Contributions of the leaves and axillary shoots to rooting in Eucalyptus granáis Hill ex Maid,stem cuttings

In one-node stem cuttings of Eucalyptus granáis the absence of leaves was fatal. The optimum leaf area per leaf for rooting was 2-5 cm2, and in this range rooting was higher in one-leaf than in two-leaf cuttings. Axillary shoots started to develop soon after the cuttings were set and their growth was promoted by the presence of the subtending leaf. Thus, in one-leaf cuttings the shoot in the leafy axil grew, at the expense of the shoot in the defoliated axil, while in two-leaf cuttings the shoot in the upper (sub-opposite) axil tended to grow more quickly. Axillary shoots were also vigorous in cuttings from decapitated and disbudded mother plants. Rapid shoot growth was associated with low rates of abscission of the subtending leaf and relatively high rooting ability. Removing axillary shoots from cuttings accelerated leaf abscission and reduced rooting. The stem of the cutting often swelled and discoloured from the base, apparently due to the demand of the cutting for water. Additional leaf wounds had no effect on rooting (but increased mortality), while immersing cuttings in water before setting, or leaving them in a loose heap on the propagation bench, promoted rooting.     

Molecular evidence for the natural hybrid origin of Phalaenopsis × intermedia Lindl.

Summary

In order to confirm the hybrid origin of Phalaenopsis intermedia Lindl., internal transcribed spacers (ITSs) of ribosomal DNA (rDNA) and three fragments of chloroplast DNA (cpDNA) were studied. Nineteen clones of ITS sequences from three accessions of P. intermedia Lindl. were aligned with nine species of sections of Phalaenopsis and Stauroglottis that were candidate parents. A phylogenetic tree, derived from the ITS data, was constructed by the Neighbor-joining (NJ) method. Two major groups were shown for 19 clones of ITS sequences of P. intermedia based on the phylogenetic tree. The average genetic distance between the aforementioned two groups and the candidate parents was calculated based on the Kimura 2-parameter method. One group had the lowest genetic distance from the ITS repeat sequences of P. aphrodite Rchb.f., and another group had the lowest distance from that of P. equestris (Schauer) Rchb.f. The results indicated that both P. aphrodite and P. equestris are parents of P. intermedia based on the ITS data. In addition, analysis of three fragments of cpDNA, namely the trnL intron, the trnL-trnF intergenic spacer (IGS), and the atpB-rbcL IGS, showed that P. intermedia had the lowest genetic distance from P. aphrodite. Both the ITS and cpDNA data, as well as reference to the effects of maternal inheritance of cpDNA, suggest that P. aphrodite was the maternal parent and P. equestris was the paternal parent of P. intermedia. Therefore, molecular evidence supports P. intermedia being a natural hybrid derived from P. aphrodite and P. equestris.     

Effect of agar,galactomannan and indole-butyric acid on in vitro rooting of the pear cultivar ‘Durondeau’ and apple rootstock cultivar ‘Marubakaido’

Summary

The influence of partial substitution of agar by galactomannans in culture media supplemented with different concentrations of indole-3-butyric acid (IBA) was studied on in vitro rooting of pear (Pyrus communis L.) cultivar ‘Durondeau’ and apple rootstock (Malus prunifolia Borkh.) cultivar ‘Marubakaido’. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum) seeds. The results obtained with mixtures of agar and galactomannan (3 g l^–1 each) were compared with those from media solidified with a standard concentration of agar (6 g l^–1). The rooting of pear shoots was enhanced significantly in the presence of a mixture of agar plus cassia galactomannan compared to medium solidified with agar only. The modified media promoted a higher number of roots than the control, and increased the percentage of rooted shoots. A maximum of 84.8% rooting was obtained on half-strength MS medium (¹?2MS) supplemented with 0.49 µM IBA and solidified with a blend of agar plus cassia galactomannan. For the apple rootstock, only the number of roots per shoot was influenced significantly by the addition of galactomannan to the rooting medium. The highest number of roots per shoot was 16.67 on ¹?2MS medium gelled with a mixture of agar plus guar galactomannan supplemented with 4.90 µM IBA. The behaviour of the agar-galactomannan gel and the possibility of reduced costs when compared with systems containing only agar, suggest new biological and commercial applications for galactomannans.     

In vitro propagation of GF677 hybrid rootstock (Prunus persica × Prunus amygdalus) from mature cotyledons

Adventitious shoot regeneration from mature cotyledons of GF677 rootstock (Prunus persica × Prunus amygdalus) was achieved in vitro. Thidiazuron (N-phenyl-N-1,2,3-thidiazol-5-yl-urea; TDZ) at 32 µM gave the highest percentage of cotyledons forming adventitious shoots (68.8%) and the highest number of shoots per cotyledon (4.8) on Quoirin and Lepoivre (QL) medium. On QL medium containing 32 µM TDZ, exposure of the proximal segments of cotyledons to darkness at the start of culture increased the percentage of cotyledons forming adventitious shoots (62.5%) when compared with those kept under light conditions (15%). A combination of 0.72 µM gibberellic acid and dark treatment resulted in at least 2.7-fold more elongated shoots than non-treated shoots. The highest rooting percentage (100%) occurred on 0.5× Murashige and Skoog medium supplemented with Gamborg (B5) vitamins and 2 mg l^?1 indole-3-butyric acid. Rooted plantlets were acclimatised under greenhouse conditions with a 70% survival rate.     

Causes of leaf-tip scorch in the cultivated Protea hybrid ‘Sylvia’

Soil and leaf samples were analysed in an attempt to identify the causes of leaf-tip scorch commonly observed in cultivated Protea sussannae and Protea eximia hybrid ‘Sylvia’. Leaf-tip scorch was hypothesised to be caused by nutritional problems. Soil and leaf mineral contents were measured and compared between healthy plants and those suffering from leaf-tip scorch. Leaf-tip scorch was found to be associated with increase electrical conductivity, total cation concentration and the concentrations of Na, K, Ca, Mg, Mn and B in the soil. It was concluded that failed attempts of growers to remedy the problem by foliar spraying of micronutrients might have artificially increased the levels of Mn and B. Leaf-tip scorch was associated with decreased foliar concentrations of Fe and increased concentrations of Na and Mn. No association between leaf-tip scorch and either ∂¹³C or ∂¹⁵N was found. It was concluded that changes in water relations and N acquisition were not associated with the symptoms. The significant decrease in the Ca concentration in the tips of leaves showing advanced leaf-tip necrosis indicated that a deficiency of Ca in the leaf tips may have been the proximal cause of the pathology. Ca deficiency in the leaf tips may in turn be caused by high soil EC.     

Pruning affects carbohydrate accumulation in the shoots and leaves of ‘Precoce de Tyrinthe’ apricot

Summary

This study was conducted in Mut-Mersin, Turkey, to determine the effects of various times of pruning (no pruning = control; only summer (July, August, or September) pruning in 1994 and 1995; only winter pruning in 1995 and 1996; both summer + winter pruning in 1994-1995 and 1995-1996) on the carbohydrate contents of annual shoots and leaves of young ‘Precoce de Tyrinthe’ apricot trees. Although carbohydrate content in annual shoots was highest in the control trees during the first year, the summer + winter pruning gave the highest values in the second year. In 1995 total sugar and sucrose contents of the leaves was greatest following winter pruning, but starch content was higher in the leaves of control trees. The content of reducing sugars was greatest in leaves of trees pruned in both September + winter. In 1996, levels of total sugars and starch were highest following September pruning, starch content was highest in the control, and; reducing sugars in the leaves were highest following August + winter pruning.     

Adaptation of the tropical hybrid Euphorbia × lomi Rauh to the exposure to the Mediterranean temperature extremes

Poysean (Euphorbia × lomi Rauh) has been introduced in the Mediterranean countries as an ornamental plant for indoor uses. When used outdoor, few information is available about its ability to withstand the Mediterranean temperature extremes. An experiment in an open stand was performed to evaluate poysean tolerance to the winter and summer temperatures of the thermo Mediterranean climate and its ornamental value for urban greening. Two genotypes of poysean (Nguen Muang, NM; and Soi Budsanin, SB) with similar bracts and flower colours were grown at either 2 or 3 plants per pot during 23 months. Number of flower racemes and leaves per plant and plant height were recorded monthly. Overall ornamental value of each pot was scored by means of a panel test (PT) performed by independent examiners. Score assignment was homogeneous between examiners. On average, NM showed a 36% lower number of flower racemes, but a 30% higher ornamental value than SB. During fall and beginning of the winter, NM showed a higher number of flower racemes per plant than SB. Increasing plant density from 2 to 3 plants per pot did not influence flower racemes and leaves per plant. From March to November of the second year, number of flower racemes in NM was similar to the first year, whereas SB flower production was markedly higher, which can be due to a genotypic difference to Mediterranean temperature tolerance. Correlation between number of flower racemes per pot and ornamental value was higher in SB than NM, which suggest that in NM flower abundance contribute less to the pot ornamental value. The present data suggest that poysean is able to withstand the Mediterranean temperature extremes and still produces flower racemes during winter, which indicate it as a suitable ornamental plant in outdoor applications.     

Evaluation of the activity of aqueous extracts of some organic waste materials on in vitro-cultured shoots of ‘M9’ apple rootstock

Summary

The present work investigated the effects of different aqueous extracts of organic waste compounds on growth, proliferation and photosynthetic activity in ‘M9’ (Malus domestica Borkh.) shoot cultures, with the aim of determining the feasibility of using in vitro cultures as a tool for the rapid evaluation of organic amendments in agriculture. Aqueous extracts of the following organic waste compounds: cow manure (CM), sugarbeet industrial waste (SB), mixed grape, poultry and municipal solid waste (GPM), and citrus pruning and industrial waste (CPI) were prepared at a rate of 1:10 (w/v) compound:distilled water. The basal media used in the proliferation phase were: (i) PM1, modified Murashige and Skoog (MS) enriched with 4.4 µM 6-benzyladenine (BA); (ii) PM2, as PM1 but with a reduced cytokinin concentration (1 µM BA) to evaluate possible hormone effects; and (iii) PM3, 4.4 µM BA with reduced salt strength (0.33 MS) to induce nutrient deficiency. Hormone-free medium with half-strength MS salts was used for rooting. All media were enriched with each extract at 0, 0.2, 2, 20 or 200 ml l^–1. Photosynthetic activity was measured on PM3 medium enriched with SB or CM. Standard culture conditions were 22° ± 2°C, with a 16 h photoperiod at 30 µmoles photosynthetically active radiation (PAR) m^–2 s^–1, but at 80 µmoles PAR m^–2 s^–1 to determine photosynthetic activity. Shoot weight increase in PM1 was not affected by the GPM and CPI extracts, while the growth trends of CM- and SB-treated shoots were described by a second-degree function with maxima at 2 ml l^–1 and 0.2 ml l^–1, respectively. Shoot proliferation for SB was represented by a quadratic curve (maximum at 2 ml l^–1), was linearly reduced as GPM increased, but was not affected by CM or CPI. Treatments did not significantly affect rooting percentage and root length; however root number was increased by SB at 2 ml l^–1.CO₂ fixation increased linearly with both SB and CM, despite reduced growth at the highest levels of extract.     

In vitro Culture of Populus × euramericana cv.Ⅰ-45

以Ⅰ-45杨带腋芽的茎段为外植体,研究其离体培养中的最佳培养基和激素配比,建立Ⅰ-45杨再生系统.结果表明:最佳诱导培养基为MS+KT 0.5 mg/L+NAA 0.02 mg/L,诱导率为82.2％;最佳继代培养基为MS+KT 1.0 mg/L+NAA 0.02 mg/L;诱导生根的培养基为1/2MS+IBA 0.05 mg/L,生根率达86.7％.     

Protocol: Streamline cloning of genes into binary vectors in Agrobacterium via the Gateway® TOPO vector system

Background

Freezing tolerance is an important factor in the geographical distribution of plants and strongly influences crop yield. Many plants increase their freezing tolerance during exposure to low, nonfreezing temperatures in a process termed cold acclimation. There is considerable natural variation in the cold acclimation capacity of Arabidopsis that has been used to study the molecular basis of this trait. Accurate methods for the quantitation of freezing damage in leaves that include spatial information about the distribution of damage and the possibility to screen large populations of plants are necessary, but currently not available. In addition, currently used standard methods such as electrolyte leakage assays are very laborious and therefore not easily applicable for large-scale screening purposes.

Results

We have performed freezing experiments with the Arabidopsis accessions C24 and Tenela, which differ strongly in their freezing tolerance, both before and after cold acclimation. Freezing tolerance of detached leaves was investigated using the well established electrolyte leakage assay as a reference. Chlorophyll fluorescence imaging was used as an alternative method that provides spatial resolution of freezing damage over the leaf area. With both methods, LT₅₀ values (i.e. temperature where 50% damage occurred) could be derived as quantitative measures of leaf freezing tolerance. Both methods revealed the expected differences between acclimated and nonacclimated plants and between the two accessions and LT₅₀ values were tightly correlated. However, electrolyte leakage assays consistently yielded higher LT₅₀ values than chlorophyll fluorescence imaging. This was to a large part due to the incubation of leaves for electrolyte leakage measurements in distilled water, which apparently led to secondary damage, while this pre-incubation was not necessary for the chlorophyll fluorescence measurements.

Conclusion

Chlorophyll fluorescence imaging is an alternative method to accurately determine the freezing tolerance of leaves. It is quick and inexpensive and the system could potentially be used for large scale screening, allowing new approaches to elucidate the molecular basis of plant freezing tolerance.     

Evaluation of the sensitivity to ionising γ-radiation of a Cymbidium hybrid

Gamma-rays are an important mutagenic agent that can induce new, useful genetic variations in plants. However, γ-irradiation can also cause damage that negatively affects the use of such mutagens in plant breeding. The aim of the present study was to investigate the effects and damage caused by γ-radiation in a Cymbidium hybrid, RB001. The relative growth rate of protocorm-like bodies (PLBs) was reduced by 50% at a γ-ray dose of approximately 40 Gy. Malondialdehyde concentrations increased significantly with increasing radiation dose. However, almost no difference was observed between untreated control PLBs and PLBs treated with 200 Gy 8 weeks after γ-irradiation. The activities of several antioxidant defence enzymes increased gradually with increasing γ-ray dose, 24 h after irradiation. These enzymes showed different responses between 1 and 4 weeks, but no difference 8 weeks after irradiation. The ‘comet assay’ and flow cytometry were performed. Clear differences in radiation-induced damage were observed between control and 200 Gy-treated PLBs at 24 h. However, PLBs had a tendency to recover from 4 weeks after irradiation, and the integrity of their DNA was similar in samples treated with 10–200 Gy. These results indicated that γ-rays caused little DNA damage and the plants could recover. This demonstrated the feasibility of using physiological responses, the ‘comet assay’, and flow cytometry to detect DNA damage after γ-irradiation.     

Characterization of bud-burst potential in Rhododendron catawbiense ‘Album’ plants from in vitro culture by measuring the manganese contents in the distal buds

Summary

It had been established that photoperiodic conditions influence morphogenesis in Rhododendron catawbiense ‘Album’ (Ericaceae). Plants developed from cuttings obtained by in vitro culture were grown in a greenhouse for one or two years, under different photoperiodic treatments. Under long days, the upper buds followed a rhythmic development. Under short days, the inhibition of the growth of the distal buds allowed the development of shoots from the plant base. When plants were placed under long days after six weeks under short days, the distal buds recovered their capacity to form a new growth unit. To find a method to characterize growth potential of vegetative buds, measurements of their mineral content were made. A relationship between the changes in manganese level of the distal buds and their growth potential during the formation of the vegetative system has been established. Under short-day conditions, manganese increased to reach more than 500 mg kg^-1 dry matter in several weeks. Under long days, the manganese level in the upper buds gradually decreased before burst. These results may not provide an explanation for the role of manganese in relation to the buds physiological activity. On the other hand, manganese measurements may be a useful test to estimate growth potential in Rhododendron buds.     

Effect of nitrogen and calcium stock plant nutrition on Petunia × hybrida leaf and anther explant growth in vitro

Petunia × hybrida Hort. Vilm.-Andr. ‘Coral Sea’ stock plants were grown with a modified Hoagland's solution to determine the effect of stock plant N and Ca nutrition on subsequent leaf explant and anther growth in vitro. In separate experiments, N was 0, 75, 150 or applied at 0, 100, 200 or 400 mg 1⁻¹ as nitrate-N, and Ca was applied at 300 mg 1⁻¹. Leaf explants from plants grown with 100 mg 1⁻¹ N produced the greatest amount of callus, while those from plants grown with 200 and 400 mg 1⁻¹ N produced the highest number of shoots. Nitrogen treatments increased the anther fresh weight and the number of shoots per culture compared to anthers derived from stock plants that did not receive N.The fresh weight of shoots produced by leaf explants decreased with increasing Ca concentrations applied to stock plants. Anthers derived from stock plants treated with 0 mg 1⁻¹ Ca produced the highest number of shoots per culture.Anatomically, both filament and anther wall tissue produced callus, but no callus production from microspores was observed. Organs produced by anthers were diploid.     

Evaluation of F1 and BC1 hybrids of Lilium lancifolium × Asiatic hybrid ‘Chianti’ by morphological analysis and fluorescence in situ hybridization

Lilium lancifolium Thunb. exhibits wide genetic diversity and numerous genetic traits within progeny populations. Parent morphology affects the distribution and assortment of progeny phenotypes. In this study, morphological analysis and fluorescence in situ hybridization (FISH) were conducted for cross progeny (F1 and BC1). Results showed that F1-1 hybrids had greater plant height than the parents; however, backcross progeny plant height (group 1) was low. Leaf number in both groups was considerably low in F1 hybrid, although high in backcrossed plants. Flower number and days to flower opening were intermediate to those of the parents for F1 hybrids, yet low in BC1 progeny. In group 1, backcross progeny showed small bulb length; however, group 2 progeny showed large bulb length. Positive and negative correlations between the phenotypic traits of parents and progeny confirmed significant variations. According to FISH results, F1-1 and F1-2 hybrids distinctly exhibited nine and eight 45S rDNA loci which were same in position with 45S loci of the parents. In backcross progeny, eight 45S rDNA signals were detected in four BC1 progeny of group 1, while 10 signals were observed in all group 2 progeny, same with the L. lancifolium karyotype. Morphological analysis and FISH helped in scrutinising progeny to obtain hybrids with desirable characteristics.     

Heme oxygenase decreases the generation of ROS in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells

AIM:To investigate the role of heme oxygenase (HO) in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells.METHODS:(1) Western blotting analysis was carried out to detect protein level of HO-1 in the tissues.(2) ［3H］-TdR, ［3H］-leucine incorporation was measured in cultured vascular smooth muscle cells.(3) 2,7-dichlorofluorescin diacetate (DCFH-DA) as an index was used to determine the cellular reactive oxygen species (ROS) level.RESULTS:(1) No significant difference in HO-1 protein expression level between AngⅡ-stimulated and control groups was observed, but HO-1 protein level in Hemin-induced group was higher than that in other two groups (P<0.01).No significant increase in HO-1 protein expression was found in zinc-protoporphyrin IX (ZnPPIX) group.(2) After AngⅡ stimulation, ［3H］-TdR and ［3H］-leucine incorporations of vascular smooth muscle cells (VSMCs) were increased.Hemin inhibited this increase.The higher concentration of Hemin, the more significant was the inhibitory effect.On the contrary, ZnPPIX promoted the increase in the effect of AngⅡ by inhibiting HO.(3) Fluorescence intensity in AngⅡ group was obviously higher than that in control groups (P<0.01).Compared with AngⅡ group, Hemin group decreased 62.7%, but ZnPPIX group increased 39.5%.CONCLUSION:Hemin induces HO-1 expression and inhibits the effect of AngⅡ to stimulate proliferation and hypertrophy of VSMCs.The mechanism may be related to its inhibition of ROS production.     

Lysophosphatidic acid (LPA) induces the proliferation of astrocytes in rat via pathway of protein kinase C-α and calcium ion in vitro

AIM: To determine if lysophosphatidic acid (LPA) regulates the proliferation of astrocytes (AS) and to approach the mechanism of the process.METHODS: The cerebral AS of the neonatal SD rats were cultured in vitro and divided randomly into control group, PKC excitomotor (PMA) group, LPA group, PKC-α inhibitor (Ro31-8220) group, Ro31-8220+PMA group and Ro31-8220+LPA group. The proliferation of the cells was detected by MTT assay and flow cytometry (FCM). The concentration of intra-cellular calcium ion of the cells (［Ca2＋］i) which were labeled with Fura-2/AM was determined by ultraviolet spectrophotometer. The change of PKC-α inside the cells was observed by Western blotting.RESULTS: LPA and PMA stimulated the proliferation of AS, they also enhanced the expression of PKC-α and increased the concentration of ［Ca2＋］i. After pretreated with Ro31-8220, the abilities of LPA that mentioned above were decreased. The change of ［Ca2＋］i was associated with the diversity of PKC-α.CONCLUSION: LPA promotes the proliferation of AS via the way of PKC-α and Ca2＋.