Showing papers in "Plant Cell Tissue and Organ Culture in 2008"
TL;DR: The effect of red- and blue-light-emitting diodes on shoot and root growth of Hybrid Franc grape, a rootstock cultivar, along with two other grape genotypes, Kadainou R-1 and Vitis ficifolia var.
Abstract: The effect of red- and blue-light-emitting diodes on shoot and root growth of Hybrid Franc grape, a rootstock cultivar, along with two other grape genotypes, Kadainou R-1 and Vitis ficifolia var. ganebu, were investigated in vitro. Plants cultured under red-light-emitting diodes produced the longest shoots with longer internodes for all genotypes. The chlorophyll content measured as SPAD value and leaf number per explant were highest on plants cultured under blue-light-emitting diodes in all the genotypes. Blue light was also responsible for a higher number of stomata in all the genotypes; however, there was no significant difference in size of stomata in all genotypes under the different light conditions tested. Different light-emitting diodes did not affect the rooting percentage of Hybrid Franc but red-light-emitting diodes gave a higher rooting percentage along with higher root numbers for the two other grape genotypes.
226 citations
TL;DR: The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin, and the transgenic nature of the transformants was demonstrated.
Abstract: Jatropha curcas contains high amounts of oil in its seed and has been considered for bio-diesel production. A transformation procedure for J. curcas has been established for the first time via
Agrobacterium tumefaciens infection of cotyledon disc explants. The results indicated that the efficiency of transformation using the strain LBA4404 and phosphinothricin for selection was an improvement over that with the strain EHA105 and hygromycin. About 55% of the cotyledon explants produced phosphinothricin-resistant calluses on Murashige and Skoog (MS) medium supplemented with 1.5 mg l−1 benzyladenine (BA), 0.05 mg l−1 3–indolebutyric acid (IBA), 1 mg l−1 phosphinothricin and 500 mg l−1 cefotaxime after 4 weeks. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l−1 BA, 0.05 mg l−1 IBA, 0.5 mg l−1 gibberellic acid (GA3), 1 mg l−1 phosphinothricin and 250 mg l−1 cefotaxime, and about 33% of the resistant calli differentiated into shoots. Finally, the resistant shoots were rooted on 1/2 MS media supplemented with 0.3 mg l−1 IBA at a rate of 78%. The transgenic nature of the transformants was demonstrated by the detection of β-glucuronidase activity in the primary transformants and by PCR and Southern hybridization analysis. 13% of the total inoculated explants produced transgenic plants after approximately 4 months. The procedure described will be useful for both, the introduction of desired genes into J. curcas and the molecular analysis of gene function.
144 citations
TL;DR: The results support the possible use of topolins as an alternative to BA for Cavendish banana tissue culture and requires a more stringent investigation as the results obtained in this investigation could have been influenced by carry-over effects from the initial cultures.
Abstract: The effect of the cytokinins mT (meta-topolin), mTR (meta-topolin riboside), MemT (meta-methoxy topolin) and MemTR (meta-methoxy topolin riboside) on micropropagation of banana cultivars ‘Williams’ and ‘Grand Naine’ was studied and compared to BA (6-benzylaminopurine). In vitro cultures, at the third sub-culture level, were purchased from African Biotechnologies (Pty) Ltd., South Africa. These were then sub-cultured on MS media containing 7.5, 15 and 30 μM of all the cytokinins tested. Results recorded after 6 weeks of growth demonstrated that there were statistically significant differences between the parameters analyzed for the treatments. Superior multiplication rates were recorded for mT and mTR treatments. This result was consistent when compared to BA at 22.2 μM (previously published standard concentration). Contrary to previous findings with other species, these cytokinins inhibited rooting. The effect on somaclonal variation was not significantly different when BA, mT and mTR were tested at the seventh multiplication cycle for ‘Williams’ banana. These results support the possible use of topolins as an alternative to BA for Cavendish banana tissue culture. The role of these cytokinins on somaclonal variation however, requires a more stringent investigation as the results obtained in this investigation could have been influenced by carry-over effects from the initial cultures.
119 citations
TL;DR: Procedures for asymbiotic seed germination and seedling acclimatization were developed for Bletia purpurea, a threatened North America native terrestrial orchid, and in vitro seedlings were readily Acclimatized to greenhouse conditions.
Abstract: Procedures for asymbiotic seed germination and seedling acclimatization were developed for Bletia purpurea, a threatened North America native terrestrial orchid. Six asymbiotic orchid seed germination media (Knudson C, PhytoTechnology Orchid Seed Sowing Medium, Malmgren Modified Terrestrial Orchid Medium, Vacin & Went Modified Orchid Medium, ½-strengh Murashige & Skoog, and BM-1 Terrestrial Orchid Medium) were examined for their effectiveness in promoting seed germination and protocorm development of B. purpurea in either a 0/24 h or 16/8 h L/D photoperiod. Germination occurred regardless of medium or photoperiod treatment. However, advanced seedling development (Stage 6) only occurred on Vacin & Went Modified Orchid Medium in the 16/8 h L/D photoperiod. Further effects of photoperiod on in vitro seedling development were also examined. Shoot length, leaf width, root number and length, and fresh weight and dry weight in the 16/8 h L/D photoperiod were all significantly different when compared to the 8/16 h and 12/12 L/D photoperiods. In vitro seedlings were readily acclimatized to greenhouse conditions. Seedlings showed high survival all potting media. Seedlings acclimatized in Fafard Mix 4 potting medium developed significantly longer roots. Corm formation occurred regardless of potting media used.
97 citations
TL;DR: The results show that transgenic expression of the transcription factor WUS can be useful to increase somatic embryogenesis in heterologous systems, however, a critical developmental stage and additional hormonal requirements are required for the induction of embryogenesis by WUS in Coffea canephora.
Abstract: Cell differentiation depends on the proper and sequential expression of key genes required for morphogenesis. Several aspects of control are required for this which include: chromatin modifications, DNA methylation, correct amount of particular transcription factors, proper nuclear arrangement, etc. During the last few years the homeobox transcription factor WUSCHEL (WUS) has been shown to cause dedifferentiation when expressed on somatic cells followed by a production of new stem cells that can lead to somatic embryogenesis or organogenesis. We found that expression of WUS in coffee plants can induce calli formation as well as a 400% increase somatic embryo production. The results show that transgenic expression of the transcription factor WUS can be useful to increase somatic embryogenesis in heterologous systems. However, a critical developmental stage and additional hormonal requirements are required for the induction of embryogenesis by WUS in Coffea canephora.
86 citations
TL;DR: High densities of endophytic bacteria were found in plant material from poplar, larch and spruce that had been micropropagated for at least 5 years and certain species or strains, especially those with a close relationship to P. humicus, seemed to accumulate under in vitro conditions without visible negative influences on the plant’s development.
Abstract: High densities of endophytic bacteria were found in plant material from poplar, larch and spruce that had been micropropagated for at least 5 years. The majority of these bacteria were assigned to the genus Paenibacillus based on the sequencing of the 16S rRNA genes. Other endophytic bacteria such as Methylobacterium, Stenotrophomonas or Bacillus could also be found but only in some tissue cultures. Certain species or strains of Paenibacillus, especially those with a close relationship to P. humicus, seemed to accumulate under in vitro conditions without visible negative influences on the plant’s development. Poplar microcuttings inoculated with the endophytic Paenibacillus isolate 22 showed significantly more roots per cutting and higher root length in comparison to the control plants after 3 weeks.
83 citations
TL;DR: In general, MS was the best medium for expression, independently of the medium previously used during induction, and the negative impact of anti-browning agents on SE potential raises a question about the role of production/accumulation of phenolics in the SE process.
Abstract: The low induction rates of somatic embryogenesis (SE) in Eucalyptus
globulus hamper scaling up the process for commercialization. We analyzed the effectiveness of several media (MS, 1/2MS, B5, WPM, DKW and JADS) during SE induction and expression. MS and B5 were the best media for SE induction and embling regeneration. In general, MS was the best medium for expression, independently of the medium previously used during induction. Several anti-browning compounds (ascorbic acid, charcoal, DTE, DTT, PVP, PVPP and silver nitrate) were added to the expression medium (MS), but all decreased SE potential and only DTE, charcoal and silver nitrate reduced explant browning. When added only during the induction period, anti-browning agents reduced accumulation of phenolics but also severely reduced SE potential. Continuous exposure completely inhibited the SE response. The negative impact of anti-browning agents on SE potential raises a question about the role of production/accumulation of phenolics in the SE process.
73 citations
TL;DR: A novel stilbene synthase gene (STS), cloned from Chinese wild Vitis pseudoreticulata and responsible for synthesis of the phytoalexin resveratrol in grapevine, was successfully transferred into V. vinifera L. cv.
Abstract: A novel stilbene synthase gene (STS), cloned from Chinese wild Vitis pseudoreticulata (W. T. Wang) and responsible for synthesis of the phytoalexin resveratrol in grapevine, was successfully transferred into V. vinifera L. cv. Thompson Seedless via Agrobacterium tumefaciens-mediated transformation. Using transformation procedures developed in the present study, 72% GFP-positive germinated embryos were produced with about 38% of transformed embryos regenerated into normal plantlets. Integration of the STS gene into the transgenic plants was verified by PCR and Southern blot analysis. Expression of the STS gene was detected by high performance liquid chromatography (HPLC), which showed that the resveratrol concentration in the transgenic plants was 5.5 times higher than that in non-transformed control plants.
71 citations
TL;DR: The tissue culture of S. africana-lutea not only favoured rapid multiplication but was also characterized by seasonal in vitro flowering that was in synchrony with that of plants growing in the wild, making it highly attractive for implementation as an in vitro conservation strategy.
Abstract: An in vitro cultivation protocol was developed for S. africana-lutea a species threatened by over collection due to its importance as an aromatic medicinal plant in the Western Cape of South Africa. Adventitious shoot induction was most successful using hypocotyls as explants for propagation on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with 4.4 μM BA only; 2.7 μM NAA and 4.4 μM BA; or 2.9 μM IAA and 9.3 μM kinetin respectively. For continuous subculture, IAA and BA (μM) at a ratio of 2.9:4.4 or 2.9:8.9 had the best regeneration potential producing approximately three plantlets per nodal explant. Plantlets had 4–5 nodes that could be utilized for the following subculture phase to induce axillary shoots. The tissue culture of S. africana-lutea not only favoured rapid multiplication but was also characterized by seasonal in vitro flowering that was in synchrony with that of plants growing in the wild. This propagation regime has the capacity for producing 2000–3000 plants from one shoot after 3 four-week long subculture cycles, making it highly attractive for implementation as an in vitro conservation strategy. The micropropagated plants were easily acclimatized (88%) within a month after rooting in vitro and planted ex vitro in a sand:soil:peat moss:vermiculite (1:1:1:1; v/v) mixture.
71 citations
TL;DR: The in vitro plants of O. vulgare ssp.
Abstract: Biomass production of micropropagated oregano was induced by inoculation with the fungus Glomus viscosum. The effects of arbuscular mycorrhizal (AM) symbiosis on morphological and metabolic variations of regenerated oregano plants were investigated at different growth stages. AM greatly increased parameters such as plant leaf area, fresh and dry weight, number of spicasters and verticillasters in infected plants. An increase of the gland density, especially on the upper leaf epidermis, was also observed following the physiological ageing of the tissues. The in vitro plants of O. vulgare ssp. hirtum described in this study provided a qualitatively and quantitatively good source of essential oils that have a chemical profile comparable to that of the control mother plants with carvacrol as the main compound.
70 citations
TL;DR: The identification results suggested that the endophytes of second and third groups were not strictly obligate or fastidious microbes but those surviving in viable but-non-culturable (VBNC) state and displaying gradual activation to cultivable form during continuous tissue culturing.
Abstract: In this article we describe the identification of endophytic bacteria belonging to three groups isolated from shoot tip cultures of banana cv. Grand Naine in a recent study (Thomas et al. 2008) based on partial 16S rRNA gene sequence homology analysis. The first group included banana stocks that displayed obvious colony growth on MS based tissue culture medium during the first in vitro passage. The second group constituted stocks that were tissue index-negative for cultivable bacteria initially but turned index-positive after a few to several (4–8) in vitro passages while the third group formed one sub-stock that turned index-positive after about 18 passages. The organisms belonged to about 20 different genera comprising of α, β, γ-proteobacteria, Gram-positive firmicutes and actinobacteria. Visibly expressing easily cultured organisms during the first in vitro passage included Enterobacter, Klebsiella, Ochrobactrum, Pantoea, Staphylococcus and Bacillus spp. Organisms of second group that were not detected or non-culturable originally constituted Brevundimonas, Methylobacterium, Alcaligenes, Ralstonia, Pseudomonas, Corynebacterium, Microbacterium, Staphylococcus, Oceanobacillus and Bacillus spp. while the third group that turned cultivable after extended in vitro culturing included mostly non-filamentous actinobacteria (Brachybacterium, Brevibacterium, Kocuria and Tetrasphaera spp.). The identification results suggested that the endophytes of second and third groups were not strictly obligate or fastidious microbes but those surviving in viable but-non-culturable (VBNC) state and displaying gradual activation to cultivable form during continuous tissue culturing. Several of the organisms isolated are known as beneficial ones in agriculture while some organisms have possible implications in human health. The use of tissue cultures for isolating uncommon endophytes is discussed.
TL;DR: A regeneration system was developed for Prunus serotina from a juvenile (F) and two mature genotypes (#3 and #4) and the effect of kanamycin concentration on adventitious shoot regeneration was also evaluated.
Abstract: A regeneration system was developed for Prunus serotina from a juvenile (F) and two mature genotypes (#3 and #4). Adventitious shoots regenerated from leaves of in vitro cultures on woody plant medium with thidiazuron (TDZ) and naphthaleneacetic acid (NAA). The best regeneration for genotype F (91.4%) was observed on medium with 9.08 μM TDZ and 1.07 μM NAA. The highest mean number of shoots (8.2) was obtained on medium containing 9.08 μM TDZ and 0.54 μM NAA. Genotype #3 had the highest regeneration (41.7%) with a mean number of shoots (4.8) on 9.08 μM TDZ and 1.07 μM NAA, whereas genotype #4 had a 38.8% regeneration with a mean of 3.3 shoots. Genotype #4 had the highest mean number of shoots (4.8) on 4.54 μM TDZ and 1.07 μM NAA. Silver thiosulphate at 60 or 80 μM increased the percent regeneration of the mature genotypes #3 (75%) and #4 (58%). Adventious shoots were rooted (70–76%) and rooted plantlets survived after acclimatization to the greenhouse. The effect of kanamycin concentration on adventitious shoot regeneration was also evaluated.
TL;DR: Attempts were made to study the effect of thidiazuron (TDZ) on adventitious shoot induction and plant development in Paulownia tomentosa explants derived from mature trees, with results indicating that shoot formation capacity was influenced by the genotype.
Abstract: Attempts were made to study the effect of thidiazuron (TDZ) on adventitious shoot induction and plant development in Paulownia tomentosa explants derived from mature trees. Media with different concentrations of TDZ in combination with an auxin were used to induce adventitious shoot-buds in two explant types: basal leaf halves with the petiole attached (leaf explant) and intact petioles. Optimal shoot regeneration was obtained in leaf explants cultured on induction medium containing TDZ (22.7 or 27.3 μM) in combination with 2.9 μM indole-3-acetic acid (IAA) for 2 weeks, and subsequent culture in TDZ-free shoot development medium including 0.44 μM BA for a further 4-week period. The addition of IAA to the TDZ induction medium enhanced the shoot-forming capacity of explants. The caulogenic response varied significantly with the position of the explant along the shoot axis. The highest regeneration potential (85–87%) and shoot number (up to 17.6 shoots/explant) were obtained in leaf explants harvested from the most apical node exhibiting unfolded leaves (node 1). An analogous trend was also observed in intact petiole explants, although shoot regeneration ability was considerably lower, with values ranging from 15% for petioles isolated from node 1 to 5% for those of nodes 2 and 3. Shoot formation capacity was influenced by the genotype, with regeneration frequencies ranging from 50% to 70%. It was possible to root elongated shoots (20 mm) in basal medium without growth regulators; however, rooting frequency was significantly increased up to 90% by a 7-day treatment with 0.5 μM indole-3-butyric acid, regardless of the previous culture period in shoot development medium (4 or 8 weeks). Shoot quality of rooted plantlets was improved not only by IBA treatment but also by using material derived from the 4-week culture period. Regenerated plantlets were successfully acclimatized in the greenhouse 8 weeks after transplanting.
TL;DR: Flow cytometry unambiguously evidenced euploid (diploid, mixoploid, triploid and tetraploid) and aneuploid papaya plantlets, indicating that in vitro culture conditions can lead the occurrence of somaclonal variation.
Abstract: Several protocols have been proposed for in vitro propagation of papaya, either based on somatic embryogenesis or shoot organogenesis. It is well-known that tissue culture-based approaches are frequently associated with somaclonal variation. Whether on the one hand this phenomenon can preclude further stages of in vitro culture, on the other hand it can generate useful genetic variability for crop improvement. However, somaclonal variation analyses are limited in papaya tissue culture. The DNA ploidy level of 250 papaya somatic embryogenesis-derived plantlets from immature zygotic embryos was analyzed by flow cytometry. In vitro-grown and greenhouse seed-derived plantlets were used as diploid standards. Flow cytometry unambiguously evidenced euploid (diploid, mixoploid, triploid and tetraploid) and aneuploid papaya plantlets, indicating that in vitro culture conditions can lead the occurrence of somaclonal variation. Additionally, the two subsequent flow cytometry analyses showed that the DNA ploidy level remained stable in all cloned papaya plantlets during the successive subcultures in the multiplication medium.
TL;DR: The endophyte species composition and plant genotype together with tissue culture conditions are the key factors for gaining plant tissue cultures with high regeneration capacity.
Abstract: Callus tissues originating from buds of mature Scots pine (Pinus sylvestris L.) trees exhibit the typical problem of browning, which leads to degeneration and death of the tissues. The effects of medium, origin (tree and location) and endophyte infection were studied on the browning and growth of bud-derived tissue cultures. The calli growing on medium with higher kinetin content and source of organic nitrogen, and originating from the southern location grew better and exhibited less browning. Endophytic microbial cells were detected in the brown callus tissues by transmission electron microscopy. The natural endophyte infection frequency of Scots pine buds was studied and found dependent on the tree, but not on the location. A well-growing, green callus line was artificially infected by an endophytic strain of Methylobacterium extorquens, and browning was not observed on solid media compared to the uninfected control clones of the same callus. However, suspension cultures started from the infected callus died faster than cultures started from the uninfected callus. The endophyte species composition and plant genotype together with tissue culture conditions are the key factors for gaining plant tissue cultures with high regeneration capacity.
TL;DR: Investigation of the relationship between ammonium ions in the medium, applied cytokinins (CKs) and CK concentrations in the induction of hyperhydricity found that CKs were capable of inducing this syndrome only when other factors in the culture system were not optimised.
Abstract: In search for the optimal culture conditions resulting in a high production of healthy plants and low occurrence of hyperhydricity in tissue cultured regenerants of Aloe polyphylla, we investigated the relationship between ammonium ions in the medium, applied cytokinins (CKs) and CK concentrations in the induction of hyperhydricity. Shoots were grown on media with different NH4
+ concentrations (10.3, 20.6 and 61.8 mM) and supplemented with N6-benzyladenine (BA), zeatin or thidiazuron (TDZ) at 0, 5 or 15 μM. Elevating the levels of NH4
+, in the absence of CKs, could not induce hyperhydricity. Similarly, very low hyperhydricity was observed when CKs were added to media containing low NH4
+ (10.3 mM). However, in the presence of higher NH4
+ concentrations, CKs increased hyperhydricity in a concentration-dependant manner, suggesting that they were capable of inducing this syndrome only when other factors in the culture system were not optimised. High numbers of healthy looking shoots were produced on media with low NH4
+ and low BA or zeatin (5 μM). The use of TDZ resulted in the formation of buds, which did not develop into shoots. Identifying the factors responsible for hyperhydricity is an important step in the successful use of the micropropagation technique for the conservation of this species.
TL;DR: The obtained result suggests that the lanthanoids can be used to effectively initiate rooting in the micropropagation and conservation of D. densiflorum.
Abstract: An efficient microprogation protocol has been developed for Dendrobiumdensiflorum Lindl. ex Wall., a traditional medicinal plant, through protocorm-like bodies (PLBs) from nodal stem segments using 6-benzylamino-purine (BAP) and the lanthanoid neodymium. The highest percentage of explants producing PLBs (72%), with an average of 15 PLBs per explant, was induced by culturing stem segments on Murashige and Skoog (MS) medium supplemented with 5.0 mg l−1 BAP. The newly formed PLBs proliferated well on the basal MS medium and completely converted into shoots on MS medium containing 2.0 mg l−1 BAP. Shoots produced an average of 22 roots per plantlet when cultured on MS medium supplemented with 2.0 mg l−1 neodymium nitrate. Healthy plantlets with well-developed roots were successfully acclimatized. The obtained result suggests that the lanthanoids can be used to effectively initiate rooting in the micropropagation and conservation of D. densiflorum.
TL;DR: While ABA always reduced the rate of secondary embryogenesis, treatments containing 4.0% sucrose significantly increased the number of secondary embryos and sucrose had little influence on maturation.
Abstract: Low efficiency of embryo maturation, germination and conversion to plantlets is a major problem in many species including Persian walnut. We studied the effects of abscisic acid (ABA) and sucrose, on the maturation and germination of Persian walnut (Juglans regia) somatic embryos. Individual globular somatic embryos were grown on a maturation medium supplemented with different combinations of ABA and sucrose for ca. 1 month, until shoot meristems and radicles had developed. White and opaque embryos in late cotyledonary stage were subjected to desiccation after the culture period on maturation media. The number of germinated somatic embryos was influenced by the concentrations of ABA in the maturation medium. The best treatment for germination, in which both shoot and root were developed contained 2 mg l−1 ABA and resulted in 41% conversion of embryos into plantlets. Regeneration was reduced at higher levels of ABA. While ABA always reduced the rate of secondary embryogenesis, treatments containing 4.0% sucrose significantly increased the number of secondary embryos. On the other hand, sucrose had little influence on maturation. Normal and abnormal embryos were verified anatomically.
TL;DR: The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling.
Abstract: Exploring the source of quiescent bacteria in tissue-cultured bananas (Musa sp.) we demonstrate here through a combination of bacterial 16S rDNA-based molecular technique, light microscopy and cultivation-based approaches the ubiquitous presence of endophytic bacteria in the field shoots of different genotypes (Grand Naine, Robusta, Dwarf Cavendish, Ney Poovan and exotic accessions) and their widespread prevalence in apparently clean tissue cultures. A portion of field shoot-tips (10–60%) showed cultivable endophytes, especially during rainy season, yielding 102–105 colony forming units g−1 fresh tissue in ‘Grand Naine’, which overtly expressed on tissue culture medium as well. The rest showed no colony development on diverse bacteriological media but proved PCR+ve to bacterial primers indicating the presence of normally non-culturable organisms, which was endorsed by microscopic observations. Such endophytes gradually turned cultivable rendering all visibly clean cultures as quiescent bacteria-harboring after a few (2–4) to several (8–20) passages, resulting in as much as 1.7 × 105 – 4.0 × 107 colony forming units g−1 tissue of ‘Grand Naine’ after ten passages, yielding different organisms. This study has thus exposed the ubiquitous and intense association existing between endophytes and bananas, including their quiescent survival in suspension cultures. The effect due to quiescent bacteria in micropropagated stocks could not be generalized. The observations question the fundamental principle of asepsis in plant tissue cultures and bring in new information on plant-endophtye association in vitro with implications in micropropagation, germplasm conservation, cell culture studies and molecular profiling. The possible involvement of unsuspected endophytic bacteria in tissue-culture associated phenomena like habituation and epigenetic and somaclonal variations are discussed.
TL;DR: Treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used, suggesting SAAT could be a promising tool for enhancing transformation efficiency in flax.
Abstract: A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO4 solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.
TL;DR: The histological events that led to somatic embryogenesis in macaw palm (Acrocomia aculeata) were described, and meristematic masses grew from procambial cells and generated somatic embryos of multicellular origin.
Abstract: The aim of this paper was to describe the histological events that led to somatic embryogenesis in macaw palm (Acrocomia aculeata (Jacq.) Lodd. ex Martius). Zygotic embryos were inoculated on Y3 medium containing 9 μM 4-amino-3,5,6-trichloropicolonic acid (picloram). Somatic embryos regenerated from nodular callus on induction medium with activated charcoal under photoperiod or without activated charcoal under dark. Many proembryos originated from the fundamental meristem after 10–20 days of culture. When transferred to medium containing activated charcoal, under photoperiod, calli regenerated into somatic embryos of unicellular origin. These embryos had protoderm, plumule and procambial strands and some of them could germinate. After 30–40 days of culture, meristematic masses grew from procambial cells. The masses generated nodular callus, and after transfer to medium without activated charcoal, under dark, they generated somatic embryos of multicellular origin. Those embryos did not regenerate into plants.
TL;DR: The morphogenic response and production of triploid plantlets by endosperm culture is discussed, which shows a high degree of chromosomal variations and polyploidy and in plants where the vegetative parts are economically useful, triploids are of good use.
Abstract: Triploid nature of endosperm is the characteristic feature of angiosperms and is formed as a result of triple fusion. Present review discusses the morphogenic response and production of triploid plantlets by endosperm culture. Both mature and immature endosperm used for culture initiation responded differently in cultures. A key factor for the induction of cell divisions in mature endosperm cultures is the initial association of embryo but immature endosperms proliferate independent of embryo. In almost all the parasitic angiosperms, endosperm shows a tendency of direct differentiation of organs without prior callusing, whereas in autotrophic taxa the endosperm usually forms callus tissue followed by differentiation of shoot buds, roots or embryos. The endosperm tissue often shows a high degree of chromosomal variations and polyploidy. Mitotic irregularities, chromosome bridges and laggards are the other important characteristics of endosperm tissues. Triploids are usually seed sterile and is undesirable for plants where seeds are commercially useful. However, in cases where seedlessness is employed to improve the quality of fruits as in banana, apple, citrus, grapes, papaya etc. the induction of triploid plants would be of immense use. Triploid plants have more vigorous vegetative growth than their diploid counterparts. Hence, in plants where the vegetative parts are economically useful, triploids are of good use. This review focuses on the progress achieved so far in endosperm culture to obtain triploid plants.
TL;DR: After the end of thermotherapy and transfer to ex vitro conditions, three clones of apple cultivar ‘Idared’ were free of both viruses according to repeated RT-PCR and ELISA and the same viruses (ACLSV, ASGV and ASPV) as in the initial field-grown trees were detected by RT- PCR in all four clones of cultivars ‘Sampion’.
Abstract: Apple cultivars ‘Idared’ and ‘Sampion’ were selected for application of in vitro thermotherapy at 39 ± 0.5°C. The presence of viruses in selected initial trees was detected by enzyme-linked immuno-sorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR) before the beginning of thermotherapy. Cultivars ‘Idared’ and ‘Sampion’ were both infected with Apple chlorotic leaf spot virus (ACLSV) and with Apple stem pitting virus (ASPV). Cultivar ‘Sampion’ was moreover infected with Apple stem grooving virus (ASGV). Both apple cultivars were successfully multiplied in in vitro cultures on Murashige and Skoog (MS) medium with 1.5 mg l−1 6-benzylaminopurine (BAP). After the end of thermotherapy and transfer to ex vitro conditions, three clones of apple cultivar ‘Idared’ were free of both viruses (ACLSV, ASPV) according to repeated RT-PCR and ELISA. On the contrary, the same viruses (ACLSV, ASGV and ASPV) as in the initial field-grown trees were detected by RT-PCR in all four clones of cultivar ‘Sampion’. Other sanitation and testing of apple cultivars will continue in the following years.
TL;DR: An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures and the low copy number integration of transgenes in transgenic plants recovered using the improved protocol was revealed.
Abstract: An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.
TL;DR: Optimization of nutrient level in the basal medium has highly significant role in obtaining maximum regeneration response from explants and callus culture.
Abstract: Basal medium constituents and their concentration play an important role in growth and morphogenesis of plant tissues cultured in vitro. In this study effect of different inorganic nutrients (CoCl2, MnSO4, ZnSO4, CuSO4 and AgNO3) on callus induction and plant regeneration in Paspalum scrobiculatum and Eleusine coracana was examined. A 5× and 3× increase in regeneration response at enhanced levels of CuSO4 was noted for kodo and finger millets, respectively. Significant improvement in plant regeneration was also observed with the increase in levels of Co and Mn. Addition of AgNO3 to the basal medium also had a stimulatory effect on callus induction and plant regeneration. Optimization of nutrient level in the basal medium has highly significant role in obtaining maximum regeneration response from explants and callus culture.
TL;DR: The described protocol for repetitive somatic embryogenesis in Eucalyptus globulus produced more somatic embryos than the primary SE protocol and data show that light may influence the quality of the process, depending on its stage.
Abstract: The described protocol for repetitive somatic embryogenesis (SE) in Eucalyptus globulus produced more somatic embryos than the primary SE protocol. Primary somatic embryos (induced on MS3NAA) were transferred to the same medium, leading to new cycles of somatic embryos, for at least 2 years. The influence of medium (MS and B5), plant growth regulators (auxins and cytokinins), and light on secondary SE was tested. The MS medium without growth regulators (MSWH) was more efficient for cotyledonary embryo formation and germination than the B5 medium. Reducing auxin (NAA) levels increased the proliferation of globular somatic embryos and allowed SE competence to be maintained on medium free of plant growth regulators. The addition of two cytokinins (BAP and KIN) to the MS medium did not improve proliferation of globular secondary embryos, but was crucial during later stages of the SE process (germination and conversion). Data also show that light may influence the quality of the process, depending on its stage. Darkness should be maintained until the cotyledonary stage is reached, after which exposure to light is recommended.
TL;DR: The triterpenoids from leaf tissue were more easily quantifiable in each phenotype than in the case of the undifferentiated cells (callus and cell suspensions), which had lower, but still quantifiable, levels of these targeted secondary metabolites.
Abstract: Two morphologically distinct phenotypes of Centella asiatica (Type-1 and Type-2) in South Africa were compared in relation to the levels of triterpenoid saponins with the aim of assessing their potential for biotechnological manipulation of triterpenoid synthesis. The metabolites investigated included madecassoside and asiaticoside and their sapogenins madecassic—and asiatic acid; produced in cultured undifferentiated cells (cell suspensions and calli) and leaves. Weight determination in plant cell suspensions and the accumulation of secondary metabolites after 16 days for Type-1 and 20 days for Type-2 were investigated since these secondary metabolites accumulate during the period that follows the active growth phase. The four triterpenoids of interest were analysed and quantified by HPLC in crude ethanolic extracts. A difference in bioactive triterpenoids was exhibited that was tissue specific and varied between the two phenotypes. The triterpenoids from leaf tissue were more easily quantifiable in each phenotype than in the case of the undifferentiated cells (callus and cell suspensions), which had lower, but still quantifiable, levels of these targeted secondary metabolites. Leaves contained the highest triterpenoid levels (ranging from 1.8 to 5% dry weight for the triterpenoid acids and their glycosides, respectively), with the free acids occurring in a ratio of approximately 1:2.5 in relation to the glycoside content.
TL;DR: It is deduces that the various organogenic capabilities of cotyledon explants from pumpkin are the result of their endogenous hormonal contents, which vary dependently on their endogenous iPA contents.
Abstract: To induce multiple shoots from pumpkin (Cucurbita moschata Duch.), cotyledon explants excised from various ages of seedlings after in vitro germination were cultured on MS augmented with different concentrations of BA (0, 0.5, 1.0 or 2.0 mg l−1). The highest frequency of shoot regeneration (63.7%) was observed from seven-day-old cotyledon explants cultured on MS containing 0.5 mg l−1 BA. The frequency and duration of shoot formation showed close correlation with the donor seedling age. By contrast, BA supply was necessary to promote shoot formation but no differences were observed in relation to different concentrations. Multiple shoots elongated on MS supplemented with 0.1 mg l−1 BA and 5–7 shoots per regenerated explant were recovered. Elongated shoots were rooted on MS, which was easier than that on 2/3MS, 1/2MS, or MS supplemented with 0.1 mg l−1 NAA. The rooted shoots were then transferred to greenhouse where they grew and flowered normally. Quantitative analysis of endogenous auxin (IAA) and cytokinins (iPA and ZR) in initial cotyledon explants of different aged seedlings showed that the regeneration ability of cotyledon explants varied dependently on their endogenous iPA contents. This study therefore deduces that the various organogenic capabilities of cotyledon explants from pumpkin are the result of their endogenous hormonal contents.
TL;DR: Attempts to genetically transform and regenerate mature plants from the economically important Navelina sweet orange cultivar by using the procedure previously established for the genetically close Pineapple sweet orange variety suggest that small changes in the procedure could induce drastic alterations in regeneration and even increase the likelihood of obtaining transformants from non-responsive genotypes.
Abstract: In citrus, production of mature transgenic plants belonging to different genotypes is an important biotechnological objective. In the present study, we tried to genetically transform and regenerate mature plants from the economically important Navelina sweet orange cultivar by using the procedure previously established for the genetically close Pineapple sweet orange variety. The use of BAP at 3 mg l−1 promoted efficient shoot organogenesis in Pineapple as expected, but not in Navelina. Furthermore, different effects were observed when the auxin α-naphtalene acetic acid (NAA) was added to BAP-containing regeneration media. Although NAA addition at 0.5 mg l−1 enhanced cambial callus formation, number of shoots and their elongation in Navelina, the contrary effect was observed in Pineapple. Moreover, transformation efficiency in Navelina rose from 0 to 3% but declined from 6 to 0% in Pineapple, indicating that BAP and BAP + NAA exerted the opposite effect in transgenic shoot regeneration from two closely related cultivars. This suggests that small changes in the procedure could induce drastic alterations in regeneration and even increase the likelihood of obtaining transformants from non-responsive genotypes. Moreover, the vigour of the starting plant material and the addition of kanamycin as selective agent were determining for the generation of mature sweet orange transgenic plants.
TL;DR: In vitro bud clusters of Calathea orbifolia (Linden) Kennedy were obtained and subcultured in semi-solid (agar) medium and temporary immersion system (TIS) for 12 weeks, and plants in TIS had substantial higher photosynthetic rates than plants in Semi-solid medium.
Abstract: In vitro bud clusters of Calathea orbifolia (Linden) Kennedy were obtained and subcultured in semi-solid (agar) medium and temporary immersion system (TIS) for 12 weeks. Uniform young plants were selected and transferred to soilless mix in a growth chamber for ex vitro acclimatization during 35 days, followed by growing in a shaded greenhouse for 65 days. Comparison of in vitro leaf anatomy, ex vitro photosynthetic behaviors and growth was made between two cultural systems. Plants in TIS produced thicker leaf chlorenchyma and aquiferous parenchyma, lower stomatal frequency and more epicuticular wax than did those in semi-solid medium. Plants from semi-solid medium had consistently lower leaf Fv/Fm values than plants from TIS. Leaf Fv/Fm value in plants from TIS decreased to 0.65 at day 7 after transfer and increased soon up to 0.76 thereafter. In contrast, leaf Fv/Fm value in plants from semi-solid medium reduced to 0.27 at day 7 after transfer and increased slowly up to 0.68 at day 35. During ex vitro acclimatization, plants in TIS had substantial higher photosynthetic rates than plants in semi-solid medium. Plants from TIS had subsequent higher leaf area, fresh and dry weights than plants from semi-solid medium.