Triploid Plants Research Articles

Triploid Formation and Heat Tolerance of Angelonia angustifolia with Various Ploidy Levels

Angelonia (Angelonia angustifolia), a popular potted flower and bedding plant, often experiences severe wilting or leaf drying during hot summers in tropical and subtropical regions. Polyploidization may enhance the heat tolerance of angelonia plants. In this study, diploid (2x) angelonia ‘Serena Purple’ and ‘Serena White’ were crossed with tetraploid (4x) lines to produce interploidy plants. More seed production was observed from 4x × 2x crosses than from 2x × 4x crosses. Despite observing triploid block in interploidy hybridization, triploid plants were successfully obtained from the interploidy seeds. Growth, flowering, and photosynthesis of diploid, triploid, and tetraploid plants were assessed at day/night temperatures of 30/25 °C and 25/20 °C. All angelonia plants exhibited increased height and stem diameter as well as reduced leaf SPAD-502 value, leaf thickness, flower number, flower length, net photosynthetic rate, and stomatal conductance at 30/25 °C compared with those at 25/20 °C. At 30/25 °C, triploid and tetraploid plants had higher leaf SPAD-502 values, leaf thickness, and net photosynthetic rates than those of diploid plants. Diploid plants had the most branches at 25/20 °C, but their branch and flower numbers decreased the most at 30/25 °C compared with those of triploid and tetraploid plants. Notably, at 30/25 °C, triploid plants exhibited the earliest f lowering and produced the most branches and flowers compared with those of diploid and tetraploid plants.

Exploring the Role of Endophytes in Cannabis sativa L. Polyploidy and Agricultural Trait Improvement

Here, we examine the effects of ploidy variation in Cannabis sativa L. cell lines on the plant host genotype-associated microbiome. The endophytic microbiome has a protocooperative role in improving plant health and productivity and represents an alternative to synthetic chemical fertilizers and pesticides in sustainable agriculture. This study assessed the effects of seed endophytes on diploid and triploid Haze hemp cultivars. Key phenotypic characteristics were evaluated, revealing significant differences in seed germination in vitro as well as vegetative growth and flowering in phytotron conditions. Endophyte-treated triploid plants exhibited significantly taller heights compared to diploids (p < 0.01). These treated triploid plants also showed longer leaves at nodes 2, 6, and 8, except at node 4, indicating a plant in transition from vegetative growth to the generative developmental stage. Additionally, triploids treated with endophytes displayed the highest number of axillary branches, while endophyte-treated diploids had the fewest (p < 0.05). Both cultivars treated with endophytes exhibited a higher number of inflorescences compared to untreated control plants. This study revealed for the first time a direct correlation between the shifts in diameter of the stem and the biomass in both tested hemp hosts, in association with endophytic microbiomes.

Characteristics of microsporogenesis in the triploid apple tree and it’s parental form in connection with usage in breeding at the polyploid level

An effective method of creating triploid apple cultivars, which are superior in their characteristics to diploid cultivars, is the crossing of heterochromosomal forms of the type: 2x×4x, 4x×2x. In order to provide a wide range of genetic diversity of riploid hybrids, it is necessary to have a large set of tetraploid donor forms of diploid gametes. A limited set of such forms is a limiting factor for this direction of breeding. And the effectiveness of work on obtaining new valuable apple varieties based on the use of the polyploidy method largely depends on their diversity Therefore, the identification of new apple tetraploids, as well as the study of cytoembryological features of their generative structures, the determination of the quality of the formed gametes necessary for successful breeding and for predicting the results of these works are relevant. Meiosis during microsporogenesis was studied in two tetraploid apple forms. To study meiosis during microsporogenesis, temporary pressed specimen were prepared by the acetohematoxylin method; the propionic-lacmoid method was used to determine the ploidy of hybrid offspring. Microscopic studies were carried out on a Nikon-50i microscope at a magnification of 10 x 105 x 40, 10 x 1.5 x 100. The analysis of the course of meiosis during microsporogenesis in apple tetraploid 34-21-39 [30-47-88(4x)×Krasa Sverdlovska(2x)] and its parent form 30-47-88 [Liberty×13-6-106 (Suvorovetz seedling)] showed that the hybrid 34-21-39 was characterized by the most correct course of meiosis. The largest percentage of deviations and the diversity of morphological types of disorders were detected in the form 30-47-88. Common types of violations were noted. Despite the presence of disorders during meiosis in tetraploid forms, microsporogenesis ended with the formation of visually normal one–dimensional pollen: 89% in the form 34-21-39 and 50% in the form 30-47-88. In combinations of crosses involving the studied forms, a high yield of triploid plants was noted. Consequently, the tetraploid apple forms 34-21-39 and 30-47-88 can be recommended for hybridization for the purpose of mass production of triploid seedlings. The results allow us to conclude that the tetraploid apple forms 34-21-39 and 30-47-88 can be used in breeding as pollinators in heteroploid crosses (2x×4x) to obtain triploid genotypes.

Understanding the Saffron Corm Development-Insights into Histological and Metabolic Aspects.

The reproduction of Crocus sativus L., a sterile triploid plant, is carried out exclusively through corms, whose size determines the saffron yield. The development of daughter corms (DC) is supported by photoassimilates supplied by the leaves as well as by the mother corms (MC). While biomass partitioning during DC development is well studied, growth dynamics in terms of cell number and size, the involved meristems, as well as carbohydrate partition and allocation, are not yet fully understood. We conducted a comprehensive study into saffron corm growth dynamics at the macroscopic and microscopic levels. Variations in carbohydrate content and enzymatic activities related to sucrose metabolism in sources and sinks were measured. Two key meristems were identified. One is involved in vascular connections between DC and MC. The other is a thickening meristem responsible for DC enlargement. This research explains how the previously described phases of corm growth correlate with variations in cell division, enlargement dynamics, and carbohydrate partitioning among organs. Results also elucidated that the end of DC growth relates to a significant drop in MC root biomass, limiting the water supply for the DC growth, and establishing the onset of leaf wilting. The lack of starch accumulation in aged leaf cells is noteworthy, as is the accumulation of lipids. We hypothesize a signaling role of sugars in DC growth initiation, stop, and leaf aging. Finally, we established a predominant role of sucrose synthase as a sucrolytic enzyme in the maintenance of the high flux of carbon for starch synthesis in DC. Together, the obtained results pave the way for the definition of strategies leading to better control of saffron corm development.

Cannabis Triploids Exhibit Reduced Fertility and Similar Growth and Flower Production Compared to Diploids

Cannabis (Cannabis sativa) grown for flowers containing cannabinoids requires all female plants, which are susceptible to seed set from exposure to pollen. Created triploids demonstrated reduced seed production compared with diploids in field and greenhouse studies in which plants were challenged with pollen from males. In the field, seed production as a percent of floral biomass ranged from 6.7% to 18.0% for triploids and from 52.6% to 57.1% for diploids. The photoperiod-insensitive triploid genotype ‘Purple Star’ × ‘Wilhelmina’ had 98.5% fewer filled (containing a developed embryo) seeds than the photoperiod-insensitive diploid genotype ‘Tsunami’ × ‘Wilhelmina’. In the greenhouse, triploid ‘Wife’ had 99.5% fewer filled seeds than diploid ‘Wife’. Plant growth and flower production were similar with eight triploid and seven diploid genotypes evaluated over three greenhouse studies. There were a few superior triploid and diploid genotypes; however, their performance was more likely attributable to the parental cultivar combination than ploidy level. The optimal cross direction for producing triploid seed in large quantities is tetraploid × diploid because the diploid × tetraploid cross exhibits triploid block caused by endosperm paternal excess. Colchicine-induced tetraploid parent plants should be tested over a prolonged period to eliminate cryptic chimeral mixoploids or tetraploid plants should be derived from seed produced by crossing two colchicine-induced putative tetraploid plants to ensure that seeds from tetraploid × diploid crosses will be triploid. The latter approach is necessary for photoperiod-insensitive cultivars because a prolonged period of ploidy testing is not possible for these plants. These findings indicate that triploid plants have significantly reduced fertility and are a suitable alternative to diploids in situations in which pollen exposure is possible.

In vitro regeneration of triploid from mature endosperm culture of Passiflora edulis "Mantianxing".

The regeneration of shoots from endosperm tissue is a highly effective method to obtain triploid plants. In this study, we elucidated the establishment of an in vitro regeneration system from endosperm culture for the production of Passiflora edulis "Mantianxing." The highest callus induction rate (83.33%) was obtained on the media supplemented with 1.0mg/L TDZ. Meanwhile, the MS medium containing 1.0mg/L 6-BA and 0.4mg/L IBA gave the optimum 75% shoot bud induction. Chromosome analysis revealed that the chromosomal count of P. edulis "Mantianxing" regenerated from endosperm tissues was 27 (2n=3x=27), which indicated that shoots regenerated from endosperm tissues were triploids. Triploid P. edulis had more drought resistance than diploid plants. Our study provided a method for breeding of passion fruit by means of a stable and reproducible regeneration system from endosperm culture, leading to the generation of triploid plants.

Grafting seedling rootstock strengthens tolerance to drought stress in polyploid mulberry (Morus alba L.)

The economically adaptable mulberry (Morus alba L.) has a long history of grafting in China, yet the physiological mechanisms and advantages in drought tolerance remain unexplored. In our study, we investigated the responses of self-rooted 2X (diploid), 3X (triploid), and 4X (tetraploid) plants, as well as polyploid plants grafted onto diploid seedling rootstocks (2X/2X, 3X/2X, and 4X/2X) under drought stress. We found that self-rooted diploid plants exhibited the most severe phenotypic damage, lowest water retention, photosynthetic capacity, and the least effective osmotic stress adjustment compared to tetraploid and triploid plants. However, grafted diploid and triploid plants showed effective mitigation of drought-induced damage, with higher relative water content and improved soil water retention. Grafted plants also improved the photosystem response to drought stress through elevated photosynthetic potential, closed stomatal aperture, and faster recovery of chlorophyll biosynthesis in the leaves. Additionally, grafted plants altered osmotic protective compound levels, including starch, soluble sugar, and proline content, thereby enhancing drought resistance. Absolute quantification PCR indicated that the expression levels of proline synthesis-related genes in grafted plants were not influenced after drought stress, whereas they were significantly increased in self-rooted plants. Consequently, our findings support that self-rooted triploid and tetraploid mulberries exhibited superior drought resistance compared to diploid plants. Moreover, grafting onto seedling rootstocks enhanced tolerance against drought stress in diploid and triploid mulberry, but not in tetraploid. Our study provides valuable insights for a comprehensive analysis of physiological effects in response to drought stress between stem-roots and seedling rootstocks.

Polyploidy in the history of breeding at the Yaltushkiv Experimental Breeding Station: new methods of differentiation and stabilisation of sugar beet tetraploids

Purpose. To analyse the history of the polyploid breeding at the Yaltushkiv Experimental Breeding Station, in particular the improvement of genetic models of new hybrids, new methods of induction of meiotic tetraploids, differentiation and selection of breeding genotypes by genome ploidy.
 Methods. Cytological analysis of metaphase chromosomes of the apical meristems of the growth points of seed plants and beets of the first year for the selection of tetraploid plants 4x = 36 chromosomes; cytophotometric analysis using computer programs AP Partec to study ploidy based on the quantitative content of nuclear DNA; field methods (selection of tetraploid seed plants according to the types of pollen grains, formation of synthetic populations of tetraploid multigerm plants on a cytogenetic basis according to the structure of bivalent conjugation of homologous chromosomes in meiosis.
 Results. In the process of tetraploid induction, cytological methods of ploidy analysis at the station were improved using computer programs of AP Partec for the selection of sugar beets 4x by the quantitative content of nuclear DNA in the cell on plants of the first year of vegetation and for the formation of tetraploid populations after colchicine induction in seed plants. In the process of studying the structure of polyploid populations by ploidy, the percentage of tetraploids varied from 82.0±2.22% in Ya/Hen pollinator to 58.1±2.81% in Ya/Man pollinator. It was established that the selection for fertility and pollen uniformity depends on the genetic characteristics of the breeding genotypes and affects the pollen-forming ability in the first generations after induction with colchicine, reducing the number of 4x IIa and IIb biotypes according to cytological classification. Using the example of the multigerm pollinator Ya/Roi, it was established that selection for sugar content and productivity had, to a certain extent, a stabilising effect on the degree of ploidy. The biotechnological methods used to deposit 4x genotypes with a high percentage of bivalent conjugation of homologous chromosomes in metaphase I of meiosis (70–90%) contributed to the cytogenetic stability of polyploid gametes and their high yield – 92.9% of 4x in the next generation of seeds. It was investigated that the productivity of triploid hybrids obtained with the use of the tetraploid pollinator Ya/DA 4x based on the germplasm of the wild species Beta patula at the early stages of vegetative development varied in triploid plants from 325 to 465 g, and the sugar content in some numbers reached 21.5%.
 Conclusions. The development of polyploid selection at the Yaltushkiv Experimental Breeding Station evolved from the development polyhybrids to the development of triploid hybrids based on CMS using tetraploid multigerm pollinators. For the formation of populations of tetraploid multigerm pollinators, the methods of fluorescent cytophotometry and computer programs AP Partec were used, as well as biotechnological methods for creating synthetic populations of tetraploid multigerm pollinators on a cytogenetic basis, which ensure the yield of tetraploids in seeds up to 92.9%.

Improvement of Citrus Rootstock Hybrids Derived by 2x × 2x Intra Crosses with the Aid of Embryo Rescue and Ploidy Detection

Diploid Citrus × Poncirus hybrids have significantly contributed to citrus rootstock evaluation. In Türkiye, common sour orange rootstock is used in many different climates and soil conditions at almost 85% of citrus plantations, but this rootstock is susceptible to Citrus tristeza virus disease. This study was conducted to improve new rootstock genotypes by traditional hybridization. Common sour orange (CSO) and Common mandarin (Citrus deliciosa Ten.) (CM) were crossed with Troyer citrange (TC) while King mandarin (KM) was crossed with Carrizo citrange (CC). Embryos obtained from crosses were taken on 110, 120 and 130 days after artificial pollination (DAP), and were germinated on MT culture media. The embryos of 120 DAP of combinations had the highest germination rate within CSO×TC, 95.15%; CM×TC, 96.25%; KM×CC, 95.23%. The trifoliate rates for each combination at subculture (CSO×TC, 17.40%; CM×TC, 11.11%; KM×CC; 6.17%) were obtained from 110 DAP embryos. Survival rates of the genotypes were ranged between 72.13% and 90.28% in subculture and varied from 40.17% and 64.71% in the greenhouse. As a result of the ploidy analysis by flow cytometry, the nuclear DNA content of diploid genotypes were found between 0.78 pg/2C and 0.93 pg/2C. One of the genotypes derived from CM×TC hybridization on 120 DAP was determinated as a triploid plant.

Triploid and aneuploid hybrids obtained from hybridization between Eriobotrya japonica and E. cavaleriei

A short ripening stage, multiple large seeds per fruit, and anthracnose disease seriously restrict the loquat (Eriobotrya japonica) industry. To obtain breeding germplasm of loquat with multiple ideal traits, a wild Eriobotrya species, E. cavaleriei (2n=2x=34), which flowers in late spring and early summer and whose fruits ripen in autumn, was used as the male parent to produce triploid plants from crosses with loquat tetraploid (2n=4x=68) lines (EjK474, EjB433 and EjB456). Fifteen progenies were obtained from three hybridizations, and 13 plants were identified as hybrids according to insertion-deletion (InDel) markers. All 13 hybrids were roughly identified as triploids by flow cytometry. Therein, 4 plants were identified as aneuploids with decreased or increased chromosome numbers based on sequencing depth according to reduced-representation genome sequencing. The sequencing depth on some chromosomes of these 4 hybrids was approximately 2/3 times or 1.41 times the average depth of all detected InDel loci. Frequencies of the loquat parent-specific alleles on increased or decreased chromosomes were approximately 50% or 75%. This verified that increased or decreased alleles in the 4 hybrids were all from tetraploid parents. The fruit flesh color genotypes of all hybrids were detected; there were two red-flesh alleles and one white-flesh allele in all hybrids. Leaves of E. cavaleriei were highly resistant to anthracnose in an in vitro assay, and the leaves of loquat B433 were sensitive. Disease spot diameters on the leaves of hybrids were significantly smaller (p < 0.01) than those of B433 after infection by spores of C. gloeosporioides. Therefore, the resistance of the hybrids was greater than that of the female parent B433. All these results showed that allotriploids can be obtained via hybridization between loquat tetraploids and E. cavaleriei, with some aneuploids as secondary products. Different flowering phases and fruit ripening stages, seedlessness and resistance to anthracnose might be combined in allotriploids. This study verified that resequencing can be used to identify the molecular karyotype of interspecific hybrids.

A female fertile triploid loquat line produces fruits with less seed and aneuploid germplasm

Most loquat cultivars are diploid (2n=2x=34) and produce numerous large seeds, affecting their edibility rate and processing. Therefore, breeding less seed triploid cultivars has always been a primary goal of loquat breeding. However, most triploid plants have low fertility and cannot meet commercial needs. Here, we identified a triploid line, Q24, that can set seeds normally under natural conditions. We statistically analyzed the fruit setting rate, seed number, and external quality of Q24. The fruit setting rate of Q24 was similar to that of diploid Q14. The number of seeds in Q24 was 79.40% less than that in Q14. Pollen viability analysis and hybridization experiments revealed that Q24 male gametes were sterile. Further cytological observations showed that male gamete sterility was caused by abnormal chromosome pairing during meiosis. Hybridization experiments also confirmed that Q24 female gametes were fertile. Finally, chromosome counts showed that the progeny of Q24 could produce aneuploids at a frequency of 92.52%. In conclusion, the triploid Q24 loquat is a valuable breeding resource, not only because it has less seed and fertile female gametes but also because most of its offspring are aneuploids. This study provides an optimal material for breeding the less seed loquat, and its large number of aneuploid progenies enrich the germplasm resources of loquat.

Comparative physiological and biochemical mechanisms in diploid, triploid, and tetraploid watermelon (Citrullus lanatus L.) grafted by branches

Seed production for polyploid watermelons is costly, complex, and labor-intensive. Tetraploid and triploid plants produce fewer seeds/fruit, and triploid embryos have a harder seed coat and are generally weaker than diploid seeds. In this study, we propagated tetraploid and triploid watermelons by grafting cuttings onto gourd rootstock (C. maxima × C. mochata). We used three different scions: the apical meristem (AM), one-node (1N), and two-node (2N) branches of diploid, triploid, and tetraploid watermelon plants. We then evaluated the effects of grafting on plant survival, some biochemical traits, oxidants, antioxidants, and hormone levels at different time points. We found significant differences between the polyploid watermelons when the 1N was used as a scion. Tetraploid watermelons had the highest survival rates and the highest levels of hormones, carbohydrates, and antioxidant activity compared to diploid watermelons, which may explain the high compatibility of tetraploid watermelons and the deterioration of the graft zone in diploid watermelons. Our results show that hormone production and enzyme activity with high carbohydrate content, particularly in the 2–3 days after transplantation, contribute to a high survival rate. Sugar application resulted in increased carbohydrate accumulation in the grafted combination. This study also presents an alternative and cost-effective approach to producing more tetraploid and triploid watermelon plants for breeding and seed production by using branches as sprouts.

Microscopical palynology: Birch woodland expansion and species hybridisation coincide with periods of climate warming during the Holocene epoch in Iceland.

Introgressive hybridisation between arctic dwarf birch (Betula nana) and European downy birch (B.pubescens) is relatively common in natural woodlands in Iceland. As dwarf birch is a diploid species and downy tree birch a tetraploid species, their hybrids are triploid. In the introgression process, triploid hybrids, which are partially fertile, can backcross with the parental species, producing progenies comprising introgressed diploid, triploid and tetraploid plants. Triploid plants produce both normal triporate pollen (with three pores) and abnormal, aborted pollen, due to dysfunctional meiosis. The type of pollen abnormality that can be detected and quantified is non-triporate pollen (with four or more pores in the pollen wall). We therefore used the presence of non-triporate pollen as a marker to trace birch hybridisation in the past. In the current study we examined fossil pollen in samples from Holocene sediments from three locations: Grímsnes (SW), Eyjafjördur (N) and Thistilsfjördur (NE Iceland). All three peat monoliths had the starting age of 10.3cal. ka BP. Ages were calibrated based on known tephra layers and by radiocarbon dating. The size of Betula pollen grains was measured, and the species proportions calculated from size. Non-triporate grains were detected in samples from all three locations and throughout the Holocene, but with different frequencies. The peaks of intense hybridisation followed birch woodland expansion in two major periods of the Holocene, both coinciding with a warming of climate. The first period occurred in the Early Holocene, around 9.5-7cal. ka BP, when the climate warmed rapidly after deglaciation. The second period occurred around 5-3.5cal. ka BP, well within the mid-Holocene Northern Hemisphere warming. A new wave of birch hybridisation appears to have started in the last few decades as the climate has warmed. Birch woodlands are likely to become more widespread. Introgressed shrub birch is likely to be more competitive over dwarf birch.

A wider spectrum of avoidance and tolerance mechanisms explained ozone sensitivity of two white poplar ploidy levels.

Polyploidization can improve plant mass yield for bioenergy support, yet few studies have investigated ozone (O3) sensitivity linked to internal regulatory mechanisms at different ploidy levels. Diploid and triploid Populus tomentosa plants were exposed to ambient and ambient plus 60 ppb [O3]. We explored their differences in sensitivity (leaf morphological, physiological and biochemical traits, and plant mass) as well as mechanisms of avoidance (stomatal conductance, xanthophyll cycle, thermal dissipation) and tolerance (ROS scavenging system) in response to O3 at two developmental phases. Triploid plants had the highest plant growth under ambient O3, even under O3 fumigation. However, triploid plants were the most sensitive to O3 and under elevated O3 showed the largest decreases in photosynthetic capacity and performance, as well as increased shoot:root ratio, and the highest lipid peroxidation. Thus, plant mass production could be impacted in triploid plants under long-term O3 contamination. Both diploid and triploid plants reduced stomatal aperture in response to O3, thereby reducing O3 entrance, yet only in diploid plants was reduced stomatal aperture associated with minimal (non-significant) damage to photosynthetic pigments and lower lipid peroxidation. Tolerance mechanisms of plants of both ploidy levels mainly focused on the enzymatic reduction of hydrogen peroxide through catalase and peroxidase, yet these homeostatic regulatory mechanisms were higher in diploid plants. Our study recommends triploid white poplar as a bioenergy species only under short-term O3 contamination. Under continuously elevated O3 over the long term, diploid white poplar may perform better.

MALE FLOWER CHARACTERISTICS OF INDUCED TETRAPLOID, MIXOPLOID, AND DIPLOID BANANA MUSA ACUMINATA (AA) CV. “PISANG REJANG”

The banana cultivar “Pisang Rejang” (Musa AA “Pisang Rejang”) is Fusarium wilt resistant and therefore important for banana breeding. Tetraploid (4x) and mixoploid (2x+4x) plants of this cultivar have been induced with oryzalin, and crosses of these two ploidy levels resulted in a triploid hybrid (3x) plant. The availability of these various ploidy levels needs to be accompanied by data on their reproductive biology for their efficient utilization for breeding. This study characterized male flowers concerning qualitative and quantitative morphology, pollen viability, pollen size, nectar volume, nectar Brix index, and acidity level of those different ploidy levels. Observations indicated no differences in qualitative morphological traits, such as, shape and color, but for quantitative morphological traits, such as, size, distinction existed among the different ploidy levels. The triploid plant has huge flower parts in all the quantitative morphological traits, except compound tepal width, followed by the tetraploid, diploid, and mixoploid. Variations also occurred in pollen viability, pollen size, and nectar characters from each level of ploidy. Pollen viability is the highest for the diploid plant, while the tetraploid plant produces bigger pollen than the diploid. The triploid plant produced a low percentage of viable pollen. The study findings indicate that ploidy level affects the flower's quantitative morphological characteristics, pollen viability, and pollen size, but not the flower's qualitative morphological characteristics.

Bioengineering and Molecular Biology of Miscanthus

Miscanthus is a perennial wild plant that is vital for the production of paper and roofing, as well as horticulture and the development of new high-yielding crops in temperate climates. Chromosome-level assembly of the ancient tetraploid genome of miscanthus chromosomes is reported to provide resources that can link its chromosomes to related diploid sorghum and complex polyploid sugarcane. Analysis of Miscanthus sinensis and Miscanthus sacchariflorus showed intense mixing and interspecific hybridization and documented the origin of a high-yielding triploid bioenergetic plant, Miscanthus × giganteus. The Miscanthus genome expands comparative genomics functions to better understand the main abilities of Andropogoneae herbs. Miscanthus × giganteus is widely regarded as a promising lignocellulosic biomass crop due to its high-biomass yield, which does not emit toxic compounds into the environment, and ability to grow in depleted lands. The high production cost of lignocellulosic bioethanol limits its commercialization. The main components that inhibit the enzymatic reactions of fermentation and saccharification are lignin in the cell wall and its by-products released during the pre-treatment stage. One approach to overcoming this barrier could be to genetically modify the genes involved in lignin biosynthesis, manipulating the lignin content and composition of miscanthus.

Recovering triploid citrus hybrids from 2x × 2x sexual crosses with the aid of embryo rescue and flow cytometry in Turkey.

Turkey is one of the major exporters of mandarins in the Mediterranean region. Seedlessness in citrus, which is one of the most desired fruit quality traits, especially in fresh mandarin export markets, can be obtained via triploidy as in many fruit species. Triploid plants can be recovered by 2x × 2x hybridizations in citrus, as well as 2x × 4x and 4x × 2x crosses. Hence, we aimed to develop local triploid hybrids by using the embryo rescue technique in five crosses using eight different citrus varieties in the present study. Embryos isolated from abortive seeds derived by 135days after pollinations were cultured on modified Murashige and Tucker (MT) medium by adding different levels of GA3 to achieve a high germination rate. A population of 574 plants was developed as a result of embryo rescue. After screening the ploidy levels of this 574-plant population with the aid of flow cytometry, 4 triploids from 'Encore' × 'Murcott', 8 triploids from 'Fortune' ×'Willow leaf', 1 triploid from 'Kiyomi' × 'Murcott', and 1 triploid from 'Ortanique' × 'Murcott' hybridization were recovered. Triploid hybrid plants and related parents were analyzed with SSR markers heterozygotic for parental mandarin varieties. In addition, we evaluated stomatal characteristics of diploid and triploid hybrids obtained from different crosses. Stomatal traits of diploid and triploid hybrids in all crosses significantly differed except the stomata index. Genotyping of triploid plants confirmed using Simple Sequence Repeat (SSR) molecular markers and five SSRs were able to identify three alleles of triploid hybrids. Selected triploid mandarin hybrids have been grafted on several rootstocks for field trials and are in the process of yield and quality performances.

Pollen Development and Viability in Diploid and Doubled Diploid Citrus Species.

Seedlessness is one of the most important agronomic traits in mandarins on the fresh fruit market. Creation of triploid plants is an important breeding strategy for development of new commercial varieties of seedless citrus. To this end, one strategy is to perform sexual hybridizations, with tetraploid genotypes as male parents. However, while seed development has been widely studied in citrus, knowledge of key steps such as microsporogenesis and microgametogenesis, is scarce, especially in polyploids. Therefore, we performed a study on the effect of ploidy level on pollen development by including diploid and tetraploid (double diploid) genotypes with different degrees of pollen performance. A comprehensive study on the pollen ontogeny of diploid and doubled diploid “Sanguinelli” blood orange and “Clemenules” clementine was performed, with focus on pollen grain germination in vitro and in planta, morphology of mature pollen grains by scanning electron microscopy (SEM), cytochemical characterization of carbohydrates by periodic acid–Shiff staining, and specific cell wall components revealed by immunolocalization. During microsporogenesis, the main difference between diploid and doubled diploid genotypes was cell area, which was larger in doubled diploid genotypes. However, after increase in size and vacuolization of microspores, but before mitosis I, doubled diploid “Clemenules” clementine showed drastic differences in shape, cell area, and starch hydrolysis, which resulted in shrinkage of pollen grains. The loss of fertility in doubled diploid “Clemenules” clementine is mainly due to lack of carbohydrate accumulation in pollen during microgametogenesis, especially starch content, which led to pollen grain abortion. All these changes make the pollen of this genotype unviable and very difficult to use as a male parent in sexual hybridization with the objective of recovering large progenies of triploid hybrids.

Cytogenetic factors decreasing the fertility of pollen and cobs during clogging of tetraploid maize with triploid grains (Zea mays L.)

Background. Clogging of tetraploid maize crops with triploid grains leads to a decrease in grain yield and the destruction of the genome’s stability. Searching for the reasons of the tetraploid genome’s decomposition as well as solving the problem of seed yield reduction in freely pollinated crops of tetraploid maize remains relevant.Materials and methods. Cultivars of tetraploid dentate (k-23427) and sweet (k-23426) maize from VIR and dentate (k-24735) and sweet maize (k-23425) hybrids served as the material of the research. The experiments were carried out in the foothill zone of Kabardino-Balkaria. Incubation and hybridization were carried out under parchment insulators. Metaphase plates of maize roots were stained with Schiff’s reagent according to Feulgen and pollen grains were stained with Lugol’s solution.Results. Triploid grains, along with diploid ones, were able to germinate and show poor fertility. As a result of the fusion between male gametes of triploid and female tetraploid plants, an imbalance in the stability of the tetraploid genome occurred, leading to increased degradation of the cultivar’s productivity with each seed reproduction. A cytological analysis and the results of test crosses 2n × 3n, 4n × 3n showed that in self-pollinated triploid plants the frequency of diploid kernel formation was 7.44%; triploid + aneuploidy, 41.78%; tetraploid, 50.74%; and in test crosses the frequency of diploid ones was 18.22%; triploid + aneuploid, 63.83%; and tetraploid, 36.15%. The analysis of segregation classes with the determination of Pearson’s χ2 criterion showed that instead of the expected segregation 1(2n) : 7(3n) + (Xn±1x) : 1(4n), diploid, triploid + aneuploid and tetraploid kernels developed on self-pollinated triploid plants in the ratio of 2 : 13 : 16, respectively, and in test crosses for the 2n and 4n genotypes splitting into diploid, triploid + aneuploid, and tetraploid grains occurred in the ratio of 7 : 18 : 14, respectively

Número de sementes em frutos e frequência de híbridos obtidos de cruzamentos com a tangerina IAC 2019 Maria

Abstract The Citrus Breeding Program of the Citriculture Center at the Agronomic Institute (IAC) has developed a mandarin cultivar IAC 2019Maria from the crossing between Murcott IAC tangor (Citrus reticulata x Citrus sinensis) and Pera IAC sweet orange (C. sinensis). The present study aimed to assess the number of seeds in fruits of IAC 2019Maria mandarin and to identify zygotic embryos and triploid plants in the crossings between IAC 2019Maria mandarin, Pera IAC sweet orange, and Ponkan mandarin (C. reticulata Blanco), in addition to IAC 2019Maria mandarin in open and self-pollination. IAC 2019Maria flowers were self-pollinated, pollinated with pollen from Pera sweet orange and Ponkan mandarin, and had no pollination. The embryos were identified using microsatellite molecular markers and ploidy was assessed by flow cytometry. The results of the treatment with no pollination suggest the variety does not produce parthenocarpic fruits. The genotyping results showed that 100% of the populations consist of zygotic embryos, suggesting that IAC 2019Maria mandarin is a plant with low polyembryony. The ploidy analysis of the hybrids allowed identifying a triploid plant from an aborted seed from the crossing with Pera sweet orange and two tetraploids, one from the crossing with Pera sweet orange and one from self-pollination.