Goldenseal (<Emphasis Type="Italic">Hydrastis canadensis</Emphasis> L.): <Emphasis Type="Italic">In vitro</Emphasis> regeneration for germplasm conservation and elimination of heavy metal contamination

Summary Hydrastis canadensis L. (Goldenseal) is an endangered medicinal plant used in the treatment of many ailments, such as gastrointestinal disturbances, urinary disorders, hemorrhage, skin, mouth and eye infections, and inflammation. Commercial preparations of wild-harvested goldenseal were found to contain heavy metal contaminants including aluminum (848 μgg⁻¹), cadmium (0.4μgg⁻¹), lead (18.7μgg⁻¹), and mercury (0.1 μgg⁻¹). As well, goldenseal is an endangered species listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix II. Therefore, the practice of wild-harvest is actually decimating natural populations of goldenseal and endangering its genetic diversity. In vitro propagation protocol by tissue culture was developed for producing high-quality tissues of goldenseal. Significantly more de novo regeneration was induced on stem explants of 3-mo.-old plants cultured on a medium containing 10 μM 6-benzylaminopurine (BA) (22 regenerants per explant) than any other treatment. Subculture of the regenerants on a medium devoid of growth regulators resulted in the development of complete plants that were acclimatized and thrived in standard greenhouse conditions. The plants regenerated in vitro contained the lowest levels of heavy metals. The findings of this study provide the first evidence that heavy metal contaminants bioaccumulate in goldenseal tissues and also provide a method for germplasm conservation, mass multiplication, and production of goldenseal tissues free from abiotic contamination.     

Shoot regeneration of dwarf dogwood (Cornus canadensis L.) and morphological characterization of the regenerated plants

Dwarf dogwoods (or the bunchberries) are the only suffrutex in Cornaceae. They are attractive ground cover ornamentals with clusters of small flowers surrounded by petaloid bracts. Little has been reported on plant regeneration of dogwoods. As a step toward unraveling the molecular basis of inflorescence evolution in Cornus, we report an efficient regeneration system for a dwarf dogwood species C. canadensis through organogenesis from rejuvenated leaves, and characterize the development of the plantlets. We used the nodal stem segments of vegetative branches as explants. Micropropogated shoots were quickly induced from axillary buds of nodes on an induction medium consisting of basal MS medium supplemented with 4.44 μM BAP and 0.54 μM NAA. The new leaves of adventitious shoots were used as explants to induce calli on the same induction medium. Nearly 65% of leaf explants produced calli, 80% of which formed adventitious buds. Gibberellic acid (1.45 μM) added to the same induction medium efficiently promoted quick elongation of most adventitious buds, and 0.49 μM IBA added to the basal MS medium promoted root formation from nearly 50% of the elongated shoots. The growth of plantlets in pot soil was characterized by the development of functional woody rhizomes, which continuously developed new aboveground vegetative branches, but not flowering branches, within the past 12 months. Potential reasons causing the delay of flowering of the regenerated plants are discussed. The establishment of this regeneration system facilitates developing a genetic transformation system to test candidate genes involved in the developmental divergence of inflorescences in Cornus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tissue culture and long-term regeneration of Phragmites australis (Cav.) Trin. ex Steud.

Phragmites australis tissue cultures were initiated from mature seeds on MS medium supplemented with 1 mgl^-1 each of 2,4-D and IAA. Cultures displayed typical embryogenic callus that was compact and bright yellow. Selection for embryogenic callus established long-term regenerable cultures. Removal of auxin from the basal medium allowed numerous complete plants to be recovered from the cultures. Histological study indicated both the presence of embryogenic-type cells and the bipolar development of regenerated plants.     

Restoration of Hydrastis canadensis: Experimental Test of a Disturbance Hypothesis After Two Growing Seasons

This paper tests the hypothesis that disturbance simulation is beneficial for restoration of Hydrastis canadensis (goldenseal) 2 years after a disturbance event. This takes into account possible delay in resource allocation to reproduction. At five deciduous woodland sites in Ontario, Canada, three disturbance simulation treatments, fertilization, soil turnover, fertilization + soil turnover, and a control were randomly applied to experimental blocks in 1999. Seeds and mature rhizomes were transplanted into each treatment plot. Seed production and seedlings were recorded in 2001. Transplants were measured and flowers and fruit recorded in 2000 and 2001. Soil turnover and fertilization + soil turnover significantly increased the production of flowers and fruit in the second year after disturbance, contrasting with lack of significance in the first year. The effect on plant size was opposite, with significantly increased cover in the first year but not the second. Soil turnover + fertilization significantly increased seed production. The disturbance treatments did not have a significant effect on number or presence of seedlings possibly due to extraordinary drought conditions. Consideration of second‐year results strongly suggests that (1) substrate disturbance, in addition to canopy gaps, is an important factor in woodland herb ecology; (2) certain woodland herbs may be rare due to lack of specific disturbances which were more prevalent in the past; and (3) mature woodlands may require management, involving disturbance simulation, for the protection and restoration of certain rare woodland flora.     

Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons

For the purpose of developing an in vitro regeneration system for chickpea (Cicer arietinum L.), an important food legume, immature cotyledons approximately 5 mm long were excised from developing embryos and cultured on B5 basal medium supplemented with 1.5% sucrose and various growth regulator combinations. Only non-morphogenic callus was formed in response to concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, 4.6, 13.7, and 45.6 M zeatin induced formation of white, cotyledon-like structures (CLS) at the proximal end of immature cotyledons placed with adaxial surface facing the agar medium. No morphogenesis, or occasional formation of fused, deformed CLS, was observed when zeatin was replaced with kinetin or 6-benzyladenine, respectively. The highest response frequency, 64% of explants forming CLS, was induced by 13.7 M zeatin plus 0.2 M indole-acetic acid (IAA). Within 20–40 days culture on zeatin, shoots formed at the base of CLS on approximately 50% of CLS-bearing explants, and proliferated upon subsequent transfer to basal medium with 4.4 M BA or 4.6 M kinetin. This regeneration system may be useful for genetic transformation of chickpea.     

Improved method of regeneration of black gram (Vigna mungo L.) through liquid culture

Summary A very rapid and efficient regeneration method of Vigna mungo L. has been established using liquid culture. A highly regenerable explant, viz., young multiple shoots obtained by germinating the seeds in 2 mgl⁻¹ (8.9μM) N⁶-benzyladenine-supplemented Murashige and Skoog (MS) medium, was used as a source of tissue to initiate the liquid culture. The liquid medium consisted of half-strength B5 or MS salts supplemented with MS organics, α-naphthaleneacetic acid (0.1 mgl⁻¹, 0.54μM) and N⁶-benzyladenine (0.5mgl⁻¹, 2.2μM). Transferring the growing tissues to fresh medium every third day resulted in ca. 142% increase in the number of shoot buds produced after 24d. Shoot buds elongated on one-third-strength MS (MS_1/3) semisolid medium and plantlets were obtained by transferring the shoots onto MS_1/3 semisolid medium supplemented with indolebutyric acid (1 mgl⁻¹, 4.9 μM).     

Somaclonal variation from Sorghum bicolor (L.) Moench cell culture

Plant Cell, Tissue and Organ Culture (PCTOC) - Sorghum bicolor (L.) Moench, plants were regenerated from 4 to 5 month old callus cultures originally derived from seedling explants. Somaclonal...     

A comparison of methods for callus culture and plant regeneration of RD25 rice (Oryza sativa L.) in two laboratories

While methodology is transferable from one laboratory to another, an exact transfer does not usually occur and even a nearly exact transfer of methods does not always result in repeatable data. Researchers should not expect that an effort to duplicate a published procedure will necessarily lead to identical results.In attempting to transfer rice tissue culture methods between laboratories in Fort Collins, Colorado, USA and Bangkok, Thailand, we discovered that a combination of the methods of each laboratory produced the best results in term of callus productions and plant regeneration. In the experiments reported here, the type of culture vessel used and the geographical location were also important variables.Supported by the USAID/Cooperative Agreement No DAN-4137-A-00-4053-00.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

Somatic embryogenesis and plant development from anther culture of Vitis vinifera (L.)

Anthers from Frumoasa alba (White beauty), Otilia, Valerien, Mission and Siegfried Rebe (FS₄) cultivars were cultured at the uninucleate stage of the microspore on Murashige and Skoog (1962) and Nitsch and Nitsch (1969) media supplemented with 2,4-dichlorophenoxyacetic acid (4.9 M) and benzyladenine (4.4 M). The primary calli were subcultured on MS medium with 6.6 M BA and 1.1 M indolylacetic acid, in order to induce their growth and plant regeneration. After seven months, vegetative buds were obtained with Frumoasa alba (2.7%), Otilia (0.3%), Valerien (4.5%), embryogenic callus was obtained with Mission and plant regeneration with Siegfried Rebe. Long term embryogenesis was maintained in Mission cv. for four years, by selection and regular transfer of the embryogenic areas of anther-derived calli. The embryogenic calli have the ability to generate abnormal somatic embryos with one, two or three cotyledons and cup or trumpet-shaped with fused cotyledons. In parallel with the embryogenic process, organogenesis with buds, leaf and shoot differentiation was regularly observed.     

Plant regeneration fromBrassicanigra (L.) Koch stem protoplasts

Summary Protoplasts ofBrassica nigra (L.) Koch were isolated from stem peels of bolting racemes and cultured in 1.5 ml of VN1 liquid medium. The protoplasts in the liquid medium were plated on top of half strength MS medium supplemented with 400 mg/liter glutamine, 15 mg/liter glutathione, 50 mg/literl-serine, 0.25 mg/liter 6-benzylaminopurine, 0.5 mg/liter 2,4-dichlorophenoxyacetic acid, 1.5% sucrose, and 5% mannitol, pH, 5.7, solidified with 0.3% agarose. Ten percent of calli obtained from the protoplasts developed into plantlets within 4 wk after transfer onto 2N regeneration medium which contains MS salts plus 200 mg/liter casein hydrolysate, 0.625 mg/liter 6-benzylaminopurine, 0.625 mg/liter kinetin, 0.625 mg/liter 6-(γ,γ-dimethylallylamino)-purine, 0.625 mg/liter zeatin, 0.5 mg/liter 1-naphthaleneacetic acid, 1.5% sucrose, and 0.4% agarose. THis is the first report of plant regeneration fromB. nigra protoplasts.     

Antimicrobial fungal endophytes from the botanical medicine goldenseal (Hydrastis canadensis)

The potential of fungal endophytes to alter or contribute to plant chemistry and biology has been the topic of a great deal of recent interest. For plants that are used medicinally, it has been proposed that endophytes might play an important role in biological activity. With this study, we sought to identify antimicrobial fungal endophytes from the medicinal plant goldenseal (Hydrastis canadensis L., Ranunculaceae), a plant used in traditional medicine to treat infection. A total of 23 fungal cultures were obtained from surface-sterilized samples of H. canadensis roots, leaves and seeds. Eleven secondary metabolites were isolated from these fungal endophytes, five of which had reported antimicrobial activity. Hydrastis canadensis plant material was then analyzed for the presence of fungal metabolites using liquid chromatography coupled to high resolving power mass spectrometry. The antimicrobial compound alternariol monomethyl ether was detected both as a metabolite of the fungal endophyte Alternaria spp. isolated from H. canadensis seeds, and as a component of an extract from the H. canadensis seed material. Notably, fungi of the Alternaria genus were isolated from three separate accessions of H. canadensis plant material collected in a time period spanning 5 years. The concentration of alternariol monomethyl ether (991 mg/kg in dry seed material) was in a similar range to that previously reported for metabolites of ecologically important fungal endophytes. The seed extracts themselves, however, did not possess antimicrobial activity.     

Fertile wheat (Triticum aestivum L.) regenerants from protoplasts of embryogenic suspension culture

We report regeneration of fertile, green plants from wheat (Triticum aestivum L. cv. Aura) protoplasts isolated from an embryogenic suspension initiated from somatic early-embryogenic callus. The present approach combines the optimization of protoplast culture conditions with screening for responsive genotypes. In addition to the dominant effect of the culture media, the increase in fresh mass and the embryogenic potential of somatic callus cultures varied considerably between the various genotypes tested. Establishment of suspension cultures with the required characters for protoplast isolation was improved by reduction of the ratio between cells and medium and by less frequent (monthly) transfer into fresh medium. A new washing solution was introduced to avoid the aggregation of protoplasts. However, the influence of the culture medium on cell division was variable in the different genotypes. We could identify cultures from cultivar Aura that showed approximately a 9% cell division frequency and morphogenic response. The protoplast-derived microcolonies formed both early and late-embryogenic callus on regeneration medium and green fertile plants were obtained through somatic embryogenesis. The reproducibility of plant regeneration from protoplast culture based on the cultivar Aura was demonstrated by several independent experiments. The maintenance of regeneration potential in Aura suspension cultures required establishment of new cultures within a 9-month period.     

Callus growth and plant regeneration in diverse cultivars of cucumber (Cucumis sativus L.)

The growth and differentiation of callus tissues derived from cotyledons of ten cultivars ofCucumis sativus L. were investigated. Cotyledonary explants from all ten cultivars formed callus tissue on Murashige and Skoog (MS) medium supplemented with 0.5 M 2,4-dichlorophenoxyacetic acid and 5 M 6-benzylaminopurine. Fresh weight of the callus tissues averaged 1 to 8 g per flask after five weeks of culture. Shoot development was achieved in three cultivars, Hukchinju, Manchoonchoungjang and Seoul, on MS medium supplemented with 0.5 M -naphthaleneacetic acid and 5 M 6-benzylaminopurine. Reducing the 6-benzylaminopurine concentration to 0.01 M resulted in root formation on callus tissues and on shoots transferred to this medium. All cultivars gave the same response in tests of root formation, but shoot regeneration from callus culture of cucumber cotyledons was dependent on genotype with cultivar Manchoonchoungjang exhibiting the best shoot differentiation capability among the genotypes examined. Examination of mitotic metaphase from the regenerants revealed that all were tetraploid.     

Tissue culture studies on species ofPassiflora

The effect of different explant sources, growth regulators and coconut water concentrations and also light radiation on tissue cultures ofPassiflora edulis var.flavicarpa was evaluated.In addition, plant regeneration was obtained from a diversity ofPassiflora germplasm. For shoot morphogenesis either cotyledonary, hypocotyledonary or leaf tissues were used to obtain cultures that formed shoots when placed on Murashige & Skoog basal medium containing 6-benzylaminopurine and coconut water. Direct organogenesis was observed.Apex cultures have been also achieved fromP. edulis var.flavicarpa, P. mollissima, P. giberti, P. maliformis andP. amethystina. Excised explants 5 mm length were placed on half-strength basal medium, free of hormones. Whole plants were recovered.Abbreviations BA 6-benzylaminopurine - CW coconut water - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

Conditions for plant regeneration from excised cotyledons of Vigna radiata were studied. Complete plant developed from the uncallused proximal ends of cotyledons on Murashige & Skoog's (MS), Gamborg's (B₅) and C (MS salts + B₅ vitamins) basal media. The basal medium C was found to be best for plant regeneration. Regeneration frequency, however, varied with genotype, size, orientation and age of explant and the different plant growth regulators combination in the medium. Addition of cytokinins induced callusing at the proximal ends of cotyledons followed by multiple shoot formation. Out of 6-benzyl aminopurine (BAP), kinetin (KIN), N (^–2 isopentyl) adenine (2iP) and adenine sulphate (AS), only BAP and KIN were found to be more effective in enhancing the frequency of shoot regeneration. BAP at 1×10^-1M induced maximum (60%) shoot regeneration whereas maximum number of shoots (8 to 9 shoots) per explant was observed with 5×10^-6M BAP. Cotyledons excised from two-day old seedlings were most regenerative. The regenerative response of cotyledons decreased when sliced into two equal parts either longitudinally or transversely. Callusing and organogenic differentiation occured only if the petiolar end of cotyledons was in contact with medium. None of the tested treatments were effective in inducing shoot bud differentiation from subcultured callus. Well developed shoots rooted when incubated on half strength MS, MS and MS basal medium supplemented with IAA (5×10^-6M). The rooted plants were transferred to pots and later established in the field with 60% success.Abbreviations AS adenine sulphate - BAP 6-benzylaminopurine - B₅ medium after Gamborg et al. [6], - C Medium with MS salts + B₅ vitamins - 2iP N (^–2 isopentyl) adenine - IAA indole-3-acetic acid - KIN Kinetin - MS medium after Murashige & Skoog [21] - NAA 1-napthaleneacetic acid     

Tissue culture in Haworthia

Summary Plants regenerated on two different media (NK and I) from the calluses of simple or cloned subcultures, which were originated from a single stock callus of Haworthia setata derived from its flower bud, were observed for eight characters, i.e., somatic chromosome number in root tips, growth vigor, leaf shape, leaf color, number of stomata per unit leaf area, esterase zymogram, chromosome association at meiotic metaphase I in pollen mother cells, and pollen fertility. From these regenerates plants with different characters from those of the parental plant were obtained. With regards to chromosomal aberrations, tetraploids, aneuploids, plants with a part of the chromosome segment deleted, with reciprocal and non-reciprocal translocations, or with paracentric inversions and those showing sub-chromatid aberrations at meiosis were obtained. The NK medium tended to regenerate more tetraploids and less plants carrying translocation than the I medium.Chromosome variabilities in somatic cells of the regenerates correlated with those of the calluses, from which they regenerated, while they did not correlate with either the meiotic irregularities (chromosome association at MI) or pollen fertility of the regenerates. From these facts, it was concluded that a rather large number of callus cells participate in the regeneration of an individual plant, although, however, only a few limited types of the cells form its germ line.Polyploidy affected growth vigor, leaf shape, stomata number and chromosome association at MI, but its effects were not detected on other characters. Chromosomal aberrations at the diploid level produced no clear changes in the regenerate's phenotype except in meiotic chromosome configuration and pollen fertility.Most chromosomal variants obtained in the present study are already reported in plants collected from wild populations, but plants with the deletion of a whole chromosome (karyotype 7L+6S) or chromosome segment (7L+1M+6S and 14L+2M+12S) have never been reported: this fact suggests that tissue culture is a powerful tool for producing plants with novel karyotypes.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 436     

Anther culture of Sorghum bicolor (L.) Moench

The sequence of pollen development from the tetrad stage to the mature tricellular grain was studied in freshly harvested anthers of Sorghum bicolor. This pattern of development was then compared with that occurring during panicle pretreatment and subsequent anther incubation in vitro. It was found that during pretreatment at 7° C mitoses of the vegetative cell were induced in up to 30% of the pollen. During anther incubation procallus development was highly polarised with contributions from both the generative and vegetative cells. After pretreatment at 14 or 20° C the generative cell became detached from the pollen wall and it was not possible to determine whether subsequent development involved only the vegetative cell or both the vegetative and generative cells.Although retarded pollen grains were observed both in vivo and in vitro, and were occasionally seen to divide in culture, they did not appear to be the source of the procalluses produced.     

Micropropagation systems for the mexican redbud (Cercis canadensis var. Mexicana L.) and other woody plants of the chihuahuan desert

Summary There are many Chihuahuan desert species that have potential as landscape plants for the arid communities of the southwestern United States [agarita, Berberis trifoliata Moric.; Mexican buckeye, Ungnadia speciosa Endl.; Texas madrone, Arbutus xalapensis var. texana (Buckl.). A. Gray]. Within these plant populations, there are superior genotypes that offer even greater interest for the landscape. However, it is difficult to clonally propagate many of these species with conventional techniques, and the seed-derived populations often do not breed true. Therefore, selection of superior genotypes in wild populations coupled with clonal propagation through tissue culture may offer an attractive option. It is relatively easy to achieve disinfestation of explants from desert plants due to a general lack of natural surface contamination by fungi and bacteria, even though interference from numerous trichomes can impede good contact with disinfesting agents. However, there is only a narrow window of time that is ideal for explant collection, because of the brief, periodic flushes of growth that characterize this unusual plant group. There may be years when, due to the harsh environment, the amount of suitable explant material is severely limited. Phenolics and exudates are also problematic in this group of plants, and acclimatization of ex vitro plantlets to the harsh desert environment is a particular challenge. For these reasons, specific adaptations and modifications were necessary to achieve success with micropropagation of desert plant species such as Mexican redbud (Cercis canadensis var. mexicana L.).