Advances and outlook of horticultural bioengineering

A Branch of modern biotechnology for creating unique relevant genotypes is bioengineering that harnesses a spectrum of plant genome modification technologies. The study aimed to analyse the current state of the art in genome modification of fruit and berry crops for more significant (vs. premium pure breeding varieties) deviations of norm in the traits and properties of biotic and abiotic resistance, productivity, fruit quality, etc. First horticultural crop transformation studies aimed at developing protocols based on selectable enzyme marker genes of phosphorylationmediated aminoglycoside antibiotics detoxification. Neomycin phosphotransferase nptII constitutes the most common system of transgenic fruit and berry crop selection. In pome crops, the transgenic selection priorities were resistance to scab (Venturia inaequalis (Wint.) Cke), rust (Gymnosporangium juniper-virginianae Schwein.) and bacterial blight (Erwinia amylovora Burrill, Winslow et al.), higher fruit quality, including bright colouring, and reduced enzymatic browning. In stone crops, it was tolerance to plum pox (PPV), papaya ringspot (PRSV) and Prunus necrotic ringspot (PNRSV) viruses. In berry crops — resistance to Sphaerotheca humuli (DC.) Burrill fungus, grey mould (Botrytis cinerea Pers.), root rot (Phytophthora cactorum (Lebert & Cohn) J.Schrot.) and powdery mildew (Oidium tuckeri Berkeley), as well as higher fruit quality. In citruses — resistance to bacterial canker (Xanthomonas citri sub sp.), citrus ulcer (Xanthomonas axonopodis pv citri), greening disease (Huanglongbing (HLB)) and fungi (Trichoderma harzianum Rifai). In tropical crops — resistance to papaya ringspot (PRSV) and banana streak (eBSV) viruses. Unique FT-phenotype transgenic fruit lines are leveraged in the new FasTrack breeding strategy. Nine fruit and berry transgenic crop lines have now been registered worldwide. Transgenic Arctic apples (Golden, Granny, Fuji), plums (Honey Sweet) and papaya (Rainbow, SunUp, Laie Gold) are industry-approved in fresh and processed form. The transgenic list regulated in the Russian Federation does not include fruit or berry crops.

Biofortification of Cereals and Pulses Using New Breeding Techniques: Current and Future Perspectives

Cereals and pulses are consumed as a staple food in low-income countries for the fulfillment of daily dietary requirements and as a source of micronutrients. However, they are failing to offer balanced nutrition due to deficiencies of some essential compounds, macronutrients, and micronutrients, i.e., cereals are deficient in iron, zinc, some essential amino acids, and quality proteins. Meanwhile, the pulses are rich in anti-nutrient compounds that restrict the bioavailability of micronutrients. As a result, the population is suffering from malnutrition and resultantly different diseases, i.e., anemia, beriberi, pellagra, night blindness, rickets, and scurvy are common in the society. These facts highlight the need for the biofortification of cereals and pulses for the provision of balanced diets to masses and reduction of malnutrition. Biofortification of crops may be achieved through conventional approaches or new breeding techniques (NBTs). Conventional approaches for biofortification cover mineral fertilization through foliar or soil application, microbe-mediated enhanced uptake of nutrients, and conventional crossing of plants to obtain the desired combination of genes for balanced nutrient uptake and bioavailability. Whereas, NBTs rely on gene silencing, gene editing, overexpression, and gene transfer from other species for the acquisition of balanced nutritional profiles in mutant plants. Thus, we have highlighted the significance of conventional and NBTs for the biofortification of cereals and pulses. Current and future perspectives and opportunities are also discussed. Further, the regulatory aspects of newly developed biofortified transgenic and/or non-transgenic crop varieties via NBTs are also presented.

Improving Nitrogen Use Efficiency Through Overexpression of Alanine Aminotransferase in Rice, Wheat, and Barley

Nitrogen is an essential nutrient for plants, but crop plants are inefficient in the acquisition and utilization of applied nitrogen. This often results in producers over applying nitrogen fertilizers, which can negatively impact the environment. The development of crop plants with more efficient nitrogen usage is, therefore, an important research goal in achieving greater agricultural sustainability. We utilized genetically modified rice lines over-expressing a barley alanine aminotransferase (HvAlaAT) to help characterize pathways which lead to more efficient use of nitrogen. Under the control of a stress-inducible promoter OsAnt1, OsAnt1:HvAlaAT lines have increased above-ground biomass with little change to both nitrate and ammonium uptake rates. Based on metabolic profiles, carbon metabolites, particularly those involved in glycolysis and the tricarboxylic acid (TCA) cycle, were significantly altered in roots of OsAnt1:HvAlaAT lines, suggesting higher metabolic turnover. Moreover, transcriptomic data revealed that genes involved in glycolysis and TCA cycle were upregulated. These observations suggest that higher activity of these two processes could result in higher energy production, driving higher nitrogen assimilation, consequently increasing biomass production. Other potential mechanisms contributing to a nitrogen-use efficient phenotype include involvements of phytohormonal responses and an alteration in secondary metabolism. We also conducted basic growth studies to evaluate the effect of the OsAnt1:HvAlaAT transgene in barley and wheat, which the transgenic crop plants increased seed production under controlled environmental conditions. This study provides comprehensive profiling of genetic and metabolic responses to the over-expression of AlaAT and unravels several components and pathways which contribute to its nitrogen-use efficient phenotype.

CLONING AND EXPRESSION OF UNIVERSAL STRESS PROTEIN 2 (USP2) GENE IN ESCHERICHIA COLI

Different types of abiotic stresses inhibit the normal growth of plants by changing their physical biochemical, morphological, and molecular traits. It links to the polygenic traits, which is controlled with the help of different genes, due to this polygenetic the manipulation of foreign genetic makeup is very difficult. Drought stress is the very major type of threat to reduce the yield of cash crops in Pakistan and as well as in all over the world. Gene manipulation is the solution to face this problem by producing genetically modified crop plants that have the ability to survive in drought conditions. Universal stress protein gene has been already identified in bacteria which showed its response under stressed conditions, by manipulation of universal stress protein gene. It was found from our study that the bacterial cells transformed with the USP2 gene isolated from cotton induced abiotic stress tolerance under heat, osmotic, and salt stress. It was suggested from our findings that the USP2 gene could be used to produce abiotic stress tolerance transgenic crop plants to enhance crop plant yield and quality.

Genetic response of growth phases for abiotic environmental stress tolerance in cereal crop plants

The yield potential and quality of main cereals crop plants including maize, wheat, rice and barley have improved through breeding and introduction of transgenic crop plants from last three decades. There has been intensive research for the improvement of resistance against biotic and abiotic environmental conditions to safe the potential of cereal crop plants. Among abiotic stresses drought and heat are two most important abiotic factors which caused major loss in yield and quality of crop plants. The heat stress leads towards drought due to loss of water from soil and plant surfaces, therefore drought and heat caused combined adverse effects on plant morphological, physiological and yield traits which leads to reduce crop plant potential. There has been always an interaction among the environmental conditions and crop plants to produce grain and restore productivity. The drought and heat stress caused changes at cellular level, molecular changes and gene expression changes in cereals at various vegetative and reproductive stages/phases of crop growth and development. A large number of genes have indentified in cereals which switch up-regulated and down-regulated during drought and heat stress conditions. However, there is a need to improve resistance in cereals at gene level to maintain potential of yield and quality under abiotic stress conditions like drought, heat, salinity, and cold.

A sexual hybrid and autopolyploids detected in seed from crosses between Neslia paniculata and Camelina sativa (Brassicaceae)

It is important to understand the probability of hybridization and potential for introgression of transgenic crop alleles into wild populations as part of pre-release risk assessment. Here we completed bidirectional crosses between the emerging crop, camelina [Camelina sativa (L.) Crantz] and its weedy relative, ball mustard [Neslia paniculata (L.) Desv.]. Ball mustard is a self-compatible annual that produces hard ball-like seeds similar to canola or mustard seed in size and shape. A total of 1593 crosses were completed and collected with camelina as the maternal parent, while 3253 crosses were successfully collected in the reverse direction. Putatively hybrid seedlings were screened with flow cytometry and species-specific nuclear ribosomal internal transcribed spacer (ITS) markers. Three plants had DNA contents close to expectations for hybrids, but only one of these, formed on camelina, had the expected ITS markers. This hybrid exhibited low fertility, and neither self-pollination nor backcrossing produced viable progeny. The other two plants, formed on ball mustard, had high pollen and seed fertility and were identified as ball mustard neoautotetraploids. Therefore, the hybridization rate between camelina and ball mustard is relatively low at one in 20 000 ovules pollinated when camelina is the maternal parent. However, autotetraploids may form frequently in ball mustard, and tetraploid populations may exist in nature.

Effect of Transgenesis on mRNA and miRNA Profiles in Cucumber Fruits Expressing Thaumatin II

Transgenic plants are commonly used in breeding programs because of the various features that can be introduced. However, unintended effects caused by genetic transformation are still a topic of concern. This makes research on the nutritional safety of transgenic crop plants extremely interesting. Cucumber (Cucumis sativus L.) is a crop that is grown worldwide. The aim of this study was to identify and characterize differentially expressed genes and regulatory miRNAs in transgenic cucumber fruits that contain the thaumatin II gene, which encodes the sweet-tasting protein thaumatin II, by NGS sequencing. We compared the fruit transcriptomes and miRNomes of three transgenic cucumber lines with wild-type cucumber. In total, we found 47 differentially expressed genes between control and all three transgenic lines. We performed the bioinformatic functional analysis and gene ontology classification. We also identified 12 differentially regulated miRNAs, from which three can influence the two targets (assigned as DEGs) in one of the studied transgenic lines (line 224). We found that the transformation of cucumber with thaumatin II and expression of the transgene had minimal impact on gene expression and epigenetic regulation by miRNA, in the cucumber fruits.