Colchicine-induced Tetraploids Research Articles

Variations in colchicine-induced autotetraploid plants of Lilium davidii var. unicolor

Colchicine-induced polyploid plants exhibit more variations not only in morphology and cytology but also in genomic DNA sequence. In this study, tetraploid Lanzhou lily (Lilium davidii var. unicolor) plants were induced by colchicine, and the factors that affected the tetraploid induction rate, including pre-culture duration and colchicine concentration, were examined. The results showed that both pre-culture duration and colchicine concentration exerted significant effects on the tetraploid induction rate. The highest tetraploid induction rate (33.33%) resulted from a 15-day-preculture treatment followed by soaking in 0.025% colchicine solution for 24 h. The ploidy levels of the regenerated plantlets were confirmed by flow cytometry analysis and chromosome number counting. The variations in morphological and cytological characteristics, as well as genomic DNA sequences of colchicine-induced tetraploids and diploids, were compared. The results indicated that tetraploids shared less leaf numbers, more leaf width, lower stomata density, and longer guard cell length. Moreover, inter-simple sequence repeat assay revealed notable genetic variations in the genomic DNA sequence between tetraploids and diploids, and the 75% polymorphism between them indicated that the variation in DNA sequence occurred during the chromosome doubling. Colchicine-induced tetraploid Lanzhou lily plants exhibited variations not only in morphology (including leaf number and leaf width) and cytology (including stomata density and guard cell length) but also in genomic DNA sequence.

Colchicine 처리에 의해 유기된 4배체 하수오의 형태 및 세포유전학적 특성

Colchicine 처리에 의해 유기된 4배체 하수오의 형태 및 세포유전학적 특성

In vitro induction of tetraploids in cassava variety ‘Xinxuan 048’ using colchicine

Polyploids have great breeding value as they could have higher vegetative yields, higher qualities and greater tolerances against biotic and abiotic stresses. This research is focusing on in vitro colchicine-induced tetraploids in cassava. The survival rate of explants decreased with the increase of colchicine concentrations. Based on the survival rate, the treatment with 0.05 g l−1 colchicine for 2 days was the best protocol for tetraploid induction in the cassava variety ‘Xinxuan 048’. The determination of ploidy levels showed that 28 autotetraploidy and 12 mixoploidy plantlets were obtained from 44 plantlets. Thus, 90.9% of the plants were variants. Significant differences in morphology and anatomy were found between the diploid and tetraploid plants. Tetraploid plants showed better photosynthetic capacities than the original diploid plants. The tetraploid cassava regenerated herein will enrich the germplasm spectrum and open a new arena for breeding novel triploids with elite cultivars by interploid crossing in the future.

In vitro induction and morphological characteristics of octoploid plants in Pogostemon cablin.

This paper describes an efficient colchicine-mediated technique for in vitro induction of octoploids in Pogostemon cablin and its confirmation by flow cytometry and chromosome numbers. The highest octoploid induction ratio was obtained by 0.05% colchicine treatment for 72 h. The chromosome number of octoploid seedlings was 2n = 8x = 128. Colchicine-induced tetraploids and octoploids planted in soil remained stable after 6 months. There were 31 lines of octoploid plants obtained. The leaf characteristics of P. cablin tetraploids and octoploids were compared. The larger leaves and stomata of transplants can be used to identify putative octoploids in P. cablin. Most octoploid lines exhibited higher patchoulic alcohol contents than the controls after 6 months of cultivation. Our results demonstrated that polyploidy induction can be beneficial in improving the medicinal value of P. cablin.

Breeding polyploid varieties of tropical acacias: progress and prospects

The paper reviews a decade of progress with breeding new polyploid varieties of Acacia mangium and other tropical acacias in Vietnam, and outlines plans for future strategies to complement conventional diploid breeding. Polyploid breeding is a means of introducing diversity into breeding populations, of making step-change improvements in wood fibre and other properties, and reducing reproductive output of these potentially weedy taxa. To date we have demonstrated that A. mangium tetraploids have larger and thicker wood fibres and we have strong preliminary evidence that the reproductive output of triploids is indeed reduced. Other potential benefits such as wind and stress tolerance and disease resistance are under field evaluation. Trials demonstrate that colchicine-induced tetraploids, whether derived from pure A. mangium seeds or from commercial hybrid clones, are substantially slower growing than diploids. While not the sole determinant of crop value, volume production is hugely important to growers, so long-term success of the program is critically dependent upon demonstration that this problem can be resolved in advanced generations. Future emphasis will be on production of triploid and tetraploids from the interspecific hybrid A. mangium × A. auriculiformis. The better rooting ability contributed by A. auriculiformis maximises prospects for operational cloning of select trees. Highly heterozygous hybrids also offer the better prospect for exploiting heterosis. A strategy for developing a diverse breeding population of neo-tetraploid F1 hybrids is presented.

Seed attributes in fruit of colchicine-induced tetraploids of Actinidia chinensis

Seed characteristics are important in kiwifruit breeding and production as they affect the efficiency of cross breeding and commercial fruit size and quality. Following a study of the size, shape and quality attributes of induced tetraploid kiwifruit, three methods for seed evaluation were compared to uncover the effect of chromosome doubling on seed development, quantity and quality in induced tetraploids of Actinidia chinensis. Results over 3 years consistently showed that colchicine-induced tetraploids had a significantly lower seed density in transverse sections of fruit, fewer normally filled seeds, and more unfertilized and aborted seeds than their diploid progenitors. The weight of filled seeds of colchicine-induced tetraploids from the commercial gold-fleshed kiwifruit cultivar ‘Hort16A’ was significantly (33%) higher than that of their diploid progenitor. In contrast, the weight of filled seeds from the fruit of colchicine-induced red-fleshed A. chinensis tetraploids was significantly lower than that of their respective diploid progenitors. Seed size may have been related mainly to the availability of pollen from nearby males of the same ploidy. The dry seed coat colour of colchicine-induced tetraploids was brown in comparison to the black seeds of diploids. This is the first time that the reproductive nature of autotetraploid kiwifruit has been systematically characterized. I have shown that chromosome doubling significantly reduces seed numbers and affects seed development inside kiwifruit.

Meiotic chromosome pairing behaviour of natural tetraploids and induced autotetraploids of Actinidia chinensis

Non-preferential chromosome pairing was identified in tetraploid Actinidia chinensis and a higher mean multivalent frequency in pollen mother cells was found in colchine-induced tetraploids of A. chinensis compared with naturally occurring tetraploids. Diploid and tetraploid Actinidia chinensis are used for the development of kiwifruit cultivars. Diploid germplasm can be exploited in a tetraploid breeding programme via unreduced (2n) gametes and chemical-induced chromosome doubling of diploid cultivars and selections. Meiotic chromosome behaviour in diploid A. chinensis 'Hort16A' and colchicine-induced tetraploids from 'Hort16A' was analysed and compared with that in a diploid male and tetraploid males of A. chinensis raised from seeds sourced from the wild in China. Both naturally occurring and induced tetraploids formed multivalents, but colchicine-induced tetraploids showed a higher mean multivalent frequency in the pollen mother cells. Lagging chromosomes at anaphase I and II were observed at low frequencies in the colchicine-induced tetraploids. To investigate whether preferential or non-preferential chromosome pairing occurs in tetraploid A. chinensis, the inheritance of microsatellite alleles was analysed in the tetraploid progeny of crosses between A. chinensis (4x) and A. arguta (4x). The frequencies of inherited microsatellite allelic combinations in the hybrids suggested that non-preferential chromosome pairing had occurred in the tetraploid A. chinensis parent.

Production of Intersectional Hybrids between Colchicine-induced Tetraploid Shashanbo (Vaccinium bracteatum) and Highbush Blueberry ‘Spartan’

With crosses between colchicine-induced tetraploid shashanbo (Vaccinium bracteatum section Bracteata) and tetraploid highbush blueberry ‘Spartan’ (Vaccinium corymbosum section Cyanococcus), intersectional hybrids were produced. The hybridity of these plants was confirmed based on DNA markers and morphological characteristics. The morphological characteristics, blooming date, and ripening period of the hybrids were intermediate between those of the parents. Ploidy analyses by flow cytometry and chromosome counting revealed that these hybrids were tetraploid. Four hybrids set fruit in the field and these two hybrids showed high pollen stainability. It was noteworthy that fruit of two hybrids had high soluble solids concentration compared with ‘Spartan’ and the fruit pulp of the hybrids was tinged with red as shashanbo. These hybrids could be useful in breeding new cultivars with high sugar content, abundant phytochemicals, extensive environmental adaptability as well as late flowering and fruit maturity.

Fruit Quality in Induced Polyploids of Actinidia chinensis

Fruit of colchicine-induced tetraploids of Actinidia chinensis were 50% to 60% larger than those of their diploid progenitors. In addition to fruit size, fruit quality is a key issue in any commercialization of these autotetraploids. We have made the first detailed study of the effects of chromosome doubling on fruit quality parameters other than size: these attributes include flesh firmness, color, soluble solid content (SSC), dry matter content (DM), vitamin C content, fruit skin thickness, and red pigmentation distribution in red-fleshed kiwifruit. Four selections from colchicine-induced tetraploids from the yellow-fleshed kiwifruit cultivar Hort16A were trialed for the stability of their fruit quality. Analysis of fruit at harvest over 3 years showed that fruit of the induced autotetraploids were significantly softer (lower flesh firmness), had lower DM, and had a less intense golden flesh color than fruit of their diploid progenitor. During development, SSC of fruit of the autotetraploid plants started to increase earlier than in the diploid ‘Hort16A’. This has been confirmed by replicated trials. No difference was found in vitamin C content between fruit of autotetraploids and diploids. Autotetraploids had significantly thicker skins than diploid ‘Hort16A’. Induced autotetraploids from three female genotypes of red-fleshed A. chinensis showed similar trends to autotetraploids of ‘Hort16A’ in fruit flesh firmness and outer pericarp flesh color, DM, SSC, and vitamin C. All the traits analyzed indicated that fruit of the autotetraploid plants matured earlier than those of their diploid progenitors. Furthermore, red pigmentation, one of the most important traits for red-fleshed kiwifruit breeding, showed a reduction in both intensity and distribution in the autotetraploids compared with their diploid progenitors. There was considerable variation among fruit of autotetraploid plants regenerated from each diploid progenitor. Therefore, selection among the regenerants may be required to achieve the best outcome after ploidy manipulation in kiwifruit breeding.

In vitro induction of tetraploids from immature embryos through colchicine treatments in Clivia miniata Regel

The immature embryos of Clivia miniata Regel. cv. Ranchang were in vitro induced with 0.00, 0.01, 0.03 and 0.05% colchicine treatment for 10, 20 and 30 d, respectively on Murashige and Skoog (MS) medium supplemented with 2.0 mg L-1 a-Naphthaleneacetic acid (NAA) and 1.5 mg L-1 6-Benzylaminopurine (BA). A total of 14 tetraploids were obtained with an average induction rate of 16.1%. The most effective treatment was 0.03% colchicine for 20 days with an induction rate of 30.0%. The stomata size (37.24 × 12.31 μm) of tetraploids was significantly larger than that of diploids (22.51 × 7.43 μm). The stomata density of tetraploids (13.80 mm2) was significantly lower than that of diploids (30.78 mm2). The chloroplast number of guard cells of tetraploids (41.53) was significantly more than that of diploids (28.00). Compared with diploids, tetraploids had thicker, wider, shorter, rougher and deeper-colorful leaves, fewer roots and slower growth. The leaf and stoma characteristics could be regarded as helpful indexes to identify colchicine-induced tetraploids in C. miniata. Key words: Clivia miniata Regel., chromosome doubling, colchicine, tetraploid, stoma, chloroplast number.

In vitro tetraploid induction and generation of tetraploids from mixoploids in Dioscorea zingiberensis.

This article describes an efficient colchicine-mediated technique for the in vitro induction of tetraploids in Dioscorea zingiberensis and its confirmation by flow cytometry. Buds immersed in 0.2% colchicine solution for 36 hours prior to culture induced as high as 35.6% tetraploid plants. Colchicine-induced tetraploids remained stable after six months in soil. Leaf characteristics of diploids and tetraploids in D. zingiberensis were compared. It was determined that the leaf sizes of glasshouse-grown plants and stomatal sizes of both in vitro and glasshouse-grown plants were suitable parameters for identifying putative tetraploids in D. zingiberensis. Besides generating tetraploids, this technique generated mixoploids in D. zingiberensis. Calli derived from mixoploid leaves were induced to form buds and shoots. Individual shoots were classed as diploid, mixoploid, and tetraploid by flow cytometry. This callus-based technique could be employed when a genome-doubling agent generated mixoploids, but no tetraploids.

Cytogenetical investigations in colchicine-induced tetraploids of Cyamopsis tetragonoloba L.

Successful induction of autotetraploidy has been achieved in five accessions of Cyamopsis tetragonoloba L. The diploid seedlings of these accessions were treated with different concentrations of aqueous colchicine using the cotton-swab method for 10–18 hours within 2–3 days. The highest percentage of success was recorded when the seedlings were treated with 0.2% colchicine for 10 h within two days. The synthesized plants showed remarkable enhancement in several morphological and floral characters making them more robust and better plants from the food and feed aspect. Cytologically, quadrivalent frequency ranging from 3.18 to 5.45 and univalent frequency ranging from 0.08 to 1.10 were characteristic of the colchicine-induced tetraploids. Among all the associations, bivalent chromosome associations were observed more frequently (2.95 to 6.04). The anaphase I and II disjunction of bivalents/chromosomes was leading more or less regularly and equally to the formation of at least few seeds from some of the synthesized plants. Significant enhancement in morphological traits as revealed in colchicine-induced tetraploid plants having a better food and feed value and normal meiotic behaviour of synthesized autotetraploids leading to a good seed set may ultimately result in the genetic improvement of Cyamopsis tetragonoloba.

In vitro tetraploid induction and generation of tetraploids from mixoploids in Astragalus membranaceus

This paper describes an efficient colchicine-mediated technique for the in vitro induction of tetraploids in Astragalus membranaceus and its confirmation by flow cytometry. Buds immersed in 0.2% colchicine solution for 36 h prior to culture induced as high as 35.3% tetraploid plants. Colchicine-induced tetraploids remained stable after 6 months in soil. Leaf characteristics of diploids and tetraploids in A. membranaceus were compared. It was determined that leaf sizes of glasshouse-grown plants and stomatal sizes of both in vitro and glasshouse-grown plants were suitable parameters for identifying putative tetraploids in A. membranaceus. As well as generating tetraploids, this technique generated mixoploids in A. membranaceus. Calli derived from mixoploid leaves, were induced to form buds and shoots. Individual shoots were classed as diploid, mixoploid and tetraploid by flow cytometry. This callus-based technique can be employed when a genome-doubling agent generates mixoploids but no tetraploid.

Comparison of Growth Characteristics between Diploid and Colchicine-induced Tetraploid Grape Rootstocks.

Autotetraploids of three grape rootstocks, 'Gloire de Montpellier'('Gloire', Vitis riparia Michx), 'Rupestris St. George'('St. George', V. rupestris Scheele), and 'Couderc 3309'('3309', V. riparia×V. rupestris), were induced by the colchicine treatment of micropropagated plantlets. Doubling of chromosomes in the selected colchicine-treated clones was confirmed by flow cytometric analysis of the immature leaves. The tetraploid leaves had larger stomata that were distributed at a lower density than those of the original diploid leaves. In the rooting culture, all tetraploids had much shorter root lengths than had the original diploids. Under rooting culture, tetraploid shoots of 'Gloire' and 'St. George' were shorter than those of diploids, but tetraploids of '3309' were not. In the acclimating stage in this experiment, the tetraploids, also had shorter shoots, internodes and roots than had the original diploid plants. In the greenhouse experiment, shoot of tetraploid rootstocks, likewise, made poorer growth than did those of the diploids, but they tended to have thicker stem diameters and the specific leaf weights (leaf dry weight/leaf area). The thicker and shorter roots of the tetraploid rootstocks made their root system more compact than the diploids.

In vitro tetraploid induction and generation of tetraploids from mixoploids in hop (Humulus lupulus L.)

This paper describes an efficient colchicine-mediated technique for the in vitro induction of hop tetraploids and its confirmation by flow cytometry. A window of conditions generated a high percentage (>20%) of tetraploid induction, with the highest induction (25.6%) achieved with 0.05% colchicine for 48 h. Colchicine-induced tetraploids remained stable after 6 months in soil. Leaf characteristics of diploid and tetraploid hops were compared, and it was determined that stomatal length and width are suitable parameters for identifying putative hop tetraploids. As well as generating tetraploids, this technique generates mixoploid hops. Calli, derived from mixoploid leaves, were induced to form shoot buds and shoots. Individual shoots were classed as diploid, mixoploid or tetraploid after screening by flow cytometry. This callus-based technique can be employed when a genome-doubling agent generates mixoploids but fails to generate tetraploids.

Chromosome doubling and fertility study of Alstroemeria aurea × A. caryophyllaea

Self-pollinations of a diploid (2n = 2x = 16) interspecific hybrid Alstroemeria aurea × A. caryophyllaea, resulted in no seeds. Backcrosses of the hybrid with parent A. aurea did not produce any seeds. In an attempt to restore the hybrid fertility, an efficient in vitro procedure has been developed and applied effectively in the chromosome doubling of the diploid hybrid. Forty-one percent of the treated plants were proven to be truly tetraploid by chromosome counts and stomatal measurements after applying 0.2 to 0.6% colchicine for 6 to 24 hours. Over 87.5% of these colchicine-induced tetraploids were stable and retained their tetraploidy after one year of growth. The fertility of the hybrid was not restored although the pollen stainability was increased from 0 to 12% after chromosome doubling. Cytological studies on the pollen mother cells (PMCs) of the sterile diploid hybrids revealed abnormal meiotic behaviors. In addition, aneuploid chromosome numbers, ranging from 2n = 1 to 2n = 18, were observed in over 45% of the PMCs examined. PMCs of the colchicine-induced tetraploids showed that meiotic chromosome pairings were normal in most cases (1.59I + 15.07II + 0.05III + 0.03IV). These results indicate that the sterility of this hybrid is not only caused by parental chromosome differences, but other complex fertility/sterility-regulating mechanisms are involved too.

Cytological Evidence for the Sterility of an Interspecific Alstroemeria Hybrid

Self-pollinations of a diploid (2n = 2x = 16) interspecific hybrid from the cross of Alstroemeria aurea × A. caryophyllaea resulted in no seed set. Pollen viability studies with the hybrid demonstrated that only 5% of the pollen grains were viable. Cytological observations with the hybrid pollen mother cell (PMC) revealed abnormal chromosome behaviors, such as no pairing in Prophase I and Metaphase I, and bridges in the Anaphase I and II. Although the development of microspores appeared normal in shape until the stage of tetrad release, some chromosomes did not remain in the nucleus after completing meiosis, formed isolated groups of 1 to 4, and remained in the cytoplasm. This genetic imbalance of the microspores could be one of the causes for the abortion of the pollen grains in the late stage of development. Additional meiotic cytological studies with colchicine-induced tetraploids (2n = 4x = 32) derived from the hybrid plants showed that chromosome pairings were normal in most cases. However, self-pollination with the tetraploid plants failed to set seeds. These studies with the tetraploids further demonstrate that the sterility of the hybrid is due not only to chromosomal differences, but also to complex genic interactions.

STOMATAL ACRILIC MICRO-REPLICATIONS TO DIFFERENTIATE DIPLOID AND TETRAPLOID WATERMELONS (Citrullus lanatus)

Early discrimination of diploids on breeding for seedless watermelon is time saving and economically important. The common method is pollen microscopic observation, it is time consuming and can not be executed on early stage. The objective was to prove the use of stomatal size and density to differentiate ploidy on plantlets. “Charleston Grey” and “Sugar Baby” diploids and colchicine-induced tetraploids of the same varieties were used. Stomata micro-replication made with acrilic varnish were used for measurements and correlated with individual ploidy. Significative differences were found on both size and density. “Charleston Grey” had larger stomata than “Sugar Baby”. Tetraploids had larger stomata and lower density regardless of genotype. Stomata micro-replications is time saving and non destructive method to differentiate ploidy on early stages.

Asp 8271 and Asp 8284 Asparagus Germplasm

Abstract Asparagus germplasm populations, Asp 8271 and Asp 8284, are the F2 generation of crosses between colchicine-induced tetraploids of garden asparagus, Asparagus officinalis L. ‘Viking’, and a wild tetraploid, A. officinalis spy. prostratus (Dumort.) Corb., both 2n = 4x = 40. They were released in Feb. 1987 from a program at Beltsville in which crosses in the genus are investigated as they relate to the gene pool available for breeding disease-resistant garden asparagus.

Genetic Progress through Hybridization of Induced and Natural Tetraploids in Crested Wheatgrass

Genetic Progress through Hybridization of Induced and Natural Tetraploids in Crested Wheatgrass