FT-NIR and linear discriminant analysis to classify chickpea seeds produced with harvest aid chemicals.

Spectroscopy and machine learning (ML) algorithms have provided significant advances to the modern food industry. Instruments focusing on near-infrared spectroscopy allow obtaining information about seed and grain chemical composition, which can be related to changes caused by field pesticides. We investigated the potential of FT-NIR spectroscopy combined with Linear Discriminant Analysis (LDA) to discriminate chickpea seeds produced using different desiccant herbicides at harvest anticipation. Five herbicides applied at three moments of the plant reproductive stage were utilized. The NIR spectra obtained from individual seeds were used to build ML models based on LDA algorithm. The models developed to identify the herbicide and the plant phenological stage at which it was applied reached 94% in the independent validation set. Thus, the LDA models developed using near-infrared spectral data provided to be efficient, quick, non-destructive, and accurate to identify differences between seeds due to pre-harvest herbicides application.

Biogenic iron oxide nanoparticles enhance callogenesis and regeneration pattern of recalcitrant Cicer arietinum L.

This study is the first report on the biosynthesized iron oxide nanoparticles (IONPs) which mediate in-vitro callus induction and shoot regeneration in economically important recalcitrant chickpea crop (Cicer arietinum L.). Here, we used leaf extract of Cymbopogon jwarancusa for the synthesis of IONPs in order to achieve a better biocompatibility. The bioactive compounds in C. jwarancusa leaf extract served as both reducing and capping agents in the fabrication process of IONPs. Field emission scanning electron microscopy (FE-SEM) revealed rods like surface morphology of IONPs with an average diameter of 50±0.2 nm. Energy-dispersive X-ray spectroscopy (EDS) depicted formation of pure IONPs with 69.84% Fe and 30.16% O2. X-ray diffractometry (XRD) and attenuated total reflectance-fourier transform infrared (ATR-FTIR) validate the crystalline structure, chemical analysis detect the presence of various biomolecular fingerprints in the as synthesized IONPs. UV-visible absorption spectroscopy depicts activity of IONPs under visible light. Thermo-gravimetric analysis (TGA) displayed thermal loss of organic capping around 500°C and confirmed their stabilization. The biosynthesized IONPs revealed promising results in callus induction, shoot regeneration and root induction of chickpea plants. Both chickpea varieties Punjab-Noor 09 and Bittle-98 explants, Embryo axes (EA) and Embryo axes plus adjacent part of cotyledon (EXC) demonstrated dose-dependent response. Among all explants, EXC of Punjab-Noor variety showed the highest callogenesis (96%) and shoot regeneration frequency (88%), while root induction frequency was also increased to 83%. Iron content was quantified in regenerated chickpea varieties through inductively coupled plasma-optical emission spectrometry. The quantity of iron is significantly increased in Punjab-Noor regenerated plants (4.88 mg/g) as compare to control treated plants (2.42 mg/g). We found that IONPs enhance chickpea growth pattern and keep regenerated plantlets infection free by providing an optimum environment for rapid growth and development. Thus, IONPs synthesized through green process can be utilized in tissue culture studies in other important recalcitrant legumes crops.

Assessment of Antioxidant Enzyme Activity and Mineral Nutrients in Response to NaCl Stress and its Amelioration Through Glutathione in Chickpea.

Salinity stress has been reckoned as one of the major threat towards crop productivity as it causes significant decline in the yield. The impact of NaCl stress (0, 1, 10, 50, 100 and 200 mg L(-1)) as well as glutathione (10 mg L(-1)) either alone or in combination has been evaluated on the induction of multiple shoots, antioxidant enzymes' activity, lipid peroxidation, relative permeability, concentration of nutrients, photosynthetic pigments, protein and proline content of nodal segments of chickpea after 14 days of culture. The antioxidant enzyme activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) were found to be increased under salt stress as well as glutathione-supplemented medium. A significant decrease in the concentrations of chlorophylls a, b, total chlorophyll and carotenoid was observed under salt stress. Concentrations of nitrogen, phosphorus, potassium, calcium, carbon, magnesium and sulphur showed an initial increase up to 10 mg L(-1) NaCl, but a decline was seen at higher NaCl levels. Proline content and malondialdehyde concentration were found to be increased under salt stress. Three isoforms of SOD, one of CAT and four of GPX were expressed during native polyacrylamide gel electrophoresis (PAGE) analysis. However, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the stressed nodal explants revealed the over-expression of several polypeptide bands related to NaCl stress. These findings for the first time suggest that glutathione (GSH) helps in ameliorating NaCl stress in nodal explants of chickpea by manipulating various biochemical and physiological responses of plants.

A new set of ESTs from chickpea (Cicer arietinum L.) embryo reveals two novel F-box genes, CarF-box_PP2 and CarF-box_LysM, with potential roles in seed development.

Considering the economic importance of chickpea (C. arietinum L.) seeds, it is important to understand the mechanisms underlying seed development for which a cDNA library was constructed from 6 day old chickpea embryos. A total of 8,186 ESTs were obtained from which 4,048 high quality ESTs were assembled into 1,480 unigenes that majorly encoded genes involved in various metabolic and regulatory pathways. Of these, 95 ESTs were found to be involved in ubiquitination related protein degradation pathways and 12 ESTs coded specifically for putative F-box proteins. Differential transcript accumulation of these putative F-box genes was observed in chickpea tissues as evidenced by quantitative real-time PCR. Further, to explore the role of F-box proteins in chickpea seed development, two F-box genes were selected for molecular characterization. These were named as CarF-box_PP2 and CarF-box_LysM depending on their C-terminal domains, PP2 and LysM, respectively. Their highly conserved structures led us to predict their target substrates. Subcellular localization experiment revealed that CarF-box_PP2 was localized in the cytoplasm and CarF-box_LysM was localized in the nucleus. We demonstrated their physical interactions with SKP1 protein, which validated that they function as F-box proteins in the formation of SCF complexes. Sequence analysis of their promoter regions revealed certain seed specific cis-acting elements that may be regulating their preferential transcript accumulation in the seed. Overall, the study helped in expanding the EST database of chickpea, which was further used to identify two novel F-box genes having a potential role in seed development.

Agronomic importance of first development of chickpea (Cicer arietinum L.) under semi-arid conditions: II. Seed imbibition.

Due to the slowness growth and weakness of the first developments of chickpea (Cicer arietinum L.), it could not combated with weeds and easily caught up by Ascochyta blight (Ascochyta rabiei (Pass) Labr.) disease. Additionally, due to biotic and abiotic stress factors, esp. at the late sowing, important seed yield losses could be happened. To be able to avoid from them is only possible to accelerate of its first development as possible as. So, one of the best solutions to is to use chemical compounds such as Humic Acid (HA) known soil regulator under the semi-arid conditions. With this aim this research was performed in a Randomized Complete Block Design (RCBD) with four replications under semi-arid field conditions during (2008/2009) and (2009/2010) in Turkiye. Two cultivars (V1 = Gokce and V2 = Ispanyol) and four seed imbibition methods (A0 = 0, A1 = Tap Water, A2 = 1/2 Tap Water + 1/2 Humic acid (HA), A3 = Full HA, as w/w) and seven yield components Plant Height (PH), Number of Branches per Plant (NBP), Number of Pods per Plant (NPP), First Pod Height (NFP), Number of Seeds per Pod (NSP), Seed Weight per Plant (SWP) and 100-Seed weight (HSW) were investigated. The PH and FPH were affected the A0, the NBP, NPP and NSP were affected the A2 and the SWP and HSW were given the varied but not clear responses according to varieties for all the parameters in A1. The A0 and A1 were encouraged the germination and top soil of the plant but, the A2 to A3 were encouraged root system's development. It was concluded that the A2 is a promising method which makes the maximum and positive effect to the first development of the chickpea agronomy under the semi-arid conditions.

Chickpea hybridization using in vitro techniques.

Tissue culture techniques play an important role in the utilization of wild Cicer species for the improvement- of cultivated chickpea. Utilization of wild Cicer species has become essential as a series of evolutionary bottlenecks have narrowed the genetic base of chickpea, thus making it susceptible to a range of diseases and pests. Crosses with wild Cicer can broaden its genetic base and introduce useful traits. Except for two wild species, none of the other Cicer species are cross-compatible. To use a range of Cicer species for the improvement of chickpea, embryo rescue and tissue culture techniques are necessary. The success of the cross with incompatible species depended on a range of techniques including the application of growth regulators to pollinated pistils and saving aborting embryos in vitro. Further, the chances of successful transfer of hybrid shoots to soil are greater if the hybrid shoots are grafted to chickpea stocks.

The immunolocation of XTH1 in embryonic axes during chickpea germination and seedling growth confirms its function in cell elongation and vascular differentiation.

In a previous work, the immunolocation of the chickpea XTH1 (xyloglucan endotransglucosylase/hydrolase 1) protein in the cell walls of epicotyls, radicles, and stems was studied, and a role for this protein in the elongation of vascular cells was suggested. In the present work, the presence and the location of the XTH1 protein in embryonic axes during the first 48 h of seed imbibition, including radicle emergence, were studied. The presence of the XTH1 protein in the cell wall of embryonic axes as early as 3 h after imbibition, before radicle emergence, supports its involvement in germination, and the fact that the protein level increased until 24 h, when the radicle had already emerged, also suggests its participation in the elongation of embryonic axes. The localization of XTH1 clearly indicates that the protein is related to the development of vascular tissue in embryonic axes during the period studied, suggesting that the role of this protein in embryonic axes is the same as that proposed for seedlings and plants.

Doubled-haploid production in chickpea (Cicer arietinum L.): role of stress treatments.

This is the first report on the production of double-haploid chickpea embryos and regenerated plants through anther culture using Canadian cultivar CDC Xena (kabuli) and Australian cultivar Sonali (desi). Maximum anther induction rates were 69% for Sonali and 63% for CDC Xena. Under optimal conditions, embryo formation occurred within 15-20 days of culture initiation with 2.3 embryos produced per anther for CDC Xena and 2.0 embryos per anther for Sonali. For anther induction, the following stress treatments were used: (1) flower clusters were treated at 4 degrees C for 4 days, (2) anthers were subjected to electric shock treatment of three exponentially decaying pulses of 50-400 V with 25 microF capacitance and 25 Omega resistance, (3) anthers were centrifuged at 168-1,509g for 2-15 min, and finally (4) anthers were cultured for 4 days in high-osmotic pressure (563 mmol) liquid medium. Anthers were then transferred to a solid embryo development medium and, 15-20 days later, embryo development was observed concomitant with a small amount of callus growth of 0.1-3 mm. Anther-derived embryos were regenerated on plant regeneration medium. Electroporation treatment of anthers enhanced root formation, which is often a major hurdle in legume regeneration protocols. Cytological studies using DAPI staining showed a wide range of ploidy levels from haploid to tetraploid in 10-30-day-old calli. Flow cytometric analysis of calli, embryos and regenerated plants showed haploid profiles and/or spontaneous doubling of the chromosomes during early regeneration stages.

Optimizing regeneration condition in chickpea (Cicer arietinum L.).

In this study, multiple shoot induction and whole plant regeneration from decapitated embryo axes of three chick peal genotypes including MCC252, MCC283 and MCC505 were evaluated on modified Murashige and Skoog's medium (MMS) which, its vitamins were replaced by vitamins of B5 medium, supplemented with varied concentration of thidiazuron (0.1, 0.2 mg L(-1)) or 6-benzylaminopurin (1,2 mg L(-1)) or zeatin (1, 2 mg L(-1)) treatments. BAP was found to be the most effective cytokinin in normal multiple shoot induction. Shoots were elongated on growth regulator-free medium and then rooted on two media containing 1/4 MMS salts and B5 vitamins + 3% sucrose + 0.8% agar with indol-3-butyric acid (0.4 or 1 mg L(-1)). The highest rooting frequency resulted in a medium including 0.4 mg L(-1) IBA. It was found that different shoot induction media also positively affected rooting, where a medium with 2 mg L(-1) BAP in MCC252/MCC505 and a medium with 2 mg L(-1) zeatin in MCC283 were the best media in shoot induction that produced high frequency, thick spread roots. Plantlets were preliminary acclimatized in liquid medium (1/4 MMS salts and B5 vitamins + 3% sucrose + 0.4 mg L(-1) IBA) for 7 to 14 days, then transferred to pots filled by cocopit: perlite (1:1) and kept in a growth chamber until their shoots and roots were well developed. This resulted in more than 70% survival rate.

Transcriptional profiling of cell wall protein genes in chickpea embryonic axes during germination and growth.

Cell wall hydrolases have been assumed to be involved in the regulation of seed germination, mostly through their contribution to the cell wall disassembly associated with endosperm cap weakening. In Cicer arietinum (a non-endospermic leguminosae seed), we have focused our research directly on the elongation process of the embryonic axes themselves during germination. The genes encoding cell wall proteins, previously implicated in the elongation of chickpea epicotyls, might also be involved in the expansion of embryonic axis cells, and the modulation of their expression could be part of the control of the germinative process. Thus, chickpea alpha-expansins and xyloglucan endotransglycosylase/hydrolase (XTH) acting on the cellulose/xyloglucan network seem to be involved in the elongation of both chickpea epicotyls and embryonic axes, although the products of different genes perform their actions on each organ. Among the four known cDNAs encoding chickpea alpha-expansins, Ca-EXPA1 was the only isoform highly expressed in embryonic axes during germination. In contrast to epicotyl elongation, the genes encoding cell wall beta-galactosidases, involved in pectin degradation, were not expressed during germination, suggesting no role in embryonic axis elongation, mainly due to the different metabolism of pectins during cell wall loosening in embryonic axis or epicotyl cells. The results concerning CanST-1 and -2, encoding two growth-related cell wall proteins, suggested that these genes were not involved in elongation of embryonic axes during germination. The transcription pattern of Cap28, which encodes a glutamic acid rich cell wall protein of unknown function, indicated a role in the development of the embryonic axes during germination.