Low cost high throughput image based root phenotyping pipeline for evaluation of sugarcane root system architecture under drought stress

Root System Architecture (RSA) plays an important role in the agronomic performance of a crop. Incorporation of these root traits in breeding program is hampered by the complexity in accessing the roots and its phenotyping. Lack of high throughput root phenotyping platforms for sugarcane is one of the major constraints in sugarcane root studies. In the present study an attempt was made to develop high throughput sugarcane root phenotyping pipeline comprising of a low cost plant cultivation platform and customized root image acquisition platform and image analyses using already available automated software. PVC tube system of specified dimension were used for plant growth and customized optical correction tank were used for imaging RSA. The acquired root images were fed into automated software GIAroots and about twenty quantitative root phenotype data were extracted and analysed. The working of the whole pipeline from plant growth to image analyses is demonstrated through comparative root phenotyping under drought using five genotypes of sugarcane wild relative Erianthus arundinaceus and three commercial sugarcane varieties.The relationships between the different root variables and genotypes in PCA biplots indicated high correlation among the different root traits. The study shows the low cost high throughput image based root phenotyping pipeline can be used to extract quantifiable root traits and analysed within a short span of time

Creation of novel alleles of fragrance gene OsBADH2 in rice through CRISPR/Cas9 mediated gene editing.

Fragrance in rice grains is a key quality trait determining its acceptability and marketability. Intensive research on rice aroma identified mutations in betaine aldehyde dehydrogenase (OsBADH2) leading to production of aroma in rice. Gene editing technologies like CRISPR/Cas9 system has opened new avenues for accelerated improvement of rice grain quality through targeted mutagenesis. In this study, we have employed CRISPR/Cas9 tool to create novel alleles of OsBADH2 leading to introduction of aroma into an elite non-aromatic rice variety ASD16. PCR analysis of putative transformants using primers targeting the flanking regions of sgRNA in the 7th exon of OsBADH2 identified 37.5% potential multi-allelic mutations in T0 generation. Sensory evaluation test in the leaves of T0 lines identified thirteen lines belonging to five independent events producing aroma. Sequence analysis of these aromatic T0 lines identified 22 different types of mutations located within -17 bp to +15bp of sgRNA region. The -1/-2 bp deletion in the line # 8-19 and -8/-5 bp deletion in the line # 2-16 produced strong aroma and the phenotype was stably inherited in the T1 generation. Comparative volatile profiling detected novel aromatic compounds viz., pyrrolidine, pyridine, pyrazine, pyradazine and pyrozole in the grains of T1 progenies of line # 8-19. This study has demonstrated the use of CRISPR/Cas9 in creating novel alleles of OsBADH2 to introduce aroma into any non-aromatic rice varieties

Genome-Wide In Silico Identification, Structural Analysis, Promoter Analysis, and Expression Profiling of <i>PHT</i> Gene Family in Sugarcane Root under Salinity Stress

The phosphate transporter (PHT) family of proteins plays an imperative role in regulating phosphorus (P) acquisition as well as in translocation from the soil into cells and organs. Phosphorus is an essential macronutrient required for plant life that is not readily available to crops, and resources are diminishing rapidly because of the huge needs of global agriculture. In this study, 23 ShPHT genes were identified in the sugarcane (Saccharum spp.) genome through a comprehensive genome-wide in silico analysis. Phylogeny, gene structure, and conserved motif analysis of PHT genes in sugarcane (ShPHTs) indicated five subfamilies (PHT1-4 and PHO1 subfamily). Gene ontology (GO) analysis revealed that the ShPHT genes were largely involved in phosphate ion transport, phosphate starvation, stimulus response, stress response, and symporter activity. Gene expression analysis under salinity stress confirmed strong induction of PHT genes in wild genotype sugarcane (IND99-907). PHT1-1, PHT1-2, and PHT1-3 members were notably up-regulated in roots under salt stress. The upstream region of PHT genes contained PHR1-binding sites (P1BS), MYB-type, and WRKY- type binding elements. Overall, the present study paves the way for a deeper understanding of the evolution of sugarcane PHT genes and their role in salinity and Pi stress tolerance in sugarcane

Genome-Wide In Silico Identification, Structural Analysis, Promoter Analysis, and Expression Profiling of PHT Gene Family in Sugarcane Root under Salinity Stress

The phosphate transporter (PHT) family of proteins plays an imperative role in regulating phosphorus (P) acquisition as well as in translocation from the soil into cells and organs. Phosphorus is an essential macronutrient required for plant life that is not readily available to crops, and resources are diminishing rapidly because of the huge needs of global agriculture. In this study, 23 ShPHT genes were identified in the sugarcane (Saccharum spp.) genome through a comprehensive genome-wide in silico analysis. Phylogeny, gene structure, and conserved motif analysis of PHT genes in sugarcane (ShPHTs) indicated five subfamilies (PHT1-4 and PHO1 subfamily). Gene ontology (GO) analysis revealed that the ShPHT genes were largely involved in phosphate ion transport, phosphate starvation, stimulus response, stress response, and symporter activity. Gene expression analysis under salinity stress confirmed strong induction of PHT genes in wild genotype sugarcane (IND99-907). PHT1-1, PHT1-2, and PHT1-3 members were notably up-regulated in roots under salt stress. The upstream region of PHT genes contained PHR1-binding sites (P1BS), MYB-type, and WRKY- type binding elements. Overall, the present study paves the way for a deeper understanding of the evolution of sugarcane PHT genes and their role in salinity and Pi stress tolerance in sugarcane

Additional file 1: of Over-expression of a NAC 67 transcription factor from finger millet (Eleusine coracana L.) confers tolerance against salinity and drought stress in rice

Table showing the list of primers used in the study. (PDF 42 kb

Joint channel assignment and routing in multiradio multichannel wireless mesh networks with directional antennas

The aggregate capacity of a wireless mesh network (WMN) is severely affected by interflow interference. In this paper, we propose a network architecture that incorporates directional antennas with multiple orthogonal channels to effectively enhance the performance of WMNs. First, a sectored connectivity graph is introduced to model multiradio multichannel WMNs with directional antennas. Next we formulate the topology design, directional interface assignment, channel allocation, and routing mathematically as a mixed integer linear programming problem. This problem is solved using an iterated local search algorithm to obtain optimized network resource allocation. Simulation results indicate that the proposed architecture can achieve higher packet delivery ratio while providing better network fairness

Controlled Over-Expression of AtDREB1A Enhances Tolerance against Drought and Salinity in Rice

Engineering transcription factors (TF) hold promise in enhancing abiotic stress tolerance in plants. In this study, one of the popular rice varieties of South India, namely ADT 43, was engineered with a TF AtDREB1A driven by a stress-inducible rd29A promoter. PCR and Southern hybridization were employed to confirm the integration and copy number of the transgene. Transgenic lines (T1) of ADT 43 showed enhanced tolerance to drought and salinity compared to the non-transgenic ADT 43. Transgenic lines were found to maintain higher RWC %, lower leaf temperature, and partially closed stomata, enabling better survival under stress conditions. qRT-PCR analysis revealed the strong induction of AtDREB1A transcripts during drought. Transgenic lines of ADT 43 exhibited increased germination and retention of chlorophyll in their leaves under salinity. Evaluation of transgenic lines under transgenic screen house conditions revealed that line # A16 exhibited on par agronomic performance against its non-transgenic counterpart under normal conditions. Under drought, non-transgenic ADT 43 showed &gt;20% reduction in the total number of spikelets per panicle, whereas transgenic line # A16 registered only a 2% reduction. Non-transgenic ADT 43 recorded 80% yield reduction under drought, whereas line # A16 recorded only 54% yield loss. The above results demonstrated the effectiveness of controlled expression of DREB1A in regulating dehydration responses in rice

Controlled Over-Expression of <i>AtDREB1A</i> Enhances Tolerance against Drought and Salinity in Rice

Engineering transcription factors (TF) hold promise in enhancing abiotic stress tolerance in plants. In this study, one of the popular rice varieties of South India, namely ADT 43, was engineered with a TF AtDREB1A driven by a stress-inducible rd29A promoter. PCR and Southern hybridization were employed to confirm the integration and copy number of the transgene. Transgenic lines (T1) of ADT 43 showed enhanced tolerance to drought and salinity compared to the non-transgenic ADT 43. Transgenic lines were found to maintain higher RWC %, lower leaf temperature, and partially closed stomata, enabling better survival under stress conditions. qRT-PCR analysis revealed the strong induction of AtDREB1A transcripts during drought. Transgenic lines of ADT 43 exhibited increased germination and retention of chlorophyll in their leaves under salinity. Evaluation of transgenic lines under transgenic screen house conditions revealed that line # A16 exhibited on par agronomic performance against its non-transgenic counterpart under normal conditions. Under drought, non-transgenic ADT 43 showed >20% reduction in the total number of spikelets per panicle, whereas transgenic line # A16 registered only a 2% reduction. Non-transgenic ADT 43 recorded 80% yield reduction under drought, whereas line # A16 recorded only 54% yield loss. The above results demonstrated the effectiveness of controlled expression of DREB1A in regulating dehydration responses in rice

Transgene-Free Genome Editing for Biotic and Abiotic Stress Resistance in Sugarcane: Prospects and Challenges

Sugarcane (Saccharum spp.) is one of the most valuable food and industrial crops. Its production is constrained due to major biotic (fungi, bacteria, viruses and insect pests) and abiotic (drought, salt, cold/heat, water logging and heavy metals) stresses. The ever-increasing demand for sugar and biofuel and the rise of new pest and disease variants call for the use of innovative technologies to speed up the sugarcane genetic improvement process. Developing new cultivars through conventional breeding techniques requires much time and resources. The advent of CRISPR/Cas genome editing technology enables the creation of new cultivars with improved resistance/tolerance to various biotic and abiotic stresses. The presence of genome editing cassette inside the genome of genome-edited plants hinders commercial exploitation due to regulatory issues. However, this limitation can be overcome by using transgene-free genome editing techniques. Transgene-free genome editing approaches, such as delivery of the RNPs through biolistics or protoplast fusion, virus-induced genome editing (VIGE), transient expression of CRISPR/Cas reagents through Agrobacterium-mediated transformation and other approaches, are discussed. A well-established PCR-based assay and advanced screening systems such as visual marker system and Transgene killer CRISPR system (TKC) rapidly identify transgene-free genome edits. These advancements in CRISPR/Cas technology speed up the creation of genome-edited climate-smart cultivars that combat various biotic and abiotic stresses and produce good yields under ever-changing conditions