Encapsulation of nodal cuttings and shoot tips for storage and exchange of cassava germplasm

We report the encapsulation of in vitro-derived nodal cuttings or shoot tips of cassava in 3% calcium alginate for storage and germplasm exchange purposes. Shoot regrowth was not significantly affected by the concentration of sucrose in the alginate matrix while root formation was. In contrast, increasing the sucrose concentration in the calcium chloride polymerisation medium significantly reduced regrowth from encapsulated nodal cuttings of accession TME 60444. Supplementing the alginate matrix with increased concentrations of 6-benzylaminopurine and alpha-naphthaleneacetic acid enhanced complete plant regrowth within 2 weeks. Furthermore, plant regrowth by encapsulated nodal cuttings and shoot tips was significantly affected by the duration of the storage period as shoot recovery decreased from almost 100% to 73.3% for encapsulated nodal cuttings and 94.4% to 60% for shoot tips after 28 days of storage. The high frequency of plant regrowth from alginate-coated micropropagules coupled with high viability percentage after 28 days of storage is highly encouraging for the exchange of cassava genetic resources. Such encapsulated micropropagules could be used as an alternative to synthetic seeds derived from somatic embryos.     

Nutrient-encapsulation of potato nodal segments for germplasm exchange and distribution

Nutrient-encapsulation technique using in vitro grown nodal segments was developed as an alternative method for distribution of potato germplasm. The nodal cuttings of two potato genotypes, Kufri Jyoti and Kufri Lauvkar, were encapsulated in calcium-free Murashige and Skoog's (MS) medium containing either 2 or 3 % sodium alginate and 0, 1, 2 or 3 % saccharose. The encapsulated segments were stored in tubes with or without semisolid MS medium, and incubated in the dark at 25 ± °C for 3 to 6 weeks. Presence of saccharose in the beads was found detrimental for regrowth of new shoots. In absence of saccharose, about 82 % and 53 % encapsulated segments initiated regrowth after 3 and 6 weeks of dark storage, respectively, in tubes containing MS medium. Storage in empty tubes deteriorated the encapsulated segments, and depressed shoot formation. For potato germplasm distribution, the encapsulated segments can be transported in small tubes containing semisolid MS medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nutrient alginate encapsulation of nodal segments of Althaea officinalis L., for short-term conservation and germplasm exchange

Abstract

In the present study, an alternate method for germplasm storage in the form of artificial seeds was standardized via nodal explants excised from in vitro proliferated shoots. The explants were encapsulated using sodium alginate and calcium chloride as gelling matrix. For development of root along with shoot, excised nodal segments were pretreated with ½ MS medium along with 20 μM IBA for 24 h and encapsulation was carried thereafter. Combination of 3% sodium alginate augmented with 100 mM CaCl₂.2H₂O was found appropriate for the formation of clear and uniform beads and subsequent conversion of encapsulated nodal segments into plantlets. Maximum (66%) encapsulated nodal segments were converted into plantlets on MS medium supplemented with 7.5 μM BA and 0.5 μM NAA after eight weeks. Regeneration frequency of auxin-pretreated encapsulated and non-encapsulated nodal segments (stored at 4 ºC) was evaluated at different storage time (0 to 6 weeks). After four weeks of storage, encapsulated propagules exhibited highest conversion response on the optimized medium after eight weeks of culture. Plantlets were hardened and established with success in ex vitro conditions. Conversion of synthetic seeds into plantlets was observed when these were directly sown in autoclaved Soilrite^TM (Keltech Energies, Bangalore, India).     

Encapsulation, cold storage, and growth of Hibiscus moscheutos nodal segments

Nodal segments of Hibiscus moscheutos (hardy hibiscus) were excised from proliferating axillary shoot cultures and encapsulated in high density sodium alginate hardened by 50 mM CaCl₂. Nodal segments 4 mm long grew as well as and were easier to encapsulate than 8 mm long nodal segments. Although nodal segments grew regardless of the concentration of sodium alginate, 2.75% was determined to produce the highest quality encapsulated nodal segments beads (sufficient alginate coating and ease of use) because of the viscosity produced by the 2.75% sodium alginate solution. When encapsulated segments were stored at 5°C they did not grow in light or darkness. During the first month on fresh proliferation medium under normal incubation conditions following 5°C storage in the dark for up to 24 weeks, root number and root and shoot elongation were inhibited linearly as storage time increased. All encapsulated nodal segments survived 24 weeks of 5°C storage in two separate experiments. In fact, 80% of encapsulated hardy hibiscus nodal segments survived refrigerated storage for 1½ years (78 weeks) and after 3 months on proliferation medium, the nodal segments produced nearly the same length axillary shoots with the same number of axillary nodes per shoot as compared to encapsulated segments either not stored at 5°C or stored for 24 weeks at 5°C. Growth from encapsulated and cold-stored ‘Lord Baltimore’ nodal segments was more vigorous than from ‘Southern Belle’ nodal segments.     

Alginate-encapsulation of nodal segments for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.)

Nodal segments obtained from in vitro proliferated shoots of Eclipta alba (L.) Hassk, were encapsulated in calcium alginate beads for large-scale clonal propagation, short-term conservation and germplasm exchange and distribution. The best gel complexation was achieved using 3% sodium alginate and 100 mM CaCl₂·2H₂O. Maximum percent response (100%) for conversion of encapsulated nodal segments into plantlets was obtained on 0.7% agar-solidified full-strength MS medium containing 0.88 μM BAP. Encapsulated nodal segments could be stored at low temperature (4°C) up to 60 days with a survival frequency of 51.2%. The well-developed plantlets regenerated from encapsulated nodal segments were hardened-off successfully with 90% survival frequency.     

Antioxidant response of Cassia angustifolia Vahl. to oxidative stress caused by Mancozeb, a pyrethroid fungicide

Seeds of Cassia angustifolia Vahl., treated with various concentrations (0, 0.1, 0.15, 0.2 and 0.25 %) of Mancozeb, a broad-spectrum contact fungicide, were sown in field conditions to study the effect of the treatments on lipid peroxidation, proline accumulation and modulation of antioxidant system of seedlings obtained. Significant increase over the control was observed in treated plants for thiobarbituric acid-reactive substances content (up to 207 %), proline content (96 %) and total glutathione content (144 %), whereas the total ascorbate content decreased by 44 %. Increased enzymatic activity was recorded for ascorbate peroxidase (63 %), glutathione reductase (154 %) and superoxide dismutase (109 %), whereas catalase activity decreased by 58 % with 0.25 % Mancozeb treatment. The changes observed were dose-dependent, showing a strong correlation with the level of treatment.     

Micropropagation and validation of genetic and biochemical fidelity amongst regenerants of Cassia angustifolia Vahl employing RAPD marker and HPLC

In vitro protocol has been established for clonal propagation of Cassia angustifolia Vahl which is an important source of anticancerous bioactive compounds, sennoside A and B. Nodal explants excised from field raised elite plant (showing optimum level of sennoside A and B) of C. angustifolia when reared on Murashige and Skoog’s medium augmented with different cytokinins, viz. N⁶-benzyladenine (BA), N⁶-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn) differentiated multiple shoots in their axils. Of the three cytokinins, BA at 5 μM proved optimum for differentiating multiple shoots in 95 % cultures with an average of 9.14 shoots per explant within 8 weeks of culture. Nearly, 95 % of the excised in vitro shoots rooted on half strength MS medium supplemented with 10 μM indole-3-butyric acid (IBA). The phenotypically similar micropropagated plants were evaluated for their genetic fidelity employing random amplified polymorphic DNA (RAPD) markers. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 36 scorable bands, ranging in size from 100 to 1,000 bp were generated amongst them by the RAPD primers. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant proving their true to the type nature. Besides, high performance liquid chromatography evaluation of the sennoside A and B content amongst leaves of the mature regenerants and the elite mother plant too revealed consistency in their content.     

In vitro shoot multiplication and plantlet regeneration from nodal explants of <Emphasis Type="Italic">Cassia angustifolia </Emphasis>(Vahl.): a medicinal plant

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0 μM TDZ and 1.0 μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the isolated shoots using MS medium with 60 μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1 week and subsequently transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.     

Encapsulation technology for short-term storage and conservation of a woody climber, Decalepis hamiltonii Wight and Arn.

An efficient protocol was developed for short-term storage and conservation of a woody medicinal climber, Decalepis hamiltonii, using encapsulated nodal segments. The encapsulation of nodal segments was significantly affected by the concentrations of sodium alginate (Na-alginate) and calcium chloride (CaCl₂·2H₂O). A gelling matrix of 4?% Na-alginate and 100?mM CaCl₂·2H₂O was found most suitable for the production of ideal Ca-alginate beads. Maximum shoot re-growth (77.00?±?2.09?%) was recorded on Murashige and Skoog (MS) basal medium supplemented with 5.0???M 6-benzyladenine (BA), 0.5???M indole-3-acetic acid (IAA) and 30.0???M adenine-sulphate (ADS). Microshoots, recovered from encapsulated nodal segments (capsule) were best rooted on half-strength MS medium containing 2.5???M ??-naphthalene acetic acid (NAA). Complete plantlets (with shoot and root) were successfully acclimatized and established in field where they grew well without any detectable variation.     

Marked enhancement of sennoside bioactive compounds through precursor feeding in Cassia angustifolia Vahl and cloning of isochorismate synthase gene involved in its biosynthesis

Plant Cell, Tissue and Organ Culture (PCTOC) - Cassia angustifolia Vahl, a chief source of anthraquinone glycosides (sennosides), extensively employed as a laxative is also reported to possess...     

Temporal cline in a hybrid zone population between Fraxinus excelsior L. and Fraxinus angustifolia Vahl

The two closely related ash species Fraxinus excelsior L. (common ash) and Fraxinus angustifolia Vahl (narrow-leaved ash) have a broad contact zone in France where they hybridize. However, little is known about the local structure of hybrid zone populations and the isolation mechanisms. We assessed the potential effect of floral phenology on the structure of a riparian ash hybrid zone population in central France. The distribution of flowering times was unimodal and lay between the flowering periods of the two species. Using microsatellite markers, we detected isolation by time, which has possibly originated from assortative mating. Multivariate analyses indicated that morphological variation is not distributed at random with respect to flowering times. Spatial autocorrelation analyses showed that temporal and spatial patterns were tightly linked. Interestingly, despite the fact that the population shows isolation by time, neighbourhood size and historical dispersal variance (sigma = 63 m) are similar to those detected in pure stands of F. excelsior where individuals flower rather synchronously and hermaphrodites are not the most frequent sexual type. Trees flowering at intermediate dates, which comprised the majority of the population, produced on average more flowers and fruits. We detected no significant differences in floral parasite infections relative to reproductive timing, although there was a tendency for late flowering trees to suffer from more gall attack. We discuss the impact of temporal variation in fitness traits and their possible role in the maintenance of the hybrid zone.     

Employment of Cassia angustifolia leaf extract for zinc nanoparticles fabrication and their antibacterial and cytotoxicity

The plant Cassia angustifolia belongs to Saudi Arabia, which is one of the native places and now cultured throughout the global countries. Medical care in the Arab world is an essential outlet for medicinal plants, both because they are crucial elements for prophetic medicine and due to their lengthy background in the Middle East. C.angustifolia is one of the medicinal plants used in the Saudi Arabia. The usage of plant extracts for synthesizing nanoparticles is conducive to other biological material, since it avoids the lengthy phase of cell culture maintenance. Silver nanoparticles attract further attention due to their strong conductivity, stability and antimicrobial activity across different metal nanoparticles. The present study was designed in the Saudi C. angustifolia leaves with the zinc synthesis of nanoparticles and its antibacterial ability. The plant extracts of C. angustifolia was used for synthesis of zinc nanoparticles, antimicrobial activities against bacterial strains have been tested along with transmission electron microscope (TEM), UV spectroscopy and antimicrobial activities have been conducted. This study showed that silver ions may be transferred from the plant extract to silver nanoparticles. AgNPs biogenic capacity to antibacterial with lovo cell with IC₅₀ ranged from 33.5 ± 0.2 μg/mL demonstrated strong antibacterial capacity to antibody. The overall absorption value for the extract was between 420 and 440 nm and the color transition to green was the plasma absorption of the AgNPs. TEM results was showed in 200,000 magnification. The uniqueness of the current study is that Cassia angustifolia leaf extract from Saudi Arabia was used to prepare the metallic nanoparticles. Additionally, ZnCl₂ may also be used as nanoparticles of mineral salt and zinc, which, since their application has been confirmed, are antimicrobial.     

Somatic embryogenesis and plantlet regeneration of Cassia angustifolia from immature cotyledon-derived callus

Plant regeneration through indirect somatic embryogenesis was attempted from the immature cotyledon-derived explant of Cassia angustifolia Vahl. — a valuable leguminous shrub. The highest frequency (90.5 %) of somatic embryos was obtained on a Murashige and Skoog (MS) medium augmented with 10.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 μM benzyladenine (BA) with the production of a maximum of 22.8 embryos per explant, of which 35.3 % germinated on the same medium after 6 weeks of culture. A half strength MS medium without plant growth regulators facilitated better conversion of embryos into complete plantlets compared to a full strength MS medium. Regenerated plantlets were successfully acclimatized in sterile Soilrite and transferred to field conditions with a 70 % survival rate. Histological studies performed at different stages of embryogenesis revealed the mode of differentiation of embryos from the callus. The content of chlorophylls (a + b) and carotenoids, and the net photosynthetic rate (P_N) in the regenerated plantlets were tested during different periods of acclimatization.     

In vitro propagation of Cassia angustifolia through leaflet and cotyledon derived calli

High efficiency shoot regeneration was achieved through leaflet and cotyledon derived calli in Cassia angustifolia - an important medicinal plant. Dark brown compact callus was induced at the cut ends of the explants on Murashige and Skoog's (MS) medium augmented with 1 μM N⁶-benzyladenine (BA) + 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Such callus pieces on transfer to cytokinins (BA or kinetin) supplemented medium differentiated shoots within 10 – 15 d. Of the two cytokinins, 5 μM BA was optimum for eliciting morphogenic response in 83.33 and 70.83 % cultures with an average of 4.16 ± 0.47 and 3.70 ± 0.56 shoots in cotyledon and leaflet derived calli, respectively. The addition of 0.5 μM α-naphthaleneacetic acid (NAA) to MS + 5 μM BA further elevated the maximum average number of shoots to 12.08 ± 1.04 and 5.37 ± 0.52 for cotyledon and leaflet calli, respectively. The excised shoots were transferred to a rooting medium containing either IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA. Nearly 95 % shoots developed an average of 5.4 ± 0.41 roots on half strength MS medium supplemented with 10 μM IBA.     

Environmental Heterogeneity Explains the Genetic Structure of Continental and Mediterranean Populations of Fraxinus angustifolia Vahl

Tree species with wide distributions often exhibit different levels of genetic structuring correlated to their environment. However, understanding how environmental heterogeneity influences genetic variation is difficult because the effects of gene flow, drift and selection are confounded. We investigated the genetic variation and its ecological correlates in a wind-pollinated Mediterranean tree species, Fraxinus angustifolia Vahl, within a recognised glacial refugium in Croatia. We sampled 11 populations from environmentally divergent habitats within the Continental and Mediterranean biogeographical regions. We combined genetic data analyses based on nuclear microsatellite loci, multivariate statistics on environmental data and ecological niche modelling (ENM). We identified a geographic structure with a high genetic diversity and low differentiation in the Continental region, which contrasted with the significantly lower genetic diversity and higher population divergence in the Mediterranean region. The positive and significant correlation between environmental and genetic distances after controlling for geographic distance suggests an important influence of ecological divergence of the sites in shaping genetic variation. The ENM provided support for niche differentiation between the populations from the Continental and Mediterranean regions, suggesting that contemporary populations may represent two divergent ecotypes. Ecotype differentiation was also supported by multivariate environmental and genetic distance analyses. Our results suggest that despite extensive gene flow in continental areas, long-term stability of heterogeneous environments have likely promoted genetic divergence of ashes in this region and can explain the present-day genetic variation patterns of these ancient populations.     

Maturation of Anthurium andraeanum cv. Eidibel somatic embryos from nodal segments

Maturation of somatic embryos of Anthurium andraeanum cv. Eidibel from embryogenic callus was evaluated. Following induction of embryogenic calli from nodal segments, tissues were transferred to 125-mL Erlenmeyer flasks containing 25 mL liquid medium, with 0, 4.52, or 9.05 μM 2,4-dichlorophenoxyacetic acid and 0, 0.47, or 2.32 μM kinetin. Callus cultures were maintained in a dark growth room at 25?±?2°C. At 45 d, the mass of embryogenic calli, number of primary and secondary somatic embryos, and percentage browning were evaluated. Nonparametric tests were used to evaluate color, texture, and somatic embryo development. The highest yield of somatic embryos was in the medium with 0.47 μM kinetin. Calli were friable, with a lower yield of secondary somatic embryos, and have minimal browning. Histology revealed polar globular somatic embryos and mature somatic embryos with defined apical and root meristematic zones, axillary buds, and primary leaves. These are important features for converting somatic embryos into plantlets.     

Cryopreservation and long-term storage of pear germplasm

Summary Germplasm collections of vegetatively propagated crops are usually maintained as plants in fields or potted in greenhouses or screened enclosures. Safety duplication of these collections, as duplicate plants or separate collections, is costly and requires large amounts of space. Cryopreservation techniques which were recently developed for long-term storage of pear germalasm may offer an efficient alternative to conventional germplasm collection maintenance. Pear (Pyrus L.) germplasm may now be stored as seeds (species), dormant buds or pollen from field-grown trees, or shoot tips fromin vitro-grown plants (cultivars). Pear germplasm may now be cryopreserved and stored for long periods (> 100 yr) utilizing slow-freezing or vitrification ofin vitro-grown shoot-tips. Dormant bud freezing, pollen, and seed cryopreservation of other lines are being developed to complete the base collection forPyrus. This cryopreserved collection provides base (long-term) storage for the field-grown pear germplasm collection at the National Clonal Germplasm Repository, Corvallis, Oregon. Based on a presentation at the 1997 Congress on In Vitro Biology held in Washington, D.C., June 14–18, 1997.