High-Throughput な迅速凍結標本の作製

Emblica officinalis Gaertn. syn. Phyllanthus emblica L., universally known as 'Amla' or 'Aonla' or 'Indian gooseberry', is a popular fruit tree belonging to the family Euphorbiaceae and order Geraniales. It is said to be the very first tree that originated on earth, as claimed by age-old Indian mythology. Almost all parts of the tree i.e., root, bark, leaf, flower, fruit and seed are utilized in Ayurvedic and Unani medicinal formulations to improve the overall digestive process, decrease fever, act as a blood purifier, relieve asthma and cough, improve heart health, etc. This tree contains major secondary metabolites like emblicanin-A and emblicanin-B, and also is an affluent source of vitamin-C. Additionally, some other secondary metabolites like tannins, gallic acid, pyrogallol, and pectin are also present in significant amounts. Conventional propagation has been improved via suitable interventions of agrotechnology both in production and protection areas. However, the rate of propagation remains slower; therefore, attempts have been made for biotechnological advancements on E. officinalis. The present review makes an attempt to highlight the botanical description, geographical distribution, ethnopharmacological importance, conventional propagation and protection of this medicinal tree, describing the in vitro-based plant organ and tissue culture methods like direct and indirect organogenesis and somatic embryogenesis along with interventions of molecular marker-based biotechnology and nanotechnology. Further, the prospect of the yet-to-be-explored biotechnological methods for secondary metabolite enhancement like cell suspension, protoplast culture, genetic transformation, etc. and their potential for enhanced emblicanin production have also been discussed in this appraisal.

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Advances in biotechnology of Emblica officinalis Gaertn. syn. Phyllanthus emblica L.: a nutraceuticals-rich fruit tree with multifaceted ethnomedicinal uses.

A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

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Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection.

Photosystem II (PSII) is an important component of the chloroplast. The PSII repair cycle is crucial for the relief of photoinhibition and may be advantageous when improving stress resistance and photosynthetic efficiency. Lethal genes are widely used in the efficiency detection and method improvement of gene editing. In the present study, we identified the naturally occurring lethal mutant 7-521Y with etiolated cotyledons in Brassica napus, controlled by double-recessive genes (named cyd1 and cyd2). By combining whole-genome resequencing and map-based cloning, CYD1 was fine-mapped to a 29 kb genomic region using 15,167 etiolated individuals. Through cosegregation analysis and functional verification of the transgene, BnaC06.FtsH1 was determined to be the target gene; it encodes an filamentation temperature sensitive protein H 1 (FtsH1) hydrolase that degrades damaged PSII D1 in Arabidopsis thaliana. The expression of BnaC06.FtsH1 was high in the cotyledons, leaves, and flowers of B. napus, and localized in the chloroplasts. In addition, the expression of EngA (upstream regulation gene of FtsH) increased and D1 decreased in 7-521Y. Double mutants of FtsH1 and FtsH5 were lethal in A. thaliana. Through phylogenetic analysis, the loss of FtsH5 was identified in Brassica, and the remaining FtsH1 was required for PSII repair cycle. CYD2 may be a homologous gene of FtsH1 on chromosome A07 of B. napus. Our study provides new insights into lethal mutants, the findings may help improve the efficiency of the PSII repair cycle and biomass accumulation in oilseed rape.

https://www.mdpi.com/1422-0067/22/4/2087/pdf

Fine Mapping and Identification of BnaC06.FtsH1 , a Lethal Gene That Regulates the PSII Repair Cycle in Brassica napus

Phyllanthus emblica L. (V. Nelli) is an important constituent of indigenous medicine and a commercially important fresh fruit species. The genomic diversity of the P. emblica germplasm in Sri Lanka has not been studied in detail. Therefore, the present study was conducted to molecularly characterize the P. emblica germplasm in Sri Lanka. Young tender leaves from 66 trees of P. emblica were collected from various parts of the country for DNA assessment. Genomic DNA was extracted using DNeasy Plant mini kit. Six microsatellite (SSR) markers and one SCAR marker developed for P. emblica were used in the PCR amplification. The PCR products were size separated by using 6% denaturing polyacrylamide gel electrophoresis. A binary scoring matrix was constructed on presence and absence of alleles in each single locus and a dendrogram based on genetic similarities was constructed. For each allele, allele frequency, heterozygosity and polymorphic information content were calculated. Cluster analysis was conducted based on marker data and association analysis was carried out to detect putative QTL associated with drupe traits. The dendrogram based on seven DNA marker alleles, showed two major clusters at 14.67% molecular similarity coefficient. A total of 51 alleles were detected in the sampled P. emblica germplasm with the six SSR markers and the SCAR marker used. Out of the six SSR markers, four were polymorphic, one was monomorphic and the other one did not produce clear bands. Heterozygosity of the tested markers ranged from 0. 44. to 0.89. Allele at 176 bp of the marker Phyll112 showed a significant association (P<0.001 and P<0.0001) with commercially important traits such as drupe height, width, weight and mesocarp thickness. The P. emblica germplasm has a higher variation in Sri Lanka and this diversity can be used to develop genetically superior varieties in the future and also to dissect underlying molecular genetic mechanisms of drupe development.

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DNA Marker Analysis Reveals Genomic Diversity and Putative QTL Associated with Drupe Traits in Phyllanthus emblica

Modified Cetyltrimethylammonium bromide (CTAB) protocol for DNA isolation was developed from leaf tissues of Phyllanthus emblica for obtaining high quality genomic DNA. Fresh leaves of three different maturity were analyzed for yield and quality of DNA. Acidity was determined in three different maturity of leaves viz. tender, intermediate and mature and their influence on DNA quality was determined. Drastic reduction of pH was the primary cause for poor quality of DNA. However, high quality DNA isolation was achieved by stabilizing the pH by addition of NaOH during different stages of DNA isolation process. The present protocol yielding high quality intact DNA for genetic fingerprinting as well as for amplification of chloroplast genes for molecular analysis.

http://www.ijbiotech.com/article_7146_62d3bfeb1f1a30b5060d0eb2d9584b40.pdf

Improved protocol for isolation of genomic DNA from leaf tissues of Phyllanthus emblica Gaertn

Micropropagated plants of Curculigo orchioides Gaertn. through five different modes of regeneration were evaluated for their genetic stability and major flavanoids content that contribute to medicinal properties of the rhizome. Leaf explants that produced callus and bulbils have produced plantlets at a higher frequency. However, direct regeneration of plants from leaf tissues without intervening callus produced a low frequency of plantlets. Explants of rhizome produced healthy plantlets directly from the terminal part of the explant. Plantlets regenerated through five different modes of regeneration did not show any morphological variation. The duration of regeneration was varying from 60 to 145 days depending upon the type of the explant. Analysis of genomic DNA among the plantlets regenerated through five different modes of micropropagation revealed about 66% polymorphism. Major flavanoids such as rutin, ferulic acid, caffeic acid, quercitrin that contributed to medicinal properties of the rhizomes differed quantitatively among the plantlets regenerated through different pathways. Present study suggests that plantlets regenerated directly from the rhizome as well as from the leaf tips of in vitro grown plants are stable genetically and phytochemically. Plant Tissue Cult. & Biotech. 24(2): 173-189, 2014 (December)

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Parameswari Alagar Alagar

Papers

Improved protocol for isolation of genomic DNA from leaf tissues of Phyllanthus emblica Gaertn

Influence of different pathways of regeneration on genetic and phytochemical instability of Curculigo orchioides