layer (tTCL) culture technique in K. parviflora. • Cytokinin-auxin (BA-IAA) synergistic effects on micropropagation was proven. • Affirmed ploidy and genetic stability via flow cytometry, CDDP, DAMD, ISSR and SCoT markers. • Bulk production of plantlets in subsequent sub-cultures via tTCL culture technique. [ABSTRACT FROM AUTHOR]

Mutation induction is a feasible and established breeding method for crop improvement and genetic diversity creation to introduce new plant cultivars. The present study was aimed at mutagenesis of four chrysanthemum cultivars ('Homa', 'Fariba2', 'Arina', and 'Delkash') using ethyl methanesulfonate (EMS) (0, 0.125, 0.25, and 0.5%) as mutagen and leaf disks as explants to obtain novel variants. In addition, genetic polymorphism among mutants and their parents was detected using inter simple sequence repeat (ISSR) and inter-retrotransposon amplified polymorphism (IRAP) molecular markers. A total of 2082 plantlets were produced through EMS induced mutagenesis under in vitro conditions and at the end 58 mutants including 28 leaf and 32 flower mutants were analyzed for phenotypic and molecular variation. The explant survival rate decreased by increasing EMS concentration. A wide range of phenotypic leaf and inflorescence variability was obtained in four studied chrysanthemum cultivars confirming the efficiency of EMS to create genetic variation and desired mutants. All generated variants with different inflorescence and leaf shape and color were maintained through cuttings and they expressed same traits in the next generation. The mutants were different in leaf size and shape, plant height, day to flowering, inflorescence head size, ray floret color and ray floret size. The used ISSR and IRAP primers could classify chrysanthemum mutants based on cultivar and somewhat based on used EMS concentration confirming their effectiveness for the discrimination of real variants that allow their earlier selection and reduction of the mutant population size. The in vitro EMS-induced mutation can be a promising tool to assist breeding programs for the generation of new chrysanthemum cultivars. Key message: New chrysanthemum mutants with distinct colors and inflorescence shape were obtained in four chrysanthemum cultivars using ethyl methanesulfonate (EMS). IRAP and ISSR could successfully classify the EMS-induced mutants and their relationship with mother plants. [ABSTRACT FROM AUTHOR]

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system.

Pterocarpus marsupium Roxb., which belongs to the Fabaceae family, is a promising herbal drug producing forest tree widely known as the 'Indian Kino'. An effective regeneration protocol using in vitro seedlings of P. marsupium under the gibberellic acid and thidiazuron regimen is described in the present study. Gibberellic acid was also tested to have an effect on in vitro rhizogenesis from microshoots. Combination of gibberellic acid and thidiazuron in culture media improved the germination percentage, multiplication and subsequent elongation of shoots. Murashige and Skoog medium containing 0.50 µM gibberellic acid with 0.50 µM thidiazuron was found to be the most effective treatment for producing the highest number of shoots per seedling (23.0) with an average shoot length 5.14 cm in 85% cultures, after 8 weeks. For in vitro rhizogenesis, 100 μM of indole 3-butyric acid was treated for 5 days in basal ends of isolated microshoots. Thereafter, pretreated microshoot was transferred to gibberellic acid (0.50 µM) supplemented MS medium. This treatment was found to be the most effective for in vitro root formation, where 10.54 roots with an average root length of 4.60 cm per microshoot in 80% cultures were recorded, after 4 weeks. During acclimatization photosynthetic traits and their attributes such as net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, and Chlorophyll content were analyzed and these attributes were found to be fluctuating initially for 35 days, thereafter, an increasing trend was recorded up to 84 days of acclimatization. The well acclimatized plantlets were shifted to fields where they grew normally without any morphological changes with an 86.7% of survival. Genetic integrity in micropropagated plants was validated by DNA-based ISSR primers. These primers were amplified in monomorphic banding pattern suggesting a high-level of genetic uniformity in micropropagated plants. Overall, an effective and high throughput plant regeneration method has been developed for obtaining true-to-type regenerants of P. marsupium. Key message: The present study revealed the high throughput regeneration of P. marsupium through in vitro seedlings method which may be an alternative tool for mass propagation and germplasm conservation of woody trees directly from seed, thus reducing an explant preparation and recovery of seedlings capable of multiple shoot formation under the regimes of gibberellic acid and thidiazuron. Both the growth regulators are involved in the improvement of plantlet regeneration processes as well as maintenance of physiological stresses during in vitro cultures. [ABSTRACT FROM AUTHOR]

A droplet-vitrification cryopreservation protocol has been successfully developed for Aranda Broga Blue orchid hybrid using protocorm-like bodies (PLBs). However, maximum growth regeneration percentage was recorded at 5% only based on previous report. Thus, to improve growth recovery of cryopreserved PLBs, cryopreservation stages were supplemented with ascorbic acid, tested at 50, 100 and 150 mg/L. However, results demonstrated that exogenous ascorbic acid was not favorable in regeneration of cryopreserved explants (maximum value of 1.67 % with 50 mg/L ascorbic acid supplementation). Total soluble protein and various antioxidant enzyme activities such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) were evaluated after each cryopreservation stages in conjunction with the application of exogenous ascorbic acid. Addition of antioxidant must be carefully evaluated and its application may not guarantee successful growth recovery. RAPD and SCoT molecular analysis confirmed the genetic stability of regenerated cryopreserved PLBs as no polymorphism was detected compared to control PLBs culture.

Crinum malabaricum , Lekhak & S.R. Yadav (Amaryllidaceae) is a critically endangered bulbous plant endemic to India. Establishing an efficient plant regeneration protocol is necessary for its conservation and large-scale propagation. In the present study, regeneration was achieved through somatic embryogenesis from callus produced on MS media supplemented with various different concentrations of 2,4-D alone and in combination with N6-benzyl-adenine (BA). Different advanced stages of embryo development (globular, torpedo and cotyledonary) and maturation were obtained on MS medium, with combinations of picloram and thidiazuron (TDZ). A high rate of somatic embryos (55.89 ± 0.60) was obtained after eight weeks. SEM examinations showed the occurrence of cell clusters (embryogenic) which converted to somatic embryos. Well-developed cotyledon embryos were successfully germinated on PGRs free full and half strength MS medium, however, 94.03% of somatic embryos germinated on half-strength MS medium supplemented with 1.0 mg L−1 gibberellic acid. The true-to-type genetic conformity of regenerated plants was examined by SCoT, ISSR and RAPD primers based molecular analysis. The amplification bands produced by SCoT, ISSR and RAPD primers applied were monomorphic across all the regenerated plants. This confirmed their genetic homogeneity compared to the mother plant and also demonstrated the reliability of our somatic embryogenesis system for C. malabaricum. A rapid method for phytochemical analysis based on LC-ESI/MS exhibited better separation and analysis of galanthamine and lycorine from methanolic extracts of in vitro raised plants derived from somatic embryogenesis. The protocol developed should be helpful in reintroduction, restoration and ex situ conservation of this valuable plant in the natural condition as well as in the pharmaceutical sectors. [Display omitted] • An efficient somatic embryogenesis system was developed in Crinum malabaricum. • Different concentrations and combinations of plant growth regulators influenced the development of somatic embryos. • The clonal fidelity of somatic embryo-derived plants was confirmed by molecular markers. • Galanthamine and lycorine were efficiently identified by using LC-ESI-MS/MS analysis. • This study will be helpful for large scale clonal propagation and production of secondary metabolites. [ABSTRACT FROM AUTHOR]

Begonias are ornamental indoor plants and also cultivated for landscaping. Begonia malabarica Lam., found naturally in the hilly regions of South India and Sri Lanka called by the tribals as Narayana Sanjeevi, i.e. possess miraculous therapeutic properties. The present study targets in vitro culture, field establishment and evaluation of its genetic uniformity using anatomical, molecular markers followed by callus induction and establishment of cell suspension. Explants such as shoot tip and leaves from in vitro germinated seeds were cultured on Murashige-Skoog medium. Maximum callus induction was obtained in Murashige-Skoog medium with 0.3 mg L-1 2, 4-D + 0.5 mg L-1 BAP, i.e. 90.8 ± 2.8% with 2.5 ± 0.1 g callus growth. In vitro suspension of cells and their growth rate were also analysed. RAPD and ISSR experiments were carried to confirm the genetic fidelity among in vitro and in vivo plants. Twelve RAPD and seven ISSR primers raised 119 amplicons. Genomic DNA amplification showed similarity between the in vivo and the in vitro generated plantlets. The overall results revealed the uniformity of the in vitro raised B. malabarica plantlets in terms of histoanatomical genetical features and substantiated the assumption that in vitro micropropagation is the secure mode for mass propagation of true to type plants without any somaclonal variations. [ABSTRACT FROM AUTHOR]

Highlights • The results obtained in the present study are a step forward to develop improved genotype through hybridization in wild pomegranate. • Phenotypic study helps in the selection of best performing individuals of wild pomegranate for domestication. • Attack of fruit borer on fruits showed that controlled pollinated fruits are more prone as compare to fruits open pollinated sources. • The important hybrids obtained were tested for their paternity using 21 RAPD markers of which thirteen primers revealed the clue for hybridity. Abstract The study was conducted in locations Tatool (L 1) district Solan, Narag (L 2) and Daron (L 3) district Sirmour, H.P. The half sibs from Narag and Daron location performed best for seedling height, minimal fruit borer incidence and oval shape. The highest titratable acidity was recorded from genotype L 3 T 3 (Daron location) with elliptical shape which is beneficial for Anardana processing. The tree genotypes bearing ovate and elliptical shaped fruits can be preferred for better fruit size selection. High heritability followed by genetic advance was observed for percent germination and seedling at six month age. In cross L 2 T 2 X L 3 T 3 , reputed fresh weight of seed without aril and germination was better whereas L 1 T 1 X L 2 T 1 cross showed better fruit quality biochemical estimates. The fruit crosses viz. L 2 T 4 X L 2 T 1 and L 2 T 2 X L 2 T 1 were found to have lower incidences of the borer attack. Maximum value in fruit traits were obtained from open pollination as compared to controlled cross combination for all the characters except fruit weight and fresh seed weight with aril. The hybrids were tested for their paternity using 21 RAPD markers of which thirteen primers revealed that 7 hybrids out of 15 hybrids have similar pattern as their parent. [ABSTRACT FROM AUTHOR]

In the present study, a simple, eco-friendly, biogenic protocol for synthesis of silver nanoparticles (AgNPs) using leaf extract of in vitro banana plantlets (Musa acuminata Colla cv. Grand Nain) was established. The synthesized AgNPs were characterized by X-ray diffraction, transmission electron microscopy, scanning electron microscopy, and fourier transform-infrared spectroscopy. Shoot tips of stevia (Stevia rebaudiana Bert.) were cultured on three forms of regeneration medium (Murashige and Skoog basal medium + 0.5 mg. L−1 6-benzyladenine) i.e. 'semi-solid', 'liquid', and 'semi-solid followed by liquid' supplemented with 100–400 µM of AgNPs to study the effect of different concentrations of AgNPs along with different forms of medium on plant growth and rebaudioside-A content in stevia under in vitro conditions. Among different concentrations, 400 µM AgNP showed the best growth response irrespective of different forms of medium. Nonetheless, the overall best growth response was recorded in 'semi-solid followed by liquid' medium. High performance liquid chromatography analysis of regenerants showed gradual incline in rebaudioside-A content with the increase in AgNP concentrations. Regenerants from medium containing 400 µM AgNPs produced the highest rebaudioside-A of 334.8 mg. g−1 dry weight (>2-fold) while control ones produced 154.3 mg. g−1 dry weight. Genetic fidelity of AgNP-treated and control regenerants was confirmed using 20 inter simple sequence repeats and start codon targeted primers. The present study can be useful for commercial-scale propagation of stevia ensuring higher fresh and dry biomass as well as ameliorated rebaudioside-A production. • First report on green synthesis and characterization of silver nanoparticles (AgNPs) from in vitro grown banana leaf. • Among three forms, 'semi-solid followed by liquid' medium showed superior growth of stevia. • Ameliorated biomass and rebaudioside-A production with rising AgNP concentrations in stevia. • AgNP-treated regenerants via ISSR and SCoT markers confirmed 100% genetic fidelity. [ABSTRACT FROM AUTHOR]

An efficient and reproducible protocol for in vitro plant regeneration was developed for Lawsonia inermis L. using cotyledonary node explant derived from axenic seedlings. Highest shoot proliferation frequency (ca 96.6%) was achieved on Murashige and Skoog's, 1962 (MS) basal medium supplemented with 8.88 μM 6-Benzyladenine (BA) + 2.68 μM Napthalene acetic acid (NAA). Up-scaling of shoots was carried out using in vitro nodes on MS medium supplemented with 4.44 μM BA. So overall, an average of 238 shoots was produced at 75 days. Of the four different forms of cotyledonary node explants evaluated, highest shoot multiplication was observed in cotyledonary node explant with two whole cotyledons. In vitro regenerated shoots were best rooted (ca 34.3 roots / shoot) on ½ MS medium devoid of any growth regulator. The plantlets were successfully acclimated in sand:soil:: 1:1and established in the garden soil. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analysis revealed a homogeneous amplification profile for all micropropagated plants validating the genetic fidelity of the in vitro-regenerated plants and supporting the regeneration protocol for economic commercial exploitation. [ABSTRACT FROM AUTHOR]

A simple and efficient regeneration protocol was developed for watermelon from cotyledonary node explants excised from 7-day-old in vitro grown seedlings. This study describes the effect of amino acids and polyamines (PAs) along with plant growth regulators (PGRs) on multiple shoot induction and rooting. The highest number of multiple shoots (46.43 shoots/explant) was obtained from cotyledonary node and they were also elongated (6.3 cm/shoot) on MS medium supplemented with 1 mg l N -Benzyladenine (BA), 5 mg l leucine, and 10 mg l spermidine. The elongated shoots developed profuse roots (23.03 roots/shoot) in MS medium containing 1 mg l indole-3-butyric acid (IBA), 5 mg l isoleucine, and 10 mg l putrescine. All the rooted plantlets were successfully hardened and acclimatized in the greenhouse with a survival rate of 98%. The present study described an efficient method to obtain a 1.5-fold increase in the number of shoots, compared with the available regeneration protocols for watermelon. The plants developed in this study showed fivefold higher photosynthetic pigments compared to the control plants. The genetic fidelity of the regenerated plants was evaluated by SCoT and RAPD marker analyses, and banding patterns confirmed the true-to-type nature of in vitro regenerated plants. [ABSTRACT FROM AUTHOR]

Somatic embryogenic system was developed in Sapindus mukorossi Gaertn. using rachis as explants from a mature tree. Explants showed callus initiation on Murashige and Skoog medium supplemented with TDZ (1-Phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea), zeatin or 6-benzylaminopurine. Induction of somatic embryogenesis was achieved on both MS basal medium and MS medium supplemented with 8.88 µM 6-benzylaminopurine. Hundred percent embryogenesis was observed on MS medium supplemented with 8.88 µM 6-benzylaminopurine with maximum intensity of embryogenesis (51.92 ± 0.40 a). Maximum maturation of somatic embryos (92.86 ± 0.34 a) was observed on induction medium supplemented with 0.0378 µM abscisic and treated for 21 days. Germination of somatic embryos was maximum (77.33 ± 0.58 a) on MS medium supplemented with 8.88 µM 6-benzylaminopurine. In vitro raised plantlets were hardened, acclimatized and transferred to the field. Survival frequency of plantlets was 80 % in field conditions. The genetic fidelity of in vitro regenerated plants was also evaluated and compared with mother plant using random amplified polymorphic DNA and inter simple sequence repeat. Both markers showed similarity in molecular profile of mother plant and in vitro regenerated plants. [ABSTRACT FROM AUTHOR]

Genetic stability of in vitro plants should be assessed in order to develop proper programs of conservation. Such conservation programs could be developed only after evaluation of genetic variability in the natural populations and the genetic stability or somaclonal variability after conservation. In case of valuable economic plants as fruit trees, vegetables, medicinal plants, the natural variability from populations should be preserved as sources of new cultivars or variations. In order to preserve endangered, vulnerable or endemic plants it is very important to evaluate the genetic variability in natural populations and to preserve this variability as well as individuals or habitats. Genetic variability or stability after plant conservation is usually assessed by DNA-based molecular markers as RAPD, SSR, ISSR, SRAP, RFLP and AFLP. [ABSTRACT FROM AUTHOR]

Low rates of shoot multiplication, clonal instability, poor rooting frequency and high cost of production are major impediments challenging the micropropagation of orchids and other ornamental plants. These problems can be solved with the proper usage of plant growth regulators (PGRs) in the medium. The highest shoot proliferation rate of 21 shoots/explant was recorded in Murashige and Skoog (MS) medium containing 1 mg/l meta-topolin and 0.8 mg/l Putrescine. To date, this is the best frequency of shoot induction for Dendrobium nobile and one of the highest among other orchids. Higher rooting frequency was also recorded with the highest rooting of 10.1 roots/shoot achieved in plants grown in half strength MS media supplemented with 2 mg/l of indole butyric acid and 0.5 mg/l of phloroglucinol. The plantlets were successfully acclimatized in the greenhouse with a survival rate of 82.3% exhibiting normal developmental patterns. The regenerated plantlets were assessed for genetic stability using the molecular markers namely SCoT and IRAP revealing high degree of genetic stability within the micropropagated plants of D. nobile . Being a highly medicinal orchid, the antioxidant potentials of the mother and the micropropagated plants were assessed using DPPH, FRAP and metal chelating assays which revealed a significantly higher antioxidant activity in the micropropagated plants. Rapid multiplication rate, higher genetic stability and significantly higher antioxidant power reported in the present study on D. nobile ensure the use of this micropropagation protocol for ex-situ conservation and commercial exploitation which could also be extended to other important orchid species. [ABSTRACT FROM AUTHOR]

A reliable and reproducible protocol for in vitro regeneration has been developed for Alhagi maurorum, a rare and medicinally important plant of family fabaceae. MS medium with BAP (2.0 mg l) proved to be the best for shoot bud induction from nodal segments. The rate of shoot multiplication was found to be influenced by a number of factors, viz., media composition, plant growth regulator's type and concentration, successive transfer of mother explant for different passage, culture vessels and gelling agents. Modified MS medium (modified having nitrates reduced to half) solidified with 0.14 % gelrite and containing BAP (0.5 mg l), IAA (0.1 mg l) and additives was found optimum for shoot multiplication which gave rise to maximum number of shoots (33.5 ± 3.43 per culture vessel). The in vitro regenerated shoots were rooted under both in vitro (on half strength MS salts with 1.0 mg l IBA + 100 mg l activated charcoal) as well as ex vitro (on sterile soilrite by treating shoot base with 250 mg l each of IBA and NOA for 4 min in green house) conditions. Thereafter, the in vitro and ex vitro rooted plantlets were hardened under green house conditions with 70 and 90 % survival rate, respectively. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity of seven tissue cultured plantlets growing in green house condition with mother plant. The amplification products were monomorphic across all the seven micropropagated plants as well as mother plant produced by all SCoT, ISSR and RAPD primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for A. maurorum. To the best of our knowledge, this is the first report on micropropagation and genetic homogeneity assessment of A. maurorum, which can be applied for large scale multiplication of elite genotypes of A. maurorum. [ABSTRACT FROM AUTHOR]

An efficient somatic embryogenesis protocol for plantlet regeneration of the commercially valuable, and nutritionally important, crop guava (Psidium guajavaL.) has been established. The immature zygotic embryos were targeted as an explant source for inducing somatic embryogenesis and plant regeneration, using four commercial cultivars (Allahabad Safeda, Lalit, Sardar and Shweta). The plantlets regenerated were assessed for genetic fidelity using random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and simple sequence repeat (SSR) molecular markers. A total of 2171 scorable bands were obtained using RAPD, ISSR or SSR, ranging from 300–3500, 250–3000 and 150–280 bp, respectively. No polymorphism was observed among the somatic embryogenesis regenerated plants, compared with respective donor mother plants. The profiles generated based on the three marker systems were found to be highly uniform and approximately 99% bands were monomorphic. This high degree of genetic uniformity (as assessed using the markers) in the somatic embryogenesis regenerated plants indicates genomic stability was maintained through the regeneration protocol. The somatic embryogenesis regenerated plants were hardened and transferred to the field for acclimatization, of which 80% plantlets survived, with all being phenotypically similar to the donor mother plants. We conclude that the RAPD, ISSR and SSR markers were informative and potentially useful in confirming the uniformity assessment of somatic embryogenesis regenerated plants. [ABSTRACT FROM AUTHOR]

A newly developed and novel DNA marker technique, i.e. start codon targeted (SCoT) polymorphic markers that target plant gene regions were used to assess genetic stability of in vitro raised plants of Cleome gynandra multiplied by enhanced bud proliferation from nodal segments. Seven randomly selected micropropagated plants, following at least 2 months of growth in the greenhouse along with mother plant were subjected to molecular analysis. Of 24 primers screened, 15 primers produced unambiguous and reproducible bands. All 15 primers generated a total of 65 fragments, with a mean of 4.3 ranging 2-7 per primer. No polymorphism was detected in regenerated plants and the mother plant, revealing the genetic fidelity of the in vitro raised plantlets. To verify the results of SCoT analysis, random amplified polymorphic DNA (RAPD) markers were also used for the assessment of genetic fidelity of tissue culture raised plants. The monomorphic banding pattern in micropropagated plants and the mother plant obtained from SCoT and RAPD analysis confirms the genetic stability of the in vitro raised plants and demonstrates the reliability of our micropropagation system for C. gynandra, an important C plant. [ABSTRACT FROM AUTHOR]

Abstract: Malabar glory lily (Gloriosa superba L.) is a medicinally potent plant species used for the production of alkaloid colchicine. With ever increasing demand, there is a pressing need to conserve it through biotechnological approaches. A large number of complete plantlets were obtained by direct regeneration from the non-dormant tuber explants on Murashige and Skoog (MS) medium supplemented with 2.0mg/l 6-benzylaminopurine (BAP)+0.5mg/l α-naphthalene acetic acid (NAA). Large number of plants can be produced in vitro under aseptic conditions, but there is always a danger of producing somaclonal variants by tissue culture technology. Thus, the genetic stability of micropropagated clones was evaluated using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. During the study a total of 80 (50 RAPD and 30 ISSR) primers were screened, out of which 10 RAPD and 7 ISSR primers produced a total of 98 (49 RAPD and 49 ISSR) clear, distinct and reproducible amplicons. The amplification products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. This is the first report that evaluates the use of genetic markers to establish genetic fidelity of micropropagated G. superba using RAPD and ISSR, which can be successfully applied for the mass multiplication, germplasm conservation and further genetic transformation assays for colchicine production to meet the ever increasing demand of this medicinally potent plant for industrial and pharmaceutical uses. [Copyright &y& Elsevier]

Bamboos (family Poaceae) are the most beautiful and useful plants on the Earth, mainly found in the tropical and sub-tropical regions of the world. Bamboos are fast growing and early maturing, but lack of proper management of bamboo resources is leading to rapid reduction of the existing bamboosetum. Bamboo propagation through seeds is limited due to long flowering cycle of upto 120 years, seed sterility and short seed viability. Infrequent and unpredictable flowering events coupled with peculiar monocarpic behaviour i.e. flowering once before culm death, and extensive genome polyploidization are additional challenges for this woody group. Similarly, vegetative propagation by cuttings, offsets and rhizomes are also inadequate to cope up with the demand of planting stock due to large propagule size, limited availability, seasonal dependence, low multiplication rate and rooting percentage. Therefore, attempts have been made to propagate bamboos through in vitro techniques. In vitro flowering has also been achieved successfully in some bamboo species. Classification systems proposed to date need further support, as taxonomic delineation at lower levels is still lacking sufficient resolution. Tremendous advancement in molecular markers holds the promise to address the needs of bamboo taxonomy (systematics and identification) and diversity studies. Successful application of molecular marker techniques has been achieved in several bamboo species although, more studies are required to understand the population structure and genetic diversity of bamboos in a better way. In addition, some efforts have also been made to clone important genes from bamboos and also for genetic transformation using Agrobacterium and particle bombardment methods. An overview of the recent developments made in improvement of bamboos through in vitro propagation, molecular marker technologies, cloning, and transformation and transgenics has been presented. The future potential of improvement of bamboos using modern biotechnological tools has also been discussed. [ABSTRACT FROM AUTHOR]

To evaluate genetic homogeneity of 1-year-old guava ( Psidium guajava L.) plants developed from in vitro somatic embryogenesis, DNA from leaf tissues of seven randomly selected plants along with the mother plant, was isolated and subjected to molecular analysis. A total of six Simple Sequence Repeat (SSR) primer pairs, producing reproducible and clear bands ranging from 100 to 300 bp in size, resulted in amplification of single band (allele), corresponding homozygous individuals. Moreover, of 10 different inter-simple sequence repeat (ISSR) primers screened, six produced resolvable, reproducible and scorable bands. All these ISSRs produced a total of 25 bands, ranging between 300 and 1,200 bp length, and the number of scorable bands, for each primer varied from three to six with an average of 4.1 bands per primer. The amplification products were monomorphic across all the micropropagated plants produced by all SSR and ISSR primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for guava. [ABSTRACT FROM AUTHOR]

The article presents a morphological and molecular analyses in micropropagated berry plants that were acclimatized under ex vitro condition. Among the berry crops are the strawberry, blueberry and lingonberry. It describes the different aspects of berry propagation in vitro, the characterization of micropropagated berry plants for morphological characters, and the employment of molecular markers in the plants to assess their genetic fidelity, uniformity and stability, among others.

The importance of neem ( Azadirachta indica A. Juss.) as a medicinal tree species has been acknowledged worldwide. Superior trees with desired traits such as high azadirachtin content have been identified and micropropagated. Somaclonal variants that may arise in vitro, however, pose limitations to large-scale micropropagation. It is, therefore, imperative to establish genetic uniformity of such plantlets by ensuring strict quality checks at various stages of in vitro culture. This is the first study that evaluates the applicability of amplified fragment length polymorphism (AFLP) markers in establishing clonal fidelity of tissue culture(TC)-raised neem plants. Seven AFLP primer combinations generated a total of 334 amplified fragments across the mother plant, TC progenies, and other neem accessions that were included as controls. Two hundred and thirty-nine amplified fragments were monomorphic across the mother tree and its TC progenies. No extra band was detected in the TC plantlets that was absent in the mother tree, indicating that the TC plantlets regenerated through nodal explants are indeed true-to-type. Ninety-five AFLP fragments were detected in the controls, which allowed their discrimination from the elite mother tree and its TC progenies. Similarity matrix based on Jaccard's coefficient revealed that the pair-wise value between the mother tree and its TC plantlets was ‘1’, indicating perfect similarity. Phenetic dendrogram based on UPGMA (unweighted pair group method of arithmetic averages) analysis further confirmed the true-to-type nature of TC progenies, since a tie was observed between the mother tree and its TC plantlets. On the contrary, the control neem accessions were distinct from the mother and its TC progenies. AFLP markers proved to be an ideal tool for routine analysis and certification of genetic fidelity of micropropagated plants prior to commercialization, especially in tree species because of their long generation time. [ABSTRACT FROM AUTHOR]

• Micropropagation and genetic study of Rhynchostylisretusa (L.) have been studied. • Half strength and quarter strength of MS medium responded best for the in vitro seed germination and protocorm development. • Full strength of MS medium supplemented by 10% coconut water was found best for the shoot development. • Fungal elicitor (CVS4) supplemented FMS medium responded best for the development of roots. • Genetic stability of in vitro developed plant materials were investigated using RAPD markers. Rhynchostylis retusa (L.) is an epiphytic orchid having both medicinal and ornamental value. The present study aimed to develop a protocol for mass propagation of R. retusa and evaluate the genetic stability of in vitro regenerants. The immature seeds obtained from capsule of mother plants (wild type) were cultured on different strength of MS medium viz. full (FMS), half (HMS), quarter (QMS) supplemented with BAP (0.5 mg/L), NAA (0.5 mg/L), and 5% or 10 % coconut water (CW). The earliest germination and protocorm development were observed on HMS and QMS media. Mature protocorms produced higher number of shoots (12.8) and longest shoots (5.3 cm) on FMS supplemented with 10 % CW. The maximum number of roots (7.3) and root length (5.0 cm) were observed on FMS when supplemented with fungal elicitor CVS4 (extracted from the stem of Vanda cristata). Ten RAPD primers were used to analyze genetic stability among six samples (five in vitro and one mother plant) produced a total of 23 fragments ranging from 275bp to 1100bp. The polymorphic information content (PIC) values ranged from 0.28 to 0.50. The amplified bands of all the samples of in vitro plants were similar to bands of mother plant. The result demonstrated the genetic stability of the micropropagated plants of R. retusa. Research reported here is indicating the applicability of tissue culture for true-to-type plant production and conservation of R. retusa. [ABSTRACT FROM AUTHOR]

In a culture method for enhanced axillary branching functional plants of Eucalyptus tereticornis and E. camaldulensis are efficiently regenerated. To assess the genetic integrity among the regenerants, we employed multiple analytical tools including cytochemical and molecular assays. The 2C DNA amounts were estimated in the meristematic zones of root and shoot tips of 250 micropropagated plants, collected at various cycles of tissue culture from multiplication to field transfer, and compared to the corresponding mother plants. The culture conditions did not induce amplification or deletion of DNA sequences, nor were there drastic change(s) in chromosome number, since all the micropropagated plants of E. tereticornis (1.2 pg) and E. camaldulensis (1.4 pg) maintained the same DNA amounts as the mother plant. Total DNA of 46 micropropagated and mother plants digested with eight restriction enzymes and hybridized to 13 nuclear, mitochondrial, and synthetic oligonucleotide DNA probes yielded 82 bands. Hybridization patterns indicated that the variation observed was minor. To further confirm the genetic fidelity, 12 arbitrary 10-base primers and six synthetic oligonucleotide sequences, successfully used to amplify genomic DNA from in vivo and in vitro materials, produced 133 fragments that were monomorphic across the plants tested. The present results demonstrate that enhanced-axillary-branching culture of mature trees could be utilized commercially for mass clonal propagation of these two important Eucalyptus species that have been recalcitrant to vegetative propagation. The results also provide novel insights into the genetic differences between E. tereticornis and E. camaldulensis. [ABSTRACT FROM AUTHOR]

• Bambusa balcooa Roxb. is a valuable bamboo species. • A stable micropropagation system was developed for B. balcooa. • No genetic or morphological variations were observed in micropropagated plants. • This study will be useful in large-scale commercial propagation to provide quality planting material. Bambusa balcooa Roxb. is one of the economically most important bamboo species cultivated in the tropical countries. The present investigation is conceded to establish an effective method for clonal propagation of B. balcooa. The morphological response from nodal cuttings was tested on Murashige and Skoog's (MS) basal medium mixed with a range of combinations and concentrations of 6-furfurylaminopurine (Kinetin) and 6-benzylaminopurine (BAP). About 96 % shoot sprouting was observed on medium containing 4.0 mg L−1 BAP, 50 mg L−1 ascorbic acid and 25 mg L−1 each of l -arginine, citric acid and adenine sulphate. These shoots were proliferated efficiently on MS medium with 4.0 mg L−1 BAP, 1.0 mg L−1 NAA, which resulted in the formation of ∼62.0 shoots per node per culture bottle. In vitro rooting was achieved on ½-strength MS basal medium with 6.0 mg L−1 NAA and 100 mg L−1 activated charcoal. About one-month-old well-rooted plantlets were exposed to the greenhouse for 4 weeks in soilrite®. The hardened plantlets exhibited 100 % survival success under the field environment after 3 months. The monomorphic pattern of inter simple sequence repeats (ISSR) and start codon targeted (SCoT) markers of in vitro raised plants matched with mother plants confirmed the genetic stability. Moreover, in vitro raised plantlest also showed histological similarties with mother plant. Hence, this method could be efficiently utilized for commercial-scale production of B. balcooa. [ABSTRACT FROM AUTHOR]