Nitrogen content determines adventitious rooting in Euphorbia pulcherrima under adequate light independently of pre-rooting carbohydrate depletion of cuttings

Root regeneration in shoot tip cuttings responds to graduated nitrogen (N) fertilization of stock plants. In pelargonium cuttings, reduced carbohydrate reserves caused by high N absorption by the donor plants and post-harvest storage of cuttings limit adventitious root formation, especially in low-light environments. In contrast, in chrysanthemum, similar carbohydrate reserves do not have this dominant effect on rooting capacity. The positive correlation between rooting capacity and internal N status is stable across a wide range of environments and is genotypically consistent for this species. However, the influence of N and carbohydrates on adventitious rooting of Euphorbia pulcherrima is unknown. We investigated the consequences of different N fertilization regimens applied to E. pulcherrima stock plants and cold and dark storage of the cuttings on N absorption, carbohydrate distribution, and rooting capacity of cuttings. Increasing time of stock plant cultivation with graduated N nutrition produced cuttings with N contents, ranging from 19 to 51 mg N g⁻¹ dry mass (DM). High N absorption resulted in low carbohydrate concentrations in cuttings, and subsequent storage decreased carbohydrate concentrations further, particularly in stems. Lower sucrose contents in leaves were correlated with reduced rooting of stored cuttings at a particular harvest date. However, despite the lower carbohydrate levels, root numbers and lengths correlated positively with internal N concentrations. These relationships remained stable in unstored and stored cuttings, even when overall rooting intensity was reduced under lower natural light during autumn. Multivariate regressions accounting for nitrogen content, sucrose content and daily light integral during rooting highlighted these relationships and explained up to 79% of rooting variances. We conclude N nutrition of stock plants and N absorption by cuttings are the dominant factors determining the rooting capacity of poinsettia when rooting occurs under sufficient light, as is commonly available during propagation. To maximize rooting capacity of poinsettia cuttings their nitrogen content should exceed a threshold of 40 mg N g⁻¹ DM.     

Light quality affects in vitro adventitious rooting and ex vitro performance of cherry rootstock Colt

The effect of light quality on in vitro rooting of cherry rootstock “Colt” was studied to evaluate adventitious root development, ex vitro acclimatization and plant growth performance. Adventitious rooting was dependent on the light quality to which the microcutting were exposed. Highest number of roots per microcuttings was recorded under dichromatic light (blue + red). This response could be ascribed to the control of a strong synergistic interaction between phytochromes and blue light photoreceptors. Red light was more effective on root elongation than blue light. In this response a strong synergistic interaction between phytochromes and blue light photoreceptors was suggested. The effect of light quality on the number of root/explant affected the plant during acclimation, scoring the highest level of plant survival in the blue-, red- and blue + red-exposed plantlets. The light quality effect was also observed under greenhouse culture conditions, as shown by growth parameters at the end of six months in plants growth. No specific role was observed for the photoequilibrium of phytochrome. The results reported in this work show that plantlets exposed to different light quality, during the in vitro rooting phase, retain the light quality effects in the subsequent plant acclimatization and greenhouse plant growth phase.     

Analysis of phenetic relationship between Citrus indica Tanaka and a few commercially important citrus species by ISSR markers

Analysis of polymorphism in ISSR amplicons revealed diverse genetic relationship between Citrus indica and five other citrus species. In a consensus UPGMA dendrogram, based on Nei and Li's distance matrices, the C. indica samples from three different sites of Meghalaya, India were clustered together with 99% bootstrap support. C. reticulata, C. sinensis, C. aurantifolia formed a cluster with 67% bootstrap separation. C. macroptera and C. maxima samples from two sites of Meghalaya formed separate clusters with respectively 100% and 98% bootstrap supports. Principal Components Analysis projected C. indica to be more closely related to C. aurantifolia than to the other citrus species of the study. PCA also exposed variations within the C. maxima and C. macroptera samples.     

Application of genomic in situ hybridization for phylogenetic study between Mangifera indica L. and eight wild species of Mangifera

The phylogenetic relationship between mango (Mangifera indica L.) and eight wild species of Mangifera were analyzed by comparing signal intensity of genomic in situ hybridization (GISH) on somatic metaphase chromosomes of M. indica, using labeled DNA of eight wild Mangifera species. The eight wild species were divided into four groups based on intensity and number of hybridization signals on chromosomes of M. indica in GISH analyses. The probe of Mangifera sylvatica Roxb. gave the highest intensities on the chromosome of M. indica, indicating a close relationship between M. indica and M. sylvatica. For the other species, classification of GISH was comparable to that of the phylogenetic analysis using AFLP markers, as previously reported ( Eiadthong et al., 2000). This suggested a possibility that GISH analysis can be effectively used in the classification of Mangifera species.     

Molecular based assessment of genetic diversity within Barbary fig (Opuntia ficus indica (L.) Mill.) in Tunisia

In this work, we report for the first time on the analysis of genetic diversity within a set of 36 Tunisian Opuntia ficus indica (L.) Mill. ecotypes using RAPD markers.     

High frequency plant regeneration through adventitious multiple shoot organogenesis in epicotyl explants of Indian gooseberry (Emblica officinalis Gaertn)

Shoots regenerated adventitiously on epicotyl segments from in vitro seedlings of Emblica officinalis var. ‘Kanchan’. Epicotyls derived from 2-week-old aseptic seedlings were most responsive and produced a maximum number of 303 shoots per explant in Murashige and Skoog (1962) medium (MS) augmented with 8.8 μM N⁶-benzyladenine (BA) + 1.425 μM indole-3-acetic acid (IAA). Shoots readily elongated in MS lacking growth regulators and rooted in half-salt-strength MS (1/2 MS) supplemented with indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). The highest rooting response was recorded in 1/2 MS containing 14.7 μM IBA. Plantlets were acclimatized inside the green house and 80% of the plantlets survived on transfer to garden soil.     

Adventitious root formation in Anacardium occidentale L. in response to phytohormones and removal of roots

Despite advances in tissue culture techniques, propagation by leafy, softwood cuttings is the preferred, practical system for vegetative reproduction of many tree and shrub species. Species are frequently defined as ‘difficult’- or ‘easy-to-root’ when propagated by conventional cuttings. Speed of rooting is often linked with ease of propagation, and slow-to-root species may be ‘difficult’ precisely because tissues deteriorate prior to the formation of adventitious roots. Even when roots form, limited development of these may impair the establishment of a cutting.     

Assessment of genetic diversity among mango (Mangifera indica L.) genotypes using RAPD markers

Knowledge about the extent of genetic diversity/relatedness in mango germplasm is vital for developing coherent strategies for future gains in productivity. The genetic diversity/relatedness among mango cultivars/genotypes developed in Pakistan has not been investigated previously. We have assessed the genetic diversity among 25 mango genotypes/cultivars using randomly amplified polymorphic DNA (RAPD). Sixty random ten-mer primers were surveyed, out of which 45 yielded amplicons in all the genotypes. Genetic similarity between genotypes/cultivars was in the range of 64–89% with an average of 74%. Similarly, the genetic relatedness among all variants derived from a mango cultivar Chaunsa was in the range of 81.18–88.63%. These coefficients were utilized to construct a dendrogram using the unweighted pair group of arithmetic means (UPGMA). The genotypes were grouped into three (A, B, C) clusters. Generally, genotypes originating from Pakistan were grouped in cluster ‘A’ while cluster ‘B’ primarily composed of southern India as well as Florida cultivars. Kensington Pride was the most distantly related genotype which grouped with Maya and Yakta, forming a distinct cluster ‘C’.     

ISSR polymorphism in Indian wild orange (Citrus indica Tanaka,Rutaceae) and related wild species in North-east India

Inter simple sequence repeats (ISSR) polymorphism in Citrus indica Tanaka (Rutaceae), an endemic and threatened wild species, was examined along with three other closely related wild taxa (C. medica L., C. latipes (Swingle) Tanaka, and C. sp. ‘Memang athur’) by analyzing 53 representative accessions sampled from North-east India. Jaccard's similarity values among 53 accessions of Citrus ranged from 0.46 to 0.97 (average = 0.75). Genetic similarity values among all the 34 accessions of C. indica were found in the range of 0.82 to 0.97 with an average of 0.90. Heterozygosity (Ht = 0.123) and Shannon's information index (I = 0.188) values estimated for C. indica revealed significantly low level of genetic variation within the species. UPGMA dendrogram grouped all 53 accessions of Citrus into four major clusters: Cluster I – C. latipes; Cluster II – C. medica; Cluster III – ‘Memang athur’ and Cluster IV – C. indica. The dendrogram placed all the 34 accessions of C. indica in five sub-clusters under Cluster IV. The placement of C. indica accessions in various sub-clusters and groups in the dendrogram was based on molecular differentiation of individual accessions rather than their geographical origin. Very low genetic diversity and destruction of its natural habitat pose serious threat to C. indica even in the Citrus Gene Sanctuary in Nokrek Biosphere Reserve (NBR) in Meghalaya. Low genetic variability, heterozygosity and Shannon's information index in C. medica, C. latipes and ‘Memang athur’ are also concerns that need to be addressed for developing appropriate strategies to conserve the genetic diversity extant in these valuable genetic resources.     

Factors affecting mango (Mangifera indica L.) protoplast isolation and culture

The major factors influencing protoplast isolation and culture of mango (Mangifera indica L.) cv. Kensington Pride were investigated. The resultant protocol was used to compare plating efficiency among 4 mango cultivars. Most responses differed between proembryonic masses (PEMs) and leaf sources. Protoplast yields of 15.22 × 10⁶ g⁻¹ from PEMs and 8.68 × 10⁶ g⁻¹ from greenhouse-derived leaves were obtained in a solution of 0.7 M mannitol CPW plus 1.5% cellulase, 1% hemicellulase and 0.75% macerozyme for PEMs or 0.5 M mannitol CPW plus 1.5% cellulose, 1% hemicellulase and 1.5% macerozyme for leaves. Culture in Ca-alginate beads with initial plating densities (IPD) of 2.5 × 10⁴ Pp mL⁻¹ for PEMs and 2.5 × 10⁵ for leaves gave the highest plating efficiencies (FPE). For PEMs 1 mg L⁻¹ 2,4-d and 3.5 mg L⁻¹ kinetin gave an FPE of 2.85% whereas lower kinetin (2 mg L⁻¹) plus 0.5 mg L⁻¹ 6-BAP was most effective for leaves (FPE of 2.12%). Most protoplast mortality occurred during the first week of culture and was more severe in liquid culture. In Ca-alginate beads, protoplast survival at 14 days was higher for PEMs (30%) than leaf (21%) as was the frequency of cell division (17.6% compared to 13.6%). PEMs protoplasts continued development through embryogenesis to in vitro plantlet regeneration whereas leaf protoplasts underwent cell division up to 40-cell colonies but failed to proceed further. For PEMs, polyembryonic cvs. Kensington Pride and Keow Savoey produced higher FPE (1.95%) than monoembryonic cvs. Tommy Atkins and Keitt (1.75%). There was no effect of cultivar for leaf protoplasts.     

Physiological and biochemical changes with special reference to mangiferin and oxidative enzymes level in malformation resistant and susceptible cultivars of mango (Mangifera indica L.)

Changes in biophysical attributes, mangiferin and polyphenol oxidase (PPO), catalase and peroxidase activities in malformation resistant mango cultivar Elaichi were studied at various stages of flower development and compared with susceptible cvs. Amrapali, Beauty Mc-lin and Dashehari. Accumulation of mangiferin was maximum (96.0 and 108.0 mg g⁻¹ FW) in Elaichi prior to flower bud differentiation (September) and at full bloom (February), while these were minimum (59.0 and 74.0 mg g⁻¹ FW) in susceptible cv. Beauty Mc-lin. Mangiferin promoted vegetative growth and exhibited inhibitory role on the occurrence of malformation. It was also found that the resistant cultivar had highest activity of PPO as compared to susceptible ones. There was no significant difference in the enzymes catalase and peroxidase activity at early stage of flower differentiation but at flower bud burst stage the catalase activity was enhanced significantly in cv. Elaichi (25.28 unit min⁻¹ g⁻¹ FW) in comparison to Amrapali (16.20 unit min⁻¹ g⁻¹ FW), Beauty Mc-lin (18.39 unit min⁻¹ g⁻¹ FW) and Dashehari (17.50 unit min⁻¹ g⁻¹ FW). The resistant cultivar had high leaf temperature (30.30 °C) and diffusion resistance (476.14 m mol m⁻² s⁻¹) during the flowering but the rate of transpiration and relative humidity (RH) were high in susceptible cultivars. Results of the present study clearly indicate that level of mangiferin could be considered as a potential biochemical indicator for screening mango genotypes to malformation.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Rooting response of shoot cuttings from three peach growth habits

Current year shoot cuttings were collected in October and August from three growth habits of peach (Compact, Pillar, and Standard) and treated with one of four concentrations of indole butyric acid (0, 250, 1250, and 2500 mg L⁻¹ IBA). Rooting response was measured after 5 weeks in the greenhouse. Little or no rooting occurred with cuttings from any growth habit that was collected in October or in August when treated with 0 and 2500 mg L⁻¹ IBA. In August, the number of shoots that rooted was greater in cuttings from Pillar (79 and 45%) than Compact (13 and 3%) treated with 250 and 1250 mg L⁻¹ IBA, respectively. Cuttings from Standard trees had intermediate rooting of 56 and 6% at 250 and 1250 mg L⁻¹ IBA, respectively. Pillar trees consistently grew more roots with greater root length per cutting than the other growth habits. It is proposed that differences in rooting response among the growth habits may be associated with differences in endogenous auxin concentration that had been found in previous studies. Within peach and possibly other fruit trees, the capacity of shoot cuttings to develop adventitious roots can vary by cultivar and successful root induction with exogenous plant growth regulators may depend, in part, on endogenous hormone levels.     

Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.)

The tips of cotyledon-stage seedlings of three crape myrtle cultivars (“Zi Wei”, “Hong Wei” and “Yin Wei”) were treated with colchicine. Various concentrations of colchicine and different treatment durations were tested. Seedlings of “Zi Wei” treated with 0.5% colchicine for 72 h and seedlings of “Yin Wei” treated with colchicine (0.2% for 96 h, 0.5% for 48 h and 0.8% for 72 h) demonstrated high rates of mutation; “Hong Wei” showed a slightly lower rate. The highest rate of morphological variation was 54.17% and this was achieved when tips were treated with 0.5% colchicine for 72 h. Putative tetraploid plants were identified with morphological and cytological variations, such as larger and thicker leaves, darker green coloration, larger stomata, lower density of stomata across the lower leaf epidermis and increased numbers of chloroplasts per stomata guard cell. Flow cytometric analysis demonstrated that the nuclear DNA content of 15 of these putative tetraploids (of which 7 were “Zi Wei”, 5 were “Yin Wei”, and 3 were “Hong Wei”) was indeed doubled relative to that of control diploid plants. The chromosome number of the tetraploid plants was 2n = 4x = 96, while that of the control diploid plants was 2n = 2x = 48. Ornamental characteristics were also enhanced in the tetraploid plants, with increased diameter of the individual flowers and greater basal length of the petals and claws. Pollen diameter and the size of capsules and seeds were also significantly greater than those of diploid plants.     

Controlled atmosphere storage at high CO2 and low O2 levels affects stomatal conductance and influence root formation in kalanchoe cuttings

Since the response of cuttings to raised CO₂ concentration is not documented, controlled atmosphere (CA) storage of kalanchoe cuttings at combinations of high carbon dioxide and low oxygen levels was investigated to study the feasibility of using CA to sustain quality of cuttings prior to planting. During storage, stomata opening and plant fresh weight (FW) were measured, and root formation (RF) was recorded post storage. Storage atmosphere composition (10/2, 15/2, or 15/5; kPa CO₂/kPa O₂), storage duration (9 or 19 days), and cutting type (rooted or un-rooted) affected stomata conductance (G_s), and influenced FW and subsequent RF in cuttings of kalanchoe ‘Yellow Josefine’. In CA stored plants, G_s was high, 60–160 mmol m⁻² s⁻¹, indicating open stomata, whereas in control plants G_s was low, 5–14 mmol m⁻² s⁻¹, indicating closed stomata. Generally G_s values were higher for un-rooted than for rooted cuttings. Overall, cutting FW was reduced by CA storage with no significant differences in FW reduction between the CA treatments. RF of un-rooted CA stored cuttings was comparable to that of controls, whereas for rooted cuttings controls grew better than CA stored cuttings. CA at 10 kPa CO₂ and 2 kPa O₂ for 2–3 weeks could sustain un-rooted cuttings, in a pests-free state whilst retaining the ability of the cuttings RF. The results showed that stomata aperture may be altered by high CO₂ concentration combined with low O₂, and results indicated that this effect was not only caused by high CO₂ but also by low O₂ concentration. In addition, the results indicated that CA storage, stomata conductance, and water stress of kalanchoe cuttings may be correlated. Monitoring G_s of leaves of cuttings could be used as a non-destructive indicator of storability and quality status. Based on the novel positive preliminary results reported here, a protocol that focuses on minimising water loss should be developed and optimised for kalanchoe.     

Chemical compositions and insecticidal effects of essential oils isolated from Achillea gypsicola, Satureja hortensis, Origanum acutidens and Hypericum scabrum against broadbean weevil (Bruchus dentipes)

The essential oils of aerial parts of Achillea gypsicola Hub.-Mor., Hypericum scabrum L., Satureja hortensis L., and Origanum acutidens (Hand.-Mazz.) Letswaart were analyzed in this study by GC and GC–MS and their oils were tested for toxicity against broadbean weevil (Bruchus dentipes). A. gypsicola oil contained camphor (40.17%), 1,8-cineole (22.01%), piperitone (11.29%), borneol (9.50%) and α-terpineol (1.56%) as major components. A total of 74 components were identified by GC–MS in H. scabrum oil, including α-pinene (9.26%), terpinen-4-ol (5.12%), camphor (5.94%), δ-cadinene (4.52%), pulegone (4.45%), γ-muurolene (4.12%), pinocarvone (3.97%) and β-caryophyllene (3.42%) as predominant components. The essential oils of O. acutidens and S. hortensis were characterized by high contents of carvacrol (86.99% and 55.74%), γ-terpinene (0.71% and 20.94%), p-cymene (1.95% and 12.30%), α-terpinene (0.13% and 2.04%) and β-caryophyllene (1.30% and 1.08%). All of the essential oils were toxic to adults of B. dentipes and insect mortality increased with increasing concentration of each oil. The oils (20 μl dose) brought about 100% mortality in 36 h. Although desirable insecticidal activities against the pest were achieved with the oils from all four plant species, S. hortensis and O. acutidens oils were more effective, particularly after 6 h of treatment. The current results concluded that the essential oils, in particular O. acutidens and S. hortensis oils, may be used as potential botanical insecticides against B. dentipes.     

Identification of Petunia hybrida cultivars that diurnally emit floral fragrances

In order to identify genetic resources for breeding fragrant petunias for use as bedding plants, volatile compounds released by day from the flowers of 40 commercial Petunia hybrida cultivars were analyzed using a solid-phase micro-extraction technique coupled with GC–MS. The three cultivars with solid deep-blue flowers that accumulate malvidin in corollas with high tissue pH were found to emit abundant iso-eugenol as the principal floral fragrance. Several other cultivars that emitted considerable amounts of methylbenzoate and/or benzylbenzoate from the flower were also identified. Association between the floral fragrance and the other floral traits such as floral anthocyanin composition and corolla-tissue pH was discussed.     

Somatic embryo,adventitious root and shoot regeneration in in vitro grown quince leaves as influenced by treatments of different length with growth regulators

The purpose of this work was to acquire more information on the capacity of in vitro grown quince (Cydonia oblonga Mill.) leaves to simultaneously regenerate somatic embryos, adventitious roots and shoots, and to evaluate the variations induced on regeneration response by treatments of different length with growth regulators. After 2 days of liquid treatment with 2,4-dichlorophenoxyacetic acid, the leaves were cultured for 0, 3, 6, 9, 12, 15, 18 and 21 days on gelled growth medium containing the basal components of Murashige and Skoog and kinetin (Kin) 4.5 μM + naphthaleneacetic acid (NAA) 0.5 μM. At the end of each treatment period, the leaves were cultured on a transfer medium in the absence or in the presence of a growth regulator combination represented by N⁶-benzylaminopurine (BA) 2.66 μM + gibberellic acid 0.58 μM + indole-3-butyric acid 0.3 μM. The culture period for all the treatments was fixed to 52 days.     

Cross-transferable polymorphic SSR loci in Prunus species

This work reports the transferability and polymorphism of previously reported SSRs in 10 Prunus species. The availability of a large number of SSRs in the genus Prunus makes marker choice random, while preventing comparison of results in fingerprinting studies. The availability of SSR markers, polymorphic in a wide sample of Prunus species, would facilitate marker choice, while allowing the comparison of results. In this work, microsatellite markers useful for analyzing 10 different Prunus species (P. persica, P. dulcis, P. armeniaca, P. domestica, P. insititia, P. salicina, P. cerasifera, P. avium, P. cersus and P. mahaleb) were searched through screening SSRs previously reported to be conserved and/or polymorphic in more than one Prunus species. A selected group of 13 SSRs, transferable to the 10 species, was analyzed in terms of their usefulness for analyzing these species. The amplification range, polymorphism and variability detected by these loci are reported. The information provided will be useful for Prunus genetic studies as well as conservation and management of Prunus germplasm resources.     

Histology of adventitious shoot and root formation on leaf-petiole cuttings of Begonia x hiemalis Fotsch ‘Aphrodite Peach’

Epidermal and subepidermal cells formed a callus at the basal portion of the petiole of Rieger Begonia cv. ‘Aphrodite Peach’ leaf-petiole cuttings. Roots arose from cells of the internal portion of the callus and also from parenchymal cells of the petiole. Shoots formed from cells on the surface of the enlarging callus. Inasmuch as many cells had divided in the callus before an organized shoot appeared, it was virtually impossible to determine from histological observations whether the cells of the new shoot were derived from a single epidermal cell of the petiole or from multiple epidermal cells. These histological observations on adventitious shoot formation are discussed relative to mutation breeding employing leaf-petiole cuttings of B. x hiemalis Fotsch.