Haploid Calli Research Articles

Improvement of Anther Culture to integrate Doubled Haploid Technology in Temperate Rice (Oryza sativa L.) Breeding.

Doubled haploid (DH) plant production, such as anther culture (AC), is an effective tool used in modern rice breeding programs. The improved efficient protocols applied can shorten the process of breeding. The effect of combinations of plant growth regulators (2.5 mg/L NAA, 1 mg/L 2,4-D and 0.5 mg/L kinetin; 2 mg/L 2,4-D and 0.5 mg/L BAP) in the induction medium were compared in AC for five rice breeding materials and combinations. Induction of calli ranged from 264.6 ± 67.07 to 468.8 ± 123.2 calli/100 anthers in AC of rice genotypes. Two basal media (MS and N6) and two combinations of growth regulators (1 mg/L NAA, 1 mg/L BAP and 1 mg/L kinetin; 1.5 mg/L BAP, 0.5 mg/L NAA and 0.5 mg/L kinetin) were used as regeneration media. The in vitro green plant production was the highest with the application of the N6NDK induction medium (NAA, 2,4-D and kinetin) and the MS-based regeneration medium (1 mg/L NAA, 1 mg/BAP and 1 mg/L kinetin) in anther culture of the '1009' genotype (95.2 green plantlets/100 anthers). The mean of five genotypes was 24.48 green plantlets/100 anthers for the best treatment. Flow cytometric analyses conducted identified the microspore origin of the haploid calli produced in AC, while the uniformity of spontaneous DH plants was checked in the DH1 and DH2 generations. Spontaneous chromosome doubling ranged from 38.1% to 57.9% (mean 42.1%), depending on the breeding source. The generated and selected DH lines were tested in micro- and small-plot field experiments to identify promising lines for a pedigree breeding program. The improved AC method was integrated in a Hungarian temperate rice pedigree breeding program.

Effect of Genotype and Growing Environment on Anther Culture in Wheat

Wheat anther culture is affected greatly by many factors,such as wheat genotype,culture medium,and the temperature during the growing period of donors.For further improving the regeneration efficiency from wheat anthers and understanding the genetic control of wheat anther culture ability,some wheat genotypes including Verry,Xinchun 9,Chinese Spring,and Ningchun 4,and their F1 hybrids by crossing each other were used for anther cultures in different years,and the callus induction frequencies were evaluated.The results indicated that haploid calli were easily induced from the anthers of Alondra,Verry,Shi 4185,Xinchun 9,and Bainong 3217.The frequency of callus production ranged from 25.3% to 51.9%.Shi 4185 and Xinchun 9 showed higher potentials for wheat haploid breeding because of their good agronomic traits and high anther culture response.Some F1 hybrids derived from Ningchun 4 had high callus production rates,indicating that Ningchun 4 had high combining ability with some crossing parents in anther culture.However,the F1 hybrids from the parents with high anther culture response were not certain to perform high callus frequency,which was more than 10.0% in majority of the crosses.This suggested that the genetic control of wheat anther culture appeared to be complicated and performed as a quantitative trait.The optimal environment conditions for the donor plants of the anthers were long vegetative growing period,moderate temperature during the tillering period,and high temperature during the late jointing period.Haploid plantlets could be induced directly from wheat anthers in the liquid media with low concentrations of auxin and glucose.It was also found that 2,4-D was better than dicamba in liquid media for callus induction.Besides,the frequency of callus induced from wheat anthers showed increase trend with the promotion of temperature during the growing period of wheat donor plants,which was caused by climate warming.

In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum

This paper describes a new protocol to develop doubled-haploid (DH) Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. In this protocol, haploid seedlings derived from microspore cultures of B. napus were used to produce haploid calli for in vitro mutation-selection. For routine screening, mutation was induced by EMS (ethylmethane sulfonate) or occurred spontaneously, and screening for resistant mutants occurred on media with added oxalic acid (OA) as a selection agent. In tests with selected lines, the optimal concentration of EMS for mutation was determined to be 0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/l (half lethal dose was 3.1 mmol/l) for the first cycle of screening. There was an accumulated effect of OA toxicity on calli over two cycles of screening, but the growth and capacity of the surviving calli for regenerating seedlings were not affected by OA. Of the 54 DH lines produced from the in vitro mutation-selection, two DH lines of resistant mutants, named M083 and M004, were selected following seedling and glasshouse tests. The resistance of M083 and M004 to S. sclerotiorum following tests with both mycelial inoculum and OA was greater than that of their donor lines and the resistant control Zhongyou 821. In both glasshouse and field disease nurseries, disease indices on M083 and M004 were less than 50% of those of the control. The time required for M083 and M004 to mature was 14 days and 10 days shorter, respectively, than that of their donor lines. Furthermore, M083 had more pods per inflorescence, a greater 1,000 seed weight and higher yield than its donor line. Random amplified polymorphic DNA characterisation showed that M083 had DNA band patterns that differed from its donor line.

Production of Double Haploids via Maize Pollination in Wheat

SUMMARY Seven good agronomic wheat varieties lacking Karnal bunt tolerance were crossed with two Karnal bunt tolerant varieties. The Fi plants from these crosses were crossed with diverse maize lines. Immediately after pollination solution containing 2,4-D (50mg/l) + GA3 (100mg/l) was injected into upper most node. After 24 hours florets of the pollinated spikes were flooded with same solution. The pollinated spikes were harvested 13 to 16 days post pollination and were taken for rescuing embryos. Rescued embryos were used to obtain callus as well as direct plant embryogenesis. The haploid plantlets and the embryoids of haploid calli were converted to double haploids (DH) using colchicine. For DH production the direct germination of rescued embryos followed by colchicine treatment of haploid plantlets figured to be more efficient method than obtaining plant via haploid callus

Effect of genotype and season on gynogenesis efficiency in Gerbera

The effects of season, genotype and their interaction on haploid production were evaluated on four genotypes responsive to gynogenesis. Naked mature unfertilised ovules, collected from April to October, were cultivated on modified MS basal medium plus 0.88 μM 6-benzyladenine and 0.57 μM indol-3-acetic acid. After 30–60 days, recovered calli were transferred to a MS basal medium with 8.8 μM BA and 0.57 μM IAA for regeneration. Genotype and season of ovule collection substantially interact in the recovery of haploid calli. Between the four genotypes analysed, two gave more calli in the spring, one in the autumn and the fourth showed only weak differences between seasons. Shoot recovery depended upon both the season of ovule collection and the genotype but no significant interaction was shown by our data. The ability to produce haploid callus is not predictive for efficient shoot regeneration.

Production of Doubled Haploid Plants Through Colchicine Treatment of Anther-derived Haploid Calli in the Asiatic Hybrid Lily 'Connecticut King'.

An effective method for producing doubled haploid plants of the Asiatic hybrid lily 'Connecticut King' was established with an in vitro colchicine treatment of haploid calli. With an increase in the concentration and duration of colchicine treatment for the haploid calli, both the survival rate and the shoot regeneration from the calli decreased, but the frequency of diploid cells in the callus increased. Treatments of 0.25 and 0.5 mM colchicine for 48 or 72 hrs induced doubled haploid cells and shoot differentiation from calli. Haploid and diploid plantlets were regenerated from colchicine-treated calli, and only haploid plantlets were formed in colchicine-free treatments. This result suggests that these diploid plantlets originated from doubled haploid cells. Double haploids developed bulblets with more scaly leaves with longer stomatal guard cells than did the haploid plantlets.

Plantlet regeneration from protoplast-derived haploid embryogenic calli of wheat

To search for an alternative method for protoplast culture, regenerable embryogenic calli were obtained from anther culture of three wheat cultivars, Karl 92, Jinghua #1, and Pavon 76. Protoplasts were isolated directly from the haploid embryogenic calli and cultured in modified PMI and LM8P media without going through cell suspension culture. After 8–11 days of subculture, the embryogenic calli produced the maximum yield of protoplasts and cell division was at the highest frequency when plated at a density of 3–4 × 105 protoplasts ml−1. Frequency of colony formation varied from 0.2% to 0.5% for Jinghua #1 and from 0.1% to 2% for Pavon 76, while Karl 92 failed to produce colonies, even though its embryogenic calli were friable and fast-growing on the maintenance medium. Green haploid plantlets of Jinghua #1 and Pavon 76 have been regenerated from protoplasts, which were cultured on a differentiation medium first and then on a rooting medium.

Pollen ontogeny in Cichorium intybus L.

Abstract Cichorium intybus L. is a highly self-incompatible species: haploids obtained in vitro through androgenesis would be of benefit in producing homozygous doubled haploids. The microsporogenesis and microgametogenesis of C. intybus is investigated using LM, TEM and SEM to obtain information which could be exploited in anther and/or microspore culture experiments and to ascertain whether the plastid change during pollen development is related to the androgenic recalcitrant behaviour of this species: anthers of this species are not induced by in vitro culture to produce haploid calli or embryos. C. intybus has a tapetum which after a limited parietal phase becomes amoeboid, losing the inner tangential and radial cell walls at the microspore stage. During pollen development the tapetum degenerates and tapetal cells have a discontinuous plasma membrane. Starch accumulation occurs only during pollen maturation, starting after the first mitotic division: at anthesis pollen grains are engorged, mainly with starch. These results are compared with observations made on other Compositae and the accumulation pattern of reserves is evaluated in relation to the androgenic recalcitrant behaviour of the species.

Haploid Calli, Shoots, and Plants from Anthers ofDigitalis lanata

Haploid Calli, Shoots, and Plants from Anthers of<i>Digitalis lanata</i>

Ploidy variation of pronamide-treated maize calli during long term culture

Anther-derived calli of corn were treated with 10 μM pronamide for 2, 3 and 4 days. The ploidy level of the calli was then evaluated using flow cytometry, at different times after the treatment. Untreated haploid calli did not change in ploidy level for 97 days but by 466 days, there were up to 50% diploid or higher ploidy cells thus showing that spontaneous doubling may occur during corn calli subculture with this genotype. Pronamide treatment did increase the percentage of diploid and tetraploid cells and by 466 days, all of the lines showed an additional change toward higher ploidy levels. This change may be due to spontaneous chromosome doubling or to differential cell cycle times of cells with different ploidy levels. The ploidy level of plants regenerated from the cultures was determined by counting the guard cell chloroplast numbers and the correlation with the ploidy level of the cultures was r(2)=0.84. These studies show that pronamide treatments can increase haploid maize callus chromosome numbers and that spontaneous chromosome doubling can occur with time in maize callus.

Effects of gamma-ray irradiation on the growth of calli in Nicotiana species.

Haploid and diploid calli of Nicotiana glauca (n=12) and N. langsdorffii (n=9) together with dlploid calli of N. tabacum (n=24) cv. Bright Yellow which were taken as control were ex-posed to various doses of 60co gamma rays at 7 days and 10 days after incubation. Calli were subsequently weighed for a period of 12-14 days after radiation (19-24 clays after incubation) and the effect of irradiation on the growth was studied. A sigmoid curve was observed for the growth rate as well as for the growth pattern. The radiosensitivlty were estimate.d on the basis of the following different two criteria: 1) the difference in the diploid callus volumes at 12 or 14 days after irradiatlon and at the time of radiation, 2) the growth rate of diploid and haploid calli during these period. In 2 species except in N. langsdorffii, calli irradiated ten days after incubation (at the beginning of the actively growing stage) were more sensitive to gamma irradiation than calli lrradiated seven days after incubation (at a relatively slow growing stage). Each species had its own distinctive color before irradiation; golden yellow, green and yellow green for N. glauca, N. langsdorffii and N. tabacum, respectively. The color of some of the calli tended to darken with increasing irradiation doses. When the rate of increase of the irradiated calli to that of the unlrradiated ones was used as an indicator of radiosensitivity, there was a significant difference in the radiosen-sitivity of the calli among the species: N. tabacum was more sensitive followed by N. glauca and N. langsdorffii, which was the least sensitive. On the other hand, when the curve of the increase of the growth rate was used as the criterion of radiosensitivity, N. langsdorffii appeared to be more sensitive than N. tabacum and N. glauca. Haploid calli were more radiosensitive than diploid ones in N. glauca, but no significant difference was observed between them in N. lanegsdorffii.

A comparative study of initiation and development of embryogenic callus from haploid embryos of several barley cultivars. II. Cytophotometry of embryos and callus

Cells of haploid barley embryos (Hordeum vulgare L. 'Bruce', 'Perth', and 'Klages') and callus cells originating from embryos were cytophotometrically examined to determine the ploidy level. Specific embryo tissues regularly exhibited predictable ploidies and smaller embryos had a higher percentage of haploid cells than larger embryos of the same age. The predominantly haploid cells of the scutellar epidermis of the embryo initiated callus which generally, at least initially, was haploid. Monitoring of the ploidy evolution of these haploid calli showed that over a 6-month period each line exhibited its own unique rate of polyploidization, although lines of the same cultivar showed similar trends. Accumulation of cells at the diploid level was often a characteristic of these cultures.