Development of interspecific somatic hybrid cell lines in cultivated jute and their early characterization using jute chloroplast RFLP marker

A strong sexual incompatibility barrier that exists between the two cultivated jute species, Corchorus capsularis and Corchorus olitorius, limits the scope for improvement through genetic introgression. Protoplast fusion was carried out to generate interspecific hybrid cell lines. Cotyledonary cell protoplasts of C. capsularis and anthocyaninpigmented hypocotyl protoplasts of C. olitorius were used in the fusion experiments, which appeared to be visually useful in the early selection of the fused products. A chloroplast DNA (cpDNA) marker was developed in jute, which showed species‐specific hybridization patterns with EcoRI‐digested total genomic DNA of C. capsularis and C. olitorius. This cpDNA marker was used in the characterization of the somatic hybrid cell lines at their early stages of growth. Evidence for the presence of both types of cpDNA in the hybrid cell lines was obtained when the total genomic DNA of 4‐ to 7‐month‐old hybrid cell lines was challenged with the chloroplast DNA marker through Southern analysis. It was shown that the early segregation of the parental chloroplasts did not occur in jute, although this is common in other plant species. The hybrid nature of the fused cell lines could also be identified through peroxidase isozyme analysis. Isozyme banding patterns were complex and varied among the hybrid cell lines.     

Somatic hybrids between navel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding

Protoplasts from cell suspension cultures of ‘Bonnaza’ navel orange (Citrus sinensis (L.) Osbeck) were electrically fused with mesophyll protoplasts isolated from seedless ‘Red Blush’ grapefruit (Citrusparadisi). After 6 months of culture, a total of 20 plants were regenerated. Root tip chromosome counting revealed that 4 of them were tetraploids (2n = 4x = 36)and the rest were diploids (2n = 2x = 18) morphologically resembling the mesophyll parent. After 6 months of transplantation into the greenhouse, 4 of the diploidmesophyll regenerants unexpectedly flowered, but this phenomenon disappeared in the next year. This is the first report of precocious flowering in citrus via protoplast fusion. RAPD analysis further confirmed that the tetraploid regenerants were somatic hybrids while the diploid regenerants were mesophyll parent type. This somatic hybrid will be utilized as a possible pollen parent for improving the seedy pummelo cultivars in China by producing triploid seedless pummelo hybrid. The mechanism of early flowering was also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Protoplast technology for the breeding of top-fruit trees (Prunus, Pyrus, Malus, Rubus) and woody ornamentals

Summary Until recently, temperate fruit trees and woody ornamentals have been regarded as recalcitrant to biotechnological breeding approaches based on protoplasts. This however should no longer be the case, as procedures are now available, not only for the regeneration of complete plants from protoplasts of various tissues of such species, but also for the exploitation of protoplast technology for their genetic manipulation. This paper will examine the recent advances and state of the art in this domain, with particular attention to the use of protoplast technology as a novel tool in the breeding of rosaceous top-fruit tree species and woody ornamentals. Problems and their solutions within the context of regenerating plants from isolated protoplasts of stone (Prunus spp.), pome (Pyrus spp., Malus spp.) and small (Rubus spp.) fruits, and of several shrubby ornamental genotypes (Lonicera spp., Weigela spp., Forsythia spp., Cotoneaster spp.) will be addressed. Interspecific (Prunus spinosa + Prunus cerasifera) and intergeneric (Forsythia spp. + Syringa spp.) somatic hybridization within this group of species, as well as the use of protoplasts for host/pathogen interaction studies (Pyrus/Erwinia amylovora) will also be discussed.     

茶树叶片和胚根原生质体的分离及PEG诱导融合

植物原生质体是细胞培养和体细胞融合等细胞水平研究及植物遗传育种的重要材料。本研究用福鼎大白茶茶树的幼嫩叶片及胚根, 分析了原生质体分离过程中的材料、酶解液组成及酶解时间、纯化方法等影响因子, 建立了最佳原生质体分离体系, 为茶树体细胞杂交等细胞水平的研究提供了高效获取大量高活力原生质体的方法。结果表明, 23°C恒温黑暗或遮光培养的茶树实生苗的5周叶龄以内的幼嫩叶片是茶树原生质体分离的最佳材料, 其次是茶树种子萌发后的幼嫩胚根; 而以茶园健康生长的5周叶龄以内的幼嫩叶片为材料时, 只能获得混有大量细胞碎片的少量具有活力的原生质体。以茶树幼嫩叶片为分离材料的酶解液组成为1.5%纤维素酶+0.1%离析酶+0.5%果胶酶+0.4 mol L^-1甘露醇+20 mmol L^-1 MES; 以茶树幼嫩胚根为分离材料的酶解液组成为1.5%纤维素酶+0.3%离析酶+0.5%果胶酶+0.4 mol L^-1甘露醇+20 mmol L^-1 MES。分离茶树幼嫩叶和幼嫩胚根原生质体时, 宜采用低速(分别为55 r min^-1和50 r min^-1)恒温(23°C)摇床振荡酶解培养, 时间分别为7 h和8 h; 最适宜采用15×g的转速, 离心4 min可纯化获得高产量和活力的原生质体。用40% PEG-6000诱导20 min后可使茶树原生质体融合, 融合率达10%。     

Production of somatic hybrid plants between Iris ensata Thunb. and I. germanica

Wide hybridization that cannot be attained through conventional sexual crosses, can now be approached by somatic hybridization. Protoplasts of I. ensata and I. germanica were fused by electrofusion. For the selection of somatic hybrids, protoplasts of I. ensata which did not form colonies in protoplast culture and protoplasts of I. germanica which had regeneration ability for only albino shoot were used in symmetric fusion. On the other hand, the protoplasts of I. ensata and I. germanica protoplasts which were inactivated by iodoacetamide (IOA) treatment were used in asymmetric fusion. Five-six months after cell fusion, green plants were obtained in the symmetric and asymmetric fusion. In the random amplified polymorphic DNA (RAPD) analysis, the green plants had bands specific to both parental species. Therefore, these plants were somatic hybrids between I. ensata and I. germanica. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of Somatic Brassies napus Hybrids by Polyacrylamide Gel Electrophoresis

Three somatic hybrids obtained by fusion of protoplasts from Brassica oleracea and B. campestris were analyzed by gel electrophoresis and compared with their respective parental species. By comparing multiple forms of esterases and phosphorylases it could be demonstrated that in all cases the hybrid plants contained one or more enzymes from each parent.     

Protoplast Fusion in the Genus Medicago and Isoenzyme Analysis of Parental and Somatic Hybrid Cell Lines

Leaf mesophyll protoplasts of Medicago arborea (2n = 32) were electrofused with cell suspension and/or callus protoplasts of Medicago sativa. Heterokaryons were identified in agarose beds by their dual fluorescein isothiocianate—chlorophyll fluorescence and their coordinates were recorded. Hybrid minicalli were manually picked up and grown first in nurse culture and then in callus induction medium. Hybrid nature of the selected calli was confirmed by isoenzyme analyses. In order to verify whether morphogenesis of somatic hybrid calli was affected by cell incompatibility, mesophyll and cell suspension protoplasts, derived from the same plant of M. sativa with high embryogenic capacity, were fused. Only callus tissues derived from mesophyll protoplasts retained the highly embryogenic character of the M. sativa genotype, while hybrid cell lines were non-morphogenic and showed isoenzyme patterns similar to tissues derived from cell suspension protoplasts. The achievement of somatic hybrid plants in the genus Medicago is discussed.     

Microprotoplast fusion technique: a new tool for gene transfer between sexually-incongruent plant species

Summary Various aspects of a microprotoplast fusion technique and the strategies followed for intergeneric partial genome transfer (one or a few chromosomes) and alien genes from sexually-incongruent donor species to recipient species are described. The essential requirements of the microprotoplast fusion technique are the induction of micronuclei at high frequencies, as well as the isolation and enrichment of sub-diploid microprotoplasts in donor species, efficient fusion of the donor microprotoplasts with normal recipient protoplasts and stable regeneration of plants from fusion products. The results on the production of microprotoplast hybrid plants between the transformed donor lines of Solanum tuberosum and Nicotiana Plumbaginifolia carrying various genetic markers, and a recipient line of Lycopersicon peruvianum or Nicotiana tabacum, and on the transfer and expression of alien genes (kanamycin resistance, -glucuronidase) are presented. The data obtained on microprotoplast hybrid plants between S. tuberosum and L. peruvianum showed that many of the hybrids contained one potato chromosome carrying nptII and GUS, and 24 or 48 L. peruvianum chromosomes (monosomic additions), and that they were male-and female-fertile. Various applications of chromosome transfer by this technique, especially for economically-important traits (e.g. disease or stress resistance) from sexually-incompatible wild species, for construction of chromosome-specific DNA libraries through microdissection and microcloning of chromosomes, or by flow-sorting of chromosomes for genome analysis, are discussed.     

Nuclear genetic control of variation in simazine tolerance in oilseed brassicas

Summary Brassica napus is a natural allotetraploid derived from the diploid species B. rapa L. (syn. campestris L.) and B. oleracea L. Somatic hybrids synthesized from highly heterozygous lines of these two diploid species were evaluated for fertility. The hybrids were obtained from two fusion experiments which differed in the B. rapa full-sibling parent used as the source of protoplasts. Both B. rapa siblings were lelf-incompatible (SI) yet contained different S-alleles; the B. oleracea species parent was self-compatible (SC). Eight tetraploid hybrids examined had very high female and male fertility; eight hybrids with higher ploidy had low fertility. Hybrids derived from one B. rapa sibling were self-incompatible, whereas those derived from the other B. rapa sibling were fully self-compatible. These data suggest that the different S-alleles of each B. rapa sibling displayed varying penetrance relative to the SC of the B. oleracea parent when combined in B. napus.Abbreviations SC self-compatibility - SI self-incompatibility     

Seed dormancy mechanisms in warm season grass species

Embryogenic protoplasts of Dancy tangerine (Citrus reticulata Blanco) were X-ray irradiated at three doses and electrofused with iodoacetic acid-treated embryogenic protoplasts of Page tangelo (C. reticulata Blanco × C. paradisi Macf.). Shoots could regenerate only from the fusion combination with the lowest irradiation dose, but were recalcitrant to rooting. In vitro grafting was applied to obtain complete plants. Chromosome examination showed that the plants contained mainly diploid and aneuploid cells, together with few tetraploid cells, indicating that they were mixoploids. Random amplified polymorphic DNA analyses with 10-mer arbitrary primers confirmed the plants as true somatic hybrids. This is the first report on regeneration of mixoploid hybrid plants via protoplast asymmetric fusion in Citrus. Negative effects of ionizing irradiation on regeneration of embryoids and plantlets and possible agronomic interest of the mixoploid plants are also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cloning and characterization of repetitive sequences and development of SCAR markers specific for the P genome of <Emphasis Type="Italic">Agropyron cristatum</Emphasis>

In previous studies, we successfully transferred the P genome of Agropyron cristatum into wheat using wide hybridization methods. In the current investigation, repetitive sequences were cloned and DNA markers specific for the P genome of A. cristatum were developed. Three P genome-specific markers, designated OPX07-1036, OPX11-817 and OPC05-1539, were identified and isolated using random amplified polymorphic DNAs. The three markers were successfully amplified in all tested materials that contained Agropyron chromatin, such as Agropyron itself and wheat-Agropyron addition lines. These RAPD markers were converted into SCAR markers to be used in detection of P genome chromatin in wheat. In situ probing with fluorescent-labeled marker DNA has shown that they are distributed in all arms of Agropyron hence they will be useful in a variety of studies on introgressions of the P-genome chromatin into wheat.     

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.     

Production and characterization of intergeneric somatic hybrids between Moricandia arvensis and Brassica oleracea

Somatic hybrids were produced between Moricandia arvensis (MaMa, 2n= 28) and Brassica oleracea (CC, 2n= 18) through cell fusion and then characterized by analysing their morphology, cytology, DNA constitution, leaf anatomy and seed fertility. Cell fusion was carried out between greenish protoplasts isolated from the mesophyll of M. arvensis and colourless ones from hypocotyls of B. oleracea. Three plants were generated from one shoot via cuttings and acclimatized in vivo. They closely resembled each other in morphology, exhibiting traits intermediate between the parental species. They were confirmed to be amphidiploids by mitotic and meiotic analyses, being 2n= 46 (MaMaCC), with pollen fertility of about 50%, which was enough to develop the subsequent progenies. Anatomical analysis of the for leaf tissue showed that the bundle sheath cells of the somatic hybrids contained some centripetally arranged organelles, like those of M. arvensis. The hybridity was also confirmed by random amplified polymorphic DNA analysis. Both chloroplast DNA and mitochondrial DNA of the somatic hybrids were estimated to be derived from M. arvensis. In leaf anatomy, the somatic hybrid showed the C₃‐C₄ intermediate trait as in M. arvensis. Many progenies resulted from backcrossing with parental species. The somatic hybrids are expected to be used as bridging plant material to introduce the C₃‐C₄ intermediate trait into Brassica crop species.     

Improved plant heterokaryon formation by surfactant-supplementation of polyethylene glycol fusogen solution

Summary PEG fusion solution for leaf protoplasts of Petunia parodii and cell suspension protoplasts of albino P. hybrida cv. Comanche was supplemented with 0.01–1.0% (w/v) Pluronic F-68. This stimulated protoplast fusion overall, including parental homokaryon formation, with increased means of 23% and 83% respectively, over appropriate controls using 1.0% (w/v) surfactant added to the standard PEG solution. Interestingly, the percentage heterokaryon formation increased near 2-fold (P<0.001) for fusogen solutions supplemented with 0.01% (w/v) Pluronic. Protoplasts regenerated to colonies in KM8P/KM8 liquid medium, indicating no adverse effects of Pluronic F-68 on viability, both in the short and longer terms.Abbreviations BA 6-benzyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - PEG polyethylene glycol     

Production and molecular characterization of Citrus intergeneric somatic hybrids between red tangerine and citrange

Somatic hybridization has been an effective and successful technique for plant improvement. In this paper, embryogenic callus protoplasts of red tangerine (Citrus reticulata Blanco) were electrically fused with mesophyll protoplasts from citrange (C. sinensis × P. trifoliata, a Chinese local strain) in an effort to produce complementary tetraploid citrus rootstocks. Regenerated embryoids grew slowly and were vulnerable to browning. Twelve plants were finally regenerated, rooted and transplanted into a greenhouse. Root‐tip chromosome counting of five randomly‐selected plants revealed most cells were tetraploid (2n = 4x = 36), but aneuploid cells also existed. Flow cytometry analysis further confirmed their tetraploid nature. Nuclear simple sequence repeat (SSR) analysis verified their hybridity. Further mitochondrial genome analysis by restriction fragment length polymorphism and cleaved amplified polymorphic sequence revealed their mtDNA banding pattern was identical to that of red tangerine, the embryogenic callus parent; while their chloroplast DNA inheritance was random as revealed by chloroplast SSR analysis, in addition to cpDNA co‐existence detected in one plant. Cytological and molecular analysis indicated that somatic hybrid plants between red tangerine and citrange had been successfully obtained.     

Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia

Summary Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used as genetic markers and chromosome morphology as a cytological marker. In this combination, development of hybrid callus was late and slow. Only a limited number of non-regenerable hybrid calli have become available. Chromosome loss could clearly be established in these hybrids from loss of markers and from chromosome cytology. Loss of markers occurred independantly.The best conditions to induce loss of chromosomal material in donor cells by irradiation were found by cytological investigations. A very drastic reduction in chromosome transfer by fusion could be effected by irradiation of plant tissue and subsequent preparation of protoplasts after a few days. Following fusion, hybrid callus was recovered with the potato genome drastically reduced. The amount of loss was deduced from the presence of a few fragments in metaphase cells or from interphase nuclei after in situ hybridization with a repetitive potato DNA probe.     

Plant Regeneration from Protoplasts of Trifolium repens and Lotus corniculatus

Protoplasts were isolated from leaves of Lotus corniculatus and Trifolium repens and from cotyledons of L. corniculatus. These protoplasts divided and produced colonies. The plating efficiency of protoplasts of both species was improved when agarose was used as a supporting medium. Plants were regenerated more regularly and in larger numbers from colonies of L. carniculatus than T. repens. The use of a culture line of T. repens that had been selected for its response in culture markedly increased the proportion of protoplast-derived cultures which regenerated shoots. One regenerant of T. repens (P6) was analysed for morphological and cytalogical variation. This plant was abnormal and highly aneuploid with a wide range of chromosome numbers.     

Analysis of protoplast-derived plants of Saintpaulia ionantha H. Wendl

Of 3272 plants regenerated from protoplasts of 10 Saintpaulia ionantha genotypes, 98.4% survived transfer to the greenhouse. The frequency of regenerants with chlorophyll deficiencies, i.e. variegated leaves or albinos, was low (1.5%). There was a higher number of polyploid, in most cases tetraploid plants, regenerated from protoplasts (16%) which were identified by their altered morphology. Measurements of stomatal length and counting the number of chloroplasts per guard cell also allowed a clear differentiation between diploid and polyploid plants. The classification was confirmed by DNA content determination using flow cytometry. Mechanisms leading to polyploidization included spontaneous protoplast fusion as well as chromosome doubling during callus growth and shoot regeneration. Two genotypes with instabilities in flower colour showed completely altered flower colours in plants regenerated from protoplasts as well as in plants regenerated on leaf explants in vitro.     

Establishing Cytoplasmic Male Sterility in Brassica napus by Mitochondrial Recombination with B. tournefortii

Fusion experiments between B. napus and X-ray-treated B. tournefortii protoplasts were carried out to develop cytoplasmic male sterility (ems) in B. napus. From the regenerants, six lines containing male sterile plants were selected; five lines segregated for male sterility, but one line (25–143) was completely male-sterile from the beginning. Molecular analyses of mitochondrial (mt) and chloroplast (cp) DNA of B. napus, B. tournefortii, B. juncea and cms juncea indicated that the original cytoplasmic donor of the cms juncea-system in B. napus was a B. tournefortii form, while the B. napus genotype used for the fusion experiments had a B. campestris cytoplasm. By analysis (it regenerated plants, line 25–143 was identified as possessing mt-DNA recombined between B. campestris and B. tournefortii. with the major part derived from B. campestris. No differences were detected between epDNAs from H. campestris and from line 25—143. The other five lines were similar to B. campestris with all the probes used. The low frequency of sterile lines from the fusion experiments and the inheritance of the cms in segregating progenies are both discussed.