Chloroplast Counts Research Articles

Chromosome Study of Six Populations of Trifolium tumens Stev. ex M. B. in IRAN

Trifolium tumens Stev. ex M. B is a perennial legume with long‐lived and deep rooting from the Vesicaria section in Trifolium genus. It is distributed in Iran. The base chromosome number (x = 8) in the population, confirmed the views of the previous researchers. In this study, the number of chromosomes in the mitotic metaphase stage in the meristematic cells of the root ends were counted in six populations of Trifolium tumens. Also, chloroplast count of stomata guard cells, morphological studies, pollen grain size and flowcytometry were studied in six accessions. Our results showed that this species has ploidy level (2n=16, 2n=32). and the tetraploid state in the species T. tumens is reported. Through flowcytometry, chloroplast count of stomata guard cells and compare the pollen grain, different characters were showed between diploid and tetraploid populations, too. Based on the results, this new cytotype is reported from Iran.

Chromosome Doubling Enhances Biomass and Carotenoid Content in Lycium chinense

Lycium chinense, a type of medicinal and edible plant, is rich in bioactive compounds beneficial to human health. In order to meet the market requirements for the yield and quality of L. chinense, polyploid induction is usually an effective way to increase plant biomass and improve the content of bioactive components. This study established the most effective tetraploid induction protocol by assessing various preculture durations, colchicine concentrations, and exposure times. The peak tetraploid induction efficacy, 18.2%, was achieved with a 12-day preculture and 24-h exposure to 50 mg L–1 colchicine. Compared to diploids, tetraploids exhibited potentially advantageous characteristics such as larger leaves, more robust stems, and faster growth rates. Physiologically, tetraploids demonstrated increased stomatal size and chloroplast count in stomata but reduced stomatal density. Nutrient analysis revealed a substantial increase in polysaccharides, calcium, iron, and zinc in tetraploid leaves. In addition, seventeen carotenoids were identified in the leaves of L. chinense. Compared to the diploid, lutein, β-carotene, neoxanthin, violaxanthin, and (E/Z)-phytoene exhibited higher levels in tetraploid strains T39 and T1, with T39 demonstrating a greater accumulation than T1. The findings suggest that the generated tetraploids harbor potential for further exploitation and lay the foundation for the selection and breeding of novel genetic resources of Lycium.

Studies on in vitro chromosome doubling of haploid derived through androgenesis in marigold (Tagetes patula L.)

Local strain of Tagetes patula L. was used for in vitro androgenesis with floret size ranging from 2 to 4.5 mm. One haploid from anther derived regenerants was isolated and confirmed by cytological analysis and chloroplast counting. The isolated haploid was further multiplied and were subjected to colchicine treatment (0.005%, 0.01%, 0.02% for 36 and 48 hours) using nodal segment explants under in vitro conditions. The results revealed that treatment T1- Colchicine (0.005%) for 36 hours exposure was found good for parameters like percent survival (81.22%), percent diploidization (48.00%), leaf length (3.77 cm), leaf width (2.49 cm), number of shoots per plant (9.89) followed by treatment T2- Colchicine (0.005%) for 48 hours exposure for parameters like percent survival (69.67%), percent diploidization (37.22%), leaf length (3.56 cm), leaf width (1.97 cm), number of shoots per plant (8.67). Cytological analysis and chloroplast counting in stomatal guard cells was done for determination of ploidy level in colchicine treated haploid plantlets. The results revealed that in cytological analysis out of 38 plants screened, 26 plants (68.4%) were tetraploids and 12 plants (31.57%) were polyploids. Whereas through chloroplast counting, 38 plants screened out of which 26 plants (68.4%) were tetraploids with mean of 16 chloroplasts in the guard cells and 12 plants (31.57%) were polyploids with mean of 18 to 22 chloroplasts in the guard cells.

Ploidy Determination in Banana Hybrids through Stomatal Studies and Chloroplast Count

Stomata are probably a viable taxonomy distinguishing feature. The study of stomata characteristics of synthetic banana hybrids and their parentage has been carried out. Ploidy determination is essential in banana breeding programs to understand the genetic makeup of hybrid plants. The purpose of this study was to determine the ploidy level of hybrids in contrast with the properties of plant stomata in synthetic banana hybrids with those of their parent plants. Therefore, by examining components such as stomata location, number of epidermal cells, number of stomata cells, stomata length, width, area, intensity and density, as well as chloroplast count, it is possible to compare and contrast the traits of synthetic banana hybrids and their parentage which have different genomes. The entire mount approach was used to make an incision for the purpose of observing stomata. By characterising the stomata traits of the synthetic hybrids and their parentage, the results were then descriptively and qualitatively assessed. Three different locations on the abaxial of the leaf were taken for the determination. The samples were collected from close to the petiole (R1), middle (R2), and distal end (R3), stomata were observed using the replica method. Results showed that more number of stomata was observed in R2of leaf abaxial. The chloroplast count in pairs of stomatal guard cells from the accessions was also measured. The chloroplast density was determined in pairs of stomatal guard cells from the accessions. Based on morphological descriptions, it was observed that 16.66% of the accessions were diploid, 50% were triploid and 33.33% were tetraploid. When considering the chloroplast count, the accessions were categorized into groups, certain accessions classified as tetraploids, others as triploids, and diploids.

Defining a diverse core collection of the Colombian Central Collection of potatoes: a tool to advance research and breeding.

The highly diverse Colombian Central Collection (CCC) of cultivated potatoes is the most important source of genetic variation for breeding and the agricultural development of this staple crop in Colombia. Potato is the primary source of income for more than 100.000 farming families in Colombia. However, biotic and abiotic challenges limit crop production. Furthermore, climate change, food security, and malnutrition constraints call for adaptive crop development to be urgently addressed. The clonal CCC of potatoes contains 1,255 accessions - an extensive collection size that limits its optimal assessment and use. Our study evaluated different collection sizes from the whole clonal collection to define the best core collection that captures the total genetic diversity of this unique collection, to support a characterization more cost-effectively. Initially, we genotyped 1,141 accessions from the clonal collection and 20 breeding lines using 3,586 genome-wide polymorphic markers to study CCC's genetic diversity. The analysis of molecular variance confirmed the CCC's diversity with a significant population structure (Phi=0.359; p-value=0.001). Three main genetic pools were identified within this collection (CCC_Group_A, CCC_Group_B1, and CCC_Group_B2), and the commercial varieties were located across the pools. The ploidy level was the main driver of pool identification, followed by a robust representation of accessions from Phureja and Andigenum cultivar groups based on former taxonomic classifications. We also found divergent heterozygosity values within genetic groups, with greater diversity in genetic groups with tetraploids (CCC_Group_B1: 0.37, and CCC_Group_B2: 0.53) than in diploid accessions (CCC_Group_A: 0.14). We subsequently generated one mini-core collection size of 3 percent (39 entries) and three further core collections sizes of 10, 15, and 20 percent (i.e., 129, 194, and 258 entries, respectively) from the total samples genotyped. As our results indicated that genetic diversity was similar across the sampled core collection sizes compared to the main collection, we selected the smallest core collection size of 10 percent. We expect this 10 percent core collection to be an optimal tool for discovering and evaluating functional diversity in the genebank to advance potato breeding and agricultural-related studies. This study also lays the foundations for continued CCC curation by evaluating duplicity and admixing between accessions, completing the digitalization of data, and ploidy determination using chloroplast count.

Development of homozygous tetraploid potato and whole genome doubling-induced the enrichment of H3K27ac and potentially enhanced resistance to cold-induced sweetening in tubers.

Polyploid plants typically display advantages on some agronomically important traits over their diploid counterparts. Extensive studies have shown genetic, transcriptomic, and epigenetic dynamics upon polyploidization in multiple plant species. However, few studies have unveiled those alternations imposed only by ploidy level, without any interference from heterozygosity. Cultivated potato is highly heterozygous. Thus, in this study, we developed two homozygous autotetraploid lines and one homozygous diploid line in parallel from a homozygous diploid potato. We confirmed their ploidy levels using chloroplast counting and karyotyping. Oligo-FISH and genome re-sequencing validated that these potato lines are nearly homozygous. We investigated variations in phenotypes, transcription, and histone modifications between two ploidies. Both autotetraploid lines produced larger but fewer tubers than the diploid line. Interestingly, each autotetraploid line displayed ploidy-related differential expression for various genes. We also discovered a genome-wide enrichment of H3K27ac in genic regions upon whole-genome doubling (WGD). However, such enrichment was not associated with the differential gene expression between two ploidies. The tetraploid lines may exhibit better resistance to cold-induced sweetening (CIS) than the diploid line in tubers, potentially regulated through the expression of CIS-related key genes, which seems to be associated with the levels of H3K4me3 in cold-stored tubers. These findings will help to understand the impacts of autotetraploidization on dynamics of phenotypes, transcription, and histone modifications, as well as on CIS-related genes in response to cold storage.

Effects of shoot tip colchicine applications on some grape cultivars

Polyploidization can provide changes in vital features such as growth, development, environmental stress tolerance in plants. Colchicine is one of the most commonly used chemicals as a polyploidization agent. In this study, 2-year-old ‘Ekşi Kara’, ‘Gök Üzüm’ and ‘Trakya İlkeren’ (2x, Vitis vinifera L.) saplings grown on their own roots were used. When the enforced shoots reached about 15 cm length, colchicine applied (0, 2.5 g L-1, 5 g L-1, 7.5 g L-1) 24 and 48 hours to the lateral shoot tips. The effects of treatments were evaluated by shoot tip viability, stoma size and density, chloroplast counts, and flow cytometry (FC) analysis, and ‘Kyoho’ (4x) used as control. The maximum stomatal variations were determined in Ekşi Kara cultivar at 2.5 g L-1 24-h application. Based on morphological differences, FC analysis was performed only in ‘Ekşi Kara’ but, there was no genomic duplication. Since the morphological differences were not sufficient in the diagnosis of polyploid in grape cultivars, FC analysis should be performed to achieve confirmed results.

Frequency of Polyploids of Solanum tuberosum Dihaploids in 2X × 2X Crosses

When breeding diploid potatoes, tetraploid progeny can result from the union of 2n eggs and 2n pollen in 2x-2x crosses. Thirty-three crosses were made to examine tetraploid progeny frequency in 2x-2x crosses. All crosses were between S. tuberosum dihaploids and diploid self-compatible donors, M6 and DRH S6-10-4P17. Using chloroplast counting for ploidy determination, the frequency of tetraploid progeny was as high as 45% in one of the 33 crosses. Based upon single nucleotide polymorphism (SNP) genotyping, the tetraploid progeny were attributed to bilateral sexual polyploidization (BSP), which is caused by the union of 2n egg and 2n pollen. Dihaploids were identified that produce lower frequencies of 2n eggs. The results of this study suggest that S. tuberosum dihaploids with a high frequency of 2n eggs should be avoided in 2x - 2x crosses for diploid breeding programs.

Polyploid induction of Eucalyptus dunnii Maiden to generate variability in breeding programs

Eucalyptus dunnii Maiden produces good quality cellulose pulp, showing good frost tolerance. However, in Uruguay, it needs more than six years to reach the reproductive stage. Genome duplication was proposed as a strategy to obtain useful variability. The application of mitosis inhibitors for chromosomal duplication, in explants growing in vitro, was evaluated. Two antimitotic agents were used: colchicine and oryzalin in different concentrations and exposure times, in two types of explants: explants growing in vitro and pre-germinated seeds. The number of chloroplasts was used as a rapid ploidy estimator and confirmed by flow cytometry. For chloroplast count, fluorescein diacetate (FDA) applied to in vitro leaves was used for staining. Oryzalin was effective for inducing plant duplication in E. dunnii from in vitro explants. In pre-germinated seeds, both antimitotic agents induced polyploids. The average number of chloroplasts was 5.5 in diploid control plants and more than 7 in tetraploids. Obtained plantlets were successfully cloned in the greenhouse. This is the first report on artificial polyploidy obtained in E. dunnii.

Induction of tetraploids in watermelon (Citrullus lanatus Thunb.) through chromosome doubling

Polyploidy is an important evolution process for the creation of new species. Up to 70% of angiosperms are reported that as polyploids. Tetraploids development in watermelon is the first most step in the seedless fruit development. In this study, the colchicine was used to generate the tetraploid watermelons. The chloroplast counting methodology was adopted to segregate tetraploids. Among the 63 plants treated, one plant showed 11- 14 chloroplasts in the guard cells. Chimeral stomatal guard cells pertaining to the number of chloroplasts also identified in this investigation.

Arabidopsis thaliana Leaf Epidermal Guard Cells: A Model for Studying Chloroplast Proliferation and Partitioning in Plants.

The existence of numerous chloroplasts in photosynthetic cells is a general feature of plants. Chloroplast biogenesis and inheritance involve two distinct mechanisms: proliferation of chloroplasts by binary fission and partitioning of chloroplasts into daughter cells during cell division. The mechanism of chloroplast number coordination in a given cell type is a fundamental question. Stomatal guard cells (GCs) in the plant shoot epidermis generally contain several to tens of chloroplasts per cell. Thus far, chloroplast number at the stomatal (GC pair) level has generally been used as a convenient marker for identifying hybrid species or estimating the ploidy level of a given plant tissue. Here, we report that Arabidopsis thaliana leaf GCs represent a useful system for investigating the unexploited aspects of chloroplast number control in plant cells. In contrast to a general notion based on analyses of leaf mesophyll chloroplasts, a small difference was detected in the GC chloroplast number among three Arabidopsis ecotypes (Columbia, Landsberg erecta, and Wassilewskija). Fluorescence microscopy often detected dividing GC chloroplasts with the FtsZ1 ring not only at the early stage of leaf expansion but also at the late stage. Compensatory chloroplast expansion, a phenomenon well documented in leaf mesophyll cells of chloroplast division mutants and transgenic plants, could take place between paired GCs in wild-type leaves. Furthermore, modest chloroplast number per GC as well as symmetric division of guard mother cells for GC formation suggests that Arabidopsis GCs would facilitate the analysis of chloroplast partitioning, based on chloroplast counting at the individual cell level.

The influential role of polyamines on the in vitro regeneration of pea (Pisum sativum L.) and genetic fidelity assessment by SCoT and RAPD markers

A proficient and reliable in vitro plant regeneration protocol was established for pea by utilizing cotyledonary node as an explants, which excised from 3-days old imbibed seeds. The present research work explains the positive role of polyamines (PAs) accompanied by plant growth regulators (PGRs) on the multiple shoot induction and rooting in pea plant regeneration. The highest multiple shoots (65.1 shoots/explant) was attained from the cotyledonary node explant on Murashige and Skoog (MS) medium accompanied with 20 mg l−1 spermidine (SPD) along with 1.5 mg l−1 6-benzyladenine (BA). Moreover, the highest elongation of multiplied shoots (10 cm length/shoot) was noted on MS medium enriched with 1 mg l−1 of gibberellic acid (GA3). The elongated shoots produced the highest number of roots (33.66 roots/shoot) on MS medium augmented with 30 mg l−1 putrescine (PUT) along with 0.6 mg l−1 1-naphthaleneacetic acid (NAA). Rooted plants were hardened and acclimatized in the greenhouse with an existence rate of 98%. The standardized procedure with the use of PAs has enhanced the multiple shoot induction to threefold higher than the plants raised from PGRs treatments. The regenerated pea plants revealed the fivefold enhanced photosynthetic pigment, twofold enhanced antioxidant profile and the substantial growth in chloroplast count have been achieved in optimized SPD and BA supplemented MS medium compared to control plant. Start Codon Targeted polymorphism and Random Amplified Polymorphic DNA molecular markers recognized the genetic purity of the in vitro regenerated pea plants for their true type of nature. An enhanced and consistence in vitro regeneration protocol was established for pea, by the application of polyamines along with PGRs, which significantly improved the multiple shooting, shoot elongation, rooting, total chlorophyll and antioxidant profile of regenerated plants, further not hampering their genetic fidelity.

Assessment of the Ploidy Level Diversity by Chloroplast Counts in Stomatal Guard Cells of Potato (Solanum tuberosum L.) Mutants

Aims: Potato (Solanum tuberosum L.) is the most important staple food in the world and plays an important role in food and nutritional security. Induced mutation generates variation within potato germplasm to widen the genetic base for breeding purposes. Polyploidy modifies both the genotype and phenotype of an organism, generating diverse changes that consequently transform the potato production. Potato has chromosomes with different ploidy levels which can be determined by counting chloroplasts in stomatal guard cells.
 Study Design: The study was carried out in completely randomized block design.
 Place and Duration of Study: Department of Biotechnology, University of Eldoret, between February 2015 and July 2016.
 Methodology: The study involved 163 potato mutants developed from three commercially grown Kenyan potato varieties; Asante, Mpya, and Sherekea irradiated using gamma rays from 60Co source under different dose rates. Three middle leaves of greenhouse-grown plants were randomly selected for chloroplast counts in ten pairs of stomata guard cells on the lower surface of the leaf. Data on the number of chloroplast counts per mutant was calculated as a percentage of the parents or control and descriptive analysis.
 Results: The results indicate that the number of ploidy level distribution was decreasing in diploids and triploids and were increasing in tetraploids from M1V1, M1V2 to M1V3 in all the potato mutant populations.
 Conclusion: This shows that mutation induction generates genetic variations from which desired mutants may be selected based on the appropriate breeding strategies.

Comparison of Methods to Distinguish Diploid and Tetraploid Potato in Applied Diploid Breeding

Diploid (2n = 2x = 24) and tetraploid (2n = 4x = 48) germplasm are commonly used in potato breeding programs. Potato breeders need to efficiently and inexpensively differentiate between diploid and tetraploid progeny in dihaploid induction crosses as well as 2x-2x crosses where 2n gametes may occur in the parents. In this study, we compared the chloroplast count, genome-wide SNP genotyping and flow cytometry methods to determine ploidy. Twenty-eight clones were used as reference samples (14 diploid lines (2n = 2x = 24), 14 tetraploid (2n = 4x = 48) varieties and advanced breeding lines) to compare the three ploidy determination methods. The chloroplast count method was used to determine the ploidy level in the 28 reference samples, and 102 potato breeding lines derived from diploid (2x-2x) crosses. The Infinium 12 K V2 Potato Single Nucleotide Polymorphism (SNP) Array was used to examine the 28 reference samples and the 102 breeding lines. The results obtained from both chloroplasts counts and SNP genotyping techniques determined that there was a total of 84 diploid lines and 18 tetraploid lines. Flow cytometry was also used to determine ploidy level in a subset of 42 lines (28 reference lines set and 14 breeding lines). All three methods of ploidy determination (chloroplast counting, SNP genotyping and flow cytometry) agreed for all samples evaluated. These results demonstrate the usefulness of chloroplast counting as an efficient and inexpensive method for breeders to differentiate ploidy between diploid and tetraploid potato in applied breeding programs.

An improved method for high-temperature induced embryo sac chromosome doubling in Populus simonii Carr. \xd7 P. nigra var. italica (Moench.) Kochne, an interspecific hybrid of Tacamahaca and Aigeiros poplars

Chromosome doubling is considered an important technique in poplar breeding, with many triploid clones being artificially induced and selected for promotion in the north and northeast of China because of their outstanding traits in vegetative growth and environmental adaption. In this study, the triploid yield of Populus simonii Carr × P. nigra var. italica (Moench.) Kochne was 23.41%, which exceeded the yield attained in our previous studies due to the use of an optimized method of chromosome doubling in the embryo sac at a high temperature. The development of the embryo sac after the pollination of this hybrid was investigated to determine the induction period. Ploidy of seedlings was identified by flow cytometry after initial filtering using the chloroplast counting method. Eleven triploids and one tetraploid were ultimately obtained, and the optimal operating conditions were exposure of female catkins to 41 °C for 2 h at 66 h after pollination (HAP). This study identified an efficient method of chromosome doubling in P. simonii × P. nigra var. italica and provided several polyploids for Populus polyploid breeding programs and subsequent studies.

The Pacific yam (Dioscorea nummularia Lam.), an under-exploited tuber crop from Melanesia

This study aims to explore the potential of Dioscorea nummularia Lam. and to gather information as a prelude to its improvement by plant breeding. After a taxonomic clarification and a review of the botanical data, its geographical areas of origin and distribution are determined. A germplasm survey conducted on nine islands of Vanuatu enabled the collection of 110 accessions corresponding to 84 cultivars. These were grouped into: (1) wild forms under domestication, (2) annual types, (3) perennial-type cultivars of D. nummularia and (4) natural interspecific hybrids with D. alata L. Within each group, remarkable morphological variation exists for stem spininess, leaf shape, emerging leaf colour, tuber shape and tuber flesh colour. Selected accessions examined for ploidy had chromosome numbers ranging from 2n = 3x = 60 to 2n = 6x = 120. Chloroplast counts conducted on stomatal guard cells confirmed the variation in ploidy levels. Eight different male cultivars were studied for pollen fertility, but <5% of the pollen grains appeared to be fertile when stained with acetocarmine. However, profuse flowering and synchrony favour pollination and result in fruit set. All accessions are resistant to anthracnose. Viruses were detected but are morphologically not discernible, with no incidence on yield. Physicochemical analyses of tubers from 16 accessions revealed that D. nummularia and interspecific hybrids have high percentages of dry matter (mean of 33.11%) and starch (82.81%). The Pacific yam could be improved through conventional hybridisation or used for interspecific crosses with related economically important species. Recommendations are made for further research.

Potential of a tetraploid line as female parent for developing yellow- andred-fleshed seedless watermelon

A tetraploid inbred line KAU-TETRA-CL-1 was developed in the watermelon cultivar Sugar Baby through treatment of seedling shoot tips with colchicine solution (0.5%) followed by selection for increased fertility in the C2, C3, C4, and C5 generations. Chloroplast count and guard cell measurement in successive generations of tetraploids were employed as criteria for detecting higher ploidy level and tetraploidy was confirmed by flow cytometry in the C5 generation. A distinct difference was observed in the stomata of diploid Sugar Baby (length: 20.52 ± 1.4 μm; diameter: 15.86 ± 1.0 μm; number of chloroplasts: 11.2-13.1) and tetraploids from the C5 generation (length: 29.12 ± 2.0 μm; diameter: 22.0 ± 0.1 μm; number of chloroplast: 20-22). These parameters were effective in identification of putative tetraploids, and selection based on number of seeds/fruit (>100) was found effective in improving seed number in advanced generations. The tetraploid line selected in the C5 generation (KAU-CL-TETRA-1) produced red-fleshed and yellow-fleshed seedless fruits when used as a female parent with diploid male parents CL-4 and CL-5. A seed yield of more than 200 per fruit was observed in crosses involving the diploid lines. The mean yield over two seasons was 12.256 kg fruits/plant for the red-fleshed KAU-CL-TETRA-1 × CL-4 and 10.168 kg fruits/plant for the yellow-fleshed KAU-CL-TETRA-1 × CL-5 seedless hybrids. The triploid hybrids have excellent fruit quality compared to diploids in terms of sweetness and absence of seeds.

In vitro androgenic response of minipaprika (Capsicum annuum L.) genotypes in different culture media

The in vitro androgenic response of minipaprika F1 hybrid cv. Vine sweet (red, yellow, and orange form) was investigated using Dumas de Vaulx (CP) and Murashige and Skoog (MS) culture media to ascertain the effectiveness of androgenesis in haploid production. The frequency of callus and embryo formation varied in anther cultures of all minipaprika forms in both culture media. Anthers in both culture media responded to form the callus without regeneration in all minipaprika forms. The anthers of ‘Vine sweet-yellow’ produced 62.5% and 46.7% normal embryo in CP and MS medium, respectively. ‘Vine sweet-yellow’ produced almost four fold more normal-looking embryos than ‘Vine sweet-red’ in both culture media, and ‘Vine-sweet orange’ showed low androgenic response to anther culture. The anthers cultured in CP medium gave more normal embryo in all minipaprika forms as compared to MS medium. Among a total of 51 embryos transferred to growth regulator free MS medium for regeneration, 45 plants were regenerated. Flow cytometry analysis revealed that 44.5%, 42.4%, and 33.3% plants were haploids, and 55.5%, 57.6%, and 66.7% plants were spontaneous diploids in red, yellow and orange forms, respectively. The chromosome number of haploid plant was 12 and that of spontaneous diploid was 24. Stomata characters and chloroplast count in the guard cell of leaf stomata were also found to be reliable and simple method to analyze the ploidy level of regenerated plants. The spontaneous diploids confirmed as doubled haploids (DHs) following the self-pollination. The obtained DH and haploid plants from anther culture would be the valuable breeding materials for heterosis breeding.

Screening different methods of tetraploid induction in watermelon [Citrullus lanatus (thunb.) Manst. and Nakai

To screen a suitable method for inducing tetraploid with high frequency, five diploid inbred lines of watermelon, WC-8C-2C, WD-2-6K, GW11, GW7, and GW2 were selected. The efficiency of chromosome doubling with three methods direct seed (DS), shoot apex (SA), and inverted hypocotyl (IH) were tested by applying two different concentrations (0.1% and 0.2%) of antimitotic agent, colchicine, during the period January, 2010 to August, 2011 at Watermelon Experiment Station, Daesan, Gochang, Jeonbuk, South Korea. Mean chloroplast counts were 12 and 19 in diploid and tetraploid, respectively. The highest rate of efficiency of generating tetraploids (29.5%) was recorded in 0.2% IH method and considered as s best method. A total of 71 new tetraploids were generated successfully using present methods and techniques. WC-8C-2C and GW7 produced maximum number of tetraploids.

Study of ploidy level of annual species ofOnobrychisin Iran

Abstract Iran has more than 63 species of genus Onobrychis. This genus has small chromosomes with different ploidy levels. Counting ploidy levels in somatic cells in the metaphase is difficult; therefore, this study aims to evaluate a method using counts of stomata guard cell chloroplasts, as well as using flow cytometry as methods for determining ploidy levels in annual species of Onobrychis genus. Three middle leaves of greenhouse-grown plants were randomly selected for chloroplast counts in 10 pairs of stomata guard cells of the lower surface of leaf cells. For determining ploidy levels, the usual method was counting chromosomes which determined in the metaphase cells of root tip meristems with having minimum of 10 metaphase plate mitosis for each species. All of the samples were also studied, to determine ploidy levels using flow cytometry, and to find the mode and ratio for each of them. The results indicated that numbers of chloroplast stomata guard cells and flow cytometry in the tetraploid samples...