Ethical and legal implications of gene editing in plant breeding:a systematic literature review

Biotechnology policies and regulations must be revised and updated to reflect the most recent advances in plantbreeding technology. New Plant Breeding Techniques(NPBT) such as gene editing have been applied to address the myriad of challenges in plant breeding, while the use of NPBT as emerging biotechnological tools raises legal and ethical concerns. This study aims to highlight how gene editing is operationalized in the existing literature and examine the critical issues of ethical and legal issues of gene editing for plant breeding. We carried out a systematic literature review(SLR) to provide the current states of ethical and legal discourses surrounding this topic. We also identified critical research priority areas and policy gaps that must be addressed when designing the future governance of gene editing in plant breeding.

Molecular Plant Breeding(MPB)

Molecular Plant Breeding(ISSN 1923-8266)is an international,open access,peer reviewed journal,journal published online by Bio Publisher.The journal publishes all the latest and outstanding research articles,letters and reviews in all areas of pant breeding,and committed to serve for transgenic breeding and marker assisted breeding in plants,

Molecular Plant Breeding(MPB)

Molecular Plant Breeding(ISSN 1923-8266)is an international,open access,peer reviewed journal,journal published online by Bio Publisher.The journal publishes all the latest and outstanding research articles,letters and reviews in all areas of pant breeding,and committed to serve for transgenic breeding and marker assisted breeding in plants,

Breeding against mycorrhizal symbiosis:Modern cotton (Gossypium hirsutum L.) varieties perform more poorly than older varieties except at very high phosphorus supply levels

Cotton (Gossypium hirsutum L.) is an importantfiber cash crop,but its root traits related to phosphorus (P) acquisition,including mycorrhizal root traits,are poorly understood.Eight cotton varieties bred in northwestern China that were released between 1950 and 2013 were grown in pots with or without one arbuscular mycorrhizal fungal (AMF) species(Funneliformis mosseae) at three P supply levels (0,50 and 300 mg P as KH_(2)PO_(4)kg^(-1)).Eleven root traits were measured and calculated after 7 wk of growth.The more recent accessions had smaller root diameters,acquired less P and produced less biomass,indicating an (inadvertent) varietal selection for thinner roots that provided less cortical space for AMF,which then increased the need for a high P fertilizer level.At the two lower P levels,the mycorrhizal plants acquired more P and produced more biomass than non-mycorrhizal plants (3.2 vs.0.9 mg P per plant;1.8 vs.0.9 g biomass per plant at P_(0);14.5 vs.1.7 mg P per plant;and 4.7 vs.1.6 g biomass per plant at P_(50)).At the highest P level,the mycorrhizal plants acquired more P than non-mycorrhizal plants (18.8 vs.13.4 mg per P plant),but there was no difference in biomass (6.2 vs.6.3 g per plant).At the intermediate P level,root diameter was significantly positively correlated with shoot biomass,P concentration and the P content of mycorrhizal plants.The results of our study support the importance of the outsourcing model of P acquisition in the root economics space framework.Inadvertent varietal selection in the last decades,resulting in thinner roots and a lower benefit from AMF,has led to a lower productivity of cotton varieties at moderate P supply (i.e.,when mycorrhizal,the average biomass of older varieties 5.0 g per plant vs.biomass of newer varieties 4.4 g per plant),indicating the need to rethink cotton breeding efforts in order to achieve high yields without very high P input.One feasible way to solve the problem of inadvertent varietal selection for cotton is to be aware of the trade-offs between the root do-it-yourself strategy and the outsourcing towards AMF strategy,and to consider both morphological and mycorrhizal root traits when breeding cotton varieties.

Pre-Breeding Genetic Diversity Assessment of Tomato(Solanum lycopersicum L.)Cultivars Based on Molecular,Morphological and Physicochemical Parameters

Appropriate knowledge of the parental cultivars is a pre-requisite for a successful breeding program.This study characterized fruit yield,quality attributes,and molecular variations of ten tomato cultivars during three consecutive generations under greenhouse conditions.Peto 86,Castle Rock,and Red Star cultivars showed the highest fruit yield(kg/plant),total phenolic compounds(TPC),and sap acidity.Principal component analysis categorized the evaluated fruit yield into three groups based on their quality attributes.A robust positive correlation appeared among traits inside each group.A positive correlation was likewise noticed between the first and the second groups.However,a negative correlation was detected between the first,the second and the third group.Molecular profiling,using seven inter-simple sequence repeat(ISSR)primers,produced 60 loci,including 49 polymorphic loci.The molecular analysis also pinpointed the highest genetic similarity(0.92)between P73 and Moneymaker,while the lowest genetic similarity(0.46)was observed between Castle Rock and Moneymaker.The cultivars P73 and Moneymaker showed the lowest genetic distance(2.24),while the highest genetic distance(5.92)was observed between Super Marmand and Peto86,on the one hand,and between Castle Rock and Moneymaker,on the other hand.The chemical analysis of fruit sap indicated the highest levels of TPC,total flavonoids,anthocyanin,ascorbic acid and total soluble solids in Peto 86 and Castle Rock cultivars.Phylogeny analysis of tomato cultivars based on morphological and molecular attributes indicated four distinct clades.Peto 86,Castle Rock,and Red star cultivars can be recommended for the tomato hybridization breeding programs in the future,with other tomato cultivars as potentially high-yielding parents.

Enemies atpeace:Recentprogressin Agrobacterium-mediated cereal transformation

Agrobacterium tumefaciens mediated plant transformation is a versatile tool for plant genetic engineering following its discovery nearly half a century ago.Numerous modifications were made in its application to increase efficiency,especially in the recalcitrant major cereals plants.Recent breakthroughs in transformation efficiency continue its role as a mainstream technique in CRISPR/Cas-based genome editing and gene stacking.These modifications led to higher transformation frequency and lower but more stable transgene copies with the capability to revolutionize modern agriculture.In this review,we provide a brief overview of the history of Agrobacterium-mediated plant transformation and focus on the most recent progress to improve the system in both the Agrobacterium and the host recipient.A promising future for transformation in biotechnology and agriculture is predicted.

Applications of cold atmospheric plasmas(CAPs)in agriculture:a brief review and the novel development of a radio-frequency CAP jet generator for plant mutation

Cold atmospheric plasmas(CAPs)have shown great applicability in agriculture.Many kinds of CAP sources have been studied in agricultural applications to promote plant growth and cure plant diseases.We briefly review the state-of-the-art stimulating effects of atmospheric-pressure dielectricbarrier-discharge(AP-DBD)plasmas,after the direct or indirect treatment of plants for growth promotion and disease control.We then discuss the special demands on the characteristics of the CAP sources for their applications in plant mutation breeding.An atmospheric and room temperature plasma(ARTP)jet generator with a large plasma irradiation area,a high enough concentration of chemically reactive species and a low gas temperature is designed for direct plant mutagenesis.Experimental measurements of the electrical,thermal and optical features of the ARTP generator are conducted.Then,an ARTP-P(ARTP for plant mutagenesis)mutation breeding machine is developed,and a typical case of plant mutation breeding by the ARTP-P mutation machine is presented using Coreopsis tinctoria Nutt.seeds.Physical and agricultural experiments show that the newly-developed ARTP-P mutation breeding machine with a large irradiation area can generate uniform CAP jets with high concentrations of chemically reactive species and mild gas temperatures,and have signiflcant mutagenesis effects on the Coreopsis tinctoria Nutt.seeds.The ARTP-P mutation breeding machine may provide a platform for systematic studies on mutation mechanisms and results for various plant seeds under different operating conditions in future research.

Advances in Transgenic Vegetable and Fruit Breeding

Vegetables and fruits are grown worldwide and play an important role in human diets because they provide vitamins, minerals, dietary fiber, and phytochemicals. Vegetables and fruits are also associated with improvement of gastrointestinal health, good vision, and reduced risk of heart disease, stroke, chronic diseases such as diabetes, and some forms of cancer. Vegetable and fruit production suffers from many biotic stresses caused by pathogens, pests, and weeds and requires high amounts of plant protection products per hectare. United States vegetables farmers are benefiting from growing transgenic squash cultivars resistant to Zucchini yellow mosaic virus, Watermelon mosaic virus, and Cucumber mosaic virus, which were deregulated and commercialized since 1996. Bt-sweet corn has also proven effective for control of some lepidopteran species and continues to be accepted in the fresh market in the USA, and Bt-fresh-market sweet corn hybrids are released almost every year. Likewise, transgenic Bt-eggplant bred to reduce pesticide use is now grown by farmers in Bangladesh. Transgenic papaya cultivars carrying the coat-protein gene provide effective protection against Papaya ring spot virus elsewhere. The transgenic “Honey Sweet” plum cultivar provides an interesting germplasm source for Plum pox virus control. Enhanced host plant resistance to Xanthomonas campestris pv. musacearum, which causes the devastating banana Xanthomonas wilt in the Great Lakes Region of Africa, was achieved by plant genetic engineering. There are other vegetable and fruit crops in the pipeline that have been genetically modified to enhance their host plant resistance to insects and plant pathogens, to show herbicide tolerance, and to improve features such as slow ripening that extends the shelf-life of the produce. Consumers could benefit further from eating more nutritious transgenic vegetables and fruits. Transgenic plant breeding therefore provides genetically enhanced seed embedded technology that contributes to integrated pest management in horticulture by reducing pesticide sprays as well as improving food safety by minimizing pesticide residues. Furthermore, herbicide-tolerant transgenic crops can help reducing plough in fields, thereby saving fuel because of less tractor use, which also protects the structure of the soil by reducing its erosion. Transgenic vegetable and fruit crops could make important contributions to sustainable vegetable production and for more nutritious and healthy food. Countries vary, however, in their market standards of acceptance of transgenic crops. Biotechnology products will be successful if clear advantages and safety are demonstrated to both growers and consumers.

Evaluation of cotton germplasm for morphological and biochemical host plant UPdates resistance traits against sucking insect pests complex

Background:Sucking insect pests cause severe damage to cotton crop production.The development of insect resistant cotton cultivars is one of the most effective measures in curtailing the yield losses.Considering the role of morphological and biochemical host plant resista nee(HPR)traits in plant defense,12 cotton genotypes/varieties were evaluated for leaf area,leaf glanding,total soluble sugars,total soluble proteins,total phenolics,tannin and total flavonoids against fluctuating populations of whitefly,thrips and jassid under field conditions.Results:The population of these insects fluctuated during the growing seas on and remained above threshold level(whitefly>5,thrips>(8-10)f or jassid>1 per leaf)during late June and early July.Strong and negative association of whitefly(r=-0.825)and jassid(r=-0.929)with seed cotton yield was observed.Mean population of insects were the highest in Glandless-1 followed by NIA-82 and NIA-M30.NIAB-Kiran followed by NI AB-878 and Sadori were the most resistant,with the mean population of 1.41,1.60,1.66(whitefly);2.24,232,2.53(thrips)and 037,0.31,036(jassid),respectively.The resistant variety NIAB-Kiran showed less soluble sugars(8.54 mg.g^(-1)),soluble proteins(27.11 mg.g^(-1))and more phenolic(36.56 mg.g^(-1))and flavonoids(13.10mg.g^(-1))as compared with the susceptible check Glandless-1.Moreover,all insect populations were positively correlated with total soluble sugars and proteins.Whitefly populations exhibited negative response to leaf gossypol glands,total phenolics,tannins and flavonoids.The thrips and jassid populations had a significant and negative correlation with these four biochemical HPR traits.Conclusion:The ide ntified resistant resources and HPR traits can be deployed against sucking in sect pests'complex in future breeding programs of developing insect resistant cotton varieties.

Cultivating potential:Harnessing plant stem cells for agricultural crop improvement

Meristems are stem cell-containing structures that produce all plant organs and are therefore important targets for crop improvement.Developmental regulators control the balance and rate of cell divisions within the meristem.Altering these regulators impacts meristem architecture and,as a consequence,plant form.In this review,we discuss genes involved in regulating the shoot apical meristem,inflorescence meristem,axillary meristem,root apical meristem,and vascular cambium in plants.We highlight several examples showing how crop breeders have manipulated developmental regulators to modify meristem growth and alter crop traits such as inflorescence size and branching patterns.Plant transformation techniques are another innovation related to plant meristem research because they make crop genome engineering possible.We discuss recent advances on plant transformation made possible by studying genes controlling meristem development.Finally,we conclude with discussions about how meristem research can contribute to crop improvement in the coming decades.

Development of new cowpea(Vigna unguiculata)mutant genotypes,analysis of their agromorphological variation,genetic diversity and population structure

Cowpea is one of the most important legume grains in the sub-Saharan region of Africa used for human consumption and animal feed,but its production is hampered by biotic and abiotic constraints raising the need to broaden its genetic basis.For this purpose,seeds of two cowpea varieties Melakh and Yacine were irradiated with 300 and 340 Gy of gamma-ray,respectively.The developed mutant populations were agromorphologically characterized from M5 to M7,while the genetic diversity of the latter was evaluated using 13 ISSR markers.Based on the agromorphological characterization,variation of flower color,pod length,seed coat color,and seed weight with 78.01,68.29,94.48,and 57.58%heritability,respectively,were recorded in the mutant lines.PCA analyses allowed to identify the elite mutants based on their agromorphological traits,while Pearson’s correlation results revealed a positive correlation between yield and yield component traits.Three subpopulations were identified through STRUCTURE analyses,but the assignment of the individuals in each group was improved using DAPC(Discriminant Analysis of Principal Components)analysis.Analysis of Molecular Variance revealed that the majority(85%)of the variance rather existed within groups than among(15%)groups.Finally,our study allowed us to select new promising mutant genotypes that could be tested for multi-locational trials to evaluate their agronomic performance.

Genetic Diversity in <i>Jatropha platyphylla</i>Accessions Based on Morphological Traits and Inter-Simple Sequence Repeats Molecular Markers

Seven accessions of Jatropha platyphylla were evaluated for their phenotypic traits and genetic diversity using inter-simple sequence repeats (ISSRs). Cluster analyses with nine traits were performed: number of branches per plant;fruit per bunch;bunch per branch;bunch per plant;total seed production;total fruit production, protein content, oil content, and fatty acid profile. Genotypes from Rosario, Sinaloa, Mexico (PR11) yielded the highest values in all traits. The correlation analysis of the quantitative traits showed high correlations between seed and total fruit production (r = 0.99). Unsaturated linoleic acid was the most abundant fatty acid (57.64% - 52.39%). Within a genetic improvement program, two of the most important variables to be considered are oil content and phenotypic characteristics of the plant. J. platyphylla has shown viable selection traits that provide a possibility of producing interspecies hybrids and giving them added value. ISSRs primers generated variable banding patterns that were found to be polymorphic;the polymorphic information content (PIC) of these loci ranged from 0.21 to 0.45 with an average of 0.34. The unweighted pair group method (UPGMA) cluster analysis of the data showed the formation of three groups, where the most divergent accession pair was the genotype from Quelite (QP11) and Rosario (PR11).

Morphological Characterization and Phenological Modeling of <i>Jatropha platyphylla</i>(Euphorbiaceae) Muell. Arg. Genotypes

Morphological characterization and phenological modeling were carried out on genotypes of Jatropha platyphylla collected from the states of Sinaloa and Durango, Mexico. The morphological characterization evidenced the existence of monoecious plants, finding individuals with male and female flowers in the same inflorescence. Fruit with four seeds was also found. The phenological study was divided into two phases and calculated in thermal requirement (°D): Vegetative [seedtime (0), germination (24), emergence (98), cotyledons (87), second (302) and fourth (524) true leaves, end of vegetative growth (302)] and reproductive [flowering (303), fructification (342), maturation (126), defoliation and senescence (450)]. The thermal constant (2558) was similar in all eight genotypes. The phenological stages and the accumulated degree days were adjusted with a third-degree polynomial (Stage = -0.0041x3 + 0.7446x2 - 8.6808x + 6.2448) (R2 = 0.99%) stage. The development of phenological models facilitates the prediction of the flowering date for the selection of varieties with high oil and protein content.

Rooting of Stem Cuttings of <i>Jatropha platyphylla</i>(Euphorbiaceae) in the Obtaining of Axillary Buds for Grafting

Jatropha platyphylla is considered as a potential source of edible protein, oil, and phenolic compounds with anti-inflammatory activity. The use of stem cutting in vegetative propagation and grafting is as indispensable tools for mass multiplication of superior genotypes and helps in improve planting yield and quality. The study was aimed to investigate the effect of different diameters (10 - 15, 16 - 25 and 26 - 35 mm) and different hormone concentrations of indo-butyric acid (0, 1.5, 3, 6 and 10 g/L), in the rooting of Jatropha platyphylla and to obtain axillary buds to performed grafts. Rooting efficiency was 80% in greenhouse conditions. Hormone concentration and diameter significantly affected the rooting and shooting ability of Jatropha platyphylla stem cuttings. Stem cuttings of 26 - 35 mm have the greatest number, length and dry root weight. 76% survival of the grafted plants was obtained. This demonstrates the necessity to improve the conventional propagation methods with appropriate scientific inputs for more economical and time efficient techniques for standard propagation protocols.

Phenotypic, Cytological and Molecular (AFLP) Analyses of the Cotton Synthetic Allohexaploid Hybrid (<i>G. hirsutum</i>×<i>G. longicalyx</i>)^²

The wild cotton diploid species (2n = 2x = 26) are important sources of useful traits such as high fiber quality, resistance to biotic and abiotic stresses etc., which can be introgressed into the cultivated tetraploid cotton Gossypium hirsutum L (2n = 4x = 52), for its genetic improvement. The African wild diploid species G. longicalyx Hutchinson and Lee could be used as donor of the desirable traits of fiber fineness and resistance to reniform nematode. However, hybridization of wild diploid species and cultivated tetraploid cotton encounters a sterility problem of the triploid (2n = 3x = 59), mainly due to ploidy. The restoration of the fertility can be done by creating an allohexaploid (2n = 6x = 78) through the doubling with colchicine of the sterile triploid chromosomes. With this method, a synthetic allohexaploid hybrid (G. hirsutum × G. longicalyx)2 has been obtained. This genotype was studied using phenotypic, cytological and molecular (AFLP) analyses in order to confirm its hybridity and its caryotype, and also to verify the expression of the desirable traits coming from G. longicalyx. The studied genotype showed a quite good level of pollen fertility (83%), and apart from larger seeds and some minor seedling anomalies, most of its morphological characteristics were intermediate between the two parental species. It had 78 chromosomes, proving its hexaploid status. Molecular analysis revealed 136 AFLP loci in this hexaploid, all from G. hirsutum and G. longicalyx, demonstrating its hybrid status. In addition, the hexaploid exhibited the useful traits of G. longicalyx with regard to its remarkable fiber fineness and its high resistance to the reniform nematode. This allohexaploid hybrid constitutes an interesting agronomic material, which can be used as a bridge for the transfer of useful agronomic traits from wild species to varieties of G. hirsutum.

Null Alleles in Gliadin Coding Loci and Wheat Allergenic Properties

Wheat gliadin proteins-an important, nutritional component of many food products may also act as allergenic proteins causing various, clinical symptoms of IgE-mediated food allergies. Gliadins are coded by six complex loci on the chromosomes 1A, 1B, 1D,6A, 6B and 6D of wheat genome. Each of the loci coding from a few to a dozen of polypeptides may spontaneously mutate to inactive gene variants called null alleles that do not code any proteins at all. The aim of the present work was to find out whether null alleles in some gliadin coding loci may decrease wheat allergenic properties. Six winter wheat genotypes: gliadin deletion lines (GDL) containing null alleles on 1D, 1B and 6B chromosomes and control lines (CL) containing active gene variants in all gliadin coding loci, were developed using plant breeding methods. Allergenic properties of the six analyzed hybrids were estimated by ELISA using polled sera of five patients allergic to gluten. Estimated immunoreactivity of GDLs was from 6% to 18% lower as compared with CLs. The obtained results evidenced that gliadin null alleles decrease wheat allergenic properties and may be used as parental forms for breeding of hypoallergenic wheat genotypes.

Constitutive basis of root system architecture:uncovering a promising trait for breeding nutrient-and drought-resilient crops

Root system architecture(RSA)plays a pivotal role in efficient uptake of essential nutrients,such as phosphorous(P),nitrogen(N),and water In soils with heterogeneous nutrient distribution,root plasticity can optimize acquisition and plant growth.Here,we present evidence that a constitutive RSA can confer benefits for sorghum grown under both sufficient and limiting growth conditions.Our studies,using P efficient SC103 and inefficient BTx635 sorghum cultivars,identified significant differences in root traits,with SC103 developing a larger root system with more and longer lateral roots,and enhanced shoot biomass,under both nutrient sufficient and deficient conditions.In addition to this constitutive attribute,under P deficiency,both cultivars exhibited an initial increase in lateral root development;however,SC103 still maintained the larger root biomass.Although N deficiency and drought stress inhibited both root and shoot growth,for both sorghum cultivars,SC103 again maintained the better performance.These findings reveal that SC103,a P efficient sorghum cultivar,also exhibited enhanced growth performance under N deficiency and drought.Our results provide evidence that this constitutive nature of RSA can provide an avenue for breeding nutrient-and drought-resilient crops.

Estimation of Genetic Parameters of Turiacu Pineapple Clones and Genetic Correlation between Traits

This study aimed to estimate genetic parameters of Turia&ccedil;u pineapple clones using mixed models;and estimate genetic correlation coefficient between traits, to provide important information to breeding programs. For this, 19 pineapple plants were visually selected and its slips were collected and planted in a single environment and nine traits were evaluated. The genetic parameters of most evaluated traits presented significant differences between the 19 clones. Some traits, such as Fusarium wilt resistance, showed high magnitudes of heritability. Clones did not differ in relation to total titratable acidity (TTA) and total soluble solids (TSS). However, these traits, which have implications to fruit quality, showed to be negatively correlated to fruit mass (-0.46 and -0.67). The results indicate that the population of Turia&ccedil;u pineapple is suitable for selection, especially for Fusarium wilt resistance. Also, Turia&ccedil;u clones can be used in breeding programs of pineapple.