Establishment of in vitro tissue cultures from Echinacea angustifolia D.C. adult plants for the production of phytochemical compounds

The establishment of in vitro cultures of Echinacea angustifolia D.C. was obtained directly from sections of flower stalks of adult plants. The shoot formation was obtained from this plant material placed on a modified MS basal medium named CH supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (BA). The in vitro propagation procedure of E. angustifolia consisted of three distinct phases: an initial regeneration phase from stalk sections (IP shoots on basal medium with 0.25 mg L⁻¹ BA), an elongation phase on active charcoal and an axillary proliferation of the shoots (AP shoots on basal medium with 0.5 mg L⁻¹ BA).Regenerating calli were established from leaves of in vitro shoots cultured on CH medium supplemented with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ indole-3-butyric acid (IBA). Developed shoots from the callus cultures were subcultured on the CH medium with 0.5 mg L⁻¹ BA (leaf regenerated shoots: LR shoots). The secondary metabolite content of the in vitro plant material was compared with that of the greenhouse growing plants. The quali-quantitative LC-DAD-ESI-MS analysis on the extracts from axillary proliferation shoots (AP shoots) showed significant production of caffeic acid derivatives while leaf callus and LR shoots, accumulated mainly alkamides. These results showed that the proper choice of the procedures for in vitro multiplication allowed us to obtain plant biomass able to produce the active compounds typical of E. angustifolia plants.     

Efficient production of transgenic plants using the bar gene for herbicide resistance in sweetpotato

Efficient production of transgenic sweetpotato (Ipomoea batatas (L.) Lam.) plants using the bar gene for herbicide resistance was achieved through the use of embryogenic suspension cultures and Agrobacterium tumefaciens-mediated transformation. Cell aggregates from embryogenic suspension cultures of sweetpotato cv. Lizixiang were cocultivated with A. tumefaciens strain EHA 105 harboring a binary vector pCAMBIA3300 with the bar gene and uidA gene. Selection culture was conducted using 0.5 mg/l PPT. A total of 1431 plants were produced from the inoculated 870 cell aggregates via somatic embryogenesis. GUS assay and PCR analysis of the regenerated plants randomly sampled showed that 86.5% of the regenerated plants were transgenic plants. Stable integration of the bar gene into the genome of transgenic plants was confirmed by Southern blot analysis and transgene expression was demonstrated by Northern blot analysis. The copy number of integrated bar gene ranged from 1 to 3. Transgenic plants exhibited functional expression of the bar gene by in vivo assay for herbicide resistance. This study also provides a simple and efficient transformation system of sweetpotato based on the use of bar gene as a selectable marker gene, which can be combined with other agronomically important genes for the improvement of sweetpotato.     

Transformation of Brassica oleracea var. capitata with bacterial betA gene enhances tolerance to salt stress

The bacterial betA gene for biosynthesis of glycinebetaine was transferred to cabbage (Brassica oleracea var. capitata) cultivar ‘Golden Acre’ through Agrobacterium-mediated transformation of hypocotyl explants. Transgenic status was established through Southern hybridization and mRNA expression in the shoots. The transformants exhibited higher tolerance to NaCl stress compared to untransformed parent plants. In physiological assessment of salinity tolerance, transgenics showed better growth response and greater stability in maintaining plant water relations at increasing levels of salinity. These results demonstrate that engineering glycinebetaine biosynthetic pathway into cabbage can lead to enhanced salt tolerance.     

Modeling individual leaf area of ginger (Zingiber officinale Roscoe) using leaf length and width

Leaf area estimation is an important biometrical observation one has to do for comparing plant growth in field and pot experiments. In this study, a leaf area estimation model was developed for ginger (Zingiber officinale Roscoe), using linear measurements of leaf length (L) and maximum width (W). Leaves from five ginger varieties (Varada, Rejatha, Mahima, Maran and Himachal) were used to develop the model in 2006–2007. The actual leaf area (LA) was measured with a leaf area meter (LI-3100, LI-COR, Lincoln, NE, USA) and taken as reference LA. The linear measurements were used to build linear (LA = a + b × L × W) and power models (LA = α × (L × W)^β) for each variety, as the modeling among variety were not different from each other, data for all five varieties have been pooled and compared with earlier models by graphical procedures and statistical criteria such as Mean Square Error (MSE), Root Mean Square Error (RMSE) and Chi-square (χ²). The selected model was validated during 2007–2008. The validation data set was used to produce a validation model for each variety by re-estimating the model parameters to develop the estimation model and the models were compared for consistency. The predicted LA (PLA) was compared with observed LA (OLA) by graphical procedures and lack of agreement was evaluated by calculating the relative bias, estimated by the mean of differences (d) and the standard deviation (SD) of the differences. Normality test was carried out by Spearman's rank correlation coefficient (r_s) and residuals were normally distributed. Finally, the proposed model for leaf area estimation of ginger is LA = −0.0146 + 0.6621 × L × W, R² = 0.997. This model can be reliably used for estimating leaf area of ginger non-destructively. The same equation can be extrapolated to all varieties and land races of ginger as it is vegetatively propagated crop with narrow genetic variability.     

Micropropagation of Dendrobium draconis Rchb. f. from thin cross-section culture

An efficient procedure is outlined for rapid and mass in vitro propagation of an orchid, Dendrobium draconis Rchb. f. through in vitro culture of thin cross-sections (TCSs) derived from young stems. The TCS explants were excised along the stem from the base to shoot tip of 6-month-old plantlets and cultured on Murashige and Skoog (MS) medium supplemented with 20 g/l sucrose and different concentrations of N⁶-benzyladenine (BA), kinetin (Kn) and 1-naphthaleneacetic acid (NAA), either individually or in combination. Protocorm-like bodies (PLBs) were directly induced from the TCS explants and completely developed into shoots within 6–7 weeks. The optimal growth regulators combination for maximal PLB development was 2 mg/l BA and 1.0 mg/l NAA, giving rise to 68% of responding explants with an average 11 PLBs per explant. Shoot development was best achieved on MS medium containing sucrose and coconut water. Plantlets, 6–8 cm height were transplanted into coconut husk peat with 92% survival rate in a nursery.     

Potential hazard characteristics of Tilia, Betula, and Acer trees removed in the Helsinki City Area during 2001–2003

In order to study decay, and to improve the management and protection of old urban trees, a total of 256 felled urban trees were examined during 2001–2003: 95 Tilia spp., 74 Betula spp., and 87 Acer spp. Most of the trees (73%) were located in the main parks and along the main streets in the downtown area of Helsinki City, Finland. The mean age of the trees was over 60 years, and the majority (64%) were old park trees. Poor condition and increasing risk of failure were the main reasons for felling in 82% of the cases. Thirty three percent of these trees were degenerated or dead, but the amenity value of 14% of the risk trees was still high. The latter were old, big trees which posed a potential hazard, but had a vital and balanced crown.Some characteristic profiles for potential failure were identified for each of the tree species studied: Ganoderma lipsiense in the butts and hollows in the stems of Tilia spp., weak fork formations together with Rigidoporus populinus on Acer spp., and degeneration together with decay in the stem on Betula spp.Decay fungi most commonly identified were R. populinus, G. lipsiense, Inonotus obliquus and Piptoporus betulinus. In addition, Kretzschmaria deusta was very common in three of the parks, and on every one of the tree species investigated.     

Clone identification and genetic relationship among vine cacti from the genera Hylocereus and Selenicereus based on RAPD analysis

Clones of Hylocereus and of Selenicereus species were distinguished from each other by the banding pattern generated by one to nine 10-mer oligonucleotide primers in the random amplified polymorphic DNA (RAPD) reaction. RAPD analysis was also applied to estimate the genetic relationship among five Hylocereus and nine Selenicereus species. A dendrogram was constructed based on a data matrix of 173 polymorphic bands originated by nine primers. Two groups were identified, one consisting of Hylocereus species and the other consisting of Selenicereus species. These results are consistent with the accepted taxonomic classification of the genera studied. The principal coordinate analysis (PCO), i.e. the plot drawn on the basis of the RAPD data, clearly distinguished between three groups, namely, Hylocereus species, S. megalanthus and the rest of the Selenicereus species studied. PCO thus strongly support the notion that the tetraploid S. megalanthus is an exception among the Selenicereus group. The RAPD results support our hypothesis regarding the allopolyploid (rather than autopolyploid) origin of this species.     

Characterization of tetraploid plants regenerated via protoplast culture of Iris fulva and their crossability with Japanese irises

Characteristics such as flower form, size and color of outer and inner perianths, anthocyanins in outer perianths, size, color and fertility of pollen and self-fertility of diploid and tetraploid lines regenerated via protoplast culture of Iris fulva were examined and compared with those of the diploid wild line. Among these characteristics, flower form, inner and outer perianth sizes of the tetraploid lines were noticeable, because these lines had upward flower forms and bigger flowers than diploid lines. Furthermore, reciprocal crosses between diploid or tetraploid lines of I. fulva and I. ensata and those of I. fulva and I. laevigata were performed. Three seedlings were obtained from the cross of tetraploid I. fulva × diploid I. laevigata through embryo rescue. One of them was identified as the interspecific hybrid between tetraploid I. fulva and I. laevigata by flow cytometoric (FCM), cytological and molecular (RAPD) analyses. This is the first report on production of hybrids from these lines. I. fulva has unique brown flowers, and this trait could be very useful for flower color breeding of I. laevigata which lacks this color. Therefore, the hybrid of I. fulva (4×) × I. laevigata may be the best available gene source for brown color breeding of this species.     

In vitro induction of the amphiploid in interspecific hybrid of blueberry (Vaccinium corymbosum × Vaccinium ashei) with colchicine treatment

Amphiploids were produced from pentaploid hybrids between Vaccinium corymbosum (4x) and Vaccinium ashei (6x) by colchicine treatment. Seeds of each parental species and those obtained by the interspecific crossing were treated with colchicine at 0, 500, 1000 and 2000 mg/l for 7 days, and the ploidy level of the seedlings was determined with flow cytometry. Either amphiploid (10x) or ploidy chimera (5x + 10x) was obtained from interspecific hybrid seeds treated with all colchicine concentrations, while no chromosome-doubled plants were obtained from both parents. Cross direction in interspecific hybridization affected the results of colchicine treatment and amphiploids were obtained only when V. corymbosum was used as the seed parent. In this cross, 17–25% of the seedlings turned to be amphidiploids or ploidy chimeras by treating with 500 mg/l colchicines for 7 days. These results indicate that susceptibility to colchicine may increase in the interspecific hybrid compared with the parental species but only when V. corymbosum was used as female parent.     

Influence of medium composition and vessel ventilation on in vitro propagation of Phillyrea latifolia L.

Micropropagation of Phillyrea latifolia L. a wild species present in Mediterranean coastal areas having drought and salt tolerance was performed using explants from adult plants. Shoots were induced from nodal explants on the Rugini’s initial medium (IM). Then these were proliferated on either Rugini olive medium (OM) or Linsmaier and Skoog (LS) medium, each supplemented with 2.22 μM 6-benzylaminopurine (BA) or 4.56 μM zeatin (Z). Rooting (66.1±11%) was induced on shoots grown in perlite soaked with half-strength Rugini olive proliferation medium (OMr) containing 2.69 μM α-naphthaleneacetic acid (NAA) and 160 mg l⁻¹ putrescine. Both shoot multiplication and rooting were performed using Magenta^® GA-7 (Sigma) vessels either non-permeable or permeable to gas exchanges. Contamination (about 40%) was observed during the first five passages notwithstanding the addition of cefotaxime to the culture medium, but a high proliferation rate (90%) of explants provided enough healthy plant material. The highest shoot proliferation was observed on LS medium and zeatin whereas the presence of the ventilated filters reduced fresh weight of explants growing on LS media and did not affect shoot growth on OM media. During rooting, the use of ventilated vessels in comparison with the closed ones enhanced development of roots, and doubled the dry weight of plantlets. The vessel ventilation combined with the artificial substrate (perlite) was beneficial for in vitro acclimatization of rooted Phillyrea plantlets.     

Effects of an in vitro maturation treatment on plant recovery from avocado zygotic embryos

An efficient protocol for in vitro maturation of very immature, <10 mm, avocado embryos has been developed. The efficiency of plant recovery as well as the quality of the resulting plants was greatly improved by including a maturation phase prior to induction of germination. The influence of different factors, such as the gelling agent, organic supplements or abscisic acid, on embryo maturation and subsequent germination was tested. Optimum conditions were met when maturation was carried out in B5m medium supplemented with the Jensen's amino acids, an extra 88 mM sucrose and 6 g l⁻¹ agar as gelling agent. At these conditions, embryos which had been collected 68 days after pollination germinated at a 65% rate in solid medium, giving rise to healthy and vigorous plantlets. Anatomical differentiation and storage product accumulation occurring during the in vitro maturation phase were studied by means of histological techniques. Results obtained revealed that, at the end of the in vitro maturation period, embryos resembled the pattern previously established for avocado embryos matured under in vivo conditions: histodifferentiation had been accomplished and starch granules and protein bodies were abundant.     

Germination and growth of sponge gourd (Luffa cylindrica) pollen tubes and FTIR analysis of the pollen tube wall

Emergence of multiple pollen tubes from single pollen grains occurred both in vitro and in vivo in sponge gourd (Luffa cylindrica (L.) Roem). The frequency with which pollen grains produced multiple pollen tubes in vivo (7.2%) was lower than that under in vitro conditions (14.9%). In pollen grains germinated in vitro, the total length of the multiple pollen tubes was greater than that of single pollen tubes, but individual tubes among the multiple tubes did not reach the same length as single tubes. Moreover, the growth of the single pollen tubes continued for a longer period in vitro than that of the multiple tubes. Fluorescence microscopy showed that callose was present throughout the pollen tube wall except in the apical part of growing pollen tubes, and nuclei moved into the longest of the multiple tubes. Results of Fourier transform infrared microspectroscopy indicated that abnormal cell wall components (peaks at 800–1000 cm⁻¹) were more frequent in multiple pollen tubes lacking nuclei, and the pectin content (1733 cm⁻¹) in multiple pollen tubes was much lower than that in single pollen tubes. These findings suggested that there were significant differences in pollen tube growth and wall composition between single and multiple pollen tubes, and that multiple pollen tubes had much less opportunity than single pollen tubes to reach the embryo sac and achieve double fertilization.     

In vitro embryo germination and proliferation of pistachio (Pistacia vera L.)

In vitro development of isolated embryos and axillary bud proliferation were studied in Pistacia vera L. Different regulator-free nutrient media were compared to determine the effects of the mineral solution, agar and sucrose concentrations on seedling development from mature embryos. Nutrient-rich MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tabacco tissue cultures. Physiol. Plant. 15, 473–479] and DKW [Driver, J.A., Kuniyuki, A.M., 1984. In vitro propagation of Paradox walnut rootstock. HortScience 19, 507–509] mineral solutions were more efficient for the development of aerial parts than nutrient-poor KN [Knop, W., 1884. Bereitung einer concentrierten nährstofflosung für pflanzen. Landwersuhssat 30, 292–294] and WT [Withe, P.R., 1936. Plant tissue cultures. Bot. Rev. 2, 419–437] solutions. Reducing the agar concentration enhanced fresh matter production and balanced seedling development. When tested at different concentrations, sucrose was found to orient mature embryo development, with the best results obtained at concentrations of 2–4%, whereas high concentrations (6 and 12%) mainly inhibited elongation of the aerial parts. Plantlets obtained previously from in vitro cultured embryos were propagated by axillary budding. High bud proliferation (six shoots per explant) was achieved when using 17.8 μM benzyladenine (BA) combined with 0.5 μM indole-3-butyric acid (IBA). The addition of 2.9 μM gibberellic acid (GA₃) to the propagation medium did not improve axillary shoot yields and on average, media with GA₃ produced 2.3–2.6 elongated stems per cultured explant. Shoots were rooted in vitro in half-strength MS medium containing 12.3 μM IBA.     

Aptitude for mycorrhizal root colonization in Prunus rootstocks

Eighteen Prunus rootstock cultivars were inoculated with three arbuscular mycorrhizal fungi under greenhouse conditions in order to evaluate their affinity for mycorrhizal colonization. The rootstocks were peach–almond hybrids, peaches, plums and cherries of Spanish, French and Italian origin. Mycorrhizal colonization was low in plants inoculated with Glomus mosseae (Nicol. and Gerd.) Gerdemann and Trappe, and Glomus etunicatum Becker and Gerdemann. In contrast, Glomus intraradices Schenck and Smith, proved to be the most infective endophyte, achieving the highest mycorrhizal colonization rate in most of the rootstocks evaluated. Species of Prunus insititia L. were the only botanical group to show a consistently high affinity for mycorrhizal colonization with G. intraradices.     

Effects of explant type, culture media and growth regulators on callus induction and plant regeneration of Chinese jiaotou (Allium chinense)

To establish an efficient protocol of shoot regeneration from callus, effects of explant type, culture media and plant growth regulators on callus induction and shoot regeneration of Chinese jiaotou (Allium chinense) were evaluated. The results showed that basal plate was the best explant for callus induction (47.5%) when cultured on B5 medium supplemented with 0.1 mg l⁻¹ 6-benzylaminopurine (BA) and 1.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), and B5 was the best medium to induce callus formation with 49.3% of the explants forming callus. The highest callus induction (65.2%) was achieved culturing basal plate on B5 medium supplemented with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ 2,4-D after 8 weeks of culture. The best callus proliferation was observed on B5 medium with 1.5 mg l⁻¹ 2,4-D. Shoots regenerated at the highest frequency of 58.8% with 4.5 shoots when calli were cultured on B5 medium with 0.1 mg l⁻¹ BA and 1.0 mg l⁻¹ a-naphthaleneacetic acid (NAA). This protocol provides a basis for future studies on genetic improvement and could be applied to large-scale multiplication systems for commercial nurseries of Allium chinense.     

Role of bioinoculants in development of salt-resistant saplings of Morus alba (var. sujanpuri) in vivo

Pot experiments were conducted for the development of salt-resistant saplings of Morus alba (var. sujanpuri) involving bioinoculants, namely arbuscular mycorrhizal (AM) fungi, Azotobacter and indole butyric acid (IBA). The IBA and sodium chloride (NaCl) concentrations were optimized prior to the experiments. By using both low and high concentrations, 15 ppm IBA and 0.05% NaCl (w/v) were found to be optimum in acting synergistically with AM fungi and Azotobacter and also for increasing all the growth parameters and microbial count in the rhizosphere. For in vivo development of salt-resistant saplings, the optimal concentration of IBA, along with AM fungi and Azotobacter in different combinations, was applied. The saplings were irrigated regularly with 0.05% NaCl water. Although growth parameters such as AM infection percentage, AM spores per 100 g soil and Azotobacter cells/g soil were affected by NaCl watering, the inoculation of both bioinoculants significantly enhanced survival percentage of saplings from 25 to 50% under salt stress. Maximum survival (55%) of saplings was found with IBA (15 ppm)+AM fungi+Azotobacter.     

Assessing Rosa persica genetic diversity using amplified fragment length polymorphisms analysis

The genetic diversity among 128 Iranian Rosa persica (R. persica) accessions in the different populations was analyzed. Amplified fragment length polymorphisms (AFLP) technique was used to produce 171 polymorphic fragments. The number of polymorphic loci ranged from 101 to 147 and the polymorphism information content (PIC) varied from 0.289 to 0.073, with an average of 0.16. This shows extreme variability and genetic diversity among the studied R. persica populations. An indirect estimate of the number of migrants per generation (Nm = 0.376) indicated that gene flow was relatively low among populations of the species. Cluster analysis using the UPGMA method grouped all accessions into six clusters. The results did not show relative agreement with the genotypes’ region of origin. Based on an analysis of molecular variance, 48% of the genetic variation of R. persica was within population and 52% was among populations. The present analysis revealed that Iranian R. persica genotypes are highly variable and genetically distinct from their origins. The apparent unique nature of the R. persica genotypes revealed by our results supports the case for the implementation of more intense characterization and conservation strategies, and provides useful information to address breeding programmes and germplasm resource management in Rosa spp.     

Phylogenetic study and molecular identification of 31 Dendrobium species using inter-simple sequence repeat (ISSR) markers

The genetic diversity of the genus Dendrobium is not well known and the phylogenetic relationship of Dendrobium species are mainly determined by studies of the comparative vegetative anatomy and plant systematics. In the present study, we used the technique of inter-simple sequence repeats (ISSRs) to evaluate genetic diversity and phylogenetic relationship among 31 Dendrobium species from Yunnan region of China. In total, 2368 bands were amplified by 17 ISSR primers, resulting from 278 ISSR loci with 100% polymorphism at genus level. Thirty-one species were unequivocally distinguished based on ISSR fingerprinting. Species-specific ISSR markers were identified in nine of 31 tested Dendrobium species. Unweighted pair-group mean analysis (UPGMA) showed that 31 Dendrobium species were grouped into six clusters, indicating the genus was polyphyletic with several well-supported lineages. The high polymorphism and reliable amplification across species demonstrated the utility of ISSR marker for species diagnosis and genetic diversity study of the genus Dendrobium.     

Isolation of arbuscular mycorrhizal fungi and Azotobacter chroococcum from local litchi orchards and evaluation of their activity in the air-layers system

Soil samples were collected from rhizosphere of litchi-growing areas of North-Western Himalayan Region (NWHR) of India, for finding qualitative and quantitative differences in arbuscular mycorrhizal (AM) fungi and Azotobacter chroococcum. These samples were taken from plants being grown in different cultivation types namely, weed control with weedicides or tillage; orchard floor either clear or with cover crops; intercropping with cereals and legumes. Qualitative and quantitative differences were noticed with different cultivation types and a marked reduction in the AM fungi was observed in orchards where chemicals were used for weed control and intensive farming system was used on the orchard floor. AM fungi were generally abundant in the soils with range pH 5.5–6.6. Among different AM fungi retrieved from the soils, Glomus spp. was most dominant. Fifteen AM fungal species were isolated, identified and characterized and along with their ability to colonize the roots. In the soil samples, a marked variation in viable bacterial count of A. chroococcum was also noticed due to varied physico-chemical characteristics of the orchard soils. The changes in AM fungal species composition can be attributed to changes in soil chemical properties resulting from cultural practices such as ploughing, application of chemical fertilizers and weedicides. An experiment was also conducted to study the comparative efficacy of four dominant and frequently occurring indigenous AM species namely, Glomus fasciculatum (Thaxter sensu Gerdemann), G. magnicaulis (Hall), G. mosseae (Nicol. & Gerd.), Gigaspora heterogamma (Nicol. & Gerd.) and two A. chroococcum strains viz., AZ₁ and AZ₂ singly and in dual combination to evaluate their effect in air-layers system. Dual inoculation of G. fasciculatum and AZ₁ increased total root length of air-layered shoots by 81.39% over uninoculated control. These studies indicated that indigenously isolated AM fungal species and A. chroococcum strains can be used for air-layering for better adaptation under specific agro-climatic and ecological zone conditions.     

Molecular cloning of three malic acid related genes MdPEPC, MdVHA-A, MdcyME and their expression analysis in apple fruits

Malic acid (MA) in apple fruit is the predominant organic acid associated with taste, flavour and juice quality. In this study, three full-length cDNAs of MdPEPC, MdVHA-A and MdcyME were cloned from apple fruit. They encoded cytosolic phosphoenolpyruvate carboxylase (MdPEPC, EC 4.1.1.31), subunit A of vacuolar H⁺-ATPase (MdVHA, EC 3.6.1.3) and cytosolic NADP-dependent malic enzyme (MdcyME, EC 1.1.1.40), respectively, for MA synthesis, transportation and degradation. Real-time quantitative PCR discovered that the expression levels of three genes varied with development stages, and that their expression patterns differed between low acid (LA) and high acid (HA) genotypes. In addition, enzyme activity assay showed that PEPC and VHA contributed positively to MA accumulation during fruit development both in LA and HA, while cyME did negatively.