Nano-PCR for the early detection of tomato leaf curl virus.

Nano-PCR is a potential tool for the early detection of plant viruses. In the current study, different concentrations of silver nanoparticles (20 nm) and magnesium oxide nanoparticles (50 nm) were included in the PCR mixture to improve the sensitivity of PCR for the detection of tomato leaf curl virus. The inclusion of nanoparticles in single or combination in PCR mixture has resulted in improvement of PCR sensitivity. Four-fold improvement was exhibited by the inclusion of 3 ng/µL silver nanoparticles, whereas the combination of silver and magnesium oxide nanoparticles (3 ng/µL and 200 ng/µL, respectively), resulted in a 4.5-fold improvement. The inclusion of 200 ng/µL of magnesium oxide nanoparticles in the PCR mixture exhibited a 7.6-fold increase in PCR sensitivity. Replacement of magnesium chloride with a combination of silver and magnesium oxide nanoparticles (3 ng/µL and 275 ng/µL, respectively) resulted in a 12-fold increase. A 13-fold improvement in PCR sensitivity was observed by the replacement of magnesium chloride in PCR buffer with 275 ng/µL of magnesium oxide nanoparticles. This could also produce detectable amplicon in PCR with a minimum of 25 cycles, resulting in a 26.5% reduction in the duration of PCR. This is the first report on the use of magnesium oxide nanoparticles in PCR for the early detection and better management of tomato leaf curl virus. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03842-2.

Development of transgenic okra (Abelmoschus esculentus L. Moench) lines having RNA mediated resistance to Yellow vein mosaic virus (Geminiviridae).

Begomovirus Yellow vein mosaic virus causes severe yield losses in okra and even the resistant lines developed through conventional breeding show susceptibility at various levels. This paper describes the development of YVMV resistant lines through RNAi strategy. A universal ihpRNA construct harbouring ßC1 ORF from the ß-satellite of the begomovirus was designed using pRNAi-LIC plasmid. Complementarity checks in sequence databases had shown no off-target effects by the target region and the success of siRNA in interference was proven using Custom Dicer-Substrate siRNA analysis. The ßC1 ORF of the begomovirus was PCR amplified and sequenced using the primer combination designed. The pRNAi-LIC vector, a derivative of pCAMBIA2300 containing duplicated CaMV 35S promoter and Nos terminator from pYL44, was SmaI digested and the amplified sense and antisense strands of the ßC1 region were cloned. E. coli transformed with the plasmid were screened for antibiotic resistance, and the plasmids confirmed for the sense and antisense regions through sequencing, were transferred to Agrobacterium tumefaciens strain GV3101. In planta transformation strategy was followed to transform a highly susceptible okra cv. Salkeerthi with ihpRNA-ßC1 cassette. Transformation success, confirmed by the amplification of sense strand using the primers VLIC1 and VLIC5, was 11.42 %. Transcription of siRNA from the ßC1 ORF in the transgenic lines was confirmed by its PCR amplification from the cDNA, using the stem loop primers designed (68 bp). When the transformed and healthy wild-type plants were co-grown with infected wild-type plants, inside an insect cage released with whiteflies and maintained within a containment facility, three of the four transgenic plants remained completely healthy throughout the crop span.

Improved transformation of Agrobacterium assisted by silver nanoparticles.

In transgenic plant development, the low transformation efficiency of Agrobacterium with exogenous DNA is the major constraint, and hence, methods to improve its transformation efficiency are needed. Recently, nanoparticlemediated gene transfer has evolved as a key transformational tool in genetic transformation. Since silver nanoparticles (AgNPs) can induce pores on the cell membrane, their efficacy in the improvement of conventional calcium chloride freeze-thaw technique of transformation of Agrobacterium was explored in this study. Agrobacterium cells in the exponential growth phase were exposed to different concentrations of AgNPs (0.01, 1, 5, 10, and 20 mg/l), and the half-maximal effective concentration (EC50) was determined via Probit analysis using the SPSS software. Transformation efficiency of AgNPs alone and in combination with calcium chloride was compared with that of the conventional calcium chloride freeze-thaw technique. AgNPs at a concentration of 0.01 mg/l in combination with calcium chloride (20 mM) showed a ten fold increase in the transformation efficiency (3.33 log CFU (colony-forming unit/microgram of DNA) of Agrobacterium tumefaciens strain EHA 105 with plasmid vector pART27 compared with the conventional technique (2.31 log CFU/µg of DNA). This study indicates that AgNPs of size 100 nm can eliminate the freeze-thaw stage in the conventional Agrobacterium transformation technique, with a 44% improvement in efficiency. The use of AgNPs (0.01 mg/l) along with 20 mM calcium chloride was found to be an economically viable method to improve the transformation of Agrobacterium with exogenous plasmid DNA.

Silver nanoparticles for biolistic transformation in Nicotiana tabacum L.

The present study reports the use of silver nanoparticles as a gene carrier, substituting gold microcarrier for biolistic gene delivery in Nicotiana tabacum L. Efficiency of biolistic transformation using silver nanoparticles (100 nm) was compared with that of gold microcarriers (0.6 micron) under varying helium pressure (450 psi, 650 psi, 900 psi and 1100 psi) and target distance (6 cm and 9 cm). Among the different concentrations (0.01-100 mgL-1) of silver nanoparticles tried, 10 mgL-1 produced the highest number of transient GUS expression (30) with statistical significance. Helium pressure of 650 and target distance of 9 cm, and 900 psi pressure and 6 cm distance resulted in the highest GUS expression with gold microcarriers and silver nanoparticles, respectively. Transformation efficiency was significantly higher with silver nanoparticles than gold microparticles as carriers resulting in a reduction up to 37.5-fold on the cost of consumables. Regeneration efficiencies of tissues bombarded with gold microcarriers and silver nanoparticles were 62.5% and 70.83%, respectively.

A novel approach for increasing transformation efficiency in E. coli DH5α cells using silver nanoparticles.

The present study is the first report on the application of silver nanoparticles for efficient bacterial transformation. EC50 value of 100 nm silver nanoparticles against E. coli DH5α cells was recorded as 4.49 mg L-1 in toxicity assay. Competency induction in E. coli DH5α cells by treatment with 100 nm silver nanoparticles at a concentration of 1 mg L-1 for 60 min and transformation using three plasmid vectors of different sizes, viz. pUC18, pBR322 and pCAMBIA resulted in tenfold increase in the bacterial transformation efficiency, i.e. 8.3 × 104, 8.0 × 104 and 7.9 × 104 cfu ng-1 of DNA, respectively, even without heat shock compared to the conventional chemical method using 0.1 M calcium chloride (2.3 × 103 cfu ng-1 of DNA).

Calcium alginate encapsulated synthetic seed production in Plumbago rosea L. for germplasm exchange and distribution.

Plumbago rosea L. (Plumbaginaceae), is a medicinal shrub commercially exploited for its naphthoquinone principle, plumbagin, extracted from the roots especially for treating skin disorders. As the plant is exploited from the wild without being replenished, conservation of the species becomes inevitable. Synthetic seeds would provide for effective conservation, germplasm exchange and distribution of this species. A reliable protocol for synthetic seed production in Plumbago rosea has been developed encapsulating the axillary buds. The axillary buds from P. rosea cultures established and multiplied using the nodal explants in Murashige and Skoog (MS) medium supplemented with Benzyl Adenine (BA) 1.5 mg/L and Indole 3-Acetic acid 1.0 mg/L, were used for synseed production. The plantlet conversion efficiency was the highest in synthetic seeds developed with sodium alginate 2.5% in modified MS with 0.4 M sucrose and CaCl2 100 mM. This combination gave the earliest bud initiation (9.19 ± 0.39 days) and maximum number of shoots per explant (2.31 ± 0.16 shoots). Microshoots from the culture, when inoculated on to MS medium supplemented with Naphthalene Acetic Acid 1.0 mg/L gave the best rooting response with 10.67 ± 0.94 roots per plant and 5.42 ± 0.29 cm root length. This is the first report of synthetic seed production in P. rosea using axillary buds as explant.

Expression of nm23-H1 protein in relation to myometrial invasion in complete hydatidiform moles.

OBJECTIVE: Although nm23-H1 protein expression has been related to invasion in many cancers, its expression and prognostic significance in complete hydatidiform moles has not yet been investigated. The search for biologic parameters in molar placentas, which are useful for identifying patients who show myometrial invasion of the tumor, is crucial. We examined the clinical significance of nm23-H1 expression in complete hydatidiform mole. METHODS: Sections of 105 cases of complete hydatidiform moles (including 25 cases of invasive mole) and 95 cases of gestational age--matched normal placentas were immunohistochemically stained with anti-nm23-H1 antibody, which recognizes the nm23-H1/NDP kinase A gene product. RESULTS: Expression of nm23-H1 was detected in the cytotrophoblasts and syncytiotrophoblasts of molar placentas and normal placentas, whereas it was not detected in stromal tissue. Expression of nm23-H1 showed a negative relation to invasion, suggesting its use as a potential marker of myometrial invasion in complete hydatidiform moles. CONCLUSION: nm23-H1 expression could be used as a marker for accurate evaluation of myometrial invasion in complete hydatidiform mole.