High efficient de novo root-to-shoot organogenesis in Citrus jambhiri Lush.: Gene expression, genetic stability and virus indexing.

A protocol for high-frequency direct organogenesis from root explants of Kachai lemon (Citrus jambhiri Lush.) was developed. Full-length roots (~3 cm) were isolated from the in vitro grown seedlings and cultured on Murashige and Skoog basal medium supplemented with Nitsch vitamin (MSN) with different concentrations of cytokinin [6-benzylaminopurine, (BAP)] and gibberellic acid (GA3). The frequency of multiple shoot proliferation was very high, with an average of 34.3 shoots per root explant when inoculated on the MSN medium supplemented with BAP (1.0 mg L-1) and GA3 (1.0 mg L-1). Optimal rooting was induced in the plantlets under half strength MSN medium supplemented with indole-3-acetic acid (IAA, 0.5-1.0 mg L-1). IAA induced better root structure than 1-naphthaleneacetic acid (NAA), which was evident from the scanning electron microscopy (SEM). The expressions of growth regulating factor genes (GRF1 and GRF5) and GA3 signaling genes (GA2OX1 and KO1) were elevated in the regenerants obtained from MSN+BAP (1.0 mg L-1)+GA3 (1.0 mg L-1). The expressions of auxin regulating genes were high in roots obtained in ½ MSN+IAA 1.0 mg L-1. Furthermore, indexing of the regenerants confirmed that there was no amplicons detected for Huanglongbing bacterium and Citrus tristeza virus. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers detected no polymorphic bands amongst the regenerated plants. This is the first report that describes direct organogenesis from the root explant of Citrus jambhiri Lush. The high-frequency direct regeneration protocol in the present study provides an enormous significance in Citrus organogenesis, its commercial cultivation and genetic conservation.

Comparative study on phenolic, flavonoids and in vitro antioxidant activity of wild edible plants from Loktak Lake wetland ecosystem under North East Indian Himalayan Region.

The present study investigates the antioxidant activity of 28 wild edible plants (WEPs) resources of Loktak Lake wetland ecosystem of Manipur, North East Indian Himalayan Region and their correlation with phenolics and flavonoids. Antioxidant activity was measured by DPPH, ABTS and FRAP assay. The antioxidant capacity was found to wide ranges of 1.71 to 263.7 µM TEAC/g fresh weight. In three assays, maximum antioxidant capacity is in same order Jussiaea repens L. > Gynura cusimbua (D. Don) Moore > Polygonum sagitattum L. ranging from 99.5 to 263.7 µM TEAC/g fresh weight. The correlation study established that all the three antioxidant assays are positively correlated and phenolics have contributed more in antioxidant activity than flavonoids. Among 28 WEPs, Jussiaea repens L. and Gynura cusimbua (D. Don) Moore were found to be most promising. These plants can be used as source of natural antioxidant additives, nutritional supplements or ingredients of functional foods.

Genetic Architecture and Anthocyanin Profiling of Aromatic Rice From Manipur Reveals Divergence of Chakhao Landraces.

Aromatic rice of Manipur popularly known as Chakhao is a speciality glutinous rice, for which protection under geographical indication in India has been granted recently. The agronomic and nutraceutical variability of the Chakhao rice germplasm is yet to be genetically characterized. To address this gap, characterization of ninety-three landraces for agro-morphological traits, grain pigmentation, antioxidant properties, and molecular genetic variation was carried out to unravel their population genetic structure. Two major groups were identified based on pericarp color, namely, purple and non-purple, which showed a significant variation for plant height, panicle length, and grain yield. Molecular marker analysis revealed three subpopulations that could be associated with pericarp pigmentation. Deep purple genotypes formed POP3, japonica genotypes adapted to hill environment formed POP1, while POP2 comprised of both indica and aus types. Liquid chromatography-mass spectrometry (LC-MS) analysis revealed two major anthocyanin compounds in pigmented rices, namely, cyanidin-3-O-glucoside (C3G) and peonidin-3-O-glucoside (P3G). The total anthocyanin content among pigmented genotypes ranged from 29.8 to 275.8 mg.100g-1 DW. Total phenolics ranged from 66.5 to 700.3 mg GAE.100g-1 DW with radical scavenging activity (RSA) varying between 17.7 and 65.7%. Anthocyanins and phenolics showed a direct relationship with RSA implying the nutraceutical benefits of deep pigmented rice such as Manipur black rice. Aromatic rices from Manipur were found to be genetically diverse. Therefore, efforts need to be made for maintaining the geographic identity of these rice and utilization in breeding for region-specific cultivar improvement.

Valorization of chicken feather waste into bioactive keratin hydrolysate by a newly purified keratinase from Bacillus sp. RCM-SSR-102.

The objective of this study is the characterization of a keratinase from Bacillus sp.RCM-SSR-102 and its application in the preparation of keratin hydrolysate from chicken feather waste. The purified KER102 keratinase was characterized as a serine-metallo protease having a molecular weight of 30 kDa with optimum pH and temperature of 10 and 50 °C respectively. The keratinase could retain 98% activity at pH 10 and above and 55% activity at 20% salt concentration. The KER102 keratinase was found to be stable in the presence of oxidizing agents, surfactants and organic solvents. The keratinase could also hydrolyze both soluble and insoluble complex protein substrates. The KER102 keratinase could hydrolyze up to 5% (w/v) feather releasing 1.7 ± 0.19 mg/mL soluble peptides. The feather keratin hydrolysate (FKH) had both antioxidant and antityrosinase activity. The IC50 value of FKH in 2, 2-diphenyl 1-picrylhydrazyl (DPPH) radical scavenging activity (1.02 ± 0.01 mg/mL), 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity (20 ± +00.04 µg/mL) and anti-tyrosinase activity (1.2 ± 0.22 mg/mL) was recorded. The FKH also had DNA protecting ability against oxidative damage. Antioxidant and anti-tyrosinase compounds have potential applications in the pharmaceutical and cosmeceutical industry. Hence, the purified keratinase can be a potential candidate for the production of antioxidant and antityrosinase compounds from chicken feather waste.

Reactive oxygen species scavenging mechanisms associated with polyethylene glycol mediated osmotic stress tolerance in Chinese potato.

Influence of polyethylene glycol (PEG) mediated osmotic stress on reactive oxygen species (ROS) scavenging machinery of Chinese potato (Solenostemon rotundifolius (Poir.) J. K. Morton) was investigated. Five genotypes of Chinese potato were raised in Murashige and Skoog (MS) basal medium containing 6-benzylaminopurine (BAP, 1 mg L-1) along with various concentrations of PEG-6000 mediated stress conditions (0, -0.2 and -0.5 MPa) and evaluated for osmotic stress tolerance in vitro. The medium containing PEG-6000 had a detrimental effect on plantlet growth and development while compared with the control. Accumulation of H2O2 was lower in Sreedhara and Subala and higher in Nidhi under PEG stress, which was evident by in situ detection in leaves. Lipid peroxidation product such as malondialdehyde (MDA) content was increased due to PEG stress which was more in susceptible genotype than that in tolerant ones. An enhanced ROS-scavenging antioxidant enzyme was observed under stress with respect to the control. The enzymes of ascorbate-glutathione cycle showed an important role in scavenging ROS. The imposition of PEG stress also increased the non-enzymatic antioxidants viz., the ascorbate and reduced glutathione content which was prominent in tolerant genotypes in comparison to susceptible. The present study indicated that, Sreedhara and Subala showed more tolerance to osmotic stress with better ROS scavenging machineries which would be the lines of interest for augmenting future breeding strategies in this climate resilient minor tuber crop.

Genetic Variability Based on Tandem Repeat Numbers in a Genomic Locus of 'Candidatus Liberibacter asiaticus' Prevalent in North East India.

The genetic variability of 'Candidatus Liberibacter asiaticus' (CLas) population associated with huanglongbing (HLB) disease of citrus in North Eastern (NE) region of India, a geographically locked region, and home for the diversity of many citrus species was analyzed on the basis of tandem repeat numbers (TRN) in variable CLIBASIA_01645 genomic loci. Fifty-five CLas strains sampled from different groves of NE Hill (NEH) region of India were in single amplicon group, but there was remarkable genetic variability in TRNs. The TRN in HLB-associated CLas strains varied from 0-21 and two novel repeat motifs were also identified. Among the NE population of CLas, TRN5 and TRN9 were most frequent (total frequency of 36.36%) followed by TRN4 (14.55%) and TRN6, TNR7 with a frequency of 12.73% each. Class II type CLas genotypes (5 < TRN ≤ 10) had highest prevalence (frequency of 60.00%) in the samples characterized in present study. Class I (TRN ≤ 5) genotypes were second highest prevalent (29.09%) in the NEH region. Further analysis of genetic diversity parameters using Nei's measure (H value) indicated wide genetic diversity in the CLas strains of NE India (H value of 0.58-0.86). Manipur CLas strains had highest genetic variability (0.86) as compared to Eastern, Southern and Central India. The R10 values (TRN ≤ 10/TRN > 10) of NE CLas population was 10.43 (73/7), higher from other regions of India. Present study conclusively reported the occurrence of high genetic variability in TRN of CLas population in North East Indian citrus groves which have evolved to adapt to the specific ecological niche.

Feather degrading, phytostimulating, and biocontrol potential of native actinobacteria from North Eastern Indian Himalayan Region.

Twelve actinobacterial strains were isolated from tomato rhizospheric soil from Manipur, a state in North East Indian Himalayan Region and screened for keratinolytic and plant growth promoting traits. Nine promising isolates were identified as Streptomyces species using partial 16S rRNA gene sequencing. Among the seven isolates showing chicken feather degradation activity, three keratinolytic strains RCM-SSR-2, -6, and -12 were found to be the most efficient feather degrading strains achieving 90% feather weight loss within 48 h of incubation. They also showed maximum keratinase and soluble peptide production. Strain RCM-SSR-2, -5, -6, -8, and -11 showed positive results for all plant growth promoting traits tested. Maximum indole-3-acetic acid production was exhibited by RCM-SSR-6. Strain RCM-SSR-1, -2, -5, -6, -9, and -11 showed antagonistic activity against three important plant pathogens. Feather hydrolysate of RCM-SSR-6 was also evaluated for in vitro seed germination test using garden pea seeds. Higher concentration of feather protein hydrolysate (3 mg ml-1 ) inhibited shoot and root length of the germinating embryo. However, lower concentration (0.01 mg ml-1 ) of feather protein hydrolysate promoted seed germination. Among the 12 strains, four isolates namely RCM-SSR-1, -2, -5, and -6 were found to be promising as multi-traits plant growth promoting rhizobacteria for development of organic fertilizer, phytostimulator, and biocontrol agents.