Micronucleation by mitosis inhibitors in developing microspores of Spathiphyllum wallisii Regel.

KEY MESSAGE : We developed an efficient protocol for chromosome scattering in Spathiphyllum microspores. The effects of plant material, developmental age, genotype and antimicrotubular toxin type, exposure and concentration were evaluated. Asymmetric hybridization through microprotoplast-mediated chromosome transfer (MMCT) is a known method for overcoming sexual breeding barriers between distantly related plant species. To obtain microprotoplasts, it is necessary to induce mass micronucleation either in somatic or gametic cells. We have tested the efficiency for micronuclei induction of five mitosis inhibitors, amiprophos-methyl (APM), butamiphos (BUT), chlorpropham (CIPC), oryzalin (ORY) and propyzamide (PRO), on developing microspores of diploid Spathiphyllum wallisii Regel. Besides the used toxins, also the effect of their concentrations and incubation period as well as plant genotypes and material was tested. We observed micronuclei (MNi) in pollen mother cells, dyads and tetrads as well as other abnormalities such as ball metaphases and chromosome bridges. The flower position on the spadix and the type of starting material (dissected anthers vs. complete spadices) did not significantly influence micronucleation frequencies. The highest micronucleation index of 86 % was obtained in microspores treated with 10 µM ORY during 72 h. All six genotypes tested formed micronuclei after this particular treatment, although the efficiency varied between cultivars. Next to ORY, CIPC was also a very efficient MNi inducer. The average number of MNi found in micronucleated cells varied between 1.67-6.44 for CIPC and 0.83-5.50 for ORY. The maximal number of MNi observed was 12 for CIPC and 9 for ORY. Our results demonstrate that CIPC and ORY can be applied for mass micronucleation on developing microspores of S. wallisii as a first step of MMCT in aroid interspecific or intergeneric breeding.

Meiotic aberrations during 2n pollen formation in Begonia.

Unreduced gametes are the driving force for the polyploidization of plants in nature, and are also an important tool for ploidy breeding. The final heterozygosity of a 2n pollen grain depends on the cytological mechanism behind 2n pollen formation. In this study, chromosome pairing and chromosome segregation during the microsporogenesis of seven Begonia genotypes were analysed using fluorescent chromosome staining on (squashed) pollen mother cells. Among the seven genotypes, five genotypes produce 2n pollen (B. 'Bubbles', B. 'Florence Rita', B. 'Orococo', B. 'Tamo' and B276) and two genotypes produce only normal n pollen (B. fischeri and B243). All 2n pollen producers showed a mechanism equivalent to first division restitution (FDR), in which chromosomes did not segregate during meiosis I but only during meiosis II. This FDR was the result of (a) an irregular chromosome pairing in B. 'Tamo', (b) stickiness of chromosomes associated with numerous chromosome bridges in B. 'Florence Rita' and B276, and (c) a combination of irregular chromosome pairing and stickiness of chromosomes in B. 'Bubbles'. The exact mechanism of the nuclear restitution in B. 'Orococo' could not be determined. Other mechanisms, such as early asymmetric cytokinesis, omission of meiosis II, parallel or tripolar spindle formation, were rather uncommon. Unpaired chromosomes (univalents) were observed in all genotypes, but they had moved to one of the poles by the end of anaphase I or II. Only B. 'Tamo' formed a high number of micronuclei. Consequently, this genotype formed a large number of malformed pollen. Obviously, chromosome behaviour during meiosis in Begonia is very dynamic, which may have important consequences for chromosome evolution and biodiversity within the genus.

Screening of remote Rhododendron species and hybrids for interspecific hybridization with R. simsii hybrids.

To introduce yellow, blue or orange pigmentation in the Rhododendron subgenus Tsutsusi (including R. simsii hybrids) by classical breeding, intersubgeneric pollination experiments involving Hymenanthes, Pentanthera and Rhododendron (including Vireya) species and hybrids were started. Prefertilization research by pollen tube staining revealed a different behaviour between bilateral crosses. Ovule culture was applied as a means to save a substantially larger amount of hybrid embryos from abortion. Though flower/seed capsule abortion eliminated many crosses in an early developmental stage, ovules of many crosses could be initiated in vitro on various time intervals after pollination. Germination was achieved on basal Rhododendron medium + 50 mg/l GA3. Green seedlings were obtained after Tsutsusi x Hymenanthes, Hymenanthes x Tsutsusi, Tsutsusi x Rhododendron, Rhododendron x Tsutsusi and Vireya x Tsutsusi pollination. Many seedlings have little growth vigour and next to green seedlings albino seedlings occur frequently, depending on the pollination type. The first true leaves of many assumed hybrids obviously lack chlorophyll. The ploidy level of Vireya x Tsutsusi and Tsutsusi x Pentanthera seedlings is intermediate, which strongly suggests their hybrid origin. Molecular analysis through SSR confirmed the interspecific charachter of seedlings in half of the crosses tested.