Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.     

Plant regeneration from mesophyll protoplasts of Solanum commersonii dun

Somatic fusion of Solanum commersonii, a frost tolerant wild potato species not crossable with Solanum tuberosum, relies on the possibility to isolate and culture protoplasts. This study was conducted to determine whether protoplasts could be isolated and plants regenerated in three S. commersonii accessions. Shoot cultures for protoplast isolation were maintained on Murashige and Skoog medium. Mesophyll protoplasts were isolated and cultured using a protocol originally described for S. tuberosum with some modifications. Differences were evident among the three accessions for protoplast yield, plating efficiency and regeneration frequency. Protoplast yield ranged from 3.0 to 8.5 × 10⁶ protoplasts per g of fresh tissue. At 1–2 × 10⁴ protoplasts ml⁻¹, which was the optimal plating density, 10–20% of plated protoplasts gave multicellular colonies. Regeneration of shoots was observed in two accessions only, the maximum regeneration frequency being 66%. In one of these accessions the reduction of sucrose concentration in regeneration media improved the regeneration frequency from 14 to 35%. About three hundred plants were rooted in vitro and successfully transferred to soil.     

Factors promoting sustained divisions of mesophyll protoplasts isolated from dihaploid clones of potato (Solanum tuberosum L.) and a cytological analysis of regenerated plants

A culture protocol has been developed for mesophyll protoplasts isolated from various dihaploid clones of potato. A special effort was made to promote the growth of initially dividing cells to form cell colonies and calli. An increase in plating efficiency in 3 different dihaploid clones and one doubled dihaploid clone was obtained after serial dilution of cultures with a suitable amount and type of medium at different stages of cell colony development. Plating on a refined semi-solid medium after 14 days of culture further improved both the yield and the quality of calli obtained. The refined plating medium also enhanced shoot regeneration ability from 67 to 90% in one of the dihaploid clones (67:9). The refined culture protocol could also be used without causing a decrease in plating efficiency at a low population density adjusted after 3 days of culture. The ploidy level of plants regenerated from dihaploid protoplasts were determined by chromosome counting and DNA analysis by flow cytometry. Most of the plants were aneuploid or tetraploid although, some dihaploid plants were obtained after protoplast culture of 2 dihaploid clones derived from the same cultivar (cv. Stina).Abbreviations BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - GA₃ gibberellic acid - NAA naphthaleneacetic acid     

Improved protoplast culture and stability of cytoplasmic traits in plants regenerated from leaf protoplasts of cauliflower (Brassica oteracea ssp.botrytis)

Nearly 1000 plants have been regenerated from leaf protoplasts of two cauliflower (Brassica oleracea ssp.botrytis) alloplasmic inbred lines. One line (7642A) carried the Ogura (R1) cms cytoplasm derived from radish; the other line (7642B) carried a normalBrassica cytoplasm and was the fertile maintainer for the cms line. The majority of regenerated plants displayed normal vegetative morphology; they formed normal cauliflower heads and retained the floral characteristics of seed-grown plants from which they were derived. We found no change in either male sterility or in the low temperature-induced chlorosis associated with the 7642A line. Mitochondrial DNA analysis by hybridization with five cloned mtDNA probes revealed no apparent alteration in 75 regenerated plants of both lines. These results indicate that cytoplasmic traits inBrassica oleracea are stable after one cycle of in vitro culture and regeneration.     

Analysis of genetic stability of plants regenerated from suspension cultures and protoplasts of meadow fescue (Festuca pratensis Huds.)

A cytological and molecular analysis was performed to assess the genetic uniformity and true-to-type character of plants regenerated from 20 week-old embryogenic suspension cultures of meadow fescue (Festuca pratensis Huds.), and compared to protoplastderived plants obtained from the same cell suspension. Cytological variation was not observed in a representative sample of plants regenerated directly from the embryogenic suspensions and from protoplasts isolated therefrom. Similarly, no restriction fragment length polymorphisms (RFLPs) were detected in the mitochondrial, plastid and nuclear genomes in the plants analyzed. Randomly amplified polymorphic DNA markers (RAPDs) have been used to characterise molecularly a set of mature meadow fescue plants regenerated from these in vitro cultures. RAPD markers using 18 different short oligonucleotide primers of arbitrary nucleotide sequence in combination with polymerase chain reaction (PCR) allowed the detection of pre-existing polymorphisms in the donor genotypes, but failed to reveal newly generated variation in the protoplast-derived plants compared to their equivalent suspensionculture regenerated materials.The genetic stability of meadow fescue plants regenerated from suspension cultures and protoplasts isolated therefrom and its implications on gene transfer technology for this species are discussed.Abbreviations PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism.     

Variation in plants regenerated from vacuolate and evacuolate protoplasts

Mesophyll protoplasts of Nicotiana tabacum were evacuolated by centrifugation through a percoll gradient. Their viability was comparable to untreated protoplasts, but the time for reformation of the cell wall as well as to the onset of the first division was delayed. Plants were regenerated from both classes of protoplast; those from evacuolate protoplasts showed higher rates of mutation than those from vacuolate protoplasts. The possible basis of this difference is discussed.     

Polyploidization in leaf callus tissue and in regenerated plants of dihaploid potato

Cytological studies on leaf callus cells and regenerated potato plants suggest that it may be possible to utilize somatic chromosome doubling to obtain tetraploids from outstanding dihaploid breeding clones. The ploidy levels found in callus-derived plants were diploid, tetraploid, and octaploid, but the proportion of these was dependent on the donor genotype. L₁ and L₃ germ layers were studied in more than 300 plants; periclinal ploidy chimerism, an undesirable feature of colchicine doubling, was not found. Leaf callus was more efficiently induced using NAA than 2, 4-D as an auxin source in the Murashige and Skoog medium. A high proportion of dividing cells in young calli were polyploid. The frequency of doubled and octaploid plants regenerated was significantly dependent on donor genotype. The extent of polyploidization was marginally higher after callus growth on a medium containing 2, 4-D than in a medium containing NAA. In some genotypes the chromosome numbers of regenerated plants were variable, being less than tetraploid (mixohypotetraploid). After tuber propagation, the original ploidy level was maintained although mixohypotetraploidy persisted. In a few somatically doubled clones, male fertility was tested and found to be satisfactory with respect to seed-setting.     

Variations in the morphology of rice plants regenerated from protoplasts using different culture procedures

Rice protoplasts were cultured using 4 different culture procedures such as agarose embedding (AE) without feeder cells and the use of filter membranes (MEM), one layer of nylon mesh (MS1), or a double layer of nylon mesh (MS2) with the inclusion of Lolium multiflorum as feeder cells. The protoplast plating efficiency was highest on the MEM, followed by MS2, MS1 and AE. However, plant regeneration frequencies were highest for MS1, followed by MS2, MEM and AE. The protoclonal plants differed in the morphology of leaves, flowers, spikelets, and panicles in comparison to seed-derived plants. They varied in almost every phenotypic characters evaluated. In many cases, the variation was significantly different in characteristics such as plant height, flag leaf length and width and ratio, and in panicle characteristics such as panicle length, number of primary branches, and number of spikelets per panicle. The number of seeds per panicle was greatly reduced in protoclonal plants when compared with seed-derived control plants. The seeds showed also significant differences in grain length and width in comparison to the control plants. Among the 4 groups of protoclonal plants derived from the 4 different culturing procedures themselves, there were also variations in almost all the phenotypic characteristics assessed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plants regenerated from mesophyll protoplasts of white mulberry

INTRODUCTIONProtoplastcultureis0neofthen1ostrapidlydevel0pingareasinp1anttissueculture,becauseofitsimportancei11plantgeneticmanipulation.However,sofar,thereareonlyafewforesttreespeciesinwhichplantregenerationfr0mprotoplastshaJsbeensuccessful,namelyLiriode…     

Development of moss plants from isolated and regenerated protoplasts

Proroplasts were enzymatically isolated from protonema of Funaria hygrometrica L. SIBTH., cultured from the spores on Knop's solid medium. The protoplasts were incubated in hanging drops in liquid Knop's medium with 0.3 M mannitol and 0.5% glucose. Isolation of protoplasts was obtained successfully, through the action of Macerozyme®, cellulase and Rohament P® mixed with mannitol. Cultured protoplasts began to regenerate walls within 24 h, followed by the enlargement and divisions within 48 h. These protoplasts started producing active and long strands of protonema within 5 days, while well-differentiated leafy gametophytes were developed just in 20–23 days.     

Regeneration of whole plants from Gracilaria asiatica Chang et Xia protoplasts (Gracilariaceae,Rhodophyta)

A large number of viable protoplasts were produced by enzymatic digestion of Gracilaria asiatica vegetative tissue. The protoplasts underwent initial division after 5–7 d in culture and developed into callus-like cell-masses. Many filaments grew from the periphery of these cell-masses and disappeared after about one month in culture. Simultaneously, the central part of the callus-like cell-masses thickened and its color deepened. The first buds appeared from the center of the cell-masses and developed into whole plants after three months in aerated culture. Many new buds formed around the first plant and more than 20 plants grew per callus-like cell-masses in less than four months. Filaments taken from callus-like cell-masses developed into young plants after about 20 d of culture.     

Highly efficlent plant regeneration from mesophyll protoplasts of Indian field cultivars of tomato (Lycopersicon esculentum)

Three Indian cultivars ofL. esculentum were assessed for shoot regeneration from protoplast-derived calli. Consistent yields of viable protoplasts (>9.0×10⁶ g f.wt.^-1) were obtained from leaflets of 14 days old cultured shoots. Protoplast viability (88–94%) and planting efficiency (55–70%) were recorded for the three cultivars. Up to 71% of the protoplast-derived tissues regenerated shoots.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - g f.wt. gram fresh weight - IAA indoleacetic acid - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid - Zea zeatin     

Isolation and regeneration of protoplasts from Porphyra dentata and Porphyra crispata

Large numbers of protoplasts (10⁶ to 3·2 × 10⁷ per gram fresh weight) were routinely isolated from two Taiwanese species of Porphyra: P. dentata and P. crispata. Intermediate and final forms of regeneration were observed, including swollen cells, monospore-like cells, filaments, callus-like structures and bladelets. Regeneration of leafy plants was achieved from protoplasts of P. crispata, whereas only conchocelis-like filaments were regenerated from protoplasts of P. dentata. In this latter species, conchosporangia and monosporangia appeared on the filaments upon cultivation at 25 °C. The variability in regenerative patterns is discussed.     

Fertile plants regenerated from suspension culture-derived protoplasts of an indica type rice (Oryza sativa L.)

Calluses were induced from immature embryos of an indica type rice and finely dispersed cell suspension cultures were initiated from the callus using modified AA medium (S₁ medium). The suspension cultures were maintained alternatively (1–2 passages in each medium) in S₁ medium and S₂ medium, the latter containing KNO₃, NH₄NO₃, proline and glutamine as nitrogen source. Protoplasts of high quality were isolated form suspension cells cultured in S₂ medium supplemented with ABA. Embedding the protoplasts in agarose blocks containing NH₄NO₃-free modified KM8P(PM₁) medium and immersing the blocks in NH₄NO₃-containing modified KM8P(PM₃) medium were most effective for obtaining protoplast division and callus formation. The protoplast-derived calluses were precultured in potato extract-aand/or ABA-containing N₆(D₁, D₂ or D₃) media and many embryo-like structures were formed. These structures developed into plantlets after being transferred to N₆ differentiation (D₄) medium. The regenerated plantlets grew into mature plants and beard seeds normally.Abbreviations AA medium amino acids based medium - ABA abscisic acid - BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DF division frequency - IAA indoleacetic acid - KIN kinetin - NAA naphthaleneacetic acid - PE planting efficiency     

Chromosome variation in protoplast-derived potato plants

Summary Chromosomes have been studied in protoplast-derived potato plants of the tetraploid cultivars Maris Bard and Fortyfold. A high degree of aneuploidy was found amongst the regenerants of both cultivars but the nature of the chromosome variation differed. The Maris Bard regenerants were characterised by high chromosome numbers, a wide range of aneuploidy (46–92) and a low percentage of plants with the normal chromosome number (2n = 48), whereas a much higher proportion of the Fortyfold regenerants had 48 chromosomes and the variants were within a more limited aneuploid range. In both cultivars chromosome variation was found between calluses, within calluses and even within shoot cultures. The origin of the chromosome variation and the differences found between the two cultivars are discussed.We regret to report the death of Emrys Thomas since the initiation of this work     

Tissue culture response in various wild and cultivated Solanum germplasm accessions for exploitation in potato breeding

The response to different in vitro methods for use in potato breeding has been evaluated in 11 genotypes of 5 Solanum species, S. etuberosum, S. lycopersicoides, S. maglia, S. rickii, and S. tuberosum. Callus induction and growth, and shoot regeneration were strongly influenced by the genotype, explant source, and medium utilized. Furthermore, considerable differences among the 11 genotypes were found both in plating efficiency and shoot regeneration from protoplast culture. Some interesting correlations were found between different tissue culture responses, suggesting linkage and/or pleiotropic effect of genes. The potential application to potato breeding of the in vitro techniques analyzed is discussed.Abbreviations BA 6-benzylaminopurine - GA₃ gibberellic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog (1962) - 2,4-d dichlorophenoxyacetic acid     

Plant regeneration from leaf protoplasts of evening primrose (Oenothera hookeri)

A protocol is presented for regenerating plants from leaf protoplasts of Oenothera. The method uses (1) embedding of isolated protoplasts at high cell densities in thin alginate layers, (2) initial culture in B5 medium containing 3 mg l^–1 α-naphthaleneacetic acid (NAA) and 1 mg l^-1 6-benzylaminopurine (BAP), (3) reduction of the osmotic pressure of the culture medium at early stages of culture and (4) plating of microcolonies recovered from the alginate onto solid B5 medium with 3 mg l^–1 NAA and 1 mg l^–1 BAP. The shortest time required from protoplast isolation to the appearance of shoot initials was 7 weeks. The efficiency of the procedure for protoplast to cell line formation is high (about 80%). Received: 17 February 1997 / Revision received: 6 November 1997 / Accepted: 15 November 1997     

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.)

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x10⁴ protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.     

Plant regeneration via somatic embryogenesis, and transient gene expression in sweet potato protoplasts

A method for regenerating plants from petiole protoplasts of the in vitro-raised sweet potato cultivar Jewel is described. Protoplast yields of 3.0–5.0×10⁶ were obtained following 4–6 h digestion of 1- to 2-cm petioles (1 g fresh weight) with 1% Cellulase-R10, 2% Macerozyme-R10, and 0.3% Pectolyase Y-23 in a washing solution with 9% mannitol. A plating density of 10⁵ protoplasts/ml was optimal for subsequent division. An initial division frequency of 12–15% was obtained in liquid or agarose-solidified KP8 culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) (0.9 μm), and zeatin (2.3 μm). Colonies consisting of 100–200 cells were formed after 4 weeks in the dark at 24±2°C. The frequency of colony formation was improved by the gradual addition of fresh liquid KP8 medium of lower osmoticum. Protocalli (1–2 mm in diameter) were formed after an additional 4–6 weeks under continuous illumination and regular dilution with fresh culture medium. Morphogenic callus formed globular and heart-shaped embryos that developed into cotyledon stage embryos, following transfer of calli onto medium containing 2,4-D (11.3 μm) and benzylaminopurine (2.2 μm). Subsequently, embryo conversion to plantlets was obtained on basal medium with 2% sucrose and 3.5 μm gibberellic acid. Regenerated plantlets were successfully transplanted in soil. Mature plants appeared phenotypically normal. The same petiole protoplast populations showed transient expression of the gusA gene introduced using electroporation. Received: 10 October 1997 / Revision received: 10 February 1998 / Accepted: 2 March 1998