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.     

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.     

An efficient protocol for <Emphasis Type="Italic">in vitro</Emphasis> regeneration and conservation of Shirui lily (<Emphasis Type="Italic">Lilium mackliniae</Emphasis> Sealy): a lab-to-land approach to save the rare endangered Asiatic lily species

An efficient protocol for direct and indirect shoot regeneration and proliferation from bulb scales of Shirui lily (Lilium mackliniae Sealy), an endangered Asiatic lily species endemic to the Shirui hill peak, Manipur, India, has been developed. Bulb scales were isolated from mature bulbs and cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin (KIN), or thidiazuron (TDZ). For direct shoot regeneration from bulb scale explants, 0.5 mg L^?1 BAP yielded the highest shoot induction (3.5 shoots per scale; a 96.7% response). For indirect de novo organogenesis, optimum callus induction was achieved with 2.0 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-D), and shoot organogenesis was higher (16.2) when subcultured onto 0.5 mg L^?1 BAP medium. Multiple shoot regeneration and pseudo-bulb formation protocols were assessed; the highest shoot proliferation (10.1) occurred with 0.5 mg L^?1 BAP and 1.0 mg L^?1 gibberellic acid (GA₃). Rooting response was 96% with 0.5 mg L^?1 1-naphthalene acetic acid (NAA), with multiple roots per shootlet. Plantlet survival was increased to 92.5% during the hardening-off process by using hydroponics with Hoagland’s solution in a mist chamber. Clonal fidelity was assessed through random amplified polymorphic DNA (RAPD) analysis comparing the mother plant and regenerated plantlets. After confirming genetic uniformity, the pseudo-bulblets with four to six leaves and three to four roots were successfully established at the Shirui hills peak. This in vitro regeneration and ex vitro conservation approach could be helpful to save this rare endangered species in a sustainable way.     

In vitro propagation,ex vitro rooting and leaf micromorphology of <Emphasis Type="Italic">Bauhinia racemosa</Emphasis> Lam.: a leguminous tree with medicinal values

A micropropagation system for Bauhinia racemosa Lam. was developed involving axillary shoot proliferation and ex vitro rooting using nodal explants obtained from mature tree. MS medium with 3.0 mg l^?1 BA (6-benzyladenine) was optimum for shoot bud induction. For shoot multiplication, mother explants were transferred repeatedly on medium containing low concentration of BA (0.75 mg l^?1). Number of shoots was increased up to two passages and decreased thereafter. Shoot multiplication was further enhanced on MS medium containing 0.25 mg l^?1 each of BA and Kin (Kinetin) with 0.1 mg l^?1 of NAA (α-naphthalene acetic acid). Addition of 0.004 mg l^?1 TDZ (thidiazuron) increased the rate of shoot multiplication and 21.81 ± 1.26 shoots per culture vessel were obtained. In vitro regenerated shoots were rooted under ex vitro conditions treated with 400 mg l^?1 IBA (indole-3-butyric acid) for 7 min on sterile soilrite. After successful hardening in greenhouse, ex vitro rooted plants were transferred to the field conditions with ≈85% of survival rate. Micromorphological changes were observed on leaf surface i.e. development of vein density and trichomes and stomatal appearance, when plants were subjected to environmental conditions. This is the first report on in vitro regeneration of B. racemosa from mature tree.     

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.     

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> preservation and micropropagation of <Emphasis Type="Italic">Oreocharis mileense</Emphasis> (W.T. Wang) M. Möller &amp; A. Weber (Gesneriaceae) through shoot organogenesis

Oreocharis mileense (W.T. Wang) M. Möller & A. Weber is endemic to China and was considered to be extinct because it had not been seen in the wild since the first collection in 1906. In 2006, the species was rediscovered in Shilin County, Yunnan Province. Oreocharis mileense was considered critically endangered for its narrow geographic range and extremely small population. An efficient method to preserve plant germplasm by in vitro culturing of O. mileense has not been reported. In this study, an orthogonal array with three factors (6-benzyladenine, BA; α-naphthaleneacetic acid, NAA; and sucrose), at four levels was performed, and shoot induction as well as shoot proliferation were recorded. The results were analyzed to determine the most significant components and the optimum combination for micropropagation of O. mileense. The results showed that: (1) organogenesis was easily induced by different combinations of plant-growth regulators and sucrose; (2) NAA and sucrose had the most significant effect on shoot induction and shoot multiplication, and (3) the optimum induction and proliferation media were 0.5 mg L^?1 BA + 0.2 mg L^?1 NAA?+?30 g L^?1 sucrose and 1 mg L^?1 BA + 0.1 mg L^?1 NAA?+?30 g L^?1 sucrose, respectively.     

Quercetin and silver nitrate modulate organogenesis in <Emphasis Type="Italic">Carissa carandas</Emphasis> (L.)

An in vitro organogenesis protocol for Carissa carandas L. was developed using an auxin transport inhibitor (quercetin) and silver nitrate (AgNO₃), an inhibitor of ethylene action, in association with cytokinins in the culture medium. This protocol produced the maximum number of shoots from aseptic seedling-derived shoot apex explants of C. carandas. The highest rate of shoot multiplication was recorded on MS medium containing 2.0 mg L^?1 6-benzylaminopurine; 0.5 mg L^?1 kinetin, and 0.75 mg L^?1 quercetin at after 4 wk of culture. Similar results were obtained when MS medium fortified with 2.0 mg L^?1 BAP, 0.5 mg L^?1 kinetin, and 1.5 mg L^?1 AgNO₃ was used. However, successful rooting was achieved on quarter strength MS medium with 0.5 mg L^?1 indole-3-acetic acid. In this study, an inhibitor of auxin transport and ethylene action maximized shoot multiplication in medium fortified with cytokinins. The established rapid micropropagation method could be used to conserve elite genotypes of C. carandas.     

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.     

Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration,production, and storage of artificial seeds in <Emphasis Type="Italic">Ceropegia barnesii</Emphasis>, an endangered plant

Approaches for in vitro regeneration and fabrication of synthetic seeds were formulated to support restoration in the wild and genetic manipulation of Ceropegia barnesii (categorized as endemic and endangered). MS medium augmented with 4 mg L^?1 benzyl adenine was most advantageous for the production of multiple shoots from nodal explants. Fabrication of synthetic seeds was accomplished by sodium alginate encapsulation of nodes from microshoots. The most favorable medium combination for the induction of multiple shoots from synthetic seeds was MS medium complemented with 4 mg L^?1 benzyl adenine and 1 mg L^?1 gibberelic acid. Following root induction promoted by half strength MS basal medium augmented with indolebutyric acid, multiple shoots were subjected to hardening. Influence of vesicular-arbuscular mycorrhizal fungi on the hardening trials was investigated and it was observed that dual inoculation of Glomus aggregatum and G. intraradices enhanced the survival rate. The encapsulated nodes of C. barnesii were tested for their capability to endure different temperatures during storage and the optimal temperature for storage was found to be 4°C. A methodology for initiation of somatic embryogenesis from C. barnesii is also reported here, but embryos could not be induced to develop further. The micropropagated plants were reintroduced in to their natural habitat. This is the first report on micropropagation of C. barnesii.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation,micromorphological studies and <Emphasis Type="Italic">ex vitro</Emphasis> rooting of cannon ball tree (<Emphasis Type="Italic">Couroupita guianensis</Emphasis> aubl.): a multipurpose threatened species

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l^?1 indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l^?1 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l^?1 each of BAP and Kinetin (Kin) + 0.5 mg l^?1 α-naphthalene acetic acid (NAA) with additives (50 mg l^?1 of ascorbic acid and 25 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l^?1 IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l^?1 of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7–8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Avocado globular somatic embryos were transformed with three binary vectors, pK7FNF2, pK7RNR2 and pK7S*NF2, harboring the marker genes gfp, DsRed and a gfp-gus fusion gene, respectively. GFP and DsRed fluorescence was detected in embryogenic lines growing in selection medium 2 months after Agrobacterium inoculation. The fluorescence signal was maintained thereafter in transgenic calli, as well as in mature somatic embryos. Red fluorescence in pK7RNR2 transgenic lines was higher and more easily observable than GFP fluorescence. Furthermore, calli transformed with pK7S*NF2, harboring gfp-gus, showed higher level of fluorescence than those transformed with pK7FNF2, containing two gfp. To improve plant recovery, maturated transgenic embryos that failed to germinate or showed an underdeveloped shoot were cultured for 4 weeks in a medium with 1 mg l^?1 TDZ and 1 mg l^?1 BA after partial removal of cotyledons. A 50% of embryos developed one or several shoots on the cut surface. These embryos were cultured for 4 additional weeks in a medium with 1 mg l^?1 BA for shoot elongation and then, shoots were grafted in vitro onto seedling rootstocks. Culture of micrografts in solid MS medium supplemented with 1 mg l^?1 BA allowed a 60–80% success rate. Young leaves from transgenic plants showed GFP or DsRed fluorescence located in the nucleus. The results obtained indicate that fluorescent marker genes, especially DsRed, could be useful for early selection of transgenic material and optimization of the transformation parameters in avocado. Furthermore, the protocol established allowed the successful recovery of transgenic plants, one of the main limiting steps in avocado transformation.     

Direct formation of axes in new plantlets of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Doty) Doty,as influenced by the use of AMPEP K<Superscript>+</Superscript>, spindle inhibitors,and plant growth hormones

In order to induce the direct formation of erect dark brown vegetative shoots of Kappahycus alvarezii, under micro-propagation conditions, the agricultural bio-stimulant “Acadian Marine Plant Extract Powder” (or AMPEP) K⁺ (a Canadian commercial extract of the brown seaweed Ascophyllum nodosum with enhanced levels of potassium) was used singly or in combination with colchicine or oryzalin at different concentrations plus (synthetic) plant growth regulators (i.e., PGR, IAA, and kinetin) over 45 days of incubation. Amongst the treatments tested, 5 mg L^?1 AMPEP K⁺ with PGR produced the longest direct axis shoots (9.6 ± 0.33 mm), followed by 0.1 mg L^?1 AMPEP K⁺ + 1.0 mg L^?1 oryzalin with PGR (8.7 ± 0.00 mm), 10 mg L^?1 AMPEP K⁺ + 0.5 mg L^?1 colchicine with PGR (8.6 ± 1.20 mm), and 0.5 mg AMPEP K⁺ only (5.3 ± 1.8 mm). The shortest, direct axis shoots measured were the following: 8.3 ± 0.33 mm with 0.1, 1 and 10 mg L^?1 AMPEP K⁺ with PGR, 6.0 ± 0.58 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 oryzalin with PGR, 5.3 ± 0.89 mm with 0.5 mg L^?1 AMPEP K⁺ + 0.1 mg L^?1 colchicine with PGR, and finally, 2.7 ± 0.33 mm with 0.1 mg L^?1 AMPEP K⁺ only. The percentage of direct shoots formed ranged from 93 to 100 % and 87 to 100 % in AMPEP K⁺ with PGR and AMPEP K⁺ only, respectively, while 62–100 % and 87–100 % were observed in AMPEP K⁺ + colchicine with PGR and AMPEP K⁺ + oryzalin with PGR, respectively. The use of AMPEP K⁺ with the PGR resulted in the longest and highest percentage formation of direct axis shoots. These observations, taken together, suggested the efficacy of AMPEP K⁺ used as a protocol to induce direct, erect shoots for the micro-propagation of K. alvarezii plantlets under laboratory conditions.     

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.     

<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.     

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.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.