High Yields Of Protoplasts Research Articles

Protoplast production and regeneration from mycorrhizal fungi and their use for isolation of mutants

Methods were developed for the reproducible production of high yields of stable protoplasts from monokaryotic and dikaryotic mycelia of Hebeloma cylindrosporum. This is a prerequisite for mutation, cell fusion, and transformation of this mycorrhizal fungus. Protoplasts from Hebeloma cylindrosporum, Hebeloma edurum, Hebeloma sinapizans, and Suillus bellini were released by using cellulase Onozuka R 10 and driselase as lytic enzymes. Production was strongly influenced by the nature and molarity of the osmotic stabilizer and by the mycelial age. In spite of the high percentage of anucleate protoplasts (42%) produced, the regeneration rate averaged 15%. Fungicide-resistant mutants from monokaryotic mycelia of H. cylindrosporum were isolated following ultraviolet irradiation of protoplasts. This indicates that protoplasts will be useful to select physiological mutants from nonsporulating mycelia and to create new strains by mating complementary mycelia or by interspecific cell fusion.

Optimal conditions for the isolation and culture of protoplasts from a cell suspension culture of an isoleucine–valine-requiring auxotroph of Datura innoxia

Conditions have been standardized for obtaining high yields of viable protoplasts from cell suspension cultures of an isoleucine–valine-requiring auxotroph (IV-1) of Datura innoxia P. Mill. Isolation of protoplasts critically required the use of a salt solution, containing sodium chloride and potassium chloride (0.125 M each), as osmotic stabilizers. The yield, viability, and divisional activity of the protoplasts isolated with mannitol or sucrose were poor. Protoplast-releasing enzymes used to isolate IV-1 protoplasts could be used twice without any loss in the yield or viability of the protoplasts. Cultured protoplasts developed cell walls and underwent sustained divisions in a modified Murashige and Skoog medium enriched with organic acids. Pretreatment of isolated protoplasts with glycine (0.1 M) and calcium chloride (0.05 M) prior to culture increased the frequency of cell colony formation. Protoplast-derived cells developed calli on transfer to agar-solidified medium.

Aseptic isolation of leaf protoplasts from Citrus, Poncirus, Citrus × Poncirus hybrids and Severinia for use in somatic hybridization experiments

An efficient protocol has been developed for the aseptic isolation of leaf protoplasts from Citrus and citrus relatives. Consistently high yields (2–6 × 10 7 protoplasts/g fresh weight tissue) of leaf protoplasts were isolated from new leaves taken from vigorously growing plants maintained in a growth chamber. Critical parameters included leaf decontamination and reconditioning, the composition of the enzyme cocktail, vacuum infiltration, incubation and clean-up of the resulting protoplasts. The use of leaf protoplasts in fusions with protoplasts isolated from embryogenic callus allowed for the visual identification of heterokaryons and suggests the possible utilization of additional physiological parameters in somatic hybridization selection schemes. Moreover, the number of genotypes which can be utilized in citrus fusion experiments has been greatly increased.

Isolation, culture and plant regeneration from mesophyll protoplasts of Digitalis obscura

High yields of protoplasts were obtained from mesophyll tissue of Digitalis obscura L. Osmotic potential of the isolation medium and Ca2+ were important in obtaining a high viability of the preparations. In different culture techniques employed, liquid‐over‐agar‐solidified medium was superior to liquid medium alone. Agar plating technique was ineffective. On Murashige and Skoog modified medium with casein hydrolysate and several indoleacetic acid and benzyladenine combinations, isolated protoplasts underwent sustained mitotic division and produced calli. The calli formed shoots when transferred to regeneration media. Regenerated shoots could be easily rooted and developed into whole plants on half‐strength Murashige and Skoog hormone‐free medium.

Techniques for enhanced release of leaf protoplasts in Populus

Microscopic examination of Populus leaf tissue following enzyme treatment revealed two factors contributing to low protoplast yields: (1) poor penetration of the enzymes into the tissue, and (2) entrapment of protoplasts in leaf debris during protoplast purification procedures. A simple combination of rapid grinding of the tissue in an Omni-mixer prior to enzyme treatment and forceful washing of leaf-debris after digestion provided high exposure of the cells, uniform digestion, and high yields of protoplasts of two Populus clones. Protoplasts exhibited cell wall regeneration and long-term viability in culture. The relative yield advantages of the techniques varied with the inherent digestibility of each clone but could produce up to 600 percent greater protoplast yields in a woody plant species in which protoplast isolation was previously limited. The techniques were also applicable to an herbaceous species, Solanum etuberosum.

Isolation and Culture of Mesophyll Protoplasts of Garden, Fodder and Sugar Beets Using a Nurse Culture System: Callus Formation and Organogenesis

Summary High yields of mesophyll protoplasts were obtained from shoot cultures grown in vitro on MS medium containing 0.25 mg · 1 −1 BAP. Mesophyll protoplasts regenerated cell walls and underwent division to produce cell colonies when cultured in either the presence of nurse cells, or nurse protoplasts from suspension cultured cells. Morphological differences such as colour and the nature of the cell colonies were used to distinguish mesophyll protoplast derived colonies from suspension cell colonies. Colonies derived from mesophyll protoplasts showed root organogenesis.

Callus production from leaf protoplasts of various cultivars of bean (Phaseolus vulgaris L.)

High yields of viable protoplasts were obtained by enzymatic treatment from cotyledonary leaves of various greenhouse grown Phaseolus vulgaris L. cultivars. The protoplasts divided and formed cell clusters in a liquid medium. Early transfer before 10 days in the same medium was necessary for the development of cell colonies. When transferred to solid medium, the colonies gave rise to proliferating green calli. Deep green patches developed on these calli but failed to form shoots.

Somatic embryogenesis and plant regeneration from cell suspension derived protoplasts of Solanum melongena (eggplant)

Cell suspension cultures of eggplant (Solanum melongena L.) were initiated from embryogenic callus cultures and maintained in medium supplemented with either 1-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Higher yields of protoplasts were obtained from cells grown in 2,4-D than in NAA. The efficiency of cell division was also greater in protoplast cultures derived from cells grown in the presence of 2,4-D. Protoplast-derived cells formed somatic embryos in modified Kao or Nagata and Takebe media which were supplemented with 1 mg/L 2,4-D. Early stages of embryogenesis were observed in liquid medium; however, these embryos and associated cell colonies were transferred onto agar-solidified medium for subsequent regeneration. Mature plants were established in soil in the greenhouse.

Regeneration of plants from mesophyll protoplasts of ground-cherry ( Physalis minimal L.)

High yields of protoplasts were obtained by enzymatic digestion of leaf tissue derived from exenic shoot cultures of Physalis minima L. Isolated protoplasts underwent sustained mitotic divisions and produced calli. Shoot and root morphogenesis were achieved from these calli. The regenerated plants were established in soil.

Influence of Lowered Temperature Storage on Growth and Protoplast Isolation of Soybean callus

Summary Protoplasts were isolated from different soybean [ Glycine max. (L.) Merr.] calli after only two days in liquid medium. Freshly started callus cultures were used for protoplast isolation after only 2–4 months culture. Storage of soybean calli at 10°C ± 1°C prevented callus growth, but after transfer to 26°C normal growth was restored after a short lag period. High yields of protoplasts were obtained when the calli were in the first part of their exponential growth phase.

Callus regeneration from Alnus incana protoplasts isolated from cell suspensions

Nagata and Takebe's (NT) medium, supllementedte with 2.5 μm 2,4-dichlorphenoxyacetic acid (2,4-D), induced development of friable calluses from leaves of axenic shoot cultures of Alnus incana. Fast-growing cell suspensions were established in the same medium without agar. Suspensions gave high yields of viable protoplasts after an overnight incubation in an enzyme mixture consisting of 1% (w/v) Onozuka R-10, 0.5% (w/v) Rhozyme HP-150, 0.03% (w/v) Macerase, CPW salts, and 13% (w/v) mannitol (pH 5.8). Protoplasts cultured on K8p medium underwent cell wall regeneration within 24 h. The optimum protoplast-derived colony formation and growth was obtained on the NT medium supplemented, as was the K8p medium, with glucose as the osmoticum, growth regulators, coconut milk and casein hydrolysate. Compared with other culture techniques, the agarose bead technique of Shillito et al. (Plant Cell Reports, 2 (1983) 244) improved cell division and colony formation frequency. Protoplast-derived macrocalluses grew under the same conditions as those used for leaf calluses.

Studies of Chlorella protoplasts. II. Isolation and fusion of protoplasts from Chlorella saccharophila (Krüger) Nadson strain 211-1a

Reproducibly high yields of protoplasts were obtained from the unicellular asexual green alga Chlorella saccharophila (Krüger) Nadson 211-1a by treating the cells with polysaccharide degrading enzymes. A maximum of 32% of the protoplasts were able to regenerate cell walls and formed colonies when plated on a regeneration medium. Chlorophyll deficient mutants were isolated after X-ray irradiation of the green wild-type cells. These mutants differed in growth requirements and light sensitivity from each other and from the wild-type cells. Intraspecific protoplast fusion of a yellow with a white pigment mutant strain was accomplished by polyethylene glycol (PEG) and Ca 2+ and green hybrid cells were obtained using selective conditions. Ultrastructural and morphological investigations carried out so far demonstrate differences between hybrid cells and the parental mutant cells as well as between hybrid cells and the green wild-type cells.

Production of protoplasts from the fungi Curvularia inaequalis and Trichoderma reesei

Protoplast formation in Curvularia inaequalis was achieved using non-commercial and commercial snail gut enzymes or Trichoderma harzianum enzymes. The cells were grown for enzyme treatment on cellophane sheets or in liquid cultures for varying periods of time. The production of T. harzianum enzymes is discussed. The highest protoplast yields were 2.6x107 protoplasts/ml enzyme solution. Protoplasts were shown to have zero to four nuclei. Protoplast regeneration was succesfully carried out in semisolid agar.

Callus formation from protoplasts of culturedSpinacia oleracea cells

Cell suspensions were initiated from plumule derived calli ofSpinacia oleracea. Some of these cell lines could be maintained in culture for at least three years without a reduced growth rate. A high yield of protoplasts was obtained from the cell suspensions. When protoplasts were cultured in Murashige and Skoog medium with naphthaleneacetic acid and 6-benzyladenine, cell wall formation was observed after three days. The cultured protoplasts produced numerous cell-clusters within two weeks. However only protoplasts isolated from suspensions which were in a rapidly dividing phase were able to divide with a high frequency and give rise to callus colonies.

Studies on a drought resistant legume: The moth bean, Vigna aconitifolia (Jacq) marechal. I. Protoplast culture and organogenesis

High yields of viable protoplasts were obtained from callus cultures derived from shoot apices of Vigna aconitifolia (JACQ) Marechal. The protoplasts divided and formed cell clusters on modified MS medium. The protoplast-derived callus formed multiple shoot buds on MS and B5 basal media without supplements, on MS medium containing supplements and on B5 medium containing charcoal (0.25%). Shoot formation occurred.

The formation of protoplasts from Mucor species

Methods were developed for the reproducible production of high yields of stable protoplasts from mesophilic and thermophilic strains of Mucor. This is a pre-requisite for the genetic analysis and manipulation of Mucor by cell fusion or transformation and is of special importance in these species since conventional genetic analysis by recombination studies is not feasible. The cell wall lytic enzyme used was the concentrated culture fluid of Streptomyces sp no. 6 grown on chitosan and chitin. Using germlings of M. circinelloides f. lusitanicus the optimum conditions for protoplast formation were determined as: 0.5–1.0 mg·ml−1 streptozyme (1.2 units chitosanase·ml−1) plus 2.0 mg·ml−1 novozym 234 or 1%(v/v) helicase in 0.5m-sorbitol, 0.01m-sodium phosphate buffer, pH 6.5 for 3–4 hours at 23°C. Treatment of 107·ml−1 germlings gave yields of up to 3×107·ml−1 protoplasts with regeneration and fusion frequencies of up to 40% and 1.7%, respectively. With the thermophilic strains growth at low temperature (25°C) was critical for the subsequent formation of stable protoplasts. Synchronous growth and germination was obtained at this temperature by a short pre-incubation (1–3 hours) of the sporangiospores at 40°C.

Plant regeneration from protoplasts of Solanum Brevidens.

Protoplasts were isolated from shoot cultures of Solanum brevidens Phil. The addition of a low concentration of 6-benzylaminopurine (BAP) to the shoot culture medium was suitable for the isolation of high yields (6 × 10 6/g) of viable protoplasts. Using a protocol modified from methods established for S. tubersum, cell division leading to callus formation was observed. Shoots initiated on protoplast-derived calluses were rooted and transferred to potting compost. Established plants flowered and set berries.

Mesophyll protoplasts of fenugreek (Trigonella foenumgraecum): Isolation, culture and shoot regeneration

High yields of mesophyll protoplasts were obtained after treatment of leaves of Trigonella foenumgraecum, a forage legume, with purified cellulase. Under appropriate conditions of culture up to 70% plating efficiency could be obtained. The protoplast-derived colonies developed into rapidly growing green calli and produced leafy shoots on a medium containing 0.1 mg/l each of benzylaminopurine (BAP) and zeatin. Addition of glutamine and asparagine to the medium was found essential for rapid cell division, callus growth and differentiation.

Isolation and growth of protoplasts from cell suspensions of Pinus contorta dougl. ex loud

Cell suspensions were initiated from embryo derived calli of Pinus contorta. Some of these cell lines could be maintained in culture for at least one year without reduced growth.A high yield of protoplasts was obtained from the cell suspensions. The protoplasts started to divide after two days and cell clusters could be observed after about two weeks. The growth phase of the cell suspensions was very important for the division of protoplasts. Only protoplasts isolated from suspensions in an actively dividing phase were able to divide with a high frequency and to give rise to cell clusters.

Mass propagation and genetic improvement of forest trees for biomass production by tissue culture

Wood derived from forest trees can serve as a major alternative source of energy and fuel because of the current energy shortage and increase in price of oil and natural gas. Normally, trees take several years to grow and produce seeds. But, in recent years, ‘test tube trees’ have been produced in large numbers (as many as 3000 plants per year) from one seedling using tissue culture by treating a few cells of a tree with specific chemical substances. Tissue culture is a promising technique for mass production of large numbers of superior trees, derived through genetic improvement, and may prove widely applicable to trees which show promise as energy sources. Three selected tree genera, viz. Sapium sebiferum (Chinese Tallow), Leucaena leucocephala (giant ipil-ipil, a tropical legume) and Copaifera multijuga (Copaiba tree from Brazil) have been studied because of their potential usefulness for biomass production. Regeneration of vegetatively produced plantlets has been achieved from embryos and callus cells grown in specific culture medium for two of the above genera. High yields of protoplasts have been obtained isolated from cells of different plant parts and grown as calli. Conditions which will enable callus derived from protoplasts to undergo in vitro regeneration, plantlet formation and eventually growth into plants are being investigated.