Polyploidy Plants Research Articles

Partially knocking out NtPDK1a/1b/1c/1d simultaneously in Nicotiana tabacum using CRISPR/CAS9 technology results in auxin-related developmental defects

The eukaryotic AGC protein kinase subfamily (protein kinase A/ protein kinase G/ protein kinase C-family) is involved in regulating numerous biological processes across kingdoms, including growth and development, and apoptosis. PDK1(3-phosphoinositide-dependent protein kinase 1) is a conserved serine/threonine kinase in eukaryotes, which is both a member of AGC kinase and a major regulator of many other downstream AGC protein kinase family members. Although extensively investigated in model plant Arabidopsis, detailed reports for tobacco PDK1s have been limited. To better understand the functions of PDK1s in tobacco, CRISPR/CAS9 transgenic lines were generated in tetraploid N. tabacum, cv. Samsun (NN) with 5–7 of the 8 copies of 4 homologous PDK1 genes in tobacco genome (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out. Numerous developmental defects were observed in these NtPDK1a/1b/1c/1d CRISPR/CAS9 lines, including cotyledon fusion leaf shrinkage, uneven distribution of leaf veins, convex veins, root growth retardation, and reduced fertility, all of which reminiscence of impaired polar auxin transport. The severity of these defects was correlated with the number of knocked out alleles of NtPDK1a/1b/1c/1d. Consistent with the observation in Arabidopsis, it was found that the polar auxin transport, and not auxin biosynthesis, was significantly compromised in these knockout lines compared with the wild type tobacco plants. The fact that no homozygous plant with all 8 NtPDK1a/1b/1c/1d alleles being knocked out suggested that knocking out 8 alleles of NtPDK1a/1b/1c/1d could be lethal. In conclusion, our results indicated that NtPDK1s are versatile AGC kinases that participate in regulation of tobacco growth and development via modulating polar auxin transport. Our results also indicated that CRISPR/CAS9 technology is a powerful tool in resolving gene redundancy in polyploidy plants.

Utilizing method of plant growth regulators induction in processing of polyploidization—A perspective crop development

Nowadays, it seems like human beings are worried about how the world will meet its food security demand urgently when they have faced on rapidly increasing population and combat climate change. Scientists and researchers are indispensably investigating to enhance food sources such developing novel cops with high yield and good quality and even coping with the adverse environment in terms of biotic and abiotic stresses. Thus, there is one valuable method it is believed that should be developed further. In fact, it is believed that human beings should continue using it as soon as possible because it can provide the world with a source of food. Polyploidization introduced by plant growth regulator induction is a good method because it is safety and easy to develop new crops with potential agronomic traits, these polyploidy plants are rare aneuploid and it contains intriguing characteristics of polyploidy plants in adapting to ecological variability. This review sheds light on 1) summarizing molecular mechanism of plant growth regulator induction for plant ploidy manipulation; 2) achieving of polyploidization through plant growth regulator induction; 3) enumerating the perspectives of polyploidization in crop development to cope with climate change. Although the role of phytohormones is underestimated, the effectiveness on physiological level of plants to make polyploidy plants is worth considering and the effects and bio-safety of that on human are also concerned.

Evolutionary Fixed Potential Agronomic Traits in Polysomic Polyploidy Plants with Special Reference to Potato

Evolutionary Fixed Potential Agronomic Traits in Polysomic Polyploidy Plants with Special Reference to Potato

AGRICULTURAL DEVELOPMENT BASED ON POLYPLOIDIZATION: A PERSPECTIVE CONTRIBUTION OF MINOR CROPS

Polyploidization is commonly known to become a vital process to succeed in developing potential crops. In the plant kingdom, the polyploids, i.e., aneuploidy and polyploidy, are generally developed through sexual and asexual pathways, resulting in totally increasing biomass, phytochemical compounds, and adaptation to detrimental environment. For more precision, thousands of polyploidy plant species adapted to various climatic and topographic conditions through genomic evolution. Autopolyploid, allopolyploid, and aneuploidy, as well as, different levels of ploidy are simply found in vegetables, such as, potatoes, and among fruits, i.e., bananas, watermelon, and so on. Contrastingly, in mammals, polyploidization causes congenital diseases and pregnancy loss, especially in human beings. This review article will first describe polyploidization in plants and then enumerate the advantages of its beneficial effects that are more valuable. The paper also intends to introduce new knowledge on polyploidization in crop breeding. Thus, it has further mentioned the polyploid like aneuploidy and polyploidy in a perspective contribution of minor crops in plant kingdoms and their beneficial and detrimental effects in the development of crop strategies.

Vinca alkaloids effects on the morphological characters of Canavalia ensiformis

Productivity and quality of crops can be increased through polyploidy plants induced by antimitotic compounds such as vinka alkaloids.The use of vinka alkaloids has never been applied to Sword bean (Canavalia ensiformis), a tropical legume belonging to the Fabaceae family. This study aimed to determine the effects of vinka alkaloids treatment on the morphological characters of Sword bean. The treatment consist of four different vinka alkaloids concentrations (0.5%, 1.00%, 1.50%, 2.0%, and untreated (0.0%) act as control. The plants were arranged in a completely randomized design. Treated plants were characterized based on morphological traits such as plant height, leaf area and dry seed weight. The results obtained revealed significant difference (p≤0.05) in the morphological traits of the treated plants when compared with the control. Overall, 2.0% vinka alkaloids concentration resulted in higher average plant height by 193.8 ± 1.86, leaf area increments by 88.6 ± 5.1 (left leaf), 115.1 ± 4.7 (middle leaf), 86.2 ± 3.8 (right leaf) and the dry seed weight by 1.7 ± 3.6. The improvement of the traits is concentration dependent and increases with increment in vinka alkaloids concentration. Thus, we suggested that 2.0% concentration should be employed in improving Sword bean growth and yield.

Growth response of diploid and tetraploid taro (Colocasia esculenta (L.) Schott) shoot culture to drought stress using polyethylene glycol

Taro genetic improvement through polyploidy induction is expected to be tolerant to abiotic stress. Several studies have shown that polyploidy plants have a higher adaptability to dry environments. In vitro selection technique for evaluation of plant tolerance to drought stress can be done by applying polyethylene glycol (PEG) as a selection agent. The aim of the research was to investigate the growth response of diploid and tetraploid taro shoot culture to drought stress using PEG. The experiment was conducted using a completely randomized design with two factors. The first factor was the concentration of PEG at 0, 5, 10, 15 and 20%. The second factor was Bentul taro clones which were 1 diploid clone and 2 tetraploid clones (clones 4.6.3 and 5.4.4). PEG was added to liquid MS medium containing 2 mg/l BAP. Observations of growth variables were carried out every week until the six weeks of culture. Fresh and dry weights, mortality percentage and proline content were determined at six weeks of culture. The results showed that the addition of PEG in liquid medium significantly affected the number of leaves, petiole length, number of roots, fresh and dry weights, as well as shoot mortality percentage. Differences in clones significantly affected the number of leaves, fresh and dry weights. The two factors tested (PEG and clones), gave an interaction on the number of leaves, fresh and dry weights. The proline content in all diploid and tetraploid clones showed an increase with increasing PEG concentration. The LC50 value in diploid clones was 9.82%, in tetraploid clones 4.6.3 and 5.4.4 were 14.14 and 15.45%, respectively. The results showed that PEG at 10% and 15% could be used for in vitro selection to drought stress both for diploid and tetraploid taro.

Transcriptome and proteome analysis suggest enhanced photosynthesis in tetraploid Liriodendron sino-americanum.

Polyploidy generally provides an advantage in phenotypic variation and growth vigor. However, the underlying mechanisms remain poorly understood. The tetraploid Liriodendron sino-americanum (Liriodendron × sinoamericanum P.C Yieh ex C.B. Shang & Zhang R.Wang) exhibits altered morphology compared with its diploid counterpart, including larger, thicker and deeper green leaves, bigger stomata, thicker stems and increased tree height. Such characteristics can be useful in ornamental and industrial applications. To elucidate the molecular mechanisms behind this variation, we performed a comparative transcriptome and proteome analysis. Our transcriptome data indicated that some photosynthesis genes and pathways were differentially altered and enriched in tetraploid L. sino-americanum, mainly related to F-type ATPase, the cytochrome b6/f complex, photosynthetic electron transport, the light harvesting chlorophyll protein complexes, and photosystem I and II. Most of the differentially expressed proteins we could identify are also involved in photosynthesis. Our physiological results showed that tetraploids have an enhanced photosynthetic capacity, concomitant with great levels of sugar and starch in leaves. This suggests that tetraploid L. sino-americanum might experience comprehensive transcriptome reprogramming of genes related to photosynthesis. This study has especially emphasized molecular changes involved in photosynthesis that accompany polyploidy, and provides a possible explanation for the altered phenotype of polyploidy plants in comparison with their diploid form.

Colchicine effects on the ploidy level and morphological characters of Katokkon pepper (Capsicum annuum L.) from North Toraja, Indonesia

BackgroundProductivity and quality of crops can be increased through polyploidy plants induced by colchicine. The use of colchicine has never been applied to Katokkon pepper, a local red pepper variety from North Toraja Indonesia. This pepper is characterized by its unique shape like the small-bell pod type of paprika and its strong spicy taste. Therefore, this study aimed to determine the effects of colchicine treatment on the ploidy level and morphological characters of Katokkon pepper. ResultsFlow cytometer analysis showed that all colchicine concentration treatments ranging from 0.025 to 0.1% with 24 h immersion time generated two plant groups based on their ploidy level; 50% of the total treatment plants was diploid while the rest plants were mixoploid. All colchicine treatment plants were significantly different in their width of stomata guard cells from the control plant. The effect of colchicine was also significantly on the plant height, thickness of fruit flesh, and the number of fruits per plant. ConclusionsThe results of this study showed that colchicine treatment had a significant effect on the ploidy level and several morphological characters of the Katokkon pepper. The colchicine treatment increased the number of fruits per plant and thicker flesh of fruits but reduced the size and weight of Katokkon pepper. Our findings provide essential information to obtain tetraploid Katokkon plants through colchicine treatment in further research. This study benefits as a preliminary step for increasing the productivity and quality of the local red peppers in Indonesia.

Regeneration of plant winter garlic <em>in vitro</em> after colchicine treatment

Relevance. The genetic diversity of winter garlic depends entirely on the natural selection of the best and highest-yielding clones. In unfavorable abiotic factors, physiological and biochemical properties allow polyploidy plants to have greater tolerance and viability than diploid species. Obtaining the source material based on polyploidy in winter garlic (Allium sativum L.) is one of the important alternative approaches to genetic improvement of this crop.Methods. From air garlic bulbs, plants were grown through in vitro culture. Pre-air bulbs were treated with 0.1% colchicine solution for 1 hour, 2 hours, 4 hours. Shoots and roots were regenerated by cultivating explants for 1/2 MS, with 3 mg / l 6-BAP and 0.1 mg/l indole-3-acetic acid and cultured in light with an intensity of 5–8 ths. LC and a photoperiod of 18 hours. Treatment of air bulbs with 0.1% colchicine solution made it possible to obtain winter garlic plants using in vitro culture.Results. In the end, we were not able to get flowering forms from garlic using the polyploidization method. But, thanks to the cultivation of winter garlic in vitro, it became possible to accelerate the selection process by obtaining one generation per year “from clove to clove”. When working on obtaining polyploidy forms, more than one variety should be used, since the reaction of varieties to colchicine treatment is ambiguous, as is their reaction to growing conditions and survival. The most responsive for obtaining plants in vitro culture was the Yubileyny Gribovsky variety.

Phenotypic and molecular characterization of polyploidy Vicia faba induced by colchicine

To induce polyploidy in diploid control plant of Vicia faba, seeds were socked in various concentrations of colchicine (0.1, 0.2, 0.3 and 0.4 %) for 9 and18 h. Putative ploidy plants were confirmed by screening morphological characters, measuring DNA content via spectrophotometer and analyzing stomatal behavior. Polyploidy plants showed slower growth rate along with increased potency and thickened large leaves. Three types of polyploidy were achieved; triploid, tetraploid and mixoploid. In induced polyploid plants, the length and width of stomata were increased, while the index of stomata was declined, in comparison to the initial diploid plants. Treatment with 0.3 and 0.4 % colchicine for 9 and 18 h were efficiently induced polyploidy. The highest stomatal size (22.53 µm) and widest stomatal aperture (9.72 µm) and the lowest stomatal index (34.24) where obtained when 0.4 % of colchicine where applied at 18hr tested time with high significant differences at p<0.05 in comparison to dipliod plants. We recommend this method for induction of polyploids in other different crops and possibly will selected these tetraploid plants for further breeding purposes.

In vitro polyploidy induction of foxtail millet (Setaria italica (L) beauv) cv. buru hotong using colchicine treatment

In vitro polyploidy induction of foxtail millet (Setaria Italica (L.) Beauv) was done using callus from the basal leaf of 10-day old seedlings. Callus was treated using three different concentrations of colchicine (0, 125, 250 and 500 ppm) and two exposure times (24 and 48 hours). Results showed that colchicine declines the survival rate and the number of shots on all combinations. Putative polyploidy plant was produced on the combination of K3T1 (500 ppm colchicine; 24 hour exposure time). Based on field experiment data, putative polyploid plant has smaller characteristics in the term of plant height, the number of tillers, and number of seed per panicle than the diploid plant. However, leaf width, panicle length and diameter, seed size, and weight of 100 seeds of putative polyploid plant bigger than the diploid plant.

In vitro selection to increase Huanglongbing tolerance of citrusderived from in vitro breeding

Until now, Huanglongbing-HLB disease caused by a simple bacterium (Ca. Liberibacter spp) and distributed by Asian citrus psyllid vector (Diaphorina citri) and clonal propagation (grafting), was a major challenge in the national citrus production even almost all citrus nation production center in Asia, Africa also at the USA. This disease attack at phloem vessels by callouses forming that covering plasmodesmata and could be crop failure. There have been no citrus varieties reported resistant to HLB attack. Plant ploidy increase derived from in vitro breeding through somatic hybridization and endosperm culture expected to increase the plant’s defense system. The aim of this study was to evaluate the tolerance of polyploidy plant to HLB attack. Tolerance of in vitro shoot of citrus regenerated from protoplast fusion (Citrus nobilis + C. unshiu) and culture of tangerine (C. nobilis) endosperm were screened by HLB pathogen suspense as selecting an agent and local lime as a negative control (HLB susceptible citrus). In vitro selection result that in vitro shoots derived from somatic hybridization shows resistance increase to HLB and characterize by recoverability after were selected.

The Impact of cDNA Normalization on Long-Read Sequencing of a Complex Transcriptome.

Normalization of cDNA is widely used to improve the coverage of rare transcripts in analysis of transcriptomes employing next-generation sequencing. Recently, long-read technology has been emerging as a powerful tool for sequencing and construction of transcriptomes, especially for complex genomes containing highly similar transcripts and transcript-spliced isoforms. Here, we analyzed the transcriptome of sugarcane, a highly polyploidy plant genome, by PacBio isoform sequencing (Iso-Seq) of two different cDNA library preparations, with and without a normalization step. The results demonstrated that, while the two libraries included many of the same transcripts, many longer transcripts were removed, and many new generally shorter transcripts were detected by normalization. For the same input cDNA and data yield, the normalized library recovered more total transcript isoforms and number of predicted gene families and orthologous groups, resulting in a higher representation for the sugarcane transcriptome, compared to the non-normalized library. The non-normalized library, on the other hand, included a wider transcript length range with more longer transcripts above ∼1.25 kb and more transcript isoforms per gene family and gene ontology terms per transcript. A large proportion of the unique transcripts comprising ∼52% of the normalized library were expressed at a lower level than the unique transcripts from the non-normalized library, across three tissue types tested including leaf, stalk, and root. About 83% of the total 5,348 predicted long noncoding transcripts was derived from the normalized library, of which ∼80% was derived from the lowly expressed fraction. Functional annotation of the unique transcripts suggested that each library enriched different functional transcript fractions. This demonstrated the complementation of the two approaches in obtaining a complete transcriptome of a complex genome at the sequencing depth used in this study.

Genotyping genome-edited mutations in plants using CRISPR ribonucleoprotein complexes.

SummaryDespite the great achievements in genome editing, accurately detecting mutations induced by sequence‐specific nucleases is still a challenge in plants, especially in polyploidy plants. An efficient detection method is particularly vital when the mutation frequency is low or when a large population needs to be screened. Here, we applied purified CRISPR ribonucleoprotein complexes to cleave PCR products for genome‐edited mutation detection in hexaploid wheat and diploid rice. We show that this mutation detection method is more sensitive than Sanger sequencing and more applicable than PCR/RE method without the requirement for restriction enzyme site. We also demonstrate that this detection method is especially useful for genome editing in wheat, because target sites are often surrounded by single nucleotide polymorphisms. Using this screening method, we were also able to detect foreign DNA‐free tagw2 mutations induced by purified TALEN protein. Finally, we show that partial base editing mutations can also be detected using high‐fidelity SpCas9 variants or FnCpf1. The PCR/RNP method is low‐cost and widely applicable for rapid detection of genome‐edited mutation in plants.

High resolution melting (HRM)-based STMS marker analysis for rapid identification of radiation induced mutants of sugarcane

Molecular marker based selection has become the method of choice to find undiscoverable natural variations and induced mutations in plants. Routine gel based molecular marker screening procedures involve complex steps which require considerable investment of time, money and efforts. Most often, genomic configuration of a plant species complicates the ultimate output of such screening programme. Therefore, a reliable and rapid high-throughput method is required to facilitate ease in identification of variants in a large number of samples. Here we demonstrate the application of a high-resolution melting (HRM) method to detect gamma radiation induced mutants of sugarcane (Saccharum spp.) which is a complex aneu-polyploidy plant. We have screened nine distinct mutants using sequence-tagged microsatellite site (STMS) markers and the analyses based on gel electrophoresis and HRM curve were compared. All the six STMS microsatellite markers coupled with HRM analysis were found highly informative and generated a unique melting curve for each of the marker tested. Of these, two markers NKSCSSR 22 and NKSCSSR 42 exhibited differential melting curve among the mutants and these results were well correlated with the gel based banding pattern. Taken together these results highlighted potential of HRM based marker screening and demonstrated reliability and robustness in detecting induced mutations in a complex polyploidy plant like sugarcane.

5-Aminolevulinic Acid Dehydratase Gene Dosage Affects Programmed Cell Death and Immunity.

Programmed cell death (PCD) is an important form to protect plants from pathogen attack. However, plants must precisely control the PCD process under microbe attacks to avoid detrimental effects. The complexity of how plants balance the defense activation and PCD requires further clarification. Lesion mimic mutants constitute an excellent material to study the crosstalk between them. Here, we identified a Gossypium hirsutum (cotton) lesion mimic mutant (Ghlmm), which exhibits necrotic leaf damage and enhanced disease resistance. Map-based cloning demonstrated that GhLMMD, encoding 5-aminolevulinic acid dehydratase and located on chromosome D5, was responsible for the phenotype. The mutant was resulted from a nonsense mutation within the coding region of GhLMMD It exhibited an overaccumulation of the 5-aminolevulinic acid, elevated levels of reactive oxygen species and salicylic acid, along with constitutive expression of pathogenesis-related genes and enhanced resistance to the Verticillium dahliae infection. Interestingly, GhLMM plays a dosage-dependent role in regulating PCD of cotton leaves and resistance to V. dahliae infection. This study provides a new strategy on the modulation of plant immunity, particularly in polyploidy plants.

Induction of polyploidy plants through colchicine treatments in balloon vine (Cardiospermum halicacabum)©

Induction of polyploidy plants through colchicine treatments in balloon vine (<i>Cardiospermum halicacabum</i>)^©

Genome-wide gene expression perturbation induced by loss of C2 chromosome in allotetraploid Brassica napus L.

Aneuploidy with loss of entire chromosomes from normal complement disrupts the balanced genome and is tolerable only by polyploidy plants. In this study, the monosomic and nullisomic plants losing one or two copies of C2 chromosome from allotetraploid Brassica napus L. (2n = 38, AACC) were produced and compared for their phenotype and transcriptome. The monosomics gave a plant phenotype very similar to the original donor, but the nullisomics had much smaller stature and also shorter growth period. By the comparative analyses on the global transcript profiles with the euploid donor, genome-wide alterations in gene expression were revealed in two aneuploids, and their majority of differentially expressed genes (DEGs) resulted from the trans-acting effects of the zero and one copy of C2 chromosome. The higher number of up-regulated genes than down-regulated genes on other chromosomes suggested that the genome responded to the C2 loss via enhancing the expression of certain genes. Particularly, more DEGs were detected in the monosomics than nullisomics, contrasting with their phenotypes. The gene expression of the other chromosomes was differently affected, and several dysregulated domains in which up- or downregulated genes obviously clustered were identifiable. But the mean gene expression (MGE) for homoeologous chromosome A2 reduced with the C2 loss. Some genes and their expressions on C2 were correlated with the phenotype deviations in the aneuploids. These results provided new insights into the transcriptomic perturbation of the allopolyploid genome elicited by the loss of individual chromosome.

OBTAINING Artemisia cina POLYPLOIDY THROUGH PLANT GROWTH REGULATOR TREATMENT IN SHOOT CULTURE

Artemisia cina is a medicinal plant species which produces bioactive compound potential to anti-tumor, antifungal and antibacterial medicines. This study was aimed to obtain A. cina polyploid plants through a treatment of growth regulators in shoot culture. The shoot were treated in 1; 15; 2; and 3 mg L -1 of 2,4-dichloro phenoxy acetic acid (2,4-D) combined with 1; 1.5; 2; 3 mg L -1 of Benzyladenine (BA) for 28 days. Chromosome analysis showed that the highest polyploidy percentage, 23%, was reached in the treatment of 2 mg L -1 of 2,4-D combined with 1 of BA mg L -1 . The polyploidy level varied, comprising 2n=3x, 2n=4x, 2n=5x, 2n=6x, with the highest polyploidy level percentage, 28.57%, and it was attained in the tetraploid (2n=4x). Polyploid plants had larger leaves area, larger stomatal size, and higher chlorophyll content than diploid plants. However stomatal density of polyploidy plants was lower than that of in diploid plants.

OBTAINING Artemisia cina POLYPLOIDY THROUGH PLANT GROWTH REGULATOR TREATMENT IN SHOOT CULTURE

Artemisia cina is a medicinal plant species which produces bioactive compound potential to anti-tumor, antifungal and antibacterial medicines. This study was aimed to obtain A. cina polyploid plants through a treatment of growth regulators in shoot culture. The shoot were treated in 1; 15; 2; and 3 mg L -1 of 2,4-dichloro phenoxy acetic acid (2,4-D) combined with 1; 1.5; 2; 3 mg L -1 of Benzyladenine (BA) for 28 days. Chromosome analysis showed that the highest polyploidy percentage, 23%, was reached in the treatment of 2 mg L -1 of 2,4-D combined with 1 of BA mg L -1 . The polyploidy level varied, comprising 2n=3x, 2n=4x, 2n=5x, 2n=6x, with the highest polyploidy level percentage, 28.57%, and it was attained in the tetraploid (2n=4x). Polyploid plants had larger leaves area, larger stomatal size, and higher chlorophyll content than diploid plants. However stomatal density of polyploidy plants was lower than that of in diploid plants.