Triploid and Hexaploid Regenerants from Hexaploid Timothy (Phleum pratense L.) via Anther Culture

Abstract

Pollen embryos were obtained from eight genotypes and viable green plants were regenerated from four genotypes in an anther-culture experiment with 165 genotypes of Phleum pratense L. Formation of proembryos inside the cultured anthers during the first 10-12 days was significantly influenced by genotypes and by the type of nutrient media. Primary embryos developed into multiple secondary embryos before regeneration of plants. Among a total of 62 plants regenerated, only 13 were albinos. Of the green regenerants, 11 were triploid while 35 were hexaploid when DNA-content was measured by flow cytometry. Eight plants with a triploid DNAcontent did possess the triploid chromosome number of 21. Triploid and hexaploid regenerants from two different parents showed simplified isozyme (GPI and PGD) banding patterns relative to that of their parents.Timothy is the main forage grass in areas north of the 62nd parallel for sown and semi-permanent pastures, because of good winter hardiness and high yield (SIMONSEN 1985). Improved dry matter production via breeding has been reported to be promising (SCHJEDLERUP 1982), with efforts being directed towards the development of cultivars with a good general performance and increased persistence (ROGNLI 1987). Breeding of quantitative traits in timothy, however, is generally hampered by the out-crossing and auto-hexaploid nature of the species (JONSSON et al. 1992).During recent decades, haploid formation, through androgenesis from pollen during anther culture, has been reported for a number of different grass species (ANDERSEN et al. 1991). In timothy, NIIZEKI and KATI (1973) have reported callus formation from cultured anthers and we now provide the first evidence for the regeneration of complete plants from pollen.Plant material for experiment I consisted of 51 different breeding clones of timothy {Phleum pratense L.) kindly provided by Danish Plant Breeding, Ltd. For experiment II, 114 seed plants from 19 different varieties of timothy were raised to the 3-4 tillering stage and divided into two ramets each. Plants for both experiments were vernalized at 1-3 °C for 10 weeks. Plants were subsequently re-potted with peat (Finnpeat B2) in 13 mm plastic pots before being grown for flowering under glasshouse conditions with 10-15°C night and 15-25°C day temperature during May to August. Spikes were harvested when they had emerged 3-4 cm above the sheath, from which those with pollen in mid-to late-uninucleate development were selected after squashing a few flowers in carmine acetic acid. These were surface-sterilized by immersing in 70 % ethanol with one drop of tween/litre, followed by 8 min in 3 % Korsolin (GmbH & Co., Hamburg) and rinsing three times with sterile water. For induction of embryos, anthers were cultured on R2M and FW U.S.

Diploid, tetraploid, and octoploid plants from anther culture of tetraploid orchard grass, Dactylis glomerata L.

“…The number of plants produced per 100 anthers from the responsive clones was, on average, 2.0 with 7.1% of the regenerated plants turning green. These results are in good agreement with similar screening studies reported on other grass species attempting to identify responsive genotypes in a random sample of plant material (Zenkteler and Misiura 1974, Olesen et al 1988, Abdullah et al 1994. Because of the few clones (28), replicated in both environments during this study with D. glomerata, it is difficult to draw conclusions regarding the effects of different genotypes on the anther culture response in this species.…”

“…The effects of the two media on be possible to prove the microspore origins of the tetraploid embryo formation were highly significant (P = 0.0021) in the and octoploid regenerants through the study of segregation of statistical analysis. From these results it is believed that R2M is genetic markers (isozymes) heterozygous in the parental plants the more suitable of the two media for further anther culture (Olesen et al 1988, Abdullah et al 1994). However, from the work in D. glomerata.…”

Genetic and nongenetic factors influencing callus induction inMiscanthus sinensis (Anderss.) anther cultures

“…The A1 medium was 190-2 medium (Wang and Hu 1984) supplemented with 0.5 mg L -1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5 mg L -1 1-naphthylacetic acid (NAA), and 30 g L -1 sucrose. The A2 and A3 media were PG-96 (A2; Guo et al 1999) and R2M (A3; Abdullah et al 1994) media, respectively, both with 1.5 mg L -1 2,4-D, 0.5 mg L -1 6-furfurylaminopurine (KIN), and 90 g L -1 maltose. The A4 medium was the C17 medium (A4; Wang and Chen 1983) modified by adding 2 mg L -1 2,4-D and 0.5 mg L -1 KIN, substituting sucrose with 90 g L -1 of maltose, and reduction of potassium dihydrogen orthophosphate (KH 2 PO 4 ) concentration from 400 to 200 mg L -1 , as described by Wêdzony (2003).…”

Androgenesis in the Bioenergy Plant Miscanthus sinensis: From Calli Induction to Plant Regeneration

“…The PG-96 medium was supplemented with 8 g L −1 agar whereas pPG-96 diff ered from PG-96 only in a lack of agar. The last medium was R2M (Abdullah et al, 1994), which is a modifi ed 190-2 (Wang and Hu, 1984) through the addition of 1.5 mg L −1 2,4-D, 0.5 mg L −1 KIN, and 90 g L −1 maltose along with an absence of 1-naphthaleneacetic acid and sucrose. All media were adjusted to appropriate pH values between 5.70 and 5.8 before the addition of solidifi ed agent, as required, and were autoclaved at 121°C for 20 min.…”