Histological and molecular insights in to in vitro regeneration pattern of Xanthosoma sagittifolium.

A study on the effect of various phytohormonal combinations on in vitro propagation of Cocoyam [Xanthosoma sagittifolium (L.) Schott] was conducted to develop an improved and efficient in vitro regeneration protocol for its mass multiplication. Histological analysis to understand the in vitro regeneration pattern and genetic fidelity assessment of regenerated plants were also carried out. Single shoots excised from in vitro established cultures of X. sagittifolium were used as explants. Among the 32 different phytohormonal combinations tested, indirect organogenesis with intervening callus phase was observed on majority of the media combinations. Meristematic clump formation was optimally achieved on all the tested media combinations with maximum 43.54 ± 0.51 shoot primordia on MS medium containing 0.2 mg/L BAP + 0.1 mg/L NAA followed by 36.44 ± 0.76 shoot primordia on MS medium having 2.5 mg/L TDZ. Micro-morphological analysis of different morphogenetic structures revealed that the regeneration of cocoyam is well executed via meristematic nodules, shoot primordia formation that may evolve in to proper shoots. Adventitious shoots (> 2 cm) were successfully (100.00 ± 0.00%) rooted on the half-strength MS medium containing IBA (0.05-1.0 mg/L) and IAA (0.05-0.5 mg/L). The number of roots ranged from 0.78 ± 0.31 on the control half-strength MS medium to 13.94 ± 0.46 on half-strength MS supplemented with 1.0 mg/L IBA. Considering somaclonal variations as a potential restriction to in vitro multiplication of plants, genetic stability was assessed using 40 ISSR primers. The PCR amplification profiles obtained from all the tested propagules (calli, meristematic clumps, regenerated plantlets) were similar to the mother plants indicating the homogeneity of the individuals raised through the regeneration protocol reported here.

Development of a real-time RT-PCR method for the detection of Citrus tristeza virus (CTV) and its implication in studying virus distribution in planta.

Tristeza is an economically important disease of the citrus caused by Citrus tristeza virus (CTV) of genus Closterovirus and family Closteroviridae. The disease has caused tremendous losses to citrus industry worldwide by killing millions of trees, reducing the productivity and total production. Enormous efforts have been made in many countries to prevent the viral spread and the losses caused by the disease. To understand the reason behind this scenario, studies on virus distribution and tropism in the citrus plants are needed. Different diagnostic methods are available for early CTV detection but none of them is employed for in planta virus distribution study. In this study, a TaqMan RT-PCR-based method to detect and quantify CTV in different tissues of infected Mosambi plants (Citrus sinensis) has been standardized. The assay was very sensitive with the pathogen detection limit of > 0.0595 fg of in vitro-transcribed CTV-RNA. The assay was implemented for virus distribution study and absolute CTV titer quantification in samples taken from Tristeza-infected trees. The highest virus load was observed in the midribs of the symptomatic leaf (4.1 × 107-1.4 × 108/100 mg) and the lowest in partial dead twigs (1 × 103-1.7 × 104/100 mg), and shoot tip (2.3 × 103-4.5 × 103/100 mg). Interestingly, during the peak summer months, the highest CTV load was observed in the feeder roots (3 × 107-1.1 × 108/100 mg) than in the midribs of symptomatic leaf. The viral titer was highest in symptomatic leaf midrib followed by asymptomatic leaf midrib, feeder roots, twig bark, symptomatic leaf lamella, and asymptomatic leaf lamella. Overall, high CTV titer was primarily observed in the phloem containing tissues and low CTV titer in the other tissues. The information would help in selecting tissues with higher virus titer in disease surveillance that have implication in Tristeza management in citrus.

Development of a SYBR Green-based RT-qPCR assay for the detection of Indian citrus ringspot virus.

The Indian citrus ringspot virus (ICRSV) that causes ringspot disease, especially to 'Kinnow mandarin' hampers the sustainability of crop production. Presently, the disease is not amenable for control through host resistance or the introduction of chemicals, hence raising virus-free plants is one of the most effective approaches to manage the disease. Consequently, it is necessary to develop rapid, sensitive, specific, and early diagnostic methods for disease control. In the present study, newly designed primers targeting a 164 bp region of the ICRSV coat protein gene were used to develop and optimize a SYBR Green-based quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay, for the detection of ICRSV. The RT-qPCR assay was evaluated and confirmed using viral RNA extracted from ICRSV infected plants maintained in screen house as well as field samples. The standard curves displayed a dynamic linear range across eight log units of ICRSV-cRNA copy number ranging from 9.48.1 fmol (5.709 × 109) to 0.000948 amol (5.709 × 102), with detection limit of 5.709 × 102 copies per reaction using serial tenfold diluted in vitro transcribed viral cRNA. The developed RT-qPCR is very specific to ICRSV does not react to other citrus pathogens, and approximately 100-fold more sensitive than conventional RT-PCR. Thus, this assay will be useful in laboratories, KVKs, and nurseries for the citrus budwood certification program as well as in plant quarantine stations. To our knowledge, this is the first study of the successful detection of ICRSV by RT-qPCR.

In-silico characterization and RNA-binding protein based polyclonal antibodies production for detection of citrus tristeza virus.

Citrus tristeza virus (CTV) is the etiologic agent of the destructive Tristeza disease, a massive impediment for the healthy citrus industry worldwide. Routine indexing of CTV is an essential component for disease surveys and citrus budwood certification for production of disease-free planting material. Therefore, the present study was carried out to develop an efficient serological assay for CTV detection based on the RNA binding protein (CTV-p23), which is translated from a subgenomic RNA (sgRNA) that accumulates at higher levels in CTV-infected plants. CTV-p23 gene was amplified, cloned and polyclonal antibodies were raised against recombinant CTV-p23 protein. The efficacy of the produced polyclonal antibodies was tested by Western blots and ELISA to develop a quick, sensitive and economically affordable CTV detection tool and was used for indexing of large number of plant samples. The evaluation results indicated that the developed CTV-p23 antibodies had an excellent diagnostic agreement with RT-PCR and would be effective for the detection of CTV in field samples. Furthermore, CTV-p23 gene specific primers designed in the present study were found 1000 times more sensitive than the reported coat protein (CTV-p25) gene specific primers for routine CTV diagnosis. In silico characterizations of CTV-p23 protein revealed the presence of key conserved amino acid residues that involved in the regulation of protein stability, suppressor activity and protein expression levels. This would provide precious ground information towards understanding the viral pathogenecity and protein level accumulation for early diagnosis of virus.

Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus.

Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India.

Haplotype distribution and association of candidate genes with salt tolerance in Indian wild rice germplasm.

KEY MESSAGE: The association of natural genetic variations of salt-responsive candidate genes belonging to different gene families with salt-tolerance phenotype and their haplotype variation in different geographic regions. Soil salinity covers a large part of the arable land of the world and is a major factor for yield losses in salt-sensitive crops, such as rice. Different gene families that respond to salinity have been identified in rice, but limited success has been achieved in developing salt-tolerant cultivars. Therefore, 21 salt stress-responsive candidate genes belonging to different gene families were re-sequenced to analyse their genetic variation and association with salt tolerance. The average single nucleotide polymorphism (SNP) density was 16 SNPs per kbp amongst these genes. The identified nucleotide and haplotype diversity showed comparatively higher genetic variation in the transporter family genes. Linkage disequilibrium (LD) analysis showed significant associations of SNPs in BADH2, HsfC1B, MIPS1, MIPS2, MYB2, NHX1, NHX2, NHX3, P5CS1, P5CS2, PIP1, SIK1, SOS1, and SOS2 genes with the salt-tolerant phenotype. A combined analysis of SNPs in the 21 candidate genes and eight other HKT transporter genes produced two separate clusters of tolerant genotypes, carrying unique SNPs in the ion transporter and osmoticum-related genes. Haplotype network analysis showed all the major and few minor alleles distributed over distant geographic regions. Minor haplotypes may be recently evolved alleles which migrated to distant geographic regions and may represent recent expansion of Indian wild rice. The analysis of genetic variation in different gene families identified the relationship between adaptive variations and functional significance of the genes. Introgression of the identified alleles from wild relatives may enhance the salt tolerance and consequently rice production in the salinity-affected areas.

Association of SNP Haplotypes of HKT Family Genes with Salt Tolerance in Indian Wild Rice Germplasm.

BACKGROUND: Rice is one of the most important crops for global food security but its productivity is adversely affected by salt stress prevalent in about 30 % of the cultivated land. For developing salt-tolerant rice varieties through conventional breeding or biotechnological interventions, there is an urgent need to identify natural allelic variants that may confer salt tolerance. Here, 299 wild rice accessions collected from different agro-climatic regions of India were evaluated during growth under salt stress. Of these 95 representative accessions were sequenced for members of HKT ion transporter family genes by employing Ion Torrent PGM sequencing platform. RESULTS: Haplotype analysis revealed haplotypes H5 and H1 of HKT1;5 and HKT2;3, respectively associated with high salinity tolerance. This is the first study of allele mining of eight members of HKT gene family from Indian wild rice reporting a salt tolerant allele of HKT2;3. HKT1;5 also showed a salt tolerant allele from wild rice. Phylogenetic analysis based on the nucleotide sequences showed different grouping of the HKT family genes as compared to the prevailing protein sequence based classification. CONCLUSIONS: The salt tolerant alleles of the HKT genes from wild rice may be introgressed into modern high yielding cultivars to widen the existing gene pool and enhance rice production in the salt affected areas.