<Emphasis Type="Italic">In vitro</Emphasis> morphogenesis and micropropagation of <Emphasis Type="Italic">Aechmea ramosa</Emphasis> var. <Emphasis Type="Italic">ramosa</Emphasis> Mart. ex Schult. f. (Bromeliaceae) from leaf explants

Aechmea ramosa Mart. ex Schult. f. is an endemic bromeliad of the Brazilian Atlantic Forest. The current habitat degradation of this hotspot biome threatens this species, which besides having an important ecological role, is also of invaluable ornamental interest. Plant tissue culture has been used in mass propagation and conservation of various bromeliads. We have established a micropropagation protocol for A. ramosa var. ramosa using leaf explants grown in MS medium supplemented with 2 μM of 1-naphthaleneacetic acid (NAA) and 2 μM of 6-benzylaminopurine (BAP) that showed higher values of shoot induction. NAA and BAP are associated with the production of proteins involved in stress response modulation, metabolic activity, and cell division, the latter being involved in inducing the differentiation of competent cells. After 120 d of culture, each explant presented 28.9 shoots with an average size of 27.8 mm, with no variation in either Stomatal Index or density of the regenerated shoots. Plantlets measuring above 15-mm height were successfully acclimatized, presenting 100% survival rate. Thus, this protocol can be used for mass propagation of A. ramosa, and to supply demand for the market of ornamental plants. Furthermore, it represents an important tool for the conservation of this species and maintenance of an in vitro germplasm.     

Growth and biochemical analysis of evergreen haloxeric tree species <Emphasis Type="Italic">Salvadora oleoides</Emphasis> and <Emphasis Type="Italic">Salvadora persica</Emphasis> under NaCl stress

In vitro growth, development, total soluble proteins and peroxidase profiles of Salvadora oleoides and Salvadora persica under NaCl stress were analysed in the present investigation. The plants are evergreen haloxeric tree species of family Salvadoraceae. Shoot apex from natural plants were initially used for screening of NaCl tolerance on MS culture medium. Shoot apex of S. oleoides and S. persica could survive optimally up to 200 and 100 mM NaCl. Axillary buds from nodal shoot segments of S. oleoides and S. persica were activated on 6 and 4 μM BAP, and were used further for extraction of total soluble proteins and peroxidases. Total soluble proteins were increased up to 150 mM NaCl in S. oleoides, but decline above 50 mM NaCl in S. persica. Peroxidase activity remained almost constant in S. oleoides at all the concentrations and duration of NaCl, but increased at 100 mM NaCl during fourth week of treatment in S. persica. Eleven peroxidase isozymes were observed in zymogram of S. oleoides. Isozymes P1, P2, P3, and P4 were slightly appeared, but P6 isozyme was lacking in S. persica. The P5 isozyme was more prominent in S. persica than S. oleoides. Isozyme P9 of S. persica was visible during the first week of NaCl treatment, but disappeared in the fourth week. Molecular biology of these plants can be useful further for the understanding of stress tolerance mechanisms for prospects.     

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.     

Plant regeneration with maintenance of the endosperm ploidy level by endosperm culture in <Emphasis Type="Italic">Lonicera caerulea</Emphasis> var. <Emphasis Type="Italic">emphyllocalyx</Emphasis>

An endosperm culture of Haskap (Lonicera caerulea var. emphyllocalyx) was established to develop polyploid plants and investigate the regeneration ability of the endosperm. Based on histological analysis of embryo and endosperm development, endosperms at the globular to early torpedo-stages of developing embryos were used to initiate an endosperm culture. Formation of shoot primordia was observed on Murashige and Skoog (MS) medium (Physiol Plant 15:473–497, 1962) containing benzyladenine and indole-3-butyric acid. Shoot primordium formation was confirmed in some genotypes with regeneration frequencies ranging between 1.9 and 10.0%. These proliferated on ½ MS medium containing 2.89 μM gibberellic acid (GA₃), and then elongated and rooted on MS medium containing 0.44 μM BA and 2.89 μM GA₃. These shoots developed into plantlets on ½ MS medium. Plantlets maintained ploidy of the endosperm following flow cytometric analysis, thus confirming that these were derived from the endosperm. These results indicated that endosperms were capable of regeneration.     

<Emphasis Type="Italic">In vitro</Emphasis> plantlet production of the endangered <Emphasis Type="Italic">Pinguicula vulgaris</Emphasis>

This study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free ¼MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.     

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 <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Vigna mungo</Emphasis> using immature cotyledonary-node explants and phosphinothricin as the selection agent

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).     

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.     

<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.     

Molecular evidence for hybridization between invasive <Emphasis Type="Italic">Solidago canadensis</Emphasis> and native <Emphasis Type="Italic">S</Emphasis>. <Emphasis Type="Italic">virgaurea</Emphasis>

Hybridization between alien and native species is biologically very important and could lead to genetic erosion of native taxa. Solidago × niederederi was discovered over a century ago in Austria and described by Khek as a natural hybrid between the alien (nowadays regarded also as invasive) S. canadensis and native S. virgaurea. Although interspecific hybridization in the genus Solidago is considered to be relatively common, hybrid nature of S. × niederederi has not been independently proven using molecular tools, to date. Because proper identification of the parentage for the hybrid Solidago individuals solely based on morphological features can be misleading, in this paper we report an additive polymorphism pattern expressed in the ITS sequences obtained from individuals representing S. × niederederi, and confirm the previous hypothesis that the parental species of this hybrid are S. canadensis and S. virgaurea. Additionally, based on variability at the cpDNA rpl32-trnL locus, we showed that in natural populations hybridization occurs in both directions.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Acacia mangium</Emphasis>

A protocol was developed for Agrobacterium-mediated genetic transformation of Acacia mangium using rejuvenated shoots as the explant. Axillary buds and shoot apices of adult trees were rejuvenated by culturing them on Murashige and Skoog (MS) medium, and stem segments of rejuvenated shoots were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI121. The selection for transgenic shoots was performed through five consecutive steps on MS medium supplemented with 1.0 mg/l thidiazuron, 0.25 mg/l indole-3-acetic acid and different concentrations of geneticin (G418; 12–30 mg/l) and timentin (T; 50–300 mg/l) in the following order: 12 mg/l G418 and 300 mg/l T for 30 days, 20 mg/l G418 and 200 mg/l T for 60 days, 30 mg/l G418 and 100 mg/l T for 30 days, 12 mg/l G418 and 50 mg/l T for 30 days, and finally 15 mg/l G418 and 5 mg/l gibberellic acid (GA₃) for 60 days. Thirty-four percent of the stem segments produced resistant multiple adventitious shoot buds, of which 30% expressed the β-glucuronidase gene. The shoot buds were subjected to repeated selection on MS medium supplemented with 2.0 mg/l 6-benzylaminopurine, 2.5 mg/l GA₃ and 20 mg/l G418. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 2.0 mg/l α-naphthaleneacetic acid, 0.1 mg/l kinetin and 20 mg/l G418. Genomic Southern blot hybridization confirmed the incorporation of the NPTII gene into the host genome.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Artemisia abrotanum</Emphasis> L. by means of somatic organogenesis

The goal of this project was to regenerate Artemisia abrotanum L., Southern wormwood, by means of organogenesis from leaves. In vitro plant propagation may greatly support the molecular characterization of the medicinal qualities of A. abrotanum. Young, intact leaves were excised from mature plants and surface sterilized. Abundant callus growth, as well as shoot formation, was produced on an MS medium supplemented with 4.44 μM BA and 0.54 μM or 0.81 μM NAA. Shoots, with some residual callus, rooted equally well in MS media with 0.49 μM IBA, 0.54 μM NAA, or without hormones. Rooted plants were best acclimated in potting soil.     

<Emphasis Type="Italic">C7</Emphasis>-prenylation of tryptophanyl and <Emphasis Type="Italic">O</Emphasis>-prenylation of tyrosyl residues in dipeptides by an <Emphasis Type="Italic">Aspergillus terreus</Emphasis> prenyltransferase

During our search for novel prenyltransferases, a putative gene ATEG_04218 from Aspergillus terreus raised our attention and was therefore amplified from strain DSM 1958 and expressed in Escherichia coli. Biochemical investigations with the purified recombinant protein and different aromatic substrates in the presence of dimethylallyl diphosphate revealed the acceptance of all the tested tryptophan-containing cyclic dipeptides. Structure elucidation of the main enzyme products by NMR and MS analyses confirmed the attachment of the prenyl moiety to C-7 of the indole ring, proving the identification of a cyclic dipeptide C7-prenyltransferase (CdpC7PT). For some substrates, reversely C3- or N1-prenylated derivatives were identified as minor products. In comparison to the known tryptophan-containing cyclic dipeptide C7-prenyltransferase CTrpPT from Aspergillus oryzae, CdpC7PT showed a much higher substrate flexibility. It also accepted cyclo-l-Tyr-l-Tyr as substrate and catalyzed an O-prenylation at the tyrosyl residue, providing the first example from the dimethylallyltryptophan synthase (DMATS) superfamily with an O-prenyltransferase activity towards dipeptides. Furthermore, products with both C7-prenyl at tryptophanyl and O-prenyl at tyrosyl residue were detected in the reaction mixture of cyclo-l-Trp-l-Tyr. Determination of the kinetic parameters proved that (S)-benzodiazepinedione consisting of a tryptophanyl and an anthranilyl moiety was accepted as the best substrate with a K _M value of 204.1 μM and a turnover number of 0.125 s^?1. Cyclo-l-Tyr-l-Tyr was accepted with a K _M value of 1,411.3 μM and a turnover number of 0.012 s^?1.     

Organogenesis from root explants of commercial populations of <Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims and a wild passionfruit species, <Emphasis Type="Italic">P. cincinnata</Emphasis> Masters

Root explants of a wild passionfruit species (Passiflora cincinnata) and three P. edulis commercial populations (‘FB 100’, ‘FB 200’, and ‘FB 300’) were incubated on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyladenine (BA) to induce shoot organogenesis. Shoots elongated in liquid medium with 2.89 μM gibberellic acid (GA₃) under agitation were rooted in coconut fiber and acclimatized followed by transfer to a greenhouse into pots containing mixture of coconut fiber and Plantmax^® (1:1). Explant samples were collected during organogenesis and submitted to light and scanning electron microscopy (SEM). Root explants of P. cincinnata responded earlier than those of P. edulis. However, on the third assessment, at 90 days, the genotype ‘FB 200’ showed shoot number significantly higher than ‘FB 100’ and ‘FB 300’, not differing from P. cincinnata. Organogenesis in P. cincinnata and P. edulis occurred via direct pathway, which was confirmed by anatomical studies and SEM. Flow cytometric analysis revealed no variation in DNA content of regenerated plantlets among all genotypes. Nuclear DNA (2C) values (pg) in regenerants of P. cincinnata (2.99 pg) and P. edulis (3.26–3.28 pg) were consistent with DNA amounts of seed-derived control plants.     

Droplet-vitrification cryopreservation of <Emphasis Type="Italic">in vitro</Emphasis>-grown shoot tips of grapevine (<Emphasis Type="Italic">Vitis</Emphasis> spp.)

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.     

An efficient <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation protocol of <Emphasis Type="Italic">Withania somnifera</Emphasis>

This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120 min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5?% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.     

Cytogenetic characterization of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. and <Emphasis Type="Italic">Amaranthus hybridus</Emphasis> subsp<Emphasis Type="Italic">. cruentus</Emphasis> (L.) Thell.

The present study is aimed to identify genetic variability between two species of Amaranthus viz., A. caudatus and A. hybridus subsp. cruentus, two economically important species, cultivated mainly for grain production. Karyomorphological studies in Amaranthus are scarce, probably due to higher number of small sized chromosomes. Karyomorphological studies were conducted using mitotic squash preparation of young healthy root tips. Karyological parameters and karyotypic formula were established using various software programs and tabulated the karyomorphometric and asymmetry indices viz., Disparity index, Variation coefficient, Total forma percentage, Karyotype asymmetry index, Syi index, Rec index, Interchromosomal and Intrachromosomal asymmetry index and Degree of asymmetry of karyotypes. The mitotic chromosome number observed for A. caudatus was 2n = 32 with a gametic number n = 16 and A. hybridus subsp. cruentus was 2n = 34 with a gametic number n = 17. In A. caudatus the chromosome length during somatic metaphase ranged from 0.8698 to 1.7722 μm with a total length of 39.1412 μm. In A. hybridus subsp. cruentus the length of chromosome ranged from 0.7756 to 1.9421 μm with a total length of 44.9922 μm. Various karyomorphometry and asymmetry indices analyzed revealed the extend of interspecific variation and their evolutionary status.     

Effects of 6-benzylaminopurine on photosystem II functionality and leaf anatomy of <Emphasis Type="Italic">in vitro</Emphasis> cultivated <Emphasis Type="Italic">Aechmea blanchetiana</Emphasis>

Cytokinins (CKs) are often used during the in vitro cultivation of plant species. However, it is not clear how CKs, such as 6-benzylaminopurine (BAP), affect photosystem PS) II functionality and leaf anatomy over a long period of in vitro plant propagation. The aim of this study was to analyze the residual effects of BAP on the photosynthetic performance and leaf anatomy of Aechmea blanchetiana after 120 d without exposure to CKs. Aechmea blanchetiana plants previously grown in vitro were transferred to Murashige and Skoog (MS) culture media containing 0, 5, 10, 15, or 20 μM BAP. After 60 d on the MS medium with BAP, explants were subcultivated twice on the MS medium without growth regulators, first in a stationary liquid medium for 60 d and then in a solidified medium with 6 g dm^-3 agar for 60 d. Leaf anatomy, pigment content, and chlorophyll a fluorescence were assessed for plants from each treatment after 120 d on the CK-free medium. Stomatal density presented a negative linear correlation with BAP concentration. Pigment content decreased in plants subjected to previous BAP exposure. An increase in absorbed energy flux per reaction center (ABS/RC) and a sharp decrease in energy transport flux (ETo/RC) followed by an increase in energy dissipation flux (DIo/RC) also occurred. Furthermore, maximum quantum yield (F_V/F_M) decreased as a function of BAP concentration. Thus, the use of BAP during in vitro propagation of A. blanchetiana induced long-term physiological defects.