Colchicine-induced Autotetraploids Research Articles

Expression dosage effects of a small number of genes after the artificial doubling of weeping forsythia

Whole genome doubling (WGD) plays a critical role in plant evolution, yet the mechanisms underlying the maintenance of overall equilibrium following an artificial doubling event, as well as its impact on phenotype and adaptability, remain unclear. By comparing the gene expression of naturally occurring weeping forsythia diploids and colchicine-induced autotetraploids under normal growth conditions and cold stress, we identified gene expression dosage responses resulting from ploidy change. Only a small proportion of effectively expressed genes showed dosage effect, and most genes did not exhibit significant expression differences. However, the genes that showed expression dosage effect were largely random. The autotetraploids had slower overall growth rates, possibly resulting from negative gene dosage effects on zeatin synthesis and multiple metabolic delays caused by other negative dosage genes. Our comparative analysis of cold response genes in diploids and autotetraploids revealed that genes related to “response to abscisic acid” and “cold acclimation” were key factors contributing to greater cold tolerance in the autotetraploids. In particular, gene expression related to “cold acclimation” might mitigate the effects of cold stress. Taken together, our findings suggested that overall gene expression equilibrium following WGD of weeping forsythia autotetraploids was achieved through the inactivation of the majority of duplicated genes. Our research provides new insights into the mechanisms regulating expression dosage balance following polyploidization events.

Effects of polyploidization on photosynthetic characteristics in three Lilium species

Photosynthesis play an important role in plant growth. Generally, it will be increased with increased DNA content per cell. However, in organ levels, the changes of photosynthesis are species dependent. So, for target breeding materials, chromosome duplication how to influence photosynthetic performances need further investigation. In this study, three lily (Lilium) species, L. rosthornii (LRI), L. primulinum var. orchraceun (LPO) and L. regale (LRE), and their colchicine-induced autotetraploids were employed in comparing photosynthetic related characteristics. For anatomic traits, the number of palisade mesophyll cells was significantly increased in LRI and LPO but decreased in LRE after polyploidization. Moreover, except the intercellular space decreased, the mesophyll cell sizes and shapes presented different changes in the three species after polyploidization. For physical-biochemical traits, the chloroplast number, thylakoid grana thickness, and photosynthetic pigments and efficiency were significantly increased in LRI after polyploidization. For LPO, the chloroplast number, and weak light absorbed and CO2 assimilated rates were significantly increased, while others showed no significant changes after polyploidization. Out of expecting, for LRE, the chloroplast number and photosynthetic pigments were significantly decreased after polyploidization. Moreover, the structures of chloroplasts were abnormal with extremely thicker thylakoid grana and more tightly arranged thylakoid layer in tetraploids than in diploids. The photosynthetic rates of LRE were similar in diploid and tetraploid in natural conditions, while the maximum photosynthetic rate (Pmax) of light-/CO2-response curves were higher in diploids than in diploids. After plants withered, larger bulblet with more developed root systems were well observed from the three tetraploid species.

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.

Hybridity and autopolyploidy increase the expressivity of apospory in diploid Paspalum rufum

Paspalum rufum Nees is a perennial grass that forms a multiploid complex. Natural populations are composed mainly of diploid (2n = 2x = 20) and tetraploid (2n = 4x = 40) cytotypes. The diploid form is sexual and highly self-sterile, while the tetraploid is pseudogamous aposporous apomict and self-fertile. Diploids can also develop aposporous sacs, and some of them complete apomixis. The objective of this work was to analyse apospory expressivity (a gametophytic apomixis component) in diploid hybrids and colchicine-induced autotetraploids of the species. One F1 family was created by crossing two diploid individuals (R6#45 × R5#49) carrying aposporous sacs in 5.8 and 13 % of their ovules, respectively. Moreover, two synthetic autotetraploids were obtained by colchicine treatment of mature seeds from R6#45. The hybrid origin of the F1s was confirmed by segregation analyses of a morphological trait and molecular markers, and the ploidy level of experimental plants was determined by flow cytometry. Apospory expressivity was estimated by embryo sacs observation at anthesis. Out of the 39 hybrids analysed, 38 showed aposporous embryo sacs. Expressivity of the trait ranged from 0 to 36 %, and some individuals differed significantly for both progenitors’ values. Both doubled-diploid plants showed 25 and 32 % of apospory expressivity, which was significantly higher than that observed in R6#45. Results presented in this work revealed a high variability in apospory expressivity of diploid hybrids and suggested that more than one allele are controlling the trait. Moreover, the new induced doubled-diploid plants showed that apospory expressivity is highly ploidy dependent.

Pollen effects on fruit attributes and seed properties in colchicine-induced autotetraploids of red-fleshed kiwifruit (Actinidia chinensis Planch.)

SUMMARYThe effect of the ploidy level of the polleniser has not been considered during the characterisation and evaluation of colchicine-induced autotetraploids of Actinidia chinensis (kiwifruit). It is therefore possible that few induced autotetraploids reach their potential size. Five genotypes of tetraploid pollenisers were used for controlled pollination of red-fleshed autotetraploids. Fruit of colchicine-induced autotetraploids from two genotypes of female red-fleshed A. chinensis plants were, on average, 25–30% larger than fruit arising from open-pollination in an orchard containing only a diploid male as polleniser. Fruit dimensions increased more in cross-section than in length, so the fruit were more round. Seed set increased up to two-fold and the number of filled seeds also increased significantly (P ≤ 0.01). The average filled-seed weighed two-to-three times more than that of seed from open-pollination with pollen from a diploid male, and were heavier than those in fruit of their diploid progenitors under open-pollination. The distribution of the red-flesh pigmentation increased, although its intensity was not significantly improved. The total soluble solids content (TSSC) increased by 1–2% and the size of the core cavity at the stylar end was significantly reduced (P ≤ 0.01) and sometimes completely. All these effects on fruit attributes would benefit the commercial cultivation of red-fleshed kiwifruit. It is therefore recommended that, as for diploids, pollenisers of tetraploid red-fleshed kiwifruit should be of matching ploidy. Two abnormal fruit with soft red cores were found on a branch of a tetraploid regenerant during the evaluation of fruit generated by controlled pollination. The possible origin and application of this mutation for novel kiwifruit breeding are also discussed.

Production of Colchicine-induced Autotetraploids in Pummelo (Citrus grandis Osbeck) through Indirect Organogenesis

Colchicine-induced stable autotetraploid plants were recovered through indirect organogenesis from stem sections of in vitro-grown zygotic seedlings of pink/red-fleshed pummelo (Citrus grandis L. Osbeck) selections cybrid Hirado Buntan (C-HBP) (pink), 5-1-99-3 (dark pink), and HBJL-5 (red), all derived from Hirado Buntan pink pummelo. Multiple shoot induction was achieved through indirect organogenesis from the callus produced from the cut ends of the treated explants. Ploidy levels of regenerated plantlets were determined through flow cytometry at a stage when recovered shoots had one to two expanded leaves. Recovered tetraploids proved to be stable after 2 years in the field. As expected, higher colchicine concentrations and treatment durations decreased the survival rates of the regenerated plantlets. Colchicine concentrations of 0.05% and 0.1% produced the most tetraploids; of the 19 total tetraploids produced, 10 were produced from the treatment with a colchicine concentration of 0.1% and six were produced from treatment with a concentration of 0.05%. After flowering, these stable pink/red-fleshed tetraploid plants generated should be useful as breeding parents in our grapefruit/pummelo improvement program, especially if any show canker tolerance or reduced furanocoumarins. Use of monoembryonic tetraploids in interploid citrus crosses eliminates the need for embryo rescue to recover seedless triploid progeny; this research expands our pool of available high-quality monoembryonic tetraploid parents.

Production of colchicine-induced autotetraploids as a basis for sterility breeding in Acacia mangium Willd

Australian acacias are widely planted as exotics and in some cases as invasive. Impact may be reduced if sterile triploid planting stock can be developed. This article reports the first step in such a breeding programme, the production of a population of tetraploid lines for inter-breeding with diploids. Three methods of polyploid induction with colchicine were compared. A conversion rate of 8.9 per cent was obtained by applying 1.5 per cent colchicine to the shoot apical meristem of seedlings. A 7 per cent conversion rate was obtained by germination of scarified seed on filter paper saturated with 0.02 per cent colchicine for 16 h and this method is recommended on logistical and safety grounds. Poor results were obtained when scarified seed were submerged in aqueous solutions of colchicine. Flow cytometry is the preferred method for ploidy determination, sampling after vegetative phase change on a minimum of two opposing phyllodes per plant. Visual classification was inaccurate due to the confounding effects of growth-retarding properties of colchicine. Size and distribution of stomata can also be used but is more time consuming than flow cytometry. At 26 months, tetraploid plants had heavier, thicker, wider and more cupped shaped phyllodes than diploids and the bark: stem diameter ratio was greater.

Growth and Fruit Quality of 'Kyoho' Grapevines Grafted on Autotetraploid Rootstocks

The growth of ‘Kyoho’ (Vitis × labruscana Bailey × V. vinifera L.) grapevines grafted on colchicine-induced autotetraploids of two grape rootstocks, ‘Riparia Gloire de Montpellier’ (‘Gloire’, V. riparia Michx.), and ‘Couderc 3309’ (‘3309’, V. riparia × V. rupestris), was compared with the original diploids. Micro-propagated rootstock and ‘Kyoho’ grapevines were grafted in vitro and rooted. During the rooting and acclimating stage, ‘Kyoho’ grapevines grafted on tetraploids had much shorter shoots and internodes than those grafted on the counterpart diploids. In the nursery period in pots, ‘Kyoho’ grapevines grafted on each tetraploid rootstock also showed weaker growth than those grafted on the corresponding diploid rootstock. After planting in the vineyard, lateral shoot growth after primary shoot-tipping, the trunk cross-sectional area and pruned cane weight of ‘Kyoho’ grapevines grafted on each tetraploid rootstock were also smaller than those grafted on the corresponding diploid rootstock. The berries of ‘Kyoho’ grapevines grafted on tetraploids showed much deeper skin coloration than those of vines grafted on diploid rootstocks.

Allotetraploid hybrids produced by protoplast fusion for seedless triploid Citrus breeding

Protoplast fusion has been used to produce novel allotetraploid hybrids for use as parents in crosses with diploids to produce easy-peel, seedless, triploid citrus cultivars. Forty three plants were produced from fusions between protoplasts of ‘Encore’ mandarin and ‘Valencia’ sweet orange, and of ‘Encore’ and ‘Caffin’ clementine mandarin in which protoplasts of each of the donors were isolated from embryogenic callus. Forty plants were confirmed as tetraploid by flow cytometry and chromosome counting. Three (less than 10% of all regenerants) were non-hybrid diploids. DNA fingerprinting confirmed that both parents contributed DNA to the hybrids, which were therefore allotetraploid. The hybrids showed morphological features typical of other tetraploid citrus, including broad, thick, dark green leaves. However, they grew much faster than colchicine-induced autotetraploids. The allotetraploid hybrids will be used in crosses with the diploid cultivar ‘Encore’ mandarin and other clementine-type mandarins in an effort to generate seedless, triploid, easy-peel cultivars with ‘Encore-type’ flavor.

Colchicine-Induced Autotetraploid in Aloe vera L.

Autotetraploid plants of Aloe vera L. were induced by colchicine treatment. Diploid shoot tips (2n=2x=14) were treated by immersion of the rhizomes in colchicine concentrations of 0.05%, 0.10% and 0.15% at exposure times of 6, 12, 18 and 24h. The highest proportion of poly-ploid plants (5.9%) with 2n=4x=28 was obtained from a 0.15% colchicine and 24h combination. Diploid/tetraploid chimerical plants were observed at the lowest colchicine concentration/time treatments. Expression of gigantism in morphological and cytological characters, such as, width and thickness of the leaves, size and frequency of juvenile foliar spots, plant weight, size of epidermal and root cells, were noted in autotetraploid plants with a significant decrease in stomatal frequency.

Chromosome substitutions and recombination in the amphiploid Lolium perenne×Festuca pratensis cv Prior (2n=4x=28)

The synthetic amphiploid cv Prior was created in the early 1970s at the Welsh Plant Breeding Station by crossing colchicine-induced autotetraploids of Lolium perenne (2n=14) and Festuca pratensis (2n=14). Meiosis in the early generations was characterized as stable, with frequent bivalent formation. In situ hybridization of a L. perenne total genomic DNA probe to mitotic chromosome spreads of 12 plants, from two extant populations of Prior, demonstrates extensive recombination between the two genomes. Recombination events occur along the whole length of chromosome arms but with a higher frequency in the medial portion. The species origins of chromosomes were assigned by the presence or absence of a fluorescent probe at the centromere. There has been a substitution of Festuca-origin chromosomes by those of Lolium-origin, resulting in a mean of 17.9 (15–21) Lolium and 9.7 (7–13) Festuca chromosomes per genotype. Mean chromatin length per genotype comprised 62.1% Lolium and 37.9% Festuca. On average 9.3 Lolium (51.1% of those present) and 3.5 Festuca (37.8%) chromosomes had no recombined segments. For chromosomes which did show recombination, fewer alien segments were observed in Lolium than in Festuca chromosomes. Festuca chromosomes in genotypes selected for drought resistance had undergone more recombination than in genotypes from an unselected population, though this difference was not statistically significant for the small sample examined.

Male and female meiosis in diploid and colchitetraploid Phlox drummondii Hook. (Polemoniaceae)

Abstract Comparative studies on male and female meiosis in diploid (2n = 2x = 14) and colchitetraploid (2n = 4x = 28) ornamentalPhlox drummondiireveal higher chiasma frequency in embryo-sac mother cells as compared with pollen mother cells in the diploid, and significant differences in the pairing properties of chromosomes in the pollen mother cells and embryo-sac mother cells of the colchitetraploid. Male meiosis in the colchicine-induced autotetraploid was abnormal with 51·70% of chromosomes associated in quadrivalents and trivalents in 96% of the cells. This was followed by discordant anaphase I disjunction. In the female meiocytes the chromosomes formed 14 bivalents in 86·66% of the cells. Quadrivalents (1–3) appeared only in 13·34% of the cells. It is concluded that meiosis in male and female cells of the colchitetraploid is governed and regulated by different controlling systems of gene(s).

In vitro Induction of Banana Autotetraploids by Colchicine Treatment of Micropropagated Diploids

Alternative breeding strategies, based on colchicine-induced autotetraploids, have been proposed as a means of introducing disease resistance into banana breeding programs. This paper describes techniques for the in vitro induction of banana autotetraploids by the use of colchicine on cultured explants. The technique can be readily applied and large numbers of autotetraploids produced. The optimum treatment involved immersing shoot tips in a 0-5% w/v colchicine solution for 2 h under aseptic conditions. Dimethyl sulfoxide (DMSO) was applied with the colchicine treatments to increase cell permeability and so absorption of colchicine, resulting in the optimum treatment unchanged at 0-5% colchicine, but including the addition of 2% v/v DMSO. Of the shoot tips treated over 30% were induced to the autotetraploid level. Methods for in vitro selection of induced tetraploids from treated diploid plantlets were also developed. Tetraploid plants were more robust with thicker pseudostems, roots and broader leaves than diploids and they could be selected on these morphological characteristics. Mean stomatal lengths of diploid banana plants growing in vitro were significantly smaller (16-0 mum) than the tetraploids (26.9 mum) and were used as a more reliable indicator of ploidy than morphological criteria alone. A root tip squash technique using carbol fuchsin was developed for positive confirmation of ploidy change by chromosome counts- Although chimerism and reversion to the diploid form occurred, it was not considered a problem because of the large number of autotetraploids induced. Stable autotetraploids were recovered and established in the field and were characterised by their large, drooping leaves and thick pseudostems. They have retained these characteristics for more than 3 years in the field.

Meiotic behaviour of induced autotetraploids in Triticum L.

Colchicine-induced autotetraploids of Triticum speltoides, T. longissimum, T. sharonense, T. bicorne, T. uniaristatum, T. monococcum, and T. tauschii were all morphologically similar to but larger than their diploid forms. Seed set was lower than in the diploids except for the autotetraploid T. speltoides. Meiotic analysis showed fewer quadrivalents and more bivalents than would be expected in all of these autotetraploids. Arm-pair switch, indicated by complex trivalents and quadrivalents, was found and involved 0.1% of total chromosomes in T. umbellulatum, 0.5% in T. longissimum, 0.7% in both T. sharonense and T. tauschii, 6.3% in T. bicorne, and 15.3% in T. uniaristatum.Key words: meiosis, chromosome association, arm-pair switch, chromosome pairing, bivalentization.

Cytogenetic studies and the occurrence of triploidy in the wild potato species Solanum commersonii Dun.

The South American wild potato species Solanum commersonii Dun. exists as two subspecies, subsp. commersonii and subsp. malmeanum (Bitter) Hawkes and Hjerting, and as both diploid and triploid cytotypes. The two subspecies were completely cross fertile but there were reciprocal differences in the number of seeds set per berry and the mean seed weight. Crosses between diploids and colchicine-induced autotetraploids yielded only a few seeds per berry, but 16 of the 18 germinated seeds were triploid or nearly so. Such a high proportion of triploids is unusual for such crosses in potatoes. Some triploids has a high percentage of stainable pollen. The origin of naturally occurring triploids is discussed in the context of 2n gametes, a triploid block, and endosperm balance numbers.

Production of colchicine-induced autotetraploid plants through micrografting in monoembryonic citrus cultivars.

Complete autotetraploid plants in three monoembryonic diploid citrus cultivars ('Clementine', 'Hassaku' and 'Hyuganatsu') were produced successfully from small young axillary buds isolated and immersed in 0.1% colchicine solution for 2-6 hours by using the micrografting technique. The induced tetraploidy was associated with the presence of broader and thicker leaves, larger stomata and flowers. Moreover, thorns were produced on the elongating shoots of all the induced tetraploid plants. The signiflcance of this thorniness for the detection of tetraploidy in citrus after colchicine treatment is discussed.

Cytogenetical studies on African rice, Oryza glaberrima Steud. 1. Colchicine-induced autotetraploid.

Cytogenetical study on the colchicine-induced autotetraploid of Oryza glaberrima was carried out. As compared with the diploid, the autotetraploid increased in stomata length, length of penultimate leaf blade, pollen diameter and seed size; decreased in plant height, first internode length, number of panicles, number of spikelets per panicle, pollen stainability and seed fertility. The differences between them were conspicuous in number of panicles, number of spikelets per panicle, seed fertility and seed size. The autotetraploid had multipored pollen grains and awned seeds, which was different qualitatively from the diploid. In pollen mother cells of the autotetraploid, 2 to 12 IV were observed at the stage of diakinesis to metaphase I, and 8IV+8 II most frequently. The mean meiotic chromosome configuration was 7.63IV+0.08 III+8.51 II+0.23 I. The meiotic chromosome configurations observed in the present study could be explained by a hypothesis postulated by Jackson and Casey (1980, 1982) with random pairing among four homologous chromosome arms and a maximum two chiasmata per bivalent.

Inheritance of Stylar Morphology and Loss of Self-incompatibility in the Progenies of Induced Autotetraploid Buckwheat

This paper deals with agronomic tiraits, chromosomal behavior, loss of incompatibility of stylar variants and inheritance of heterostyly in the progenies of colchicine-induced autotetraploid. Because of a small amount of aneuploids and low frequency of multivalent formation in M1, autotetraploids resulted neither terrible reduction of pollen fertility nor decline of seed set. Grain yield, therefore, increased rather than the original diploid strain and some native varieties. Two types of homostyled variants, namely one reduced style length in pin (PH) and the other increased style length in thrum (TH), were found in the progenies. Of these types, PH plants lost self-incompatibility and, therefore, set selfed seeds. Genetic analysis of heterostyly and incompatibility was attempted in connection with cytological evidences of autotetraploid.

Consequences of artificial and natural chromosome doubling on DNA, RNA and protein contents inCochlearia (Brassicaceae)

The effects of chromosome doubling on macromolecular composition, i.e. DNA, RNA and protein, were examined in diploidCochlearia pyrenaica DC., its established natural autotetraploidC. officinalis L., and their newly colchicine-induced autotetraploid and autooctoploid derivatives. DNA, RNA and protein contents increase from lower to higher ploidy levels (2x > 4x > 8x). The established natural autotetraploids and newly induced autotetraploids also differ, as DNA, RNA and protein have been reduced during the course of establishment. Net synthesis of RNA and protein per unit DNA does not change significantly neither from lower to higher ploidy levels nor between the autotetraploids of newly induced and established natural origins.

Mutation breeding of autotetraploid Achimenes cultivars

Colchicine-induced autotetraploids of three Achimenes cultivars were irradiated with X-rays or fast neutrons. The results were compared, in one cultivar, with those of the irradiated diploid form. The mutation frequency after irradiation of the autotetraploid was 20–40 times higher than for the corresponding diploid. These results may open new possibilities for mutation breeding, though they are hard to explain. Several promising mutants were selected.