<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.     

Induction of <Emphasis Type="Italic">Gentiana cruciata</Emphasis> hairy roots and their secondary metabolites

Gentiana cruciata L. (cross gentian) is a medicinal and ornamental plant. The root extracts of this species are known to exhibit many curative properties. The natural Gentiana populations are exposed to great danger because of their uncontrolled usage. In this study, hairy roots from Gentiana cruciata L. stem and leaf explants belonging to three different clones were induced by inoculation with four different Agrobacterium rhizogenes wild strains namely A4, 15834, 8196 and R1000. Induction of the root transformation was significantly dependent on the explant type used. On the other hand, the genotype and bacterial strain had no significant effect on hairy root formation. Hairy root formation percentages of the explants varied between 5.6–33.3% in the stem explants, and between 0.0–6.7% in the leaf explants. Transformations of the hairy roots were confirmed by PCR using rolC specific primers, and revealed the absence of contaminating A. rhizogenes with virC primers. Total of twelve hairy root clones were obtained, and their secondary metabolite content was also analyzed by HPLC. Quantitative results exhibited that gentiopicroside was the most abundant compound in all root samples. Furthermore, metabolites such as loganic acid, swertiamarin, and sweroside were also identified and quantified in the samples.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the medicinal plant <Emphasis Type="Italic">Centaurea montana</Emphasis>

An efficient transformation system was developed for Centaurea montana by co-cultivation of leaf explants with Agrobacterium tumefaciens strain AGL1 that contained a plasmid harboring the isopentenyl transferase gene under the control of the developmentally regulated Atmyb32 promoter of Arabidopsis thaliana and the gene encoding for hygromycin resistance under the control of the Cauliflower Mosaic Virus 35S (CaMV35S) promoter. A total of 990 explants were infected with Agrobacterium, and 18 shoots were regenerated resulting in an overall transformation efficiency of 1.8%. Molecular analyses, including PCR, Southern blotting and RT-PCR, were performed on T₀ and T₁ plants to confirm chromosomal integration and expression of the transgene in the phenotypically normal transformed plants. Transformation of C. montana was also performed using A. tumefaciens supervirulent strain EHA105 harboring the β-glucuronidase (GUS) reporter gene. Expression of the GUS gene in the putative transgenics was confirmed using a histochemical GUS assay.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from the immature seeds of <Emphasis Type="Italic">Cymbidium faberi</Emphasis>

An in vitro plant regeneration protocol of Cymbidium faberi from immature seeds was established. The immature seeds of 50 days old started to form rhizomes 4 months after they were cultured on hormone free medium. The rhizomes multiplied 5 times when subcultured on the medium containing 1.0 mg l^–1 -naphthalene acetic acid (NAA) for 40 days and more than 90% of the rhizomes initiated shoots within 60 days on the media containing 0.5 or 1.0 mg l^–1 NAA plus 2.0 or 5.0 mg l^–1N⁶-benzylaminopurine (BA). Plantlets were regenerated when the shoots were planted on the basal medium amended with 1 g l^–1 activated charcoal for 50 days and the plantlets grew normally after transplanting.     

Lignan accumulation in two-phase cultures of <Emphasis Type="Italic">Taxus</Emphasis> x <Emphasis Type="Italic">media</Emphasis> hairy roots

The biosynthetic potential for six lignans accumulation in two lines of Taxus x media hairy roots was investigated. The cultures of KT and ATMA hairy root lines were supplemented with precursors: coniferyl alcohol (CA 1, 10 or 100 µM) and/or l-phenylalanine (100 µM PHEN) and/or methyl jasmonate (100 µM MeJa). Moreover the two-phase in vitro cultures supported with perfluorodecalin (PFD) as a gas carrier and in situ extrahent were used. The hairy root lines differed in lignan production profiles. In the control untreated cultures KT roots did not accumulate secoisolariciresinol and lariciresinol while ATMA roots did not accumulate matairesinol. In ATMA roots the treatment with CA (1 or 10 µM) resulted in the production of lariciresinol and secoisolariciresinol whereas solely lariciresinol was present after 100 µM CA application. Elicitation with 1 µM CA and MeJa yielded with hydroxymatairesinol aglyca and lariciresinol glucosides with their highest content 37.88 and 3.19 µg/g DW, respectively. The stimulatory effect of simultaneous treatment with 1 µM CA, PHEN and MeJa on lignan production was observed when the cultures were supplemented with PFD-aerated or degassed. In ATMA root cultures these applied conditions were the most favourable for matairesinol content which amounted to 199.86 and 160.25 µg/g DW in PFD-aerated and PFD-degassed supported cultures, respectively. In KT root cultures solely, hydroxymatairesinol and coniferin/CA content was enhanced with their highest yield 59.29 and 134.60 µg/g DW in PFD-aerated and PFD-degassed cultures, respectively.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of a medicinal plant <Emphasis Type="Italic">Taraxacum platycarpum</Emphasis>

Dandelion plants, the genus Taraxacum, are used in herbal medicine owing to their choleretic, diuretic and anti-carcinogenic activities and several medicinal compounds have been isolated from the roots of these plants. Metabolic manipulation of secondary metabolite biosynthesis is a potential strategy to improve the production of high-value secondary metabolites. The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is known to control a key regulatory step in the isoprenoid pathway. We report an efficient transformation protocol for stable introduction of HMGR into dandelion plants (Taraxacum platycarpum H. Dablstaed), which is essential for the biotechnological approach. The Agrobacterium tumefaciens strain EHA105 containing the binary vector, pCAMBIA1301, with GUS and HMGR genes, showed high transformation efficiency after 3–5 week hygromycin selection. Southern blotting, GUS staining and RT-PCR analyses demonstrated stable integration of one copy of the HMGR gene into the dandelion genome. Expression of the integrated genes was particularly eminent in root tissues of primary transformant plants. The establishment of an efficient transformation method may facilitate the improvement of medicinal plant in terms of the accumulation levels of secondary metabolites.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Fraxinus pennsylvanica</Emphasis> hypocotyls and plant regeneration

A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were transformed in the presence of 100 μM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l⁻¹ was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with 13.3 μM 6-benzylaminopurine, 4.5 μM thidiazuron, 50 mg l⁻¹ adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse. This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Stimulation of saponin production in <Emphasis Type="Italic">Panax ginseng</Emphasis> hairy roots by two oligosaccharides from <Emphasis Type="Italic">Paris polyphylla</Emphasis> var. <Emphasis Type="Italic">yunnanensis</Emphasis>

Two oligosaccharides, a heptasaccharide (HS) and an octasaccharide (OS), isolated from Paris polyphylla var. yunnanensis, stimulated the growth and saponin accumulation of Panax ginseng hairy roots at 5–30 mg l⁻¹. HS and OS at 30 mg l⁻¹, fed separately to hairy root cultures at 10 days post-inoculation, increased the root biomass dry weight by more than 70% to ∼20 g l⁻¹ from 13 g l⁻¹ and the total saponin content of roots by more than 1-fold to ∼3.5% from 1.6% (w/w). The results suggest that the two oligosaccharides may have plant growth-regulatory activity in plant tissue cultures.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Population genetic structure of the endangered and endemic medicinal plant <Emphasis Type="Italic">Commiphora</Emphasis><Emphasis Type="Italic">wightii</Emphasis>

Commiphora wightii is a medicinally important endangered species endemic to the Thar Desert of Rajasthan, India and adjoining areas of Pakistan. The populations of this species are declining sharply because of its extensive use as a natural herb. Random amplified polymorphic DNA analysis was conducted to find the genetic variation among 7 populations of C. wightii. Of the 100 random primers screened, 44 primers yielded 220 loci. Statistical analysis indicated low genetic diversity (H _pop = 0.0958; I = 0.1498; mean polymorphic loci = 14.28%), and high genetic differentiation among the populations (G _ST = 0.3990; AMOVA Φ _ST of 0.3390; Bayesian θ ^(II) = 0.3002). The low genetic diversity may be due to geographic isolation and restricted gene flow (N _m = 0.7533) between the fragmented populations. Unsustainable utilization of the plant has fragmented the population continuum which served the purpose of genetic exchange between populations. Mantel’s test was performed which revealed a highly significant positive correlation between genetic and geographic distance (r ² = 0.614, P = 0.023) among the populations studied. Low variation can also be attributed to poor seed setting and the slow growth pattern of the species, which is also an apomict. In UPGMA dendrogram the Commiphora wightii samples were divided into two major and one minor cluster. These findings can serve as a guide to preserving the genetic resources of this medicinal plant species.     

High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> transformed roots of the parasitic plant <Emphasis Type="Italic">Triphysaria versicolor</Emphasis> retain parasitic competence

Parasitic plants in the Orobanchaceae invade roots of neighboring plants to rob them of water and nutrients. Triphysaria is facultative parasite that parasitizes a broad range of plant species including maize and Arabidopsis. In this paper we describe transient and stable transformation systems for Triphysaria versicolor Fischer and C. Meyer. Agrobacterium tumefaciens and Agrobacterium rhizogenes were both able to transiently express a GUS reporter in Triphysaria seedlings following vacuum infiltration. There was a correlation between the length of time seedlings were conditioned in the dark prior to infiltration and the tissue type transformed. In optimized experiments, nearly all of the vacuum infiltrated seedlings transiently expressed GUS activity in some tissue. Calluses that developed from transformed tissues were selected using non-destructive GUS staining and after several rounds of in vivo GUS selection, we recovered uniformly staining GUS calluses from which roots were subsequently induced. The presence and expression of the transgene in Triphysaria was verified using genomic PCR, RT PCR and Southern hybridizations. Transgenic roots were also obtained by inoculating A. rhizogenes into wounded Triphysaria seedlings. Stable transformed roots were identified using GUS staining or fluorescent microscopy following transformation with vectors containing GFP, dsRED or EYFP. Transgenic roots derived from both A. tumefaciens and A. rhizogenes transformations were morphologically normal and developed haustoria that attached to and invaded lettuce roots. Transgenic roots also remained competent to form haustoria in response to purified inducing factors. These transformation systems will allow an in planta assessment of genes predicted to function in plant parasitism. Alexey Tomilov and Natalya Tomilova made an equal contribution in the paper.     

Induction and origin of adventitious roots from chimeras of <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Adventitious roots were induced from shoots and leaves of the chimera plant TCC (LI-LII-LIII = TCC; T = Tuber mustard, C = Red Cabbage), previously developed by in vitro grafting of tuber mustard (Brassica juncea) and red cabbage (B. oleracea). The regeneration frequency of adventitious roots from TCC shoots and leaf sections was markedly higher than that obtained from the parents TTT (tuber mustard) and CCC (red cabbage). Moreover, levels of α-naphthaleneacetic acid in the culture medium had lower effects on rooting efficiency of TCC chimeras compared to those of TTT and CCC. The number and fresh weight of adventitious roots per TCC shoot, 13.11 roots and 0.274 g, respectively, were also significantly higher than those of the parents. This demonstrated that replacing the histogenic LI layer (the outermost apical cell layer) with a different genotype might improve adventitious root induction capability of these vegetative tissues due to likely synergistic effects between LI and the other two histogenic layers, LII and LIII. Following polymerase chain reaction analysis and histological investigation, it was found that these adventitious roots originated from the LIII histogenic layer.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated transformation of <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> and production of flavonoids in hairy roots

Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A₄GUS, R1000 LBA 9402 and ATCC11325. The A₄GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15–20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B₅ medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A₄GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1–30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).     

<Emphasis Type="Italic">In vitro</Emphasis> shoot induction and plant regeneration from flower buds in <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchids

Paphiopedilum species are recalcitrant in tissue culture, and no explant from mature plants has been successfully mass propagated in vitro. This study was aimed at inducing shoots and regenerating plants from the flowering plants of a sequentially flowering Paphiopedilum Deperle and a single floral Paphiopedilum Armeni White. By using cross-sectioned flower buds (FBs), we found that in both species, only sections that contained the base tissue of FBs were able to produce shoots and plants. We have also found that sections of FBs between 1.5 and 3.0 cm from Paphiopedilum Deperle were able to produce shoots, but only sections of FBs >2.5 cm from Paphiopedilum Armeni White were regenerable. Our microscopic observations revealed that the small bract at the FB base harbored a new miniature FB, which further harbored a primitive FB with dome-shaped meristem-like tissues that presumably led to the plant induction. The reiteration of this pattern resulted in a scorpioid cyme inflorescence architecture in the multifloral Paphiopedilum species, and its failure to reiterate resulted in a single flower. The induction rates were 57–75%, and all plants survived in a greenhouse. This method is potentially applicable for the micropropagation and conservation of slipper orchids.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.     

Protoplast isolation and plant regeneration of different genotypes of <Emphasis Type="Italic">Petunia</Emphasis> and <Emphasis Type="Italic">Calibrachoa</Emphasis>

Protoplasts were isolated from leaves of in vitro-grown shoot cultures of different Petunia and Calibrachoa genotypes by enzyme digestion with 0.6% macerozyme R-10 and 2.0% cellulase R-10. Shoot regeneration was achieved in five out of nine Calibrachoa and three out of four Petunia genotypes. Protoplast yield and frequency of shoot regeneration varied among experiments and genotypes. Among all regenerants, few morphological changes, such as chlorophyll deficiency, variegated leaves, and polyploidization, were observed.