The improvement of woody fruit species by traditional plant breeding techniques has several limitations mainly caused by their high degree of heterozygosity, the length of their juvenile phase and auto-incompatibility. The development of new biotechnological tools (NBTs), such as RNA interference (RNAi), trans-grafting, cisgenesis/intragenesis and genome editing tools, like zinc-finger and CRISPR/Cas9, has introduced the possibility of more precise and faster genetic modifications of plants. This aspect is of particular importance for the introduction or modification of specific traits in woody fruit species while maintaining unchanged the general characteristics of a selected cultivar. Moreover, some of these new tools give the possibility to obtain transgene-free modified fruit tree genomes, which should increase consumer’s acceptance. Over the decades biotechnological tools have undergone rapid development and there is a continuous addition of new and valuable techniques for plant breeders. This makes it possible to create desirable woody fruit varieties in a fast and more efficient way to meet the demand for sustainable agricultural productivity. Although NBTs have a common goal i.e. precise, fast and efficient crop improvement, individually they are markedly different in approach and characteristics from each other. In this review we describe in detail their mechanisms and applications for the improvement of fruit trees and consider the relationship between these biotechnological tools and the EU biosafety regulations applied to the plants and products obtained through these techniques.

/pdf/new-biotechnological-tools-for-the-genetic-improvement-of-3c4h9y6rro.pdf

New Biotechnological Tools for the Genetic Improvement of Major Woody Fruit Species

The novelty of this study was the use of a subcritical water unit to recover phenolic compounds from papaya seeds, a relevant agroindustrial waste. The effect of temperature conditions on subcritical water extraction (SWE) was compared to Soxhlet extraction (SOX) in terms of total yield, phenolic content (TPC), Maillard reaction products (MRPs), and antioxidant activity, detected by DPPH and β-carotene bleaching methods. TPC and MRPs values were maximized by SWE at 150 °C for 5min, which provided the best antioxidant potential by DPPH method. Liquid chromatography analysis of papaya seed extracts identified 30 phenolic compounds within the samples tested. The main compounds were ferulic acid, mandelic acid, and vanillic acid. It was also found that the subcritical water equipment was successfully applied for the recovery of bioactive compounds from papaya seeds in a simple and effective method.

Recovery of bioactive phenolic compounds from papaya seeds agroindustrial residue using subcritical water extraction

Calcium (Ca2+) ion is a critical ubiquitous intracellular second messenger, acting as a lead currency for several distinct signal transduction pathways. Transient perturbations in free cytosolic Ca2+ ([Ca2+]cyt) concentrations are indispensable for the translation of signals into adaptive biological responses. The transient increase in [Ca2+]cyt levels is sensed by an array of Ca2+ sensor relay proteins such as calmodulin (CaM), eventually leading to conformational changes and activation of CaM. CaM, in a Ca2+-dependent manner, regulates several transcription factors (TFs) that are implicated in various molecular, physiological, and biochemical functions in cells. CAMTA (calmodulin-binding transcription activator) is one such member of the Ca2+-loaded CaM-dependent family of TFs. The present review focuses on Ca2+ as a second messenger, its interaction with CaM, and Ca2+/CaM-mediated CAMTA transcriptional regulation in plants. The review recapitulates the molecular and physiological functions of CAMTA in model plants and various crops, confirming its probable involvement in stress signaling pathways and overall plant development. Studying Ca2+/CaM-mediated CAMTA TF will help in answering key questions concerning signaling cascades and molecular regulation under stress conditions and plant growth, thus improving our knowledge for crop improvement.

/pdf/ca-2-calmodulin-complex-triggers-camta-transcriptional-3kb4hy0ovg.pdf

Ca 2+/Calmodulin Complex Triggers CAMTA Transcriptional Machinery Under Stress in Plants: Signaling Cascade and Molecular Regulation

Genetic engineering based on Agrobacterium-mediated transformation has been a desirable tool to manipulate single or multiple genes of existing genotypes of woody fruit crops, for which conventional breeding is a difficult and lengthy process due to heterozygosity, sexual incompatibility, juvenility, or a lack of natural sources. To date, successful transformation has been reported for many fruit crops. We review the major progress in genetic transformation of these fruit crops made in the past 5 years, emphasizing reproducible transformation protocols as well as the strategies that have been tested in fruit crops. While direct transformation of scion cultivars was mostly used for fruit quality improvement, biotic and abiotic tolerance, and functional gene analysis, transgrafting on genetically modified (GM) rootstocks showed a potential to produce non-GM fruit products. More recently, genome editing technology has demonstrated a potential for gene(s) manipulation of several fruit crops. However, substantial efforts are still needed to produce plants from gene-edited cells, for which tremendous challenge remains in the context of either cell's recalcitrance to regeneration or inefficient gene-editing due to their polyploidy. We propose that effective transient transformation and efficient regeneration are the key for future utilization of genome editing technologies for improvement of fruit crops.

/pdf/agrobacterium-mediated-transformation-of-tree-fruit-crops-azdz5cj57s.pdf

Agrobacterium-Mediated Transformation of Tree Fruit Crops: Methods, Progress, and Challenges

Interplay between hydrogen sulfide and calcium/calmodulin enhances systemic acquired acclimation and antioxidative defense against nickel toxicity in zucchini

Papaya ( Carica papaya L.) is an important tropical fruit crop. The fruit is consumed fresh and used in the pharmaceutical, rayon and food industries. Papaya improvement for stress tolerance and qualitative traits using conventional breeding has been difficult due to the narrow germplasm pool in the Carica genus and sexual incompatibility problems encountered during intergeneric hybridization with other genera in the Caricaceae family. Genetic engineering is an important tool in papaya improvement for modifying one or more traits in elite cultivars without altering existing characteristics. Advances in genetic engineering have been facilitated by concerted efforts for genome sequencing of papaya, development of papaya regeneration systems and efficient gene insertion techniques for transfer of desirable traits. Papaya regeneration via organogenesis and somatic embryogenesis has been refined during the past 3 decades. Early efforts to optimize gene insertion protocols utilized a number of reporter and selectable marker genes, viral- and bacterial-derived regulatory sequences and functional genes for biotic and abiotic stress tolerance. Transgenic plants were routinely produced with several cultivars. One of the best success stories in the commercialization of a genetically modified fruit crop has involved the development of transgenic papaya ring spot virus (PRSV) resistant Rainbow and SunUp cultivars, which saved the Hawaiian papaya industry. Additionally, genetically modified papayas with traits for disease resistance and extended shelf life have been extensively screened in field tests. The papaya genome sequence was published in 2008 and has opened new avenues for papaya improvement by precision breeding, which involves the use of regulatory and functional gene sequences from related genera of the Caricaceae family, and is a logical extension of conventional breeding and genetic transformation. The application of precision breeding technology for papaya can pave the way for the development of consumer and eco-friendly cultivars that would be developed in ways similar to conventional breeding while causing fewer GMO-related concerns.

Advances in papaya biotechnology

A grapevine-derived VvMybA1 transcription factor was evaluated for its efficiency as a reporter gene by comparing it with existing reporter genes, the green fluorescent protein (GFP) and β-glucuronidase (GUS). Embryogenic cultures of Vitis cultivars Thompson Seedless and Bronx Seedless were initiated from leaves of in vitro grown micropropagation cultures and somatic embryos were used as explants for Agrobacterium-mediated transformation. Transient and stable expression of the MybA1 gene was characterized by intense red pigmentation in co-cultivated explants, callus tissues and secondary embryos lines compared to GFP and GUS that exhibited green fluorescence and blue coloration following a substrate assay. No differences were observed in transient gene expression frequencies between the MybA1 gene and GUS among the two compared reporter genes. Visual levels of stable gene expression were higher in GFP and GUS expressing cultures compared to MybA1 expressing cultures. The presence of the inserted genes and their expression in regenerated plant lines was confirmed by PCR and RT-PCR. Embryo and plant lines expressing the MybA1 gene accumulated varying levels of anthocyanin pigment in plant tissues and organs, and were characterized by slower growth compared to plant lines expressing GFP and GUS. Scanning electron microscopy analyses revealed a significant change in abaxial and adaxial leaf epidermal cells of MybA1-expressing plant lines compared to those expressing GFP and GUS, and non-transformed control plants. The study demonstrated the utility of the VvMybA1 transcription factor as a reliable reporter gene for identification of gene insertion events in cell culture and regeneration of modified plants.

Evaluation of a grapevine-derived reporter gene system for precision breeding of Vitis

Evaluation of plant-based reporter systems for improvement of cold-hardy grape cultivars.

Development of novel plant phenotypes using plant pigment-associated genes©

Stimuli-induced fluctuations in intracellular free calcium (Ca2+) serve as secondary messenger signals that regulate diverse biochemical processes in eukaryotic cells, such as developmental transitions and responses to biotic and abiotic stresses. Stimuli-specific Ca2+ signals are manifested as spatially and temporally defined differential Ca2+ signatures that are sensed, decoded, and transduced to elicit distal responses via an array of Ca2+ binding proteins (CBPs) that function as intracellular Ca2+ sensors. Calmodulin (CaM), the most important eukaryotic CBP, senses and responds to fluctuations in intracellular Ca2+ levels by binding to this ubiquitous second messenger, and transducing given Ca2+ signatures that differentially activate distal effector (target) proteins regulating a broad range of biochemical responses. Ca2+/CaM targets include an increasing number of proteins whose functions continue to be elucidated. Hundreds of reports have highlighted the importance of CaM, and other CBPs, in the transduction of Ca2+-mediated signals involved in transcriptional regulation, protein phosphorylation/dephosphorylation, and metabolic shifts. Other Ca2+-binding proteins are known to play significant functional roles in plant cells as well. This review is primarily focused on the role of CaM in some key plant processes, and discusses recent advances in understanding the pivotal role of CaM in an ever-increasing number of plant cell functions and biochemical responses. We also discuss recent work highlighting the emerging importance of CaM in nuclear and organellar signaling.

/pdf/the-role-of-calmodulin-and-related-proteins-in-plant-cell-npcpl1xteu.pdf

The Role of Calmodulin and Related Proteins in Plant Cell Function: An Ever-Thickening Plot

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