Number Of Chloroplasts In Guard Cells Research Articles

In vivo polyploidy induction of Phalaenopsis amabilis in a bubble bioreactor system using colchicine

Abstract Phalaenopsis amabilis Blume var. grandiflora Bateman is economically important as cut and pot flower. Polyploidy is considered as a valuable tool in improvement and evolution of ornamental plants. Protocorm-like bodies (PLBs) of P. amabilis were cultured on Murashige and Skoog medium containing 0.20 mg L-1 IBA together with 2.00 mg L-1 KIN and 1.00 g L-1 activated charcoal and grown for a period of five months. Fully-developed plantlets from in vitro grown PLBs were immersed in a bubble reactor filled with half-strength Hoagland solution containing the antimitotic agent colchicine (0.05%, 0.10% and 0.15%, w/v) for 72 h with a few drops (1 mL of 0.1%) of octylphenoxypolyethoxyethanol or Nonidet (P-40) as a surfactant. Plantlets were aerated to prevent hypoxia. Colchicine-treated and untreated plantlets were transferred to pots for a period of 60 days. Tetraploidy was successfully induced by 0.15% colchicine. Polyploidy levels were firstly detected using flow cytometry and then confirmed by cytological and morphological observations. The chromosome number was 2n = 2x = 38 in diploids and 2n = 4x = 76 in tetraploid. Incubation of plantlets in liquid medium containing 0.15% colchicine induced the maximum recovered tetraploids with minimum frequency of survival (50%). The tetraploid plants were more compact and exhibited round and thick leaves with darker green color than diploids. Stomata size in tetraploids were larger with less density than diploids. Chloroplast number in guard cells of tetraploids was about two times more than that of control. These results indicate that induction of tetraploids are a reliable and powerful tool for generation of novel phenotypes with ornamental and horticultural value for genetic improvement and breeding. Produced tetraploids in current study have potential in the ornamental/floriculture trade.

Morphological, anatomical, physiological, and cytological studies in diploid and tetraploid plants of Ispaghul (Plantago ovata Forsk.)

Ispaghul (Plantago ovata Forsk.) as an important medicinal plant has obtained a remarkable reputation due to therapeutic applications of seed mucilage. To determine the effect of in vitro-induced polyploidy on various characteristics of P. ovata, the terminal bud of two true leaves seedlings were separately treated with colchicine [0.1, 0.3 and 0.5% (w/v) for 6, 12 and 24 h] and trifluralin [7.5, 15 and 22.5% (w/v) for 24, 48 and 72 h] solutions. The ploidy level of induced tetraploids was determined via chromosome counting of root tip cells, and then confirmed through flow-cytometric analysis. Comparison the morphological, physiological, anatomical features of intact diploids and induced tetraploids revealed that tetraploids had considerable more height, thicker leaf, larger spike and seed, larger pollen grain and more seeds per spike. Moreover, the amount of chlorophyll (a, b, and total) and carotenoids, as well as chloroplast number in guard cells was further in tetraploids than diploids. Unlike density, stomata size in tetraploids was bigger than that one in diploids. It was also observed that seeds of tetraploids had more mucilage than diploids. In summary, we firstly developed the P. ovata tetraploids and suggested 0.3% colchicine for 24 h and 22.5% trifluralin for 72 h as the optimum treatments for inducing tetraploidy in P. ovata.

Morphological, anatomical, physiological, and cytological studies in diploid and tetraploid plants of Plantago psyllium

Plantago psylliumis is under development as the source of mucilage, but has not been entirely domesticated. Since mucilage content is low and variable in the medicinal plant, it is imperative to apply breeding approaches to accelerate the domestication process and improve the mucilage content in the seeds. One approach to achieve these purposes is polyploidy induction, which can increase the size of plants and their phyto-medicines. To conduct this approach and evaluate the effect of in vitro-induced polyploidy on different traits of P. psylliumis, a gradient of colchicine concentrations, 0.1, 0.3 and 0.5% (w/v), was used to treat the terminal buds for 6, 12 and 24 h. The terminal buds were separately treated with different trifluralin concentrations, 7.5, 15 and 22.5% (w/v), for 12, 24 and 48 h. The optimal treatments of 0.5% colchicine for 24 h, and of 22.5% (w/v) trifluralin for 72 h resulted in an induction efficiency of 23% and 19%, respectively. Chromosome counting revealed successful chromosome duplication in tetraploids (2n = 4x = 24), in contrast with intact diploids (2n = 2x = 12). Duplication of DNA size in tetraploids was also proved by flow-cytometry analysis. The tetraploids size was larger than their intact diploids for spike, seed and pollen grain, leaf thickness, and plant height. The stomata of tetraploids were larger with lower density than diploids. The chloroplast number in guard cells, along with chlorophyll and carotenoid content all were higher in tetraploids. In final, our study suggests that tetraploidization could be useful to improving the seed mucilage content for commercial use. We firstly fulfilled the in vitro induced tetraploidy in P. psyllium by 0.5% colchicine for 24 h and 22.5% (w/v) trifluralin for 72 h, and improved the mucilage content in the seeds.

In Vitro Segregation of Tetraploid and Octoploid Plantlets from Colchicine-induced Ploidy Chimeras in Echinacea purpurea L.

Echinacea purpurea L. is one of the important ornamental and medicinal plant species. Ploidy manipulation is a valuable tool for improving plant quality or production in E. purpurea as well as in many other plants. To study the segregation of pure ploidy plantlets from colchicine-induced ploidy chimeras in E. purpurea, we used a chimera plantlet that consisted of 1.93% diploid, 35.04% tetraploid, and 63.03% octoploid cells as the source material for experiments. The results showed that three factors significantly influenced the segregation, i.e., the component ratios of different ploidy cells in the chimera, the number of sequential passages, and the methods of segregation culture of the chimera plantlets. Other factors, such as explant types (i.e., leaf, petiole, or root) and 6-benzyladenine (BA) concentrations (i.e., 0.2, 0.4, 0.8, and 1.2 mg·L−1) occasionally influenced the segregation. Pure chromosome-doubled polyploids are not easily obtained in various plant species, so segregation culture of ploidy chimeras may potentially be more effective. The morphological characteristic and content of cichoric acid were compared among diploid, tetraploid, and octoploid plants. Results indicated that tetraploid and octoploid plants had more stunted growth, larger stomata, lower stomata frequency, more chloroplast number in guard cells, and higher cichoric acid content than original diploid lines.

Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea (L.)

Echinacea purpurea (L.) is one of the important medicinal plant species. To obtain the tetraploid plants of Echinacea purpurea with improved medicinal qualities, the root tips of two true leaves seedlings were imbibed in 0.25 % (w/v) colchicine solution for 24, 48, 72, 96 and 168 h. The ploidy level of plants was determined by chromosome counting of root tip cells, and confirmed by flow cytometric analysis. Tetraploid induction occurred in seedlings treated for 24, 48 and 72 h at colchicine solution. The morphological, physiological, cytological, and phytochemical characteristics of diploid and colchicine-induced tetraploid plants were compared. Results indicated that tetraploid plants had considerable larger stomata, pollen grain, seed and flower. Moreover, chloroplast number in guard cells, amount of chlorophyll (a, b, and a + b), carotenoids as well as width and thickness of leaves were increased in tetraploids. However, stomata frequency, leaf index, plant height, and quantum efficiency of photosystem II in tetraploid were lower than diploid plants. High-performance liquid chromatography analysis showed that leaves of the tetraploid plants had more cichoric acid (45 %) and chlorogenic acid (71 %) than diploid plants. It was concluded that morphological and physiological characteristics can be used as useful parameters for preliminary screening of putative tetraploids in this species.

Induction and identification of polyploidy in basil (Ocimum basilicum L.) medicinal plant by colchicine treatment

Basil (Ocimum basilicum) is one of the important medicinal plant species. In order to produce an autotetraploid population of basil (Ocimum basilicum) by colchicine, different concentrations (0.00, 0.05, 0.10, 0.20, 0.50 and 0.75%) and four treatment methods were examined (seed, the growing point of seedlings at the emergence of cotyledone leaves stage and emergence of true two type leaves stage, and root treatment) to determine the best treatment for the induction of tetraploid plants. Autotetraploid plants were produced only by treatment of growing point of seedlings, at the emergence of cotyledone leaves stage, and treatment with 0.5% proved to be the most effective in producing autotetraploids. The induced tetraploids in basil was accompanied by larger stomata and pollen grains, increase in chloroplast number in guard cells and decrease in stomata density, compared to diploid control plants. In order to distinguish the induced colchicine tetraploid plants from the diploids, morphological changes and techniques as stomata size, number of chloroplasts per guard cell, pollen grain diameter and flow cytometry were considered and proved that these methods are suitable, quick and easy methods for identification the ploidy level of Ocimum basilicum in various stages of the plant development of these species and among this methods flow cytometry as found to be the most efficient method for detecting induced changes in ploidy level.

Doubling the chromosome number of Salvia hains using colchicine: Evaluation of morphological traits of recovered plants

This study demonstrates a protocol of polyploidy induction in Salvia hains, in order obtained tetraploid five concentrations (0.1, 0.3, 0.4, 0.5 and 0.7%) of colchicine were used to determine the best treatment for the induction of tetraploid plants. Treatment seeds with colchicine for 24 h product tetraploid plants. Ploidy level of the resulting plantlets was determined by morphological study, chromosome counting and flow cytometry. Tetraploid plants were obtained from S. hains after applied concentrations 0.3, 0.4 and 0.5% of colchicine. Plants obtained from 0.1% colchicine were all diploids. The induced tetraploid in S. hains was accompanied by larger stomata increase in chloroplast number in guard cells and decrease in stomata density, compared to diploid (control) plants. The techniques used to induce tetraploid S. hains plants by colchicine were successful and could be used in breeding programs. Key words: Autotetrapolid, flowcytometry, polyploid, Salvia hains, chromosome counting.

Chloroplast number in guard cells as ploidy indicator of in vitro-grown androgenic pepper plantlets

The number of chloroplasts per guard cell pair and the stomatal length of seventeen anther-derived plants obtained from three pepper donor genotypes were recorded. The haploid plants showed significantly fewer chloroplasts and shorter stomatal length than the diploid plants. Although both characters were positively correlated with the ploidy level, chloroplast number resulted a more reliable method compared to stomatal length to distinguish haploid from diploid in vitro-grown plantlets. The stomatal length values showed either significant variation among androgenic plants with the same ploidy or overlapping values between haploid and diploid individuals. On the contrary, the values for chloroplast number were more stable permitting discrimination of haploid from diploid plantlets regardless of their genetic background. Early ploidy estimation based on chloroplast counts of in vitro-grown plantlets may hasten pepper breeding programs utilizing anther-derived doubled haploids.

Rapid estimation of ploidy levels in in vitro-regenerated interspecific Arachis hybrids and fertile triploids

A significant correlation among chromosome number, chloroplast number and pollen grain size was observed using interspecific hybrids (2n=30,60), Arachis hypogaea (2n=40), A. stenosperma (PI 338280) (2n=20), A. batizocoi (PI 468329) (2n=20) and A. villosulicarpa Hoehne (PI 336984) (2n=20), representing four ploidy groups. Both chloroplast number in guard cells and pollen grain size were found to be positively correlated with ploidy, and provided a reliable method to distinguish diploid, triploid, tetraploid and hexaploid plants from each other regardless of their taxonomic backgrounds. These methods in combination with root-tip chromosome counts enabled us to confirm ploidy determinations on all three dissue layers, L1, L2 and L3 (guard cells, microsporocytes and root meristematic cells, respectively), and to detect the chimeric nature of some colchicine-treated tissue culture-derived plants. Screening pollen grains by size and the detection of highly stainable and viable, 30-chromosome pollen grains have enhanced the use of triploid hybrids in peanut breeding programs.

The control of chloroplast number in Solanum muricatum Ait. and Cyphomandra betacea (Cav.) Sendt. and its value as an indicator of polyploidy

Guard cell chloroplast numbers were counted in the tamarillo (Cyphomandra betacea (Cav.) Sendt.), the pepino (Solanum muricatum Ait.), and in wild relatives and polyploids of these species. Chloroplast number was found to be a reliable indicator of the euploid level in both species, but in aneuploids the effects of different chromosomes were variable. Some had no effect but in others the effects werepositive and sometimes significantly different from the diploids. Comparison of DNA amounts and plastid numbers in wild Cyphomandra species did not reveal a high correlation between these two characters. These results argue against a purely nucleotypic control of chloroplast number in guard cells in favour of a more complex genotypic system.

Chromosomal instability in cell- and tissue cultures of tomato haploids and diploids

The effect of the tissue culture system, the genotype and the ploidy level of the plant material used as explant source on the stability of the ploidy level of plants regenerated fromcell and tissue cultures of tomato was investigated. In addition the use of the chloroplast number in guard cells as a measure for ploidy level was evaluated. Haploids of tomato were very instable, which instability was observed already in somatic root tip and leaf cells. The number of regenerated plants that retained the original ploidy level differed significantly between the tested haploids. The plants that were regenerated from leaf explants of diploids were predominantly diploid in contrast to the plants regenerated from established callus cultures and protoplast where the majority was tetraploid.