Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

Histological study of embryogenesis and organogenesis from anthers ofVitis rupestris du Lot cultured in vitro

Summary Anthers ofVitis rupestris du Lot were cultured in vitro at the uninucleate stage of the microspore, in order to investigate the histology of embryogenic and organogenic processes in this genotype. Microspores divided in the anther loculi resulting in the formation of globular structures with a ruptured exine. Somatic embryogenesis and, occasionally, caulogenesis and rhizogenesis occurred in calli produced from all anther tissues except the endothecium. The initial cell of the embryoid was surrounded by a jacket layer when situated deep within the callus. When the embryoid's initial cell was situated in the peripheral callus, a cutinized wall was present and the three-celled proembryoid was almost always segmented, showing the same embryonal type as the zygotic proembryo. Root differentiation and elongation and cap differentiation occurred during the growth phase in liquid medium. The mature root was diarch and contained cells with calcium-oxalate raphides, as seen in vivo. No starch or tannin deposition was ever observed in the mature embryoids.Abbreviations 6-BAP 6-benzylaminopurine - CC3 Nitsch and Nitsch (1969) basal medium - CS cross section - 2,4-D 2,4-dichlorophenoxy-acetic acid - LS longitudinal section     

Spontaneous extensive chromosome elimination in somatic hybrids between somatically congruent species Nicotiana tabacum L. and Atropa belladonna L.

Summary Mesophyll protoplasts of the kanamycin-resistant nightshade, Atropa belladonna, were fused with mesophyll protoplasts of the phosphinothricin resistant-tobacco, Nicotiana tabacum. A total of 447 colonies resistant to both inhibitors was selected. Most of them regenerated shoots with morphology similar to one of the earlier obtained and described symmetric somatic hybrids Nicotiana + Atropa. However, three colonies (0.2%) regenerated vigorously growing tobacco-like shoots; they readily rooted, and after transfer to soil, developed into normal, fertile plants. Unlike their tobacco parental line, BarD, the obtained plants are resistant to kanamycin [they root normally in the presence of kanamycin (200 mg/1)] and possess activity of neomycin phosphotransferase (NPT II) with the same electrophoretic mobility as the one of the nightshade line. According to Southern blot hybridization analysis carried out with the use of radioactively labeled cloned fragments of the Citrus lemon ribosomal DNA repeat, as well as with Nicotiana plumbaginifolia genus-specific, interspersed repeat Inp, the kanamycin-resistant plants under investigation have only species-specific hybridizing bands from tobacco. Cytological analysis of the chromosome sets shows that plants of all three lines possess 48 large chromosomes similar to Nicotiana tabacum ones (2n = 48), and one small extra chromosome (chromosome fragment) similar to Atropa belladonna ones (2n = 72). Available data allow the conclusion that highly asymmetric, normal fertile somatic hybrids with a whole diploid Nicotiana tabacum genome and only part (not more than 2.8%) of an Atropa belladonna genome have been obtained without any pretreatment of a donor genome, although both these species are somatically congruent.     

Modulation of auxin-binding proteins in cell suspensions

Summary Cultured cell lines from carrot (Daucus carota L.) with little or no embryogenic potential were examined for the auxin-binding capacity of their membranes. The lines belonged to different classes: (a) wild-type lines kept in culture for different periods (the longer the period, the lower being their embryogenic potential); (b) variants, isolated after mutagenesis, showing normal growth but a lack of embryogenic response; (c) auxin-resistant lines, isolated as colonies on solid media containing 45 M 2,4-d; (d) a previously described tumorous line (E9) isolated because of its resistance to hypomethylating drugs. All of these lines showed alterations in auxin-induced, auxin-binding capacity (modulation), i.e. in the non-embryogenic lines the addition of auxin increased the auxinbinding capacity to a very small degree, or removal of the hormone did not produce the proper decrease in that capacity, or both defects could be simultaneously present. Both types of defects were shown to be correctable: after treatments designed to increase the amplitude of modulation, embryogenic capacity was restored in a number of lines.     

石刁柏体细胞胚胎发生过程中蛋白质及同工酶变化的研究

本文报道石刁柏胚性愈伤组织的可溶性蛋白质含量与组分、过氧化物酶和酯酶的活力及同工酶带均比其体细胞胚的要少。而在体细胞胚胎发生过程中,过氧化物酶和酯酶活力、可溶性蛋白质含量均以球形胚为最低,子叶分化期胚为最高而呈递增趋势;可溶性蛋白质组分以子叶分化期胚、成熟胚为最多,球形胚、香蕉形胚为最少;过氧化物酶同工酶带以梨形胚为最多,子叶分化期胚、成熟胚为最少;酯酶同工酶则以子叶分化期胚为最多,成熟胚为最少。胚性愈伤组织与体细胞胚均有其特异性可溶性蛋白质及同工酶带,可作为体细胞胚胎发生的分子标记。     

Chromosomal and isozyme studies of Nicotiana tabacum — Glycine max hybrid cell lines

Summary The chromosomal stability of a number of somatic hybrids derived from soybean (Glycine max (L.) Merr.) and Nicotiana tabacum var. Xanthi were investigated. Several of the hybrid cell lines retained more than half the complement of N. tabacum chromosomes after 7 months of culturing. A number of chromosomal abnormalities were observed. The hybrids were positively identified by employing isozyme analysis of several dehydrogenases and aspartate aminotransferase.NRCC No. 20130     

Chromosomal instability in Lathyrus sativus L.

Summary In Lathyrus sativus (2n=14), variety LSD-1 shows an instability of somatic chromosome number which can be observed in root tip and shoot tip mitoses. In this variety, approximately 54% of the seedlings showed intra-individual variation in chromosome number ranging from 2n=14–3. This variability in chromosome number was recorded in approximately 60% of the dividing cells. Two seedlings were triploid with 21 chromosomes. Variation in chromosome number in somatic cells within individual plants is possibly controlled by genetic factors, which result in spindle abnormalities, chromosome degradation and minute chromosomes. The variation in chromosome number is probably responsible for the pollen polymorphism noted in this particular strain. The possible mechanism of intra-individual variability and the occurrence of the phenomenon vis-a-vis its applications are discussed.     

Somatic hybrids between unilateral cross-incompatible Petunia species

Summary Somatic hybrid plants regenerated following the fusion of leaf mesophyll protoplasts of Petunia parodii with those isolated from a cell suspension of albino P. inflata. These two species exhibit a unilateral cross-incompatability with a pre-zygotic mode of reproductive isolation preventing hybridizations with P. inflata as the maternal parent. Selection of somatic hybrids relied on the fact that unfused or homokaryon protoplasts of P. parodii did not develop beyond the cell colony stage while those of the putative somatic hybrids and albino P. inflata parent produced callus. Green somatic hybrid calluses were readily identified against the white background of P. inflata following complementation to chlorophyll synthesis proficiency and continued growth in hybrid cells. Shoots, and ultimately flowering plants, were identified as somatic hybrids based on their floral morphology and colour, chromosome number and the fact that they segregated for parental characters. The frequency of somatic hybrid production was comparable to that previously established for two sexually compatible Petunia species.     

Effects of cryopreservation techniques on the preservation of ear skin – An alternative approach to conservation of jaguar,Panthera onca (Linnaeus, 1758)

Currently, it has been observed that a considerable segment of the jaguar population is declining mainly because of hunting, and destruction and fragmentation of habitat. Given this scenario, efforts of the scientific community have been concentrated on the development of conservation strategies, such as the formation and use of somatic sample banks. We aimed to assess the effects of cryopreservation techniques of the ear skin of jaguar [slow freezing (SF) or direct vitrification in cryovials (DVC) or solid-surface vitrification (SSV)] on the morphological analysis and cell ability during the culture. All cryopreserved fragments regardless of the technique used, showed a reduction in the dermis and total thickness of the skin. Although a collagen matrix similar to the control group (fresh) has been observed only for the fragments from SF and SSV groups, all cryopreserved techniques were able to maintain normal patterns of the fibroblasts. Moreover, DVC and SSV methods maintained the proliferative activity of the tissues even after warming. After the culture, SF and SSV techniques were efficient for the recovery of the somatic cells according to most of the evaluated parameters, especially with regard to the duration of culture and cell metabolic activity. In conclusion, SSV was found to be a more efficient technique for cryopreserving jaguar skin when compared to DVC and SF. These results are relevant for the formation of somatic resource banks of this species, directed at cryopreserving adequate samplings of different individuals and generations for future applications in regenerative medicine, and assisted reproductive technologies.     

Glycoalkaloid Profile in Potato Haploids Derived from Solanum tuberosum–S. bulbocastanum Somatic Hybrids

Cultivated and wild potato species synthesize a wide variety of steroidal glycoalkaloids (GA) that may affect either human health or biotic stress resistance. Therefore, GA composition must be a major criterion in the evaluation of breeding products when species genomes are merged and/or manipulated. This work reports the results of GA analysis performed on unique haploid (2n=2x=24) plants obtained from tetraploid (2n=4x=48) Solanum bulbocastanum–S. tuberosum hybrids through in vitro anther culture. Glycoalkaloids were extracted from tubers and analyzed by HPLC. Haploids generally showed the occurrence of parental GA. However, in several cases loss of parental GA and gain of new GA lacking in the parents was observed. It may be hypothesized that new GA profiles of our haploids is the result of either genetic recombination or combinatorial biochemistry events. To highlight differences between haploids and parents, soluble proteins and antioxidant activities were also determined. Both were always higher in haploids compared to their parents. The nature of the newly formed GAs will be further investigated, because they may represent new metabolites that can be used against pest and diseases, or are useful for human health.