In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP?+?1.4 µM GA₃ enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.

     

Camptothecin from <Emphasis Type="Italic">Nothapodytes nimmoniana</Emphasis>: review on biotechnology applications

Since the first report on Camptothecin detection in Nothapodytes nimmoniana by Govindachari and Viswanathan (Phytochem 11:35–29, 1972), considerable work has been done on biotechnology and its applications on the species. Plant tissue culture techniques have applications in clonal propagation, CPT production, and conservation of N. nimmoniana. Discovery of CPT production by endophytes existing in symbiotic association with N. nimmoniana has provided new insights into finding alternative sources of the alkaloid. Development of molecular markers such as RFLP, RAPD, ISSR, and AFLP has facilitated understanding of population ecology and genetics of the species. Molecular information generated from these studies is promising in establishing strategies for conservation and sustainable use of N. nimmoniana populations under overexploitation pressure. The advances in instrumentation in the 20th century, such as desorption electrospray ionization mass spectrometry allowed CPT analysis in tissues without sample pretreatment. Other ancient techniques for qualitative and quantitative analysis such as chromatography, spectroscopy, and H1-NMR are applied in the detection of CPT due to variable sensitivity to the alkaloid. The review covers work on plant tissue culture for clonal propagation and CPT production in N. nimmoniana. Besides symbiotic endophyte sources of CPT in N. nimmoniana, population genetics studies and instrumentation analysis of the alkaloids are reviewed.     

Antigen-independent induction of Tim-3 expression on human T cells by the common γ-chain cytokines IL-2, IL-7, IL-15, and IL-21 is associated with proliferation and is dependent on the phosphoinositide 3-kinase pathway

T cell Ig mucin domain-containing molecule 3 (Tim-3) is a glycoprotein found on the surface of a subset of CD8(+) and Th1 CD4(+) T cells. Elevated expression of Tim-3 on virus-specific T cells during chronic viral infections, such as HIV-1, hepatitis B virus, and hepatitis C virus, positively correlates with viral load. Tim-3(+) cytotoxic T cells are dysfunctional and are unable to secrete effector cytokines, such as IFN-γ and TNF-α. In this study, we examined potential inducers of Tim-3 on primary human T cells. Direct HIV-1 infection of CD4(+) T cells, or LPS, found to be elevated in HIV-1 infection, did not induce Tim-3 on T cells. Tim-3 was induced by the common γ-chain (γc) cytokines IL-2, IL-7, IL-15, and IL-21 but not IL-4, in an Ag-independent manner and was upregulated on primary T cells in response to TCR/CD28 costimulation, as well as γc cytokine stimulation with successive divisions. γc cytokine-induced Tim-3 was found on naive, effector, and memory subsets of T cells. Tim-3(+) primary T cells were more prone to apoptosis, particularly upon treatment with galectin-9, a Tim-3 ligand, after cytokine withdrawal. The upregulation of Tim-3 could be blocked by the addition of a PI3K inhibitor, LY 294002. Thus, Tim-3 can be induced via TCR/CD28 costimulation and/or γc cytokines, likely through the PI3K pathway.     

HIV delays IFN-α production from human plasmacytoid dendritic cells and is associated with SYK phosphorylation

Plasmacytoid dendritic cells (pDC) are the major producers of type I interferons (IFNs) in humans and rapidly produce IFN-α in response to virus exposure. Although HIV infection is associated with pDC activation, it is unclear why the innate immune response is unable to effectively control viral replication. We systematically compared the effect of HIV, Influenza, Sendai, and HSV-2 at similar target cell multiplicity of infection (M.O.I.) on human pDC function. We found that Influenza, Sendai, HSV-2 and imiquimod are able to rapidly induce IFN-α production within 4 hours to maximal levels, whereas HIV had a delayed induction that was maximal only after 24 hours. In addition, maximal IFN-α induction by HIV was at least 10 fold less than that of the other viruses in the panel. HIV also induced less TNF-α and MIP-1β but similar levels of IP-10 compared to other viruses, which was also mirrored by delayed upregulation of pDC activation markers CD83 and CD86. BDCA-2 has been identified as an inhibitory receptor on pDC, signaling through a pathway that involves SYK phosphorylation. We find that compared to Influenza, HIV induces the activation of the SYK pathway. Thus, HIV delays pDC IFN-α production and pDC activation via SYK phosphorylation, allowing establishment of viral populations.     

Cultural conditions affect somatic embryogenesis in Catharanthus roseus L. (G.) Don

We established an efficient plant regeneration system for Catharanthus roseus L. (G.) Don through somatic embryogenesis. Embryogenic callus was induced from hypocotyl of seed germinated in vitro. Somatic embryogenesis in Catharanthus has been categorized into three distinct stages: (1) initiation and proliferation of embryo; (2) maturation, and; (3) germination or plantlet conversion. Beside plant growth regulators, various stages of embryogenesis were screened for their response to a wide variety of factors (pH, gelrite, light, sugar alcohols, polyethyleneglycol and amino acids), which affect embryogenesis. All of the tested factors had a small to marked influence on embryogeny and eventual conversion to plantlets. The plantlets were acclimatized successfully in a greenhouse. To our knowledge, this is the first report describing a detailed study of various cultural factors which regulate embryogenesis in C. roseus. The results discussed in this paper may be used in mass propagation to produce medicinal raw material, and the embryo precursor cells could be used in genetic modification programmes that aim to improve the alkaloid yield as well.     

Secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies,approaches, and limitations to achieving higher yield

Biotechnology is playing a vital alternative role in the production of pharmaceutical plant secondary metabolites to support industrial production and mitigate over-exploitation of natural sources. High-value pharmaceuticals that include alkaloids, flavonoids, terpenes, steroids, among others, are biosynthesized as a defensive strategy by plants in response to perturbations under natural environmental conditions. However, they can also be produced using plant cell, tissue, and organ culture techniques through the application of various in vitro approaches and strategies. In the past decades, efforts were on the clonal propagation, biomass and secondary metabolites production in the in vitro cultures of medicinally important plants that produce these molecules. In recent years, the effort has shifted towards optimizing culture conditions for their production through the application of cell line selection, elicitation, precursor feeding, two-phase co-culture among cell, tissue, and organ culture approaches. The efforts are made with the possibility to scale-up the production, meet pharmaceutical industry demand and conserve natural sources of the molecules. Applications of metabolic engineering and production from endophytes are also getting increasing attention but, the approaches are far from practical application in their industrial production.     

Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

Effect of growth regulators and ethylmethane sulphonate on growth,and chlorophyll,sugar and proline contents in <Emphasis Type="Italic">Dracaena sanderiana</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

A high efficient four step protocol (callus initiation, regeneration, shoot elongation and rooting) for in vitro propagation of Dracaena sanderiana Sander ex Mast was developed. Callusing was achieved from nodal stem segment explants treated with various concentrations of ethylmethane sulphonate (EMS) on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 1.5 g m⁻³). A significant increase in callus induction percentage and biomass production was noticed from lower EMS treated lines (ET₁ and ET₂) comparatively to control and other (ET₃, ET₄ and ET₅) lines. Calli of ET₁ line showed high regeneration potential on MS medium with N ⁶-benzylaminopurine (BAP; 1.75 g m⁻³). Length of microshoots, which was reduced by EMS, restored by addition of gibberellic acid (GA₃; 0.4 g m⁻³). A marked increase in rooting with increasing EMS concentration was noticed on MS medium fortified with 3-indolebutyric acid (IBA; 1.5 g m⁻³).