Regeneration and transformation system in Mirabilis jalapa

Protocols for in vitro regeneration and production of in vitro-propagated plants and a transformation system were developed for Mirabilis jalapa (Nyctaginaceae). Among the types of explants and the different media tested, consistent shoot regeneration was obtained only from nodal segments grown in a regeneration medium consisting of Murshashige and Skoog medium supplemented with 2 mg l⁻¹ 6-benzyladenine, 2 mg l⁻¹ zeatin and 1 mg l⁻¹ indole acetic acid. Regeneration efficiency was dependent on the type of plant – white or pink flowers – used as the source of explants. Stable transformation was obtained following inoculation of nodal segments with Agrobacterium tumefasciens strain EHA105, which harbours the binary plasmid pAD1339 containing both nptII and gus genes under the control of the 35S promoter. Transformation was confirmed by PCR and Southern blot analysis of genomic DNA from mature regenerated plants. β-Glucuronidase (GUS) activity was observed only in tissues regenerated from in vitro-grown plants and not in tissues originating from greenhouse-grown plants. GUS expression was not uniform in regenerated leaves and showed a chimera pattern.     

Effect of different lifting dates and different lengths of cold storage on plant vitality of silver birch and common oak

Woody ornamental bare-root plants transplanted in urban areas frequently show poor establishment and occasionally high mortality. Tolerance to different stress factors during transplanting is dependent on plant vitality. The significance of lifting date, in combination with different lengths of time in cold storage, was investigated with 2-year old plants of silver birch (Betula pendula Roth.) and common oak (Quercus robur L.). Plants were grown on a field site in Southern Sweden and were lifted on four occasions, at intervals of 4 weeks, during autumn and early winter. After lifting, plants were stored in a cold store for 90, 135 and 180 days and then (a) xylem water potential in roots and shoots, (b) root growth potential (RGP), (c) mortality and die-back, and (d) shoot and leaf growth were assessed. Lifting bare-root plants of silver birch and common oak too early in the autumn resulted in 62% mortality in birch and 43% in oak, and 31 and 26% die-back in birch and oak, respectively. Plants lifted at a later date showed no mortality in birch and <10% in oak, and almost no die-back. Plants lifted too early in autumn showed decreased root, shoot and leaf growth, compared with those lifted later in the autumn. A longer time in cold storage had a further negative effect on plant vitality in plants lifted too early. Plants lifted later in autumn showed little or no difference in mortality or die-back, for different periods of cold storage, and showed an increase in growth with longer periods of cold storage. Birch plants lifted too early had lower water potential, both in roots and shoots, compared to plants lifted at a later date, while oak plants had the lowest water potential when lifted late. Plants lifted on the first occasion had not been exposed to temperatures below +5°C before lifting, while plants lifted 4 weeks later had been exposed for 67 h to a mean temperature below +5°C. Plants lifted on the first occasion had high mortality and more die-back, while those lifted 4 weeks later showed almost no damage at all. These observations indicate that accumulated exposure to low temperature is probably an important factor in the development of stress tolerance in bare-root plants of silver birch and common oak.     

Investigating the relationship between various measuring methods for determination of establishment success of urban trees

Establishment is a key concept in urban forestry but it is currently inconsistently defined and measured. Thus, several different methods are being used to determine establishment success but their consequences and applications are rarely discussed. With this paper we would like to stimulate an increased discussion regarding these concepts both in relation to a theoretical definition but also to their practical use. The problem was approached through an experiment using sweet cherry (Prunus avium L.) and northern red oak (Quercus rubra L.) trees and the most common methods used for determination of establishment success. The trees were studied during the first three years after transplant and the association between the different measuring methods was examined. A Principal Component Analysis showed that terminal and lateral shoot length were strongly correlated, and that midday- and pre-dawn shoot water potential, and stomatal conductance were strongly correlated. We developed an index for nightly recovery of water status, which showed that terminal shoot growth was not related to nightly recovery until the third year after transplanting. Our results suggest that successful tree establishment is determined differently depending on which method is used for determination but that the differences might decrease with time. The lack of a firm definition of the term establishment may complicate communication, both within the scientific community and in practice.     

In vitro propagation of ‘Troyer’ citrange from epicotyl segments

Isolated epicotyl, root meristem and root segment tissues of ‘Troyer’ citrange [Poncirus trifoliata (L.) Rat. × Citrus sinensis (L.) Osbeck] were established in continuous culture to compare their regeneration potential. Callus was obtained from these explants on a Murashige—Skoog (MS) medium containing NAA (10 mg l^?1) and BAP (0.1–10 mg l^?1). Formation of shoots from root segments was direct without callus formation on MS medium containing BAP (10 mg l^?1) and NAA (1 mg l^?1). Shoot formation from epicotyl callus occurred on MS medium containing 0.25 mg l^?1 BAP and 0.1 mg l^?1 NAA. Formation of shoots from epicotyl segments occurred on MS medium containing BAP (0.5 mg l^?1) and NAA (0.1–1.0 mg l^?1), while rooting of regenerated shoots occurred in treatments containing 2.0 mg l^?1 NAA alone. This system provides a rapid method for propagation of ‘Troyer’ citrange.     

Vegetative propagation of carnation in vitro through multiple shoot development

Vegetative shoot cuttings of 6 cultivars of carnation (Dianthus caryophyllus L.) were irradiated with 30 or 60 Gy X-rays. Subsequently, from both control and irradiated cuttings, explants were excised at 2 positions of the stem and cultured in vitro. The effects of X-ray dose, genotype and explant position on multiple shoot development of in vitro-cultured nodal stem explants, and on root formation of sub-cultured shoots, were examined. The in vitro propagation method resulted in the production of 2220 plantlets from 209 explants, 4 months after incubation.     

Seeing the oakscape beyond the forest: a landscape approach to the oak regeneration in Europe

Context

Intensification and specialisation of agriculture and forest use has led to profound structural and compositional changes in European landscapes. In particular, sharp, narrow edges adjacent to relatively homogenous vegetation types progressively replace transitional habitats, crucial for a plethora of species and ecological processes. Quercus robur and Q. petraea regeneration niches make them best adapted to such transitional habitats. However, contemporary oaks’ importance, including their regeneration, is usually considered within limits of forest habitats.

Objective

Defining habitats, landscape patterns and processes fostering oak regeneration and ‘oakscape’ development.

Methods

We assessed the state-of-the art of the topical literature with respect to various aspects of oak regeneration based on a refined list of 234 titles from the Web of Science database.

Results

The review confirmed that the vast majority of studies focus on forest habitats, disregarding the fact that substantial part of acorns are being carried away and seeded by birds in non-forest habitats.

Conclusions

The common acceptance of the simplistic landscape mosaic model, based on segregated homogenous vegetation categories and clear-cut lines separating patches, impedes proper assessment of landscape changes, referring to ‘untypical’, transitional habitats—the true oaks’ domain. Hence, restoring and sustaining European ‘oakscape’ should result from the overall landscape management, based on a better adapted gradient approach to landscape studies. Applying such an approach, we identified a set of habitats fostering successful oak regeneration and recruitment without direct human support, contributing to the contemporary ‘oakscape’, represented mostly by non-forest, either natural or anthropogenic habitats.

     

Growth and stability of deep planted red maple and northern red oak trees and the efficacy of root collar excavations

Trees with root systems established well below grade due to deep planting or soil disturbance are common in urban landscapes, yet the long term effects of buried trunks and subsequent remediation strategies, such as root collar excavation are poorly documented. We evaluated the consequences of deep planting over a 10-year period on tree growth and stability, with and without root collar excavation, for red maple [Acer rubrum L. Red Sunset^® (‘Franksred’)] and Northern red oak (Quercus rubra L.) planted at grade or 30-cm below grade. Sleeves to prevent soil-trunk contact were installed around trunks on a subset of deep trees. Root collar excavations were made during the 6th growing season for both species and trees were grown for an additional 4 and 3 growing seasons for red maples and Northern red oaks, respectively. Within two weeks of root collar excavations, pulling tests compared the effect of treatments on stability of red maples. Deep planting generally slowed growth of red maple but had no clear effect on Northern red oak. Root collar excavation had no lasting effect on growth of either species. Approximately 55% of deep red maples and 33% of deep Northern red oaks had roots crossing and in intimate contact with buried trunks, suggesting a potential for future girdling roots. Approximately 25% of deep maples had substantial adventitious rooting. All deep Northern red oaks had new roots emerging just above the first original structural roots but none were clearly adventitious. Trunk sleeves had no effect on growth for either species. Neither deep planting nor root collar excavation resulted in a loss of tree stability compared to trees planted at grade, although failure patterns varied among treatments. Overall, the biggest long term concern for deep-planted trees is the potential for girdling root formation.     

Fertility-mycorrhizal-isolate interactions in production of containerized pin oak seedlings

Slow-release fertilizer rates (0, 1.1 and 4.5 kg/m³ of 14 N-6 P-11.6 K Osmocote) and isolates of the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker and Couch affected growth and mycorrhizal development on pin oak (Quercus palustris Meunchh.) seedlings. Mycorrhizae developed on all inoculated seedlings after 8 weeks regardless of fertility regime. Through the first 14 weeks, the best mycorrhizal development occurred on all inoculated seedlings fertilized at the lower fertilizer rate. The higher fertilizer rate delayed mycorrhizal development. However, after 18 weeks there were no significant differences in mycorrhizae on inoculated seedlings receiving fertilizer. Inoculated, non-fertilized seedlings had significantly less mycorrhizae than fertilized seedlings throughout the experiment. Only one isolate increased seedling growth, and only at the higher fertilizer rate. No differences in plant height were found among non-inoculated control and inoculated seedlings at the low fertilizer rate.     

Effects of supplementary light to mother plants on adventitious shoot formation in flower peduncle segments of Begonia × hiemalis Fotsch in vitro

Plants of two Begonia × hiemalis cultivars (‘Schwabenland’ and ‘Nixe’) were grown in a greenhouse and continuously illuminated (24 h a day) from late October to early March, with light from warm white fluorescent lamps (L“WW”), high pressure mercury lamps (HPI/T) or high pressure sodium lamps (SON/T), each at three irradiance levels (5, 10 or 15 W m^?2). The control received light continuously (0.8 W m^?2) from incandescent lamps (INC). Every third week, plant material (flower peduncles) was collected for in vitro culture. The adventitious shoot formation was strongly dependent on cultivar, length of mother plant illumination and light source. The use of L“WW”, HPI/T or SON/T improved the regeneration ability of flower peduncle segments compared to control. Prolonged irradiation lowered the regeneration in ‘Schwabenland’, especially when HPI/T lamps were used.     

Callus induction and plant regeneration in Cardiospermum halicacabum Linn. an important medicinal plant

Cardiospermum halicacabum Linn. is an important medicinal twining herb belonging to the family sapindaceae. A method for rapid micropropagation of C. helicacabum through plant regeneration from leaf and nodal explant derived calli has been developed. The nodal and leaf segments were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D; 0.5–9 μM) for callus induction. Callus production was highest at 5 μM 2,4-D where 96 and 90% of cultured leaf and nodal cuttings produced callus, respectively. The viable calli were maintained at reduced concentration of 2,4-D (2 μM). These calli were transferred to MS medium supplemented with various concentrations of 6-benzyladenine (BA; 2–10 μM) or kinetin (2–10 μM) alone or in combination with indole 3-acetic acid (IAA; 0.2–1.0 μM) for shoot regeneration. The addition of low concentrations of IAA into BA or kinetin containing medium significantly increased the frequency of shoot regeneration in both nodal cuttings and leaf-derived calli. The highest number of adventitious shoots (28 per callus) formed at 8 μM Kin and 0.5 μM IAA. For rooting of the shoots, half-strength MS medium supplemented with different concentrations of indole 3-acetic acid, indole 3-butyric acid (IBA) and (alpha)-naphthalene acetic acid (NAA) 1–5 μM was tried. The optimal result was observed on half-strength MS medium supplemented with 2.5 μM IBA, on which 91% of the regenerated shoots developed roots with an average of 4.2 roots per shoot within 45 days. The in vitro raised plantlets were acclimatized and transferred to soil with 90% success. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this medicinal plant.     

Climatic response and impacts of drought on oaks at urban and forest sites

This study focuses on the climate and growth response of the sessile oak (Quercus petraea (Matt.) Liebl.) and the northern red oak (Quercus rubra L.) using dendrochronological methods. Tree-ring series of street trees in the city of Dresden, Germany and in a nature reserve close to the urban area were analysed, and the climate and growth response and moving correlation functions (CF) were computed. Moreover, the impact of 13 years of drought was examined by superposed epoch analysis (SEA). Our results show that differences emerge from peculiarities of the different site conditions, indicating that city oaks contain a higher level of noise in their ring width data due to human activities that degrade the strength of growth-climate associations. Importantly, climatic conditions during the previous summer and autumn and during the current spring season display significant correlations with annual growth. The stability of the climate–growth relationship was tested via moving correlation functions. A strong increase in the correlation with the temperature and precipitation in April was found for the trees of northern red oak in forests. Drought years induced significant reductions in radial growth for the northern red oak only. Nevertheless, the long-term impact of drought years must be considered as marginal because of the fast recovery of the radial growth after one to two years. In the future, the growth of the northern red oak may be restricted by an increased frequency of droughts and warmer and drier conditions in April.     

Age estimation of different tree species using a special kind of an electrically recording resistance drill

The determination of tree age is an important issue for urban green planning, forestry and dendrology; finding non-destructive and quasi-non-destructive methods for this purpose is of great theoretical and practical importance. The resistance drilling method is quasi-non-destructive because the average diameter of an opening that remains after drilling does not exceed 3 mm. Do electrically recording resistance drills allow precise assessment of tree age? The aim of this study is to assess the accuracy of determining the number of tree rings based on an examination of this special kind of drilling resistance profiles for three tree species, the pine Pinus sylvestris L., the oak Quercus robur L., and the birch Betula pendula ROTH. In 2015 and 2016, 15 pine trees, 15 oak trees, and 15 birch trees were randomly selected. For each studied tree, a measurement was conducted using the electrically recording resistance drill IML-Resi E400 with a flat-tipped 1.5/3 mm steel needle (research sample), and an increment core was taken (reference sample). The drill used was not a real Resistograph^®. The analysis of the E400-profiles underestimated the number of tree rings; the mean bias error (MBE) values were –6.5, –2.5, and –6.0 years for pine, oak, and birch, respectively. The proportion of investigated trees with less than five years difference between the research and reference samples varied from 38.4 (birch) to 66.7 (oak) percent. The accuracy of tree age determination was lowest for birch and highest for oak. The binomial generalised linear model (GLM) revealed that the most accurate tree age assessments were obtained from tree rings wider than 2 mm. The measurements clearly showed that the electrically recording resistance drill IML-Resi E400 enables a quick, although approximate, tree age assessment. Future research should concentrate on electronically regulating and recording drills, providing a higher spatial and signal resolution, and a stronger correlation to wood density.     

核桃离体培养和植株再生

 以5个核桃品种(株系) 10年生植株的茎段为外植体, 进行了离体培养和植株再生的研究。结果表明, 附加2 g·L^-1聚乙烯吡咯烷酮的DKW + 6-BA 1.0 mg·L^-1 + IBA 0.01～0.10 mg·L ^-1是诱导茎段萌发最适宜的培养基, 5个品种(株系) 间的萌发率存在差异; DKW + 6-BA 110 mg·L^-1 + IBA 0.01mg·L^-1 + 蔗糖40 g·L^-1是最适宜的增殖培养基; 采用二步生根法最高生根率为41.67% , 生根试管苗移栽于草炭土∶河沙= 1 ∶1的基质中, 控制温度20～30 ℃, 保湿20 d, 期间遮荫, 40 d后成活率95%。     

Bioactivities of Some Wild Fruits Grown in Turkey

In this study some bioactive properties of extracts of wild pear (Pyrus elaegnifolia), hawthorn (Crataegus spp.), oriental hackberry (Celtis tournefortii), oleaster (Elaeagnus angustifolia), Japanese crabapple (Malus floribunda), rosehip (Rosa canina), pedunculate oak (Quercus robur), service tree (Sorbus domestica) and firethorn (Pyracantha coccinea) wildly growing in Turkey were investigated. The fruit extracts were analysed in terms of total phenolic content (TPC), antioxidant activities (DPPH and phosphomolybdenum methods) and antimicrobial activity. In the results, TPC, IC50 value of DPPH and IC50 value of phosphomolybdenum for pedunculate oak were 454.35?mg GAE/g d.w., 340.52?mg AAE/g d.w. and 96.52%, respectively. Service tree and firethorn exhibited the lowest TPC (4.11?mg GAE/g d.w.) and IC50 value of DPPH (53.32?mg AAE/g d.w.), respectively. Moreover, antimicrobial activity of oleaster was 2.46%. The hackberry fruit extract had no inhibitory effect against the tested microorganisms whereas pedunculate oak extract (with 10%) had the highest inhibitory effect against Aeromonas hydrophila. Additionally, all the fruit extracts had no antimicrobial activity against the Saccharomyces cerevisiae. In conclusion, wild edible fruit extracts can be used as bioactive material due to their antioxidant and antimicrobial activity in food industry and human nutrition.

     

The assessment of mulch sheets to inhibit competitive vegetation in tree plantations in urban and natural environment

Weed control is often crucial for successful tree establishment. Removal or suppression of competitive vegetation has most often been done by either mowing or the use of herbicides. The Institute of Forestry and Game Management in Flanders (Belgium) has been conducting a three-year experiment (as part of a five-year programme) to assess 18 treatments for newly planted deciduous trees established on ex-agricultural sites. On grass-covered sites mortality of English oak(Quercus roburL.) decreased significantly when mulch sheets were applied. Mulch sheets increased growth of all species in a pasture. An unequivocal relation was found between the diameter of the weed-free area around the tree and the Relative Growth Rate of Common alder (Alnus glutinosa (L.) Gaertn.). Rigid sheets, such as those made of polypropylene could severely damage the trunk of the trees. Bark mulch decreased growth of English oak in the first year, but increased it significantly after 3 years. The application of bark mulch was time-consuming compared to the other treatments. Usage of herbicides on a clay soil was nefarious to both survival and growth of the treated trees. Mowing, often used by the Flemish Forestry Service, had no significant effect on the growth of the trees.     

The importance of native valley oaks (Quercus lobata) as stopover habitat for migratory songbirds in urban Sacramento,California, USA

Urban development occupies over 375,000 ha (6%) of California's Central Valley, and expansion continues to displace natural and agricultural landscapes. The value of urban areas as habitat for native wildlife and the characteristics that determine its value, however, remain little studied. Many Neotropical migrant passerine bird species are declining due to changes in breeding, migratory, and wintering habitats and climatic conditions. During 2010–2013, we evaluated the importance of native valley oak (Quercus lobata) as stopover foraging habitat used by Neotropical migrant birds in urban areas of the Sacramento region in California, USA. Over 3 years, we surveyed spring and late summer-early fall migrant songbirds and measured tree canopy cover within 31 c.0.91 ha transects in Curtis Park, an older residential neighborhood. We detected 607 individuals from 20 migrant species, but four wood warblers comprised the bulk of observations: black-throated gray (Setophaga nigrescens), Wilson's (Cardellina pusilla), orange-crowned (Oreothlypis celata), and yellow warblers (Setophaga petechia). Migrant abundance was closely correlated with valley oak canopy abundance and increased linearly with oak canopy especially during fall migration. Migrants were nearly absent from areas lacking oak canopy. Migrant bird species as a group also foraged in valley oak substantially more often (74%) than would be expected based on its 15% relative canopy cover (χ²_1d.f. = 924, p < 0.0001), as did all species whose selectivity could be tested. These results are important in demonstrating previously undocumented migrant use of urban areas with remnant valley oak canopy and suggest that protecting existing valley oaks and increasing their use in future urban forestry and landscape plantings in the Central Valley could provide substantial habitat benefits for native migratory birds.     

Plant regeneration potential in fly ash ecosystem

Ability of certain plant species to regenerate by adaptive growth can address the harsh conditions of fly ash dumps in fly ash ecosystem. In the present study, we examined the status of regeneration potential of planted tree species in three life forms at fly ash ecosystem of tropical zone. On the basis of importance value index Prosopis juliflora (Sw.) DC., Pithecellobium dulce (Roxb.) Benth. and Pongamia pinnata (L.) Pierre have been found as dominant species in fly ash ecosystem. In this regeneration status study, the above three tree species (68.91%) have been found in good regeneration category which can be used for revegetation of new fly ash dumping sites.     

Multiple plantlets in lateral bud and leaf explant in vitro cultures of pineapple

Plantlets were obtained from usually dormant axillary buds, excised from the crown of pineapple (Ananas comosus L. Merr.) and grown in culture. Multiple shoots arose from single buds grown on Murashige and Skoog medium supplemented with auxins and kinetin. Shaking culture-flasks during growth increased the number of multiple shoots formed, when compared with stationary liquid cultures. Leaf explants excised from in vitro plantlets developed into a callus capable of plantlet regeneration. Subjecting developing buds to surgical segmentation also resulted in multiple shoot formation. Such shoots, when excised and grown on Murashige and Skoog medium supplemented with auxins, developed roots and grew into complete plantlets capable of being grown in soil.     

Plant regeneration of Nerium oleander L. from alginate-encapsulated shoot explants after short-term cold storage

In this study, an efficient protocol for the regeneration of encapsulated explants of oleander (Nerium oleander L.) has been developed. Shoot tips and 1st nodal segments below the shoot tip, from in vitro-derived oleander microshoots, were encapsulated in 2.5% sodium alginate prepared in liquid MS sucrose-free nutrient medium and hardened in 50 mM of calcium chloride producing solid beads, uniform in shape. These artificial seeds, irrespective of their maintenance under light or in darkness, germinated at frequencies of 38.8–42.2%, producing 3.0–3.3 microshoots per bead. In the case of using 100 mM of calcium chloride for hardening, the beads were firm, of uniform globular shape and suitable for handling, exhibiting a germination response of 68.9%. Encapsulated shoot tip explants, following storage at 4°C for 8 weeks, exhibited a higher regeneration response (60.0%) than non-encapsulated similar explants stored under the same conditions (11.1%). Microshoots, excised from cold-stored encapsulated explants after germination, rooted easily in agar-solidified MS medium with 2 μΜ IBA and after their transplantation into a peat-perlite substrate (3:1, v/v), were acclimatised successfully and established in the greenhouse with minimal losses. The present encapsulation procedure could be applied as an alternative method of micropropagation of desirable elite clones of oleander.     

Chromosome doubling of Lychnis spp. by in vitro spindle toxin treatment of nodal segments

Lychnis (Caryophyllaceae) consists of about 30 species distributed throughout the temperate regions of the Northern Hemisphere, from East Asia to Europe. Many Lychnis spp. have high ornamental value and cultivated as pot or garden plants. In the present study, in vitro chromosome doubling of several Lychnis spp. was examined in order to widen their variability in horticultural traits. Initially effect of various spindle toxin treatments [100, 500 or 1000 mg l⁻¹ colchicine (COL), 10, 20 or 50 mg l⁻¹ oryzalin (ORY), or 1, 5, 10 mg l⁻¹ amiprophos-methyl (APM)] of nodal segments of a triploid genotype of L. senno (3x) was investigated on survival of nodal segments and chromosome doubling in nodal segment-derived plantlets. Significantly higher percentage (75.0%) of surviving segments after spindle toxin treatment was obtained in 10 mg l⁻¹ ORY treatment. Flow cytometry (FCM) analysis of leaf tissues showed that 9.4–13.8% of plantlets, which were derived from 10 to 20 mg l⁻¹ ORY, or 5 mg l⁻¹ APM treatments, were hexaploid (6x) or ploidy chimera (3x + 6x, 4x + 6x, 5x + 6x, 3x + 4x + 6x). The results obtained by FCM analysis were confirmed by chromosome observation in root tip cells. Thus 10 mg l⁻¹ ORY treatment of nodal segments is suitable for in vitro chromosome doubling of triploid L. senno. Efficient chromosome doubling was also achieved in diploid L. fulgens (2x) and L. sieboldii (2x) by treating nodal segments with 10 mg l⁻¹ ORY: 68.9–88.7% of nodal segments survived after ORY treatment, and 24.7–26.5% of plantlets derived from ORY-treated nodal segments were tetraploid (4x) or ploidy chimera (2x + 4x) in both species. These results indicate that the in vitro chromosome doubling method established for triploid L. senno may be applicable to a wide range of Lychnis spp. Tetraploid L. fulgens and L. sieboldii showed a compact plant form, and had thick stems and deep green leaves compared with the diploid mother plants. On the other hand, hexaploid L. senno showed very poor growth and died before flowering.