A. Mujib

Somatic embryogenesis and plant regeneration in Catharanthus roseus

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.

Catharanthus roseus alkaloids: application of biotechnology for improving yield

Catharanthus roseus (L.) G. Don. is a well known medicinal plant. It produces several phyto-compounds many of which show anticancerous properties. The yields of these compounds are however, very low. In this present article, the current development of secondary metabolite synthesis in C. roseus involving biotechnology has been reviewed keeping in mind the various basic factors that influence yield. The importance of cell culture, the role of culturing conditions and other approaches aiming at higher production of alkaloids have been discussed. The genes encoding important enzymes, proteomics, expressed sequence tag and transcription factors in relation to alkaloid yield have also been summarized in order to understand the regulatory mechanisms of C. roseus.

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.

Influence of freezing and non-freezing temperature on somatic embryogenesis and vinblastine production in Catharanthus roseus (L.) G. Don.

Catharanthus roseus (L.) G. Don. (Apocynaceae) is an important dicotyledonous medicinal plant. It produces vinblastine and vincristine, two alkaloids that are being used against a variety of cancers. In the present study, the freezing (−196, 4, 15°C) and non-freezing (25°C) temperature was imposed on embryogenic cultures, and later in vitro embryogeny and vinblastine production in C. roseus was studied. Somatic embryo (SE) production was maximum at 15°C, but the SE maturation was high at 4°C. The SEs, grown at 25°C, showed highest germination and plantlet conversion. Quantitative estimation of vinblastine was carried out using high-performance liquid chromatography in various in vitro raised tissues (embryogenic callus), embryo stages (proliferated, matured and germinated embryos)], and SE-derived plantlets (leaf, shoot, root and whole plant) after various freezing- and non-freezing temperature treatments. Vinblastine synthesis was temperature dependent in C. roseus that has been discussed in this present article.

In vitro regeneration of Solanum nigrum with enhanced solasodine production

High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Skoog (MS) medium without any callusing stage. Shoot forming ability was more pronounced on leaves positioned dorsally. For shoot induction, 2.0 mg dm−3 benzylaminopurine and 1.5 mg dm−3 kinetin were observed to be the most effective plant growth regulators (PGRs). The present paper also describes first successful induction of in vitro flowering in S. nigrum. The leaf derived shoots were excised and treated with various root promoting PGRs and 0.25 mg dm−3 indole-3-butyric acid produced maximum number of roots (15.2 per plant). Plants were later transplanted in field with 100 % survival. Solasodine content was higher in in vitro raised shoots and leaf derived callus, compared to ex vitro grown shoots.

Alliin obtained from leaf extract of garlic grown under in situ conditions possess higher therapeutic potency as analyzed in alloxan-induced diabetic rats.

Context: Garlic, Allium sativum L. (Liliaceae), possesses high therapeutic and pharmacological properties. Hypoglycemic activity is attributed to alliin (S-allyl cysteine sulfoxide), the main active principle localized in garlic cloves. Objective: To compare the production and therapeutic efficiency of alliin extracted from garlic leaves of plants grown under ex situ and in situ conditions. Materials and methods: Alliin content of leaves was quantified and aqueous leaf extracts (from ex situ and in situ grown plants) were given to normal and alloxan-induced diabetic rats for five weeks. Results: Alliin production noted ~50% enhancement in leaves from plants grown under in situ conditions. Serum glucose, triglycerides, total lipids, total cholesterol, low-density lipoprotein (LDL)-, and very low-density lipoprotein (VLDL)-cholesterol in diabetic rats treated with alliin produced from in situ grown plants noted significant reduction of ~54%, 15%, 14%, 20%, 24%, and 15%, while 35%, 14%, 10%, 12%, 17% and 11% reduction was noted in diabetic rats treated with alliin produced from ex situ grown plants in comparison with those administered with distilled water. High-density lipoprotein (HDL)-cholesterol did not show any significant change. Leaf extract of plants lowered serum enzyme levels (alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase) toward the norm better than glibenclamide. The histopathological alteration in pancreas caused by alloxan was also reduced by leaf extract. Discussion and conclusion: These findings demonstrate leaf extract obtained from plants grown under in situ condition possess higher therapeutic efficiency in comparison with leaf extract obtained from plants grown under ex situ condition. Studies suggest that environmental factors influence production of alliin and its therapeutic potential.

In vitro propagation and camptothecin production in Nothapodytes nimmoniana

Nothapodytes nimmoniana is an endemic endangered tree and convenient source for large scale isolation of anticancer alkaloid camptothecin. Due to indiscriminate collection of its wood chips for camptothecin, population of the species is under heavy exploitation pressure in India. Plant tissue culture techniques offer alternative strategies for clonal propagation and camptothecin production to meet demand of the alkaloid along with conservation of the species. Several strategies are employed to enhance in vitro response through seed germination, culture medium optimisation for camptothecin production and clonal propagation. Cryopreservation of embryonic axes is an approach employed in short term preservation of the species. Scale up for the production of camptothecin from N. nimmoniana in bioreactor is a future possibility to meet pharmaceutical market demand and mitigate exploitation of natural population. In this article, we reviewed progress made so far on clonal propagation and camptothecin production in N. nimmoniana. We assessed the challenges and status of camptothecin production in N. nimmoniana by application of in vitro techniques.

In vitro Production of Secondary Metabolites Using Elicitor in Catharanthus roseus : A Case Study

Secondary metabolites are mainly derived from plants and are used by humans from time immemorial. A plant cell, tissue, and organ culture has an inherent capacity to manufacture valuable chemical compounds as the parent plant does in nature. In vitro plant materials are one of the good sources for the production of secondary metabolite and elicitation can be used as one of the important tool in order to improve the synthesis of these compounds. In a variety of plant cell cultures, elicitors have increased production of terpenoid indole alkaloids, isoflavonoid phytoalexins, serquiterpenoid phytoalexin, coumarins etc. Although elicitation has been carried out in large number of medicinal plants, we extensively studied it in Catharanthus roseus, because it is an important source of anticancer compounds Vinblastine (VLB) and Vincristine (VCR). The use of elicitor is also important in order to meet the market demands, for reducing production costs and for in-depth investigation of biochemical and metabolic pathways. This information helps us in manipulation of biosynthetic pathways which can be used as a powerful tool to make natural product-like compounds.

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.

Camptothecin from Nothapodytes nimmoniana: 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.