Modified Modified Murashige And Skoog Medium Research Articles

Effects of culture methods and plant growth regulators on in vitro propagation of Brassavola nodosa (L.) Lindl. hybrid

Brassavola nodosa (L.) Lindl. is an epiphytic orchid with great potential for the ornamental plant industry. The lack of information on propagation and production techniques limits the development of large-scale commercial production. Furthermore, this species is experiencing a reduction in population due to habitat destruction, the impact of climate change, and over-collection from native habitats. This study aimed at developing an efficient protocol for micropropagation of B. nodosa, which could be valuable towards the large-scale commercial production as well as for conservation of this species. Six different concentrations of plant growth regulators (BA or IBA, with or without adenine sulfate) were evaluated in modified Murashige and Skoog (MS) medium for shoot multiplication. In addition, two concentrations of either NAA or IBA were evaluated for rooting. Explants were cultured under three different culture media conditions: semi-solid medium, liquid medium (partial immersion), and liquid medium (complete immersion). Results indicate that B. nodosa could be successfully micropropagated in liquid culture with partial immersion. The modified MS medium supplemented with 2.0 mg L−1 BA and 30.0 mg L−1 adenine sulfate resulted in higher multiplication rates. Rooting was obtained using either 0.5 mg L−1 NAA or 1.0 mg L−1 IBA with no significant differences between both rooting treatments. Plantlets achieved 100% ex vitro survival after 30-d acclimatization.

Rescue and in vitro culture of herbaceous peony immature embryos by organogenesis

Herbaceous peony (Paeonia lactiflora Pall.) is a landscaping plant that has considerable development prospects worldwide. However, its embryo dysplasia severely limits the breeding of high-quality P. lactiflora cultivars by the cross-breeding method. In this study, the immature natural hybrid embryos of P. lactiflora ‘Zi Fengyu’ with double flower were used to study its rescue and in vitro culture by organogenesis. The results showed that the best medium for adventitious bud induction was modified Murashige and Skoog (MS) medium supplemented with 1.0mg/L thidiazuron (TDZ), 600mg/L acidic hydrolysis casein (ACH), 10% coconut water and 100g/L sucrose. The best medium for adventitious bud multiplication was modified MS medium supplemented with 4.0mg/L TDZ, 15% coconut water, 60g/L sucrose and 0.1% activated charcoal (AC). Modified MS medium containing 0.5mg/L gibberellic acid (GA3), 100g/L sucrose and 0.1% AC was the more suitable medium for adventitious bud growth. For root regeneration, indole-3-acetic acid (IAA) was a more effective plant growth regulator (PGR), and 1/2 MS medium supplemented with 0.5mg/L IAA, 30g/L sucrose and 0.1% AC was the more suitable medium, resulting in a rooting rate of up to 92.12%, and the survival rate was 70.8%. Additionally, histological observation confirmed that the rescue and in vitro culture of P. lactiflora immature embryos by organogenesis was successful. These results provide useful technical support for breeding high-quality P. lactiflora cultivars by the cross-breeding method.

A selection strategy in plant transformation based on antisense oligodeoxynucleotide inhibition

Antisense oligodeoxynucleotide (asODN) inhibition was developed in the 1970s, and since then has been widely used in animal research. However, in plant biology, the method has had limited application because plant cell walls significantly block efficient uptake of asODN to plant cells. Recently, we have found that asODN uptake is enhanced in a sugar solution. The method has promise for many applications, such as a rapid alternative to time-consuming transgenic studies, and high potential for studying gene functionality in intact plants and multiple plant species, with particular advantages in evaluating the roles of multiple gene family members. Generation of transgenic plants relies on the ability to select transformed cells. This screening process is based on co-introduction of marker genes into the plant cell together with a gene of interest. Currently, the most common marker genes are those that confer antibiotic or herbicide resistance. The possibility that traits introduced by selectable marker genes in transgenic field crops may be transferred horizontally is of major public concern. Marker genes that increase use of antibiotics and herbicides may increase development of antibiotic-resistant bacterial strains or contribute to weed resistance. Here, we describe a method for selection of transformed plant cells based on asODN inhibition. The method enables selective and high-throughput screening for transformed cells without conferring new traits or functions to the transgenic plants. Due to their high binding specificity, asODNs may also find applications as plant-specific DNA herbicides.

Protuberance-mediated high frequency of adventitious shoot regeneration from sweetpotato leaf explants [ Ipomoea batatas (L.) Lam.

In this report, a novel, reproducible and efficient protocol for protuberance-mediated adventitious shoot regeneration has been established for sweetpotato [Ipomoea batatas (L.) Lam.]. The leaf explants were cultured on a modified Murashige and Skoog (MS) medium containing various concentrations (0.5, 1.0, 2.0, 5.0 or 10 μM) of Thidiazuron (TDZ). The adventitious shoots were developed from the base of leaf explants on an MS medium supplemented with 2.0, 5.0 or 10.0 μM TDZ. The responsive explants were transferred to a freshly modified MS medium containing TDZ. Green-colored, oval, or cone-shaped shoot-forming protuberances were developed from explants. It appeared that the frequency of protuberance development depended on the concentration of TDZ. However, this did not further improve, when the concentration of TDZ increased from 5.0 μM to 10.0 μM. Adventitious shoot buds were developed from protuberances, and the histology of protuberances revealed a direct origin of shoot buds. In vitro raised shoots were rooted either on a modified MS medium alone or on a modified MS medium containing indole-3-butyric acid (IBA). The regenerated plants were acclimatized and successfully established in a greenhouse.

In Vitro Micrografting of the Almond Cultivars “Texas”, “Ferrastar” and “Nonpareil”

ABSTRACTA successful micrografting technique for the almond cultivars (cvs) “Texas”, “Ferrastar” and “Nonpareil” was developed using in vitro germinated seedlings as rootstocks and axenic shoot cultures established from mature tree sources as microscions. In vitro germinated seedlings, which developed 14 days after culturing in the modified Murashige and Skoog (MS) medium, were decapitated and used as rootstock. Shoot culture initiation from three almond cvs (“Texas”, “Ferrastar” and “Nonpareil”) was successfully achieved by culturing mature shoot tips from forced nodal buds, about 4–6 mm, on a modified MS medium containing 1 mgL−1 benzyl adenin (BA). Slit micrografting on epicotyl and on hypocotyls were equally successful (83.3% to 100%). Grafting success was dependent on the rootstock type and lenght of the scion. Grafting success varied between 83.33% and 100% depending on the cultivar, when the scion contained 1, 2, and 3 nodes. When almond scions, about 1.5 cm long, were micrografted on germinated seedling and cultured on proliferation medium (PM), the mean shoot length was 19.84 mm, 16.50 mm, 26.93 mm for the cvs “Texas”, “Ferrastar” and “Nonpareil” respectively. Micrografts could be easily cultured on a hormone-free semi-solid MS medium and were potted out after 4 to 6 weeks of culture growth. Rooted micrografted plantlets were successfully acclimatized and transferred to potting mix with 100 % survival. Although low percentages of variation were obtained in tested cvs (3.70%, 6.25% and 10.2% in “Texas”, “Ferrastar” and “Nonpareil”), molecular analysis showed that the developed micrografting technique produces genetically stable plantlets, at least up to 6 months of sub-culturing in cvs “Ferrastar” and “Nonpareil”. The described micrografting technique could be usedfor rejuvenation ofshoot explants of mature elite almond cultivars and it also has potential use in the commercial production of other almond cultivars.

The micropropagation of chrysanthemums via axillary shoot proliferation and highly efficient plant regeneration by somatic embryogenesis

Protocols for axillary shoot proliferation and somatic embryogenesis were developed for Dendranthema × grandiflora (Ramat.) Kitamura cv. Palisade White. Shoot tips were cultured on a modified Murashige and Skoog (MS) media supplemented with benzyl aminopurine (BA) and gibberellic acid (GA 3 ) or BA, kinetin (Kin) and indole-3-acetic acid (IAA). The auxins indole -3-butyric acid (IBA) and IAA were used to induce rooting. Direct somatic embryogenesis was induced from leaf, internode’s stem and for the first time for chrysanthemums from petiole explants. Modified MS medium supplemented with 1 mg/L naphthalene acetic acid (NAA) or 2,4- dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L BA, 200 mg/L casein hydrosylate (CH) and 290 mg/L proline was used for induction. Proliferation rate of 3.2 new microshoots per one inoculated was achieved when BA (0.1 mg/L) was used in combination with GA 3 (0.5 mg/L). The number of roots per shoot was higher using IBA (0.5 mg/L), but IAA (2 mg/L) promoted longer roots. A high percentage of embryogenesis was induced by both combinations of plant growth regulators (PGRs). Leaf explants were most responsive, demonstrating the highest percentage of embryogenesis (97.9%), followed by petiole and internode’s stem explants (56.3 and 35.1%, respectively). The number of somatic embryos per embryogenic explant was also the highest on leaf explants; however, the best conversion rate (53.8%) of somatic embryos to plantlets was observed from petiole explants. For this reason, petiole explants are the most suitable type of explants for plant regeneration of chrysanthemum cv. Palisade White through somatic embryogenesis. Key words : Chrysanthemum, Dendranthema × grandiflora (Ramat.) Kitamura cv. Palisade White, micropropagation, direct somatic embryogenesis, explant type.

Regeneration of Anthurium andraeanum from Leaf Explants and Evaluation of Microcutting Rooting and Growth under Different Light Qualities

This study established a new method for regenerating Anthurium andraeanum Lind. and evaluated effects of different wavelengths from light-emitting diodes (LEDs) on rooting and growth of adventitious shoots. Callus occurred in leaf explants of A. andraeanum ‘Alabama’ and ‘Sierra’ cultured on a modified Murashige and Skoog (MS) basal medium supplemented with four concentrations of N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ). Adventitious shoots were induced from callus pieces (≈1 cm3) cultured on the modified MS medium containing 6-benzyladenine (BA) with kinetin (KN), BA, and/or KN with 3-indolebutyric acid (IBA) or α-naphthalene acetic acid (NAA). Results showed that 1.82 μM TDZ induced 83.3% and 77.8% of leaf explants of ‘Alabama’ and ‘Sierra’ to produce callus and 24.9 and 24.7 adventitious shoots were produced per callus piece of ‘Alabama’ and ‘Sierra’ cultured on the modified MS medium containing 0.89 μM BA, 2.32 μM KN, and 0.98 μM IBA, respectively. Adventitious shoots were cut and rooted in the modified MS medium containing 0.98 μM IBA and grown under the same light level but with different light qualities. All adventitious shoots rooted; root numbers, root lengths, root fresh and dry weights, and leaf area of plantlets grown under red plus blue light were comparable to those grown under conventional fluorescent white light. Shoot height was the greatest in monochromic blue light followed by red light. Shoot fresh and dry weights of plantlets grown under red plus blue light, however, were significantly greater than those grown under the other light qualities. Plantlets grown under red plus blue light had 22.7% greater total dry weight and more balanced root-to-shoot ratio than those grown under fluorescent white light. These results suggested the use of complex of red plus blue LED could be an option for improving growth of A. andraeanum plantlets in vitro.

Micropropagation from apical and nodal segments of Bituminaria bituminosa and the furanocoumarin content of propagated plants

SummaryAn efficient micropropagation protocol has been developed for Bituminaria bituminosa, a species considered to be of pharmaceutical interest for its furanocoumarin (FC) content. Four accessions of B. bituminosa with different FC contents were used: variety albomarginata (“albo-tedera”), variety bituminosa (“bitu-Calnegre”), var. albomarginata× var. bituminosa (“albo-hybrid”), and var. bituminosa × var. albomarginata (“bitu-hybrid”). Different concentrations of sodium troclosene solution were evaluated for in vitro establishment of apical and nodal explants cultured on modified Murashige and Skoog (MS) medium. The effects of the type of agar and the addition of activated charcoal (AC) were also evaluated. The best treatment for surface sterilising the explants was 3.5 g l–1 sodium troclosene. The lowest frequency of hyperhydrated shoots was achieved using 0.8% (w/v) American bacteriological agar. Apical explants resulted in greater shoot proliferation than nodal explants. The addition of AC increased the quality and development of regenerated shoots. The highest average numbers of shoots ≥ 5 mm per explant were obtained from apical explants of “albo-tedera” (7.8 shoots) and “albo-hybrid” (7.6 shoots) on a modified MS medium supplemented with plant growth regulators (T2 media) and for “bitu-Calnegre” (6.3 shoots) and “bitu-hybrid” (5.3 shoots) on a modified MS medium without plant growth regulators (T1 media). The regenerated shoots rooted efficiently on different media depending on the plant material. Plantlets were acclimatised and maintained in a greenhouse until they were transferred to the experimental field. The total FC contents in micropropagated field grown plants were found to be higher or equal to those in the non-micropropagated mother plants. The accession “bitu-Calnegre” was found to have higher, or equivalent FC levels to those in other species such as Psoralea corylifolia and Ruta graveolens, reported as sources of FC. All micropropagated plants maintained the characteristics of the non-micropropagated mother plants.

Myo-inositol increases the plating efficiency of protoplast derived from cotyledon of cabbage (Brassica oleracea var. capitata)

This study describes the effect of myo-inositol on sustained cell division and plant regeneration from cotyledon-derived protoplast of cabbage (Brassica oleracea var. capitata). Freshly isolated protoplasts were cultured in modified Murashige and Skoog (MS) medium removed ammonia ions and containing <TEX>$0.4\;mg\;l^{-1}$</TEX> thiamine HCl, <TEX>$100\;m</TEX><TEX>g\;l^{-1}$</TEX> myo-inositol, <TEX>$2\;mgl^{-1}$</TEX> 2,4-D, <TEX>$0.5\;mgl^{-1}$</TEX> BA, <TEX>$30\;gl^{-1}$</TEX> sucrose and several concentrations of myo-inositol (2, 4, 6, 8, 10% (w/v)) as an osmotic stabilizer. After 3 weeks of culture in the dark at <TEX>$25^{\circ}C$</TEX>, the plating efficiency of cabbage protoplasts reached to <TEX>$22.5{\pm}2.9%$</TEX> when cultured in modified MS medium supplemented with <TEX>$2\;mgl^{-1}$</TEX> 2,4-D, <TEX>$0.5\;mgl^{-1}$</TEX> BA, <TEX>$30\;gl^{-1}$</TEX> sucrose and 8% (w/v) of myo-inositol at a density of <TEX>$2{\times}10^5$</TEX> protoplasts/ml. Rapidly growing cell colonies after 3 weeks of culture were transferred to the same culture medium removed osmoticum. To induce shoot regeneration from calluses, calluses with about 2 mm in diameter were transferred to the MS medium containing <TEX>$2\;mgl^{-1}$</TEX> BA and <TEX>$0.5\;mgl^{-1}$</TEX> NAA. After further three weeks of incubation onto the medium in the light, green shoots were formed on the surface of calluses at a frequency of 30%. Upon transfer to half-strength MS basal medium, roots were formed onto the bottom of regenerated shoots without auxin treatments. These regenerated plantlets were successfully acclimatized to soil transfer, grown to normal mature plants. The cabbage protoplast culture system established in this study could be applied for production of somatic hybrids or cybrids by asymmetric protoplast fusion and mass proliferation of elite somatic clones of cabbage.

Microspore embryogenesis and plant regeneration from anthers of barley cultured through meiosis

A technique has been developed which allows the culture of barley cv. Igri anthers in isolated spikes and spikelets through meiosis until the mid-late uninucleate microspore stage. Light microscopical analyses and measurements of the duration of the individual substages of meiosis and microsporogenesis revealed a developmental sequence indistinguishable from that observed in vivo. Embryogenesis was induced in microspores at the mid-late uninucleate stage, when anthers from cultured spikes and spikelets were plated on induction medium and green plants were regenerated from the embryoids. Among different media tested for their ability to sustain a normal development of the microsporocytes and the microspores, modified Murashige and Skoog (MS) and Kao media solidified with agarose yielded optimal growth of the isolated spikes and spikelets. On a modified MS medium with 100 g/1 maltose and 1 mg/1 6-benzylaminopurine (BAP) 62 % of the anthers survived. Fifty-one percent of these anthers responded with microspore embryogenesis and a mean of 2.4 green plants were regenerated per responding anther. The albino to green plant ratio was 0.5. The significance of this culture system for establishing a transformation system with gene targeting mediated by homologous meiotic recombination is discussed.

Detection of DNA methylation changes during somatic embryogenesis of Siberian ginseng ( Eleuterococcus senticosus)

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation during somatic embryogenesis in Eleuterococcus senticosus, using two methods to evaluate DNA methylation rates: (1) direct determination of 5-methyl-deoxycytidine (5mdC) amounts in genomic DNA by HPLC separation and quantification of nucleosides and (2) methylation-sensitive amplification polymorphism (MSAP) technique. The tissue assayed include embryogenic and non-embryogenic callus. Leaf segments were cultured on modified Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D). Opaque and friable embryogenic callus formed on modified MS medium with 2.26 μM 2,4-D. Initiation and development of somatic embryos occurred in the same medium containing 2.26 μM 2,4-D. Non-embryogenic callus failed to regenerate even after prolonged maintenance and subculture on to the same media. Dark treatment significantly increased the number of mature somatic embryos. HPLC analyses on genomic DNA from embryogenic and non-embryogenic callus showed that global DNA methylation rates were significantly lowered in embryogenic calli. Similarly, 16.99% of 5′-CCGG-3 sites in the genome of non-embryogenic callus were cytosine methylated, where as 11.20% were methylated in case of embryogenic callus tissue, as detected by MSAP technique. Hypermethylation of DNA in non-embryogenic callus compared with embryogenic callus reflects the marked expression of this molecular feature, which may well contribute to the developmental gene expression.

Meristem culture and virus eradication in Alstroemeria

Stem apical meristems, rhizome apical meristems and rhizome axillary meristems excised from Alstroemeria plants were grown in vitro on modified Murashige and Skoog (MS) media containing different concentrations of gibberellic acid and 6-benzylaminopurine (BA). Plantlets developed from stem apical meristems never regenerated a rhizome and eventually died. The highest regeneration rate (74.1%) of plantlets with a rhizome was observed when rhizome axillary meristems were grown on modified MS medium containing μM 8.9 of BA. Alstroemeria mosaic potyvirus (AlMV) could be eradicated from infected Alstroemeria plants through meristem culture. The rate of virus eradication was 73.7 and 14.7% for plantlets developing from explants measuring 0.7 mm and 2.0 mm, respectively. Greenhouse evaluation of virus-negative and AlMV-infected Alstroemeria plants showed that healthy plants produced more floral stems, more vegetative stems, longer floral stems and gave a higher fresh weight than infected plants.

In Vitro Culture of Lingonberry (Vaccinium vitis-idaea L.)

Abstract Shoots of three lingonberry (Vaccinium vitis-idaea L.) cultivars, ‘Regal’, ‘Splendor’, and ‘Erntedank’ and two clones from natural stands in Newfoundland were initiated in vitro from shoot tip and nodal explants on modified Murashige and Skoog (MS) medium containing the plant growth regulators N6-[2-isopentenyl]adenine (2iP) (12.3 μM) or zeatin (5.7 μM). Zeatin was more effective than 2iP, and induced proliferation of 2 to 3 times as many shoots in ‘Regal’ as 2iP. Out of four media tested for shoot proliferation, modified MS medium was found more effective than the Woody Plant Medium for shoot multiplication. With all three cultivars, a reasonable balance in terms of shoot multiplication rate and desirable growth characteristics was attained in a new medium. Nodal explants of the two clones cultured on the modified MS medium with 12.3 μM 2iP produced 4 to 6 healthy axillary shoots per explant in clone 1 and 2 to 4 shoots in clone 2. Shoots rooted well ex vitro directly on a 2 peat: 1 perlite (v/v) medium after dipping in 39.4 mM indole-3-butyric acid. The new protocol proposed from this study is expected to be adapted for propagating a wide range of lingonberry germplasm.

Naphthoquinone contents of in vitro cultured plants and cell suspensions of Dionaea muscipula and Drosera species

In vitro cultured carnivorous plants were grown on a hormone-free medium. They produced the following naphthoquinones: Dionaea muscipula (plumbagin: 5.3%), Drosera rotundifolia (7-methyljuglone: 0.6%), D. binata (plumbagin: 1.4%), and D. capensis (7-methyljuglone: 0.5%). A red, slow-growing suspension culture of D. muscipula was maintained in a modified McCowns Woody Plant (McC) medium and produced plumbagin (2.59%) after 30 days growth. A suspension culture of D. rotundifolia grew slowly as multicoloured small aggregates only in a modified Murashige and Skoog (MS) medium. No quantifiable amounts of naphthoquinones were produced. Several cell lines of D. capensis were developed. Green aggregates grown in a modified MS medium contained 7-methyljuglone (0.33%) and differentiated into plants when placed onto hormone-free medium. Pink cultures grown in modified McC medium contained 7-methyljuglone (1.24%), while dark red cultures produced ca. 1% in both modified McC and MS media. Though the latter medium was significantly better with regard to biomass production, cells excreted a mucin when cultured in both media (0.21 g dry mucin/g dry cells in McC) and (0.16 g dry mucin/g dry cells in MS). Effects of the presence or absence of light during the growth period of 30 days showed that there was no effect on biomass and only slight effects on mucin production and naphthoquinone contents.

Direct differentiation of ears and tassels from cultured shoot apices of maize

In vitro morphogenesis of inflorescences from the cultured corn seedling shoot tips was obtained on modified Murashige and Skoog (MS) medium in complete darkness. Some shoot tip meristems excised from seedlings of inbred line 515, inbred line 8112 and their filial generations would directly give rise to florets on modified MS medium supplemented with 2.0 mg/L N(6) -bezyladenine (6-BA) in five or six weeks. On the medium with 1.0 mg/L 6-BA and 0.2 mg/L 2, 4-dichlorophenoxy acetic acid (2,4-D), the explants swelled first, and produced multiple shoot clumps, then the culture of the shoot tips from all of the six inbred lines in experiment would ultimately initiate to develop ears and tassels accompanied by multiple shoot clumps developing on the medium with 1.0 mg/L 6-BA and 0.2 mg/L indole-3-butyric acid (IBA). The developmental patterns of the corn inflorescences were similar to the controls of normal plants in the field, but the number of the ears was much more than that of the tassels in vitro. It seemed that proper kinds and concentrations of exogenous hormones were necessary for the initiation and development of the inflorescences. The genotype of explants was responsible for the response to culture conditions. It could be inferred that there were different control mechanisms existing in the vegetative and reproductive developmental processes of maize, because the cultured shoot tips could directly produce the reproductive organs.

Preliminary studies of micropropagation of Hopea odorata, a dipterocarp tree

Plantlet development from in vitro cultures of Hopea odorato Roxb. is described. Embryos excised from seeds and cultured on Gamborg's B5 or modified Murashige and Skoog (MS) medium with benzyladenine (BA, 2.2–22.2 μM) produced axillary shoots at cotyledonary and/or stem nodes. Shoot production was greatest in germinated embryos on modified MS medium with 8.9 μM BA. Excised axillary shoots formed few buds when cultured on medium with BA and limited root development occurred on Woody Plant Medium with naphthaleneacetic acid. Nodal explants from aseptically grown plantlets sprouted axillary shoots in modified MS medium with BA.

890 PB 472 SOMACLONAL VARIATION IN PINEAPPLES

The objective of this project is to propagate disease-free clones of fresh market pineapples for growers. To expedite the distribution process, sterile cultures were obtained from the USDA/ARS Germplasm Repository, Hilo, Hawaii of pineapple clones 119, 155 and 159. Plantlets were placed on modified Murashige and Skoog (MS) medium with one half of the salts and 2 uM BAP and 1 uM IAA. The cultures were transferred to modified MS medium with one half of the salts, no growth regulators, and solidified with Phytagel. Plantlets were transplanted at one-inch size into peatlite mix. At 4-6 inch size, the plants were transplanted in growers fields. Early observations indicate difference between clones in kinds and frequency of “off-types.” Compared with surrounding plants, “off-types” were categorized as being dwarfed, variegated, thorny leaved, and pigmentation modified.

Preparation of protoplasts from callus derived from buds of mature Scots pine and subsequent induction of cell proliferation

Callus cultures were established from shoot tip explants excised from buds of mature, 10–40-year-old Scots pine (Pinus sylvestris L.) trees. Protoplast yield and viability were highest when the callus cells were plasmolyzed for 1.5 h in osmoticum (0.35 M mannitol, 0.15 M sorbitol, 1.5 mM CaCl2, 0.7 mM KCl, pH 5.8) before being enzymatically digested in osmoticum containing 0.5% cellulase Onozuka R-10, 0.5% macerase, 0.5% hemicellulase, and 0.5% bovine serum albumin. Bovine serum albumin markedly increased viability of isolated protoplasts. Protoplasts were purified by flotation on either osmoticum alone or osmoticum containing 9% Nycodenz®. Cell proliferation was initiated by spreading purified protoplasts on a filter impregnated with a modified Murashige and Skoog (MS) medium supplemented with 3.5 g casein hydrolysate, 1.3 mM L-glutamine, 0.2 M glucose, 10.7 μM naphthalene acetic acid (NAA), and 4.4 μM benzylaminopurine (BAP). The filter was placed on a solid, modified MS medium containing pine callus cells as nurse tissue and supplemented with 0.9 μM BAP and 1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The first microcalli were observed after three weeks, and viability of subcultured cells was about 40% after six weeks.

Micropropagation of Eucalyptus Species

Two cold-tolerant species (Eucalyptus macarthurii Deane et Maiden and E. smithii R.T. Baker), a cold-tolerant hybrid (E. macarthurii×E. grandis Hill ex Maiden), and E. saligna Sm. were propagated in vitro from nodal explants collected from field-grown seedlings and from clonal hedges. Shoot growth was initiated on modified Murashige and Skoog (MS) medium containing BA at 0.1 mg·liter-1. Modified MS medium with BA (0.2 mg·liter-1) and NAA (0.01 mg·liter-1) was most effective in promoting shoot proliferation. Root initiation was achieved on half-strength modified MS medium with 2 mg IBA/liter. Rooted plants were hardened and established in the field. Chemical names used: N-(phenylmethyl)-1EZ-purin-6-amine (BA); 2-(1-naphthyl)acetic acid (NAA); 1H-indole-3-butyric acid (IBA).

Plant Regeneration from Leaf Mesophyll Protoplasts of Broussonetia kazinoki Sieb. (Paper Mulberry)

Summary Leaf mesophyll protoplasts of Broussonetia kazinoki Sieb. were enzymatically isolated from in vitro grown shoot cultures. The protoplasts showed first divisions between 10-20 days in culture and formed colonies within 4 weeks in modified Murashige and Skoog (MS) medium. Reduction of ammonium concentration and use of glucose as a carbon source were essential for sustained cell divisions and colony formation. Shoot buds were produced from protoplast-derived callus on modified MS medium containing 0.1 mg · 1 −1 NAA and 1 mg · 1 −1 BA.