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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]

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

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

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

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

Fast-growing forest species with multiple uses, like bamboo, have aroused interest for their silvicultural applications. Bamboo species are a valuable source of renewable raw material, and Bambusa vulgaris is an economically important species. However, there are limitations to large-scale cloning of adult-selected genotypes. This study aimed to evaluate the in vitro cloning of Bambusa vulgaris in different culture systems, sucrose and activated charcoal supplementation by the inter-simple-sequence repeat (ISSR) molecular markers. In vitro bud multiplication and shoot elongation were evaluated in three cultivation systems: semi-solid and liquid culture media, and temporary immersion bioreactor (TIB). The sucrose concentrations, 0 and 30 g L−1 were evaluated in the stages. Both the culture media were supplemented with 2.0 mg L−1 benzylaminopurine (BAP) and 0.5 mg L−1 α-naphthalene acetic acid (NAA). The absence and presence of activated charcoal (100 mg L−1) were evaluated in the in vitro rooting. MS culture medium was supplemented with 2 mg L−1 indole-3-butyric acid (IBA), 1.0 mg L−1 NAA, and 0.5 mg L−1 BAP. Semi-solid culture medium supplemented with 30 g L−1 of sucrose presented superior emission of bud per explant. Liquid culture medium supplemented with 30 g L−1 of sucrose presented the most elongated shoots. Activated charcoal in the culture medium did not influence the adventitious rooting. Micropropagated plants showed genetic fidelity and were clones of the adult selected plant.

The present study was focused towards determining the factors governing direct organogenesis in kiwifruit cultivar 'Allison'. Using in vivo buds or nodal segments as explants, the maximum 75.5% uncontaminated explants were obtained upon consecutive individual disinfection treatment with 1% carbendazim (3 min), 0.1% HgCl2 (1 min), and 2% NaOCl (2 min), respectively. The maximum shoot regeneration response of 84.81% was recorded on Murashige and Skoog (MS) medium fortified with 0.45 μM BA, 0.1 μM IBA, and 0.13 μM GA3. The months of May displayed the highest in vitro explant regeneration response for two consecutive years. The highest shoot proliferation rate was 1:6 with a 5.34 mean number of shoots and a maximum mean shoot length of 6.32 cm on MS medium containing 0.22 μM BA, 0.1 μM IBA, and0.69 μM GA3. A two-step or sequential approach for in vitro root induction showed good quality rooting response of 91.03% producing a higher number of long (2.23 cm) roots per shoot, and afterwards, the plantlets were hardened successfully with a higher survival rate (95%). Also, ISSR markers confirmed the genetic fidelity of the in vitro-raised regenerants. [ABSTRACT FROM AUTHOR]

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

Muntingia calabura L. is a potent medicinal plant. Because of that several researchers reported phytochemical and pharmacological studies, but only minimal information is obtainable on tissue culture and genetic stability evaluation studies. Therefore, in the present investigation we attempted to establish a reliable direct and indirect regeneration study through leaf and node explants on MS medium containing cytokinins BAP and KN (0.5-3.0 mg l-1) in combination with auxins IAA and NAA (0.5 mg l-1). The breakage of bud and shoot initiation were noticed with the initiation of callus at lower concentrations of auxin alone at 0.5 mg l-1. 2,4-D was found to be good in callus induction from leaf and nodal explants. A problem of browning callus was prevented by regular subculturing of callus cultures. Leaf explants exhibited maximum number of shoots (32±0.88a) with shoot length (7.6±0.17a) and nodal explants obtained optimal number of shoots (26±0.88a) with shoot length (9.6±0.30a) on MS medium supplemented with BAP (2 mg l-1) and IAA (0.5 mg l-1). Half strength MS medium fortified with IBA (2.0 mg l-1) was effective and achieved 70% of rooting. These well-developed plantlets were shifted to pots containing soil and vermicompost in 1:1 ratio for acclimatization. The acclimatized plants were field transferred with survival rate of 85%. The ISSR and RAPD markers analysis revealed the genetic stability of in vitro regenerated plants with the mother plant.

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]

Muntingia calabura L. is a potent medicinal plant. Because of that several researchers reported phytochemical and pharmacological studies, but only minimal information is obtainable on tissue culture and genetic stability evaluation studies. Therefore, in the present investigation we attempted to establish a reliable direct and indirect regeneration study through leaf and node explants on MS medium containing cytokinins BAP and KN (0.5-3.0 mg l-1) in combination with auxins IAA and NAA (0.5 mg l-1). The breakage of bud and shoot initiation were noticed with the initiation of callus at lower concentrations of auxin alone at 0.5 mg l-1. 2,4-D was found to be good in callus induction from leaf and nodal explants. A problem of browning callus was prevented by regular subculturing of callus cultures. Leaf explants exhibited maximum number of shoots (32±0.88a) with shoot length (7.6±0.17a) and nodal explants obtained optimal number of shoots (26±0.88a) with shoot length (9.6±0.30a) on MS medium supplemented with BAP (2 mg l-1) and IAA (0.5 mg l-1). Half strength MS medium fortified with IBA (2.0 mg l-1) was effective and achieved 70% of rooting. These welldeveloped plantlets were shifted to pots containing soil and vermicompost in 1:1 ratio for acclimatization. The acclimatized plants were field transferred with survival rate of 85%. The ISSR and RAPD markers analysis revealed the genetic stability of in vitro regenerated plants with the mother plant. [ABSTRACT FROM AUTHOR]

RAPD and ISSR molecular markers were employed to evaluate genetic fidelity of shoots of Achras sapota L. var. 'Cricket Ball' raised through nodal bud cultures. Out of 53 RAPD and 60 ISSR primers screened, 34 RAPD and 20 ISSR primers generated a total of 270 and 120 clear and scorable amplification products, respectively. All the shoot cultures analyzed at different culture passages showed similar RAPD and ISSR profiles as compared to that of mother plant. No genetic variation was observed in in vitro shoot cultures. The results indicated the genetic stability of the tissue culture raised shoot cultures of A. sapota and corroborate the assumption that axillary multiplication is the safest mode for multiplication of true to type plants without any somaclonal variation. [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]

Orthosiphon stamineus has been widely used as traditional remedy for various illnesses and diseases, such as cardiovascular diseases and epileptic seizures. In this study, direct regeneration through nodal segment of this species was attempted using Kinetin (6-Furfurylaminopurine) and IAA (indole-3-acetic acid). Optimum regeneration media was identified as MS media supplemented with 2.0 mg L−1 Kin plus 0.5 mg L−1 IAA. This yielded the highest number of shoots (5.57 ± 0.42) and leaves (20.53 ± 1.91) per explant. Acclimatization of the resulting in vitro regenerants was successful in all potting mixtures tested. However, potting mixture PF (1:1:1 ratio of black soil/red soil/compost) was identified as the best medium for acclimatization of this species, as it yielded 100% survival percentage after 90 days of acclimatization. Ten in vitro regenerants of O. stamineus were randomly collected after the third subculture and subjected to genetic variation analysis using inter-simple sequence repeat (ISSR) markers. Out of 20 ISSR markers tested, 10 working primers were observed to produce satisfactory amplification of bands, with an average of 7.11 bands per primer. A total of 610 bands were produced by the 10 primers. The percentage of polymorphism was observed to be very low, yielding only 7.32% polymorphism among all samples. Jaccard dissimilarity analysis was also conducted and very low genetic distance (about 0.1) was found among the in vitro regenerants and between the regenerants with the mother plant, thus ascertaining the clonal nature of the plantlets produced in this study.

The aim of this study was to produce sugarcane plantlets from cell suspension culture and study its genetic fidelity using molecular markers. The study was carried out using sugarcane varieties Co 86032 and Q117. Callus cultures of both the varieties were optimized using six different callus induction media. After screening the growth response of callus on six different callus inductionmedia, itwas observed that medium no. VI supplemented with 500 mg l-1 of each PVP, Casein hydrolysate and MES buffer showed high amounts of callus in Co 86032 (79.66 ± 0.44%) and Q117 (82.83 ± 1.69%). Addition of PEG 8000 at 2.5% to this medium had a profound impact on inducing somatic embryogenesis in Co 86032 (54.66 ± 1.76%) and Q117 (66.66 ± 2.60%) as compare to control (24.33 ± 1.76%) and (27.33 ± 2.73%), respectively. Cell suspension cultures were established by culturing embryogenic calli in liquid medium showed well established suspension cultures with fever cell aggregates. There was negligible cell division during initial 2 days of incubation and cell count increased rapidly between 2 and 8 days. Further incubation beyond 8 days resulted in a decrease in cell viability. Enhanced callus proliferation in Q117 while enhanced shoot regeneration in Co 86032 was observed from cell suspension culture. The clonal fidelity of in vitro regenerated plants was assessed by using RAPD and ISSR markers. Analysis of the ten RAPD markers indicated that 90.48 and 86.95%true-to-type regenerated plantlets in Co 86032 and Q117, respectively. However, in the ISSRmarkers, Co 86032 did not show any polymorphism and in the Q117, 92.18% true-to-type plantlets were found. These results confirmed that somaclonal variation occurs during the process of indirect organogenesis and RAPD and ISSR marker based molecular analysis is a suitable method for an early detection of variation in sugarcane. [ABSTRACT FROM AUTHOR]

In the present study we have developed an efficient and effective method of synthetic seed production and its two-way germination system ofVitex trifolia, for easy transport of the propagules and efficient utilization of itsin vitroregeneration system. Nodal segments harvested from 8-week-oldin vitrocultures were encapsulated in calcium alginate beads. Three percent (w/v) Na-alginate polymerized in 100 mmol/L CaCl2.2H2O for 30 min produced clear and uniform beads. Germination of encapsulated beads with shoot and roots was achieved on Murashige and Skoog (MS) medium augmented with 6-furfurylaminopurine (KN, 2.5 µmol/L) + α-naphthalene acetic acid (NAA, 1.0 µmol/L). For multiple shoot production, synseeds were incubated on 6-benzyladenine (BA, 5.0 µmol/L) + NAA (0.5 µmol/L) augmented MS medium followed byin vitrorooting on MS + indole-3-butyric acid (IBA, 1.0 µmol/L). The synseeds produced retained about 90% regeneration potential even after 4 weeks of storage at 4°C. Genetic stability of the regenerated plants was evaluated using 13 inter simple sequence repeats (ISSR) primers. The study thus provides an efficient system for production of synthetic seeds, their storage and subsequent conversion into genetically identical plants. [ABSTRACT FROM PUBLISHER]

A viable and simple method for the large scale production of breadfruit (Artocarpus altilis) using tissue culture is used at the Food and Agricultural Research Extension Institute in line with the Food Security policy of Mauritius. Shoot tip buds of Artocarpus altilis are initiated and multiplied on basal micropropagation medium supplemented with 4.4 µM Benzyl adenine (BA). Direct systems of regeneration through the culture of organized meristems normally produce true-to-type plants. However, variations among in vitro raised plants have been widely reported. The aim of this study was to use ISSR markers to confirm the clonal fidelity of in vitro derived plantlets of breadfruit. DNA extraction of Artocarpus altilis was performed using a modified CTAB method and then purified using RNase and phenol treatment. The presence of DNA was verified on agarose gel electrophoresis and its yield and quality were determined by spectrophotometry. An average yield of 307.3 µL DNA was obtained but with some proteins, phenols and polysaccharides contamination was noted. The 63 markers generated by Inter Simple Sequence Repeat (ISSR) markers revealed a mean of 22.2% genetic variation and 77.7% genetic fidelity in the 15 week cultured plantlets and a mean of 1.9% genetic variation and 98.2% genetic fidelity in the 38 weeks cultured plantlets. These findings indicate that shoot meristems can be used as explants for ensuring clonal fidelity of micropropagated A. altilis and confirm the usefulness of ISSR markers as a certification tool for monitoring genetic stability during the regeneration process. [ABSTRACT FROM AUTHOR]

An in vitro protocol has been established for clonal propagation of Nothapodytes nimmoniana which is an important source of Camptothecin (CPT). Elite source was identified based on the chemical potency to accumulate the optimum level of CPT. Different types and concentrations of plant growth regulators were used to study their effect on inducing multiple shoots from the explants regenerated from embryos of N. nimmoniana. Of these, a combination of N6-benzyladenine (0.2 mg L) and Indole-3-butyric acid (IBA) (0.1 mg L) proved optimum for differentiating multiple shoots in 90.6 % of the cultures with an average of 10.24 shoots per explant obtained within 8 weeks of inoculation. Nearly, 92 % of the excised in vitro shoots rooted on half strength Murashige and Skoog (MS) medium containing 0.05 % activated charcoal, supplemented with 1-naphthaleneacetic acid and IBA at 0.1 mg L each. The micropropagated plants were evaluated for their genetic fidelity by employing inter simple sequence repeats (ISSR) markers. Ten individuals, randomly chosen from a population of 145 regenerants, were compared with the donor plant. The regenerated plants were also evaluated for their chemical potency using high-performance liquid chromatography (HPLC) analysis of CPT content. The true-to-type nature of the micropropagated plants was confirmed based on their monomorphic banding profiles with that of the mother plants using ISSR markers. Besides, HPLC evaluation of the CPT content confirmed the existence of chemical uniformity among the regenerated plants and the elite mother plant. [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]

Highlights: [•] We have optimized the concentration of sodium alginate and calcium chloride for the production of synthetic seeds in cucumber for efficient conversion of encapsulated shoot tips into plantlets. [•] First application of slow growth storage techniques in cucumber with assessing influences of different medium was developed. [•] In vitro flowering were induced making the method more effective. [•] We have also hardened and developed true to type plantlets re-grown from synthetic seed as assessed by molecular marker (RAPD and ISSR). [Copyright &y& Elsevier]

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]

The present study has investigated the efficiency of regrowth of Bacopa monnieri (L.) Pennell after 6 months of storage along with its clonal fidelity checking. Shoot tips and nodal segments of in vitro grown plantlets were encapsulated and stored at different temperatures and the highest regeneration was achieved by storing shoot tips at 4 °C for a period of 6 months. The response of shoot tips was found to be much better when compared to nodal segments in the case of forming healthy shoots and roots. The genetic stability of regrown plantlets was analysed by using RAPD and ISSR markers. Twenty ISSR primers produced 130 clear and reproducible amplicons, of which 125 bands were monomorphic. A total of 115 bands were produced by 25 RAPD markers with 94% monomorphism. This corresponds to a very low genetic variation pertaining to the clonal nature of the regrown plantlets with that of the mother plant. By means of HPLC biochemical analysis, bacoside A content in the regrown plants was also found to be comparable with that of the mother plant. Therefore, the protocol derived can be efficiently utilised for storing the encapsulated shoot tips of B. monnieri for about 6 months. [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]

Genetic stability and phytochemical analysis of in vitro established plants of Picrorhiza kurroa Royle ex Benth, have been carried out. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) markers were used to assess the genetic fidelity of tissue culture products including three adventitious shoots from three calli and 6 months old tissue culture raised plants growing in green house condition with mother plant. Apparent genetic variation was detected in the five types of plant materials. The percentage of polymorphic bands in the RAPD and ISSR analysis were 16.25 and 14.54 %, respectively. The genetic similarity was calculated on the basis of RAPD and ISSR data among the five types of plant materials and were ranged from 0.5 to 1.0 (mean 0.75) and 0.47 to 1.0 (mean 0.73), respectively. The similarity coefficient by both RAPD and ISSR analysis revealed that differences between tissue culture raised plants and mother plant was not remarkable, but notable differences were observed among three adventitious shoots regenerated from three calli. The phytochemical analysis of tissue culture raised products showed higher secondary metabolite (picrotin and picrotoxinin) content as compare to mother plant. The information gained on genetic stability/variability will be valuable for the large scale propagation and secondary metabolite production of P. kurroa. [ABSTRACT FROM AUTHOR]

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 μM BA, 2.5 μM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4-5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 μM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant. [ABSTRACT FROM AUTHOR]

In vitro grown axillary micro shoots of Glycyrrhiza glabra were encapsulated in alginate beads. Following 6 months of normal storage at 25 ± 2°C the re growth of encapsulated G. glabra micro shoots, reached 98% within 30 days of incubation on MS medium supplemented with 0.1 mg/l IAA. Re growth was characterized by the development of both shoot and root from single encapsulated micro shoot. Healthy plants were established to glass house with 95% survival. The genetic fidelity of plants obtained after conversion of alginate beads was ascertained through 10 RAPD and 13 ISSR primers. Of the 10 RAPD primers tested, 6 of them produced 14 clear and reproducible amplicons with an average of 2.3 bands per primer out of which 28.57% were polymorphic generated by only two primers. Eight ISSR primers produced total 37 bands ranging between 300 and 3,500 bp length. Number of scorable bands for each primer varied from 3 to 8 with an average of 4.6 bands per primer. Cluster analysis from ISSR and RAPD showed that all the tested plants including the mother plant distributed in two major groups with similarity coefficient ranging from 0.91 to 0.96 for RAPD and 0.89 to 0.97 for ISSR. [ABSTRACT FROM AUTHOR]

An efficient in vitro leaf regeneration protocol has been established in Woodfordia fruticosa L. (Lathyraceae), a threatened woody shrub. The leaf segments derived from in vitro-grown plants were cultured on Murashige and Skoog's medium supplemented with Thidiazuron (2.27, 4.54, 6.81, and 9.08 μM) and 6-Benzyladenine (4.4, 8.90, 13.30, and 17.70 μM) alone or in combination with Indole-3-acetic acid (1.14 and 2.28 μM). Maximum number of shoots (15.60 ± 1.15) with highest shoot length (2.90 ± 0.24) were regenerated directly from the leaf explants with a combination of TDZ (4.54 μM) and IAA (2.28 μM), whereas the intervening callus phase was observed in the media supplemented with TDZ or BA alone. The in vitro-regenerated shoots were rooted (100%) on half-strength MS salts fortified with 4.90 μM IBA. The regenerated plantlets hardened in the greenhouse showed an 85% survival rate. ISSR PCR of in vitro-regenerated plantlets reveals their genetic fidelity with the mother plant. [ABSTRACT FROM PUBLISHER]

Mucuna pruriens L. is an important medicinal plant reported to contain L-3,4-dihydroxy phenylalanine (L-DOPA). The present report describes a simple protocol for plant regeneration through callus morphogenesis in four strains belonging to two different varieties of Mucuna pruriens. Friable, soft and nodular callus was induced from nodal and internodal segments of in vitro-grown seedlings on modified Murashige and Skoog's (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). For regeneration of shoots, the callus tissues were transferred to medium supplemented with various levels of cytokinin. Highest shoot regeneration from the callus tissue was achieved in modified MS medium fortified with BAP at 1.33 μM level. The regenerated shoots were rooted in vitro in half-strength of liquid MS medium supplemented with various levels of NAA. The regenerates were acclimatized for 2-3 weeks and showed about 80 % survival rate after transferring to the field. Cytological analysis revealed chromosome number stability of all the regenerates (2n = 22) with complete absence of aneuploidy. Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) analyses of total genomic DNA supported stability at the molecular level among all the clones of these strains when compared with their mother plants. [ABSTRACT FROM AUTHOR]

Origanum majorana (L.) (family Lamiaceae) is a multipurpose medicinal plant with high demand across the pharmaceutical, cosmetic, food, beverage, and perfume industries that has raised concern for its mass propagation through in vitro culture techniques. The present report describes an effective in vitro propagation system for an aromatic medicinal plant Origanum majorana L. The Murashige and Skoog's (MS) medium supplemented with 1.0 mg L-1 6-benzylaminopurine (BAP) initiated a maximum of 4.4 ± 0.22 micro-shoots from the nodal segment explants. The regenerated shoots were further proliferated (98.0 ± 0.22 shoots per explant/culture vessel) on MS medium augmented with 0.5 mg L-1 BAP and 0.25 mg L-1 α-naphthalene acetic acid (NAA). Individual shootlets were rooted on a half-strength MS medium with 1.0 mg L-1 indole-3 butyric acid (IBA) (4.8 ± 0.20 roots/shoots). The rooted plantlets were transplanted into eco-friendly paper cups containing soilrite®: manure; sand (2:1:1) and maintained in the greenhouse for hardening. Inter simple sequence repeats (ISSR) molecular markers were utilized to investigate the somaclonal variations between in vitro developed plants and the mother plant of O. majorana. The band patterns of regenerated plantlets have shown genetic uniformity with the donor plant. The foliar micro-morphological features under in vitro and ex vitro conditions were compared with the mother plant and elucidated morphologically stable plantlets development through gradual acclimatization. This is the foremost report on ISSR molecular marker-based genetic fidelity studies and foliar micro-morphological evaluation of micropropagated plants of O. majorana. • Large-scale in vitro propagation method is developed for Origanum majorana. • During shoot multiplication, 98 shoots were obtained on MS medium fortified with 0.5 mg L−1 BAP and 0.25 mg L−1 NAA. • Genetic homogeneity of regenerants was confirmed by ISSR markers. • Foliar micro-morphology of in vitro propagated plants revealed the development of stable plantlets. [ABSTRACT FROM AUTHOR]

• Andrographis echioides L., is an important medicinal plant which is famously known for pharmaceutical compounds found in its leaf. • Due to the low availability of seeds and its erratic germination; the propagation and multiplication of Andrographis echioides is limited. • High frequency of shoot regeneration was carried on Murashige and Skoog media with a combination of thidiazuron and indole-3-acetic acid. • Rooting of in vitro raised shoots was induced on the combination of indole-3-acetic acid and indole-3-butyric acid. • Genetic fidelity of regenerated plantlets was confirmed by molecular markers and DNA barcoding primers. There are several studies about the phytochemical investigations and pharmacological activities of Andrographis echioides (L.), but there is scanty information about its in vitro propagation. Hence the present study is undertaken for in vitro micropropagation of Andrographis echioides (L.) through direct organogenesis. Initially, aseptic leaf explants of in vitro seedlings were excised and inoculated on to plant growth regulators (PGR's) supplemented in the Murashige and Skoog (MS) media. Among the individual and combinations of PGR's tested, the combination of thidiazuron (TDZ) along with indole-3-acetic acid (IAA) at 0.5 and 0.2 mg/L concentrations respectively, was proved to be best for high frequency (24.48 ± 1.54) of direct shoot regeneration of A. echioides. Rooting of in vitro raised shoots with the highest rooting percent of 90.42 was carried on IAA (1.0 mg/L), and indole-3-butyric acid (IBA) (1.0 mg/L) amended MS media. in vitro plantlets of A. echioides were hardened in poly cups possessing a 1:2 ratio of compost and garden soil. Initially, hardened plantlets were maintained in a greenhouse for four weeks by irrigating with half-strength MS salt solution and later transferred to field conditions with 80 % survivability. Genetically clonal nature of regenerated plantlets was confirmed by carrying polymerase chain reaction (PCR) based molecular analysis using inter simple sequence repeats (ISSR) and ribulose bisphosphate carboxylase large chain (rbcLa) primers. [ABSTRACT FROM AUTHOR]

Helicteres isora L., is an endangered valuable medicinal tree species. Direct shoot regeneration was initiated from cotyledonary node (CN) and axillary nodal explants (AN) of in vitro raised seedlings using the plant growth regulators (PGRs) and organic additives. A combination of 4.92 μM N6- (2- isopentenyl) adenine (2iP), 9.08 μM thidiazuron (TDZ) and 2.69 μM naphthaleneacetic acid (NAA) in Murashige and Skoog (MS) medium resulted with 16.1 and 11.4 shoots from CN and AN explants respectively. Maximum shoot numbers (CN: 21.3; AN: 16.9) were recorded by addition of 342.1 μM glutamine with 4.92 μM 2iP, 9.08 μM TDZ and 2.69 μM NAA combination. Basal callus associated shoot regeneration problems were successfully overcome through altering sucrose concentration (20 g l−1) in culturing media. Pre-treated microshoots were elongated (10.9 and 9.8 cm) when cultured on half-strength MS medium fortified with a combination of 4.92 μM 2iP and 1.44 μM gibberellic acid (GA 3). Elongated shoots were best rooted (12.9 per shoot), in half-strength MS medium containing 4.90 μM indole-3-butyric acid (IBA). Plantlets were successfully acclimatised (71.4%) in poly tunnel chamber. Genetic homogeneity was successfully confirmed by SCoT and ISSR marker systems. In both techniques 63 and 27 monomorphic bands were resolved from SCoT and ISSR primers respectively. Developed micropropagation protocol could be a reliable method for clonal propagation of H. isora. • N6- (2- isopentenyl) adenine (2iP) was found suitable cytokinin to enhance the direct shoot regeneration in H. isora. • Microshoots were successfully regenerated into well-developed shoots suitable for root induction. • SCoT and ISSR, the two different genetic marker systems were employed to ensure genetic fidelity of regenerants. [ABSTRACT FROM AUTHOR]

Orthosiphon stamineus has been widely used as traditional remedy for various illnesses and diseases, such as cardiovascular diseases and epileptic seizures. In this study, direct regeneration through nodal segment of this species was attempted using Kinetin (6-Furfurylaminopurine) and IAA (indole-3-acetic acid). Optimum regeneration media was identified as MS media supplemented with 2.0 mg L−1 Kin plus 0.5 mg L−1 IAA. This yielded the highest number of shoots (5.57 ± 0.42) and leaves (20.53 ± 1.91) per explant. Acclimatization of the resulting in vitro regenerants was successful in all potting mixtures tested. However, potting mixture PF (1:1:1 ratio of black soil/red soil/compost) was identified as the best medium for acclimatization of this species, as it yielded 100% survival percentage after 90 days of acclimatization. Ten in vitro regenerants of O. stamineus were randomly collected after the third subculture and subjected to genetic variation analysis using inter-simple sequence repeat (ISSR) markers. Out of 20 ISSR markers tested, 10 working primers were observed to produce satisfactory amplification of bands, with an average of 7.11 bands per primer. A total of 610 bands were produced by the 10 primers. The percentage of polymorphism was observed to be very low, yielding only 7.32% polymorphism among all samples. Jaccard dissimilarity analysis was also conducted and very low genetic distance (about 0.1) was found among the in vitro regenerants and between the regenerants with the mother plant, thus ascertaining the clonal nature of the plantlets produced in this study. [ABSTRACT FROM AUTHOR]