Tetraploid Seedlings Research Articles

POLYPLOID INDUCTION VIA COLCHICINE TREATMENT IN LOQUAT

Tetraploid and triploid seedlings of ‘Algerie’ loquat were obtained using colchicine. Colchicine treatments at different doses and exposure times were applied to shoot apex, whole plants and non-germinated seeds. No stable tetraploids were obtained by treating the shoot apex of 30-days-old in vitro-grown seedlings with one drop of 0.01 and 0.05% (w/v) colchicine solution during one, two or three consecutive days. In vitro whole plants totally submerged in 0.1% (w/v) colchicine solution during 15, 30, 45 or 60 min promoted the highest percentage of tetraploid induction. During the acclimation stage, plants turned necrotic and finally died. Non-germinated seeds immersed in 0.5% (w/v) colchicine solution for 24 and 48 h resulted in two triploids and one tetraploid, respectively. Nuclear DNA content was analyzed by flow cytometry using leaf samples, and five biological replicates per plant. Chromosome counts using samples taken from fully developed leaves and root tips confirmed the ploidy obtained. A minimum of five metaphases per leaf or root, and a minimum of five leaves and roots were observed in each plantlet. The chromosome number of a diploid loquat plant is 2n=2x=34. Chromosome counts were 2n=3x=51 in triploids 2n=4x=68 in tetraploids.

Higher seed size and germination rate may favour autotetraploids ofVicia craccaL. (Fabaceae)

The phenotypic effect of increased cell size in polyploid angiosperms has been repeatedly described; the ecological consequences of the gigas effect are, however, relatively poorly understood. Here, we investigated the effect of cytotype, seed weight, and inter-population variation on seedling germination and growth in diploid and autotetraploid Vicia cracca L. in a common garden experiment. Seeds used in this study originated in the contact zone of the cytotypes in Central Europe. Tetraploids had heavier seeds than diploids and greater germination rates irrespective of seed size. Both seed weight and germination rate displayed high inter-population variation. Further, tetraploids seem to germinate earlier and deposit fewer reserves into the seed bank than diploids. Mean above-ground biomass and seedling height were similar in the two cytotypes of V. cracca. Nonetheless, the tallest tetraploid seedlings were taller than the tallest diploid seedlings, which may be advantageous under strong competition in dense vegetation. This study thus demonstrates that tetraploids of V. cracca may have superior competitive ability to diploids in certain habitats. It also suggests the necessity of studying multiple populations per cytotype when comparing diploids and polyploids, as the effect of population may be of similar or even higher magnitude than the effect of cytotype. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113, 57–73.

Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment.

Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation. Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of 'Tardivo di Ciaculli' mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype ('Carrizo' citrange; Citrus sinensis × Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries. Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area. Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks.

INHERITANCE OF 2N POLLEN FORMATION IN AN F1 AND F2 POPULATION OF BEGONIA HYBRIDS

Inheritance of 2n pollen formation was investigated in progenies of B. 'Orococo' by analysing the microsporogenesis and pollen size, which is correlated to the ploidy level. After crosses between B. 'Orococo', B. 'Art Hodes' and B. solimutata, an F 1 progeny of 359 seedlings was established of which 298 were triploid and 61 diploid. All 50 analysed triploid seedlings produced a variable frequency of monads, dyads or triads. Consequently, all triploid seedlings produced large putative 2n pollen, except for those which had formed no pollen or only bad pollen. In contrast, only 14 of the 31 diploid seedlings investigated had formed monads, dyads or triads; 11 seedlings produced only tetrads or polyads. The remaining six seedlings formed no pollen mother cells at all. F 2 progeny of 15 tetraploid seedlings was established by using a triploid seedling that produced unreduced pollen as the pollinator. Of the 10 tetraploid seedlings analysed, 8 produced a variable frequency of monads, dyads or triads while 2 did not. One of these 2 seedlings, which produced only normal n gametes, was self-pollinated and resulted in a F 3 population of 121 tetraploid seedlings. The results show that the inheritance of 2n pollen is very high and is probably modulated by one dominant gene and some modifier genes.

Donors of diploid gametes in apple breeding at the polyploid level

An agrobiological and cytoembryological assessment of eight apple tetraploid varieties and a number of tetraploid seedlings, the donors of diploid gametes, is given. A high breeding value of seedlings from hybridization at the polyploid level is noted. A brief economic and biological characterization of 13 new apple varieties of different ploidy obtained from intervalent crosses is given.

Oxygen requirement of root meristems in diploid and autotetraploid rye

The oxygen requirement of the root meristems of diploid and tetraploid rye seedlings has been determined. It was found that the polyploid seedlings require 40–50 per cent higher oxygen concentration at the root surface than the diploid seedlings for normal (maximum) respiration in the temperature interval 15–30° C. The critical oxygen concentration, when respiring air atmosphere, occurred at about 22° C in the diploid meristem and at about 15° C in the tetraploid meristem. A hypothesis is presented according to which the radial oxygen diffusion into the root meristems is one of the factors determining the geographical distribution of polyploid plant species.

Induction of tetraploidy in meristematically active seeds of Japanese barberry ( Berberis thunbergii var. atropurpurea) through exposure to colchicine and oryzalin

Japanese barberry ( Berberis thunbergii DC) is an invasive shrub, widely naturalized across the United States, whose numerous cultivars remain an important horticultural commodity. Maintaining this crop for the future necessitates the development of sterile clones. Exposure to the mitotic inhibitors colchicine and oryzalin is a traditional method for inducing tetraploidy in breeding lines as a precursor to creating sterile genotypes. Treatments utilized pre-germinated B. t. var. atropurpurea seeds with emerged radicles. Seeds were immersed in aqueous solutions of colchicine (.02%, .05%, .1% and .2%) and oryzalin (.002%, .005%, .01% and .02%) dissolved in 1% DMSO for 6, 12 and 24 h durations. Seedling ploidy level was determined via flow cytometry following 6 and 52 weeks of growth in the greenhouse. Both anti-mitotic chemicals proved effective at inducing tetraploidy and produced comparable efficiency rates. The survival rate of treated seeds decreased in response to both increased mitotic inhibitor concentration and longer exposure duration. While exposure to oryzalin produced greater seed mortality than colchicine, most seedlings that survived had altered ploidy levels. The most efficient oryzalin concentration was 0.002% with a rating of 28%, while the most efficient colchicine concentrations were in the range from 0.05% to 0.2%. Duration of exposure to mitotic inhibitor was not a significant factor over the range from 6 to 24 h. Reversion of tetraploid plants to the diploid state occurred at a low frequency following a dormancy period. Some tetraploid seedlings derived from exposure to both chemicals displayed foliar abnormalities including irregular leaf margins and mottled lamina. The primary advantage of colchicine was low seedling toxicity, while oryzalin was notable for its ability to induce tetraploidy at low concentrations.

Colchicine-induced chromosome doubling in Platanus acerifolia and its effect on plant morphology

We report the production of tetraploid plants of Platanus acerifolia, with the ultimate aim of improving the ornamental qualities of this important urban landscaping tree. Chromosome doubling was achieved by the application of colchicine to either pre-soaked seed or to the apical meristems of young seedlings. Treatment of the ungerminated seed was the more efficient method in terms of numbers of tetraploid seedlings (up to 40%, as determined by chromosome counting of the root-tip nuclei) but this method produced no mature tetraploid plants due to the deleterious effect of colchicine on subsequent root growth. When colchicine was applied directly to the apical growing tip of cotyledon-stage seedlings, leaf and stem growth was temporarily affected but the plants eventually recovered. We conducted a preliminary screen for putative tetraploids based on the observation in other plant species of a correlation of stomatal size and distribution with ploidy. Plants containing significantly larger stomata and at a lower density across the lower leaf epidermis, were selected for further analysis by flow cytometry and chromosome counting. These techniques confirmed that, of the 12 putative polyploids, four were tetraploid, five were mixoploid and three were, in fact, diploid. Morphological differences of the tetraploids included a more compact growth habit and broader, thicker leaves. These plants are being grown to full maturity in order to test their potential for use in a breeding programme aimed at producing sterile triploid lines.

Production of 2n pollen of Asiatic hybrid lilies by nitrous oxide treatment

Tetraploid varieties of lilies have superior agronomic traits such as large flowers and resistance to physiological disorders. In the present study, we attempted to induce 2n pollen of Asiatic hybrid lilies by arresting the meiotic process with nitrous oxide (N2O) gas. To determine which meiotic stage is optimal for induction of 2n pollen, plants with attached buds at different meiotic stages were treated with N2O for 24 h in a pressure-tolerant cylinder. A few 2n pollen grains were induced using plants with anthers in prophase I, whereas mixed pollen grains of differing size were produced using plants undergoing meiotic metaphase predominantly in anthers. Although normal lily pollen grains are elliptical, nitrous oxide exposure induced giant pollen grains that appeared spherical. Flow cytometry analysis showed that the giant pollen grains were diploid. When mixed pollen that included normal and giant pollen was crossed to tetraploid cultivars, the resulting seedlings were tetraploid and aneuploid, indicating that the giant pollen grains were diploids that could generate tetraploid seedlings through fusion to diploid eggs supplied from a tetraploid female parent. Thus, treatment with N2O is useful for the production of 2n lily pollen and may provide a new approach for tetraploid lily breeding.

Reproductive Behavior of Induced Allotetraploid ×Chitalpa and In Vitro Embryo Culture of Polyploid Progeny

×Chitalpa tashkentensis Elias & Wisura is a sterile intergeneric hybrid [Catalpa bignonioides Walt. × Chilopsis linearis (Cav.) Sweet]. To restore fertility in ×Chitalpa the following were evaluated: 1) oryzalin as a polyploidization agent, 2) fertility of induced polyploids, and 3) in vitro culture methods for embryo rescue of interploid crosses. Meristems of ×Chitalpa `Pink Dawn' were submerged in an aqueous solution of 150 μm oryzalin for 0, 6, 12, or 24 hours and ploidy analyzed via flow cytometry. As treatment duration increased, recovery of diploids decreased as mixoploids and shoot mortality increased. Two tetraploid shoots occurred in the 24-hour treatment. Four tetraploids and two cytochimeras were stabilized in total. Tetraploids flowered sparsely; however, cytochimeras flowered profusely and these were used to study fertility at the tetraploid level. Diploid ×Chitalpa `Pink Dawn' pollen was essentially nonviable, but cytochimera pollen stained and germinated equal to or greater than pollen of C. bignonioides and C. linearis `Bubba'. Cytochimera ×Chitalpa were selfed yielding tetraploid seedlings, crossed with C. bignonioides to yield triploids, but failed in reciprocal crosses with C. linearis `Bubba' and `Burgundy Lace'. To increase recovery of triploids, germination of triploid and tetraploid embryos was investigated, as either intact ovules or excised embryos, on Schenk and Hildebrandt (SH) basal salts supplemented with sucrose at 20, 40, and 80 g·L-1, presence or absence of 2% coconut-water, and gibberellic acid (GA3) at 0, 1, 2, or 4 μm, and harvested weekly beginning 2 weeks after pollination (WAP). Germination of triploids (cytochimera ×Chitalpa × diploid C. bignonioides) and tetraploids (selfed cytochimera ×Chitalpa) were greatest with excised embryos at 7 WAP on SH supplemented with sucrose at 20 g·L-1 and ≥1 μm GA3. Germination of triploids (diploid C. linearis × cytochimera ×Chitalpa) was <5% at 4, 5, or 6 WAP on the same medium as above. Oryzalin effectively induced polyploidy and restored fertility in ×Chitalpa `Pink Dawn'. Successful crosses between hybrid and parental taxa of different ploidy levels, coupled with embryo culture will facilitate a ×Chitalpa breeding program. Chemical names used: 4(dipropylamino)-3,5-dinitrobenzenesulfonamide (oryzalin).

Comparative study of chlorophyll content in diploid and tetraploid black wattle (Acacia mearnsii)

The effects of the number of chromosome sets on chlorophyll content in diploid and tetraploid black wattle were assessed through chlorophyll absorbance. Seedlings of ∼8 months of age, 1-year-old trees in polybags and 2-year-old field material were selected for this investigation. Three families per ploidy level comprising of 10 half-sibs per family were used in the analysis. An analysis of variance revealed significant differences (P < 0.05) in chlorophyll absorbance between diploid and tetraploid materials (⁠A¯ = 0.375 and A¯ = 0.653, respectively). Significant differences (P < 0.05) between treatments (seedling, bagged material and field material) within each ploidy level were recorded, where chlorophyll absorbance increased from diploid bagged trees (⁠A¯ = 0.343) to seedlings (⁠A¯ = 0.375) to field trees (⁠A¯ = 0.408). Similar significant increases were recorded for the tetraploid seedlings (⁠A¯ = 0.629) to bagged trees (⁠A¯ = 0.644) to field trees (⁠A¯ = 0.686). Significant differences (P < 0.05) were also recorded between genetic lines within both ploidy levels. However, when comparing families within each ploidy level, with respect to treatment differences, in most cases, two of the three families showed no significant differences (P > 0.05). Within each family, significant differences (P < 0.05) were recorded between each of the three treatments tested, with the field material consistently having significantly (P < 0.05) greater amounts of chlorophyll.

INTERPLOIDY CROSSES IN ROSES: USE OF TRIPLOIDS

Interspecific crosses between diploid rose species and tetraploid cultivars yield triploids. Using such triploids as pollen parents and tetraploid plants as seed parents resulted in relatively high seed set. Although efficiency was lower, reciprocal crosses with the triploid plant as a seed parent also yielded some seedlings. Screening of the progeny showed that seedlings obtained after a cross between a tetraploid seed parent and a triploid pollen donor are mostly tetraploid. In the reciprocal cross, both triploids and tetraploid seedlings are found. These results indicate that triploid rose plants form both haploid and diploid pollen and egg cells. However, in pollen production, the diploid fraction seems to have a competitive advantage over the haploid. Molecular marker (AFLP) data show that markers can be passed on from the diploid species to a tetraploid F2 generation via a triploid F1.

A tetraploid hybrid plant from 4x × 2x crosses in Vitis and its origin

To study the origin of unreduced (2n) gametes in diploid Vitis cultivars, we surveyed the occurrence of tetraploid hybrid seedlings from 40 interploid crosses with five tetraploid and seven diploid cultivars. A total of 250 seedlings from the interploid crosses were established through embryo culture and by seed sowing. In 20 2x × 4x crosses, no tetraploid hybrid seedlings were derived from 8,902pollinations. In 20 4x × 2x crosses, two tetraploid hybrid seedlings were obtained from 8,057 pollinations. Investigation of isozyme genotypes of the two tetraploid seedlings using three variable enzyme systems indicated that one of the two seedlings resulted from the union of a diploid egg with a 2n male gamete and that failure of second meiotic division resulted in the formation of the 2n male gamete. The percentage of giant pollen grains in `Muscat Bailey A', a pollen parent of the tetraploid seedling, was relatively high(about 5.9%) and 10.9% of the giant pollen grains germinated on agar medium. These results suggested that 2n pollen of diploid cultivars are useful for breeding tetraploid grape.

‘土佐文旦’ב水晶文旦’の倍数性交雑実生の染色体構成に基づく対合染色体と非還元配偶子の推定

To clarify why polyploids were generated from the cross of the diploids 'Tosa-Buntan' and 'Suisho-Buntan' pummelo (Citrus grandis [L.] Osb.), the chromosome compositions of their di-, tri-, and tetraploid progenies, based on CMA staining, and their chromosome parings were observed. 1. Thirteen kinds of chromosome composition were manifested by 38 diploid seedlings indicating that the chromosome compositions of seedlings are highly variable. 2. Among the 13 kinds of diploid chromosome composition, the number of chromosome varied 1 to 3 in chromosome type-A, 0 to 1 in type-B, 3 to 5 in type-C, 2 to 4 in type-D, and 8 to 10 in type-E. The number of chromosome type-A varied 1 to 2 among the chromosome compositions with chromosome type-B. From these results, the paring set of the chromosome type in 'Tosa-Buntan' was estimated as AB+CC+CC+CD+DE+EE+EE+EE+EE or AB+CC+CC+DD+CE+EE+EE+EE+EE. 3. Among six triploid seedlings, three kinds of chromosome composition exist which suggested that they resulted from unreduced female gamete at the first or second meiotic division, or unreduced male gamete at the second meiotic division. 4. Among three tetraploid seedlings, two kinds of chromosome composition exist. Because the chromosome compositions had two type-B chromosomes and an even number of chromosome of any other type, they probably resulted from the combination of an unreduced female with unreduced male gametes at the second meiotic division. These results showed that the triploid seedlings from diploid 'Tosa-Buntan'×'Suisho-Buntan' crossing also originated from an unreduced male gamete as well as from an unreduced female gamete.

Transmission of bacterial wilt resistance by First Division Restitution (FDR) 2n pollen via 4x×2x crosses in potatoes

Resistance to bacterial wilt (Pseudomonas solanacearum) found in tuber-bearing Solanum species was transferred into a diploid potato breeding population. Simultaneous selections were made for agronomic characters, production of first division restitution (FDR) 2n pollen, and resistance. Diploid resistant genotypes were identified via inoculation with a virulent isolate (CIP-204) of race 3 of Pseudomonas solanacearum. These resistant diploid genotypes were crossed to susceptible tetraploid potatoes. An investigation was made to assess whether resistant diploid genotypes transmit resistance to bacterial wilt, which is a quantitatively inherited trait, to tetraploid potatoes via FDR 2n pollen. Tetraploid seedlings from 4x×2x crosses were inoculated with the same isolate CIP-204, and the percentage of surviving seedlings was scored. Some 4x×2x families from resistant diploid genotypes demonstrated a high level of survival rate. The transmission of bacterial wilt resistance was achieved by the use of FDR 2n pollen. It was speculated that a female x male interaction effect on the survival rate of the evaluated progeny may exist. Selecting proper 4x and 2x parents would be important for obtaining a higher frequency of transmission of resistance to bacterial wilt in the progeny.

Citrus Pentaploids from Small Seeds of Diploid × Diploid Crosses

Seedlings from some small seeds of diploid × diploid crosses using `Miyauchi iyokan' (Citrus iyo Hort. ex Tanaka) as the seed parent were found to be pentaploid (2n = 5x = 45). The growth of pentaploid seedlings was extremely weak on their own roots, but was much more vigorous when micrografted on seedlings of tetraploid trifoliate orange [Poncirus trifoliata (L.) Raf.]. Diploid `Miyauchi iyokan' produced hexaploid seedlings from small seeds in addition to triploid and tetraploid seedlings from normal seeds when crossed with tetraploid `Funadoko' (C. funadoko Hort. ex Y. Tanaka) and `Trovita' sweet orange [C. sinensis (L.) Osbeck]. Based on this phenomenon, the origin of pentaploids is discussed.

Gene Dose Effects on Cane Thorn Density and Cotyledonary Gland Number in Tetraploid Blackberries1

Abstract Fourteen tetraploid seedling populations of blackberry (Rubus sp.), representing quadruplex, (TTTT), triplex (TTTt), duplex (TTtt), simplex (Tttt), and nulliplex (tttt) genotypes for the major gene conferring thorniness, were evaluated for segregation of cane thorn density and cotyledonary gland number. Comparisons of seedling distribution curves, means and variances of segregating and non-segregating populations did not show a gene dose effect on gland and thorn occurrence. Inheritance of cotyledonary glands and cane thorns in blackberry was qualitative with the density of glands and thorns apparently controlled by several modifying genes.

Root peroxidase isoenzymes as an aid in Citrus breeding and taxonomy

A rapid method for differentiating between Citrus clones as well as between zygotic and nucellar plants has been developed. It is based on determination of peroxidase isoenzyme patterns in small root samples. Ten to 14 bands could be distinguished, in contrast to the small number of bands present in leaves. Tetraploid seedlings gave uniform patterns closely resembling the diploid, while triploids showed segregation in isozyme pattern.

Rapid Procedure for Isolation of Polyhaploids in Bromegrass1

Cotyledons of Bromus inertnis Leyss. were fixed and decolorized in Carnoy's solution for 24 hours, immersed in 70% alcohol, and softened in lactic acid at 60 C for 15 min. The whole leaf was then mounted in lactic acid. Mean stomatal lengths of 41.8 and 43.4 µ were obtained for adaxial and abaxial surfaces of cotyledons of tetraploid seedlings compared to 62.3 and 65.1 µ for octoploid seedlings. This simple technique provides a rapid and economical method of screening for polyhaploids in populations of octoploid Bromus inermis.

A Tetraploid Gerbera1

Abstract A tetraploid of Gerbera jamesonii Bolus ex Hook, obtained from colchicine treatment of seed is characterized by increased vegetative and flower size. The tetraploid plant is fertile and produces tetraploid seedlings following self-pollination.