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De novo root regeneration is the process by which adventitious roots form around the wound site from wounded or detached plant organs. The de novo root regeneration process has been widely exploited in cutting technology used for vegetative propagation. Here, we employed detached leaf explants from Arabidopsis thaliana to form adventitious roots for studying the process of de novo root regeneration. GUS staining showed that the expression of CLAVATA3/EMBRYO SURROUNDING REGION-RELATED26(CLE26) was gradually increased surrounding the wound site of leaf explants during adventitious root formation. Semi-thin sections further showed that the expression pattern of CLE26 was closely linked to the formation of adventitious roots. Next, genetic analyses confirmed that the CLE26 gene was involved in de novo root regeneration. Furthermore, RNA sequencing (RNA-seq) of the leaf explants revealed that stress-related genes might be involved in CLE26-mediated adventitious root formation. Specifically, genes associated with the hydrogen peroxide catabolic process and oxidative stress response were predominantly upregulated in the cle26 mutant. In contrast, genes involved in the response to salicylic acid were largely downregulated in the cle26 mutant. Overall, our study indicates that the mutation in CLE26 might upregulate the expression of genes involved in reactive oxygen species metabolism or suppress the expression of genes associated with salicylic acid synthesis, thus promoting the formation of adventitious roots. These findings suggest that CLE26 is a potential candidate for the genetic improvement of adventitious rooting in cuttings. [ABSTRACT FROM AUTHOR]

Tetrastigma hemsleyanum is a perennial evergreen vine of the Vitaceae. The entire herb is used in traditional Chinese medicine as a broad-spectrum plant-based antibiotic, so it has high economic and social value. Wild T. hemsleyanum resources are scarce, so it has been declared an endangered and rare medicinal plant. Seed yield is low and vegetative propagation by cuttings results in limited plant production, so development of the T. hemsleyanum industry requires optimized propagation protocols and the development of new biotechnologies to proliferate this plant in commercial quantities. In this study, shoot organogenesis was successfully induced from leaves and petioles. Two plant growth regulators, 6-benzyladenine (BA) and thidiazuron, induced callus and adventitious shoots, but the ideal adventitious shoot induction medium was Murashige and Skoog (MS) medium containing 1.0 mg L−1 BA and 0.1 mg L−1 α-naphthaleneacetic acid (NAA). This resulted in a shoot proliferation coefficient (SPC) of 6.73 within 30 d at a light intensity of 100 µmol m−2 s−1. When light intensity was increased from 50 to 200 µmol m−2 s−1, SPC (7.35), chlorophyll a (Chl a), Chl b, and total Chl (a + b) content increased. On MS medium containing 0.1–2.0 mg L−1 NAA or indole-3-butyric acid, 100% of adventitious shoots formed adventitious roots. Plantlets showed no obvious morphological variation, and their survival exceeded 98% on a substrate of peat and river sand (v:v = 2:1). This study's protocols allow for the mass production of adventitious shoots for conservation purposes, and potentially for the commercial propagation of T. hemsleyanum. [ABSTRACT FROM AUTHOR]

Hieracium lucidum subsp. lucidum is a critically endangered endemic taxa of the Sicilian flora. It is a relict of the Tertiary period surviving on the cliffs of Monte Gallo (NW-Sicily). This research focused on finding the best protocols for seed germination and vegetative and in vitro propagation to contribute to ex situ conservation. Seed germination tests were carried out using constant temperatures of 15 ℃, 20 ℃ and 25 ℃ in continuous darkness and an alternating temperature of 30/15 ℃ (16 h/8 h, light/dark). The seeds had no dormancy, and a high germination capacity (70-95%) was obtained at all tested thermoperiods. The possibility of vegetative propagation of the taxon was evaluated through the rooting capacity of stem cuttings treated or not treated with indole-3-butyric acid (IBA). All cuttings were treated with IBA rooted within 2 months, while only 50% of the untreated cuttings were rooted within a longer time. An efficient protocol for rapid in vitro propagation from leaf portions was developed. The response of explants was tested on hormone-free Murashige and Skoog (MS) basal medium and MS enriched with different types of cytokinins: 6-Benzylaminopurine (BAP) and meta-Topolin (mT) in combination with naphthaleneacetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) at the same concentration. The combination of mT (2 mg L-1) and 2,4-D (1 mg L-1) in the medium was the most effective and showed the highest percentage of callus induction and the mean number of regenerated shoots. The maximum rate of root regeneration and the maximum number and length of roots were obtained on hormone-free MS and MS enriched with IBA at concentrations of 1 mg L-1. From the results obtained, it can be concluded that H. lucidum subsp. lucidum can be successfully propagated using one of the tested techniques, subject to the availability of the material for reproduction. [ABSTRACT FROM AUTHOR]

Two models of Arabidopsis thaliana rhizogenesis in vitro were studied: (1) from the callus and (2) leaf explants petioles on the Murashige and Skoog one-tenth strength hormone-free nutrient medium and also with the addition of a growth regulator, such as indole-3-butyric acid. Morphological and anatomical studies show significant changes in the structure of the roots formed de novo in vitro from callus tissue, while the organs from leaf explant petioles were similar to those formed from seeds (primary). By the Sabinin–Kolosov method, a decrease in the percentage of the active root surface was established. The occurrence of structural changes during in vitro rhizogenesis and their effect on root functionality are discussed. [ABSTRACT FROM AUTHOR]

Nematanthus wettsteinii (Fritsch) H. E. Moore is one of the most popular ornamental species in China, and is known for its peculiar "goldfish" shape flower and is usually propagated by cuttings. This study aimed to establish in vitro protocols for N. wettsteinii regeneration. In this study, leaves and shoot-tips were used as explants, and the effects of adventitious shoot induction, rooting medium, and plant growth regulators (PGRs) on regeneration from explants were evaluated. Murashige and Skoog (MS) medium was used as the basic substrate. Among the PGRs tested, when leaves were used as explants, 0.1 mg L−1 α-naphthaleneacetic acid (NAA) and 2.0 mg L−1 thidiazuron (TDZ) induced the largest number of adventitious shoots (22.5 per leaf) with a 93.5% induction rate. When shoot-tips were used as explants, 3.0 mg L−1 benzyl adenine (BA) induced the fewest adventitious shoots (2.6 per shoot-tip), while 0.1 mg L−1 NAA and 2.0 mg L−1 TDZ yielded the largest number of adventitious shoot (20.3 per shoot-tip). The adventitious shoots were then transferred to MS medium containing NAA, BA, and gibberellin A3 (GA3) for shoot proliferation and elongation. When half-strength MS medium was supplemented with 0.3 mg L−1 indole-3-butyric acid (IBA), shoot rooting was essentially 100%. After transplanting the plantlets to garden soil mixed with river sand and vermiculite (1:1:1, v/v) substrate, 100% of the plantlets survived after 3 wk. These reliable regeneration protocols constitute a significant advance towards the large-scale propagation and conservation of this famous horticultural species. [ABSTRACT FROM AUTHOR]

This study was conducted to establish an efficient in vitro plantlet regeneration protocol using the ex vitro leaves as explants for Haworthia retusa. Leaf tissues were cultured on liquid full-strength Murashige and Skoog (MS) medium supplemented with 2.0 mg/L indole 3-butyric acids (IBA) for callus induction, followed by sub-cultured to solid medium for callus proliferation. Callus was then transferred to a fresh medium supplemented with 6-benzyl amino adenine (BA) for shoot development. The result showed that the maximum rate of shoot regeneration (100%), number of shoots per explant (43), and shoot height (9.4 mm) were recorded on the solid MS medium supplemented with 1.0 mg/L BA and 30 g/L sucrose. IBA improved rooting, whereas, NAA (naphthaleneacetic acid) causes calli to form at the base of the shoots. The half-strength MS medium supplemented with 0.5 mg/L IBA provided the best rooting response for the shoot. This medium formulation resulted in the highest rooting rate (100%) and the highest mean root number (5 roots/explant). The result of the present study would be helpful for the mass propagation of commercially important H. retusa. [ABSTRACT FROM AUTHOR]

This study was conducted to develop a protocol for in vitro shootmultiplication and callus induction of various mung bean varieties to obtain enhanced phytochemical content with the help of elicitors. For shoot multiplication, two types of explants (shoot tips and nodal tips) of three varieties of mung bean (Mung NCM-13, MgAT-7, and MgAT-4) were used. Both types of explants from in vitro and in vivo sources were cultured on the MS medium supplemented with different concentrations (0.25-3.0 mg/L, increment of 0.5 mg/L) and combinations of BAP and IBA as independent treatments. For callus induction, leaf explants (in vitro source) were cultured on MS medium supplemented with 2,4-D (1-3 mg/L) alone or in combination with BAP or NAA (0.5 and 1.0 mg/L). For the enhanced production of phenolics and glycosides, calli were cultured on MS media supplemented with zinc oxide (0.5 mg/L) and copper oxide nanoparticles (0.5 mg/L) as nano-elicitors. Results showed that in vitro explants responded better in terms of shoot length, number of shoots, and number of leaves per explant when compared to in vivo explants. Moreover, shoot tips were better than nodal explants to in vitro culturing parameters. All three varieties showed the optimized results in the MS medium supplemented with 1 mg/L BAP, while roots were produced only in cultures fortified with 1 mg/L IBA. The leaf explants of in vitro and soil-grown plantlets showed a maximum callogenic response of 90 and 80%, respectively, on MS medium supplemented with 2,4-D (3 mg/ml). Maximum phenolic content (101.4 µg of gallic acid equivalent/g) and glycoside content (34mg of amygdalin equivalent/g of plant material) was observed in the calli cultured on MS medium supplemented with 3 mg/L of 2,4-D. Furthermore, the addition of zinc oxide (0.5 mg/L) and copper oxide (0.5 mg/L) nanoparticles to the callus culture medium significantly enhanced the phenolic content of Mung NCM-13 (26%), MgAT-7 (25.6%), and MgAT-4 (22.7%). Glycosidic content was also found to be increased in Mung NCM-13 (50%), MgAT-7 (37.5%), and MgAT-4 (25%) varieties when compared to the control. It is suggested that elicitation of in vitro cultures of mung beans with nanoparticles could be an effective strategy for the enhanced production of secondary metabolites. [ABSTRACT FROM AUTHOR]

The aim of the present investigation was to optimize protocols for micropropagation and adventitious shoot regeneration from leaf explants of two wild ecotypes of red flesh apple Malus niedzwetzkyana for future breeding programs. At the proliferation stage, different concentrations of sodium nitroprusside (SNP) and triacontanol (TRIA) were compared. To optimize shoot regeneration from leaf explants, interactive effects of 1-phenyl-3-(1,2,3-thidiazol-5-yl)-urea – thidiazuron (TDZ), indole-3-butyric acid (IBA) and two explant types were investigated. At rooting stage, the effect of exposure time of microshoots to darkness and exposure time to different concentrations of IBA and α-naphthalene acetic acid (NAA) were compared. The results showed that SNP affected the growth rate significantly and the maximum multiplication rates per explant (9.6 in the first ecotype and 8.8 in the second) were produced in the Quoirin and Lepoivre medium containing 17 SNP µM, in addition to 4 µm 6-benzylaminopurine (BAP) and 3 µm gibberellic acid (GA3). IBA and TDZ affected the adventitious shoot regeneration from leaf explants significantly, the highest number of regenerated shoots (18.3 per explant) was obtained from the basal section of the leaves cultured on the medium containing 2 μM IBA and 15 μM TDZ. At rooting stage, the maximum rooting (88.6%) was obtained in the result of one weak exposure to darkness on medium containing 3 μM IBA. [ABSTRACT FROM AUTHOR]

An efficient in vitro plant regeneration method through somatic embryogenesis has been established in Haworthia retusa. Somatic embryos were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and α-naphthaleneacetic acid (NAA) either alone or in combination with 4 μM thidiazuron (TDZ). Of the four auxins studied, IBA was found to be the most promising in terms of somatic embryo induction, followed in decreasing frequency by 2,4-D, IAA, and NAA. The highest somatic embryo induction (60.7%), with a mean of 20.7 embryos per leaf explant, was observed on MS medium amended with 20 μM IBA. The inclusion of 4 μM TDZ to the auxin-containing medium significantly (p < 0.0001) increased the somatic embryo induction frequency as well as the number of somatic embryos. The best combination for somatic embryogenesis was IBA + TDZ. The highest incidence of somatic embryo induction (100%), with a mean of 55.8 somatic embryos, was obtained on a culture medium containing 16 μM IBA + 4 μM TDZ. Somatic embryos germinated best on MS medium supplemented with 2 μM gibberellic acid. Morphological variations were observed among the regenerated plantlets. Well-developed plantlets obtained from germination media were acclimatized in the greenhouse. [ABSTRACT FROM AUTHOR]

An efficient method for direct somatic embryogenesis and callogenesis from date palm (Phoenix dactylifera L. cv. Estamaran) was established using leaf explants. The effect of pretreating leaf explants with three antioxidant combinations on the browning was studied. The results showed that both the pre-treatments were effective against the browning of explants. Surface sterilization of explants, without pre-treatment, enhanced the browning. After recognizing the importance of antioxidants, we cultured the explants on an MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.5, 1, 2, and 5 mg/l, thiadiazuron (TDZ) at 0 and 5 mg/l and 6-benzyladeninepurine (BAP) at 0, 5, and 10 mg/l. Callogenesis and direct embryogenesis on the surface of the leaf explants were observed. Only on the medium containing 5 mg/l 2,4-D, 10 mg/l BAP, and 5 mg/l TDZ globular embryos were formed directly on the leaf explants. The percentage of callus formation increased with an increase in the 2,4-D concentration; however, high concentrations of cytokinin caused a reduction of callogenesis. A medium supplemented with 5 mg/l 2,4-D, 5 mg/l TDZ, and 5 mg/l BAP, and 2 mg/l 2,4-D, and 5 mg/l BAP produced the highest number of calli per replication (2.7) on the leaf surfaces and leaf margins. Along with the advantages of direct somatic embryogenesis, this protocol facilitated the prospect of micropropagation without resorting to the intermediary callus stage, an important aspect to commercial production, and without sacrificing the plant by excising the shoot tips, which are commonly used for date palm tissue cultures. [ABSTRACT FROM AUTHOR]

To preserve and propagate valuable Chinese cabbage (Brassica rapa L.) using mature vegetative organs without seeds, we established a high efficiency regeneration system from heading leaf explants and further explored its application in genetic transformation. Our results showed that maximum shoot regeneration of cultivars ‘Beijing New No. 3’ (88.46 ± 1.65%) and ‘Chengyangqing’ (73.77 ± 1.73%) were obtained in the presence of 17.76 μM 6-benzylaminopurine (BA) and 5.37 μM α-naphthylacetic acid (NAA). The addition of silver nitrate (AgNO3) at 11.77 μM was essential for shoot regeneration from heading leaf explants. Genotypic differences in shoot regeneration frequencies (from 47.24 ± 3.65 to 88.46 ± 1.65%) were observed amongst eight Chinese cabbage cultivars, in addition to the number of shoots per explant (from 1.31 ± 0.02 to 2.02 ± 0.05) in Murashige and Skoog medium containing 17.76 μM BA, 5.37 μM NAA and 11.77 μM AgNO3. Low temperature (4 °C) had an effect on in vitro preservation of leafy heads with a delay in leaf wilting, and there were no significant differences in shoot induction frequency within 24 h for cultivars ‘Beijing New No. 3’ and ‘Chengyangqing’. In the genetic transformation experiments using selection with kanamycin (17.17 μM), a transformation efficiency of 0.6-1.2% was achieved, as assessed from PCR and Southern blot results. The above results suggested that the heading leaf explants can not only achieve efficient seed-independent propagation of Chinese cabbage, but also provide a feasible platform for genetic transformation. [ABSTRACT FROM AUTHOR]

Incorporation of nanoparticles into a number of manufacturing products raised the concern of environmental release via deliberate or accidental routes. Here, experiments were performed to examine the effect of copper oxide nanoparticles (CuO NPs), and polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG) impregnated CuO NPs on seed germination and growth of Trigonella foenum‐graecum L. as well as on callus induction through tissue culture technique. Seed germination frequency, length, and weight parameters did not inhibit at higher extent by application of NPs; however, copper acetate, PVP, and PEG significantly decreased the values of all parameters. In all the cases, negative effects were observed concentration‐dependent. PVP and PEG impregnated CuO were found less toxic for calli fresh and dry weight induced from leaf and stem explants. The 2, 2‐diphenyl‐1‐picrylhydrazyl reagent‐free radical scavenging activity, total antioxidative potential, and total reducing power potential along with total flavonoid and phenolic contents are found elevated in root when compared with shoot. Furthermore, impregnation of PVP and PEG on CuO NPs increases the oxidative response. The results conclude that impregnation of organic molecules on nanoparticles does not reduce the toxicity though can be exploited for enhanced production of secondary metabolites for medicinal purposes. [ABSTRACT FROM AUTHOR]

Black ash ( Fraxinus nigra) is an endangered hardwood tree species under threat of extirpation by the emerald ash borer (EAB), an aggressive exotic phloem-feeding beetle. We have developed an efficient regeneration system through adventitious shoot organogenesis in F. nigra using in vitro-derived leaf explants. Two types of leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth regulators to induce callus and adventitious shoot bud formation. Significant effects of explant, and plant growth regulator interactions were found. The frequency of callus formation ranged from 77.8 to 94.4% and 88.9-100% from single leaflets and intact compound leaves, respectively, with no significant difference between treatments. For adventitious shoot bud induction, however, 22.2 µM 6-benzylaminopurine (BA) combined with 31.8 µM thidiazuron (TDZ) was the best treatment regardless of the initial leaf explant type, showing 21.1 and 28.8% shoot bud induction, with 1.5 and 1.9 adventitious shoots per explant, from single leaflets and intact compound leaves, respectively. The regenerated shoot buds were elongated on MS medium supplemented with Gamborg B5 vitamins plus 2 mg L glycine (MSB5G), 13.3 µM BA, 1 µM indole-3-butyric acid (IBA), and 0.29 µM gibberellic acid. The elongated shoots were continuously micropropagated through nodal stem sectioning until used for rooting. An average of 85.2% of the microshoots were successfully rooted in woody plant medium containing 5.7 µM indole-3-acetic acid plus 4.9 µM IBA with a 10-day initial dark culture, followed by culture under a 16-h photoperiod. Rooted plantlets were acclimatized to the greenhouse and showed normal plant growth and development with 100% survival. This regeneration protocol would be useful for mass propagation for conservation of F. nigra and for use in genetic transformation for EAB resistance. [ABSTRACT FROM AUTHOR]

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant ofSwertia corymbosahas been developed for the first time. In the present study,in vitroderived leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) individually and in combination with cytokinins for its effectiveness to induce the differentiation of somatic embryos. The antioxidant activities of methanolic extracts from non-embryonic callus (NEC), pre-embryoid masses (PEM), somatic embryos at globular stage (SEG), somatic embryos at heart-shaped stage (SEHS), and cotyledonary embryos (SEC) ofS. corymbosawere evaluated using threein vitroassays: scavenging of free radicals (DPPH and ABTS) and ferric reducing antioxidant test. In all cases, the methanolic extract from SEG demonstrated better antioxidant activity than those taken from other tested samples. Higher amounts of swertianin (1), methylswertianin (2), and 1,2- dihydroxy-6-methoxyxanthone-8-O-β-D-xylopyranosyl (3) were found in SEG stage followed by SEHS and PEM when compared to NEC and SEC. [ABSTRACT FROM PUBLISHER]

Somatic embryogenesis is a powerful tool for clonal propagation and plant breeding in many plant species, but its application is limited by the genotype response based on the interaction between gene expression and culture conditions. In this context, the recalcitrance of the nonembryogenic cherry rootstock Prunus cerasus cultivar CAB 6P was investigated via the analysis of the candidate genes PiABP19, Picdc2, and PiSERK3 in comparison with the embryogenic genotype Prunus incisa cultivar N°131. Semiquantitative RT-PCR was employed to assess the transcript level of these genes in leaf explants and derived calli of the two genotypes cultured on picloram-containing MS medium during 30 days of culture. Results showed that only Picdc2 and PiSERK3 gave differential expression profiles between genotypes N°131 and CAB 6P. Concerning N°131, transcripts levels were low at the beginning of culture and then increased to reach peaks at the 15th (PiSERK3) and at the 25th (Picdc2 and PiSERK3) days of culture. Contrarily, with the CAB 6P cultivar, transcripts levels that were high at the beginning of the culture considerably decreased afterwards. This decrease may explain the recalcitrance of the cultivar CAB 6P to somatic embryogenesis, at least in our experimental conditions. [ABSTRACT FROM AUTHOR]

Water hyssop (Bacopa monnieri (L.) Pennell) is a medicinal plants. Its upper and lower halves of leaf explants were incubated in Murashige and Skoog (MS) medium supplemented with 0.25, 0.50 and 1.0 mg/L benzylaminopurine (BA) for 8 weeks; the explants were exposed to white (W) and red and blue (R and B, respectively) light-emitting diodes (LEDs), at 4:1, 3:1, 2:1 and 1:1 R and B light ratios, respectively. Shoot regeneration (100%) was achieved from all explants at all applied concentrations of BA and LED types. All explants showed different BA concentration requirements for regeneration of the maximum number of shoots. Longer shoots were obtained on medium with 0.25 mg/L BA. The W LED lighting system was found to be more effective for regenerating the maximum number of shoots (26.11) per explant (on the upper half of the leaf). Conversely, longer and shorter shoots were generated under 1:1 R:B and W LEDs, respectively. The number of shoots per explant ranged from 9.67-24.0 (full leaf), 6.33-25.92 (lower half of the leaf) and 7.33-27.33 (upper half of the leaf), respectively, in response to BA and LED light. Shoot length ranged from 0.94-1.90 cm (full lamina), 0.70-2.11 cm (lower half of the leaf) and 0.93-1.83 cm (upper half of the lamina) in response to BA and LED lifght. Regenerated shoots were successfully rooted using indole-3-butyric acid (IBA) and acclimatized in the aquarium provided with tap water. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis (SE) inEucalyptusspp. has been limited to germinated seeds, flowers, lignotubers or zygotic embryos. The low yield of somatic embryos from leaf explants has hampered progress, even though leaves offer a more viable source of clonal explants from superior selected genotypes. It was hypothesised that SE from leaf explants could be enhanced through pairing of synergistic exogenous plant growth regulators, such as natural auxins with natural cytokinins. Callus and embryo induction using 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthalene acetic acid (NAA), indole acetic acid (IAA), and indole butyric acid (IBA), each at either 1.0 or 3.0 mg L−1, indicated that IAA and IBA favoured significantly higher numbers of embryos compared with 2,4-D or NAA. Hence, IAA and IBA were used for subsequent experiments, combining them (at 1.0 mg L−1) with either the synthetic cytokinin, kinetin, or the natural cytokinin,trans-zeatin, both at 0.1 mg L−1. The combination oftrans-zeatin and either IAA or IBA resulted in a significant increase in SE (e.g. 86 ± 17.2% and 23 ± 3.2% for IAA withtrans-zeatin and kinetin, respectively), compared with kinetin, or with these auxins alone. Embryo maturation and plantlet regeneration was highest in those calli that were induced with IAA andtrans-zeatin, indicating that maturation was dependent on auxin depletion, based on the stability of the analogue used for induction. For theE. grandisclone under study, the use of synergistic plant growth regulators significantly enhanced SE from leaf explants, thus presenting the opportunity to benefit from the advantages that SE offers over conventional organogenesis. [ABSTRACT FROM PUBLISHER]

A simple and effective somatic embryogenic system was established for watermelon ( Citrullus lanatus) cv. 'Arka Manik'. Embryogenic callus was obtained from leaf explants of 20-d-old in vitro-grown seedlings cultured on embryogenic callus induction medium. The highest frequency of embryogenic callus induction (96.8%) occurred on Murashige and Skoog (MS) medium supplemented with 2.44 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.27 μM thidiazuron (TDZ). Transfer of embryogenic calluses with proembryogenic masses to embryo maturation medium led to the asynchronous development of somatic embryos (SEs), which progressed from the globular stage to the cotyledonary stage. The maximum number of SEs/explant (16.1 ± 0.24) was obtained on MS medium supplemented with 2.44 μM 2,4-D, 2.27 μM TDZ, and 30 g L sucrose. Plantlet conversion from cotyledonary-stage SEs was tested on different strengths of MS medium (quarter-, half-, and full-strength) lacking plant growth regulators. The highest frequencies of germination (91.5%) and survivability (82.1%) of plantlets were achieved on full-strength MS medium. Transverse sections of embryogenic callus revealed SE development from callus cells near the epidermis. Secondary SEs occasionally formed from globular-shaped primary embryos. Genetic fidelity of mother plants and ex vitro plants was confirmed by inter-simple sequence repeat (ISSR) markers. The present study is the first report on the use of molecular markers in in vitro culture of watermelon. The developed protocol facilitates rapid production of true-to-type watermelon plants by somatic embryogenesis and thus could serve to generate effective target material for genetic transformation protocols. [ABSTRACT FROM AUTHOR]

The PDS1000-He biolistic gun was used to bombard plasmid DNA harbouring gus and nptII genes into tender young leaves of in vitro grown shoots of Camellia sinensis (tea). Out of a total of 500 bombarded leaves, 217 (43.4 %) showed callusing after 5 weeks on selection medium containing 1.71 µM kanamycin. Only 15 of these regenerated into indirect shoot buds. Only 7 out of 15 putative transformants showed the expected 400 bp signal with gus gene specific primers during PCR analysis. On the other hand, all the 15 putative transformants tested positive with nptII gene specific primers. In Southern hybridization with nptII specific gene probe, all the six randomly selected PCR positive plants showed stable integration of nptII gene. Both the transgenic and not-bombarded control plants showed phenotypic similarity under polyhouse conditions. Although their growth parameters were significantly at par, significantly lesser shoot height was recorded in transgenic plants. The reproductive behavior of the transgenics was also depressed. Thus, floral bud and flower abscission, fruit drop as well as empty seed production was higher in the transgenics as compared to control. Viability and germination of transgenic seeds was also significantly lower than control. Survival of the transgenic seedlings was also negligible (about 3 %). Hence, the chances of germ-line transmission of the transgenes were remarkably reduced in case of gus-transgenics. Tea being a vegetatively propagated plant, the method described in the present paper is an important approach for developing transgenics of elite tea plants from leaf explants. [ABSTRACT FROM AUTHOR]

A somatic embryogenesis system was developed for Sapindus mukorossi Gaertn. from leaf explants obtained from fresh flushes of a mature tree. Callus was induced from the midrib region of leaf explants on Murashige and Skoog (MS) medium containing different concentrations of 2,4-dichlorophenoxyacetic acid or 6-benzylaminopurine. Callus induction and somatic embryogenesis was significantly influenced by the size, physiological age, and orientation of leaf explants on the culture medium and plant growth regulators. Adaxial-side-up orientation of leaf explants significantly promoted embryogenesis in comparison with abaxial-side-up orientation. Maximum number of somatic embryos was induced on MS medium supplemented with 8.88 μM 6-benzylaminopurine. Scanning electron microscopy of embryogenic callus revealed somatic embryo origin and the development of globular-, heart-, and cotyledonary-stage somatic embryos. The frequency of maturation as well as germination of somatic embryos was higher on MS medium containing 8.88 μM 6-benzylaminopurine than on medium without 6-benzylaminopurine. Plantlets which developed from somatic embryos were acclimatized successfully with 90 % survival. [ABSTRACT FROM AUTHOR]

Transformation approach is a useful tool for the study of gene function, the mechanism of molecular regulation, and increase usefulness of components by reverse genetic approach in plants. In this study, we developed a stable and rapid method for Agrobacterium-mediated transformation of a medicinal plant Chelone glabra L. using leaf explants. Stable transformants were obtained using Agrobacterium tumefaciens strains GV2260 and GV3101 that harbored the binary vector pBI121 and contained the neomycin phosphotransferase gene ( NPT II) as a selectable marker and a reporter gene β-glucuronidase ( GUS). Putative transformants were identified by kanamycin selection and a histochemical assay. PCR and Southern blot analysis confirmed the integration of the GUS gene into transformed genomes as well as detected stable expression of the β-glucuronidase gene (GUS) by RT-PCR. Resulting transformed plants had morphologically normal phenotypes. This method requires two changes of medium and few leaf explants as well as the transformation efficiency of 2-8 % after 2-3 months of inoculation. This method can provide a quick and economical transformation method for reverse genetic approach to change the secondary metabolic pathway to increase useful components in C. glabra. [ABSTRACT FROM AUTHOR]

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum. [ABSTRACT FROM AUTHOR]

The first step in Agrobacterium tumefaciens infection of plants is the establishment of cell-cell contact between the two partners. However, failure to establish such contact makes many plants and explants recalcitrant to A. tumefaciens infection. Tea is one such example where even the popular inducer, acetosyringone failed to facilitate A. tumefaciens infection due to the presence of high amounts of bactericidal/bacteriostatic polyphenols. Quinones are formed as a result of polyphenols oxidation. They cause tissue browning and necrosis during the process of transformation, and in turn prevent A. tumefaciens infection. Compounds such as polyphenol adsorbents, i.e., polyvinylpyrrolidone and charcoal, and antioxidants like cysteine and ascorbic acid were screened to overcome tissue browning. Although these compounds enhanced the growth of A. tumefaciens, these failed to facilitate the infection of the leaves of either Kangra Jat, Tocklai Variety-1, UPASI-9, UPASI-10, and Stock-449 cultivars of tea. On the contrary, the presence of filter sterilized l-glutamine and l-glutamic acid in the co-cultivation medium facilitated successful A. tumefaciens infection of recalcitrant tea leaves. l-Glutamine and glutamic acid form harmless adducts by binding to quinones. Therefore, their presence in the co-cultivation medium allowed the tea leaves to remain living and appealing to the infecting A. tumefaciens. Successful A. tumefaciens infection of tea leaves was confirmed by positive signals in GUS assay, PCR, and Dot blot. [ABSTRACT FROM AUTHOR]

The paper described the scheme of sterilization of silver birch leaf explants for callus culture establishment, and the dependence of morphogenesis from the composition of growth regulators and culture conditions. Noticeable effects of long-term maintenance of callus cultures and individual differences between initial forms on its regeneration capacity have shown. [ABSTRACT FROM AUTHOR]

An efficient and reproducible Agrobacterium-mediated genetic transformation of Withania coagulans was achieved using leaf explants of in vitro multiple shoot culture. The Agrobacterium strain LBA4404 harboring the binary vector pIG121Hm containing β-glucuronidase gene ( gusA) under the control of CaMV35S promoter was used in the development of transformation protocol. The optimal conditions for the Agrobacterium-mediated transformation of W. coagulans were found to be the co-cultivation of leaf explants for 20 min to agrobacterial inoculum (O.D. 0.4) followed by 3 days of co-cultivation on medium supplemented with 100 μM acetosyringone. Shoot bud induction as well as differentiation occurred on Murashige and Skoog medium supplemented with 10.0 μM 6-benzylaminopurine, 8.0 μM indole 3-acetic acid, and 50.0 mgl kanamycin after three consecutive cycles of selection. Elongated shoots were rooted using a two-step procedure involving root induction in a medium containing 2.5 μM indole 3-butyric acid for 1 week and then transferred to hormone free one-half MS basal for 2 weeks. We were successful in achieving 100 % frequency of transient GUS expression with 5 % stable transformation efficiency using optimized conditions. PCR analysis of T transgenic plants showed the presence of gusA and nptII genes confirming the transgenic event. Histochemical GUS expression was observed in the putative transgenic W. coagulans plants. Thin layer chromatography showed the presence of similar type of withanolides in the transgenic and non-transgenic regenerated plants. A. tumefaciens mediated transformation system via leaf explants developed in this study will be useful for pathway manipulation using metabolic engineering for bioactive withanolides in W. coagulans, an important medicinal plant. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis is a useful tool of plant breeding. In this context, a procedure for inducing somatic embryogenesis in Prunus incisa leaf explants had been previously developed. The original in vitro protocol relies on picloram treatments and exposure to darkness as inductive conditions, the best frequency of embryogenesis being obtained on the second leaf (F) exposed to 4 μM picloram during 30 days. The morphological and biochemical changes observed during somatic embryogenesis occur in response to alterations in gene expression regulation patterns. A molecular study was conducted in order to provide deeper insight into the fundamental biological factors involved in the induction of this process using a gene candidate strategy and semi-quantitative reverse transcription polymerase chain reaction analysis. So far, no sequence data related to somatic embryogenesis has been available in cherry. In the present study, we cloned and sequenced cDNA fragments of putative genes encoding auxin-binding protein, cell cycle regulator and somatic embryogenesis receptor kinase. Time-course differential transcript accumulations were observed for all investigated genes in leaves or derived callus tissues during the observation period (first month of culture). Their possible involvement in the sequential steps of the embryogenic pathway (dedifferentiation, cell proliferation, differentiation through somatic embryogenesis) is presented and discussed. [ABSTRACT FROM AUTHOR]

Viola patrinii DC. (Violaceae) petiole explants were used for inducing calli. Significant callus proliferation was observed on MS medium supplemented with 16.12 µM NAA and 13.33 µM BA. Shoot regeneration was achieved upon transferring the green friable calli to MS medium with a 2-fold dilution of potassium dihydrogen phosphate supplemented with 23.25 µM Kn and 2.68 µM NAA. Maximum shoot regeneration was achieved in 4 weeks. Multiple shoots were separated and further cultured in half-strength MS medium supplemented with 9.85 µM IBA with 2% sucrose. Profuse root development was observed after 20-25 days of culturing. Regenerated plants were successfully transferred to soil, showing an 85% survival rate. The genetic fidelity of the micropropagated plants was analysed using random amplified polymorphic DNA (RAPD) markers. The RAPD profile of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrating the homogeneity of the micropropagated plants. This protocol could be successfully used for the mass multiplication and germplasm conservation of this medicinal plant. [ABSTRACT FROM AUTHOR]

The induction of somatic embryogenesis from shoot apices and leaf explants of shoot cultures derived from 6- to 7-year-old white oak ( Quercus alba L.) trees is reported in this study. Embryogenic response was obtained in two out of the three genotypes evaluated with embryo induction frequencies up to 50.7% for WOQ-1 and 3.4% for WOQ-5 genotypes. The embryogenic explants formed translucent nodular structures and cotyledonary-stage somatic embryos, which developed from callus tissue, indicating an indirect embryogenesis process. An efficient procedure was developed for WOQ-1 material on the basis of the most appropriate leaf developmental stage. Growing leaves excised from two nodes below the shoot apex showed the highest embryogenic induction index. These leaves contain cells in an undifferentiated state, as shown by the presence of precursor cells of stomata, absence of intercellular spaces and low starch content in the mesophyll cells. Nodular structures and/or somatic embryos began to arise 7-8 weeks after culture initiation, although most emerged after 9-12 weeks in culture. The sequence of application of media for somatic embryo induction was optimized with a two-step procedure consisting of culturing the explants in medium supplemented with 21.48 μM NAA and 2.22 μM BA for 8 weeks and transfer of explants into plant growth regulator-free medium for another 12 weeks. Clonal embryogenic lines were established and maintained by secondary embryogenesis. Embryo germination (30%) and plantlet conversion (16.6%) were achieved after cold storage for 2 months. [ABSTRACT FROM AUTHOR]

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, 'Attar of Roses' and 'Atomic Snowflake', grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for 'Attar of Roses' and 'Atomic Snowflake', respectively. [ABSTRACT FROM AUTHOR]

Viola baoshanensis is one of the most rare cadmium (Cd) hyperaccumulators, however, it is hard to propagate. Micropropagation has been applied to solve the problems with propagation of a few heavy metal hyperaccumulators. Therefore there is a high likelihood that micropropagation may offer a suitable method for large-scale propagation of V. baoshanensis. To test this hypothesis, three types of explants were used for shoot regeneration and various combinations of four plant growth regulators were used to improve shoot regeneration efficiency from leaflet of V. baoshanensis. Best shoot regeneration efficiency was obtained by incubating leaflet in a 1/2 MS medium supplemented with 2.5 μM BA + 2.5 μM IBA, therein shoot regeneration rate was 70.9% and the number of shoots formation per explant was 22.4. Rooting was achieved from almost all regenerated shoot growing on 1/2 MS medium without plant growth regulator. Micropropagated seedlings were acclimatized under greenhouse conditions and 95% of them survived and showed no visible morphological variation compared to their donor plant. Furthermore, there were no significant differences between regenerated and seed-germinated V. baoshanensis in Cd tolerance and accumulation. These results suggested that an efficient and rapid micropropogation system was successfully developed for V. baoshanensis. [ABSTRACT FROM AUTHOR]

Carbenicillin, a well-known antibiotic, has been reported to have growth regulator-like activity in vitro for some plant species. In the present paper we add horseradish ( Armoracia rusticana) to the list of plants exhibiting such responses. This project began as an effort to eliminate latent bacterial contamination among established in vitro horseradish plants. Carbenicillin (100 mg L) added to regeneration medium eliminated all visible bacterial contaminants. Unexpectedly, carbenicillin-grown explants regenerated adventitious shoots faster (14 days) than those on control medium (21 days). In addition eight of 11 horseradish cultivars grown on carbenicillin produced more adventitious shoots per explant than control. At much higher levels (2,000 mg L) carbenicillin was found to retard somatic embryogenic callus induction. Based on these observations we suggest that carbenicillin at moderate levels enhances shoot development in horseradish. The mode of action of carbenicillin’s growth regulator-like activity needs to be investigated. [ABSTRACT FROM AUTHOR]

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 µM acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured on woody plant medium supplemented with 9.08 µM thidiazuron, 1.07 µM napthaleneacetic acid, 60 µM silver thiosulphate, 3% sucrose, plus 200 mg l-1 timentin in darkness for 3 weeks. Regenerating shoots were selected 27 days after initial co-culture, on Murashige and Skoog medium with 3% sucrose, 8.88 µM 6-benzylaminopurine, 0.49 µM indole-3-butyric acid, 0.29 µM gibberellic acid, 200 mg l-1 timentin, and 30 mg l-1 kanamycin for five subcultures. After 5-6 months of selection, transformation efficiencies were determined, based on polymerase chain reaction (PCR) analysis of individual putative transformed shoots relative to the initial number of leaf explants tested. The transformation efficiency was 1.2%. Southern blot analysis of three out of four PCR-positive shoots confirmed the presence of the neomycin phosphotransferase and AG genes. Transgenic shoots were rooted (37.5%), but some shoot tips and leaves deteriorated or died, making acclimatization of rooted transgenic plants difficult. This transformation, regeneration, and rooting protocol for developing transgenic black cherry will continue to be evaluated in future experiments, in order to optimize the system for several mature black cherry genotypes. [ABSTRACT FROM AUTHOR]

We examined the effects of cooling applied for 4 to 20 weeks on donor cultures of four dwarfing apple rootstocks (P16, P22, P59 and M26). Our aim includes increasing their competence for in vitro adventitious shoot regeneration from the leaves. Donor cultures were maintained on a shoot multiplication medium at 4°C in the dark for 4 months, followed by subculture on a fresh medium for 4 weeks. The cooling of the cultures caused an increase in the adventitious shoot number and a decrease in the starch content and an increase in the soluble sugar content (monosaccharides, raffinose and stachyose). The accumulation of stachyose in response to cold is a new observation, and it suggests that raffinose and stachyose play important role in the acclimation of dwarf apple rootstocks to low temperatures. [ABSTRACT FROM AUTHOR]

This report describes an efficient plant regeneration system for the medicinal plant Lythrum salicaria via direct adventitious shoot development from leaf and stem explants. Leaf explants were much more responsive to regeneration than stem segments. Of the hormonal combinations tested, those involving thidiazuron (TDZ; 0.1, 0.3 or 0.5 mg dm-3) were more effective than the combinations of other hormones and 0.1 mg dm-3 TDZ combined with either indole-3- acetic acid (IAA) or indole-3-butyric acid (IBA) was the most productive. Rooting was readily achieved when multiple shoots were singled out and cultured on medium containing different auxins. IAA was the most effective on root development in terms of both the number of roots per shoot and the frequency of rooted shoots. More than 90 % of the regenerants survived after hardening for four weeks at gradually decreased air humidity. [ABSTRACT FROM AUTHOR]

The morphology of horseradish (Armoracia rusticana P. Gaertn., B. Mey & Scherb.) leaves varies through the growing season. The leaves range from laminate (complete) in the summer to pinnate (fern-leaf) toward the end of the growing season in the fall, with intermediate types appearing regularly. The causes of these changes are not understood. To determine whether leaf morphology affects their ability to regenerate adventitious shoots in vitro, laminate, pinnate, and intermediate leaves of 10 horseradish cultivars were established in vitro. Laminate leaves were more likely to regenerate shoots (77%) than other leaves (23%). It is recommended that laminate leaves be used as explants to regenerate horseradish plants in vitro. [ABSTRACT FROM AUTHOR]

Long-sized oligogalacturonides (OGs) are cell wall fragments that induce defence and developmental responses. The Ca2+-dependent “egg-box” conformation is required for their activity, and polyamines may prevent them from adopting this conformation. Although OGs are known to inhibit auxin-induced growth processes, their effect on cytokinin-induced ones requires investigation. In the present work OGs were shown to promote cytokinin (benzyladenine, BA)-induced vegetative shoot formation from tobacco leaf explants, independent of the presence of CaCl2 in the medium and of auxin (indoleacetic acid, IAA) supply. The effect of polyamines, putrescine (PU) and spermidine (SD) supplied with/without their biosynthetic inhibitors (DFMO, CHA) was also investigated, and showed that spermidine enhanced adventitious vegetative shoot formation, but only on medium containing Ca2+ and IAA. Treatments with inhibitors blocked this promotive effect. OGs did not alter free polyamine concentrations, but caused a moderate increase of conjugated ones, and exhibited an early inhibitory effect on polyamine biosynthetic gene expression. OGs, but not SD, caused long-term changes in calcium-associated epifluorescent signals in the cell walls, and, later, inside the cells of specific tissues. Electron microscopy analysis (ESI system) demonstrated that calcium accumulated in the cell walls and vacuoles of OG-cultured explants. The relationship between OGs, cytokinin, calcium, and polyamines in adventitious vegetative shoot formation is discussed. [ABSTRACT FROM AUTHOR]

The direct induction of adventitious shoots from leaf explants obtained from adult plants of Zhanhua winter jujube, an elite variety of Zizyphus jujuba Mill., is reported. The percentage of leaf explants producing shoots and the average number of shoots per explant were significantly improved when 10-day-old leaves were explanted onto Woody Plant Medium and maintained initially in the dark. The plant growth regulator thidiazuron (TDZ) was effective in stimulating shoot regeneration from leaf explants of Zhanhua winter jujube. The highest efficiency of shoot formation was observed with a 20-day culture in the dark on WPM containing 4.54 μ M TDZ and 2.85 μ M indoleacetic acid (IAA). The regenerated shoots were transferred to MS medium containing 0.89 μ M benzyladenine and 5.77 μ M gibberellic acid for growth. When the shoots were about 2 cm in height, they were transferred to Nitsch medium supplemented with 1.14 μ M IAA and 2.46 μ M indolebutyric acid to induce rooting. This system of adventitious shoot production from leaf explants of adult plants could be useful for the genetic engineering and polyploidization of winter jujube. [ABSTRACT FROM AUTHOR]

Protocols elaborated with the objective of achieving valuable material for selection procedure of variants with virusresistance traits in tomato genotypes are presented. Preliminary results are demonstrated in the domain of testing for variability in somaclones obtained through indirect adventitous organogenesis initiated on leaf explants of cultivated tomato ( Lycopersicon esculentum Mill.). Somaclones were grown in greenhouse conditions and variation of their symptoms upon infection with tomato mosaic (ToMV) or cucumber mosaic (CMV) respectively was observed. Tests for resistance to the local isolates of the above cited viruses were performed using enzyme linked immunosorbent assay and back inoculation onto diagnostic plants. Screening data are presented. Desirable variants were selected from cultivars ‘Moneymaker’, ‘Potentat’ and ‘Rutgers’. Some of the ‘Moneymaker’ somaclones exhibited increased tolerance to cucumber mosaic virus, a few seemed to be even fully resistant though most were susceptible as donor plants. The most favourable somaclonal lines are actually further tested and monitored for changes in horticultural characteristics. The described procedure of searching for resistance trait in specific pathogen-free (SPF) plants regenerated from infected tissue looks promising and thus can serve as aid in attaining appropriate objectives of breeding programme. Additionaly experiments were initiated to obtain somaclones from cultivars ‘Beta’, ‘Krakus’ and Stevens Rodade hybrid via regeneration of isolated protoplasts. To this end the callus stage was obtained from all donors. [ABSTRACT FROM AUTHOR]

Leaf explants of Sinningia speciosa were cultured in vitro on Murashige and Skoog (MS) basal medium with various growth substances in order to regenerate shoots. On MS medium supplemented with indoleacetic acid (IAA) and kinetin, 80% of the explants produced green callus and 25 to 30 shoots with roots per explant. On MS supplemented with IAA and N6 benzyladenine (BA), 80% of the explants produced green callus and 40 to 50 shoots per explant but lacked roots. After 3–4 mo., these shoots were removed from the initial explants and transferred separately onto MS supplemented with indolebutyric acid for their elongation and successive rooting (3 mo.). Histological studies showed that the callus was associated with mesophyll cell layers, primarily with the spongy parenchyma. The shoots regenerated at the callus surface and were associated with newly differentiated vascular areas. Recurrent regenerations were obtained from leaf explants or apical meristems excised from shoots of the previous subcultures. These explants, as compared to initial cultures, had a high frequency of regeneration and also produced more shoots per explant. Chromosome numbers of root tip cells of the mother plant and of all in vitro-regenerated plants remained constant: 2n=26. [ABSTRACT FROM AUTHOR]

According to the hormonal conditions, after one month of culture shoots or somatic embryos could be obtained from leaf explants of Helianthus smithii. Well-shaped embryos developed on media containing a combination of auxin and cytokinin, while on media containing only cytokinin shoots were observed. The primary leaves of these shoots resembled cotyledons. A detailed histological study of the regeneration process on three media, containing only cytokinin or auxin, or a combination of both, allowed the origin and development of the observed structures to be determined. All three conditions induced somatic embryos, which then developed differently and, within one month, finally gave rise to the two types of structures which were initially observed. [ABSTRACT FROM AUTHOR]

Adventitious shoots were successfully regenerated from leaf explants of Gypsophila paniculata L. The efficiency of shoot regeneration for cv. Arbel was tested on 18 media based on Murashige and Skoog basal medium containing different concentrations of thidiazuron or 6-benzylaminopurine in combination with naphthaleneacetic acid. Both explant age and that of the cuttings used as leaf donors affected the regeneration efficiency. The highest efficiency of adventitious shoot regeneration was obtained with the oldest leaves originating from the youngest cutting analyzed; on thidiazuron-containing medium, shoots regenerated on average from 67% of the leaves, with an average of seven shoots per explant. This regeneration procedure was suitable for all six commercial cultivars studied. Regenerated shoots elongated, rooted and successfully acclimatized to the greenhouse where they were grown to flowering. [ABSTRACT FROM AUTHOR]

High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 μg/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants. [ABSTRACT FROM AUTHOR]

Regeneration was investigated in non-sterile leaf explants taken from 87 Begonia lines after culturing for up to 10 weeks in vitro on Knop's mineral salts agar medium with no exogenous growth substances present. For many lines, only very limited supplies of leaf samples were available; nevertheless, in 66 lines de novo regeneration of roots and buds occurred and potted plants were recovered from 49 lines. Additionally in 13 Begonia lines, buds were excised from stock plants and cultured under non-sterile conditions on Knop's agar. In seven of these the buds rooted and gave rise to potted plants. In six lines organogenic cultures (derived from sterile leaf explants) have been established in long-term culture on Murashige & Skoog medium supplemented with growth substances and potted plants have been recovered. Sterile explants from a further 12 lines showed regeneration and have been sub-cultured. The establishment of such sterile cultures, with continued regenerative capacity, is a significant step in the development of a programme for the conservation of rare and endangered begonias. [ABSTRACT FROM AUTHOR]

The main challenge of plant chemical diversity exploration is how to develop tools to study exhaustively plant tissues. Their sustainable sourcing is a limitation as bioguided strategies and dereplication need quite large amounts of plant material. We examine if alternative solutions could overcome these difficulties by obtaining a secure, sustainable, and scalable source of tissues able to biosynthesize an array of metabolites. As this approach would be as independent of the botanical origin as possible, we chose eight plant species from different families. We applied a four steps culture establishment procedure, monitoring targeted compounds through mass spectrometry-based analytical methods. We also characterized the capacities of leaf explants in culture to produce diverse secondary metabolites. In vitro cultures were successfully established for six species with leaf explants still producing a diversity of compounds after the culture establishment procedure. Furthermore, explants from leaves of axenic plantlets were also analyzed. The detection of marker compounds was confirmed after six days in culture for all tested species. Our results show that the first stage of this approach aiming at easing exploration of plant chemodiversity was completed, and leaf tissues could offer an interesting alternative providing a constant source of natural compounds. [ABSTRACT FROM AUTHOR]

A two-step procedure was used for plant regeneration from in vitro grown leaf strips (2-3 mm wide) of cv. Bintje. Step I medium was designed with 2,4-dichlorophenoxycetic acid (2,4-D) at 0.0 or 9.0 μM, in combination with 2.28 μM kinetin (K), benzyl adenine (BA), zeatin (Z) or zeatin riboside (ZR). Step II media were 2,4-D-free media containing 5.78 μM gibberellic acid (GA3) and growth regulators similar to those of step I media. Leaf explants cultured in medium I containing zeatin riboside or zeatin for 6 days and then subcultured in medium II containing zeatin riboside produced numerous shoots without callus formation. Zeatin riboside containing step I and II media caused shoot regeneration in a high number (97.5±2.2) of explants. Approximately, 33.7±8.4 shoots were regenerated from each leaf explant. [ABSTRACT FROM AUTHOR]

Transgenic technology is a powerful tool for gene functional characterization, and poplar is a model system for genetic transformation of perennial woody plants. However, the poplar genetic transformation system is limited to a number of model genotypes. Herein, we developed a transformation system based on efficient Agrobacterium-mediated transformation for the hybrid poplar Populus Alba × Populus glandulosa Uyeki, which is a fast-growing poplar species that is suitably grown in the northern part of China. Importantly, we optimized many independent factors and showed that the transformation efficiency was improved significantly using juvenile leaf explants. Explants were infected by an Agrobacterium suspension with the OD600 = 0.6 for 15 min and then co-cultured in dark conditions for 3 days. Using the improved transformation system, we obtained the transgenic poplar with overexpression of β-glucuronidase (GUS) via direct organogenesis without callus induction. Furthermore, we analyzed the GUS gene in the transgenic poplars using PCR, qRT-PCR, and GUS staining. These analyses revealed that the GUS gene was efficiently transformed, and it exhibited various expression levels. Taken together, these results represent a simple, fast, and efficient transformation system of hybrid poplar plants. Our findings may facilitate future studies of gene functions in perennial woody plants and tree breeding via transgenic technology assisted design. [ABSTRACT FROM AUTHOR]