Development and characterization of microsatellite markers in Abies firma and interspecific amplification in other Japanese Abies species

Abies firma is a dominant coniferous tree species endemic to Japan. We isolated eight microsatellite loci from needles of this tree species and tested their polymorphism among 26 A. firma individuals. Six of them showed polymorphism, with two to 16 alleles per locus. Their expected heterozygosities ranged from 0.075 to 0.922. Moreover, interspecific amplification among Abies sachalinensis, Abies mariesii and Abies veitchii was successful in majority of the isolated loci, suggesting that these loci may be useful for characterization of other fir species.     

Somatic hybrids between<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> and cytoplasmic male-sterile radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>)

Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.Abbreviations CMS Cytoplasmic male sterilityCommunicated by M.R. Davey     

Spatiotemporal distribution of fruitbodies of ectomycorrhizal fungi in an Abies firma forest

 Spatial associations between ectomycorrhizal (ECM) fungi and their presumed host trees, and spatiotemporal associations among ECM fungi were surveyed for 3 years in an Abies firma-dominated forest in central Japan. A total of 39 species in 13 genera of ECM fungi were recorded, with more species in the Russulaceae than any other family. Russula ochroleuca, Russula sp.1 and Strobilomyces confusus tended to produce their fruitbodies on the forest floor directly under the crown of A. firma, whereas those of Inocybe cincinnata, Gomphus floccosus and G. fujisanensis were aggregated in limited areas outside the A. firma crown. Interspecific spatial associations were analysed for Russula sp.1, which was the most dominant species, and three other frequent species, I. cincinnata, S. confusus and R. ochroleuca. Pairwise, Russula sp.1 with I. cincinnata, with S. confusus or with R. ochroleuca showed an association which was exclusive, overlapping or independent, respectively. Fruiting phenologies differed in that S. confusus showed a peak density in the summer, whereas the other three species peaked in the autumn. These results suggest that the formation of ECM fruitbodies can be partitioned among the species both spatially and temporally. Accepted: 7 July 1998     

The spatial pattern and canopy-understory association of trees in a cool temperate,mixed forest in western Japan

The spatial distribution pattern of trees and the association between canopy and understory individuals were examined with reference to the distribution of tree crowns in a cool temperate, mixed forest in Ohdaigahara, western Japan. Line transect and contact sampling methods were used to examine the pattern over various spatial scales. These methods are useful to detect patterns over a large study area. The dominance ofChamaecyparis obtusa on steep slopes forming large patches suggested that the distribution of this species is a consequence of landslides. UnderstoryFagus crenata showed a clumped distribution, and the relative coverage of this species was larger in canopy gaps than under a closed canopy. Understory individuals ofAbies homolepis showed a positive association with canopy trees ofF. crenata but a negative association with conspecific canopy individuals. These patterns suggested thatF. crenata regenerates in canopy gaps and is replaced byA. homolepis. The dynamics of these two species are consistent with the process of gap dynamics. The effects of both small- and large-scale disturbance must be evaluated to understand the mechanisms of patch formation and the coexistence of forest tree species.     

Allelic sequence polymorphisms in the intron region of the nuclear-encoded GapC gene preceded the speciation of three closely related Abies species (Pinaceae)

The DNA sequences of GapC intron 8 were determined for three closely related Abies species, Abies firma, Abies homolepis and Abies veitchii, and ten alleles were identified. Although, in most cases, an allele was specifically detected in one species, some rare alleles were found in two species. The phylogenetic analysis of those alleles showed that they trace back to different ancestral sequences, and that a species possessed the different originated alleles. The polymorphism of the GapC gene observed in the three Abies species seemed to have preceded their evolutionary divergence. Received: 1 February 2000 / Accepted: 10 May 2000     

DIFFERENTIATION OF MITOCHONDRIAL DNA POLYMORPHISMS IN POPULATIONS OF FIVE JAPANESE ABIES SPECIES

Mitochondrial DNA polymorphism of 40 populations of five Abies species was investigated using PCR-amplified coxI and coxIII gene probes. Using four combinations of probe and restriction enzyme, we detected three major haplotypes and 15 total haplotypes. We also found varied levels of gene diversity for the different species: 0.741, 0.604, 0.039, 0.000, and 0.292 for A. firma, A. homolepis, A. veitchii, A. mariesii, and A. sachalinensis, respectively. The marginal and southern populations of A. firma and A. homolepis have unique haplotypes, especially the Kyushu, Shikoku, and Kii Peninsula populations, which inhabit areas coinciding with probable refugia of the last glacial period and possess high levels of mtDNA genetic diversity. The haplotypes in some populations suggested mtDNA capture also occurred between species through introgression/hybridization. The strong mtDNA population differentiation in Abies is most likely due to the maternal inheritance of mitochondria and restricted seed dispersal. A phenetic tree based on the genetic similarity of the mtDNA suggests that some species are polyphyletic. Based on mtDNA variation, the five Abies species could be divided roughly into three groups: (1) A. firma and A. homolepis, (2) A. veitchii and A. sachalinensis, and (3) A. mariesii. However, we found that all these Abies species, except A. mariesii, are genetically very closely related according to an analysis of their cpDNA sequences. This showed that the chloroplast rbcL gene differed by only one base substitutions among the four species. We believe that the mtDNA variation and cpDNA similarity clearly reflect relationships among, and the dissemination processes affecting these Abies species since the last glacial period.     

Patterns of on-shoot positioning of seed cones in relation to shoot length and position in the crowns of young Picea mariana

Summary Patterns of distribution of seed cones along the length and around the circumference of shoots were determined for young black spruce [Picea mariana (Mill.) B.S.P.] trees aged 9–17 years. Seed cone production averaged 5, 30, 62, 95, and 112 per tree of ages 9, 11, 13, 15, and 17 years, respectively. Cone distribution was restricted to distal and medial 1-year-old branches at age 9 and spread to distal 3-year-old branches by age 17. Sequentially, shoots of 0–25, 26–100, 101–250, 251–325, and 326–375 mm bore more cones in terminal, distal, medial, proximal, and basal positions along shoots, respectively. The uppermost and longest distal 1-year-old shoots bore most of their cones proximally and medially. On the shortest and innermost proximal order 2 shoots, cones were often terminal. The percentages of cones borne on upper, lower, and each of the two side surfaces of shoots were 31, 22, and 21, respectively, and 5% of the cones were terminal. Cones on lower surfaces were most common on the longest shoots near the apex of trees. Cones on upper surfaces became prevalent downward and outward in the crown. On-shoot positioning therefore varied in a predictable manner with shoot length, and thus with pattern of shoot-length distribution in the crown.     

Herbivory and tree mortality across a pinyon pine hybrid zone

We examined the abundances of three common insect herbivores on pure and hybrid pinyon pines along a 250-km transect in west-central Arizona, United States. Using six morphological traits, we developed a hybrid index to classify trees as pure Pinus californiarum, hybrid, or pure Pinus edulis. The insects (the stem-boring moth, Dioryctria albovittella, the scale insect, Matsucoccus acalyptus, and several species of pitch moths that produce wounds on the trunk and branches) exhibited different distributional patterns across tree types. Stem-boring moths were significantly more abundant on trees at hybrid sites compared to trees at pure sites. In addition, within hybrid sites, hybrids supported significantly more moth larvae than pure trees of either species. These two patterns support the hybrid susceptibility hypothesis in which hybrid breakdown results in increased susceptibility to herbivory. In contrast to stem-borers, there were significantly more pitch moth wounds on trees at pure P. californiarum sites than at hybrid and pure P. edulis sites. Within the hybrid zone, pitch moth abundance was equal on pure P. californiarum and hybrids, and both were significantly greater than on pure P. edulis. These within-site comparisons support the dominance hypothesis where hybrid resistance differs from one tree species, but not the other. Scale insects exhibited the most restricted distribution; over the 250 km transect they were found only in the hybrid zone. This supports the hybrid susceptibility and/or the stress hypothesis (i.e., species at the edge of their range suffer greater stress and are more susceptible to herbivory). We summed the mean numbers of these three common herbivores across sites and found that hybrid sites supported 2.1 and 3.9 times more herbivores than pure P. californiarum and P. edulis sites, respectively. Furthermore, tree mortality was on average, 35 times greater within the hybrid zone compared to pure zones of each species and was associated with the cumulative abundance of herbivores (r ²=0.646). Regardless of whether this mortality is due to insect infestation, stress or a combination of both, these results suggest that hybrid zones are important arenas of natural selection.     

Responses of crown architecture in Betula pendula to competition are dependent on the species of neighbouring trees

We measured the growth responses of individual shoots and branches of Betula pendula when growing next to trees of the same species or Pinus sylvestris, Larix sibirica or Alnus glutinosa. We used the three-dimensionally digitized response variables and the size and distance of trees growing within a 5-m radius of the study trees to establish a relationship between tree performance and the effect of competing neighbouring tree species on crown architecture. B. pendula was able to modify its crown architecture and thus alter its strategy to compete with different neighbours. Trees of B. pendula growing beside species counterparts had the highest growth of new long shoots in relation to the already existing branch length [growth vigour (GV)], while GV was the lowest next to L. sibirica. With B. pendula or P. sylvestris as its main neighbour, B. pendula invested in short shoots by growing them rather densely in short branches with limited numbers, whereas with L. sibirica the number, length and angle of the branches were high. The competitive response was also strongly dependent on tree ontogeny and the shoot and branch characteristics were significantly affected by their location inside the crown. B. pendula was able to respond to the challenges posed by its neighbours, which was also reflected in the GV. The ability to maintain steady growth with alternative crown designs in different neighbourhoods reflects plasticity in the crown responses.     

Hybrid Weakness in Phaseolus vulgaris L. II. Disruption of Root-Shoot Integration

We have been examining the importance of the root system on shoot growth and development using a developmentally disabled hybrid of the common bean Phaseolus vulgaris L. Parental cultivars (P. Vulgaris cv. Redkloud of Mesoamerican origin, and P. vulgaris cv. Batt of Andean origin) grow normally, but crosses produce F1 hybrids exhibiting hybrid weakness associated with reduced root and shoot growth. In this study, applications of benzylaminopurine (BAP) to roots of F1 hybrids increased the number of root tips and leaves. Reciprocal grafting was used to study the effects of the root system on shoots. Grafting of roots of the Mesoamerican cultivar onto shoots of F1 hybrids increased the cytokinin concentrations in leaves of F1 hybrids and removed the characteristics associated with hybrid weakness. To determine whether factors in the xylem sap enhanced leaf growth, leaf discs were incubated on sap collected from Mesoamerican and Andean cultivars. Sap from Mesoamerican plants enhanced the growth of leaf discs excised from F1 hybrids more than sap collected from Andean cultivars. Estimates of the transport of zeatin riboside (ZR)–type cytokinins from roots of F1 hybrids indicated that transport out of hybrid roots was reduced compared with those transported out of Mesoamerican or Andean roots. Results suggest that ZR-type cytokinins are involved in hormonal integration between roots and shoots of P. vulgaris and that one of the barriers to hybridization between Andean and Mesoamerican landraces is related to hormone transport. Received October 15, 1998; accepted May 12, 1999     

Multiple shoot formation and plant regeneration from stem nodal explants of <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchids

Summary Stem nodal explants of Paphiopedilum philippinense hybrids (hybrid PH59 and PH60) directly formed shoots when cultured on a modified half-strength Murashige and Skoog (1962) basal medium supplemented with a combination of 2,4-dichlorophenoxyacetic acid (2,4-D: 4.52 and 45.25 μM) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ; 0.45 and 4.54 μM) for 6 mo. On hormone-free basal medium, the percentages of explants with shoots were 33.3% and 0% and the shoot numbers per explant were 1 and 0 in hybrid PH59 and hybrid PH60, respectively. In hybrid PH59, 4.52 μM 2,4-D plus 0.45μM TDZ induced a higher percentage of explants with shoots and shoot number per explant than did the hormone-free treatment. In hybrid PH60, although 4.52 μM 2,4-D and 0.45 μM TDZ promoted shoot formation, the highest shoot number was found with 4.52 μM 2,4-D alone. Plantlets, each having several roots, were obtained from regenerated shoots after transferring onto hormone-free basal medium for 3 mo. The plantlets were potted in sphagnum moss, and acclimatized well in a greenhouse.     

Relationship between reproductive behavior and new shoot development in 5-year-old branches of olive trees (Olea europaea L.)

The development of new shoots plays a central role in the complex interactions determining vegetative and reproductive growth in woody plants. To explore this role we evaluated the new shoots in the olive tree, Olea europaea L., and the effect of fruiting on new shoot growth and subsequent flowering. Five-year-old branches served as canopy subunits in order to obtain a global, whole-tree view of new shoot number, size and morphological origin. The non-bearing trees had many more shoots than the fruit-bearing trees, and a greater number of longer shoots. In both bearing conditions, however, the majority of shoots were less than 4 cm long, with shoots of progressively longer lengths present in successively decreasing frequencies. Six major shoot types were defined on the basis of apical or lateral bud origin and of parent shoot age. On fruit-bearing trees, the new shoots originated predominantly from the shoot apex, while on non-fruiting trees, they formed mainly from axillary buds, but in both cases, they tended to develop on younger parent shoots. The previous bearing condition of the tree was the main determinant for subsequent inflorescence development, which was independent of both shoot type and length. Thus, reproductive behavior strongly affected both the amount and type of new branching, but subsequent flowering level was more influenced by previous bearing than by the potential flowering sites on new shoots.     

Annual and spatial variation in shoot demography associated with masting in Betula grossa: comparison between mature trees and saplings

Backgrounds and Aims

Shoot demography affects the growth of the tree crown and the number of leaves on a tree. Masting may cause inter-annual and spatial variation in shoot demography of mature trees, which may in turn affect the resource budget of the tree. The aim of this study was to evaluate the effect of masting on the temporal and spatial variations in shoot demography of mature Betula grossa.

Methods

The shoot demography was analysed in the upper and lower parts of the tree crown in mature trees and saplings over 7 years. Mature trees and saplings were compared to differentiate the effect of masting from the effect of exogenous environment on shoot demography. The fate of different shoot types (reproductive, vegetative, short, long), shoot length and leaf area were investigated by monitoring and by retrospective survey using morphological markers on branches. The effects of year and branch position on demographic parameters were evaluated.

Key Results

Shoot increase rate, production of long shoots, bud mortality, length of long shoots and leaf area of a branch fluctuated periodically from year to year in mature trees over 7 years, in which two masting events occurred. Branches within a crown showed synchronized annual variation, and the extent of fluctuation was larger in the upper branches than the lower branches. Vegetative shoots varied in their bud differentiation each year and contributed to the dynamic shoot demography as much as did reproductive shoots, suggesting physiological integration in shoot demography through hormonal regulation and resource allocation.

Conclusions

Masting caused periodic annual variation in shoot demography of the mature trees and the effect was spatially variable within a tree crown. Since masting is a common phenomenon among tree species, annual variation in shoot demography and leaf area should be incorporated into resource allocation models of mature masting trees.     

Comparison of somatic and sexual incompatibility between Datura innoxia and Atropa belladonna

After protoplast fusion somatic hybrid calli were obtained by complementation selection between an albino mutant of Datura innoxia and the wildtype of Atropa belladonna (Krumbiegel and Schieder, 1979. Planta 145, 371–375). In the present study experiments are described concerning leaf and shoot induction on several media supplemented with different combinations and concentrations of hormones. Except for fleshy leaves and embryos, no well-formed shoot could be obtained. However, under standard culture conditions after one and a half years, one line produced numerous green shoots, showing a reduced number of chromosomes from Atropa belladonna. The loss of some chromosomes decreased the degree of somatic incompatibility. The additional appearance of shoots with albino sectors, of total albino shoots, and of green shoots showing a different phenotype, demonstrated that the elimination of the chromosomes occurred not only once, but several times. At least one shoot nearly stable in chromosome content and green subline could be obtained possessing only 6 chromosomes of Atropa belladonna and the original chromosome number of Datura innoxia. Experiments were carried out to test the feasibility of producing sexual hybrids through in vivo and in vitro methods by cross pollination. However, no embryos, seeds, or plantlets were obtained, thus demonstrating that protoplast fusion is the only possibility for obtaining hybrids between these two species.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlor-phenoxyacetic acid - IAA indoleacetic acid - NAA -naphtaleneacetic acid - SDS sodiumdodecylsulfate     

Shoot damage by Tomicus sp. (Coleoptera: Scolytidae) and effect on Pinus yunnanensis resistance to subsequent reproductive attacks in the stem

Abstract 1 In South‐western China, Yunnan pines Pinus yunnanensis, suffer considerable damage from an undescribed Tomicus sp. previously thought to be T. piniperda. 2 To assess the effect of shoot maturation feeding (during which an aggregation process appears to occur) on host resistance to attacks on the bole, the relationships between shoot damage, bole attack density and tree survival were studied. 3 Attack distribution in the crown and in the stem did not vary between killed and surviving trees, indicating that mortality is determined by the quantity of attacks. 4 The level of shoot damage and bole attack density were positively and linearly correlated. This can be explained by the fact that bole attacks are caused by beetles coming from the crown of the same tree. 5 A critical threshold of bole attack density (around 80 attacks/m²) above which trees die was observed. However, because attacks continue after this threshold is reached, the density of failed attacks on the killed trees was used as an estimator of the threshold density. It decreased when shoot damage increased. 6 The existence of a critical threshold of shoot damage (60% damaged shoots) was also demonstrated. Above this threshold, stem attack density was always sufficiently high to kill trees. 7 The results emphasize that concentration of shoot attacks is the main reason for the extensive tree damage observed in China. 8 A model of relationships between shoot and stem attacks is proposed, suggesting that management to reduce shoot attacks would protect trees from dying by both decreasing the number of bole attacks and raising the threshold for successful attack density on the bole to levels that could not be attained.     

Direct shoot regeneration from basal leaf segments of Lilium and assessment of genetic stability in regenerants by ISSR and AFLP markers

Here, we report a widely applicable procedure for direct shoot regeneration via basal leaf segments of Lilium. Leaf segments (0.8–1.0 cm long and 0.4 cm wide) were excised from leaves on shoot nodes 3 to 6 of 4-wk-old in vitro stock shoot cultures. The segments were wounded by three transverse cuts across the midvein on the abaxial side, with 1 mm between cuts, and cultured with the abaxial side in contact with a shoot regeneration medium composed of half-strength Murashige and Skoog medium supplemented with 1 mg/l naphthaleneacetic acid, 0.5 mg/l thidiazuron, 30 g/l sucrose, and 7 g/l agar (pH?5.8). The cultures were incubated for 4 wk under a 16-h photoperiod at 23?±?2°C for adventitious shoot regeneration. With this procedure, a mean shoot regeneration frequency of 92–100% and mean number of shoots of 4.7–7.0 per segment were obtained in five Lilium species and hybrids, which represent diverse genotypes of Lilium and are commercially popular lilies. Histological studies with Lilium Oriental hybrid “Siberia” revealed that meristemoids initiated from subepidermal cells on the adaxial side of the explant and eventually developed into adventitious buds, without callus formation. In an assessment of genetic stability in the regenerants of “Siberia”, no polymorphic bands were detected by intersimple sequence repeat and only 0.73% polymorphic bands were detected by amplified fragment length polymorphism. The morphologies of the regenerants were identical to those of the control. These results demonstrated that the regenerants were genetically and morphological stable. Thus, this procedure has great potential application for micropropagation, genetic transformation, and preparation of shoot tips for cryopreservation and cryotherapy for virus eradication of Lilium.     

Within-tree and among-tree variation in growth of Epirrita autumnata on mountain birch leaves

Abstract.

• 1 We studied among-tree and within-tree variation in the growth of larvae of a geometrid, Epirrita autumnata, on mountain birch (Betula pubescens ssp. tortuosa) leaves at different hierarchical levels: among trees (genets), among ramets within trees, among branches within ramets, among shoots within branches and among leaves within shoots. We used only short shoot leaves, which burst simultaneously in spring.
• 2 Trees accounted for most of the variation in larval growth rate, but there was substantial variation also among ramets within trees, among branches within ramets, and among short shoots within branches. Variation among leaves within short shoots was negligible. When the probabilities from different experiments were combined, the differences were statistically significant at the tree, ramet and branch levels, and approached significance at the shoot level.
• 3 In different experiments, larval growth was from 9% to 54% lower on the worst tree than on the best tree of the experiment. On average, larval growth rate was in different experiments from 11% to 32% lower on the worst ramet than on the best ramet within tree, from 8% to 18% lower on the worst than on the best branch within ramet, and from 12% to 30% lower on the worst than on the best shoot within branch.
• 4 The amount of among-and within-tree variation shown by our results may have ecological and evolutionary implications: among-tree variation should select for discrimination by ovipositing females and dispersing larvae, within-tree variation should select also for optimal foraging behaviour of larvae.

     

The effect of weaver ants Oecophylla smaragdina on the shoot borer Hypsipyla robusta on African mahoganies in Australia

• 1 African mahogany Khaya senegalensis is a high‐value timber tree species widely grown in central Africa, south‐east Asia and northern Australia. Pilot plantings show that the tree grows well in the wet‐dry tropical areas of northern Australia, and the shoot borer Hypsipyla robusta (Lepidoptera: Pyralidae) is a potential pest of the tree. The weaver ant Oecophylla smaragdina is an efficient biocontrol agent in some horticulture crops. To investigate whether the ants control shoot borers, field experiments were conducted at two sites near Darwin, Australia from April 2006 to January 2009.
• 2 In the weaver ant treatments, the overall percentage of trees damaged by shoot borers was 0–2.7% at Berrimah Farm and 0–4.2% at Howard Springs, and the damaged trees were attacked once only. In the treatments without weaver ants, however, 9.9–52.1% trees were damaged at Berrimah Farm, and 6.3–64.6% at Howard Springs, and the damaged trees were generally attacked more than once.
• 3 At both sites, significantly fewer trees on each monitoring occasion were damaged in weaver ant treatments than in treatments without weaver ants.
• 4 The mean percentage of overall flushing shoots damaged by the pest at both sites was significantly lower in weaver ant treatments compared with treatments without weaver ants.
• 5 Fewer shoots were damaged per damaged tree in weaver ant treatments compared with treatments without weaver ants.
• 6 The data obtained suggest that weaver ants were effective biological control agents of the shoot borer, and that the ants can be used to manage the pest on African mahogany trees.

     

Needle morphological evidence of the homoploid hybrid origin of Pinus densata based on analysis of artificial hybrids and the putative parents,Pinus tabuliformis and Pinus yunnanensis

Genetic analyses indicate that Pinus densata is a natural homoploid hybrid originating from Pinus tabuliformis and Pinus yunnanensis. Needle morphological and anatomical features show relative species stability and can be used to identify coniferous species. Comparative analyses of these needle characteristics and phenotypic differences between the artificial hybrids, P. densata, and parental species can be used to determine the genetic and phenotypic evolutionary consequences of natural hybridization. Twelve artificial hybrid families, the two parental species, and P. densata were seeded in a high‐altitude habitat in Linzhi, Tibet. The needles of artificial hybrids and the three pine species were collected, and 24 needle morphological and anatomical traits were analyzed. Based on these results, variations in 10 needle traits among artificial hybrid families and 22 traits among species and artificial hybrids were predicted and found to be under moderate genetic control. Nineteen needle traits in artificial hybrids were similar to those in P. densata and between the two parental species, P. tabuliformis and P. yunnanensis. The ratio of plants with three needle clusters in artificial hybrids was 22.92%, which was very similar to P. densata. The eight needle traits (needle length, the mean number of stomata in sections 2 mm in length of the convex and flat sides of the needle, mean stomatal density, mesophyll/vascular bundle area ratio, mesophyll/resin canal area ratio, mesophyll/(resin canals and vascular bundles) area ratio, vascular bundle/resin canal area ratio) relative to physiological adaptability were similar to the artificial hybrids and P. densata. The similar needle features between the artificial hybrids and P. densata could be used to verify the homoploid hybrid origin of P. densata and helps to better understand of the hybridization roles in adaptation and speciation in plants.     

Standardization of a protocol for micropropagation of Eupatorium glandulosum L. an important medicinal plant

The presence of residual female fertility in most of the parthenocarpic banana accessions encourages the banana breeder to develop new hybrids through conventional breeding. Desirable trait can be fixed in the first generation of hybrid progenies, but the evaluation of these hybrids in field is the time-consuming process owing to non-availability of uniform suckers/planting material. This can be overcome by developing multiple shoots from single embryo in a short period of time through embryo culture. A protocol for in vitro multiplication of plantlets from zygotic embryos was standardized in seeded accessions. Multiple shoots from zygotic embryos were achieved up to 55.2% and 64.1% in seeded accessions of Musa acuminata and M. velutina respectively in medium supplemented with 17.76 µM of BAP. The Single shoot derived (only germination) from zygotic embryos was decapitated and the apical meristem were disturbed for further multiple shoot formation in media supplemented with 17.76 µM of BAP. Present studies revealed that in total 75% and 91% of the M. acuminata and M.velutina embryos were able to produce multiple shoot from single embryo by manipulating the media composition and decortications technique. The above protocol was applied for zygotic embryos obtained from controlled pollination (18 cross combinations) and open pollination (nine accessions) of various genomic groups (ABB, AAB, AA). The multiple shoots derived from zygotic embryos and plantlet germinated from zygotic embryos was examined for genetic fidelity analysis by SSR markers.