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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]

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]

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]

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]

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]

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]

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]

This study described a protocol for genetically stable synseed production of Azadirachta indica using shoot tips of in vitro grown plantlets. For this, the shoot tips from in vivo grown tree were cultured on Murashige and Skoog (MS) media. The highest shoot response, number of buds per shoot and longest shoot were 94.44%, 5.07 and 3.73 cm, respectively, with 0.5 mg L−1 benzyladenine (BA) and 0.1 mg L−1α-naphthaleneacetic acid in combination. These shoots were further enhanced by subculturing and the second subculture yielded both maximum shoots per micro shoot (6.08) and longer shoot length (4.64 cm) on the same media composition. The shoot tips of this subculture were employed for encapsulation. The 3% sodium alginate and 75 mM calcium chloride with an anion exchange period of 20 min was found to be best for synseed production which displayed a quick response (3.17 days) for germination with 100% germination frequency. The maximum in vitro root response, the highest number of roots per shoot and longest root length were 91.67%, 4.78 and 3.18 cm, respectively, in 1/2 strength MS media with 0.5 mg L−1 indole-3-butyric acid and 0.1 mg L−1 BA in combination. The 24 °C stored synseeds showed more germination frequency and regeneration potentiality in all storage periods as compared to 4 °C stored synseeds. The soil: organic manure (1:1) exhibited the best result of plant survival (91.18%) for primary hardening after 1 month and it displayed 85.9% after two months in the vermiculite-soil mixture (2:1) after secondary hardening. This investigation certified the genetic fidelity of synseed-derived plantlets after storage at 4 °C and 24 °C using ISSR markers. None of the ISSR markers exposed polymorphism for hardened plants. Key message: This study reported first time the development of synseed of neem. The genetic fidelity of regenerants were performed after storage at 4°C and 24°C for different period of times (Days) using ISSR markers. [ABSTRACT FROM AUTHOR]

Many tree species are an excellent source of a wide range of bioactive compounds of pharmaceutical importance. However, overexploitation of medicinal trees to fulfil the demand for plant-based herbal drugs puts pressure on its natural population. Therefore, many tree species are declining continuously, particularly those used in pharmaceutical industries. In addition, limited production of secondary metabolites from a specific part and only at a particular developmental stage and sometimes very-low yield are some major bottlenecks when secondary metabolites are isolated directly from a tree species. Tissue culture-based biotechnological interventions for propagation, in vitro conservation and secondary metabolite production in medicinally important tree species have been practiced during the last two–three decades. Many medicinally important trees successfully propagated in vitro through different modes of regeneration i.e., axillary shoot proliferation, adventitious organogenesis and somatic embryogenesis. Success in in vitro propagation of most of the medicinal trees has been achieved through axillary shoot proliferation. Recent studies on medicinal trees showed that ex vitro rooting is an ideal method of rooting of microshoots. Gene targeted molecular markers have now been preferred for genetic fidelity of tissue culture-raised plants of medicinal trees. In recent years, newly developed droplet-vitrification and cryo-plate methods increased the applicability of cryopreservation for the long-term conservation of many medicinal trees. Several bioactive compounds of pharmaceutical importance are produced from trees via in vitro culture technique. There are a few success stories of producing secondary metabolites at a commercial scale from medicinal trees i.e., taxol, camptothecin and azadirachtin. This review paper presents the recent progress on plant tissue culture-mediated biotechnological advances in medicinal trees, emphasizing different aspects of in vitro propagation, conservation, and production of bioactive compounds of pharmaceutical importance. [ABSTRACT FROM AUTHOR]

Cryopreservation is vital to the preservation of genotypic diversity of plant species. In the present study, an efficient procedure for cryopreservation of Chrysanthemum cv. Chuju shoot tips was established for the first time. The adventitious shoot clusters of Chuju were transferred to MS medium fortified with 100 µM melatonin (MLT) and cultured at 4 °C for 4 days. Then, the shoot tips were excised and precultured on MS medium supplemented with 0.4 M sucrose for 4 days at 4 °C in the dark. After that, the shoot tips were loaded in 60% plant vitrification solution 2 (PVS2) for 20 min at 0 °C, followed by immersion in 100% PVS2 solution for 60 min. Then, put the shoot tips into liquid nitrogen (LN, − 196 °C). After at least 1 h, the LN-stored shoot tips were rapidly rearmed for 2 min at 39 °C, and liquid MS medium containing 1.2 M sucrose was used as the unloading solution for 15 min. Finally, the cells were postcultured in recovery medium for shoot regeneration. Under these conditions, 65.7% of cryopreserved shoot tips survived and regenerated. Further biochemical and histological analysis showed that exogenous MLT could improve the survival rate by regulating the level of oxidative stress and maintaining the morphological structure of shoot tip cells, which provided a new method for exploring the mechanism of MLT in cryopreservation. Intersimple sequence repeat analysis did not reveal any polymorphic bands in regenerants recovered from the shoot tips of Chuju. We expect that our results will facilitate the successful application of MLT in the cryopreservation of rare and commercially important plant germplasm. Key message: An efficient cryopreservation procedure for Chrysanthemum cv. Chuju was established. By enhancing the activity of antioxidant system and cell integrity in this process, exogenous melatonin improved the regeneration ability of shoot tips. [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]

Genetic engineering can add new capabilities or traits and direct method using biolistic particle delivery holds key for rapid, routine and efficient transformation of chickpea. Regeneration efficiencies of five different explants derived from BAP pre-treated breeders' chickpea seeds (cv. DCP 92-2) raised in phytohormones combinations (BAP and KIN for shoot primordia induction; GA3 for shoot elongation and NAA for rooting) were compared. Best response was obtained using the embryonic axis (EAX) explants with 86.69% regeneration efficiency followed by epicotyl (EPI) explants (78.69%). Direct genetic transformation were demonstrated in two responding explants by bombarding with pre-treated tungsten, coated with plant expression cassette (harboring Bt and nptII gene) from a distance of 4 cm with 1100 psi helium pressure. Transgenic chickpea lines with multiple and single copy integrations were obtained with transformation frequency of 0.72% for EPI explants and 1.21% for EAX explants, significantly higher than Agrobacterium tumefaciens mediated transformation of same genotype (0.076%). Southern blot based analyses of seven single copy transgenic chickpea lines exhibited presence and transmission to subsequent generations (T1 and T2). Presence of Bt protein were detected in the leaves of transgenic chickpea lines at pre-flowering (6.63–11.95 ng/mg TSP) and post flowering stages (4.85–8.93 ng/mg TSP). Genetic fidelity analysis using genome wide SSR markers of ten independent transgenic lines indicated true to type with original genotype. Taken together, this study describes a protocol that can be adapted for direct genetic transformation of chickpea with high efficiency. Key message: Routine protocol for direct transformation of grain legume, chickpea. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis is an important tool for crop improvement through transgenic approach and even for gene editing. It has been hypothesized regularly for large-scale propagation of banana which necessitates basic data on genetic fidelity and field performance of the plants towards ensure the commercial feasibility of the technique. Plantlets regenerated from embryogenic cell suspension (ECS) cultures established using immature male flower buds were examined for genetic fidelity using Inter Simple Sequence Repeats (ISSR) markers. Results showed that the primers UBC 808, UBC 811 and UBC 841 each generated one polymorphic band with an overall variation in banding pattern of 3.34 and 2.09% in cvs. Grand Naine and Rasthali respectively. Field evaluation of the ECS derived plants showed that there were no negative effects on the vegetative and yield parameters. Remarkably no phenotypic off-types were observed in this field trial. The level of genetic variation observed in this study is not an obstacle for further uptake of this novel propagation technique. Key message: Field performance of ECS derived plants being on par with shoot tip cultured plants concludes that somatic embryogenesis could be successfully employed for commercial propagation of banana plantlets. [ABSTRACT FROM AUTHOR]

An advanced micropropagation protocol has been developed for the global spice crop Vanilla planifolia using meta-topolin [mT, 6-(3-hydroxybenzylamino) purine] for the first time. Among the two types of cytokinins [mT and BAP (6-benzylaminopurine)] experimented, healthy shoot development and growth in terms of shoot numbers (5.0 ± 0.20/explant) with shoot length (4.3 ± 0.17 cm) were noted on agar-gelled Murashige and Skoog's (MS) medium supplemented with 1.0 mg L−1mT. The regenerated shoots were multiplied in liquid medium using the continuous shake-flask culture method in an incubator shaker. The liquid MS medium containing 0.5 mg L−1mT + 0.25 mg L−1 α-Naphthalene acetic acid (NAA) agitated at 60 rpm resulted in the production of 62.0 ± 0.31 shoots (3.8 ± 0.11 cm length) per explants per culture vessel after 3rd subculture, and this is the best rate of shoot proliferation among the prevailing reports on V. planifolia. Further, the transverse sections of the leaves revealed that the mT medium derived leaves were physically sturdy and physiologically more active with developed anatomical parameters, such as higher proportions of palisade and spongy parenchyma tissue, xylem and phloem elements, and abundant chloroplasts than the shoots raised on BAP. The leaves collected from the shoots of mT containing medium possessed the higher amount of chlorophylls (380.0 μg g−1 fresh weight) and carotenoids (52.0 μg g−1 fresh weight) under in vitro conditions. The concurrent ex vitro rooting and acclimatization method has been adopted to reduce the cost and time in large scale cloning of V. planifolia. All the shoots (100%) were rooted in soilrite™, when pulse treated with 100 mg L−1 NAA for 4 min. The ex vitro rooted plantlets were successfully acclimatized in a greenhouse. The regenerated clones' genetic uniformity and stability with the donor plant were tested using Start codon targeted polymorphism and detected no somaclonal variation. All the plantlets were survived in the soil transplantation, confirming the production of genetically, physically, and physiologically stable plantlets of V. planifolia. Key message: An improved in vitro regeneration protocol was developed for Vanilla planifolia. It is the first report revealing the positive effect of meta-topolin and liquid medium (shake-flask culture method) for enhanced proliferation of shoots in this orchid. The mT derived leaves showed better anatomical features and retained high level of photosynthetic pigments. The in vitro raised shoots were rooted ex vitro and successfully transplanted in the field. [ABSTRACT FROM AUTHOR]

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]

An efficient method of rapid micropropagation of Solanum khasianum Clarke was successfully established from the leaf, petiole, and nodal explants. The morphogenetic response of different concentrations of TDZ and BAP individually or in combination with auxins (IAA/IBA/2,4-D) was tested. Friable callus was obtained on different concentrations of BAP alone or in combination with IAA/IBA/2,4-D. Rapid multiple shoot induction was achieved from friable callus on MS medium supplemented with varying concentrations of TDZ and IBA. The leaf explants exhibited a high frequency of multiple shoots than petiole and nodal explants with an optimal percentage of response (92.73%), mean shoot number (53.5 ± 0.47), and shoot length (11.2 ± 0.53 cm) on MS medium augmented with TDZ (1.5 mg l−1) and IBA (1.5 mg l−1). Maximum rooting efficiency was achieved on MS medium with 1.5 mg l−1 IBA with 12.8 ± 0.36 mean number of roots. The in vitro rooted plants were acclimatized with a survival rate of 80%. The genetic fidelity of the regenerants assayed by the ISSR and the SCoT markers showed no genetic variation. The study examined the micropropagation responses of S. khasianum in the presence of various growth regulators and provided a simple and more suitable protocol adapted for the mass propagation of clones in this species. Key message: We have established a highly efficient micropropagation system for large scale production in Solanum khasianum. Evaluation of clonal fidelity by using ISSR and SCoT markers detected no somaclonal variations. The present study helps to the enhancement of potential alkaloids (solasodine) with the help of biotechnological tools. [ABSTRACT FROM AUTHOR]

The present study reports an optimized protocol for high frequency in vitro plant propagation through direct and indirect organogenesis, phytochemical accumulation, molecular profiling and antioxidant evaluation for micropropagated Swertia minor, a promising alternative to industrially important Swertia chirayita. Moreover, the study also aimed at enhancing the production of antidiabetic and anti-obesity drug swertiamarin using an alternative technology of elicitated cell suspension cultures. Different types, concentrations and combination of cytokinins and auxins showed their effects during various in vitro growth stages. A combination of BAP (3.0 mg/l) and TDZ (1.0 mg/l) had a dominant role in promoting multiple shoot proliferation with production of an average of 19.1 ± 0.95 shoots/node in 85% response. MS medium added with IBA (2.0 mg/l) showed optimal response for in vitro rooting (9.2 ± 0.56 roots/shoot). In order to establish genetic stability, molecular marker-based profiling of micropropagated plants were done and 'monomorphic banding pattern were identical to the mother plants. 2,4-D (2.0 mg/l) supported the maximum callus induction and proliferation rate (95%). The wild-grown plants showed higher polyphenols content and antioxidant activities as compared to callus and in vitro derived plantlets. However, chitosan-treated (25 ppm) methanolic extract of cell biomass accumulated in cell suspension cultures produced higher contents of swertiamarin (1.45 mg/g DW) than salicylic acid and methyl jasmonate. The described protocol can be effectively used for the large-scale propagation, exploitation of active compounds and will serve as potential alternative to S. chirayita for fulfillment of over-growing industrial requirements. Key message: The present investigation addressed in vitro regeneration, callus culture, somatic embryogenesis, molecular profiling, secondary metabolite production, cell suspension culture studies for first time in Swertia minor. [ABSTRACT FROM AUTHOR]

High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l−1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l−1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l−1 BAP along with 0.05 mg l−1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l−1), GA3 (1.0 mg l−1) and BAP (1.0 mg l−1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l−1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation. Key message: An efficient and reproducible in vitro regeneration protocol was established for chickpea. Application of different concentrations and combinations of PGRs was found to enhance multiple shoot induction, shoot elongation, rooting and acclimatization of in vitro regenerated plants in field conditions, and further evaluated genetic fidelity using molecular markers. [ABSTRACT FROM AUTHOR]

A proficient and reliable in vitro plant regeneration protocol was established for pea by utilizing cotyledonary node as an explants, which excised from 3-days old imbibed seeds. The present research work explains the positive role of polyamines (PAs) accompanied by plant growth regulators (PGRs) on the multiple shoot induction and rooting in pea plant regeneration. The highest multiple shoots (65.1 shoots/explant) was attained from the cotyledonary node explant on Murashige and Skoog (MS) medium accompanied with 20 mg l−1 spermidine (SPD) along with 1.5 mg l−1 6-benzyladenine (BA). Moreover, the highest elongation of multiplied shoots (10 cm length/shoot) was noted on MS medium enriched with 1 mg l−1 of gibberellic acid (GA3). The elongated shoots produced the highest number of roots (33.66 roots/shoot) on MS medium augmented with 30 mg l−1 putrescine (PUT) along with 0.6 mg l−1 1-naphthaleneacetic acid (NAA). Rooted plants were hardened and acclimatized in the greenhouse with an existence rate of 98%. The standardized procedure with the use of PAs has enhanced the multiple shoot induction to threefold higher than the plants raised from PGRs treatments. The regenerated pea plants revealed the fivefold enhanced photosynthetic pigment, twofold enhanced antioxidant profile and the substantial growth in chloroplast count have been achieved in optimized SPD and BA supplemented MS medium compared to control plant. Start Codon Targeted polymorphism and Random Amplified Polymorphic DNA molecular markers recognized the genetic purity of the in vitro regenerated pea plants for their true type of nature. Key message: An enhanced and consistence in vitro regeneration protocol was established for pea, by the application of polyamines along with PGRs, which significantly improved the multiple shooting, shoot elongation, rooting, total chlorophyll and antioxidant profile of regenerated plants, further not hampering their genetic fidelity. [ABSTRACT FROM AUTHOR]

Morphological variation, genotypic stability and genetic distances among micropropagated tea [Camellia sinensis (L.) Kuntze] cv. 'Kolkhida' plantlets after 7 years of propagation in vitro and among plantlets regenerated from callus were compared. Fifteen individuals, randomly chosen amongst a set of 100 plantlets regenerated from callus, and ten individuals, randomly chosen from a set of 100 micropropagated plants were used in the study. Morphological descriptors showed higher variability among callus-derived regenerants than in micropropagated plants. Flow cytometry analysis showed no significant variability of nuclear DNA content among micropropagated plants, however changes in DNA indicated aneuploidy in three out of ten callus-derived regenerants. ISSR analysis showed lesser genetic distances among micropropagated plantlets and maternal cultivar 'Kolkhida' compared with callus derived plantlets. Yet, there is some degree of genetic instability after long-term micropropagation. Results confirmed the hypothesis of genetic instability of some species during long term in vitro conservation. Key message: Tea vegetative explants plants were propagated in vitro for 7 years. A comparison of tea plantlets derived from callus with plantlets from long-term in vitro propagation revealed genetic variability and confirmed the hypothesis of genotype instability of tea plant. [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]

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 × 10 protoplasts (Pp) per gram fresh weight with 94 % viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5 % cellulase Onozuka R10, and 1 % pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3-5 × 10 Pp ml. Protoplast-derived microcalli became visible after 3-4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2-3 weeks, with an overall shoot organogenesis efficiency of 78-93 %. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62 %. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings. [ABSTRACT FROM AUTHOR]

Sixty six plants raised via direct shoot bud organogenesis from pre-plasmolysed leaf explants of Catharanthus roseus were assessed under in vivo conditions for their physio-morpho traits, tryptophan metabolism, genetic fidelity and alkaloid profile. Morphologically all plants were parent like except the three morpho-types i.e. broad cup-shaped leaves, dwarf phenotype and enlarged pink corona. Vindoline was detected in most of the plants in substantial amount while ajmalicine, ajmaline and catharanthine were detected in few plants only. More than eightfold increased vindoline (0.08 % dry wt.) was showed by plant no. 1, 4 and 39. Except plant no. 54, which accumulated the highest alkaloid content of 3.09 % dry wt. with tryptophan content of 0.0283 % dry wt., majority of the plants showed a negative correlation between total alkaloid content and tryptophan accumulation and strong positive correlation between tryptophan content and 5-methyltryptophan tolerance. ISSR and RAPD profile of seventeen randomly selected, field established plants was generated by using 10 ISSR and 60 RAPD primers. In RAPD, a total of 753 bands were detected, out of which 624 (82.87 %) were monomorphic. In ISSR profiling, a total of 205 bands were detected out of which 200 bands were monomorphic (97.56 %) and only 5 bands were found to be polymorphic. Highly significant data for the monomorphic banding pattern across all the three genotypes ( p < 0.01) was observed. Principal components, AMOVA analysis and multivariate analysis using Nei and Li's coefficient further validate the monomorphism. Thirteen plants having superior traits were grown in Random Block Design in field. The relevance of physiological and epigenetic changes occurred in the light of morpho-types and alkaloid profile of directly regenerated plants during in vitro to in vivo acclimatization in C. roseus is discussed. [ABSTRACT FROM AUTHOR]

The escalating loss of biological diversity throughout the world has become a major concern for the conservation biologists. Like other threatened plant species, the natural populations of the orchids are also severely threatened. Dendrobium thyrsiflorum is one such representative of the family Orchidaceae whose natural populations are getting destroyed at an alarming rate and deserves special conservation attention. Both direct shoot organogenesis (DSO) and indirect shoot organogensesis (ISO) pathways were experimented and the highest regeneration frequency for DSO and ISO pathways were found to be 86.2 and 96 % respectively. The regenerated shoots were best rooted in half-strength MS medium supplemented with 1 mg/l indole butyric acid (IBA) and 0.5 mg/l phloroglucinol. The genetic stability of the acclimatized plants derived from ISO and DSO pathways was assessed using Inter Simple Sequence Repeats (ISSR) and Start Codon Targeted (SCoT) molecular markers. SCoT proved to be a superior marker over ISSR in detecting clonal variability. The phytochemical analysis of the micropropagated plants also revealed a comprehensively higher yield of various secondary metabolites with significantly higher antioxidant potentials in both ISO- and DSO-derived plants over the mother plant. However, the ISO-derived plants were more phytochemically enriched compared to the DSO-plants. The rapid multiplication rate, higher genetic stability and secondary metabolite production ensures the utility of this micropropagation method for D. thyrsiflorum in the ex situ conservation and commercial exploitation of other important orchid species. [ABSTRACT FROM AUTHOR]

The effects of subculture frequency on genetic variations of Tetrastigma hemsleyanum callus under a long-term tissue culture were evaluated by means of flow cytometry (FCM), inter simple sequence repeat (ISSR), and sequence-related amplified polymorphism (SRAP) markers to clarify the variation pattern and frequency of somatic variation in the course of the subculture. The effect of subculture frequency on endogenous hormone content was also assessed by Enzyme-linked immunosorbent assay to explore the correlation within the differentiation rate, somaclonal variation frequency of callus and the endogenous hormone content. Moreover, the flavonoid content in the callus of different subculture times was compared and the antitumor activity of them was evaluated by using MTT cell proliferation and cytotoxicity assay. FCM confirmed similar ploidy level in mother plant and all callus, but some changes were observed in the ISSR and SRAP patterns, of which 10.3 and 6.06 % were polymorphic, respectively. The somatic variation frequency tended to increase with the increasing subculture time in the early stage of subculture, with a peak in the 4-6 times and then kept nearly at a constant after 8 times. High IAA content may be one reason for this variation by this period. The flavonoid content and antitumor potential increased and remained at high level during the 2nd and 6th subculture. Though there was a significant decline after the 6th subculture, they still had about the same flavonoid content and antitumor potential as those of mother plant. The results suggested that the in vitro callus of T.hemsleyanum is potentially useful for pharmaceutical uses. For supplying true-to-true seedlings, germplasm conservation and further genetic transformation, subculture frequency of callus should be controlled within 4 times to obtain clonally identical plantlets. However, for the development of germplasm improvement of the species, subculture frequency should be controlled in 6-8 times to make better use of somatic variations. [ABSTRACT FROM AUTHOR]

A protocol for long-term in vitro conservation of multiple shoot cultures of Lavandula officinalis and regeneration of true-to-type plants from them is described here. Multiple shoots were developed from apical bud explants on a modified vitamin-enriched Murashige and Skoog's basal medium supplemented with 2.5 mg/l Kinetin. The cultures were subsequently conserved in vitro for 6 years under slow growth conditions imposed by lowering the sucrose level from 3 to 2.0 % and increasing the agar concentration from 0.8 to 1.2 % in this medium. Complete plant regeneration from in vitro conserved shoots was achieved through axillary shoot proliferation on original 2.5 mg/l Kinetin containing medium followed by rooting of individual shoots in a hormone-free half strength MS basal medium. The genetic fidelity of the regenerated plants was tested by random amplified polymorphic DNA analysis. Twenty one arbitrary decamer primers produced a total of 64 scorable bands (1-6 bands/primer) in the size range of 100-5,148 bp. All DNA fingerprints with these primers displayed monomorphic band profiles indicating homogeneity among the regenerated progeny and their uniformity with the donor parent. Head-space gas chromatographic analysis of the leaf tissue excised from mother as well as regenerated plants also revealed a similar qualitative and quantitative profile of volatile terpenes. The utility of the developed protocol for long-term ex situ conservation and quality plant production for cultivation of this high value aromatic herb is discussed. [ABSTRACT FROM AUTHOR]

An efficient propagation system of pitaya with diverse genetic background was established by using tender stem as explants. Various types of plant growth regulators (PGRs) were used to determine the most effective hormone combination for shoot proliferation. Zeatin (ZT), thidiazuron (TDZ), 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 6-benzylaminopurine (6-BA) alone could induce multiple shoots. In that case, the most shoots per explant i.e. 4.6 (13.68 µM ZT), 4.8 (0.11 µM TDZ), 4.3 (17.76 µM 6-BA) and 3.2 (0.23 µM 2, 4-D) were obtained. The best PGRs combination was MS medium with 3.0 µM ZT and 0.5 µM IBA producing the most shoots per explant and the most vigorous shoots. Of the two varieties and six superior selections cultured on the best PGRs combination, more than 6.0 shoots per explant were obtained. No polymorphism was detected among in vitro-derived plantlets selected at random after 11 sub-cultures. Those results showed that MS media with 13.68 µM ZT and 2.46 µM IBA was suitable for shoot propagation of diverse pitaya varieties and selections. The protocol can be used for large-scale propagation of pitaya to meet the demand of increasing commercial cultivation. [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 efficacious regeneration system was developed from shoot apex explants of Zea mays using a two-step culture procedure. Seventeen Indian genotypes were assessed for their regeneration potential. The maximum response of shoot induction was obtained from explants cultured on Murashige and Skoog medium supplemented with 4.5 µM thidiazuron and 26.7 µM glycine. Maximum mean number of shoots (17.2) was observed in genotype COH (m)-5 while NPK was the least responsive (6.7). Shoot clumps transferred from shoot induction medium to multiplication (second) medium amended with 1.1 µM thidiazuron and 0.88 µM N-benzylaminopurine showed increased number of shoots in COH (m)-5 (36.1 shoots); NPK was the least responsive with an average of 9.5 shoots. The best response in root induction, with a larger number of roots (10.5) and longer roots (6.6 cm), was observed in Murashige and Skoog medium supplemented with 7.3 µM indole-3-butyric acid and 7.9 µM phloroglucinol. Analysis of variance indicated that plant regeneration response varied greatly among the genotypes. In vitro raised plants were successfully transferred to the field after hardening, with a 99 % survival rate. Inter simple sequence repeats analysis revealed that the similarity matrix pair-wise value was 1, the Mantel test value was p 1.0; Analysis of molecular variance genetic variances were 93 % within the population and 7 % between populations; Principal component jolliffe cut off was 0.15, Principal component and Principle coordinate analysis % variance was 13.19. These values were congruent for both the mother and the in vitro-raised plants, confirming genetic integrity. [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]

A new technique to regenerate caper plants ( Capparis spinosa L. subsp. rupestris) starting from flower explant is reported. In vitro plant regeneration was attempted using stigma, anthers and unfertilized ovules of unopened flowers collected in the field. Plant regeneration was achieved from unfertilized ovules on MS medium supplemented with 88 mM sucrose and 13 μM 6-benzyladenine (BA). New individuals obtained from unfertilized ovules were used as source material for micropropagation and multiple shoots were obtained on MS medium supplemented with the adeninic cytokinin BA and the auxin indole-3-butyric acid (IBA). Explants obtained in micropropagation step were used for rooting step under several treatments. The best results (100% of rooted explants) were obtained when explants were dipped for 10 min in 50 μM IBA solution and successively maintained in growth regulator free medium. New plants were vigorous, of good quality and presented phenotypic characters similar to mother plants. Furthermore genetic stability of regenerants was verified through flow cytometric analysis and two different DNA-based techniques. [ABSTRACT FROM AUTHOR]

In vitro grown microshoots of Picrrhiza kurrooa were encapsulated in the alginate beads. Regrowth of encapsulated microshoots, using alginate encapsulation, of P. kurrooa reached 89.33% following 3 months of storage. Amongst developing plantlets, 42.66% exhibited formation of multiple shoots at the onset of regrowth and 21.43% demonstrated simultaneous formation of shoots and roots. Healthy root formation was observed in plantlets following 2 weeks of their transfer to half-strength Murashige and Skoog medium containing 1 μM α-naphthalene acetic acid. Plants were transplanted to the greenhouse in three batches with 95% frequency of survival. The genetic fidelity of P. kurrooa plants growing out after storage in encapsulated form was ascertained by random amplified polymorphic DNA (RAPD) analysis. Molecular analysis of randomly selected plants from each batch was conducted using 45 random decamer primers. Of 45 primes tested, 14 produced scorable amplified products. Total 68 bands were observed amongst them 7.35% bands were polymorphic. Cluster analysis of the RAPD profile revealed an average similarity coefficient of 0.966 thus confirming genetic stability of plants derived from encapsulated microshoots following 3 months of storage. [ABSTRACT FROM AUTHOR]

The genetic fidelity of in vitro-raised gerbera clones was assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Out of 35 RAPD and 32 ISSR primers screened, only 12 RAPD and 10 ISSR primers produced clear, reproducible and scorable bands. The 12 RAPD primers produced 54 distinct and scorable bands, with an average of 4.5 bands per primer. The number of scorable bands for ISSR primers varied from 3 (ISSR-14) to 9 (ISSR-07), with an average of 5.5 bands per primer. The number of bands generated per primer was greater in ISSR than RAPD. All banding profiles from micropropagated plants were monomorphic and similar to those of the mother plant. A similarity matrix based on Jaccard's coefficient revealed that the pair-wise value between the mother and the in vitro-raised plantlets was 1, indicating 100% similarity. This confirmed the true-to-type nature of the in vitro-raised clones. [ABSTRACT FROM AUTHOR]

gave tequilana is a species of great economic importance for the Mexican society. Like most agaves, it can be propagated either sexually or asexually. However, plants originating from rhizomes are mainly used for commercial production. The aim of this study was to determine the genetic fidelity of plants that were obtained from the field and propagated from rhizomes and of those propagated by in vitro culture methods (somatic embryogenesis and proliferation of axillary buds). We used an inverse ISTR (sequence-tagged repeat) molecular marker based on retrotransposon sequences to detect variability among related individual plants. Cluster analysis showed that plants could be grouped according to the propagation method employed. Plants propagated by the same in vitro method were found to be distinct from those propagated in the field. Even within each method, plants were not genetically identical. Genomic changes were evidenced not only in plants subjected to somaclonal variation in vitro but were also evident in those propagated through the natural asexual process, despite the fact that they were considered true clones. The differences observed in plants confirm the existence of asexual genetic variability. [ABSTRACT FROM AUTHOR]