Anther Culture Research Articles

Agronomic performance, yield stability and selection of doubled haploid rice lines in advanced yield trials

<abstract> <p>Anther culture is a widely utilized technique in rice breeding because of its simplicity and effectiveness in rapidly obtaining pure lines in the form of doubled haploid plants. The selection of doubled haploid (DH) rice lines derived from anther culture in advanced yield trials is an important step for obtaining superior DH lines. We aimed to determine agronomic performance, including yield and yield stability in order to select lowland DH rice lines that are high yield and have good agronomic performance based on the selection index method. The research was conducted in Indonesia at three locations, i.e., Bogor (West Java), Indramayu (West Java) and Malang (East Java) from July to December 2022. The genotypes tested were 29 DH lines and three check varieties (Inpari-42 Agritan GSR, Inpari-18 Agritan and Bioni63 Ciherang Agritan) using a randomized complete block design (RCBD) with genotypes as a single factor and three replications. High heritability values are found in all agronomic characters, except the percentage of filled grain/panicle, the percentage of empty grain/panicle and productivity. The yield stability based on the Kang method showed that 15 lines were stable and had high productivity. Phenotypic correlation analysis showed that the number of productive tillers, days to flowering, days to harvesting, number of filled grains/panicle and percentage of filled grains all had positive values and significantly correlated with productivity. Phenotypic path analysis showed that the character of days to harvesting, number of filled grains/panicle, number of productive tillers and percentage of filled grains/panicle directly affected the productivity. Based on the weighted selection index, 12 DH lines were selected due to having a positive and higher index (8.54 to 0.28) than the Bioni63 Agritan and Inpari 18 check varieties. Among those lines, 9 DH lines were also stable based on the Kang Method.</p> </abstract>

Effect of different concentrations and combinations of some plant growth regulators on Punica granatum anther culture

Plant growth regulators (PGR), also known as plant hormones, are a variety of chemical compounds that have a significant impact on the development and differentiation of plant cells. This work aimed to study the effect of various combinations of Plant Growth Regulators (PGRs) for example [6-Benzyladenine (BA), 2,4-dichlorophenoxyaceticg acid (2,4-D), Naphthalene acetic acid (NAA) and indol-3-ylbutyric acid (IBA)] on callus induction and shoot regeneration from anther culture of S1 progenies resulted from Manfalouty cultivar open pollinated. The results showed that highest value of percentage of callus induction was 10.6% on MS medium supplemented with 1 mg / l of NAA and 2 mg / l of BA also highest value of percentage of shoot formation 60 % on MS medium supplemented with 3 mg/ l of NAA and 2 mg /l of BA.

ПУТИ МОРФОГЕНЕЗА В КУЛЬТУРЕ IN VITRO ИЗОЛИРОВАННЫХ ПЫЛЬНИКОВ ПШЕНИЦЫ: РОЛЬ БАЛАНСА ФИТОГОРМОНОВ

The study is aimed to the study of the peculiarities of the induction of morphogenesis pathways in vitro of morphogenetically competent anther cells - microspores in the isolated anther culture of a number of spring soft wheat genotypes, recommended for cultivation in the climatic conditions of the Republic of Bashkortostan. The content of IAA in seedlings of the studied genotypes of spring sof wheat was analyzed to determine the class of the genotype – characterized by high or low auxin content. Such morphogenesis pathways in vitro as embryoidogenesis, polyembryoidogenesis and callusogenesis have been identified and studied. Morpho-histological assessment of androclinic structures is given. It was established that the induction of a certain morphogenesis pathway in vitro is determined mainly by the balance between the content of endogenous auxin IAA in anthers before inoculation on a nutrient medium and the concentration of exogenous synthetic auxin 2,4-D in the composition of the nutrient medium. The fundamental possibility of regulation of morphogenesis pathways in vitro in isolated anther culture of wheat by selecting for each genotype an adequate balance of endogenous and exogenous auxins for the induction of a specific pathway has been shown.

Cannabis sativa: Applications of Artificial Intelligence AI and Plant Tissue Culture for Micropropagation

This review paper highlights about the important applications of Artificial Intelligence (AI) and in vitro micropropagation of Cannabis. Cannabis micropropagation has largely been an underground effort with few peer reviewed studies. This lack of insight concerning in vitro cannabis techniques has limited the biotechnological utility of Cannabis crop. This is mainly due to the fact that Cannabis found to be recalcitrant under in vitro conditions, restrictions, long legacy of prohibition and stigmatization surrounding this Indian origin medicinal plant. Machine Learning (ML) and Deep Learning (DL) are two of the most exciting technological areas of Artificial Intelligence (AI). Data is a power today, and artificial intelligence (AI) can help Cannabis businesses to gather and analyze data in a wide variety of ways. Artificial Intelligence (AI) technology has enhanced Cannabis crop production and improved real-time monitoring, harvesting, processing and marketing. These technologies saves the excess use of water, pesticides, herbicides, maintains the fertility of the soil, and also helps in the efficient use of man power and elevated the productivity and improved the quality of Cannabis products. Artificial neural networks (ANNs) are widely used in science and technology, and have been successfully applied in Cannabis plant tissue cultures. Furthermore, Artificial neural networks (ANNs) can also simulate the growth of plants under different in vitro conditions. However, very few and limited in vitro regeneration protocols have been developed in Cannabis and existing protocols highlights only organogenesis. Therefore, there is a golden opportunity for the development of new in vitro regeneration protocols particularly induction of somatic embryogenesis, cryopreservation, protoplast isolation and culture, genetic transformation, production of synthetic seeds, and anther culture for the production of haploids in Cannabis.

Origem, evolução e estratégias para o melhoramento genético de fisális

ABSTRACT: Physalisperuviana L. (physalis) has significant economic potential by virtue of the unique flavor of its fruit. However, the productivity of Brazilian plantations is low because of the limited number of varieties or cultivars available. The main obstacle in the selection of superior genotypes is the lack of information about genetic variability within- and between- populations and limited genetic basis that has likely resulted from evolutionary, domestication and selection processes of the natural or artificial populations. Physalis currently cultivated in Brazil is tetraploid, and such polyploidy may have led to the reproductive isolation of the species, preventing the occurrence of intraspecific hybridization. Moreover, cultivated populations derive from a common gene pool and have undergone a long process of domestication and selection carried out empirically by farmers. In Colombia and other Andean countries there are wild populations that exhibit genetic diversity which; although, fundamental for the conservation of the species, have low potential for the development of genotypes with superior agronomic traits. In order to create and expand the genetic variability of physalis, breeders have employed various strategies including induction of mutation, chromosome duplication, and interspecific and intraspecific hybridization. Furthermore, the production of double haploid lines from in vitro anther cultures has shown good results in the selection of hybrids. The mutant genotypes and/or hybrids obtained using these methods in association with those of wide genomic selection can generate cultivars with superior agronomic traits.

Doubled Haploid Rice Lines Production through Anther Culture of F1 Derived from Abiotic Stress Tolerant Parents

Anther culture is one of the feasible technologies for accelerating rice breeding programs. This research aimed at determining the anther culture ability and doubled-haploid plants production of eight F1s derived from single crosses between indica rice parents that had a single or multiple abiotic stresses tolerance. The experiment was conducted using completely randomized design (CRD) with 15 replications. Medium for callus induction was based on N6 + 2.0 mg L-1 NAA +0.5 mg L-1 kinetin + 1 mM putrescine, while the regeneration medium was based on MS + 0.5 mg L-1 NAA + 2.0 mg L- kinetin 1 + 1 mM putrescine. Medium for rooting was MS + 0.5 mg L-1 IBA. The results indicated that all F1 populations gave varied responses to callus induction and plant regeneration. The F1populations from HS4-11-1-2/B13926E-KA-23 had higher anther culture ability compared to other seven F1 populations as indicated by the efficiency in callus formation and plant regeneration. Overall, the number of doubled haploid individuals (DHs) obtained was 158. F1 populations using HS4-11-1-2 as female parent, HS4-11-1-2/B13926E-KA-23 and HS4-11-1-2/CG8-93-1-1 contributed 45.4% and 34.9% doubled haploid individuals, respectively. The developed DHs lines need to be morphologically and agronomically characterized as well as screened under different combinations of abiotic stress to identify high-yielding and abiotic stress tolerant individuals for further rice breeding program.

Metabolomic Changes as Key Factors of Green Plant Regeneration Efficiency of Triticale In Vitro Anther Culture

Green plant regeneration efficiency (GPRE) via in vitro anther culture results from biochemical pathways and cycle dysfunctions that may affect DNA and histone methylation, with gene expression influencing whole cell functioning. The reprogramming from gametophytic to sporophytic fate is part of the phenomenon. While DNA methylation and sequence changes related to the GPRE have been described, little attention was paid to the biochemical aspects of the phenomenon. Furthermore, only a few theoretical models that describe the complex relationships between biochemical aspects of GPRE and the role of Cu(II) ions in the induction medium and as cofactors of enzymatic reactions have been developed. Still, none of these models are devoted directly to the biochemical level. Fourier transform infrared (FTIR) spectroscopy was used in the current study to analyze triticale regenerants derived under various in vitro tissue culture conditions, including different Cu(II) and Ag(I) ion concentrations in the induction medium and anther culture times. The FTIR spectra of S-adenosyl-L-methionine (SAM), glutathione, and pectins in parallel with the Cu(II) ions, as well as the evaluated GPRE values, were put into the structural equation model (SEM). The data demonstrate the relationships between SAM, glutathione, pectins, and Cu(II) in the induction medium and how they affect GPRE. The SEM reflects the cell functioning under in vitro conditions and varying Cu(II) concentrations. In the presented model, the players are the Krebs and Yang cycles, the transsulfuration pathway controlled by Cu(II) ions acting as cofactors of enzymatic reactions, and the pectins of the primary cell wall.

The study of the efficiency of anther culture <i>in vitro</i> of bread wheat varieties and hybrids <i>(Triticum aestivum L.)</i>

Anther culture is the most effective and not technically complex tool for obtaining double wheat haploids. The current study is devoted to the research of the influence of the genotype and concentration of the growth regulator 2.4-D on the efficiency of androgenesis in vitro of spring bread wheat. The purpose of the study was to estimate the efficiency of the anther culture of hybrids of the first and second generation and their parental varieties and to study its dependence on different concentrations of the growth regulator 2.4-D. There has been carried out the estimation of the traits of the F1hybrids, the F2population, and the parental varieties ‘Obskaya 2’ and ‘Novosibirskaya 15’ when 2.4-D was added to the induction medium at a concentration of 1 and 2 mg/l. There has been established that the F2population and the parental variety ‘Novosibirskaya 15’ are characterized by the largest indicators. The average values were significantly surpassed by the F2population in the traits ‘number of productive anthers per 100 anthers’ and ‘number of all regenerants per 100 anthers’ at 1 mg/l of 2.4-D. The variety ‘Novosibirskaya 15’ surpassed the average values in traits ‘number of sprouts per 100 anthers’ and ‘number of all regenerants per 100 sprouts’ at 2 mg /l of 2.4-D. The trial has shown that the best responsiveness of the anther culture for the studied genotypes was when the growth regulator 2.4-D was added to the induction medium at a concentration of 1 mg/l. The variety ‘Novosibirskaya 15’ has been characterized by good responsiveness in anther culturein vitroand could be used as a donor of valuable alleles in wheat breeding programs using DH technologies.

Somatic Embryogenesis, Genetic Modification, and GMOs at the Department of Plant Biotechnology and Cytogenetics at the IHAR

AbstractAt the Plant Breeding and Acclimatization Institute, National Research Institute (IHAR), research on plant tissue cultures began in 1970s. Some in vitro methods, such as anther cultures or isolated microspore cultures, have been directly used to produce homozygous lines in plant breeding. These methods are also a prerequisite for genetic manipulation for both scientific and practical purposes. Here, we describe the achievements of the Department of Biotechnology and Cytogenetics over the last few decades in the area of somatic embryogenesis of cereals, genetic modifications, GMO detection and identification, and GMO legislation. This long-term research on plant regeneration systems has led to the development of the first transgenic triticale plants in the world and the first transgenic plants in Poland, which was followed by a multi-generation study of triticale transgene stability. The gene flow of transgenic triticale and transgenic maize investigated in field studies under Polish environmental conditions, as well as the evaluation of triticale pollen flow, provided scientific data for the development of coexistence measures and GMO risk assessment in case of plant authorization for cultivation. Based on the experience gained over the years, a GMO Controlling Laboratory was established to support the official control of GMOs in Poland and the EU. It was later nominated as one of the National Reference Laboratories collaborating with the Joint Research Center (JRC) of the European Commission, providing scientific and technical support in areas related to health and consumer protection. The GMO Controlling Laboratory is accredited by the Polish Center for Accreditation according to the ISO 17025 standard, ensuring high-quality analyses and flexible accreditation scope. It participates in the ongoing debate in Poland and the EU on the legal aspects of new genomic techniques in plant breeding through publications, lectures at scientific conferences, and by providing expertise to relevant bodies.

PROTOCOL FOR IN VITRO ANTHER CULTURE IN TOMATO AND THE CHARACTERIZATION OF REGENERATED PLANTS

This paper presents a methodology of obtaining whole tomato plants from anther culture and their characterization. Year 1: the initiation of anther cultures and in vitro development of the new regenerated structures. Year 2: phenotypic characterization of plantlets acclimatized to ex vitro conditions, and their molecular analysis with SSR markers. Year 3: morphological characterization of plants obtained by germination of seeds from plants that fruited in the previous year and their molecular analysis with SSR markers. The phenotype descriptors and the SSR markers revealed differences among the regenerants and the donor variety. The uniformity of the genetic profiles with SSR markers in plants obtained from seeds of androgenic plants, but with several differences in comparison to anther-donor plants, indicates that some of the regenerated plantlets may have microspore origin

Improvement of Anther Culture to integrate Doubled Haploid Technology in Temperate Rice (Oryza sativa L.) Breeding.

Doubled haploid (DH) plant production, such as anther culture (AC), is an effective tool used in modern rice breeding programs. The improved efficient protocols applied can shorten the process of breeding. The effect of combinations of plant growth regulators (2.5 mg/L NAA, 1 mg/L 2,4-D and 0.5 mg/L kinetin; 2 mg/L 2,4-D and 0.5 mg/L BAP) in the induction medium were compared in AC for five rice breeding materials and combinations. Induction of calli ranged from 264.6 ± 67.07 to 468.8 ± 123.2 calli/100 anthers in AC of rice genotypes. Two basal media (MS and N6) and two combinations of growth regulators (1 mg/L NAA, 1 mg/L BAP and 1 mg/L kinetin; 1.5 mg/L BAP, 0.5 mg/L NAA and 0.5 mg/L kinetin) were used as regeneration media. The in vitro green plant production was the highest with the application of the N6NDK induction medium (NAA, 2,4-D and kinetin) and the MS-based regeneration medium (1 mg/L NAA, 1 mg/BAP and 1 mg/L kinetin) in anther culture of the '1009' genotype (95.2 green plantlets/100 anthers). The mean of five genotypes was 24.48 green plantlets/100 anthers for the best treatment. Flow cytometric analyses conducted identified the microspore origin of the haploid calli produced in AC, while the uniformity of spontaneous DH plants was checked in the DH1 and DH2 generations. Spontaneous chromosome doubling ranged from 38.1% to 57.9% (mean 42.1%), depending on the breeding source. The generated and selected DH lines were tested in micro- and small-plot field experiments to identify promising lines for a pedigree breeding program. The improved AC method was integrated in a Hungarian temperate rice pedigree breeding program.

Ploidy level of goldenberry, <em>Physalis peruviana</em>, plants obtained by anther culture

The goldenberry, Physalis peruviana L., is an important fruit in Colombia due to its export value and nutritional quality. However, commercial crops face challenges of fruit heterogeneity and the presence of diseases that reduce fruit yield and quality. These drawbacks could be handled through different breeding methods to develop uniform cultivars, for example, through anther culture, which is used to rapidly produce homozygous lines. However, the ploidy level may change when using this technique. Therefore, in this study, the level of ploidy of parental and the plants obtained by anther culture was determined by cytogenetics, flow cytometry, and single-simple repeat or microsatellites (SSR).Additionally, the homozygous condition of obtained plants and the degree of heterozygosity of parental plants were evaluated using SSR. Cytogenetic analysis showed parental plants with 48 chromosomes and anther culture generated plants with 24, 32 and 48 chromosomes, and mixoploids and average nuclear DNA content between 5.04 and 20.08 pg. Diploid, tetraploid, hexaploidy, and octoploid plants were identified by flow cytometry; the highest levels of ploidy (6x and 8x) correspond to mixoploid plants found by cytogenetics. The SSRs did not allow identifying ploidy due to the lack of a particular band pattern, however, they showed heterozygosity in most of the plants obtained by anther culture. It was concluded that the anther culture modified the ploidy level with respect to the parental plants, and that flow cytometry is efficient, precise, and less laborious, compared to cytogenetics, to determine the ploidy level of goldenberry in the laboratory.

Anther Culture-Derived Haploids of Citrus aurantium L. (Sour Orange) and Genetic Verification of Haploid-Derived Regenerated Plants.

Citrus plants are important fruit tree species; however, the breeding of high-quality varieties of citrus species is a time-consuming process. Using haploid-derived plants from anther culture may reduce the time required for obtaining purebred lines. This study aimed to genetically verify whether anther culture-derived sour orange (Citrus aurantium L.) plants developed from somatic embryos or haploid tissues. Sour orange anthers were cultured in N6 and MS media to induce calli and somatic embryos. N6 liquid medium supplemented with 1 mg·L-1 gibberellic acid and 200 µM spermidine resulted in a 10% increase in callus and embryo induction rates. Regenerated plants were validated using simple sequence repeat markers. Out of the 109 regenerated plants, ploidy analysis identified 99 diploids, two haploids, and eight putative aneuploids; out of the 99 diploid plants, 33 were haploid-derived homozygous diploids. The chromosomal analysis confirmed most plants as diploids, whereas some were identified as aneuploids (19-21 chromosomes). Furthermore, phylogenetic analysis confirmed that the resultant homozygous or heterozygous plants were haploid-derived. This is the first report of haploid-derived homozygous diploid and aneuploid sour orange plants obtained through anther culture. Moreover, the anther cultivation technique described herein can be applied to other citrus varieties.

The Production of Helianthus Haploids: A Review of Its Current Status and Future Prospects.

The genus Helianthus comprises 52 species and 19 subspecies, with the cultivated sunflower (Helianthus annuus L.) representing one of the most important oilseed crops in the world, which is also of value for fodder and technical purposes. Currently, the leading direction in sunflower breeding is to produce highly effective heterosis F1 hybrids with increased resistance to biotic and abiotic stresses. The production of inbred parental lines via repeated self-pollination takes 4-8 years, and the creation of a commercial hybrid can take as long as 10 years. However, the use of doubled haploid technology allows for the obtainment of inbred lines in one generation, shortening the time needed for hybrid production. Moreover, it allows for the introgression of the valuable genes present in the wild Helianthus species into cultivated sunflowers. Additionally, this technology makes it possible to manipulate the ploidy level, thereby restoring fertility in interspecific hybridization. This review systematizes and analyzes the knowledge available thus far about the production of haploid and dihaploid Helianthus plants using male (isolated anther and microspore cultures) and female (unpollinated ovaries and ovules culture) gametophytes, as well as by induced parthenogenesis using γ-irradiated pollen and interspecific hybridization. The genetic, physiological, and physical factors influencing the efficiency of haploid plant production are considered. A special section focuses on the approaches used to double a haploid chromosome set and the direct and indirect methods for determining the ploidy level. The current analyzed data on the successful application of haploid sunflower plants in breeding are summarized.

In Vitro Doubled Haploid Production of Bacterial Blight Resistant Plants from BC2F1 Plants (Ranbir Basmati X Pau148) Through Anther Culture

Doubled haploid plants are very important for the development of complete homozygous plants from heterozygous parents in one generation as they possess duplicate copy of haploid chromosome. Haploid production is easily obtained from in vitro anther culture. The present study was undertaken with the objective to develop doubled haploids using anthers for in vitro induction of callus on N6 medium supplemented with various combinations and concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5-2.5 mg/L), Kinetin (0.5-1.0 mg/L) and Naphthalene acetic acid (NAA) (2.0 mg/L) as callus induction medium (CIM). The highest callus induction frequency was obtained when N6 medium fortified with 2,4-D (2.5 mg/L), Kinetin (0.5 mg/L) and NAA (2 mg/L) of 10.07 per cent. The induced callus was sub cultured for shoot regeneration on Murashige and Skoog medium (MS) supplemented with growth regulators: Kinetin and NAA (0.5 mg/L each) in combination with BAP (0.0 - 2.5 mg/L). MS medium supplemented with NAA (0.5 mg/L), Kinetin (0.5 mg/L) and BAP (1.5 mg/L) was most responsive exhibiting regeneration frequency of 28.1 per cent which resulted in maximum regeneration of green plantlets and only 5.21 per cent of albinos. Individual plantlets were separated and immersed in liquid MS medium augmented with NAA (0.5-1.0 mg/L) and BAP (0.5-1.0 mg/L). Maximum rooting was observed in MS medium with NAA (0.5 mg/L) and BAP (1.0 mg/L). The survival rate of in-vitro raised plants was 51.51 per cent. Of these surviving plants, 21 plants were observed to have the sterility percentage above 50 percent and hence can be considered as the doubled haploid plants. Plant DH8 is susceptible and DH20 is heterozygous for gene Xa21. Two plants are susceptible for gene xa13

Anther culture performances of selected <em>Indica</em> rice varieties in Sri Lanka: ability of callus induction and plant regeneration

Tropical Agricultural Research and Extension is a peer-reviewed international scientific journal covering a wide range of subject areas in tropical and subtropical agriculture published quarterly by the Faculty of Agriculture, University of Ruhuna, Sri Lanka. The journal is also available on the Faculty of Agriculture, University of Ruhuna website https://www.agri.ruh.ac.lk/tare/index.htm .

Comparison of tissue culture-induced variation in triticale regenerants obtained by androgenesis and somatic embryogenesis

Triticale is becoming an increasingly important livestock crop production. This is evidenced by increasing triticale-producing areas and by improved yields. In addition, meeting the increasing demand for cereals involves the introduction of high-yielding and stress-resistant varieties into breeding. In vitro culture techniques can accelerate the development of new varieties. Therefore, it seems extremely important to develop efficient plant regeneration methods through in vitro cultures and to understand the mechanisms involved in gaining regenerants. Obtaining regenerants of triticale through somatic embryogenesis and androgenesis may lead to tissue culture-induced variation. In the present study, we compared regenerants obtained in both regeneration systems (anther and immature zygotic embryo cultures), considering the level of genetic and epigenetic changes observed in different DNA sequence contexts for methylated cytosine (CG, CHG, CHH). The changes concerning the DNA sequence (so-called sequence variation) and the changes concerning the DNA methylation patterns, i.e., the removal of methylated cytosine (DNA demethylation) and the introduction of methylation to cytosine (de novo DNA methylation), were analyzed. We observed that regenerants derived via somatic embryogenesis and androgenesis differ notably for demethylation in the symmetrical CG sequence context and de novo methylation in the asymmetrical CHH context. These changes may be related to the reprogramming of microspore development from gametophytic to sporophytic and lack of such process in zygotic embryos.

Inheritance of androgenesis response in pepper.

Anther culture has become an important part of pepper breeding. Response to haploidy via androgenesis is highly genotype-specific. However, studies on the inheritance of response to anther culture are lacking. Therefore, the study aimed to determine the inheritance of androgenesis. The plant material included crosses involving Capsicum annuum L. (253A, and Inan3363) X C. chinense PI 159,236 populations. To estimate the heritability of the trait, two pepper lines with very high androgenesis responses were crossed with PI 159,236, a non-responsive accession. The androgenesis response was phenotyped using the parents, F1, F2, BC1P1, and BC1P2/P3 created through reciprocal crosses. Using variance components, the number of genes controlling the trait, broad (H2 = 0.97), and narrow sense heritabilities (h2 = 0.20), genetic variance (VG=113.8), the additive (VA=23.9), and the dominance gene variances (VD = 89.9), as well as the environmental variance (VE = 3.6) were calculated. Additive and dominant gene effects were 21% and 79%, respectively. Results derived from two different populations showed that the number of genes controlling the trait was between 1.96 and 2.46, and H2 = 0.96-0.97, h2 = 0.20-0.65, VG=91.8-113.8, VA, = 23.9-62.7, VD = 29.1-89.9, and VE=3.5-3.6 were calculated. The X2 analysis indicated that the most suitable one is the 9: 3: 4 epistatic genetic model (X2 = 2.13, P = 0.343, N = 155). Results obtained from two different populations indicate the existence of a few major genes for response to androgenesis in pepper. Elucidating the inheritance of androgenesis is expected to pave the way for tagging the gene(s) in the pepper genome.

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice.

Lycopene epsilon-cyclase (LcyE) is a key enzyme in the carotenoid biosynthetic pathway of higher plants. Using the CRSPR/Cas9 and the geminiviral replicon, we optimized a method for targeted mutagenesis and golden SNP replacement of the LcyE gene in rice. We have exploited the geminiviral replicon amplification as a means to provide a large amount of donor template for the repair of a CRISPR-Cas-induced DNA double-strand break (DSB) in the target gene via homology-directed repair (HDR). Mutagenesis experiments performed on the Donggin variety achieved precise modification of the LcyE loci with an efficiency of up to 90%. In HDR experiments, our target was the LcyE allele (LcyE-H523L) derived from anther culture containing a golden SNP replacement. The phenotype of the homologous recombination (HR) mutant obtained through the geminiviral replicon-based template delivery system was tangerine color, and the frequency was 1.32% of the transformed calli. In addition, the total carotenoid content of the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines was 6.8–9.6 times higher than that of the wild-type (WT) calli, respectively. The reactive oxygen species content was lower in the LcyEsg2-HDR1 and LcyEsg2-HDR2 lines. These results indicate that efficient HDR can be achieved in the golden SNP replacement using a single and modular configuration applicable to different rice targets and other crops. This work demonstrates the potential to replace all genes with elite alleles within one generation and greatly expands our ability to improve agriculturally important traits.

Microsporogenesis in faba bean (Vicia faba L.) grown in Mersin, Turkey

Legumes are an alternative to animal foods due to their high protein content. Like other legumes, Vicia faba (faba bean) has high protein content. However, faba bean breeding with classical methods is challenging due to inbreeding depression, self-incompatibility, and abortive embryo formation. The inadequacy of classical breeding methods due to the problems in the fertilization biology of the plant limits the production of new varieties. Therefore, the importance of using in vitro haploidization technique in legume breeding is increasing. Anther culture is a widely preferred tissue culture technique for obtaining haploid plants. The initial and most crucial stage in anther culture is the identification of anthers containing microspores in the appropriate developmental stage. In the study, anther samples with single and triple-nucleated microspores were cultured in MS nutrient medium containing 2,4-D (0.5 mg L-1) and Kinetin (2.5 mg L-1). In the first month of culture, embryo and embryoid-like structures were obtained at the heart stage from anthers containing microspores in the mononuclear stage, while anther samples containing microspores in the trinucleated stage were observed to darken and did not develop.