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Grapefruit is considered as a minor citrus crop in Pakistan and its annual production is less than 0.5% of total citrus production. Grapefruit industry is depending upon pink flesh cultivar ‘Shamber’ and need varietal diversification. Mutation breeding has played a pivotal role in grapefruit crop improvement and most of the commercial cultivars are bud sports or induced mutants which were selected and later released as new cultivars. Flesh color enhancement, seedlessness and low furanocoumarin level have been main objectives for grapefruit breeders. An overview of potential of mutation breeding in grapefruit and leading mutants produced is presented in the following sections. Though physical mutagens have been more successful, breeders’ interest is rising in more precise and targeted mutagenesis technologies including CRISPR/Cas9 which has enormous potential in genome editing and could shorten breeding and selection cycle. The available leading grapefruit cultivars were screened for horticultural traits and potential candidate varieties has been selected for diversification and selection of better parents for breeding programs. Several putative mutants have been developed using gamma irradiated plant material. The effect of irradiation on plant growth, morphology and biochemical properties has been evaluated and salient findings are discussed. The developed genetically diverse material could be useful for future biotechnology applications.

MADS-box gene family members play multifarious roles in regulating the growth and development of crop plants and hold enormous promise for bolstering grain yield potential under changing global environments. Bread wheat (Triticum aestivum L.) is a key stable food crop around the globe. Until now, the available information concerning MADS-box genes in the wheat genome has been insufficient. Here, a comprehensive genome-wide analysis identified 300 high confidence MADS-box genes from the publicly available reference genome of wheat. Comparative phylogenetic analyses with Arabidopsis and rice MADS-box genes classified the wheat genes into 16 distinct subfamilies. Gene duplications were mainly identified in subfamilies containing unbalanced homeologs, pointing towards a potential mechanism for gene family expansion. Moreover, a more rapid evolution was inferred for M-type genes, as compared with MIKC-type genes, indicating their significance in understanding the evolutionary history of the wheat genome. We speculate that subfamily-specific distal telomeric duplications in unbalanced homeologs facilitate the rapid adaptation of wheat to changing environments. Furthermore, our in-silico expression data strongly proposed MADS-box genes as active guardians of plants against pathogen insurgency and harsh environmental conditions. In conclusion, we provide an entire complement of MADS-box genes identified in the wheat genome that could accelerate functional genomics efforts and possibly facilitate bridging gaps between genotype-to-phenotype relationships through fine-tuning of agronomically important traits.

Increased temperature caused by climate change is exerting negative impacts on productivity of cotton crop. Therefore, breeding cultivars tolerant of high temperature are need of the time. Realizing the situation, 154 accessions of upland cotton were sown under alpha lattice design in three replications in two sowing dates. The first sowing date was planned to coincide the flowering stage with maximum annual temperature (± 48 °C) of the region. The data were recorded at appearance of first flower for physiological traits like viability of pollen grains and cell membrane thermostability. Enzymatic and non-enzymatic antioxidants, i.e., peroxidase activity and proline contents were also quantified along with hydrogen peroxide. K-means cluster and biplot analysis revealed the differential response of genotypes. FH-Lalzar, IUB-13, GH-Mubarak and Shahkar exhibited higher values for antioxidants and physiological traits. The yield and fibre quality of these genotypes were also superior as compared to others. It was suggested that diversity in germplasm for aforementioned traits can be utilized in further breeding programs.

Summer Cypress (Bassia scoparia) is a large annual herb belonging to the family Amaranthaceae native to Eurasia. It has been introduced in many other countries of the world. In Pakistan, summer cypress is also known as kochia and grown as an ornamental plant for its red fall foliage for landscapes. During October, 2017 a survey was conducted in Punjab Province, Pakistan, where 100 wilted plant samples were collected from 30 different plantations of Faisalabad district. Up to 50% loss of plantation was noted in all visited locations. Lower parts of the plants were affected first presenting with necrosis of leaf tips surrounded by a chlorotic zone (Fig. 1. A). Then necrosis of apical margins of the plant parts occurred, followed by stem discoloration and wilting of entire herbaceous branches, leading to the partial wilting of the plants. Ultimately, whole plant wilted and died, (Fig. 1. B) appearing as though they had been scorched. Diseased tissues from lower stem (crown portion) were sampled, surface sterilized in 70 % ethanol for 30 s, and cultured on to Potato Dextrose Agar (PDA) medium. Petri dishes were incubated at 25 ˚C with alternating 12-hour periods of light and dark. Frequently observed, fast growing whitish grey fungal cultures with black pin head points were obtained after 7 days (Fig. 1. C). Young conidia were one-celled, yellow to orange in color and turned brown to black (Fig. 1. D & E), ranged in size from 11 μm to 16 μm x 9.5 μm to 12 μm (Fig. 1. F), and were ellipsoidal at maturity (Fig. 2. A). Hyphae were branched, septate and dark brown in color while conidiophores were flexuous, branched and ranged between 3.5 μm to 4.5 μm in diameter and 14.5 μm to 26.5 μm in length. Based on morphology (Ellis, 1971), the pathogen was identified as Nigrospora oryzae and submitted to the Westerdijk Collection of Fungi, Netherland (CBS 146145/RNOEG30). Total DNA of isolate EG30 was extracted and portions of the Internal transcribed spacer (ITS) region and beta-tubulin (βt) gene were amplified using the universal primers ITS1F and ITS4 (White et al., 1990) and βt2a and βt2b (Glass and Donaldson, 1995). The generated ITS (GenBank Accession No. MG745331.1 491 bp) and βt (GenBank Accession No. MN629896 408 bp) sequences were searched against GenBank using BLASTn and were 99% homologous to ITS (KX986074 525 bp ; MN341493 550 bp) and 100% homologous to βt (MK262852 409 bp) gene region from Nigrospora oryzae (Wang et al., 2017; Zhang, 2019). For pathogenicity tests, ten healthy two-month-old summer cypress plants were inoculated by soil drenching of a spore suspension (106-107 spores/mL) of the fungal isolate EG30 while five plants were treated with sterilized water and used as control treatments. Plants were incubated at 60 to 75% relative humidity (RH) and 25 ˚C in a greenhouse. Leaf necrosis and partial to whole plant witling (Fig. 2. B & C) were observed in the inoculated plants after 21 days. No symptoms appeared in control plants. A fungus was re-isolated from the lower stem (crown portion) parts of the inoculated plants that was identical in morphology to isolate EG30. No fungus resembling EG30 was isolated from the control plants. To the best of our knowledge, this is the first report of summer cypress wilt caused by Nigrospora oryzae (Berk. and Broome) Petch, a known pathogen of several important crops in China, Australia, India, Canada, and Pakistan (Sharma et al., 2013).

Climate change is the change in the statistical distribution of weather patterns that lasts for an extended period. Climate change and agriculture are interrelated processes and affect in many ways. Citrus fruits are one of the largest fruit crops in the world. Yield loss at a drastic level due to abiotic stress annually in which temperature and water stress are the main environmental factors. These factors cause biochemical, anatomical, physiological, and genetic changes in plant structure and lead to defective growth, development, and reproduction, which ultimately cause a reduction in the economic yield of the crop. An increase in temperature and water stress at critical phenological stages of citrus results in reduced tree fruit set, decrease in fruit growth and size, increase in fruit acidity, low tree yield, reduced fruit peel thickness, and pre-harvest fruit drop. Stomatal conductance and net carbon dioxide assimilation in citrus leaves can be reduced by super optimal leaf temperature. Water deficit reduces the transpiration rate, stomatal conductance by stomatal closure associated with ABA content and causes an abrupt decrease in photosynthesis and CO2 assimilation in citrus which reduce trees overall growth and production. Interventions in agronomic practices, breeding strategies, and biotechnological approaches can mitigate climate change effects on citrus. The groundwork against climate change is compulsory for better global livelihood and food security.

Date palm is dioecious, characterized by unisexual flowers located on separate male or female trees. Dioecy has always been an issue in date palm breeding because the sex of the plant cannot be known until it reaches its reproductive stage, which takes 5‒9 years to first flowering. Hence, early sex identification can enhance breeding and genetic improvement success in date palm. The breeding programs help in the selection and identification of superior seedlings of important traits. However, the sex of dioecious plants is usually tedious to identify. Sex determination may not only enhance genetic resources but also gear up breeding programs. No doubt, substantial progress has been made in understanding mechanisms of sex identification in date palm using morphological, physiological, biochemical, and cytological procedures but the discrimination in male and female plants by these techniques is not reliable. Sexuality in plants is regulated by genetic, epigenetic, and physiological mechanisms, and the most specialized mechanism of sex determination is the sex chromosomes. Molecular markers have been applied accurately in date palm breeding programs to differentiate male and female seedlings, but the development of more reliable molecular markers is needed. Hence, this chapter is focused on an understanding of floral biology, genetics of sex, and methods applied for sex determination in date palm.

Rice is an important cereal crop that serves as staple food for more than half of the world population. Abiotic stresses resulting from changing climatic conditions are continuously threating its yield and production. Genes in APETALA-2 (AP2) family encode transcriptional regulators implicated during regulation of developmental processes and abiotic stress responses but their identification and characterization in indica rice was still missing. In this context, twenty-six genes distributed among eleven chromosomes in Indica rice encoding AP2 transcription-factor subfamily were identified and their diverse haplotypes were studied. Phylogenetic analysis of OsAP2 TF family-members grouped them into three clades indicating conservation of clades among cereals. Segmental duplications were observed to be principal route of evolution, supporting the higher positive selection-pressure, which were estimated to be originated about 10.57 to 56.72 million years ago (MYA). Conserved domain analysis and intron-exon distribution pattern of identified OsAP2s revealed their exclusive distribution among the specific clades of the phylogenetic tree. Moreover, the members of osa-miR172 family were also identified potentially targeting four OsAP2 genes. The real-time quantitative expression profiling of OsAP2s under heat stress conditions in contrasting indica rice genotypes revealed the differential expression pattern of OsAP2s (6 genes up-regulated and 4 genes down-regulated) in stress- and genotype-dependent manner. These findings unveiled the evolutionary pathways of AP2-TF in rice, and can help the functional characterization under developmental and stress responses.

Background Rice is a drought susceptible crop. A symbiotic association between rice and mycorrhizal fungi could effectively protect the plant against sudden or frequent episodes of drought. Due to its extensive network of hyphae, the endophyte is able to deeply explore the soil and transfer water and minerals to the plant, some of them playing an important role in mitigating the effects of drought stress. Moreover, the endophyte could modify the expression of drought responsive genes and regulate antioxidants. Results Three rice genotypes, WC-297 (drought tolerant), Caawa (moderately drought tolerant) and IR-64 (drought susceptible) were inoculated with Piriformospora indica (P. indica), a dynamic endophyte. After 20 days of co-cultivation with the fungus, rice seedlings were subjected to 15% polyethylene glycol-6000 induced osmotic stress. P. indica improved the growth of rice seedlings. It alleviated the destructive effects of the applied osmotic stress. This symbiotic association increased seedling biomass, the uptake of phosphorus and zinc, which are functional elements for rice growth under drought stress. It boosted the chlorophyll fluorescence, increased the production of proline and improved the total antioxidant capacity in leaves. The association with the endophyte also up regulated the activity of the Pyrroline-5-carboxylate synthase (P5CS), which is critical for the synthesis of proline. Conclusion A mycorrhizal association between P. indica and rice seedlings provided a multifaceted protection to rice plants under osmotic stress (− 0.295 MPa). Electronic supplementary material The online version of this article (10.1186/s12284-018-0226-1) contains supplementary material, which is available to authorized users.

Abiotic stress, particularly heat stress, affects various parts of the cotton plant and ultimately impacts the seed cotton yield. Different portions of a single cotton plant of a cultivar exhibit variable responses to stress during reproductive and vegetative phases. To test this hypothesis, physiological and morphological traits related to heat stress were observed for two flowering positions in 13 genotypes of upland cotton. These genotypes were sown in field conditions in triplicate following a randomized complete block design. Data were collected for pollen germination, pollen viability, cell membrane thermostability, chlorophyll content, boll weight, and boll retention for both the top and bottom branches of each genotype. The collected data were analyzed for the identification of variability within and between genotypes for these two flowering positions. Tukey’s test was applied to estimate the significance of differences between genotypes and positions within each genotype. Results showed that the two positions within the same plant statistically varied from each other. The bottom branches of the genotypes performed significantly better for all traits measured except boll weight. The genotype AA-933 performed best for pollen germination and boll retention, while CYTO-608 exhibited maximum pollen viability in both the bottom and top flower positions compared with other genotypes. Overall, MNH-1016 and CIM-602 showed better cell membrane thermostability and chlorophyll content, respectively. This intra-plant variability can be further exploited in breeding programs to enhance the stress tolerance capabilities of the resulting varieties.

Hot pepper (Capsicum annum L.) is a major spice crop and is used worldwide for its nutritional value. In the field, its plant is susceptible to various fungal diseases, including fusarium wilt, caused by soil-borne fungus Fusarium oxysporum f. sp. capsici, which can survive in the soil for several years. The infected plant can be recognized by the yellowing of older leaves and downward curling of apical shoots, followed by plant wilting and ultimately the death of the plant. The resistance mechanism in plants is controlled by a single dominant gene, and conventional plant breeding techniques are used to develop a wilt-resistant germplasm. Non-conventional techniques such as gene pyramiding and expression enhancement of antifungal genes could be used to shorten the time to develop resistance against fusarium wilt in hot peppers. In this review, we discuss different aspects of the disease and the molecular basis of resistance in chili/hot pepper plants. Furthermore, this review covers the scope of conventional and non-conventional breeding strategies and different management approaches used to tackle the disease.

High-temperature stress resulting from global warming is a major limiting factor for agriculture around the globe. Although cotton is a warmer-season crop, consistent high temperatures above optimal levels lead to various changes in morphological, physiological and biochemical characteristics of the plants that reduce the yield and quality of fibre. Breeding for thermotolerant cultivars is one of the best possible strategies to mitigate the adverse effect of heat stress. For this purpose, sufficient knowledge of plant response to heat stress, the mechanism of high-temperature tolerance and possible breeding strategies is imperative. All the stages of plant growth are affected by heat stress but the level of heat threshold varies at different stages. Heat stress may inhibit or reduce seed germination, causing poor seedlings and wilted roots at early stages, whereas at later stages it may adversely affect vegetative growth, reproductive growth, photosynthesis rate and cell membrane stability. Heat stress may also modulate the level of metabolites, hormones and reactive oxygen species (ROS). The expression of different heat shock proteins (HSP) varies with increase in temperature. In addition to HSPs, the cotton plant has various other mechanisms to cope with heat stress such as production of antioxidants for scavenging of ROS, maintenance of membrane stability, accumulation of compatible solutes and activation of various stress-responsive genes. A comprehensive understanding of traditional and advanced breeding and biotechnological approaches is required to improve heat tolerance in plants. There are many examples of plant improvement using traditional breeding approaches, but few achievements by utilizing biotechnological tools are reported, which is the result of very limited knowledge of molecular approaches and the genes related to heat tolerance. Conventional breeding approaches combined with biotechnological tools can provide opportunities to identify and incorporate genes related to stress tolerance for the development of new cultivars that can withstand high temperatures.

Chickpea (Cicer arietinum L.) is a highly rich source of protein and is documented as the second most valuable food legume worldwide. In rain-fed areas, the chickpea productivity is strictly threatened by abiotic stress; notably among them is terminal drought stress. Drought stress causes reduction in photosynthesis and stomatal conductance that leads to the biosynthesis of ABA. Consequently, the plant faces oxidative stress, which is produced by ROS: H2O2, O2−, O−, and HO−. As a result, the plant defense system is activated in the form of antioxidants (CAT, APX, POD, etc.) and scavengers (e.g., proline). In this scenario, the integration of conventional breeding with omics approaches is the ideal approach to increase the worth of the breeding program. The breeding program based on omics approaches, that is, genomics, transcriptomics, proteomics, metabolomics, ionomics, and phenomics, is the quickest and efficient way to develop drought-tolerant chickpea accessions. Moreover, the availability of high-throughput sequencing tools accelerates the working efficiency and quality of these omics approaches. Drought-responsive genes, regulatory TFs, and metabolic pathways can be identified through RNA-Seq. The worth and efficiency of the breeding program will be increased by exploiting the omics-based breeding strategies in chickpea against drought stress.

Engineered nucleases have emerged as a powerful tool for site specific gene manipulation in plants. Based on Clustered Regularly Interspersed Short Palindromic Repeats/CRISPR associated (CRISPR/Cas) system, engineered Cas9:gRNA complex can be used to cleave specific DNA sequences in the genome. In the present study, Nicotiana benthamiana Phytoene Desaturase (NbPDS) gene was targeted by CRISPR/Cas9 system. The plant codon optimized (pcoCas9) along with guided RNA (gRNA) was cloned in plant expression vector pGreen0029, to target NbPDS gene. The NbPDS gene was disrupted transiently by agroinfiltration of pcoCas9-gRNA complex. Visible albino spots were observed on agro-infiltrated leaves of N. benthamiana plants after 7 days of infiltration. The observed albino spots were analyzed through PCR amplification of gRNAtarget, fluorescent microscopy and chlorophyll contents measurements. Our results support the notion that CRISPR/Cas9 system is a swift, robust and useful tool for targeted gene disruption, deletion and editing. © 2016 Friends Science Publishers

Thirteen premium quality commercial mango cultivars grown in Pakistan were assayed with seven SSR primer series viz., mMiCIR, MiSHRS, MIAC, MITGIT, LMMA, UBC and MiIIHR. Among the 120 primers used, 101 produced bands and revealed a narrow genetic base ranging from 0.62-0.8 with maximum similarity among the cvs. ‘Anwar Ratole’ and ‘Sensation’ while the maximum divergence was between cvs. ‘Sindhri’ and ‘Sensation’. Chaunsa was genetically more similar (74.2%) and ‘Kala Chaunsa’ was more dissimilar (31.2%) respectively when compared with all the other cultivars. Of 101 SSR primers, we have identified 30 (29.7%) highly informative primers (PIC value ≥0.6), which could be useful in the molecular characterization of mango germplasm. The primer series MIAC, UBC and MiIIHR had the maximum percentage of highly informative primers in descending order. The UPGMA based Euclidian dendrogram constructed with similarity indices placed all the cultivars according to regions of their origin and magnitude of diversity among them. The morphological characterization of the commercial cultivars was also done using 11 quantitative traits; however, none of the traits except fruit weight was informative for the assessment of genetic diversity. SSR based molecular characterization appeared to be reliable, reproducible and cost effective.

Mango being famous as ‘King of Fruits’ is one of the excellent and admired fruit crops of tropical and subtropical areas around the globe. Characterization and evaluation of mango is an imperative step for germplasm conservation and utilization in the breeding programmes. Moreover, characterization of available germplasm is very crucial to identify desired traits or genes. For this purpose morphological and molecular markers are used. Morphological markers generally include those pivotal features that can be scored and distinguished by naked eye and easily be expressed in all environmental conditions. In contrast, molecular markers are used at molecular level and are more suitable for precise characterization, which in turn can successfully be utilized in the development of new cultivars with premium and desired fruit traits. Moreover, preservation of germplasm for future utilization in crop improvement needs accurate information. Consequently, there is an extreme need for integration of the precious available scattered information on various aspects of mango characterization. So, an attempt has been made to review all possible available literature comprehensively to provide essential details for identification of mango cultivars, characterization of desired traits or genes and evaluation of valuable germplasm with morphological as well as molecular markers. Consequently, the desired traits can be used successfully in future breeding programmes to boost quality mango production throughout the world.

The plant cellulose synthase gene superfamily belongs to the category of type-2 glycosyltransferases, and is involved in cellulose and hemicellulose biosynthesis. These enzymes are vital for maintaining cell-wall structural integrity throughout plant life. Here, we identified 78 putative cellulose synthases (CS) in the soybean genome. Phylogenetic analysis against 40 reference Arabidopsis CS genes clustered soybean CSs into seven major groups (CESA, CSL A, B, C, D, E and G), located on 19 chromosomes (except chromosome 18). Soybean CS expansion occurred in 66 duplication events. Additionally, we identified 95 simple sequence repeat makers related to 44 CSs. We next performed digital expression analysis using publically available datasets to understand potential CS functions in soybean. We found that CSs were highly expressed during soybean seed development, a pattern confirmed with an Affymatrix soybean IVT array and validated with RNA-seq profiles. Within CS groups, CESAs had higher relative expression than CSLs. Soybean CS models were designed based on maximum average RPKM values. Gene co-expression networks were developed to explore which CSs could work together in soybean. Finally, RT-PCR analysis confirmed the expression of 15 selected CSs during all four seed developmental stages.

Wild crop relatives are important source of genetic diversity. Plant tissue culture provides proficienct means of conservation of endangered wild plants sustaining biodiversity, improving plant traits by virtue of induced somaclonal variation and trangensis. The objective of study was to establish direct in vitro regeneration and biolistic transformation protocol for wild tobacco N. plumbaginifolia using uidA gene. For direct in vitro regeneration, six combinations of IAA and kinetin were used. Then, biolistic transformation was carried out using BioRad PDS 1 000 (He) gun and 1 100-psi rupture discs. Transformation was confirmed through histochemical gus assay. A proficient and reproducible in vitro regeneration system was standardized for wild tobacco named as Nicotiana plumbaginifolia . In present study 1 mg/L kinetin in combination with 0.25 mg/L IAA proved best for direct in vitro shoot regeneration from mature leaf-based explants. Success in direct in vitro regeneration circumvented the callogensis step, accelerating the process of in vitro regeneration and increasing the chances of obtaining true to type plants. Individual shoots were placed on MS medium with/without IBA for rooting. Individual shoots were successfully rooted on MS medium without IBA. Then, explants were transformed with uidA gene using gene gun. Transient assay of bombarded calli showed gus activity on histochemical assay even after a week. Analysis of gus expression has been found as a principal method of plant transformation optimizations.

RNA interference (RNAi) cassettes for gene silencing in plants consist of an inverted repeat sequence of the targeted gene and a spacer region separating sense and antisense fragments. The sequences are generally placed under the control of a strong promoter and terminator and produce hairpin RNA structures that generate small interfering RNAs (siRNAs). These siRNAs destroy the mRNA of the matching gene in the cytoplasm. The number and efficiency of siRNAs depend upon the sequence length of inverted repeats and the region in the gene. In the present study, our objective was to downregulate three members of the Glucan Synthase-Like family in wheat. Initially, a 230-nucleotide fragment was cloned under the maize ubiquitin-1 promoter and nos terminator in both sense and antisense directions, and separated by a spacer including the Escherichia coli uidA (GUS) gene. RNAi constructs were prepared for TaGSL8 and TaGSL10. Biolistic delivery of RNAi constructs for TaGSL8 and TaGSL10 into immature zygotic embryos (IZEs) of wheat did not yield any transgenic plants. The experiment was repeated and comparisons were made with a control bar gene construct. IZEs bombarded with a bar selection cassette alone generated healthy calli on selection medium and transgenic plants were recovered. IZEs co-bombarded with the combination of RNAi and bar gene constructs produced only unhealthy calli when cultured on selection medium and no transgenic plants were recovered. This indicated that the RNAi constructs with 230-bp long inverted repeats of TaGSL8 or TaGSL10 were inhibiting regeneration. In the second experiment, the length of the inverted repeats was reduced to 150 and 122 nucleotides for TaGSL3 and TaGSL8, respectively. Two independent transgenic plants were recovered for each of the TaGSL3 and TaGSL8 target genes. These transgenic plants showed transcription of the introduced RNAi constructs, and the transcript levels of the corresponding endogenous genes were reduced. RNAi transgenic lines of TaGSL3 and TaGSL8 showed slightly reduced resistance against Fusarium graminearum compared to nontransgenic control plants.

Cultivated guava (Psidium guajava L.) is an important commercial fruit crop. One hundred and thirty two accessions were collected from 12 regions in Pakistan for study of genetic diversity. A total of 33 traits (18 qualitative and 15 quantitative) enabled an assessment of the genetic variability and structure of this guava germplasm. The measured traits of fruit acidity, fruit diameter, seed weight, non-reducing sugars, thickness of outer flesh, number of seeds, fruit sweetness, longitudinal grooves, leaf twisting, fruit skin color, fruit shape at the stalk, longitudinal ridges, and flesh color were found highly variable. Many of these traits are of significant economic importance and could be used as breeding targets to increase fruit yield and fruit quality. There were strong positive correlations detected among the 15 quantitative traits related to fruit yield and fruit quality. These included fruit length and diameter, fruit weight and diameter, length and width of the leaf blade, number of seeds and seed weight, fruit weight and diameter of the fruit cavity, and seed weight and fruit weight. On the other hand, there were some negative correlations among the 18 qualitative traits studied. The 2D PCA plot successfully grouped the samples according to their phenotypic resemblance and morphological characteristics. The morphological dendrogram generated from agglomeration hierarchical clustering (AHC) grouped the 132 accessions into 3 major clusters.

A panel of 94 diverse hexaploid wheat accessions was used to map quantitative trait loci (QTL) underlying the yield related traits on chromosome 3A. Population structure and kinships were estimated using unlinked SSR markers from all 21 chromosomes. Analysis of variance revealed significant difference among accessions; however, genotype × year interaction was non-significant for majority of yield related traits. A mixed linear model (MLM) approach identified six QTLs for four traits that individually accounted for 10.7 to 17.3% phenotypic variability. All QTLs were consistently observed for both study years. New putative QTLs for the maximum fertile florets per spike and spike length were identified. This report on QTLs for yield related traits on chromosome 3A will extend the existing knowledge and may prove useful in marker-assisted selection (MAS) for development of high yielding cultivars.

A serious wilt disease has recently been found on Prosopis cineraria (Ghaf) in Oman and on Dalbergia sissoo (Shisham) in Pakistan. Disease symptoms on both these native, leguminous hosts include vascular discolouration and partial or complete wilt of affected trees. A species of Ceratocystis was consistently isolated from symptomatic material. Morphological comparisons and analyses of DNA sequence data of the ITS, β-tubulin, and EF 1-α gene regions showed that the Ceratocystis isolates obtained from both tree species represent C. manginecans. This is the same pathogen that is causing the devastating mango sudden decline disease in Oman and Pakistan. This is also the same pathogen that has been reported causing a wilting disease on Acacia mangium in Indonesia. Cross inoculation with C. manginecans isolates from P. cineraria, D. sissoo and mango showed that the fungus can cause disease on all three trees.

Cotton is cultivated worldwide for its white fiber, of which around 90% is tetraploid upland cotton (Gossypium hirsutum L.) carrying both A and D genome. Since centuries, yield increasing efforts for the cotton crop by conventional breeding approaches have caused an extensive erosion of natural genetic variability. Mutation based improvement strategies provide an effective way of creating new allelic variations. Targeting Induced Local Lesions IN Genomes (TILLING) provides a mutation based reverse genetic strategy to create and evaluate induced genetic variability at DNA level. Here, we report development and testing of TILLING populations of allotetraploid cotton (G. hirsutum) for functional genomic studies and mutation based enrichment of cotton genetic resources. Seed of two cotton cultivars “PB-899 and PB-900” were mutagenized with 0.3 and 0.2% (v/v) ethyl methanesulfonate, respectively. The phenotyping of M1 and M2 populations presented numerous mutants regarding the branching pattern, leaf morphology, disease resistance, photosynthetic lesions and flower sterility. Molecular screening for point mutations was performed by TILLING PCR aided CEL1 mismatch cleavage. To estimate the mutation frequency in the mutant genomes, five gene classes were TILLed in 8000 M2 plants of each var. “PB-899” and “PB-900.” These include actin (GhACT), Pectin Methyl Esterase (GhPME), sucrose synthase (GhSUS), resistance gene analog, and defense response gene (DRGs). The var. PB-899 was harboring 47% higher mutation induction rate than PB-900. The highest rate of mutation frequency was identified for NAC-TF5 (EU706348) of DRGs class, ranging from 1/58 kb in PB-899 to 1/105 kb in PB-900. The mutation screening assay revealed the presence of significant proportion of induced mutations in cotton TILLING populations such as 1/153 kb and 1/326 kb in var. “PB-899” and “PB-900,” respectively. The establishment of a cotton TILLING platform (COTIP) and data obtained from the resource TILLING population suggest its effectiveness in widening the genetic bases of cotton for improvement and utilizing it for subsequent reverse genetic studies of various genes.

The okra germplasm was screened for salinity tolerance at the seedling stage and during plant ontogeny. Substantial variation existed in okra for salinity tolerance at the seedling stage. An 80 mmol/L NaCl concentration was suitable for discriminating tolerant and non-tolerant okra genotypes. The pooled ranking of the genotypes, based on individual rankings for each trait (root and shoot length, germination percentage, and relative Na(+) and K(+)) in individual NaCl concentrations, was effective for selecting tolerant genotypes. Genotypes selected at the seedling stage maintained their tolerance to NaCl during plant ontogeny, suggesting that screening of the germplasm entries and advanced breeding materials for salt tolerance at the seedling stage is effective. Among 39 okra genotypes, five were identified as the most tolerant genotypes and showed potential for use in breeding programs that focus on the development of salt-tolerant, high-yield okra cultivars.

Citrus Greening Disease (CGD) was critically reviewed with respect to its history, origin, symptomatol - ogy, host range, pathogenic association, identification and detection, transmission and management. It is evident that the citrus disease has emerged as a potential serious threat to citrus production in all the citrus growing countries of the world including Pakistan. It is has been proved that in Pakistan the greening disease is the major cause of citrus decline; hence it is necessary to formulate definite planning and strategies.

In Pakistan citrus groves in general are facing a serious problem of decline that is attributed to different causes. The major cause, however, is the prevalence of citrus virus and virus-like diseases; Citrus tristeza virus (CTV) is of utmost concern. Although CTV has been identified and characterized on the basis of serological and physical proper - ties, no information is available on the strains of CTV in Pakistan. The identification of CTV strains will be helpful in developing strategies to control the decline of citrus trees to a great extent. Many citrus growing countries have success- fully used the technique of cross protection to minimize the drastic effect of severe CTV strains. By pre-immunization of the citrus tree with mild strains, the decline can be controlled to increase the life span of the citrus tree. In this study we focus on the possibility of establishing a cross protection technique in Pakistan against the CTV strains.

Although cotton is sun loving crop but its production is adversely affected due to biotic and abiotic stress, heat stress contributed significantly in this loss around the globe. The present research work on heat tolerance in cotton is a step forward to understand the genetic basis of heat tolerance in upland cotton. The presence and identification of genetic variation for certain traits is one of the pre-requisite to start research work. There are several techniques for the screening of germplasm for heat tolerance. Relative cell injury was used for the identification of the most heat tolerant and susceptible cotton genotypes i.e. VH-259, VH-142 and DNH-40, VH-282 respectively. The identified genotypes were used for the development of F1, F2, BC1 and BC2 populations for genetic studies. The biometrical analysis revealed that selected traits i.e. relative cell injury, chlorophyll contents, boll shedding percentage, plant height, number of bolls per plant, number of seeds per boll, seed cotton yield were controlled by additive and non-additive type of gene action including epistatic effects. It is suggested that selection in these populations should be done in later generations for the development of new germplasm with enhanced heat tolerance.

Powdery mildew of wheat, caused by Erysiphe graminis f.sp. tritici is a serious threat to wheat grain production in many parts of the world. Efforts have been made to engineer resistance into wheat using single gene technology which culminated in the improved resistance efficiency but never a complete control. Some efforts of expressing genes for disease resistance under the control of constitutive promoter resulted in the production of abnormal plants. We used double gene technology and over expressed a couple of antifungal genes i.e., chitinase and chitosanase from Trichoderma harzianum simultaneously into winter wheat genotype Florida . They were expressed separately under the constitutive promoter ( Ubiquitin- 1) from Maize and stress cum disease inducible promoter ( Vst -1) from Vitis vinefera . Six lines achieved under constitutive promoter and five lines under inducible promoter showed the co-integration and expression of chitinase and chitosanase genes. All these lines showed multiple copy integration of both these genes except one line which showed single copy integration of chitinase under ubiquitin promoter. The copy number varied for Chitinase (1-3) and Chitosanase (2-10). Different promoters did not seem to have any impact on transformation efficiency. Pathological analysis done with E.graminis showed a decrease in the susceptibility for both types of transgenic plants. A decrease in susceptibility was seen upto 75% when the transgenics were under inducible promoter. While in transgenics under constitutive promoter the decrease in susceptibility of upto 60% was seen. All the primary transforments with the exception of a couple showed normal growth.

Salicornia spp is one of the most salt-tolerant vascular plants and is native to salt marshes and estuaries. We developed expressed sequence tag derived-simple sequence repeat (EST-SSR) markers for estimating genetic diversity and marker-assisted Salicornia breeding. Six polymorphic EST-SSRs of 40 detected 27 alleles, ranging from three to five alleles per locus. The average number of alleles per locus was 4.33 and 4.17, and the major allele frequency at locus DY529765 was high, being 0.859 and 0.857 in S. bigelovii and S. europea, respectively. Gene diversity, heterozygosity and polymorphism information content were highest at locus DY529950 and similar in these two species. Gene diversity increased with increase in the number of alleles that had a low major allele frequency at a locus. Six polymorphic loci effectively discriminated 46 taxa into three clusters via different analyses. Significant deviation of F(ST) from zero in three suggested populations for six loci indicated population differentiation and limited gene flow among them. A reduced median network established that taxon SB65 is primitive. SMART (simple modular architecture research tool) analysis of peptide sequences of six EST-SSRs showed that loci DY529765, DY529950 and EC906203 contained transmembrane, TLC, AgrB and NTR domains and might be involved in salinity stress tolerance. These EST-SSRs are a valuable resource for marker development and may be useful in marker-assisted Salicornia breeding.

Improvement in leaf hydraulics is directly related to the improvement of plant tolerance to drought stress. Therefo re, a field and pot experiment was carried out to determine the type of genetic variability and selection of parental types on the basis of combining ability for leaf hydraulics. Genotypes showed similar performance in both experiments; higher values were shown by drought tolerant genotypes in all traits except for osmotic potential, which drought tolerant genotypes maintained lower. Osmotic adjustment in pot experiment showed the highest magnitude of additive type of genetic variability. Female showed a higher and significant contribution of general combining ability effects as compared to male; it suggests that within genotypes female rather than male mostly contribute for additive genes. AMES-10103 showed the highest general combining ability effects for traits such as turgor pressure and osmotic adjustment.

Microsatellite markers containing simple sequence repeats (SSRs) are a valuable tool for genetic analysis. Date palm is a dioecious and slow flowering and is very difficult to identify the gender of the trees until it reaches the reproductive age (5–10 years). A total of 12 microsatellite primers were used with 30 date palm samples, 14 parents (8 male + 6 females) and 16 progeny (developed from parents breeding) which showed that microsatellites were highly polymorphic, having a great number of alleles. A total of 124 alleles were characterized in 12 SSR loci. On average, there are 9.08 alleles per locus, with a range from 5 to 16 alleles, for primers mpdCIR15 and mpdCIR57, respectively. These primers produced 15 polymorphic loci specifically in male date palm samples and the seedlings harboring the unique fragments were further characterized as male plants. Increasingly, 38.46 % of these loci were scored as homozygous alleles while 61.53 % heterozygous allelic loci were determined. Primer mpdCIR48 produced a specific locus (250/250) in all male samples whereas the same locus was absent in female samples. Similarly, a locus of 300/310 bp reoccurred in 5 date palm male samples using marker DP-168 which indicated that these are the promising candidate marker to detect the sex in date palm seedlings at early stage. The data resulted from combination of 12 primers enabled the 16 seedling samples progeny (developed from parents breeding) of date palm cultivars to divide into two groups i.e., male and female regarding their sex expression comparative to the parents (male + female) using the principle coordinate analysis.