Mature Nodal Research Articles

Development of an efficient micropropagation system for Dioscorea bulbifera L. and phytochemical profile of regenerated plants

BackgroundThe present study was designed to develop an improved in vitro regeneration system for Dioscorea bulbifera using mature nodal explants. Direct organogenesis from nodal segments was achieved by culturing the nodal explants on Murashige and Skoog medium supplemented with 3.5 mgl−1 6-benzylaminopurine (BAP). Shoot multiplication was widely affected by the kind and concentration of plant growth regulators, and the sub-culturing of in vitro regenerated shootlets on fresh medium. After incubation for 4 weeks at optimum BAP concentration, cultures were transferred to secondary medium with BAP (optimized concentration) supplemented with different auxins. ResultsOn medium with 3.5 mgl−1 6-benzylaminopurine, maximum regeneration (87 ± 1.85%) with an average shoot number of 2.0 ± 0.08, and length of 3.5 ± 0.04 cm were observed after 4 weeks of incubation. Maximum number of shoots (3.9 ± 0.21) was observed with 3.5 mgl−1 BAP in combination with 0.75 mgl−1 indole-3-acetic acid. The best root formation was observed on ½ MS medium supplemented with 0.75 mgl−1 α-naphthalene acetic acid, with 4.7 ± 0.03 roots per shoot. The well-rooted plantlets were successfully acclimatized in with 100% survival rate. The plantlets grew well with normal growth, flowering and showed, by High performance thin layer chromatography, almost same number of phytoconstituents compared with the mother plant. ConclusionsThis is the first study on comparative phytochemical analysis of wild growing and in vitro regenerated plants of D. bulbifera which validates the medicinal and nutritional properties of in vitro raised plants.

B-cell lymphoma-2 downregulation is a useful feature supporting a neoplastic phenotype in mature T-cell lymphomas

Normal T cells express high levels of B-cell lymphoma-2 (BCL2) protein, and data regarding BCL2 expression status and its diagnostic utility in T-cell lymphoma are scarce. We evaluated BCL2 expression in a series of mature T-cell lymphoproliferations (TCLs) including indolent and more recently recognized entities (follicular helper T-cell [TFH] lymphomas). Sixty-six neoplastic biopsies (60 patients) representing mature nodal, extranodal, and leukemia T-cell neoplasms were collected from three institutes (2 US and 1 Japan) and were compared with reactive T cells in 8 benign tissues/blood and 9T cell-rich B-cell proliferations. BCL2 immunostaining was performed and scored based on intensity-weighted H-score (0-300). Next-generation sequencing (NGS; 5 cases), BCL2 gene sequencing, and real-time polymerase chain reaction (PCR; 3 cases) were conducted. Association of H-score with overall survival (using proportional hazards modeling) was assessed in nonleukemic TCLs. Most TCLs showed significantly downregulated median BCL2 H-score (125, range: 18-300) with the exception of T-cell prolymphocytic leukemia and hepatosplenic T-cell lymphoma, both of which showed uniform strong retention of BCL2 as did the 8 reactive tissues (median H-score: 280; p=0.000). Notably all TFH lymphoma CD4 neoplastic T cells, subcutaneous panniculitis-like T-cell lymphoma, CD8 adipocyte-rimming T cells, and T-cell large lymphocyte leukemia with pathogenic STAT5B and TP53 mutation showed BCL2 downregulation. No BCL2 mutations were observed by NGS or sequencing with decreased BCL2 mRNA transcripts by real-time PCR. BCL2 downregulation is pervasive among many TCLs and unrelated to any mutations. There is utility for BCL2 immunostaining in some challenging situations as discussed in this article.

Synergetic effects of TDZ with various phytohormones on high-frequency plant regeneration from mature nodal explants of Capparis decidua and their ex vivo implications

Synergetic effects of TDZ with various phytohormones on high-frequency plant regeneration from mature nodal explants of Capparis decidua and their ex vivo implications

Alteration of media enables efficient in vitro cloning of mature Elaeocarpus serratus L. (Ceylon olive): a commercially important fruit tree.

Elaeocarpus serratus is a fruit tree able to propagate through conventional vegetative means to a limited extent restricts its wide cultivation by the farmers. In the present report, we have developed an efficient in vitro propagation protocol using mature nodal explants from a 17-year-old tree for the first time with 6.6 shoots/culture. Explants cultured on agar (0.8%) gelled standard Murashige and Skoog (MS) medium, ½ MS, ¾ MS, White's, Gamborg's B5 or woody plant medium (WPM) supplemented with 2.5µM benzyl adenine (BA) and 0.1µM α-naphthalene acetic acid (NAA) showed the superiority of ½ MS medium in terms of explant response and number shoots (6.6). Further optimization of ½ MS medium by altering nutrient elements (macros, micros, vitamins and Fe EDTA) were undertaken, and MS medium composed of half-strength major salts, original strength of minor salts and vitamins were supplemented with BA (2.5µM) and NAA (0.1µM), produced enhanced axillary bud proliferation (8.88/explant) and shoot elongation (3.83cm). Reculturing of original explant on this medium after IV passages produced more than 16 healthy shoots per culture which attained a length of 4.13cm. Microshoots raised through this way were rooted (86.11%) ex vitro by pulse treatment with 2mM indole-3-butyric acid (IBA) for 5min followed by planting in nursery pots containing a 1:1:1 (v/v/v) mix of sand, soil, and farmyard manure. The hardened plants were successfully planted in the fruit tree garden of the Department. Genetic fidelity of micropropagated and mother plants were tested using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers which showed a high degree of monomorphism thus supported morphological uniformity of micropropagated plants.

Development of an efficient micropropagation system for Tecoma stans (L.) Juss. ex Kunth using thidiazuron and effects on phytochemical constitution

The present study was designed to investigate the stimulatory effects of different doses (0.1 to 2.5 μM) of thidiazuron (TDZ) on in vitro shoot induction and proliferation of mature nodal explants of Tecoma stans. Of the tested concentrations, 2.0 μM TDZ proved to be optimal for maximum regeneration (91%) with a mean shoot number of 5.6 ± 0.67, and length of 2.38 ± 0.08 cm, after 4 wk of incubation. To determine the negative effects of prolonged TDZ exposure, after 4 wk of incubation at optimized level of TDZ, the cultures were transferred to a secondary medium either lacking plant growth regulators or supplemented with benzyladenine (BA) alone, or in combination with different auxins (indole-3-acetic acid, indole-3-butyric acid, or α-naphthalene acetic acid; NAA). Among the tested concentrations, 2.5 μM BA in combination with 0.5 μM NAA yielded the maximum mean shoot number (16.60 ± 0.40), and average shoot length (4.76 ± 0.15 cm) after 4 wk of culture. The best rhizogenesis (93%) was achieved on ½ MS medium containing 1.5 μM NAA, with a mean root number of 7.60 ± 0.40 and length of 4.11 ± 0.23 cm, after 4 wk of incubation. The micropropagated plantlets were successfully acclimatized and hardened off in Soilrite™ with a 90% survival rate. The plantlets grew well with normal growth, flowering and showed, by gas chromatography–mass spectroscopy, an increase in the number of bioactive compounds compared with the donor plant. This is the first report on T. stans in vitro regeneration using TDZ.

Influence of meta-topolin on in vitro organogenesis in Tecoma stans L., assessment of genetic fidelity and phytochemical profiling of wild and regenerated plants

Comparative effect of meta-topolin and other cytokinins was assessed to develop an efficient and reliable regeneration protocol for Tecoma stans, using mature nodal explants. The morphogenic effect of benzyl adenine (BA), kinetin (Kin), meta- topolin (mT) and 2-iP (2-iso pentenyl adenine) at various concentrations (1.0–10 µM) was studied individually or in combination with auxins (IAA, IBA or NAA). Superior multiplication rates were achieved on MS medium supplemented with mT and NAA. Of the tested combinations, maximum shoot regeneration (95%), mean shoot number (19.6 ± 0.60) and length (5.26 ± 0.73 cm) was recorded on MS medium supplemented with 7.5 µM mT + 0.5 µM NAA after 8 weeks of incubation. Among the different auxins employed for in vitro root induction, 92.5% microshoots rooted on MS medium enriched with 1.0 µM IBA with 10.8 ± 0.20 mean root number and 5.62 ± 0.17 cm length after 4 weeks of incubation. The acclimatized plants grew well in green house with 90% survival rate. The gas chromatography–mass spectrometry (GC–MS) analysis of ethanol leaf extract of in vitro-raised plants yielded a higher number of compounds than control plant. The assessment of genetic fidelity among regenerants, using ISSR markers did not reveal any somaclonal variation. Therefore, the protocol developed appears to be simple and reliable for mass production of clones with higher diversity of secondary metabolites. Our studies based on interaction of mT with auxins for better shoot proliferation will undoubtedly be helpful in understanding many developmental processes in plant tissue culture. The regenerated plantlets survived well under field conditions and no somaclonal variation was detected. This study proved that in vitro raised plants are superior for the production of higher diversity of secondary metabolites than in vivo plant. Thus, our findings are helpful in generating an efficient micropropagation system for mass scale production of plants with higher diversity of bio-active molecules without losing their genetic stability.

Ex vitro rescue, physiochemical evaluation, secondary metabolite production and assessment of genetic stability using DNA based molecular markers in regenerated plants of Decalepis salicifolia (Bedd. ex Hook.f.) Venter

To ensure replenishment, a refine protocol for micropropagation of Decalepis salicifolia (Bedd. ex Hook.f.) Venter a critically endangered and endemic medicinal plant was developed using mature nodal explants. A high frequency shoot regeneration system was obtained on Murashige and Skoog (MS) medium comprised of 6- benzyladenine (BA) (5.0 µM) + α-naphthalene acetic acid (NAA) (0.5 µM) + adenine sulphate (ADS) (30.0 µM) corresponds to a highest mean number of 9.97 ± 0.01 shoots/explants with maximum shoot length of 6.46 ± 0.1 cm. Successful rooting in microshoots was achieved on half strength MS medium supplemented with indole-3-butyric acid (IBA) (2.5 µM). A maximum of 6.10 ± 0.07 roots/microshoot with average root length of 2.30 ± 0.06 cm was obtained. As much as 90% plantlets survived when Soilrite™ was used as planting substrate and finally established in soil without any casualty and morphological variation. Acclimatized plantlets were screened for pigment content, net photosynthetic rate (PN), stomatal conductance (Gs) and transpiration rate (E) during subsequent days of acclimatization as well as the changes in antioxidant was also evaluated. A steady rise was observed in the activity of superoxide dismutase (SOD) for initial 21 days and then after a decrease was found showing improved acclimatization efficiency of the plant. Similarly, the activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) enzyme shows reliable increase as the days of acclimatization advanced which play their precautionary role against oxidative damage to the plant. The genetic fidelity of the in vitro raised plantlets with that of mother plant was further confirmed using random amplified polymorphic DNA (RAPD) and inters simple sequence repeats (ISSR) analysis. Additionally, the effect of acclimatization on the biosynthesis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) in the root system was also evaluated in relation to their biomass production. Maximum fresh weight (4.9 g/plant), dry weight (0.65 g/plant) of roots and 2H4MB content (6.8 µg ml−1 of root extract) was obtained after 10 weeks of acclimatization. Accelerated multiplication rate with the stability of genetic virtue, physiological and biochemical parameter assure the efficacy of the protocol developed for the propagation of this critically endangered medicinal plant.

Micropropagation of Caralluma adscendens var. fimbriata-An Indigenous Medicinal Plant of India

Present study describes micro-propagation of traditionally important medicinal plant in northern interior part of Karnataka, Caralluma adscendens var. fimbriata, for its conservation. Results showed that the survival ability of regenerated plants was 84 per cent. Proliferation of C. adscendens var. fimbriata shoots was achieved on medium supplemented with various concentrations of different hormones. The effective combination of 6-Benzylaminopurine (BA) 2.0 mg/l along with 0.5 mg/l kinetin (Kn) and 0.2 mg/l Indole-3-acetic acid (IBA) produced highest number of shoot sprouting frequency (91%) with a mean of 2.60 ± 0.17 shoot number per explants and a mean shoot length of 3.88 ± 0.20 cm from the mature nodal explants. A good frequency of callus was induced in 1.5 to 2.0 mg/l of 1-Naphthaleneacetic acid (NAA) and in 2.0 mg/l of 2,4-dichlorophenoxy acetic acid (2, 4-D) were transferred to root induction media. The NAA showed positive response of rooting when compared with IAA and IBA in the present study. The best root and shoot sprouted plants were successfully acclimatized in the greenhouse and transferred to open field conditions. This micropropagation protocol can be successfully used for large-scale multiplication and conservation of germplasm of this threatened plant.

In vitro plant regeneration, flowering and fruiting from nodal explants of Andrographis lineata nees (Acanthaceae)

In the present study, in vitro propagation of tribal endemic medicinal plant, Andrographis lineata has been established using mature nodal explants. High frequency of regeneration (91.4%) was achieved on MS medium containing BA (3.0 mg L–1) along with IAA (0.2 mg L–1). Strikingly, irrespective of the season and collection period, we observed axillary flower induction and fruit formation from the above in vitro cultures supplemented with BA and NAA. Transition from the vegetative to the reproductive phase occurred within 2 months of culture, and importantly was influenced by factors such as sucrose and cytokinins. In vitro-regenerated flowers were morphologically identical to in vivo flowers. Furthermore, our scanning electron microscopic studies revealed that pollen external morphology of both in vitro and in vivo flowers were similar. The flowers self-fertilized and produced fruits in vitro. Elongated shoots rooted well on half-strength MS basal medium, and were successfully acclimatized to the garden conditions. Altogether, our established protocol can be utilized in plant breeding for the purpose of ex situ conservation, quick flowering, and fruit set.

In vitro clonal propagation and evaluation of genetic fidelity using RAPD and ISSR marker in micropropagated plants of Cassia alata L.: a potential medicinal plant

Present study reports successful in vitro clonal propagation of a potential medicinal plant, Cassia alata using mature nodal explants. Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5, 10.0 and 12.5 μM) of 6-benzyladenine (BA), kinetin and 2-isopentenyl adenine (2-iP) singly as well as in combination with different auxins, α-naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA) or Indole-3-acetic acid (IAA) (0.1, 0.5 and 1.0 μM) were used. MS medium enriched with 7.5 μM BA and 0.5 μM NAA yielded the highest regeneration frequency (92 %) with maximum multiple shoots (12.3 ± 0.6) and shoot length (4.7 ± 0.1 cm) after 12 weeks of culture. Shoots were rooted best on full MS containing 0.5 μM IBA. Ex vitro rooting of in vitro derived microshoots was also achieved in 150 μM IBA treatment for 20 min followed by transfer to thermocol cups containing sterile Soilrite™. About 85 % plantlets survived acclimatization procedure to the field. The genetic fidelity of in vitro regenerated plants was analyzed using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. Of the 20 RAPD primers, 18 primers produced clear, reproducible and scorable bands while out of 13 ISSR primers screened, only ten generated well-defined and scorable bands in all the tested plants. A total of 69 and 71 bands were scored with an average of 3.8 and 7.1 bands per primer for RAPD and ISSR primers respectively. All banding profiles from micropropagated plants were monomorphic and similar to of the mother plant, thus confirming the true-to-type nature of the in vitro-raised clones.

Assessment of Genetic Fidelity Using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats Markers of Lawsonia inermis L. Plants Regenerated by Axillary Shoot Proliferation

An efficient, reliable and reproducible plant regeneration protocol was developed for Lawsonia inermis L. using mature nodal explants. Shoot proliferation (81.6 %) with 7.8 shoots/explant was achieved on Murashige and Skoog’s (MS) medium supplemented with 1.0 mg l−1 6-benzyladenine. Shoot numbers were up-scaled by inducing multiple shoots from axenic nodal segments derived from the primary shoots on the shoot regeneration medium. Thus the authors could achieve ca. 129–134 shoots from single nodal explant. Ninety-three percent rooting of in vitro regenerated shoots was achieved on growth regulator free half-strength MS medium. Regenerated plantlets were successfully transferred to soil with 85 % survival rate. Genetic stability analyses of the in vitro regenerated plants using random amplified polymorphic DNA and inter simple sequence repeat markers revealed a homogeneous amplification profile for all micropropagated plants. This is the first report that evaluates the use of molecular markers to establish genetic fidelity of micropropagated L. inermis for the rapid clonal multiplication and true-to-type production of plant for attaining the ever increasing demand in pharmaceutical industries.

In vitro adventitious organogenesis and histological characterization from mature nodal explants of Citrus limon

An efficient and robust system of shoot organogenesis for lemon has been developed as an initial step in the transformation and in vitro mutagenesis protocols. In vitro adventitious organogenesis was studied and histologically characterized using mature nodal explants of two important cultivars of Citrus limon, ‘Verna 51’ and ‘Fino 49’. The effects of several plant growth regulator concentrations, incubation conditions, basal medium, explant type and position along the shoot, and ethylene inhibitors were determined. The presence of 1–3 mg L−1 N 6-benzyladenine, in combination with 1 mg L−1 gibberellic acid, was essential for the development of adventitious buds, with the optimum concentration depending on the cultivar, while organogenesis was lowest when N 6-benzyladenine was used in the medium without gibberellic acid. When the effect of different basal media was studied, the best results for percentage of responsive explants were obtained with Murashige and Skoog medium for both cultivars, while the number of buds per regenerating explant was less than 2 in Woody Plant medium. Attempts were made to identify the type of explant that had the highest regeneration ability using mature shoots of C. limon. When different types of explants (nodal segments with the buds completely removed or internodal segments) were compared, the highest percentage of responsive explants was obtained with nodal segments, while with internodal segments, the regeneration percentage was very poor. Histological analysis showed that nodal segments were more reactive than internodal ones and those structural changes occurred sooner in nodal segments. The existence of a morphogenetic gradient along the shoot was also observed and nodal segments from the apical region exhibited the highest regeneration ability. An initial culture period of 2–4 wk in the dark, depending on the cultivar, clearly improved the regeneration percentage and the number of buds for lemon adult explants. Silver thiosulphate increased the regeneration percentage in adult explants of both cultivars, and the best results were obtained with 20 μM silver thiosulphate.

Micropropagation of Semecarpus anacardium L. from mature tree-derived nodal explants

A protocol was established for micropropagation of Semecarpus anacardium L. from mature tree-derived twigs. Sixty percent of aseptic cultures were obtained by surface sterilization with Bavistin, liquid detergent, and cefotaxime. Elongated twigs collected before flowering were optimum for in vitro culture initiation. Meristematic activity was triggered at all concentrations of thidiazuron (TDZ) incorporated into Woody Plant Medium. TDZ suppressed elongation of axillary buds, resulting into swollen meristems and upon its elimination multiple shoot primordia formation and differentiation were noted. Differentiation and shoot elongation were slower in explants pre-cultured with higher concentrations of TDZ. Swollen axillary meristems pre-cultured on TDZ (9.08 and 13.62 μM) failed to differentiate, whereas TDZ at 2.27 μM was optimal for shoot differentiation and elongation. Multiple bud induction was favored by 4.45 μM of TDZ. Differentiation of multiple shoot primordia by repeated subculturing on growth regulator-free medium and rooting was 100% in filter-paper supported half-strength liquid medium containing 7.38 μM IBA. Rooting was 90% in shoots placed directly in half-strength liquid medium with 2.46 μM IBA. Rooted plantlets hardened in soil:sand mixture (1:1) were transferred to green house. Genetic uniformity of in vitro raised clones with mother plant was confirmed by Inter-Simple Sequence Repeat markers.

Clinical, Pathological, and Prognostic Characteristics Of Mature Nodal Or Extranodal T-Cell and NK-Cell Non-Hodgkin´s Lymphoma (NHL) In a Single Mexican Institution

BackgroundMature nodal or extranodal T-cell and NK-cell NHL are a rare and heterogeneous group of NHL with aggressive behavior and poor clinical outcome. Their incidence varies according to geographical region and racial characteristics. Mexico is included in those countries known to have a high incidence of extranodal T/NK-cell lymphoma, type nasal (NKTCL). ObjectiveTo evaluate the outcome and prognosis of patients with mature nodal or extranodal T-cell or NK-cell NHL in a single institution in Mexico City. MethodsClinicopathological characteristic, treatment, outcome, and prognosis of patients admitted to our institution between August, 1991 and December 2009 were analyzed. Prognostic Index T-cell (PIT) was used in all subtypes of lymphomas except in NKTCL subtype. All tissue biopsies and immunophenotypic markers were reviewed by an expert hematopathologist and reclassified according to the WHO 2008 classification. Univariate analysis using log-rank test was used to determine the correlation between clinical features and overall survival (OS). Multivariate analysis using Cox proportional hazard models were performed. A p value < 0.05 was considered significant. ResultsA total of 67 patients were analyzed. Median age was 37 years. B symptoms were presented in 83.6%, 74.6% had at least one site with extranodal disease, 73.1% advanced clinic stage, 32.8% high risk by International Prognostic Index (IPI) and 47.5% high risk by PIT. According to WHO 2008 classification the most common subtype was peripheral T-cell lymphoma not otherwise (PTCL NOS) specified in 38 patients (56.7 %), angioimmunoblastic T-cell lymphoma (AITL) and NKTCL were the second most common subtypes with 8 cases in each group (11.9 %), anaplastic large cell lymphoma (ALCL) kinase-positive (ALK-positive) was identified in 3 patients (4.5 %), ALCL ALK-negative in 2 cases (3.0 %), lymphoblastic lymphoma and subcutaneous panniculitis-like T-cell lymphoma (SPTCL) with 3 patients in each group (4.5 %), hepatosplenic T-cell lymphoma (HSTL) and aggressive NK-cell leukemia with one case in each group (1.5 %). CHOP-like therapy was used in 71.6 % of patients. Nine percent of patients did not receive treatment. The response was evaluated in 53 patients in whom overall response was 71.7 % with 44.8 % achieving complete remission (CR). Median OS was 2760 days (CI 95 % 1153.145-4366.855). Histopathology subtype did not predict OS. Both prognostic scores, IPI and PIT, were able to identify 4 groups of patients with different outcomes. The analysis failed to demonstrate any advantage of adding etoposide to the chemotherapy schedule. Multivariate analysis showed that, IPI, PIT, and CR were predictive for OS (Table 1). ConclusionPrevious publications in Mexican population, with larger number of patients included, were particularly focus on clinical characteristics and prognosis of NKTCL. Our series provides data of mature nodal or extranodal T-cell and NK-cell NHL in Mexico. The current study confirms the poor prognosis of aggressive forms of mature nodal or extranodal T-cell and NK-cell NHL regardless of the chemotherapy schedule employed. Disclosures:No relevant conflicts of interest to declare.

Evaluation of clonal integrity in desert date tree (Balanites aegyptiaca Del.) by inter-simple sequence repeat marker assay

Axillary shoot bud multiplication as a safest mode of micropropagation to obtain clonal progeny was revealed through the application of molecular marker technique in Balanites aegyptiaca. Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic constancy of micropropagated plantlets chosen from a clonal collection of shoots that originated from mature nodal explants (mother plant). Out of 20 ISSR primers screened, ten primers yielded reliable and reproducible patterns of amplified products in all the tested plants. In this study, on an average, 11.7 bands were amplified per primer. A total of 117 bands were scored for the tissue culture-raised plantlets; 115 amplification products were monomorphic and 2 bands were polymorphic. Based on the ISSR band data, 98.2 % genetic uniformity was detected among the regenerants. Thus, the amplification products validated that the plantlets were true-to-type in morphological or growth characteristics when compared with the mother plant.

Lipid peroxidation, H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; content, and antioxidants during acclimatization of Abrus precatorius to ex vitro conditions

An efficient, rapid, and reproducible plant regeneration protocol was successfully developed for Abrus precatorius L. using mature nodal explants excised from a 5-year-old field grown plant. The highest shoot regeneration frequency (87 %) with maximum number of multiple shoots (15.0) and shoot length (4.8 cm) were recorded on Murashige and Skoog (MS) medium amended with 2.5 μM thidiazuron, 120 mg dm−3 polyvinylpyrrolidone, and 0.5 μM α-naphthalene acetic acid. The best treatment for maximum root (4.0) induction was half strength MS medium supplemented with 1.5 μM indole-3-butyric acid. The in vitro plantlets with well-developed shoots and roots were successfully transferred into plastic cups with Soilrite and acclimatized in a culture room under photon flux density (PFD) of 150 μmol m−2 s−1, thereafter transferred to a greenhouse with PFD of 300 μmol m−2 s−1, and finally to a field with 70 % survival rate. During the acclimatization period (0–49 d), leaf chlorophyll and carotenoid content increased whereas malondialdehyde and H2O2 content decreased probably due to increasing activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase). Our work suggests that micropropagated plants developed an antioxidant enzymatic protective system to avoid oxidative stress during establishment under ex vitro environment.

High efficiency in vitro organogenesis from mature tissue explants of Citrus macrophylla and C. aurantium

A simple and efficient protocol for obtaining organogenesis from mature nodal explants of Citrus macrophylla (alemow) and Citrus aurantium (sour orange) has been developed by optimizing the concentrations of the plant growth regulators, the incubation conditions, the basal medium and by the choice of explant. In order to optimize the plant growth regulator balance, explants were cultured in the regeneration medium supplemented with several N6-benzyladenine (BA) concentrations or with 2 mg l−1 BA in combination with kinetin (KIN) or 1-naphthaleneacetic acid (NAA). The presence of BA was found to be essential for the development of adventitious buds; the best results were obtained using BA at 3 and 2 mg l−1 for alemow and sour orange, respectively. The combination of BA with KIN or NAA in the culture medium decreased the regeneration frequency, with respect to the use of BA alone. The effect of three different basal media was rootstock-dependent. For C. macrophylla the best results were obtained with Woody Plant Medium or Driver and Kuniyuki Walnut Medium (DKW). However, for C. aurantium, although high percentages of regenerating explants were obtained independently of the basal medium used, the highest number of buds per regenerating explants was obtained with DKW medium. Attempts were made to identify the type of explants which had a higher regeneration ability using particular regions along the mature shoots of C. macrophylla. When nodal segments, where the buds were completely removed, and internode segments were compared, the highest percentage of responsive explants was obtained with nodal segments. The existence of a morphogenetic gradient along the shoot was observed and the organogenic efficiency was highest when explants from the apical zone were used. Incubation in darkness for 3 or 4 wk was essential for regeneration process in both rootstocks.

In vitro clonal propagation of Gardenia latifolia Ait.: a toy making woody tree

An efficient, in vitro clonal propagation protocol has been established for Gardenia latifolia Ait. using mature nodal explants on Murashige and Skoog (MS) medium fortified with cytokinins (BA/Kn/2-iP) (1.0–5.0 mg l−1) in combination with auxin IAA (0.5 mg l−1). Maximum bud break (87 %) with shoot number (7.2 ± 0.26) observed on MS medium supplemented with BA (4.0 mg l−1) and IAA (0.5 mg l−1). Maximum number of shoots (30 ± 0.46) with shoot length of (0.9 ± 0.03 cm) observed on MS medium supplemented with BA (2.0 mg l−1), Kn (2.0 mg l−1) and IAA (0.5 mg l−1). Further elongation of shoots (3.5 ± 0.06 cm) was achieved on MS medium supplemented with BA (1.0 mg l−1) and IAA (0.1 mg l−1). About 70 % of root induction occurred in half-strength MS medium supplemented with IBA (4.0 mg l−1) in 4–6 weeks. Further elongation of roots with average length (9.0 cm) was achieved in culture bottles containing vermiculite and ¼ strength MS salts. After their partial hardening in these bottles for 30 days they were transferred to pots containing a mixture of soil and vermicompost (1:1) for acclimatization. The acclimatized plantlets were established in the field successfully with 85 % survival rate.

Quantification of Psoralen in Plant Parts of Psoralea Corylifolia growing in vivo and in vitro and Enhancement of Psoralen by Organic Elicitors

Psoralen were evaluated from in vivo and in vitro plant parts of Psoralea corylifolia by HighPerformance Liquid Chromatography (HPLC). The high amount of psoralen (3038 μg/g fresh weight) in invivo condition were obtained in brown seeds. Psoralen content in plant parts growing in vitro were lower thanthose growing in vivo. Quantitative analysis on psoralen in callus derived from different plant parts showedthat maximum psoralen (2601.8 μg/g fw) was recorded in callus derived from cotyledons. Estimation of psoralenin shoots derived from different callus were indicated high amount of psoralen (572.3 μg/g fw) in plant drivatedfrom node callus. Identification of psoralen in the callus of Psoralea corylifolia is reported for first time. In theother section attempts were done to enhance of psoralen by some organic elicitors including; yeast extract,proline, myzo-inositol and sucrose.in different medium. For the first time a comparative study of mature nodal,juvenile nodal explants and cotyledonary callus cultures were evaluated. These explants elicitated with differentorganic elicitor. Results revealed that maximum quantity of psoralen were present in cotyledonary callus cultures,mature and juvenile nodal cultures, respectively. Also, results indicated that there were significant variations inthe psoralen content in mature nodal, juvenile nodal and cotyledonary callus cultures, when their mediumsupplemented with different elicitors in the wide range of concentrations. Interestingly, high amount of psoralencontent (2881.9 μg/g fw.) even among all of the organic elicitors tried was found at 300 mg/l yeast extract incotyledonary callus cultures.

Direct shoot regeneration from nodal explants of Sida cordifolia Linn

A method was developed to initiate multiple shoots from mature nodal explants of Sida cordifolia Linn. High frequency of regeneration was achieved on Murashige and Skoog (MS) medium supplemented with 2.0 mg l−1 6-benzylaminopurine, 0.5 mg l−1 α-naphthalene acidic acid, 1.0 mg l−1 adenine sulfate, and 10% (v/v) coconut milk. Multiple shoots were initiated within 21 d and the above media was capable of inducing the formation of more than 20 shoots from each explant. Regenerated shoots were successfully rooted on half-strength MS medium supplemented with 2.0 mg l−1 indole-3-butyric acid and 3% (w/v) sucrose. Rooted plantlets were established in soil. The regenerated plantlets showed no morphological differences from the parent material. This protocol could be useful for germplasm conservation, cultivation, and genetic improvement of S. cordifolia.