Neoxanthin from Helianthus,Taraxacum and Impatiens

Neoxanthin has been isolated from petals of Helianthus annuus, Taraxacum officinale and Impatiens nolitangere. It is identical with authentic neoxanthin from Euglena gracilis. MS and IR spectra were recorded, showing typical fragmentation patterns of neoxanthin, and an allene group. A pigment with the properties of deepoxineoxanthin and polar xanthophylls, which absorbed at very short wavelengths, were also isolated.     

Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale

Endemic Muscari muscarimi Medikus is the most fragrant plant among Muscari species and has a high ornamental potential. The natural populations of M. muscarimi, are severely affected by increased environmental pollution and urbanization. There is a need to develop a micropropagation method that should serve effectively for commercial propagation and conservation. Therefore, the study targeted to set up a strategy for efficient in vitro bulblet regeneration system of M. muscarimi using twin scale bulb explants on 1.0 × MS medium containing 4.44, 8.88, 17.76 μM BAP (6-Benzylaminopurine) plus 2.685, 5.37, 10.74 μM NAA (α-Naphthalene acetic acid). Maximum number of 19 daughter axillary bulblets and 16 daughter adventitious bulblets per twin bulb scale explant was regenerated on 1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA and 17.76 μM BAP plus 2.685 μM NAA respectively. The daughter bulblets regenerated on twin bulb scales on 8 out of 9 regeneration treatment could be easily rooted on 1.0 × MS medium containing 4.9 μM IBA (Indole-3-butyric acid). The daughter bulblets regenerated on 9th treatment (1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA) were transferred to 1.0 × MS medium containing 30 g/l sucrose to break negative carry over effect of this dose of BAP–NAA, where they grew 2–3 roots of variable length. Daughter bulblet diameter was increased by culturing them on 1.0 × MS medium containing 4.44 μM BAP plus 5.37 μM NAA. The results verified that both age and the source of explants had significant effect on regeneration. In another set of experiments, twin scales were obtained from in vitro regenerated daughter bulblets, although they induced bulblets, yet their bulblet regeneration percentage, mean number of bulblets per explant and their diameter were significantly reduced. In vitro regenerated bulblets were acclimatized in growth chamber under ambient conditions of temperature and humidity on peat moss, where they flowered. The study provides important information about selection of suitable micropropagation medium, strategies to improve bulblet diameter and rooting of M. muscarimi which offers a scope for commercial propagation.Abbreviations: MS medium, Murashige Skoog medium; BAP, 6-Benzylaminopurine; NAA, α-Naphthalene acetic acid; IBA, Indole-3-butyric acid     

In vitro propagation and withaferin A production in Withania ashwagandha,a rare medicinal plant of India

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15 mg/g of DW) production was obtained on MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0 μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90 % survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0 μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

In vitro propagation and characterization of phenolic content along with antioxidant and antimicrobial activities of Cichorium pumilum Jacq.

Cichorium pumilum, a member of Asteraceae, is widely used as a traditional medicinal herb. An efficient protocol for callus formation and whole plant propagation has been developed. Callus cultures were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with 1.5?mg?l^?1 6-Benzyladenine (BA) and 0.5?mg?l^?1 Naphthalene acetic acid (NAA). Maximum numbers of shoots were obtained from calli transferred to shoot regeneration medium containing MS basal medium with 1.5?mg?l^?1 BA or Kinetin (Kin). The shoots were effectively rooted on MS medium supplemented with different concentrations of Indole-3-butyric acid. In the present study, the antibacterial activity of C. pumilum extracts was assayed in vitro by agar disc diffusion and agar well diffusion methods against 10 different bacterial species. The results showed effect on the growth of 50 and 70% of the tested bacterial species using methanol and ethanol extracts respectively. Klebsiella pneumoniae was susceptible to the ethanolic and methanolic extracts of wild plants and in vitro tissues, whereas Enterococcus faecalis was resistant to all the extracts. This study verified that the methanol extracts have strong antioxidant activities with high levels of phenolic compounds. The antioxidant activity and total phenol content of callus cultures and in vitro plantlets were lower than those of the wild plants. The results obtained confirm the therapeutic potency of Cichorium used in the traditional medicine, in addition, the efficient in vitro production system developed in this study provide sterile and consistent tissues for the investigation of phytochemical and pharmacological effects and germplasm conservation of C. pumilum.     

Synergistic effect of BAP and GA3 on in vitro flowering of Guizotia abyssinica Cass.-A multipurpose oil crop

Apical and axillary buds of Guizotia abyssinica Cass., isolated from seedlings raised in vitro, were cultured. High frequency of shoot regeneration was achieved on MS medium with BAP (1 mgl⁻¹). Effect of BAP, Kn and GA₃ applied successively in culture on shoot regeneration and flower bud formation has been studied. The shoots differentiated in cultures elongated on this medium. These rooted subsequently on half strength MS medium. The shoots flowered in vitro on MS medium with a combination of BAP (0.1mgl⁻¹) + GA₃ (0.1 mgl⁻¹). The plantlets thus formed were successfully hardened with 90 % survival.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

A Rapid In Vitro Morphogenesis and Acclimatization Protocol for Balanites aegyptiaca (L) Del — a Medicinally Important Xerophytic Tree

The callus mediated regeneration system for Balanites aegyptiaca (L) Del is reported here. Different explants like apical buds, young thorns and cotyledon pieces from mature tree and root segments from in vitro raised seedlings were used for callus induction on MS medium supplemented with 2.23 μM 2,4-Dichlorophenoxyacetic acid. Seven to eight weeks old calli were transferred on hormone free MS medium in order to get regeneration. Shoot morphogenesis was achieved only from cotyledon-derived callus. The shoots so produced rooted well, when cultured on B5 medium supplemented with 9.84 μM Indole-3-butyric acid. Plantlets have been transferred to the field after two-phase hardening and are performing well.     

In vitro plant regeneration through direct organogenesis in Populus deltoides clone G48 from petiole explants

A rapid and efficient protocol is developed for in vitro plantlet regeneration of Populus deltoides clone G48 using petiole explants. The highest frequency of shoot regeneration (74.75%) from petiole was obtained on MS medium supplemented with 0.50?mg/l BAP and 0.20?mg/l IAA. The regenerated shoots started turning brown and necrotic after 10?C15?days in culture. To overcome the browning problem, the explants along with the developing shoot buds were transferred to modified MS medium containing 0.50?mg/l BAP, 0.20?mg/l IAA, 15?mg/l AdS, 0.1% PVP, 100?mg/l casein hydrolysate, 50?mg/l L-glutamine, 250?mg/l (NH₄)₂SO₄ and 0.5% agar. Shoot multiplication and elongation took place on the same medium. Indole-3-acetic acid at 0.10?mg/l was most effective for root regeneration. Using the current protocol, it took 2?months to regenerate plantlets. The in vitro regenerated plantlets were successfully acclimatized and established in greenhouse conditions. This regeneration system using petiole explants provides a foundation for Agrobacterium-mediated genetic transformation of P. deltoides clone G48 for incorporation of various silviculturally important traits.     

An efficient in vitro regeneration of Ceropegia noorjahaniae: an endemic and critically endangered medicinal herb of the Western Ghats

An efficient protocol was developed for the rapid in vitro multiplication of an endemic and critically endangered medicinal herb, Ceropegia noorjahaniae Ans., via enhanced axillary bud proliferation from nodal explants. The effects of phytohormones [6-benzylaminopurine (BAP), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)] on in vitro regeneration were investigated. The highest number of shoots (18.3 ± 1.3), maximum shoot length (10.1 ± 0.8 cm) and the highest response of shoot induction (95 %) were recorded on MS medium supplemented with 2.0 mg/l BAP. Rooting was best achieved on half-strength MS medium augmented with IBA (1.0 mg/l). Half-strength MS medium supplemented with BAP (4 mg/l) and sucrose (5 %, w/v) produced an average of 5.6 flower buds per microshoots with highest (90 %) flower bud induction response. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing sterile sand and coco peat (1:1) and grown in a greenhouse with 85 % survival rate. The regenerated plants did not show any detectable morphological variation. The developed method can be successfully employed for large-scale multiplication and conservation of C. noorjahaniae.     

Evaluation of phenolic profile,antioxidant and anticancer potential of two main representants of Zingiberaceae family against B164A5 murine melanoma cells

Background

Curcuma longa Linnaeus and Zingiber officinale Roscoe are two main representatives of Zingiberaceae family studied for a wide range of therapeutic properties, including: antioxidant, anti-inflammatory, anti-angiogenic, antibacterial, analgesic, immunomodulatory, proapoptotic, anti-human immunodeficiency virus properties and anticancer effects. This study was aimed to analyse the ethanolic extracts of Curcuma rhizome (Curcuma longa Linnaeus) and Zingiber rhizome (Zingiber officinale Roscoe) in terms of polyphenols, antioxidant activity and anti-melanoma potential employing the B164A5 murine melanoma cell line.

Results

In order to evaluate the total content of polyphenols we used Folin-Ciocâlteu method. The antioxidant activity of the two ethanolic extracts was determined by DPPH assay, and for the control of antiproliferative effect it was used MTT proliferation assay, DAPI staining and Annexin-FITC-7AAD double staining test. Results showed increased polyphenols amount and antioxidant activity for Curcuma rhizome ethanolic extract. Moreover, 100 μg/ml of ethanolic plant extract from both vegetal products presented in a different manner an antiproliferative, respectively a proapoptotic effect on the selected cell line.

Conclusions

The study concludes that Curcuma rhizome may be a promising natural source for active compounds against malignant melanoma.     

In vitro regeneration and Agrobacterium mediated genetic transformation of Artemisia aucheri Boiss

In the present study, we developed an efficient protocol for in vitro plant regeneration and genetically transformed root induction in medicinal plant Artemisia aucheri Boiss. Leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2, 4-dichlorophenoxyaceticacid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.05 mg/l NAA plus 2 mg/l BA (96.3 %) and MS medium supplemented with 0.5 mg/l IAA plus 2 mg/l BA (88.3 %). Root induction was obtained on MS medium supplemented with 0.5 mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. aucheri Boiss in short period via adventitious shoot induction approach. Also, an efficient genetically transformed root induction for A. aucheri was developed through Agrobacterium rhizogenes-mediated transformation by four bacterial strains, A4, ATCC15834, MSU440, and A13 (MAFF-02-10266). The maximum frequency of hairy root induction was obtained using MSU440 (93 %) and ATCC15834 (89 %) bacterial strains. Hairy root lines were confirmed by PCR using the rolB gene specific primers and Southern blot analysis.     

An in vitro propagation protocol of two submerged macrophytes for lake revegetation in east China

Thirty-six programs have been set up to revegetate the degraded lake wetlands in east China since 2002. Most projects however faced deficiency of submerged macrophyte propagules. To solve the problem, alternative seedling sources must be found besides traditional field collection. This paper deals with an in vitro propagation protocol for two popularly used submerged macrophytes, Myriophyllum spicatum L. and Potamogeton crispus L. Full strength Murashige and Skoog-based liquid media (MS) plus 3% sucrose in addition to 0–2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0–1.0 mg l⁻¹ indoleacetic acid (IAA) were tried for shoot regeneration. Meanwhile, full, half or quarter strength MS in addition to 0, 0.1 or 0.2 mg l⁻¹ naphthaleneacetic acid (NAA) were tested for root induction, respectively. Results indicated that both species had the ability of regeneration from stem fragments in MS without further regulators. However, the addition of 2.0 mg l⁻¹ BA with 0.2 or 1.0 mg l⁻¹ IAA in MS drastically stimulated the regeneration efficiency of M. spicatum, while the addition of 2.0 mg l⁻¹ BA with 0.2 or 0.5 mg l⁻¹ IAA in MS significantly stimulated that of P. crispus. For root induction, full strength MS in combination with 0.1or 0.2 mg l⁻¹ NAA was preferred by M. spicatum, and the same MS without or with 0.1 mg l⁻¹ NAA was preferred by P. crispus. Seedlings of each species produced from tissue culture room had a 100% survival rate on clay, sandy loam or their mixture (1:1) in an artificial pond, and phenotypic plasticity was exhibited when the nutrient levels varied among the three types of sediments. This acclimation of seedlings helped develop the shoot and root systems, which ensured seedling quality and facilitated the transplantation. Our study has established an effective protocol to produce high quality seedlings for lake revegetation programs at a larger scale. Since the two species we tested represent different regeneration performances in nature but shared similar in vitro propagation conditions, this study has indicated a potentially wide use of the common media for preparing seedlings of other submerged macrophytes.     

Ectopic expression of Atleafy in Brassica juncea cv. Geeta for early flowering

High temperature stress during pod filling severely affects the yield of Brassica juncea. Early flowering can evade the terminal heat stress and result in early maturity of the crop. In this study, a regeneration and transformation protocol has been standardized for B. juncea cv. Geeta. Hypocotyl from 5-day-old seedlings were used as explants. Of the various combinations of auxins and cytokinins tried along with Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium, MS + IAA (0.2 mg/l) + BA (3 mg/l) proved best for shoot regeneration with 89.9 % regeneration efficiency. To induce early flowering Leafy gene from Arabidopsis thaliana was transformed using Agrobacterium mediated transformation method. After 12 weeks transgenic plants showed flowering in vitro whereas their untransformed counterpart did not flower even after 16 weeks. The maximum transformation frequency was 4 %.     

In vitro regeneration of Eucalyptus camaldulensis

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l⁻¹ benzyl aminopurine (BAP) and 0.1 mg l⁻¹ naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l⁻¹ BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l⁻¹ Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60–148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.     

The influence of Agrobacterium rhizogenes on induction of hairy roots and ß-carboline alkaloids production in Tribulus terrestris L.

We have developed an efficient transformation system for Tribulus terrestris L., an important medicinal plant, using Agrobacterium rhizogenes strains AR15834 and GMI9534 to generate hairy roots. Hairy roots were formed directly from the cut edges of leaf explants 10–14 days after inoculation with the Agrobacterium with highest frequency transformation being 49 %, which was achieved using Agrobacterium rhizogenes AR15834 on hormone-free MS medium after 28 days inoculation. PCR analysis showed that rolB genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. Isolated transgenic hairy roots grew rapidly on MS medium supplemented with indole-3-butyric acid. They showed characteristics of transformed roots such as fast growth and high lateral branching in comparison with untransformed roots. Isolated control and transgenic hairy roots grown in liquid medium containing IBA were analyzed to detect ß-carboline alkaloids by High Performance Thin Layer Chromatograghy (HPTLC). Harmine content was estimated to be 1.7 μg g⁻¹ of the dried weight of transgenic hairy root cultures at the end of 50 days of culturing. The transformed roots induced by AR15834 strain, spontaneously, dedifferentiated as callus on MS medium without hormone. Optimum callus induction and shoot regeneration of transformed roots in vitro was achieved on MS medium containing 0.4 mg L⁻¹ naphthaleneacetic acid and 2 mg L⁻¹ 6-benzylaminopurine (BAP) after 50 days. The main objective of this investigation was to establish hairy roots in this plant by using A. rhizogenes to synthesize secondary products at levels comparable to the wild-type roots.     

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.     

Hybridization rate and genotypic diversity of apomictic hybrids between native (<Emphasis Type="Italic">Taraxacum japonicum</Emphasis>) and introduced (<Emphasis Type="Italic">T. officinale</Emphasis>) dandelions in western Japan

Hybridization between the introduced and native plants may enhance invasiveness, especially in asexually reproducing species. Hybrid apomictic dandelions between native (Taraxacum platycarpum and T. japonicum) and exotic (T. officinale) species are distributed widely throughout Japan. To estimate the origin(s) and dispersal of the hybrids, we investigated the hybridization rate and genotypic diversity in mixed populations of T. japonicum, T. officinale and their hybrids at two green parks in western Japan. Among the plants identified as exotics from flower morphology, 86–96% were hybrids by genetic analysis. Genetic data with simple sequence repeat markers revealed a high clonal diversity of the hybrid both within and between populations, indicating multiple origins. A hybrid seed was found from among the 1891 seeds collected from T. japonicum in the parks, indicating ongoing hybridization in the field. T. officinale and hybrids were genetically differentiated between the two parks independent of the ploidy level; the allele frequency of T. officinale and tri- and tetraploid hybrids were similar within each park but different between the two parks. This suggests that the origins of hybrids were similar within the park but different between the parks. Overall, our results suggest that hybridization, including backcross, is an ongoing process, and that genetically diverse hybrids with various origins have been spreading in western Japan, probably because hybridization enhanced invasiveness at native habitat.     

Plantlet regeneration through different morphogenic pathways in pommelo tissue culture

Pommelo (Citrus grandis Osbeck) plantlets were regenerated through different morphogenic pathways in culture. Multiple shoot regeneration through de novo organogenesis was obtained with epicotyl segments and root cultures. Shoot regeneration was observed in 84% of the midtal epicotyl segments cultured in Murashige and Skoog's medium (MS) with 2.2 M benzyladenine (BA) and 83% of the middle and proximal epicotyl segments cultured on basal medium. Isolated root segments cultured on medium containing 0.089 M BA showed best shoot regeneration at 71% with an average of 3.3 shoots per segment. Callus tissues derived from cotyledon and leaf explants regenerated shoots on BA-enriched medium. Shoots were also obtained at high frequencies from shoot-tip and nodal explants. Roots developed when regenerated shoots were excised and cultured on half strength MS medium with 2.5 M indolebutyric acid.Abbreviations BA 6-Benzyladenine - IBA Indole-3-butyric acid - MS Murashige and Skoog medium - NAA I-Naphthaleneacetic acid - 2,4-d 2,4-Dichlorophenoxyacetic acid