Tetraploid Plants Research Articles

Competitive ability, neopolyploid establishment and current distribution of a diploid–tetraploid plant complex

Polyploidy is a pervasive phenomenon in nature and has significantly contributed to the adaptive evolution of plants. The conditions necessary for the spread of neopolyploids in populations of the diploid progenitor are limited; however, the superior competitive ability of neopolyploids may promote its establishment. Here, we assess the contribution of polyploidisation to the divergence of plant traits affecting competitive response, which could explain the successful establishment and current geographic distribution of polyploids. We conducted an intraspecific competition experiment using diploids, neotetraploids and established tetraploids of Jasione maritima var. maritima to determine whether cytotypes differ in phenological, growth and physiological traits and competitive response. Cytotypes respond differently under different competition scenarios with implications for cytotype establishment and distribution. Competition impacted all cytotypes, but neotetraploids were least affected by competition, and the tetraploids were the most impacted. Thus, competitive advantage may have contributed to the displacement of diploid populations and colonisation of new areas by neotetraploids but might have been lost afterwards. Evolutionary changes after polyploidisation have also been detected, and tetraploids invested more in belowground biomass, suggesting that root development might also play a role in colonising southernmost locations. Interestingly, diploids and both tetraploids seem to have different life strategies, the first investing in growth while the latter investing in reserves for the next season. Overall, polyploidisation seems to provide immediate changes that confer an advantage under competition that, together with other factors, may have allowed the establishment of neotetraploids.

Production of Tetraploid Plants from Cotyledons of Diploid Melia volkensii Gürke

Polyploidy was induced in Melia volkensii (Mukau), a valuable native tree from the semi-arid regions of East Africa. Cotyledons of diploid M. volkensii (2n = 2x = 28) were treated with oryzalin for 0 (control), 1, 2, or 3 h with or without pretreatment with 1.1 µM thidiazuron. Cotyledons treated with 10 mg·L−1 oryzalin for three hours yielded 40% tetraploids. Pretreatment of cotyledons with thidiazuron for 18 days followed by treatment with oryzalin increased tetraploid plant production to 52.5%, but this also yielded more mixoploids. Compared to diploid M. volkensii, the tetraploid in vitro and young potted plants were compacter, with thicker stems, wider leaves, and a low density of longer and wider stomata. In the coming years, tetraploid M. volkensii plants will be observed in field trials and serve as a basis for further breeding efforts.

The protective role of tetraploidy and nanoparticles in arsenic-stressed rice: Evidence from RNA sequencing, ultrastructural and physiological studies

Genome doubling in plants induces physiological and molecular changes to withstand environmental stress. Diploid rice (D-2x) and its tetraploid (T-4x) plants were treated with 25 μM Arsenic (As) and 15 mg L−1 TiO2 nanoparticles (NPs), and results indicated decreased growth and photosynthetic activity with high accumulation of reactive oxygen species (ROS) due to the As-toxicity in rice lines, significantly in D-2x rice plants. The treatment of As-contaminated rice with TiO2 NPs resulted in increased root length (8.17%) and chlorophyll AB (13.28%) and decreased electrolyte leakage (21.76%) and H2O2 (17.65%) contents than its counterpart diploid rice. Moreover, TiO2 NPs improved the activity of peroxidase, catalase, glutathione, and superoxide dismutase and reduced lipid peroxidation due to lower ROS production in D-2x and T-4x under As toxicity. Transcriptome analysis revealed abrupt changes in the expression levels of key signaling heat shock proteins, tubulin, aquaporins, As, and metal transporters under As toxicity in T-4x and D-2x lines. The KEGG and GO studies highlighted the striking distinctions between rice lines under As-stress in glutathione metabolism, H2O2 catabolic process, MAPK signaling pathway, and carotenoid biosynthesis terms, revealing consistency between physiological and molecular results. Root cells from D-2x rice were significantly more distorted by As poisoning than those from 4x rice, and cell organelles, such as mitochondria and endoplasmic reticulum, were changed or deformed. These findings proved the superiority of tetraploid rice lines over their diploid counterpart in coping with As-stress.

Polyploidy affects responses to Nickel in Ni-hyperaccumulating plants: Evidence from the model species Odontarrhena bertolonii (Brassicaceae)

Compared with their diploid progenitors, polyploid plants often display enhanced vigor and adaptive ability to extreme conditions. Previous studies investigated polyploid fitness under various forms of environmental stress, but whether the whole-genome duplication can affect growth, accumulation ability and tolerance levels to trace metals in metal-accumulating species remains obscure. We investigated this topic using an experimental approach and Odontarrhena bertolonii as model system. This obligate serpentine endemic includes diploid and tetraploid accessions with allopatric distribution in Tuscany and Liguria. Three diploid (2 n = 2x = 16) and three natural autotetraploid (2 n = 4x = 32) accessions were compared for growth, leaf traits, photosynthetic parameters and metal accumulation after cultivation on natural serpentine soil and in Ni-containing hydroponic solutions. In soil cultivation, shoot Ni concentration was similar in diploid and tetraploid plants, but the metal content per plant was significantly higher in polyploids because of their higher biomass production. The latter was mainly due to the production of larger leaves, coupled to a lower specific leaf area, which pointed to a better adaptation of the tetraploids to serpentine soil. In hydroponics, all the accessions showed an hormetic-like Ni effect, with stimulation of growth and photosynthesis in the low-dose zone. The data fitting to the hormetic model displayed significant differences in some parameters, with the tetraploid plants showing higher maximal length of roots and shoots and higher requirement of both external and internal Ni concentrations for optimal growth, in respect to diploid plants. On the other hand, Ni tolerance to both external and internal metal concentrations was similar in the plants of the two ploidy levels. Our results suggest that whole-genome duplication can affect the potential for growth and Ni-hyperaccumulation in obligate Ni-accumulator plants like O. bertolonii. This points to the opportunity of selecting polyploid instead of diploid accessions for environmental restoration and metal cropping practices, when the candidate species includes both chromosome races.

Optimal protocol for in vitro polyploid induction of Cymbidium aloifolium (L.) Sw

BackgroundCymbidium aloifolium is a popular ornamental flower in Thailand with both economic and medical values. Polyploid induction techniques are used to improve plant quality. This study identified polyploidy levels of C. aloifolium induction by colchicine. Protocorms of C. aloifolium were treated on solid New Dogashima Medium (NDM) with various concentrations of colchicine (0, 0.01, 0.02, 0.03, 0.04 and 0.05% w/v) for 2, 4 and 8 weeks.ResultsColchicine effectively induced plant polyploidy. Tetraploid plants were found after treatment with 0.03% and 0.04% colchicine for 8 weeks, while at increased concentration and duration, survival, response and growth performance decreased. Tetraploid plants showed the lowest growth performance but highest size of stomatal and epidermal cells. Growth performance and leaf surface anatomy data were analyzed by Pearson’s correlation and PCA. Results showed that stomatal and epidermal cell sizes had strongly negative correlations with other variables, while HCA revealed that stomatal and epidermal cell sizes of tetraploid plantlets were larger but stomatal and epidermal cell densities decreased when compared with the diploids.ConclusionColchicine at suitable concentrations and duration produced polyploid plants with alteration of morphological and anatomical traits. This study provides potential information to support orchid quality production as ornamental plants and a source of pharmaceutical raw materials.

ZIP4 is required for normal progression of synapsis and for over 95% of crossovers in wheat meiosis.

Tetraploid (AABB) and hexaploid (AABBDD) wheat have multiple sets of similar chromosomes, with successful meiosis and preservation of fertility relying on synapsis and crossover (CO) formation only taking place between homologous chromosomes. In hexaploid wheat, the major meiotic gene TaZIP4-B2 (Ph1) on chromosome 5B, promotes CO formation between homologous chromosomes, whilst suppressing COs between homeologous (related) chromosomes. In other species, ZIP4 mutations eliminate approximately 85% of COs, consistent with loss of the class I CO pathway. Tetraploid wheat has three ZIP4 copies: TtZIP4-A1 on chromosome 3A, TtZIP4-B1 on 3B and TtZIP4-B2 on 5B. Here, we have developed single, double and triple zip4 TILLING mutants and a CRISPR Ttzip4-B2 mutant, to determine the effect of ZIP4 genes on synapsis and CO formation in the tetraploid wheat cultivar 'Kronos'. We show that disruption of two ZIP4 gene copies in Ttzip4-A1B1 double mutants, results in a 76-78% reduction in COs when compared to wild-type plants. Moreover, when all three copies are disrupted in Ttzip4-A1B1B2 triple mutants, COs are reduced by over 95%, suggesting that the TtZIP4-B2 copy may also affect class II COs. If this is the case, the class I and class II CO pathways may be interlinked in wheat. When ZIP4 duplicated and diverged from chromosome 3B on wheat polyploidization, the new 5B copy, TaZIP4-B2, could have acquired an additional function to stabilize both CO pathways. In tetraploid plants deficient in all three ZIP4 copies, synapsis is delayed and does not complete, consistent with our previous studies in hexaploid wheat, when a similar delay in synapsis was observed in a 59.3 Mb deletion mutant, ph1b, encompassing the TaZIP4-B2 gene on chromosome 5B. These findings confirm the requirement of ZIP4-B2 for efficient synapsis, and suggest that TtZIP4 genes have a stronger effect on synapsis than previously described in Arabidopsis and rice. Thus, ZIP4-B2 in wheat accounts for the two major phenotypes reported for Ph1, promotion of homologous synapsis and suppression of homeologous COs.

12-Oxophytodienoate Reductase Overexpression Compromises Tolerance to Botrytis cinerea in Hexaploid and Tetraploid Wheat.

12-Oxophytodienoate reductase is the enzyme involved in the biosynthesis of phytohormone jasmonates, which are considered to be the major regulators of plant tolerance to biotic challenges, especially necrotrophic pathogens. However, we observe compromised tolerance to the necrotrophic fungal pathogen Botrytis cinerea in transgenic hexaploid bread wheat and tetraploid emmer wheat plants overexpressing 12-OXOPHYTODIENOATE REDUCTASE-3 gene from Arabidopsis thaliana, while in Arabidopsis plants themselves, endogenously produced and exogenously applied jasmonates exert a strong protective effect against B. cinerea. Exogenous application of methyl jasmonate on hexaploid and tetraploid wheat leaves suppresses tolerance to B. cinerea and induces the formation of chlorotic damages. Exogenous treatment with methyl jasmonate in concentrations of 100 µM and higher causes leaf yellowing even in the absence of the pathogen, in agreement with findings on the role of jasmonates in the regulation of leaf senescence. Thereby, the present study demonstrates the negative role of the jasmonate system in hexaploid and tetraploid wheat tolerance to B. cinerea and reveals previously unknown jasmonate-mediated responses.

SCREENING FOR BIOMORPHOLOGICAL AND CYTOGENETIC FEATURES OF POLEMONIUM CAERULEUM L. AFTER COLCHICINE APPLICATION

Background. Currently, it is quite relevant in breeding to create varieties with high yields of raw materials and the content of biologically active compounds.
 This work aim is to identify promising forms of P. caeruleum L. is for further inclusion in the breeding process based on biomorphological, anatomical, cytogenetic studies and the bioproductivity of colchicine-induced and wild-growing forms in comparison with the Lazur variety.
 Materials and methods. To obtain polyploids in 2018, the seeds of the P. caeruleum L. the Lazur variety were germinated in Petri dishes on filter paper at room temperature. When the roots reached the length of the seed, they were placed in different concentrations of colchicine solution 0.05 %, 0.1 %, 0.2 % and 0.52 % for 12 and 24 hours, then washed in running water for three hours.
 Results. The article for the first time presents data on a comparative study of three forms of Polemonium caeruleum L. in terms of morphological characteristics, microscopy of the leaf epidermis and cytogenetic analysis. It was established that the raw materials productivity of the underground part of the plants tetraploid forms in the C1 generation significantly exceeded the diploid and wild forms of plants by 32 %, as well as the yield of seeds by 22 %. In the C2 generation, the yield of raw materials (rhizomes with roots) in tetraploid plants increases in relation to the control by 56 %, seeds by 34 %. Quantitative determination of the amount of triterpene saponins in terms of β-escin in the plant material of three forms of P. caeruleum L. showed an excess of this indicator in the colchicine form by 24 % in the grass and by 36 % in rhizomes with roots, in the wild-growing form - by 15 % in the aerial part and decrease in the underground part by 9 % in relation to the control. It was found that plants of colchicine and wild forms differ in the presence of larger stomata compared to the control. Based on the pattern of DAPI-bandin and the distribution of 45S rDNA and 5S rDNA genes, a generalized species idiogram of blue cyanosis was constructed, taking into account polymorphic variants of DAPI-bandin patterns and indicating the location of the sites of 45S rDNA and 5S rDNA genes. The cytogenetic stability of the resulting tetraploid form was revealed.
 Conclusion. The article presents comparative studies on morphological characteristics, bioproductivity, microscopy of the leaf epidermis and cytogenetic analysis of the tetraploid form (colchicine) of P. caeruleum L. in comparison with the variety Lazur (diploid form) and the wild form of P. caeruleum L. plants.

Manipulation of ploidy for blueberry breeding: in vitro chromosome doubling of diploid Vaccinium duclouxii (Lévl.) Hand.-Mazz by trifluralin

The diversification, evolution, domestication, and breeding of many plant species have all been greatly aided by polyploidization. In this study, we reported a successful induction of autotetraploids of Vaccinium duclouxii (Lévl.) Hand.-Mazz, which was an important germplasm for developing highbush blueberry cultivars. The isolated stems of V. duclouxii were soaked in different concentrations of trifluralin solution, and then the ploidy of the seedlings sprouted from stems were identified by flow cytometry and chromosome counting analyses. The optimal condition for inducing tetraploid of V. duclouxii leaf bud was 0.002 % trifluralin for 48 h, which the induction rate reached 8.33 %. The morphological and anatomical characteristics of induced tetraploid seedlings were significantly different from those of the original diploid plants. The average stem diameter, leaf length, leaf width, and leaf thickness of the tetraploid plants showed significantly larger and thicker compared with the diploid progenitor plants. The stomata number of lower epidermis on the tetraploid seedling leaves only was 64.20 % of the diploid progenitors’, while the average stomatal area was increased by 166.67 %. In addition, the length and width of guard cells were 128.57 % and 141.18 % of its diploid progenitor, separatively, and the chloroplast numbers were in guard cells increased by 157.36 %. The main leaf vein diameter, the upper epidermal thickness, palisade tissue and sponge tissue of induced tetraploid leaves were all significantly increased in comparison to its diploid progenitor, indicating that these indexes can be used for screening tetraploid seedlings during breeding program.

Morpho-genetic characterization of diploid and tetraploid taro (Colocasia esculenta L. Schott) cv. Kaliurang - An Indonesian local cultivar

Kaliurang is one of the local taro cultivars in Indonesia, potentially due to its high productivity and delicacy. This study analysed morpho-genetic variation between one diploid and three tetraploid Kaliurang taro clones from <i>in vitro</i> polyploid induction after plantlet acclimatization in the greenhouse for 6 weeks. Plants’ morphological characters were analysed using the unweighted pair group method with arithmetic mean (UPGMA) method based on 50 characters according to the taro descriptor from the International Plant Genetic Resources Institute (IPGRI). Furthermore, a morphometric characterization based on the Euclidean distance of 17 digitations from mature leaves, was analysed with the Analysis Phylogenic and Evolution package in R software. DNA band pattern was also performed with ISSR to analyse the clone’s genetic variance. The results showed morpho-genetic character diversity in diploid and tetraploid plants. A plant morphological study revealed that 22 out of 50 characters were distinct, with similarity coefficients ranging from 0.35 to 0.78. Leaf geometric morphometric analysis showed changes in five digitations and formed three clusters. Twelve ISSR primers out of twenty amplified the sequence of the Kaliurang taro genome to yield two clusters with a similarity coefficient of 0.71–0.83. Based on these three characterization approaches, tetraploid clones (K2 and K3) consistently differed morphologically from diploid clones (K0).

Does competitive asymmetry confer polyploid advantage under changing environments?

Abstract Competitive interactions drive critical ecological processes in plant communities. Yet, how competitive interactions are influenced by polyploidy that has a widespread incidence in plants remains largely unknown. To evaluate the hypothesis of competitive asymmetry between polyploids and diploids, we competed tetraploid and diploid plants of perennial herbaceous Chrysanthemum indicum L. (Asteraceae) at different relative frequencies under contrasting soil water contents. We quantified the interaction intensity between competing plants of the same (intraploidy) and different ploidy levels (interploidy), and measured functional traits related to gas exchange and plant water use to understand the underlying mechanisms. The stronger competitive effect of tetraploids on diploids than that of diploids on tetraploids provided evidence for the competitive asymmetry. As a stronger competitor, tetraploids were limited more by individuals of their own than by diploids. Such competitive asymmetry was not only maintained under reduced soil water content, but also translated into higher above‐ground biomass of tetraploids. Tetraploids showed more resource‐acquisitive traits than diploids under high soil water content and more resource‐conservative traits under reduced soil water content. As such, the higher trait plasticity in tetraploids than diploids likely explained the competitive asymmetry. Synthesis. These results elucidate the nature and magnitude of species interactions between polyploid and diploid plants under changing environments and the underlying mechanisms, and provide important insights into the prevalence and persistence of polyploid plants under a changing climate.

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.

Physiological and biochemical determinants of drought tolerance in tetraploid vs diploid sour orange citrus rootstock

ABSTRACT Citrus plants face various abiotic stresses like drought during their life span which significantly affect their active growth and development. Tetraploid (4×) plants are more adaptable to environmental constraints than diploid (2×) plants by inducing critical physiological and biochemical processes. In this study, tetraploid and their corresponding diploid sour orange rootstocks were subjected to drought stress for 12 days. Results revealed that drought stress significantly affected plant physiology by reducing photosynthesis rate, stomatal conductance, transpiration rate, and leaf colour, which was prominent in diploid plants compared to tetraploid plants. In diploid plants, phenotypic differences (i.e. wilting and rolling of leaves) were also significant. Diploids with more accumulation of malondialdehyde and hydrogen peroxide in their leaves and roots exhibited more oxidative damage. It was observed that tetraploid plants had higher activities of peroxidase and catalase enzymes, while lower superoxide dismutase was recorded in the leaves and roots of plants. Higher glycine betaine, proline, total soluble protein, total phenolic content, and antioxidant activities were also observed in the leaves and roots of tetraploids. Overall, results suggest that tetraploids in citrus can have better defence mechanisms that help them to sustain under water deficit scenarios.

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.

ORYZALIN-INDUCED TARO (COLOCASIA ESCULENTA L.) TETRAPLOID AND DIPLOID ASSESSMENT FOR GROWTH AND AGRONOMIC TRAITS

Taro (Colocasia esculenta L. cv. Pontianak) is a popular and widely cultivated cultivar in the Indonesian farming community. In taro Pontianak, manipulating the ploidy level by chromosome doubling can enhance its genetic diversity. The latest study aimed to evaluate the oryzalin-induced polyploid taro cv. Pontianak compared with its diploid in terms of growth, anatomical, cytological, morphological, and agronomic traits in the greenhouse and the field. In vitro shoots of taro cv. Pontianak were soaked using a liquid medium supplemented with oryzalin with a concentration of 30 and 60 μM for one day. After five times of subcultures, flow cytometry analysis proceeded to determine their ploidy levels. The diploid (2n = 2x) and tetraploid (2n = 4x) plantlets were then acclimatized in the greenhouse for six to eight weeks and hardened for two months before planting in the field for 10 months. Squashing the root tip to ensure the ploidy levels of taro seedlings helped measure the chromosome numbers. Observing the growth, anatomical, and agronomic traits of diploid and tetraploid plants occurred during the hardening process until harvest time. The 30 μM oryzalin treatment produced tetraploid and mixoploid shoots; none from the 60 μM treatment. Oryzalininduced tetraploid taro exhibited some morphological characteristics of tetraploid plants, such as, longer stomata size and low density. Tubers of tetraploid plants tended to have increased protein, ash, and water content compared with their diploid counterpart. However, tetraploid taro showed a slower growth rate both in the greenhouse and the field. Additionally, it gave lower yields than diploids in some agronomic characteristics, including delayed flowering time, lower plant fresh weight, and smaller tubers. This study provides information that oryzalin-induced tetraploid taro could exhibit increasing and decreasing nutritional-agronomic traits (as mentioned above) compared with its diploid.

ORIGIN OF RECOGNITION COMPLEX 3 controls the development of maternal excess endosperm in the Paspalum simplex agamic complex (Poaceae).

Pseudogamous apomixis in Paspalum simplex generates seeds with embryos genetically identical to the mother plant and endosperms deviating from the canonical 2(maternal):1(paternal) parental genome contribution into a maternal excess 4m:1p genome ratio. In P. simplex, the gene homologous to that coding for subunit 3 of the ORIGIN OF RECOGNITION COMPLEX (PsORC3) exists in three isogenic forms: PsORC3a is apomixis specific and constitutively expressed in developing endosperm whereas PsORCb and PsORCc are up-regulated in sexual endosperms and silenced in apomictic ones. This raises the question of how the different arrangement and expression profiles of these three ORC3 isogenes are linked to seed development in interploidy crosses generating maternal excess endosperms. We demonstrate that down-regulation of PsORC3b in sexual tetraploid plants is sufficient to restore seed fertility in interploidy 4n×2n crosses and, in turn, its expression level at the transition from proliferating to endoreduplication endosperm developmental stages dictates the fate of these seeds. Furthermore, we show that only when being maternally inherited can PsORC3c up-regulate PsORC3b. Our findings lay the basis for an innovative route-based on ORC3 manipulation-to introgress the apomictic trait into sexual crops and overcome the fertilization barriers in interploidy crosses.

Enhanced Na+ and Cl− sequestration and secretion selectivity contribute to high salt tolerance in the tetraploid recretohalophyte Plumbago auriculata Lam.

Enhanced secretion of Na+ and Cl- in leaf glands and leaf vacuolar sequestration of Na+ or root retention of Cl-, combined with K+ retention, contribute to the improved salt tolerance of tetraploid recretohalophyte P. auriculata. Salt stress is one of the major abiotic factors threatening plant growth and development, and polyploids generally exhibit higher salt stress resistance than diploids. In recretohalophytes, which secrete ions from the salt gland in leaf epidermal cells, the effects of polyploidization on ion homeostasis and secretion remain unknown. In this study, we compared the morphology, physiology, and ion homeostasis regulation of diploid and autotetraploid accessions of the recretohalophyte Plumbago auriculata Lam. after treatment with 300mM NaCl for 0, 2, 4, 6, and 8 days. The results showed that salt stress altered the morphology, photosynthetic efficiency, and chloroplast structure of diploid P. auriculata to a greater extent than those of its tetraploid counterpart. Moreover, the contents of organic osmoregulatory substances (proline and soluble sugars) were significantly higher in the tetraploid than in the diploid, while those of H2O2 and malondialdehyde (MDA) were significantly lower. Analysis of ion homeostasis revealed that the tetraploid cytotype accumulated more Na+ in stems and leaves and more Cl- in roots but less K+ loss in roots compared with diploid P. auriculata. Additionally, the rate of Na+ and Cl- secretion from the leaf surface was higher, while that of K+, Mg2+, and Ca2+ secretion was lower in tetraploid plants. X-ray microanalysis of mesophyll cells revealed that Na+ mainly accumulated in different cellular compartments in the tetraploid (vacuole) and diploid (cytoplasm) plants. Our results suggest that polyploid recretohalophytes require the ability to sequester Na+ and Cl-(via accumulation in leaf cell vacuoles or unloading by roots) and selectively secrete these ions (through salt glands) together with the ability to prevent K+ loss (by roots). This mechanism required to maintain K+/Na+ homeostasis in polyploid recretohalophytes under high salinity provides new insights in the improved maintenance of ion homeostasis in polyploids under salt stress.

Morphological, cariological, and phytochemical studies of diploid and autotetraploid Hippeastrum papilio plants

The polyploidization of Hippeastrum papilio influences its primary and secondary metabolism including the biosynthesis of bioactive alkaloids. Hippeastrum papilio is an ornamental plant that has advantages in comparison to the currently used plants for the extraction of galanthamine, a natural compound used for the cognitive treatment of Alzheimer's disease. In the present study, an autotetraploid line of H. papilio was induced for the first time, after treatment with 0.05% colchicine for 48h. The chromosome number in diploids was found to be 2n = 2x = 22 and for autotetraploids 2n = 4x = 44. The flow cytometric analyses detected a DNA C-value of 14.88 ± 0.03pg (1C) in diploids and 26.57 ± 0.12pg in autotetraploids. The morphological, cytological, and phytochemical studies showed significant differences between diploids and autotetraploids. The length and width of stomata in autotetraploids were 22.47% and 17.94%, respectively, larger than those observed in the diploid leaves. The biomass of one-year-old autotetraploid H. papilio plants was reduced by 53.99% for plants' fresh weight, 56.53% for leaves' fresh weight, and 21.70% for bulb diameter. The GC-MS analysis of methanol extracts from one-year-old diploid and autotetraploid H. papilio plants revealed over 60 primary and secondary metabolites including alkaloids, phenolic acids, sterols, saccharides, and alcohols, among others. Principal component analysis of the metabolite profiles indicates a divergence of the metabolism between diploid and autotetraploid plants. The content of galanthamine and haemanthamine was found to be 49.73% and 80.10%, respectively, higher in the leaves of autotetraploids, compared to the diploid ones. The biosynthesis of the saccharides shows a tendency to be upregulated in tetraploid plants, while that of phenolic acids was downregulated. Polyploidization of H. papilio creates possibilities for further crop improvement aimed at high-galanthamine-producing genotypes.

Colchicine-mediated in vitro polyploidization in gerbera hybrid.

An efficient in vitro protocol for high-frequency polyploidization for the first time in gerbera hybrid (BGC-2019-01) was developed in the present study. Two-week-old in vitro-developed shoots (tips) were treated individually with 0.1%, 0.25% and 0.5% (w/v) colchicine solutions for 4, 6, 8, and 12h. The colchicine-treated shoot tips were then inoculated on Murashige and Skoog (MS) medium fortified with 1.5mg/l meta-Topolin for multiple shoot proliferation and later transferred into 1.5mg/l indole-3-acetic acid-fortified MS medium for rooting of shoots. The ploidy levels of the colchicine-treated and regenerated plantlets along with the non-treated oneswere confirmed via flow cytometry analysis and metaphasic chromosome count. The highest frequency oftetraploid plantlets (50%) were obtained when shoot tips were treated with 0.1% colchicine for 4h. Morphological observations revealed that inducedtetraploidplantlets exhibited delayed fresh shoot initiation, fewer but longer shoots, as well as fewer but broader leaves. Likewise, the studyof stomata revealed that in comparison to their diploid counterparts, the tetraploidplantlets exhibited less frequent yet significantly larger stomata, and higher number of chloroplasts. The tetraploids were recorded with significantly higher chlorophyll, carotenoid, and anthocyanin content during the photosynthetic pigment analyses. During ex vitro acclimatization and field growth, the tetraploid plants exhibited delayed proliferation but with higher vigor and thickened broad leaves. The genetic uniformity among the diploid and the tetraploidplants was confirmed using conserved DNA-derived polymorphism(CDDP), directed amplification of minisatellite-region DNA(DAMD), inter simple sequence repeats(ISSR), and start codon targeted(SCoT) polymorphism marker systems. The tetraploids developed in the present study would be of immense importance for the genetic improvement of gerbera as far as its ornamental values are concerned.

Effects of Polyploidy on Physiological Performance of Acclimatized Solanum betaceum Cav. Plants under Water Deficit

The urgent need to identify stress-tolerant genotypes and understand their inherent genetic plasticity is one of the major targets of research and breeding programs. Species that are cultivated in areas that are prone to drought need to be able to tolerate water stress (WS) while still displaying features that are economically valuable. Tamarillo (Solanum betaceum) is a solanaceous fruit crop with increasing agronomic interest due to the nutritional properties of its edible fruits and its biotechnological potential. Several protocols have been established for the in vitro culture of this species and controlled hybridization, as well as for the induction of tetraploidy. Nevertheless, the impact of WS on S. betaceum performance has been poorly studied, and nothing is known about the role of ploidy status on this response. Since no morphological differences were noticed between diploids and tetraploids at the end of the acclimatization period, we hypothesized that ploidy level may have a role in plant drought responses. Thus, micropropagated and acclimatized tamarillo diploid (2n = 2x = 24) and tetraploid (4n = 4x = 48) plants were exposed to WS, and several physiological parameters were evaluated, such as plant growth, water potential, photosynthetic performance, sugars, proline, and MDA levels. Water stress did not affect plant growth in both diploids and tetraploids, but it induced stomatal closure and reduced the net CO2 assimilation rate. Water stress also reduced the photosynthetic efficiency of PSII, but no differences were found in the total chlorophyll content. From all the parameters analyzed, tetraploid plants showed a better response under water shortage conditions when considering water potential (WP). Metabolite analysis indicated no significant differences in the accumulation of soluble sugars and MDA in WS plants but a significant increase in proline accumulation in diploids exposed to WS. These observed differences in parameters such as WP and proline accumulation point to mechanisms of osmoregulation and stress signaling that differ between diploid and tetraploid plants, particularly in WS conditions, demonstrating that tetraploids can adapt better to water shortage conditions than their diploid counterparts.