Efficient transformation and regeneration of transgenic plants in commercial cultivars of Citrus aurantifolia and Citrus sinensis.

Citrus is one of the major horticultural crops with high economic and nutraceutical value. Despite the fact that conventional research has developed numerous improved varieties, citriculture is still susceptible to various stresses and requires innovative solutions such as genetic engineering. Among all the currently available modern approaches, Agrobacterium-mediated transformation is the most efficient method for introducing desired traits in citrus. However, being a non-host for Agrobacterium, various citrus species, including Citrus aurantifolia and Citrus sinensis, are recalcitrant to this method. The available reports on Agrobacterium-mediated transformation of commercial citrus cultivars show very low transformation efficiency with poor recovery rates of whole transgenic plantlets. Here, we provide an efficient and reliable procedure of Agrobacterium-mediated transformation for both C. aurantifolia and C. sinensis. This protocol depends on providing callus-inducing treatment to explants before and during Agrobacterium co-cultivation, using optimum conditions for shoot regeneration and modifying in-vitro micrografting protocol to combat the loss of transgenic lines. As transgenic citrus shoots are difficult to root, we also developed the ideal conditions for their rooting. Using this protocol, the whole transgenic plantlets of C. aurantifolia and C. sinensis can be developed in about ~ 4 months, with transformation efficiency of 30% and 22% for the respective species.

PRpnp, a novel dual activity PNP family protein improves plant vigour and confers multiple stress tolerance in Citrus aurantifolia.

Under field conditions, plants are often simultaneously exposed to several abiotic and biotic stresses resulting in significant reductions in growth and yield; thus, developing a multi-stress tolerant variety is imperative. Previously, we reported the neofunctionalization of a novel PNP family protein, Putranjiva roxburghii purine nucleoside phosphorylase (PRpnp) to trypsin inhibitor to cater to the needs of plant defence. However, to date, no study has revealed the potential role and mechanism of either member of this protein group in plant defence. Here, we overexpressed PRpnp in Citrus aurantifolia which showed nuclear-cytoplasmic localization, where it functions in maintaining the intracellular purine reservoir. Overexpression of PRpnp significantly enhanced tolerance to salt, oxidative stress, alkaline pH, drought and two pests, Papilio demoleus and Scirtothrips citri in transgenic plants. Global gene expression studies revealed that PRpnp overexpression up-regulated differentially expressed genes (DEGs) related to ABA- and JA-biosynthesis and signalling, plant defence, growth and development. LC-MS/MS analysis validated higher endogenous ABA and JA accumulation in transgenic plants. Taken together, our results suggest that PRpnp functions by enhancing the endogenous ABA and JA, which interact synergistically and it also inhibits trypsin proteases in the insect gut. Also, like other purine salvage genes, PRpnp also regulates CK metabolism and increases the levels of CK-free bases in transgenic Mexican lime. We also suggest that PRpnp can be used as a potential candidate to develop new varieties with improved plant vigour and enhanced multiple stress resistance.

Nanoclay based study on protein stability and aggregation and its implication in human health.

Protein aggregation is the major cause of several acute amyloid diseases such as Parkinson's, Huntington's, Alzheimer's, Lysozyme Systemic amyloidosis, Diabetes-II etc. While these diseases have attracted much attention but the cure is still unavailable. In the present study, Human Serum Albumin (HSA) and Human Lysozyme (HL) were used as the model proteins to investigate their aggregations. Nanoclays are hydrous silicates found in clay fraction of soil and known as natural nanomaterials. They have long been used in several applications in health-related products. In the present paper, the different types of nanoclays (MMT K-10, MMT K-30, Halloysite, Bentonite) were used to inhibit the process of HSA and HL aggregation. Aggregation experiments were evaluated using several biophysical tools such as Turbidity measurements, Intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS), Thioflavin T (Th T), congo red (CR) binding assays and Circular dichroism. Results demonstrated that all the nanoclays inhibit the DTT-induced aggregation. However, bentonite and MMT K-10 were progressively intense and potent as they slowed down nucleation stage which can be perceived using several biophysical techniques. Hence, nanoclays can be used as an artificial chaperone and might provide effective treatment against several protein aggregation related disorders.