Diploid Regenerants Research Articles

In vitro chromosome doubling potential of colchicine, oryzalin, trifluralin, and APM in Brassica napus microspore culture

The potential of colchicine and the microtubule depolymerizing herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling during B. napus microspore culture was studied. Colchicine was administered during the first 6, 12 or 24 h of culture with 8 different concentrations up to 3 mM, and herbicides at 6 different concentrations up to 30 μM for 12 h. Treatments with moderate concentrations of colchicine (3–100 μM) produced a small increase in embryo production, while concentrations above 300 μM were toxic. Colchicine treatment for 12 h resulted in higher embryo production than treatment for 6 and 24 h. Duration of treatment and concentration of colchicine both had a significant effect on the chromosome doubling. The highest diploidization rates (94% diploid regenerants) were seen after 24 h treatment with 1 mM colchicine. All three herbicides were similar to colchicine in terms of their effect on embryo formation and chromosome doubling comparable to the one of colchicine, but at concentrations approximately 100 times lower. APM was less toxic than trifluralin and oryzalin, but no significant difference in chromosome doubling efficiency was detected between the compounds. The 12 h treatment resulted in a maximum of approximately 65% diploid regenerants with all three herbicides, but APM may have an advantage because of its less toxic effects. Prolonged treatment with APM (20–24 h) may produce 95–100% diploid regenerants.

Identification of tetraploid regenerants from cotyledons of diploid watermelon cultured in vitro

Adventitious shoots were obtained from the diploid watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] cultivars Dixielee, Jubilee II, Mickylee, Minilee, and Royal Sweet by culturing excised cotyledons on shoot regeneration medium for six weeks. Tetraploid and diploid regenerants were identified by counting the number of chloroplasts per guard cell pair from leaves of regenerated plants. Cross fertilization of putative tetraploids with diploid pollinators and the production of triploid seed confirmed the efficacy of this approach. The mean number of chloroplasts for tetraploid regenerants was 19.1 whereas diploids averaged 11.2. These values were similar to tetraploid and diploid plants from seed. Ovary diameter, petal, and anther diameter of male flowers, and leaf length by width ratio were also good indicators of plant ploidy. Progeny obtained from self-fertile tetraploids of ‘Mickylee’ were crossed with various diploid pollinators to produce triploid hybrid seed. All triploid plants from tissue culture-derived tetraploids produced fruit comparable in quality to fruit produced by currently-available triploid hybrids, demonstrating that in vitro tetraploid induction can be used to produce high quality tetraploid plants for use in triploid hybrid seed production.

A new One-step Anther Culture Method which Allows Short Duration of Culture for Regeneration of Rice Plant thorough Somatic Embryogenesis.

With the aim of making anther culture more applicable to rice breeding, a new method of anther culture has been developed. The method consists of the pretreatment of anthers with phytohormone before inoculation on a hormone-free medium and regeneration of plants without transfer of the calli to a regeneration medium. The best results were obtained when anthers were pretreated with NAA or NAA plus BA. This method (one-step culture) has the following features compared with the conventional two-step culture method; 1) a higher frequency of callus formation and plant regeneration, 2) a much shorter duration of culture for plant regeneration (a half of the two-step culture method), 3) a higher ratio of haploid to diploid regenerants, 4) no segregation within A2 lines. The histological observation showed that plants in the one-step culture method were regenerated through somatic embryogenesis.

Cytology of Explants, Calli and Regenerants in Five Inbred Lines of Pearl Millet, Pennisetum glaucum (L.) R Br.

Cytological stability of the 6 and 12 week old embryogenic callus initiated from immature inflorescence explant was investigated in five inbred lines of Pennisetum glaucum (L.) R. Br. Three different types of embryogenic cells have been observed. Different types of chromosomal abnormalities in the embryogenic cell populations include hypo-haploidy, aneuploidy and polyploidy. The extent of chromosomal variability depended upon the age of the callus, 2, 4-D concentration and genotype. No specific dose-response relationship was observed between the 2, 4-D concentration and the frequency of abnormalities in the calli. The regenerants were all diploids. The operation of a selection mechanism within the embryogenic callus favouring the development of normal diploid regenerants has been inferred.

Regeneration of plants from protoplasts of rapid cycling Brassica oleracea L.

Rapid cycling Brassica species have great potential in plant genetic research because of their short life cycles and their minimal space requirements. Rapid cycling B. oleracea can be grown with up to six generations per year. Protoplast culture of this genotype can be applied for gene transfer by direct DNA uptake and by protoplast fusion. We here report on fast regeneration of flowering plants from protoplasts of rapid cycling B. oleracea. Regeneration frequencies of 27-65% were achieved with multiple shoots developing from individual calli. The regenerated plants were grown to maturity, and flowering and other morphological characteristics were monitored. The regenerants flowered within a similar time frame as plants grown from seeds. The ploidy level of regenerated and seed-grown plants was measured by flow cytometry. Many (20-45%) of the regenerants were tetraploid. Although only few seeds could be obtained from the tetraploids, large numbers of seeds with good germination were recovered from the diploid regenerants.

Heterozygous diploid plants regenerated from anther culture of F1 rice plants

Plants derived from anther culture are theoretically haploid, but diploid plants are also known to arise. Anther culture-derived diploid plants are usually homozygous and are believed to be due to spontaneous doubling of chromosomes in either microsporocytes or callus cells during the culture process. However, heterozygous diploid regenerants may also arise from a) regeneration from cultured somatic cells, b) mutation occurring during or after a spontaneous doubling event, c) fusion of unlike haploid cells in chimeric callus, and d) regeneration from diploid microsporocytes resulting from aberrant meioses. This study was designed to elucidate the frequency and origin of diploid regenerants from rice anther culture. Regenerants were obtained from 11 F1 genotypes. Progeny testing detected heterozygosity in 7 out of 211 regenerants. Each of the heterozygous regenerants were from ‘Calrose 76’/waxy ‘M-101’, Half of the diploid regenerants from this cross were heterozygous. No heterozygous regenerants arose from the other 10 F1 genotypes. Progeny testing indicated that two of the heterozygous regenerants were as heterozygous as the F1 plants for three parental characters. The other five regenerants exhibited decreased levels of heterozygosity. One of the heterozygous regenerants exhibited evidence of mutation for a non-parental character. However, mutation is an unlikely cause of the observed high levels of parental-type heterozygosity. No evidence for the occurrence of chimeric callus was detected, making this an unlikely cause as well. The most likely origin of the observed partial heterozygosity is regeneration from diploid microspores, which could also produce plants exhibiting complete parental-type heterozygosity.

Diploidization in Haploid Tissue Cultures of Sorghum

AbstractConditions for the effective experimental regulation of ploidy level in regenerants from callus cultures derived from young, undifferentiated leaves and panicles of haploid sorghum were established. Diploidization depended on the ontogenetic age of the explant and the 2,4‐D concentration in the medium. With a low 2,4‐D concentration (0.5 mg/1) and segments of young panicles (< 35 mm long) the cultures produced only haploid regenerants. Diploid plants were formed from cultures derived from more mature panicles ( 35 mm long) and young leaves (15–65 mm long). Under a high 2,4‐D concentration (2.5 mg/1) diploid plants were regenerated from cultures derived from young panicles (less than 35 mm) except the most young ones (5–15 mm). The majority of the diploid regenerants contained mutations, mainly affecting male fertility and plant height.

Isolation and characterization of potato diploid clones generating a high frequency of monohaploid or homozygous diploid androgenetic plants

Cultured microspores of diploid potato clones lead with high frequency to diploid regenerants. In this paper we report on the genetic variability for in-vitro monohaploid production from anthers of diploid plants. Three diploid genotypes have been isolated which combine the capacity to regenerate monohaploid plants with outstanding embryoid production. A trait of the anther-donor clones associated with the generation of monohaploid plants is the low production of 2n pollen grains. When present in anthers of diploid genotypes, diploid unreduced microspores are, in fact, derived mainly from a first division restitution mechanism leading to high heterozygosity of the derived embryoids, a state which apparently supports superior growth in-vitro. Also, reduced microspores have been found capable of generating diploid regenerants and the adoption of the RFLP technique allowed the isolation of such diploid plants, which can be considered to be pure lines. Donor clones with a low capacity to generate monohaploids are, as expected, poor producers of homozygous diploid plants. The selection of an anther donor producing a sufficient number of monohaploid or homozygous diploid regenerants fulfills the requirements of the first part of the analytical breeding scheme, i.e., the production of homozygous diploid clones.

Biotechnology and plant breeding: relevance of cell genetics in potato improvement

SynopsisThe adoption of emerging biotechnological methods in plant breeding is presented discussing specifically the relevance of cell genetics to potato improvement. The finding and use of efficient anther plant producing strains of potato (EAPP clones) is first discussed. This is followed by the presentation of recent progress toward the obtaining of diploid potato strains producing a high percentage of monohaploids from anthers. The selection of homozygous diploid regenerants (potato ‘pure lines’) by using an RFLP approach is also presented. A summary chart is presented, containing the description of an integrated breeding procedure that may be considered for future breeding of this species.

Chloroplast Density in Guard Cells of Leaves of Anther-derived Potato Plants Grown in Vitro and in Vivo

A technique was developed to count chloroplasts in guard cell pairs of leaflets of anther-derived potato (Solanum phureja Juz. and Buk) plants grown in vitro to determine how they compare to chloroplast counts for similar plants grown in vivo. The mean chloroplast density in guard cells of in vitro leaflets was visualized using fluorescein diacetate (FDA) stain and observed under a microscope. A consistently higher mean was observed for in vitro than for in vivo plants but monoploid could readily be distinguished from diploid regenerants whether grown in vitro or in vivo. A rapid method of ploidy estimation based on chloroplast counts on in vitro leaflets may hasten identification of monoploids to be used for in vitro applications to genetic reconstruction of potato.

Insert copy number, chromosome number, pollen stainability, and crossability ofAgrobacterium-transformed diploid potato

Two 2n-pollen producing cultivated diploid, 2n = 24, potato genotypes DM56.4 and US-W5337.3 were inoculated with binaryAgrobacterium vectors. The T-DNA contained a neomycin phosphotransferase II coding region fused to a nopaline synthase promoter providing gene expression in plants and resistance to kanamycin. A total of 28 clones regenerated as adventitious shoots from callus tissue, growing on medium with kanamycin, was analyzed for chromosome number, pollen stainability, and crossability. Several clones were tested and found to retain crossability with diploid and tetraploid clones. Nine out of 10 of the regenerants from DM56.4 had been doubled; seven of these were tetraploids with 2n = 48, one clone had 2n = 49, and another 2n = 46. Five of 14 regenerants of US-W5337.3 had been doubled, and three of these had fewer than 2n = 48. Pollen stainability revealed that the tetraploid regenerants from DM56.4 were male sterile, whereas tetraploids from US-W5337.3 had stainable pollen. All of the diploid regenerants produced 2n pollen. Crossability of several diploid and tetraploid regenerants was high. The number of T-DNA inserts revealed by Southern blot analysis was also determined. Six, 10, and 9 regenerants had 1, 2, and 3 inserts of T-DNA, respectively. There were single regenerants each with 5, 6, or 7 inserts. Selection for higher insert number among regenerants should be feasible based on the range of insert number found. Identical restriction patterns within three groups of clones indicated that the 28 clones analyzed emerged from only 21 cells derived from independent transformation events. The variability in chromosome number and pollen stainability suggests that variation in other traits would be obtainable.

Colchicine-induced polyploidy in Citrus embryogenic cultures, somatic embryos, and regenerated plantlets

Two morphologically distinct Citrus sinensis (L.) Osbeck embryogenic cultures were grown for 4 or 12 weeks on Murashige and Tucker (MT) medium amended with 0.01% or 0.10% colchicine. Ploidy level frequencies were determined in treated cultures and untreated controls, as well as somatic embryos and regenerated plantlets, by chromosome counts. Mortality was widespread after treatment. The frequency of polyploid cells and the number of ploidy classes was increased by colchicine treatment; the frequency of non-diploid cells in untreated cultures was less than 10%. One line partially reverted back to diploidy, but the other line remained cytogenetically unstable; however, colchicine-induced cytogenetic variation was attenuated through the regeneration process. Although 2x, 3x, 4x, 6x, and 8x cells and somatic embryos were observed, only diploid and tetraploid plantlets were regenerated from treated cultures. Untreated cultures yielded only diploid regenerants.

The Effect of Carbohydrate Composition and Concentration on Anther Culture Response in Barley (Hordeum vulgare L.)

AbstractCulture medium composition is critical for the successful induction of microspore division in vitro. The present experiments have focused on a relatively neglected area of cell and tissue culture research, namely the carbohydrate component used in the medium. Three spring barley genotypes were cultured on a medium which was modified by replacing sucrose with the following carbohydrates (6 % w/v): maltose, fructose, malt extract, galactose and a glucose (3 % w/v)/fructose (3 % w/v) mixture. Both maltose and malt extract were superior to sucrose in their capacity to induce green plantlet differentiation from microspores. The concentrations of both sucrose and maltose were also varied. Overall the response of anthers on maltose based media was higher than on sucrose based media. Furthermore, a concentration of maltose m the range 6—12 % w/v produced a higher frequency of green plants than a low concentration (1—3 % w/v). The effect of maltose based media on germplasm of direct relevance to barley breeders was also tested. The cultivar ‘Blenheim’ was shown to be very responsive and this genetic factor was transmitted to the F1 hybrid. The frequency of haploid to diploid regenerants was not consistent over genotypes, but in general there were more haploid than diploid regenerants. The implications of these results for barley breeding are discussed.

Relative performance of monohaploid potato clones and their diploid parents at plant level and after protoplast isolation and subsequent fusion

Plant growth performance was studied in 118 potato monohaploids and in their diploid parents. Of these monohaploids 76 were also investigated at the protoplast level and eight of these were used in protoplast fusion experiments as well. No correlation was found between relative performance of greenhouse grown and in vitro grown plants. No or only weak correlations were found between different in vitro characteristics such as plant growth, protoplast yield per gram plant material, plating efficiency and callus growth. This indicates the unpredictability of these characters.The protoplast fusion experiments indicated that only in some genotype combinations increased callus growth rates may be found. However, it is not clear whether such calli were hybrids or not. In protoplast monocultures only diploid and tetraploid regenerants were obtained. After fusion, tetraploids but also some triploids could be regenerated. The finding of triploids indicates that monoploid protoplasts were involved in fusion. Isozyme analysis and morphological assessment of the plants pointed out that the majority of the fusion regenerants were hybrids. The implications of these results are discussed.

Elimination of Solanum phureja nucleolar chromosomes in S. tuberosum + S. phureja somatic hybrids

The karyotype of the dihaploid SVP1 line of S. tuberosum (2n=2x=24) showed two nucleolar chromosomes with differently sized satellites. The diploid SVP5 line (2n=2x=24) and tetraploid regenerants of S. phureja had larger but similar satellites. Somatic hybrids between the diploid lines of these potato species with genome combinations 4 tub + 2 ph (plants 1-3), 2 tub + 4 ph (plants 4-7) and 4 tub + 4 ph (plant 8) had lost 2 phureja nucleolar chromosomes if 4 phureja genomes were present. One phureja nucleolar chromosome of plants 1-3 and both of plants 5 and 7 had rearranged satellites. Elimination of the two nucleolar chromosomes occurred preferentially, was under genetic control, and probably took place during early callus development. NOR activity resulting in rear-rangements between NORs may have caused the elimination.

Ploidy Variation inSolanum brevidensPlants Regenerated from Protoplasts Using an Improved Culture System

Plating efficiency of cultured protoplasts of Solanum brevidens increased to c. 10% after the addition of 5⋅0 mol m−3 glutamine to the culture medium. Growth of protoplast-derived colonies at densities of 20 colonies cm−3 was obtained by lowering the auxin content alter 4–6 d. A sample of 50 protoplast-derived plants was examined for chromosomal variation. Twelve plants were diploid (2n=24), 26 were tetraploid (2n=48) and 12 were aneuploid at the tetraploid level (2n=48±). Tetraploid and aneuploid plants had broader leaves and set fewer or no seeds compared to the diploid regenerants which were similar in gross morphology and seed set to control plants.

Cytogenetic characterization of embryogenic callus and regenerated plants of Pennisetum americanum (L.) K. Schum

Embryogenic calli were derived from cultured segments of immature inflorescences of Pennisetum americanum (pearl millet). The original explants as well as the embryogenic calli and the plants regenerated via somatic embryogenesis were examined cytogenetically. Embryogenic calli were predominantly diploid (2n=14) after one month and six months in culture (92% and 76%, respectively). Tetraploid and aneuploid cells were observed in the original explant (2.5% and 1.2%) as well as in one (4.0% and 4.0%) and six-month-old calli (10.0% and 14.0%). Plants were regenerated from calli that had been in continuous culture for two, four and six months. Of the 101 regenerants, 100 were diploid and 1 was tetraploid. The tetraploid was an albino as were three of the diploid regenerants. Examination of 30 of the regenerants in meiotic diakinesis, anaphase I, anaphase II and quartet stages revealed no cytogenetic differences between control and regenerated plants. Gel electrophoresis for total protein content and alcohol dehydrogenase and malate dehydrogenase activity also did not reveal any differences between the controls and regenerants. The results of this study show that a slight shift toward aneuploidy and polyploidy may occur in embryogenic cultures, but there also is a strong selection in favor of plant regeneration from cytogenetically normal cells.

Cytogenetics of tissue culture regenerated hybrids of Triticum tauschii × Secale cereale

Immature inflorescences of Triticum tauschii (Coss.) Schmal. × Secale cereale L. cv. 'Puma' hybrids were induced to callus on Kao's medium supplemented with 5 mg/L 2-4-dichlorophenoxyacetic acid (2.4-D). Plantlets with diploid and tetraploid chromosome numbers were subsequently regenerated from the callus. Chromosome pairing in the diploid regenerants and tetraploid amphiploids was incomplete and the plants were sterile.Key words: Triticum, Secale, chromosome pairing, callus, tissue culture.