Source Of Protoplasts Research Articles

Characterization of chromosome instability in interspecific somatic hybrids obtained by X-ray fusion between potato (Solanum tuberosum L.) and S. brevidens Phil

Asymmetric somatic hybrids between Solanum tuberosum L. and S. brevidens Phil. have been obtained via the fusion of protoplasts from potato leaves and from cell suspension culture of S. brevidens. The wild Solanum species served as donor after irradiation of its protoplasts with a lethal X-ray dose (200 Gy). Selection of the putative hybrids was based on the kanamycin-resistance marker gene previously introduced into the genome of Solanum brevidens by Agrobacterium-mediated gene transfer. Thirteen out of the 45 selected clones exhibited reduced morphogenic potential. The morphological abnormalities of the regenerated plantlets were gradually eliminated during the extended in vitro culture period. Cytological investigations revealed that the number of chromosomes in the cultured S. brevidens cells used as protoplast source ranged between 28-40 instead of the basic 2n=24 value. There was a high degree of aneuploidy in all of the investigated hybrid clones, and at least 12 extra chromosomes were observed in addition to the potato chromosomes (2n=48). Interand intraclonal variation and segregation during vegetative propagation indicated the genetic instability of the hybrids, which can be ascribed to the pre-existing and X-ray irradiation-induced chromosomal abnormalities in the donor S. brevidens cells. The detection of centromeric chromosome fragments and long, poly-constrictional chromosomes in cytological preparations as well as non-parental bands in Southern hybridizations with restriction fragment length polymorphism (RFLP) markers revealed extensive chromosome rearrangements in most of the regenerated clones. On the basis of the limited number of RFLP probes used, preferential loss of S. brevidens specific markers with a non-random elimination pattern could be detected in hybrid regenerants.

Advances in cereal protoplast research

Beginning in 1986, plants have been regenerated from protoplasts of all of the important cereal species, including wheat, rice, maize, and barley, and grasses such as sugarcane. In addition, somatic hybrids/cybrids as well as transgenic plants with introduced useful agronomic traits have been obtained in several instances. This rapid and impressive progress in the genetic manipulation of cereals has been made possible by two critical technical advances during the past decade: the establishment of embryogenic suspension cultures as a source of totipotent protoplasts and the direct delivery of DNA into protoplasts for genetic transformation.

Expression of self-incompatibility and fertility of Brassica napus L. resynthesized by interspecific somatic hybridization

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.

Somatic embryogenesis in protoplast derived calli of cultivated jute, Corchorus capsularis L.

Protoplasts were isolated from cotyledon, hypocotyl and mesophyll cells of Corchorus capsularis L., a major fibre crop, by one step enzyme digestion method. They were further cultured successfully on modified KM-8p (Kao and Michayluk, 1975) medium to form microcalli. The required cultural conditions could be used to achieve 34% to 78% plating efficiency, depending upon the source of protoplasts. Hypocotyl protoplasts gave the highest plating efficiency. On transfer to regeneration medium, somatic embryos developed at high frequency. The present success is a significant step forward in the development of meaningful plant cell culture methods for application in jute.

Callus formation from cultured protoplasts of banana ( Musa sp.)

Protoplasts were isolated from cell suspension initiated from calli of immature seeds of Musa acuminata ssp. burmannica cv Long Tavoy (AA). When co-cultured at high density with a reliable feeder culture, the protoplasts underwent sustained divisions and formed callus. Cytological studies showed that the cells of protoplast-derived calli had embryogenic characteristics as the cell suspensions used as protoplast source. Analysis of isoenzymes, in particular phosphoglucomutase (PGM) and alcohol dehydrogenase (ADH), distinguished the Lolium feeder cells from the protoplast-derived calli of banana and confirmed the Musa nature of these calli. The response of protoplasts depended upon the specificity of the feeder-physical barrier interaction. As a matter of fact, Lolium feeder induced a high rate of growth of protoplast-derived calli when combined with the use of Millipore membrane, used as a physical barrier keeping separate the protoplast culture from the feeder. Banana feeder gave similar results if nylon mesh was used as a physical barrier. The addition of dimethylsulfoxide (DMSO) to the feeder culture neither increased the proliferation of nurse cells nor affected the growth of protoplast-derived calli of banana.

Transient foreign gene expression in polyethylene/glycol treated or electropulsated Eucalyptus gunnii protoplasts

Conditions for optimal transient gene expression of reporter genes (chloramphenicol acetyl transferase and β-glucuronidase) by polyethylene glycol or electrical treatment of Eucalyptus gunnii protoplasts derived from callus or cell suspension cultures were investigated. The effciency of electropermeabilisation depended on several factors including electrical parameters, pH and the source of protoplasts. Polyethylene glycol mediated DNA uptake was highly stimulated by heat shock pretreatment and was also more efficient than the electrical treatment. For both treatments the nature of the promoter associated with the reporter gene was very important. A promoter corresponding to a protein synthesis elongation factor gene was more effective than the classical promoter of the 35 S transcript from cauliflower mosaic virus.

Plant Regeneration from Haploid Suspension and Protoplast Cultures from Isolated Microspores of Maize

Summary Isolated maize microspores were used to initiate haploid suspension and protoplast cultures capable of plant regeneration. Exposure of 10-day induced microspores to a medium containing 9.0 µM 2,4-D resulted in the proliferation of androgenic proembryos into mucilaginous cultures that, after several cycles of selective sub-culture, were enriched for densely cytoplasmic cell clusters. Suspension cultures ranging from yellowish brown, coarse textured, thick, and slow growing to bright yellow, finer textured, and rapidly growing were obtained from the same population of microspores. Re-plating onto callus induction medium resulted in the formation of nodular, embryogenic callus from which haploid plants could be recovered upon transfer to regeneration medium. Enzymatic digestion of suspension cultures followed by density gradient centrifugation yielded totipotent protoplasts. The results from this study expand the list of available maize germplasm amenable to protoplast culture to include a Lancaster-related genotype and add the microspore to the limited list of explants capable of being used directly as a source of embryogenic suspension cultures and totipotent protoplasts.

Plant regeneration from protoplasts of Solanum species with potential agricultural value (S. hjertingii, S. polyadenium, S. capsicibaccatum)

Protoplasts have been isolated from three tuber-bearing Solanum species, S. hjertingii, S. polyadenium and S. capsicibaccatum, that are sexually incompatible with S. tuberosum, but possess potentially useful characters. For isolating protoplasts from leaves of in vitro shoot cultures of S. hjertingii and S. capsicibaccatum growth was improved by including silver thiosulfate in the medium. However, for S. polyadenium, leaves of pot-grown plants were the best source for protoplasts. Following protoplast division and culture, plants were regenerated from protoplasts of each of the species. The pattern of chromosome variation in regenerants was similar to that observed for other diploid and tetraploid Solanum species. The results indicate that it should be possible to introduce the potentially useful germplasm from these wild species into somatic hybrids with S. tuberosum by protoplast fusion.

Analysis of chloroplast and mitochondrial segregation in three different combinations of somatic hybrids produced within Brassicaceae

Mitochondrial and chloroplast DNA were characterized in three different combinations of somatic hybrids produced between different species within Brassicaceae. The fusions were made between B. campestris and B. oleracea, B. napus and B. nigra and between B. napus and Eruca sativa. The combinations represent interspecific hybridizations, but the phylogenetic distance between the species used in each instance is different. Whereas the B. campestris (+) B. oleracea and the B. napus (+)B. nigra hybrids are both examples of intrageneric hybrids, B. campestris is more closely related to B. oleracea than B. napus is to B. nigra. The fusion of B. napus and E. sativa represents an intergeneric hybridization. Since hybrids were produced with reproducible and uniform fusion and culture methods, a comparison of chloroplast and mitochondrial segregation and mitochondrial DNA (mt-DNA) rearrangements could be made between the combinations. The segregation of both chloroplasts and mitochondria was biased in the B. napus (+)B. nigra and the B. napus (+)E. sativa combination. The nonrandom segregation of chloroplasts and mitochondria could be due to the different ploidy levels of the fusion partners and/or reflect differences in organelle replication rate. Furthermore, segregation of mitochondria was correlated to the differences in phylogenetic distance between the species used in the fusions. However, mitochondrial segregation, in contrast to chloroplast segregation, could in all combinations also have been affected by the cell type used as protoplast source in the fusions. All different chloroplast types could be established within each combination. Hybrids containing chloroplast from one parent together with mitochondria from the other parent were found in two of the combinations, although the majority of the hybrids had mt-DNA that was altered compared to the parental species. The rearranged mt-DNA found in most hybrids was an effect of the heteroplasmic state following protoplast fusion rather than of the tissue culture methods, since no mt-DNA rearrangements were found in B. napus plants regenerated from protoplast culture. The mtDNA restriction patterns of the hybrids with rearranged mt-DNA indicated that specific regions of the mt-DNA were involved in the rearrangements following protoplast fusion.

ASSESSMENT OF THE LIMITATIONS OF SOMATIC HYBIDIZATION

This study was designed to assess the general limitations of somatic hybridization as one of the key technologies for genetic manipulation in plants. The limits of somatic hybridization against different taxonomic backgrounds, intraspecific to interfamilial, were also assessed. Protoplast culture studies provided essential information relating to the species cultural and morphogenetic capacity. several #elect Ion strategies for the recovery of somatic hybrid colonies/plants were developed and assessed using various combinations of protoplast sources and species in the genera Petunia, Nicotiana, Salpiglossis and Chrysanthemum. Morphological, cytological and biochemical analyses were performed to confirm the hybridity of plants or cell lines recovered following protoplasm fusion (using 4-5 methods) and selection.The somatic hybrid callus/plants were obtained at intraspecific to interfamilial levels by complementation to chlorophyll proficiency, together with media selection or complementation of nitrate reductase deficient mutants as follows; P. Hybrida var. Monsanto (+) P. hybrida cv. Blue Lace (intraspecific), P. hybrida var. Monsanto (+) P. inflata and P. parviflora (interspecific), P. parviflora (+) N. tabacum (intergeneric), S. sinuata (+) P. hybrida var. Monsanto, P. parodii and N. tabacum (intertribal), and C. morifolium (+) S. sinuata.From this study, it appeared that there were no taxonomic limits to the production and proliferation of somatic hybrid cell lines. However, obtaining morphologically normal hybrid plants met with limited success as the taxonomic relationships became more distant. The regeneration capacity of somatic hybrids seemed to be controlled by both parental species. Somatic incompatibility mechanism was also shown to operate on chromosome elimination. Such chromosome elimination may well be advantageous in plant improvement.

Selection of interspecific somatic hybrids of Medicago by using agrobacterium-transformed tissues

Protoplast fusion was investigated as a means of obtaining somatic hybrids between alfalfa, Medicago sativa L., and two sexually incompatible annual species, M. intertexta L. (Mill.) and M. scutellata L. (Mill.). A selection scheme was developed using genetically stable transgenic fusion partners, each carrying a different antibiotic resistance gene. A kanamycin-resistant transgenic alfalfa plant (RS-K2A5051a) that was phenotypically and karyotypically normal was used as a source of mesophyll protoplasts. Because M. intertexta and M. scutellata do not regenerate to plants from culture, A. rhizogenes was used to produce root cultures of each that were resistant to hygromycin B. The root cultures exhibited chromosome stability and were used for protoplast isolation. Double antibiotic selection for somatic hybrid callus was completed 3–5 weeks after polyethylene glycol fusion. Southern blot analysis with a ribosomal DNA probe confirmed that the selected calli were nuclear hybrids. The regenerability of alfalfa from culture was expressed only in the alfalfa (X) M. intertexta hybrid calli, where embryos and several plantlets were obtained.

Regeneration of Fertile Plants from Protoplasts of Elite Inbread Maize.

We have obtained friable, non-mucilaginous embryogenic callus from cultured immature embryos of an elite maize inbred line derived from Iowa stiff stalk synthetic germplasm. This callus was used to generate embryogenic cell suspensions containing compact clumps of densely cytoplasmic cells. Plants were readily regenerated from the suspensions, which were also an excellent source of protoplasts. The suspension cultures maintained their plant regeneration capacity through many months. The cultures were cryopreserved in liquid nitrogen and embryogenic callus and suspension cultures were routinely recovered from cryopreserved cells. Protoplasts were isolated in large numbers from both primary and cryopreserved suspension cultures. Cultured protoplasts divided and formed embryogenic callus both when plated in agarose and on nurse cultures. Hundreds of plants were regenerated from protoplast-derived calli. Plants with viable progeny were obtained although many of the regenerates showed morphological and reproductive abnormalities.

High Rate of Plant Regeneration from Cultured Protoplasts of two Medicinal Plants: Solanum laciniatum Ait. and Solanum khasianum C. B. Clarke

Summary Solanum laciniatum and S. khasianum plants were propagated by subculturing leafy single node cuttings. The sources of protoplasts were lamina and stems for S. laciniatum, and only lamina for S. khasianum. They were taken from 4-week old plants. Lamina gave the best yield of protoplasts. The first division of protoplasts occurred after 3–5 days of culture in KM8p + 0.2 mg · l −1 2,4-D + 0.5 mg · l −1 zeatin + 1 mg · l −1 NAA. At day 7, the division frequency was estimated at 39.0 % and 26.7 % for lamina and stem protoplasts of S. laciniatum, respectively, and 23.0 % for S. khasianum. After 20–30 days, a dilution of the cultures to 5 times with zeatin or kinetin-containing medium resulted in the formation of meristematic nodules in S. laciniatum with a frequency of 95 %, but only in an increase of callus growth in S. khasianum without any formation of organized structures, especially at a low level of cytokinin (0.1 mg · l −1). The nodules were composed of very small meristematic cells. They were partly covered with an epidermis and still connected with the callus. No bipolar structures were observed. The nodules produced only shoots on MS + 0.5 mg · l −1 BAP with a frequency of nearly 90 % and irrespective of protoplast sources. On MS + 2 mg · l −1 zeatin + 0.1 mg · l −1 IAA, more than 80 % of S khasianum calli regenerated buds which further developed into plantlets when transferred onto MS + 0.1 mg · l −1 BAR

Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.)

An embryogenic suspension culture of orchardgrass (Dactylis glomerata L.) consisting of small, embryogenic cell clusters was obtained from callus formed on basal sections of young leaves through a process of selective enrichment. These suspensions were used as a source of protoplasts. The isolated protoplasts divided at a frequency of 0.5-10% when plated in an agarose solidified culture medium. Conditioned medium, in which embryogenic Dactylis suspension cultures had been grown, was found to increase the rate of cell colony formation. Protoplast-derived colonies grew rapidly in a bead-type culture system of floating agarose slabs in liquid medium. New suspension cultures formed as the colonies grew out of the agarose. These cultures were embryogenic and formed green plantlets when plated on a solid medium lacking auxin. The plantlets were established in soil and grown to mature plants.

Relation between protoplast division, cell-cycle stage and nuclear chromatin structure

Using different sources of protoplasts and two complementary techniques, flow cytometry and image analysis, to study the cell-cycle phases, we sought to define the particular protoplast state associated with the disposition to divide. Both inPetunia and inNicotiana plumbaginifolia, tissues with a higher G2 frequency (from different aged plants) yielded protoplasts capable of increased cell division. InSorghum, the age of the plant does not modify the proportion of G2 nuclei in leaf protoplasts, and we used root protoplasts to increase G2 frequencies. InHelianthus annuus, leaf protoplasts did not divide; however, hypocotyl protoplast preparations with relatively high 4C DNA frequencies do divide. Moreover, image analysis of chromatin structure indicated that leaf nuclei were in the G0 phase, unlike those from hypocotyls which were in G1. A high frequency of protoplasts with G2 nuclei appears to be correlated with the ability of a given preparation to undergo division; conversely, the differentiated G0 state is not conducive to division.

Regeneration of somatic hybrid plants formed between Lycopersicon esculentum and L. pennellii

Somatic hybrid plants have been regenerated following polyethylene glycol mediated fusion of leaf mesophyll protoplasts from tomato and protoplasts from Lycopersicon pennellii callus. Three different cultivars of tomato were used as sources of protoplasts: Early Girl, Manapal, and UC82B. Fusions were performed between protoplasts of these tomato cultivars and protoplasts of L. pennellii, and between protoplasts of the cultivars and protoplasts of L. pennellii that had been exposed to 3 or 6 krads of gamma radiation. Somatic hybrid plants were identified on the basis of heterozygous isozyme banding patterns, and leaf and flower morphology. Somatic hybrid plants were regenerated following fusion of tomato protoplasts with either untreated or irradiated L. pennellii protoplasts. All were heterozygous for isozyme loci on five different chromosomes. Regenerated somatic hybrids showed inheritance of either or both parental chloroplast genomes, but predominantly the L. pennellii mitochondrial genome. The regenerated somatic hybrid plants exhibited reduced fertility, less than 20% viable pollen. A total of 34 somatic hybrid calli were identified. Of these, 21 regenerated shoots, and 7 produced seed following manual pollinations.

Variations in Morphology and Nicotine Alkaloid Accumulation in Protoplast-derived Hairy Root Cultures of Nicotiana rustica

A suspension culture was produced by the phytohormone treatment of hairy roots resulting from infection of Nicotiana rustica with Agrobacterium rhizogenes . This was used as a source of protoplasts for the production of single-cell-derived clones. After conditions for protoplast release were optimised, it was possible to induce high rates of cell division from these protoplasts and regenerate hairy roots from the protoplast-derived calli following return to hormone-free medium. Regenerated roots were assessed for nicotine alkaloid accumulation and their transformed nature was confirmed by DNA analysis. Variation between clones was observed in alkaloid synthesis, morphology and T-DNA structure, with some of the clones showing improved alkaloid synthesis characteristics relative to the parent line.

Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.)

This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1(-1) zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1(-1). Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.

Use of a transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation

Plasmid DNA was transfected into tobacco mesophyll protoplasts by electroporation. Transfection efficiency was estimated, using a transient expression assay based on the measurement of chloramphenicol transacetylase activity or by scoring colonies expressing resistance to paromomycin, an aminoglycoside related to kanamycin. Under conditions of cell survival superior to 50% after electroporation, transient expression signals and transformation efficiencies were found to be proportional. Factors affecting the efficiency of transformation were studied. A clear-cut optimum voltage (250–300 V/cm) was detected. Among various salts tested, potassium chloride was the best electrolyte. No improvement of electroporation efficiency was obtained by a heat - shock (45°C/5 min) treatment prior to electroporation or by the presence of polyethylene glycol in the electroporation medium. The physiological state of plants used as the protoplast source significantly affected the transfection ability of the resulting protoplasts. These results are discussed and compared to previously published procedures.

High frequency plant regeneration from rice protoplasts by novel nurse culture methods

Novel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%–50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes.