Brassica napus Haploid and Double Haploid Production and Its Latest Applications.

Rapeseed is one of the most important oil crops in the world. Increasing demand for oil and limited agronomic capabilities of present-day rapeseed result in the need for rapid development of new, superior cultivars. Double haploid (DH) technology is a fast and convenient approach in plant breeding as well as genetic research. Brassica napus is considered a model species for DH production based on microspore embryogenesis; however, the molecular mechanisms underlying microspore reprogramming are still vague. It is known that morphological changes are accompanied by gene and protein expression patterns, alongside carbohydrate and lipid metabolism. Novel, more efficient methods for DH rapeseed production have been reported. This review covers new findings and advances in Brassica napus DH production as well as the latest reports related to agronomically important traits in molecular studies employing the double haploid rapeseed lines.

Quality of fish eggs and production of androgenetic and gynogenetic doubled haploids (DHs).

Induced development of haploid embryos (H) with only paternal (androgenesis) or maternal (gynogenesis) chromosomes requires irradiation of eggs before fertilization or activation of eggs with irradiated spermatozoa, respectively. To provide doubled haploids (DHs), androgenetic and gynogenetic haploid zygotes need to be subjected to the thermal or high hydrostatic pressure (HHP) shock to suppress the first mitotic cleavage and to double paternal or maternal haploid set of chromosomes. Androgenesis and mitotic gynogenesis (mito-gynogenesis) result in the generation of fully homozygous individuals in a single generation. DHs have been utilized in selective breeding programs, in studies concerning the phenotypic consequences of recessive alleles and to evaluate the impact of sex chromosomes on the early ontogeny. Moreover, the use of DHs for the NGS approach radically improves de novo the assembly of the genomes. However, reduced survival of the doubled haploids limits the wide application of androgenotes and gynogenotes. The high mortality of DHs may be only partly explained by the expression of recessive traits. Observed inter-clutch variation in the survival of DHs developing in eggs originating from different females make it necessary to take a closer look at the quality of the eggs used during induced androgenesis and gynogenesis. Moreover, the developmental competence of eggs that are subjected to irradiation before fertilization in order to deactivate maternal chromosomes when undergoing induced androgenesis and exposed to the physical shock after fertilization that leads to the duplication of the zygotes in both mito-gynogenesis and androgenesis may be also altered as irradiation and sublethal values of temperatures and hydrostatic pressure are considered as harmful for the cell organelles and biomolecules. Here, recently provided results concerning the morphological, biochemical, genomic, and transcriptomic characteristics of fish eggs showing high and low competence for androgenesis and mito-gynogenesis are reviewed.

Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture.

BACKGROUND: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley. RESULTS: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, 'Jersey' and 'Mercada' that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. 'Mercada' that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating 'Mercada' embryos contained a low number of plastome copies whose replication was not always completed. Contrary to 'Mercada', cv. 'Jersey' that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in 'Jersey' regenerants. CONCLUSIONS: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

Chromosome doubling methods in doubled haploid and haploid inducer-mediated genome-editing systems in major crops.

The doubled haploid technique aims to generate pure inbred lines for basic research and as commercial cultivars. The doubled haploid technique first generates haploid plants and is followed by chromosome doubling, which can be separated in time or overlapped, depending the procedure for each species. For a long time, much effort has been focused on haploid production via androgenesis, gynogenesis, or parthenogenesis. The obtention of haploid plants has frequently required more optimization and has lagged behind research and improvements in chromosome doubling methods. Nevertheless, chromosome doubling has recently been of renewed interest to increase the rates and efficiency of doubled haploid plant production through trialing and optimizing of different procedures. New antimitotic compounds and application methods are being studied to ensure the success of chromosome doubling once haploid material has been regenerated. Moreover, a haploid inducer-mediated CRISPR/Cas9 genome-editing system is a breakthrough method in the production of haploid plant material and could be of great importance for species where traditional haploid regeneration methods have not been successful, or for recalcitrant species. In all cases, the new deployment of this system will demand a suitable chromosome doubling protocol. In this review, we explore the existing doubled haploid and chromosome doubling methods to identify opportunities to enhance the breeding process in major crops.

Androgenic-Induced Transposable Elements Dependent Sequence Variation in Barley.

A plant genome usually encompasses different families of transposable elements (TEs) that may constitute up to 85% of nuclear DNA. Under stressful conditions, some of them may activate, leading to sequence variation. In vitro plant regeneration may induce either phenotypic or genetic and epigenetic changes. While DNA methylation alternations might be related, i.e., to the Yang cycle problems, DNA pattern changes, especially DNA demethylation, may activate TEs that could result in point mutations in DNA sequence changes. Thus, TEs have the highest input into sequence variation (SV). A set of barley regenerants were derived via in vitro anther culture. High Performance Liquid Chromatography (RP-HPLC), used to study the global DNA methylation of donor plants and their regenerants, showed that the level of DNA methylation increased in regenerants by 1.45% compared to the donors. The Methyl-Sensitive Transposon Display (MSTD) based on methylation-sensitive Amplified Fragment Length Polymorphism (metAFLP) approach demonstrated that, depending on the selected elements belonging to the TEs family analyzed, varying levels of sequence variation were evaluated. DNA sequence contexts may have a different impact on SV generated by distinct mobile elements belonged to various TE families. Based on the presented study, some of the selected mobile elements contribute differently to TE-related SV. The surrounding context of the TEs DNA sequence is possibly important here, and the study explained some part of SV related to those contexts.

Application of artificial intelligence models and optimization algorithms in plant cell and tissue culture.

Artificial intelligence (AI) models and optimization algorithms (OA) are broadly employed in different fields of technology and science and have recently been applied to improve different stages of plant tissue culture. The usefulness of the application of AI-OA has been demonstrated in the prediction and optimization of length and number of microshoots or roots, biomass in plant cell cultures or hairy root culture, and optimization of environmental conditions to achieve maximum productivity and efficiency, as well as classification of microshoots and somatic embryos. Despite its potential, the use of AI and OA in this field has been limited due to complex definition terms and computational algorithms. Therefore, a systematic review to unravel modeling and optimizing methods is important for plant researchers and has been acknowledged in this study. First, the main steps for AI-OA development (from data selection to evaluation of prediction and classification models), as well as several AI models such as artificial neural networks (ANNs), neurofuzzy logic, support vector machines (SVMs), decision trees, random forest (FR), and genetic algorithms (GA), have been represented. Then, the application of AI-OA models in different steps of plant tissue culture has been discussed and highlighted. This review also points out limitations in the application of AI-OA in different plant tissue culture processes and provides a new view for future study objectives. KEY POINTS: • Artificial intelligence models and optimization algorithms can be considered a novel and reliable computational method in plant tissue culture. • This review provides the main steps and concepts for model development. • The application of machine learning algorithms in different steps of plant tissue culture has been discussed and highlighted.

Development and innovation of haploid induction technologies in plants.

Haploid induction is one of the main techniques for breeding new varieties of major crops, and its key steps are improving the haploid induction rate and simplifying the induction procedure. With the development and innovation of plant haploid induction technologies, haploid breeding has been widely used in varietal improvement of important crops, showing the advantages of rapid homozygosity of heterozygous genes, shortening breeding period, and improving breeding efficiency. The combination of haploid breeding with crossing breeding, mutation breeding, reverse breeding, and molecular marker-assisted selection will greatly improve the effectiveness of crop breeding. Haploids and doubled haploids have demonstrated their usefulness in production of genetic populations, characterization of gene functions, and transgenic and cytological studies in plants. In this review, we summarize the progress of haploid induction technologies in view of various haploid induction techniques and applications of haploids and double haploids. In particular, the advances on the haploid induction in several major crops by genome editing were briefly described. Finally, we discuss current issues and future perspectives in this field, so as to promote the application of the haploid induction techniques, especially the techniques of creating haploid inducer lines by genome editing in crop breeding.

Embryogenic competence of microspores is associated with their ability to form a callosic, osmoprotective subintinal layer.

Microspore embryogenesis is an experimental morphogenic pathway with important applications in basic research and applied plant breeding, but its genetic, cellular, and molecular bases are poorly understood. We applied a multidisciplinary approach using confocal and electron microscopy, detection of Ca2+, callose, and cellulose, treatments with caffeine, digitonin, and endosidin7, morphometry, qPCR, osmometry, and viability assays in order to study the dynamics of cell wall formation during embryogenesis induction in a high-response rapeseed (Brassica napus) line and two recalcitrant rapeseed and eggplant (Solanum melongena) lines. Formation of a callose-rich subintinal layer (SL) was common to microspore embryogenesis in the different genotypes. However, this process was directly related to embryogenic response, being greater in high-response genotypes. A link could be established between Ca2+ influx, abnormal callose/cellulose deposition, and the genotype-specific embryogenic competence. Callose deposition in inner walls and SLs are independent processes, regulated by different callose synthases. Viability and control of internal osmolality are also related to SL formation. In summary, we identified one of the causes of recalcitrance to embryogenesis induction: a reduced or absent protective SL. In responding genotypes, SLs are markers for changes in cell fate and serve as osmoprotective barriers to increase viability in imbalanced in vitro environments. Genotype-specific differences relate to different responses against abiotic (heat/osmotic) stresses.

Alterations in the rainbow trout (Oncorhynchus mykiss) eggs exposed to ionizing radiation during induced androgenesis.

Ionizing radiation (IR) is applied to inactivate nuclear genome in the salmonid eggs to induce androgenetic development. However, it has been considered that doses of IR used to damage maternal chromosomes may also affect morphology of the eggs and decrease their developmental potential. Thus, the main goal of the present research was to assess alterations in the rainbow trout (Oncorhynchus mykiss) eggs caused by the high dose of IR administered during androgenesis. In the present research, rainbow trout eggs were irradiated with 350 Gy of X-rays, inseminated and exposed to the high hydrostatic pressure (HHP) shock to develop as androgenetic doubled haploids (DHs). The distribution of lipid droplets in the irradiated and non-irradiated rainbow trout eggs, survival rates and morphology of larvae from androgenetic and control groups were compared. It has been observed that non-irradiated and irradiated eggs exhibited altered distribution of lipid droplets. Most of the eggs before IR treatment displayed rather equal distribution of the oil droplets. In turn, majority of eggs studied after irradiation had coalesced lipid droplets, a pattern found in eggs with reduced quality. Incidences of abnormally developed larvae were more frequently observed among fish that hatched from the irradiated eggs. Observed changes suggest X-rays applied for the genetic inactivation of rainbow trout eggs may lead to decrease of their developmental competence.

Sex mosaics in the honeybee: how haplodiploidy makes possible the evolution of novel forms of reproduction in social Hymenoptera.

Hymenoptera are haplodiploid: females arise from fertilized, diploid eggs, while males arise from unfertilized, haploid eggs. The cytogenetic mechanisms underlying haplodiploidy enable remarkable phenomena including female cloning, male cloning and gynandromorphy (sex mosaics). We collected 11 newly emerged putative gynandromorph honeybees from a single colony, assessed the sex of various tissues morphologically and determined the genetic origin (maternal or paternal) of each tissue by genotyping. Ten bees were gynandromorphs with one to three distinct paternal origins. Remarkably, one bee carried no maternal alleles. This bee had female organs throughout, and arose from the fusion of two sperm nuclei. This is the first reported case in the Hymenoptera of sperm fusion resulting in a female, emphasizing the flexibility for social insect reproduction and potentially novel colony-level social structures.

Male contraceptive efficacy of poly herbal formulation, contracept-TM, composed of aqueous extracts of Terminalia chebula fruit and Musa balbisiana seed in rat.

CONTEXT: Terminalia chebula Retz (Combretaceae) and Musa balbisiana Colla (Musaceae) have a traditional reputation as a male contraceptive. OBJECTIVE: To determine the hypo-testicular activity of aqueous extracts of Terminalia chebula (fruit) and Musa balbisiana (seed) separately, and in composite manner at the ratio of 1:1 named as 'Contracept-TM' compared to cyproterone acetate (CPA), for developing a polyherbal contraceptive. MATERIALS AND METHODS: The separate extract of above said plants or 'Contracept-TM' at the dose of 40 mg/100 g body weight of rat/day or CPA at 2 mg/100 g body weight of rat/day was administered for 28 days. Spermiological, androgenic and oxidative stress sensors, LD50 and ED50/100 g body weight values were measured. RESULTS: Treatment of individual, 'Contracept-TM' or CPA resulted significant decrease in the count of spermatogonia A (36.36-49.09%), pre-leptotene spermatocyte (19.11-55.30%), mid-pachytene spermatocyte (28.65-47.28%) and step 7 spermatid (29.65-51.59%). Activities of testicular Δ5, 3ß (21.25-48.02%),17ß-hydroxysteroid dehydrogenases (29.75-55.08%), catalase (19.06-43.29%) and peroxidase (30.76-62.82%), levels of testosterone (28.15-63.44%), testicular cholesterol (19.61-49.33%), conjugated diene (29.69-84.99%) and thiobarbituric acid reactive substances (41.25-86.73%) were elevated compare to the control. The ED50 and LD50 values were 40 mg and 5.8 g (T. chebula), 48 mg and 6.3 g (M. bulbisiana), 40 mg and 6.0 g ('Contracept-TM'), respectively. DISCUSSION AND CONCLUSION: The said spermiological and androgenic sensors' levels were decreased significantly by 'Contracept-TM' than its constitutional individual plant extract and it may be comparable to standard anti-testicular drug like CPA. So, it may be concluded that above polyherbal formulation is potent for inducing hypo-testicular activity.

Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

Genetic uniformity and long-distance clonal dispersal in the invasive androgenetic Corbicula clams.

The clam genus Corbicula is an interesting model system to study the evolution of reproductive modes as it includes both sexual and asexual (androgenetic) lineages. While the sexual populations are restricted to the native Asian areas, the androgenetic lineages are widely distributed being also found in America and Europe where they form a major aquatic invasive pest. We investigated the genetic diversity of native and invasive Corbicula populations through a worldwide sampling. The use of mitochondrial and nuclear (microsatellite) markers revealed an extremely low diversity in the invasive populations with only four, undiversified, genetic lineages distributed across Europe and America. On the contrary, in the native populations, both sexual and androgenetic lineages exhibited much higher genetic diversity. Remarkably, the most abundant and widely distributed invasive forms, the so-called form A and form R found in America and Europe respectively, are fixed for the same single COI (cytochrome c oxydase subunit I) haplotype and same multilocus genotype. This suggests that form R, observed in Europe since the 1980s, derived directly from form A found in America since the 1920s. In addition, this form shares alleles with some Japanese populations, indicating a Japanese origin for this invasive lineage. Finally, our study suggests that few androgenetic Corbicula individuals successfully invaded the non-native range and then dispersed clonally. This is one striking case of genetic paradox raising the issue of invasive and evolutionary success of genetically undiversified populations.

Correlation of stages of microsporogenesis with bud and anther morphology in pepper genotypes through DAPI staining with different levels of mordant in cytological fixative.

The haploid and doubled haploid plants serve as valuable tools for breeders due to their ability to expedite the mapping of genes of agronomic importance, as well as accelerate the breeding cycle for generation of novel hybrids and improved homogenous varieties. Successful anther/microspore culture largely depends on the use of microspores at appropriate developmental stages at the time of culture, which can be specific for each plant species and genotype. In the present study, we described the visible morphological characteristics of flower buds and anthers at different developmental stages to identify the optimal microspore stage within the anther/buds of two pepper hybrids, Indra and Lakshmi. This information enabled us to predict the suitable microspore stage for successful haploid production. To enhance the visualization of nuclei in the pepper microspores, different concentrations of FeCl3 were employed as a mordant to Carnoy's fixative I, followed by DAPI staining. A clear and distinct nucleus was observed using DAPI staining procedures in the pepper microspores when fixed in Carnoy's solution containing ferric chloride (40-90 µl) as mordant. The use of mordant thus facilitated the efficient cytological analysis of the pepper microspores. Present results indicate that, to achieve efficient haploid production, flower buds with an average length of 4.4 to 5.02 mm for the hybrid Indra and 5.15 to 5.40 mm for the hybrid Lakshmi should be utilized. Additionally, these buds should have a calyx covering approximately 80-90% of the total bud length. We observed that in such buds, microspores are in the late-uninucleate and early binucleate stage which has been reported to be the most conducive stage for androgenesis induction in pepper.

Metabolic changes during wheat microspore embryogenesis induction using the highly responsive cultivar Svilena.

Androgenetically-derived haploids can be obtained by inducing embryogenesis in microspores. Thus, full homozygosity is achieved in a single generation, oppositely to conventional plant breeding programs. Here, the metabolite profile of embryogenic microspores of Triticum aestivum was acquired and integrated with transcriptomic existing data from the same samples in an effort to identify the key metabolic processes occurring during the early stages of microspore embryogenesis. Primary metabolites and transcription profiles were identified at three time points: prior to and immediately following a low temperature pre-treatment given to uninuclear microspores, and after the first nuclear division. This is the first time an integrative -omics analysis is reported in microspore embryogenesis in T. aestivum. The key findings were that the energy produced during the pre-treatment was obtained from the tricarboxylic acid (TCA) cycle and from starch degradation, while starch storage resumed after the first nuclear division. Intermediates of the TCA cycle were highly demanded from a very active amino acid metabolism. The transcription profiles of genes encoding enzymes involved in amino acid synthesis differed from the metabolite profiles. The abundance of glutamine synthetase was correlated with that of glutamine. Cytosolic glutamine synthetase isoform 1 was found predominantly after the nuclear division. Overall, energy production was shown to represent a major component of the de-differentiation process induced by the pre-treatment, supporting a highly active amino acid metabolism.

Androgenesis is a maternal trait in the invasive ant Wasmannia auropunctata.

Androgenesis is the production of an offspring containing exclusively the nuclear genome of the fathering male via the maternal eggs. This unusual mating system is generally considered a male trait, giving to androgenetic males a substantial fitness advantage over their sexually reproducing relatives. We here provide the first empirical study of the evolutionary outcomes of androgenesis in a haplo-diploid organism: the invasive ant Wasmannia auropunctata. Some of the populations of this species have a classical haplo-diploid sexual mating system. In other populations, females and males are produced through parthenogenesis and androgenesis, respectively, whereas workers are produced sexually. We conducted laboratory reciprocal-cross experiments with reproductive individuals from both types of populations and analysed their progenies with genetic markers, to determine the respective contribution of males and females to the production of androgenetic males. We found that androgenesis was a parthenogenetic female trait. A population genetic study conducted in natura confirmed the parthenogenetic female origin of androgenesis, with the identification of introgression events of sexual male genotypes into androgenetic/parthenogenetic lineages. We argue that by producing males via androgenesis, parthenogenetic queen lineages may increase and/or maintain their adaptive potential, while maintaining the integrity of their own genome, by occasionally acquiring new male genetic material and avoiding inbreeding depression within the sexually produced worker cast.

Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning.

Induction of embryogenesis from isolated microspore cultures is a complex experimental system where microspores undergo dramatic changes in developmental fate. After ~40 years of application of electron microscopy to the study of the ultrastructural changes undergone by the induced microspore, there is still room for new discoveries. In this work, high pressure freezing and freeze substitution (HPF/FS), the best procedures known to date for ultrastructural preservation, were used to process Brassica napus microspore cultures covering all the stages of microspore embryogenesis. Analysis of these cultures by electron microscopy revealed massive processes of autophagy exclusively in embryogenic microspores, but not in other microspore-derived structures also present in cultures. However, a significant part of the autophagosomal cargo was not recycled. Instead, it was transported out of the cell, producing numerous deposits of extracytoplasmic fibrillar and membranous material. It was shown that commitment of microspores to embryogenesis is associated with both massive autophagy and excretion of the removed material. It is hypothesized that autophagy would be related to the need for a profound cytoplasmic cleaning, and excretion would be a mechanism to avoid excessive growth of the vacuolar system. Together, the results also demonstrate that the application of HPF/FS to the study of the androgenic switch is the best option currently available to identify the complex and dramatic ultrastructural changes undergone by the induced microspore. In addition, they provide significant insights to understand the cellular basis of induction of microspore embryogenesis, and open a new door for the investigation of this intriguing developmental pathway.

The kin selection theory of genomic imprinting and modes of reproduction in the eusocial Hymenoptera.

Genomic imprinting is known from flowering plants and mammals but has not been confirmed for the Hymenoptera even though the eusocial Hymenoptera are prime candidates for this peculiar form of gene expression. Here, the kin selection theory of genomic imprinting is reviewed and applied to the eusocial Hymenoptera. The evidence for imprinting in eusocial Hymenoptera with the typical mode of reproduction, involving the sexual production of diploid female offspring, which develop into workers or gynes, and the arrhenotokous parthenogenesis of haploid males, is also reviewed briefly. However, the focus of this review is how atypical modes of reproduction, involving thelytokous parthenogenesis, hybridisation and androgenesis, may also select for imprinting. In particular, naturally occurring hybridisation in several genera of ants may provide useful tests of the role of kin selection in the evolution of imprinting. Hybridisation is expected to disrupt the coadaptation of antagonistically imprinted loci, and thus affect the phenotypes of hybrids. Some of the limited data available on hybrid worker reproduction and on colony sex ratios support predictions about patterns of imprinting derived from kin selection theory.

Induction of Triticale (×Triticosecale Wittmack) In Vitro Androgenesis in Anther Cultures of F1 Hybrid Combinations, Varieties and Homogeneity Testing of Offspring Generation.

In cereal breeding, in vitro androgenesis methods are frequently applied to achieve doubled haploid (DH) plants. The aim of this study was to determine the effects of genotype (three registered varieties and eight F1 crossing combinations) and induction medium (W14mf and P4mf) on anther cultures (ACs) of triticale (×Triticosecale Wittmack). Androgenesis was induced in the treatment of each tested genotype, and the genotype significantly influenced the efficiency of AC, including in embryo-like structures (ELSs), albinos, green plantlets, and transplanted plantlets. The utilized medium also had a significant effect on the number of ELSs, albinos, and transplanted plantlets. Both media were suitable for AC in triticale DH plant production. The efficiency of AC was higher when using the P4mf medium (103.7 ELS/100 anthers, 19.7 green plantlets/100 anthers) than when using the W14mf medium (90.0 ELS/100 anthers, 17.0 green plantlets/100 anthers). However, the green plantlet regeneration efficiency of microspore-derived structures was 18.0% when using the W14mf medium, while this value was 15.9% in the case of ELSs induced with the P4mf medium. After nursery seed evaluation and propagation (DH1), the genetic homogeneity of the offspring generation (DH2) was tested using a molecular genetic method. Most of the tested DH lines showed homogeneity and were progressed into a breeding program after agronomic selection. Some DH lines showed inhomogeneity, which could be explained by the outcross inclination of triticale. We would like to call breeders' attention to the outcross character of triticale and emphasize the vigilant propagation and maintenance of the triticale DH lines in breeding programs. Due to the outcross nature of triticale, even in self-pollinated genotypes, breeders should focus on careful maintenance, along with isolation in the case of line propagations, in triticale breeding programs.

Haploid Induction in Indica Rice: Exploring New Opportunities.

Haploid plants are of significant interest to crop breeders due to their ability to expedite the development of inbred lines. Chromosome-doubling of haploids, produced by either in vitro or in vivo methods, results in fully homozygous doubled haploids. For nearly five decades, in vitro methods of anther and microspore culture have been attempted in many crops. In rice, in vitro methods are used with some success in japonica cultivars, although indica types have remained recalcitrant to a large extent. This review aims to explore the reasons for the lack of success of in vitro methods in indica rice and discuss new advancements in in vivo haploid induction protocols in other cereals and their relevance to rice. In particular, the current level of understanding of in vivo haploid inducer systems that utilize MTL and CENH3 mutants is analyzed in detail. One notable advantage of in vivo haploid induction systems is that they do not require tissue culture competence. This makes these methods more accessible and potentially transformative for research, offering a pragmatic approach to improving indica rice cultivars. By embracing these in vivo methods and harnessing the power of gene editing technologies like CRISPR/Cas9 systems, breeders can reshape their approach to indica rice improvement.