Rapid and highly efficient callus induction and plant regeneration in the starch-rich duckweed strains of <Emphasis Type="Italic">Landoltia punctata</Emphasis>

The starch-rich duckweed Landoltia punctata is a valuable aquatic plant in wastewater purification, bioenergy production, and many other applications. A highly efficient callus induction and plant regeneration protocol is desirable so that biotechnology can be used to develop new varieties with added value and adaptation. We studied both known and unknown factors that influence callus induction in L. punctata and obtained almost 100 % induction rate in 30 days. The optimum medium for callus induction was MS basal medium supplemented with 1 % sorbitol, 15 mg/L 2,4-D, and 2 mg/L 6-BA. Green fragile callus was induced from the meristematic region in the budding pouches. The optimum photoperiod for callus induction was 16-h day, and the optimum explant orientation was dorsal side down on the medium. The optimum medium for callus subculture was WPM basal medium supplemented with 2 % sorbitol, 4 mg/L 2,4-D, and 0.5 mg/L TDZ. Green callus could be maintained by subculture once every 4 weeks. However, when the subculture cycle was prolonged to 6 weeks or longer, yellow fragile embryogenic callus was obtained. The optimum plant regeneration medium was MS medium supplemented with 0.5 % sucrose, 1 % sorbitol, and 1.0 mg/L 6-BA with frond regeneration rates of approximately 90 %. The regenerated fronds rooted in Hoagland’s liquid medium in 1 week. The callus induction and frond regeneration protocol was tested for its efficiency in geographically distinct strains 5502, 8721, and 9264. Thus, we obtained a rapid and efficient protocol for callus induction and frond regeneration of L. punctata, which takes only 9 weeks.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.     

Invasive alga <Emphasis Type="Italic">Caulerpa racemosa</Emphasis> var. <Emphasis Type="Italic">cylindracea</Emphasis> makes a strong impact on the Mediterranean sponge <Emphasis Type="Italic">Sarcotragus spinosulus</Emphasis>

Here we present the first observation of the impact of the invasive Caulerpa racemosa var. cylindracea on native photophilic sponge species in the Adriatic Sea, with special focus on Sarcotragus spinosulus. Caulerpa racemosa var. cylindracea is able to completely overgrow the sponge, developing an exceptionally thick canopy with a maximum measured density of 1,887 m of stolons m⁻² and 40,561 fronds m⁻². Necrosis of the sponge surface was significantly correlated with the algal dry biomass, frond number and stolon length. Dense algal canopy, penetration of the algal stolon and rhizoids into the sponge oscula and covering of the ostiae probably diminishes the seawater circulation through the sponge and consequently results in its smothering and even death. We suggest that chemotropism is the reason why C. racemosa penetrates the sponge oscula and establishes such dense canopy on the sponge.     

Assignment of <Emphasis Type="Italic">Lemna gibba</Emphasis> L. (duckweed) bioassay for <Emphasis Type="Italic">in situ</Emphasis> ecotoxicity assessment

To narrow the differences between the results obtained from radionuclides and heavy metal ecotoxicity investigations in the laboratory and in the abandoned uranium mines, a few standardised plant bioassay procedures were selected from the literature for testing with Lemna gibba L. The bioassay procedures were tested in situ and ex situ. The laboratory culturing was performed in batch and semicontinuous modes. The results revealed that most of the standardised plant bioassay procedures require modification for the L. gibba bioassay to predict the actual effects under field conditions. L. gibba performed relatively better in the field than laboratory batch cultures despite that the batch cultures had many-fold higher nutrient concentrations than in the field. For instance, the phosphorus concentration of the mine tailing water was 0.13 ± 0.09 μg l⁻¹ in the field, while the literature range for phosphorus in the laboratory culture media is 13.6–40 mg l⁻¹. L. gibba growth in the laboratory batch culture was influenced by speciation changes due to consumption of nutrients, CO₂ and O₂ phase exchanges, and excretion of organic substances by the test plants. Semicontinuous culture modes performed significantly better than batch cultivation even after 10× dilution of the nutrient solution. The growth behaviour revealed that L. gibba exhibited intrapopulation and probiotic interaction for best performance. Growth performance of L. gibba was influenced by the anions that balanced essential cations despite equal cation concentration in the culture media; e.g., the best growth was observed in culture media that had more SO₄²⁻ than Cl⁻. Water samples from the field had higher SO₄²⁻ concentrations than Cl⁻. The test vessel material, sterilisation and axenic culturing procedures also influenced the sensitivity of the bioassay. These, for instance, and a few others are neither described nor reported in most standard Lemna tests or the literature. Thus, this work presents results of a series of tests conducted on the selected methods. Common and possible errors and corrective measures in assigning L. gibba bioassay from laboratory population levels to field community levels are discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation from young and mature explants of thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> and <Emphasis Type="Italic">T. longicaulis</Emphasis>) resulting in genetically stable shoots

An efficient in vitro propagation protocol, applicable both to young and mature explants of two Thymus spp., results in genetically stable plantlets. In vitro-grown shoot tips of Thymus vulgaris L. were exposed to cytokinins (6-benzyladenine, kinetin, and thidiazuron) alone or in combination with auxins, gibberellic acid (GA₃) and/or silver nitrate in order to optimize in vitro shoot proliferation. Optimum shoot proliferation (97% regeneration rate, with 8.6 shoots produced per explant) was obtained when semi-solid Murashige and Skoog (MS) medium was supplemented with 1 mg L⁻¹ kinetin and 0.3 mg L⁻¹ GA₃. Rooting of the shoots was easily obtained on semi-solid MS medium that was either hormone-free or supplemented with auxins. However, the best root apparatus (92.5% rooting rate, with 19 adventitious roots per shoot) developed on MS medium supplemented with 0.05 mg L⁻¹ 2,4-dichlorophenoxyacetic acid. Genetic stability was confirmed in the in vitro-germinated mother plant as well as the shoots that underwent two, four, six, eight, or ten cycles of in vitro subculturing by random amplified polymorphic DNA (RAPD) analysis. When applied to the micropropagation of mature shoot tips of T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas, the optimized in vitro propagation protocol resulted in a 97.5% shoot regeneration rate, with five shoots formed per explant, and 100% rooting. Rooted plantlets of both species were transferred to 250-mL plastic pots and successfully acclimatized by gradually reducing the relative humidity.     

Somatic embryogenesis and plant regeneration from leaf and petiole explants of <Emphasis Type="Italic">Campanula punctata</Emphasis> Lam. var. <Emphasis Type="Italic">rubriflora</Emphasis> Makino

A simple and efficient protocol was developed for somatic embryogenesis from leaf and petiole explants of Campanula punctata Lam. var. rubriflora Makino. Somatic embryos (SE) were obtained with greater frequency from petiole explants than from leaf explants when cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L⁻¹ 6-benzyladenine (BA). On this medium, a mean number of 19.5 and 31.2 SE were developed per leaf and petiole explants, respectively. Embryos were induced both light and dark conditions but culturing the explants 2 weeks in the dark followed by 3 weeks under light resulted in high frequency of embryo formation. Globular embryos germinated best on MS medium supplemented with 0.3% (w/v) activated charcoal (AC) and 1.0 mg L⁻¹ GA₃. The germinated plantlets grew further on MS medium containing 0.3% AC. Plantlets were successfully acclimatized in the greenhouse with 94% survival rate. This is the first report on induction of somatic embryogenesis in this genus and also has implications for genetic transformation, and mass clonal propagation.     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.     

Tissue culture and plant regeneration of the salt marsh monocots <Emphasis Type="Italic">Juncus roemerianus</Emphasis> and <Emphasis Type="Italic">Juncus gerardi</Emphasis>

Summary Tissue culture and plant regeneration protocols for the salt marsh plants Juncus roemerianus Scheele and Juncus gerardi Loisel, were developed. J. roemerianus callus was induced from mature seeds cultured on Murashige and Skoog (MS) medium supplemented with 2.22 μM 6-benzylaminopurine (BA), 5.37 μM α-naphthaleneacetic acid (NAA), 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and 50 ml l⁻¹ coconut water (callus induction medium). The callus was subcultured on MS medium containing 2.22 μM BA, 5.37 μM NAA, and 9.05 μM 2,4-D for callus maintenance. Shoot regeneration occurred 2 wk after transferring the callus onto shoot regeneration medium, which consisted of MS medium containing BA or thidiazuron. A high frequency of shoot regeneration was obtained when the medium contained 13.3 μM BA. Regenerated shoots were transferred to MS medium supplemented with 10.7 μM NAA for root production. Rooting did not occur in the shoots regenerated on the thidiazuron-containing media. The callus induction medium for J. roemerianus was also effective in inducing callus of J. gerardi from young inflorescences. The same medium was also used for callus maintenance. Shoot regeneration occurred 10 d after transferring the callus onto MS medium supplemented with 0.44 μM BA and 0.57 μM indole-3-acetic acid. Root regeneration occurred after transferring the shoots onto MS medium plus 0.44 μM BA and 14.8 μM indole-3-butyric acid. The regenerated plants of both J. roemerianus and J. gerardi grew vigorously in potting soil in the greenhouse. J. roemerianus regenerants also grew well in a saltwater-irrigated field plot. Tissue culture-produced plants of J. roemerianus and J. gerardi can be used for planting in created or restored wetlands.     

Effects of plant growth regulators and <Emphasis Type="SmallCaps">l</Emphasis>-glutamic acid on shoot organogenesis in the halophyte <Emphasis Type="Italic">Leymus chinensis</Emphasis> (Trin.)

The halophyte Leymus chinensis (Trin.) is a perennial rhizome grass (tribe Gramineae) that is widely distributed in China, Mongolia and Siberia, where it is produced as a forage product. In this report, we establish a highly reproducible plant regeneration system through somatic embryogenesis. Two explants, mature seeds and leaf base segments were used; these parts displayed different responses to combinations of growth factors that affect embryogenic callus induction, callus type optimization and plant regeneration. The highest callus induction frequency was obtained on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of 5.0 mg l⁻¹ l-glutamic acid. The inclusion of 5.0 mg l⁻¹ l-glutamic acid was found to significantly promote primary callus induction, embryogenic callus formation and callus status improvement. Subculturing on maintenance medium for 1–2 months before plant regeneration was found to be essential for the optimization of callus type and the maturation of embryogenic callus. Callus relative water content and growth rate were simultaneously investigated during callus maintenance, and found to possibly be related to callus type. Shoots were differentiated from the embryogenic callus on the optimal medium with MS salts containing 0.2–0.5 mg l⁻¹ α-naphthalene acetic acid (NAA), 2.0 mg l⁻¹ kinetin (Kn) and 2.0 g l⁻¹ casamino acids in 71.0 and 69.2% of wild-type (WT) and Jisheng No.1 (JS) plants, respectively. Plant regeneration was variable depending on NAA levels, and the addition of casamino acids stimulated the maturation of embryogenic callus and plant regeneration. Transferring callus with shoots onto half-strength MS medium resulted in rooting within 1 week. The growth of regenerated plants was also surveyed in the field. This is the first report of plant regeneration through somatic embryogenesis from mature seeds and leaf base segments of L. chinensis.     

<Emphasis Type="Italic">CDPK</Emphasis> gene expression in somatic embryos of <Emphasis Type="Italic">Panax ginseng</Emphasis> expressing <Emphasis Type="Italic">rolC</Emphasis>

A somatic embryogenesis protocol for plant regeneration of northern red oak (Quercus rubra) was established from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium (MS) containing 3% sucrose, 0.24% Phytagel™, and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) after 4 weeks of culture in darkness. A higher response (66%) of embryogenic callus was induced on 0.45 μM 2,4-d. Higher numbers of globular- (31), heart- (17), torpedo- (12), and cotyledon-stage (8) embryos per explant were obtained by culturing embryogenic callus on MS with 3% sucrose, 0.24% Phytagel™, and devoid of growth regulators after 8 weeks culture in darkness. Continuous sub-culturing of embryogenic callus on medium containing 2,4-d yielded only compact callus. Desiccation of embryos for 3 days in darkness at 25 ± 2°C followed by cold storage at 4°C in darkness for 8 weeks favored embryo germination and development of plantlets. Cotyledon-stage embryos subjected to desiccation and chilling treatment cultured on MS with 3% sucrose, 0.24 Phytagel™, 0.44 μM 6-benzylaminopurine (BA), and 0.29 μM gibberellic acid germinated at a higher frequency (61%) than with 0.44 μM BA alone and control cultures. Germinated plantlets developed a shoot and root, were acclimatized successfully, and maintained in a growth room for plantlet development.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of Persian poppy (<Emphasis Type="Italic">Papaver bracteatum</Emphasis>)

Persian poppy (Papaver bracteatum Lindl.) is an important commercial source of medicinal opiates and related compounds. In this research, calli were induced from seeds, roots, cotyledons and hypocotyls of P. bracteatum at a high efficiency. The optimized callus induction media consisted of the Murashige and Skoog (MS) basic media supplemented with 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin and 15 mg/L ascorbic acid. The concentrations of 2,4-D and ascorbic acid were found critical to callus induction and proliferation. Subsequent subcultures resulted in excellent callus proliferation. Ascorbic acid at concentration 15 mg/L increased the callus proliferation significantly. Maximum callus growth was achieved when the explants were incubated at 25°C. MS salts at full strength were found inhibitory for callus induction, while ľ MS salts were found to favor callus induction. Shoot regeneration of calli in vitro was achieved on ľ MS medium containing 0.5 mg/L benzylamine purine and 1.0 mg/L naphthalene acetic acid. Analysis of alkaloid extracts from Persian poppy tissues by high-performance liquid chromatography showed that thebaine accumulated in the tissues of plants. The thebaine alkaloid profile of the Persian poppy is a well-defined model to evaluate the potential for metabolic engineering of thebaine production in P. bracteatum.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.     

FISH analysis of<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> heterochromatin using BACs and<Emphasis Type="Italic"> P</Emphasis> elements

The heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster contains about 30 essential genes defined by genetic analysis. In the last decade only a few of these genes have been molecularly characterized and found to correspond to protein-coding genes involved in important cellular functions. Moreover, several predicted genes have been identified by annotation of genomic sequence that are associated with polytene chromosome divisions 40, 41 and 80 but their locations on the cytogenetic map of the heterochromatin are still uncertain. To expand our current knowledge of the genetic functions located in heterochromatin, we have performed fluorescence in situ hybridization (FISH) mapping to mitotic chromosomes of nine bacterial artificial chromosomes (BACs) carrying several predicted genes and of 13 P element insertions assigned to the proximal regions of 2R and 3L. We found that 22 predicted genes map to the h46 region of 2R and eight map to the h47 regions of 3L. This amounts to at least 30 predicted genes located in these heterochromatic regions, whereas previous studies detected only seven vital genes. Finally, another 58 genes localize either in the euchromatin-heterochromatin transition regions or in the proximal euchromatin of 2R and 3L. Edited by: B. McKeeN. Corradini and F. Rossi contributed equally to this work     

Cytokinin-induced growth in the duckweeds <Emphasis Type="Italic">Lemna gibba</Emphasis> and <Emphasis Type="Italic">Spirodela polyrhiza</Emphasis>

Duckweeds, quick-growing aquatic plants, have been recently recognized as promising hosts for the large-scale production of recombinant proteins and as an ideal biomass feedstock for biofuel production. These possible wide-spread industrial uses of duckweeds intensified research aimed at understanding the mechanisms that control duckweed growth. Here, we describe how the hormone cytokinin affects growth. We performed a number of standard cytokinin growth- and physiological-response assays using sterile-grown colonies of Lemna gibba and Spirodela polyrhiza. Similar to land plants, cytokinin inhibited root elongation in duckweeds. Surprisingly, and in contrast to land plants, cytokinin promoted growth of aerial organs in both duckweed species, suggesting that the cytokinin growth response fundamentally differs between aquatic and land plants.     

In vitro hormone-regulated growth and floral induction of <Emphasis Type="Italic">Cuscuta reflexa</Emphasis>: a parasitic angiosperm

The role of different growth regulators in callus induction, shoot regeneration, floral induction and chlorophyll content of the obligatory parasitic plant Cuscuta reflexa has been studied. Callus development was excellent from the nodal part of the shoot explants in modified Murashige and Skoog (MMS) media supplemented with 2 mg L⁻¹ benzyl adenine (MMS1c). Supplementation of 2 mg L⁻¹ naphthalene acetic acid (NAA) along with MMS1c (MMS2c) was responsible for estimable shoot induction and development in callus. 2,4-Dichloro acetic acid (2,4-D) played a crucial role in the floral induction of C. reflexa in vitro. MMS supplemented with 2 mg L⁻¹ NAA and 2 mg L⁻¹ 2,4-D (MMS3b) supported floral induction after shooting in vitro. MMS supplemented with 3 mg L⁻¹ 2,4-D (MMS4a) rapidly induced flower directly from the stem explants without showing any elongation of shoot. MMS1c along with MMS3b (MMS5a) showed callus proliferation followed by shoot elongation and floral induction. In vitro MMS5a grown plants show a sharp increase in the chlorophyll contents. Cytokinin treatment further increases the chlorophyll level of the plant.     

Long-term cultured callus and the effect factor of high-frequency plantlet regeneration and somatic embryogenesis maintenance in <Emphasis Type="Italic">Zoysia japonica</Emphasis>

In this study, we have demonstrated that Zoysia japonica callus induced from mature seeds can produce high frequencies of plant regeneration and somatic embryogenesis, even following a prolonged period of subculturing. Initial callus cultures were induced from mature seeds of Japanese lawngrass (Z. japonica Steud.) incubated on a medium containing major N₆ medium salts, minor Murashige and Skoog (MS) medium salts, and modified MS medium organic elements supplemented with 3 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.01–0.02 mg L⁻¹ 6-benzyladenine. Compact callus were selected and subcultured monthly on a medium containing 2 mg L⁻¹ 2,4-D, 0.5 mg L⁻¹ kinetin, 500 mg L⁻¹ casein hydrolysate, 500 mg L⁻¹ proline, and 500 mg L⁻¹ myoinositol. Callus maintained in vitro for 18 mo could be induced to regenerate plantlets with a frequency of >90%. By contrast, 36-mo-old callus cultures failed to produce normal shoot regeneration. However, the addition of CuSO₄ to the subculture media maintained >90% regeneration frequencies in such long-term callus cultures. Histological observations revealed that plant regeneration occurred both through somatic embryogenesis and organogenesis pathways. The ability to sustainable regeneration in long-term callus cultures will be valuable to the program of genetic transformation and somaclonal variant selection.