Micropropagation, <Emphasis Type="Italic">in vitro</Emphasis> flowering,and tuberization in <Emphasis Type="Italic">Brachystelma glabrum</Emphasis> Hook.f., an endemic species

Brachystelma glabrum Hook.f. is an endemic plant species of Eastern Ghats, India. In this study, efficient protocols for in vitro micropropagation, flowering, and tuberization of this plant were developed. Sterilized shoot tip and nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs) and additives for shoot induction and multiplication. Both shoot tip and nodal explants showed the best response (90 and 100%, respectively) on MS medium supplemented with thidiazuron (TDZ) at 1.0 mg L^?1. The microshoots multiplied best on MS + TDZ (1.0 mg L^?1) in combination with α-naphthaleneacetic acid (NAA) at 0.5 mg L^?1 and coconut water (CW) at 25%. The highest number of in vitro flowers (4.0 flowers per microshoot) was observed on MS medium supplemented with a combination of N6-benzyladenine (BA) and indole-3-butyric acid (IBA), each at 1.5 mg L^?1. In vitro-derived shoots produced aerial tubers on MS + TDZ (2.0 mg L^?1) + IBA (0.5 mg L^?1) and basal tubers on MS + TDZ at 2.0 mg L^?1. In vitro shoots were best rooted on half-strength (½) MS + NAA at 0.5 mg L^?1. The rooted plantlets were successfully acclimatized in pots with 70% survival after a hardening period of 1 mo. This protocol provides an effective method for the conservation of this endemic plant species.     

Efficient plant regeneration of the endangered medicinal orchid, <Emphasis Type="Italic">Coelogyne cristata</Emphasis> using protocorm-like bodies

An efficient method of Coelogyne cristata mass propagation was developed using segment of protocorm-like bodies (PLBs) (3 mm² in size). It was observed that ½ MS medium showed to be more effective to induce shoots through PLBs segment. The explants when cultured on ½ MS media containing TDZ and CP showed relatively superior effect on shoot regeneration as compared to the media containing TDZ alone or in combination with BP. Addition of BP and CP to the medium containing NAA and BA combinations proved distinctly better for shoot multiplication than that of the medium with NAA and BA combinations alone. The highest percentage of explants producing shoots, with a maximum average of 8.1 per explant, was induced on the medium supplemented with 1.0 mg l^?1 NAA and 0.5 mg l^?1 BA with CP. Shoots produced an average of 15 roots per explant on ½ MS medium supplemented with 2.0 mg l^?1 IBA and BP. The 4 cm height plantlets with well-developed roots were successfully acclimatized. The results suggest that CP and BP can be used effectively to initiate shooting and rooting of Coelogyne cristata. Ploidy analysis of regenerated plants using flow cytometry revealed the same ploidy level (diploid). This efficient and reliable protocol could be useful for mass multiplication and germplasm conservation of the wild medicinal orchid.     

Efficient regeneration system for rapid multiplication of clean planting material of <Emphasis Type="Italic">Ensete ventricosum</Emphasis> (Welw.) Cheesman

Enset (Ensete ventricosum (Welw.) Cheesman) is an economically important staple food crop in Ethiopia, especially in the southern and southwestern regions. It is called “false banana” due to its resemblance to banana, but inability to produce any edible fruit. The crop is clonally propagated using field-grown suckers. This study reports the development of a robust regeneration technique to propagate large numbers of plantlets using corm discs containing intercalary meristematic tissues. Hundreds of shoot buds were induced from corm discs of enset cultivar ‘Bedadeti’ cultured on Murashige and Skoog (MS) medium supplemented with 1.5 mg L^?1 2,4-dichlorophenoxyacetic acid, 0.216 mg L^?1 zeatin, and 2 g L^?1 activated charcoal. The shoot buds were regenerated into complete plantlets when transferred onto MS medium supplemented with 1 mg L^?1 6-benzylaminopurine and 2 g L^?1 activated charcoal. More than 100 plantlets were generated in 4 mo from corm discs isolated from a single in vitro mother plantlet. Well-rooted plantlets were acclimatized in soil with 100% success, and did not show any apparent phenotypic abnormalities under glasshouse conditions. This efficient regeneration system could be very useful for the rapid multiplication of clean pathogen-free planting material.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Effect of over-expression of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> PINORESINOL LARICIRESINOL REDUCTASE (<Emphasis Type="Italic">LuPLR</Emphasis>) gene in transgenic <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l^?1, kan 50 mg l^?1 and cephotaxime 62.5 mg l^?1. The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l^?1, kan 50 mg l^?1. Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid ½ MS medium containing 0.7 mg l^?1 indole 3-butyric acid (IBA) and 5 mg l^?1 kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3–0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.     

Micropropagation of <Emphasis Type="Italic">Asparagus macrorrhizus</Emphasis>, a Spanish endemic species in extreme extinction risk

Asparagus macrorrhizus: is a new species, which has been recently described. It is limited to the area surrounding the “Mar Menor” lagoon, in Murcia (Spain), and is the only “Critically Endangered” species of the genus Asparagus. Despite being protected, the number of plants has decreased in the last years due to the urbanization of its natural habitat. This species is a valuable genetic resource for asparagus breeding because of its special characteristics. So, the development of a micropropagation protocol is crucial to its conservation and use in breeding programs. The micropropagation protocol from asparagus rhizome buds previously developed by our research group has been adapted for A. macrorrhizus. Rhizome buds of A. macrorrhizus were extracted, disinfected, and then cultured on Asparagus Rhizome Bud Medium (ARBM) consisting of MS medium supplemented with 0.3 mg l^??1 NAA, 0.1 mg l^??1 KIN, 2 mg l^??1 ancymidol and 6% sucrose. A percentage of 69.7?±?8.0% of the rhizome buds developed shoots, but only 17.4?±?7.9% of them rooted. To increase this low rooting rate, the shoots were cultured on Macrorrhizus Rooting Media (MRM) supplemented with three different concentrations of IBA. The highest rooting rate (55.0?±?7.9%) was reached when shoots were incubated in MRM-2 consisting of MS medium supplemented with 2 mg l^??1 IBA and 4% sucrose. The acclimatization rate of the micropropagated plantlets was 90%. The method developed in this study allows the micropropagation of A. macrorrhizus, offering a new option to preserve this almost extinct species.     

In vitro shoot tip multiplication of bambara groundnut [<Emphasis Type="Italic">Vigna subterranea</Emphasis> (L.) Verdc.]

A rapid, simple and efficient protocol for direct in vitro multiple shoot induction and plantlet recovery was achieved from shoot tip explants of Bambara groundnut [Vigna subterranea (L.) Verdc.]. Shoot tips, were isolated from in vitro-grown seedlings and cultured on Murashige and Skoog basal medium (MS) containing Gamborg’ vitamins (B5) and supplemented with different concentrations of the plant growth regulators Thidiazuron (TDZ), N⁶-benzylaminopurine (BAP), Kinetin (KIN) and Adenine sulfate (ADS). TDZ 0.45 μM was found to be best for shoot multiplication with a mean of 11shoots per explant. Among the carbon sources tested, the best response in terms of mean number of shoots per explant was obtained with sucrose (11). The mean number of shoots per explant induced among the studied landraces of Bambara groundnut varied from 9 to 13. Individual shoots, aseptically excised, produced normal roots within 2 weeks on the basal MS medium supplemented with Indole Butyric Acid (IBA) or Naphthalene acetic acid (NAA). The highest number of roots per shoot and the longest roots were obtained on MS medium with 2 mg/L IBA. Rooted plantlets were successfully hardened under greenhouse conditions and subsequently established in the field conditions, with a recorded survival rate of 70 and 80?%, respectively. The transferred plants in the field were morphologically normal and fertile. This protocol can be efficiently used for mass propagation, germplasm preservation and probably also for gene transfer of Vigna subterranea (L.).     

High responsiveness in <Emphasis Type="Italic">de novo</Emphasis> shoot organogenesis induction of <Emphasis Type="Italic">Passiflora cristalina</Emphasis> (Passifloraceae), a wild Amazonian passion fruit species

The aim of the present study was to establish a regeneration system via de novo organogenesis from different types of non-meristematic explants of Passiflora cristalina. Leaf, hypocotyl, root segments, cotyledons, and endosperm of P. cristalina seeds were inoculated in Murashige and Skoog (MS)-basal medium, supplemented with different concentrations of 6-Benzyladenine (BA), Thidiazuron (TDZ), or Kinetin (KIN). BA was found to be the most efficient cytokinin in induction of de novo organogenesis from most the explants used in the study. The highest frequencies of adventitious bud formation in the hypocotyl and cotyledon explants were observed in medium supplemented with 1.0 mg L^?1 BA. For leaf and endosperm segments, the best concentration was 2.0 mg L^?1 BA; while for root segments, the highest mean values were observed with 1.0 mg L^?1 KIN. The different morphogenetic responses obtained from each explant source were characterized using light microscopy. P. cristalina revealed a remarkable organogenic potential, with superior production of adventitious shoots compared with the other Passiflora species evaluated elsewhere. These results will be helpful to establish a reproducible and reliable micropropagation protocol, as well as to implement conservationist and biotechnological-based genetic breeding strategies for this wild Passiflora species.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration and cryopreservation of <Emphasis Type="Italic">Arachis glabrata</Emphasis> (Fabaceae) using leaflet explants

Arachis glabrata Benth (perennial peanut) is a rhizomatous legume with high forage value and great potential for soil conservation as well as it displays valuable plant genetic resources for the cultivated edible peanut improvement. In this study, we developed for the first time successful protocols for micropropagation and cryopreservation of A. glabrata. First fully expanded leaflets from greenhouse-growing plants were efficiently established in vitro (93%) and displayed high frequency of bud induction (58%) on MS medium with 6 mg L^?1 1-fenil-3-(1,2,3-tiadiazol-5-il)urea [TDZ]. Whole plant regeneration was achieved via direct organogenesis by transferring the induced buds to MS media. Immature unexpanded leaves from micropropagated plants were effectively cryopreserved by using the droplet-vitrification technique. Maximum survival (~ 70%) and further regeneration (60–67%) were obtained by preconditioning immature leaves on semisolid MS with 0.3 M sucrose (1 d), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (30 min) followed by glycerol-sucrose plant vitrification solution PVS3 (150 min in ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on cryoplates. Tissues were rewarmed by plunging the aluminum foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and plant regeneration were efficiently achieved via shoot organogenesis, and somatic embryogenesis by culturing cryostored explants on MS added with 6 mg L^?1 TDZ. Genetic stability of plants derived from cryopreserved leaves was confirmed by random amplified polymorphic DNA markers. The protocols established in this study have great potential for rapid multiplication and conservation of selected A. glabrata genotypes.     

Optimizing micropropagation of drought resistant <Emphasis Type="Italic">Pyrus boissieriana</Emphasis> Buhse

The present study concentrated on introducing a micropropagation protocol for a drought resistant genotype from Pyrus boissieriana, which is the second most naturally widespread pear species in Iran with proper physiological and medicinal properties. Proliferating microshoot cultures were obtained by placing nodal segments on MS medium supplemented with BAP and IBA or NAA. The highest number of shoots (27 shoots per explant) were obtained with 1.5 mg l^?1 BAP and 0.05 mg l^?1 IBA, but this combination did not produce shoots of desirable length (>1.7 cm). Combination of 1.75 mg l^?1 BAP and 0.07 mg l^?1 IBA was the best for the shoot multiplication in P. boissieriana with a sufficient number of shoot production (22.33 shoots per explant) and relatively more appropriate shoot length. The larger and greenish leaves were obtained when PG was added to the best multiplication treatment. Microshoot elongation was carried out in 1/2 and 1/4 MS medium containing 50–100 mg l^?1 PG with different concentrations of IBA or NAA at intervals of 30–60 days. Significant increase in shoot length was detected after 45–60 days of culture in the presence of PG. The highest shoot length (8 cm) was recorded on 1/2 MS medium supplemented with 0.5 mg l^?1 IBA and 100 mg l^?1 PG. GA₃ negatively affected number and length of shoots and generally caused generation of red leaves. The highest percentage of root induction (100%) and root length (9 cm) were obtained on 1/6 strength MS medium supplemented with 0.005 mg l^?1 IBA. All plantlets were hardened when transferred to ex vitro conditions through a period of 25–30 days. The results suggest axillary shoot proliferation of P. boissieriana could successfully be employed for propagation of candidate drought resistant seedling.     

Application of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> (Chlorophyceae) as supplement and/or an alternative medium for the in vitro cultivation of <Emphasis Type="Italic">Schomburgkia crispa</Emphasis> (Orchidaceae)

Schomburgkia crispa Lindley (Orchidaceae) is an epiphytic species found in gallery forests and dry vegetation in the Brazilian Cerrado. It is typically unable to germinate or exhibits low germination because of dependency on mycorrhizal associations. In vitro cultivation techniques have helped circumvent difficulties involved in propagation from seeds. Alternative media and organic biostimulant substances that reduce costs and promote satisfactory in vitro growth are constantly sought. This study evaluated in vitro multiplication and rooting of S. crispa in a modified culture medium containing extract of the microalga Chlorella sorokiniana. We analyzed supplementation of WPM (Woody Plant Medium) with microalgae suspended in NPK medium, or as the supernatant resulting from the centrifugation of a culture in NPK medium. The extracts were added to WPM instead of distilled water. The compounds 6-benzylaminopurine (BAP) and indolebutyric acid (IBA) were used as reference in the in vitro multiplication and rooting of S. crispa, respectively. Both growth regulators were tested at 0, 2.5, and 5.0 mg L^?1. During in vitro multiplication of S. crispa, WPM supplemented with 5.0 mg L^?1 BAP favored the formation of more sprouts, whereas WPM containing 2.5 mg L^?1 IBA supplemented with microalgae extract stimulated in vitro rooting. Schomburgkia crispa explants cultivated in medium supplemented with microalgae suspension or the supernatant of C. sorokiniana showed growth similar to explants cultivated in WPM alone. Therefore, it is possible to use the microalga C. sorokiniana as a supplement and/or alternative to WPM for the in vitro cultivation of S. crispa.     

Efficient transformation of <Emphasis Type="Italic">Actinidia arguta</Emphasis> by reducing the strength of basal salts in the medium to alleviate callus browning

An efficient transformation system for high-throughput functional genomic studies of kiwifruit has been developed to overcome the problem of necrosis in Actinidia arguta explants. The system uses Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pART27-10 to inoculate leaf strips. The vector contains neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) (uidA) genes. A range of light intensities and different strengths of Murashige and Skoog (MS) basal salt media was used to overcome the problem of browning and/or necrosis of explants and calli. Callus browning was significantly reduced, resulting in regenerated adventitious shoots when the MS basal salt concentration in the culture medium was reduced to half-strength at low light intensity (3.4 μmol m^?2 s^?1) conditions. Inoculated leaf strips produced putative transformed shoots of Actinidia arguta on half-MS basal salt medium supplemented with 3.0 mg l^?1 zeatin, 0.5 mg l^?1 6-benzyladenine, 0.05 mg l^?1 naphthalene acetic acid, 150 mg l^?1 kanamycin and 300 mg l^?1 Timentin^®. All regenerated plantlets were deemed putative transgenic by histochemical GUS assay and polymerase chain-reaction analysis.     

Improvement of shoot proliferation and comparison of secondary metabolites in shoot and callus cultures of <Emphasis Type="Italic">Phlomis armeniaca</Emphasis> by LC-ESI-MS/MS analysis

Phlomis armeniaca Willd. is a medicinal plant in the Lamiaceae family endemic to Turkey. The present study describes efficient plant regeneration and callus induction protocols for P. armeniaca and compares phenolic profiles, total phenol and flavonoid contents, and free radical scavenging activity of in vitro-derived tissues. Stem node explants from germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with 75 plant growth regulator (PGR) combinations. The highest shoot number per explant, frequency of shoot proliferation, and frequency of highly proliferated, green, compact callus were obtained on MS medium containing 0.25 mg L^?1 thidiazuron (TDZ) and 0.25 mg L^?1 indole-3-acetic acid (IAA). The best root formation was on MS basal medium (control). Methanol extract of leaves obtained from regenerants contained higher total phenol and flavonoid contents than the callus extract. The callus extract showed stronger free radical scavenging activity than leaves with IC₅₀ [concentration inhibiting 50% of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical] values of 4.30 ± 0.08 and 2.21 ± 0.04 mg g^?1 dry weight in leaves and callus, respectively. Apigenin, caffeic acid, p-coumaric acid, luteolin, rutin hydrate, vanillic acid, ferulic acid, salicylic acid, sinapic acid, and chlorogenic acid were detected by liquid chromatography–electrospray ionization multistage tandem mass spectrometry (LC-ESI-MS/MS) analysis in in vitro-grown leaves and callus tissue. Rutin hydrate, p-coumaric acid, and vanillic acid were found at approximately tenfold higher levels in callus than in leaves. This new micropropagation protocol, the first for P. armeniaca, could be used in industrial production for new herbal tea and germplasm conservation.     

Construction of high efficiency regeneration and transformation systems of <Emphasis Type="Italic">Pyrus ussuriensis</Emphasis> Maxim

The purpose of the present study was to establish a simple and efficient protocol for pear regeneration and transformation. Major factors that influence multiplication, rooting, regeneration, and transformation (concentrations of plant growth regulators, pre-culture or co-culture times, and kanamycin concentration) were examined in wild Pyrus ussuriensis Maxim ‘Shanli’. The results showed that the best multiplication index (5.1) was obtained on Murashige and Skoog (MS) medium containing 1.5 mg L^?1 N6-benzyladenine (BA) and 0.2 mg L^?1 indole-3-butyric acid (IBA) with healthy and strong growth. On 1/2 MS medium supplemented with 1.0 mg L^?1 IBA or 0.2 mg L^?1 indole-3-acetic acid (IAA), the rooting percentage was up to 94.4%. The highest regeneration rate (100%) and number of adventitious shoots per explant (6.30) were obtained on Nitsch and Nitsch 1969 (NN69) medium containing 2.0 or 3.0 mg L^?1 thidiazuron (TDZ) and 0.2 mg L^?1 IAA with leaves as explants. Transformation was successfully achieved using the following protocol: cut-wounding leaves pre-cultured for 5 days were co-cultivated with the Agrobacterium tumefaciens strain EHA105 harboring 35S::DHN3 plasmid for 2 days, then were transferred to regeneration medium containing 15 mg L^?1 kanamycin to select the resistant plants, and followed by being cultured on rooting medium supplemented with 5 mg L^?1 kanamycin. PCR analysis revealed that 27 independent transgenic lines were obtained, with the transformation rate up to 11.72%. We postulate that the regeneration and transformation system in this study is an alternative method for pear breeding.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration and enhanced synthesis of plumbagin in root callus of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> L.—an important medicinal herb

Plumbago zeylanica L., an important medicinal herb, possesses plumbagin, a valuable secondary metabolite. Roots of this plant, collected from four locations in Himachal Pradesh, India, were screened for plumbagin content with high-performance liquid chromatography. The chemotype collected from Hamirpur yielded the highest content (26.47?±?0.63 mg g^?1 dry weight). Callus cultures were established from nodal explants of this chemotype on Murashige and Skoog (MS) medium augmented with α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, (2,4-D), 6-benzyladenine (BA), isopentenyl adenine (2iP), or thidiazuron, (TDZ). After 45 d, 98% of the cultures induced bright-green, compact callus on MS?+?5 μM TDZ. Upon subculturing, this callus differentiated an average of 4.08?±?1.16 shoots in 62.5% of the cultures. After elongation on basal MS medium, excised shoots were transferred to indole-3-acetic acid, NAA, or IBA supplemented MS medium. A maximum of 4.3?±?1.36 roots with an average length of 15.31?±?2.76 cm were recorded on 5 μM IBA. Rooted plantlets were successfully acclimatized in a greenhouse, and their genetic fidelity was evaluated using inter simple sequence repeats and start codon targeted molecular markers, which revealed 97% similarity. A significant increase in plumbagin content (6.5- and 3.4-fold) was achieved in root callus employing 100 mg L^?1 yeast extract (YE) and 25 μM salicylic acid (SA), respectively. This is the first report of large-scale propagation of P. zeylanica and an increase of plumbagin through in vitro root callus.     

Thidiazuron: an efficient plant growth regulator for enhancing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Petunia hybrida cv. Mitchell. Leaf explants of petunia were cultured on Murashige and Skoog (MS) medium with different concentrations of thidiazuron (TDZ) without auxin. The highest frequency of shoot regeneration (52.1%) and mean number of shoots per explant (4.1) were obtained on medium containing 2 mg l^?1 TDZ. Leaf explants inoculated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring ß-glucuronidase (uidA) and hygromycin resistance genes developed putative transformant shoots. The highest frequency of shoot regeneration (22.5%) and mean number of transformant shoots per explant (2.4) were obtained on a selection medium consisting of the above described regeneration medium and containing 25 mg l^?1 hygromycin as the selection agent. Approximately 95% of putative transformant shoots expressed the uidA gene following histochemical ß-glucuronidase (GUS) assay. These were confirmed to be transgenic by PCR analysis and Southern blot hybridization.     

Efficient organogenesis and plantlet regeneration in the timber species <Emphasis Type="Italic">Cunninghamia lanceolata</Emphasis> (Lamb.) Hook

Two efficient regeneration systems were developed in Cunninghamia lanceolata, the most important conifer for industrial wood production in China. Cotyledons and hypocotyls derived from greenhouse-grown seedlings were used as initial explants in our research. A high frequency (95.1?±?1.84%) of adventitious buds were initiated directly from cotyledons cultured on Douglas-fir cotyledon revised (DCR) medium supplemented with 1 mg l^?1 benzyladenine (BA), 0.1 mg l^?1 α-naphthaleneacetic acid (NAA), and 0.004 mg l^?1 thidiazuron (TDZ) with a maximum mean number of adventitious buds per cotyledon explant of 3.76?±?0.08. In contrast, a high percentage (93.73?±?0.55%) of adventitious buds regenerated via callus produced from hypocotyls cultured on DCR medium supplemented with plant growth regulators with a maximum number of adventitious buds per explant (16.71?±?0.34). Adventitious buds elongated on DCR medium supplemented with 0.2 mg l^?1 BA and 0.02 mg l^?1 NAA. After liquid pretreatment with 50 mg l^?1 indole-3-butyric acid (IBA), over 95% of the shoots successfully rooted on ½ DCR medium supplemented with 0.3 mg l^?1 IBA. The innovated systems reported in this study will be useful tools for future genetic manipulation of C. lanceolata and may be adapted for large-scale propagation in other conifers.     

Triploid plant regeneration from immature endosperms of <Emphasis Type="Italic">Melia azedazach</Emphasis>

Melia azedazach, a plant for forestation, is popular in many countries. Development of triploid M. azedazach varieties will provide additional advantages, such as faster growth, higher biomass, and; therefore, increased productivity. In this study, we aimed to develop triploid M. azedarach L. by immature endosperm tissue culture. After 22 days of initiation of cultures, calli of the endosperm were visible. After 50 days cultured on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l NAA and 1.0 mg/l BAP, maximum of callus induction rate from the immature endosperm with seed coat was obtained at 55.9%. The highest frequency of shoot induction from endosperm-derived callus was 98% and average of 16.7 shoots per explant on the medium supplemented with 1.5 mg/l BAP and 0.5 mg/l NAA after 42 days. A single shoot was detached from the multi-shoots and transferred to the rooting medium supplemented with 0.5 mg IBA, inducing root formation with 96.6% and with average of 5.8 roots per plantlet after 28 days. The plantlets transferred to polythene hycotrays containing soil and sand (mixture 1:1) in greenhouse showed 100% survival after transplantation. The endosperm-derived plantlets were 100% triploids as evidenced by flow cytometry analysis. Creating triploid M. azedazach plants by regenerating directly from endosperm (3n) described in this work required only 5 months whereas the traditional method of generating triploids through crossing between tetraploid (4n) and diploid (2n) plants could take up to 12 years.     

Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.