Plantlet Survival Research Articles

액체배지 첨가에 의한 Anthurium andreanum ′Atlanta′의 기내생육 촉진

In order to enhance shoot elongation and rooting of Anthurium andreanum `Atlanta` in vitro, 15mL of liquid media containing various concentrations of activated charcoal, sucrose and MS salts were added in same vessels after small shoots were induced from the calli on mudium supplemented with 10.0mg/L BA and 0.1mg/L 2.4-D. The post-supplying of 15mL liquid medium containing MS macro and micro elements, 30g/L sucrose and 5.0∼10.0g/L activated charcoal was significantly stimulated the shoot elongation and rooting of regenerated shoots from calli. The medium addition was also resulted in the enhanced soil survival, elongation and rooting of plantlets in cultural soil mixed with perlite and vermiculite(1 : 1)

세포 배양을 통한 가시오갈피(Eleutherococcus senticosus) 묘목의 대량 생산 시스템 개발

Immature zygotic embryos of Eleutherococcus senticosus seeds matured rapidly within one month when the seeds comprising zygotic embryos were pieced to small size and cultured on 1/2 MS medium. Frequency of somatic embryos formation was declined rapidly when the zygotic embryos germinated and grew to plantlets. Embryogenic cells were induced by consecutive subculture of somatic embryos on MS medium with 1.0mg/L2,4-D. After heart-shaped somatic embryos were induced by suspension culture, these embryos were plated onto petri dish to support maturation of embryos. Germination of embryos occurred on medium with 5mg/L GA<TEX>$_3$</TEX>and transferred to culture bowl to stimulate the further growth. Frequency of soil survival of plantlets was influenced by soil mixture (perlite and peatmoss). The suitable combination of perlite and peatmoss was 1:5, and the soil survival rate was 78% after 4 months. The soil transferred plantlets were over-wintered in field condition after defoliation. New year sprouting of plants was achieved successfully and they grew to adult plants. These results indicate that the systematic procecure of plant production in E. senticosus for micro propagation.

Plant regeneration from callus cultures of Maclura tinctoria, an endangered woody species

Some native species produce seeds with a low frequency of germination accompanied with a period of dormancy. These features make it difficult to produce new phenotypes through sexual propagation. Maclura tinctoria has been considered an endangered species due to extensive use of its wood and low frequency of seed germination. The objective of the present study is to establish an in vitro propagation system for this species. Organogenic friable callus formation from nodal segments has been obtained using woody plant medium (WPM) supplemented with 10.74 μM 1-naphthaleneacetic acid (NAA)+4.43 μM 6-benzylaminopurine (BA). Results indicate that the highest frequency of shoot formation is observed when WPM supplemented with 4.03 μM NAA+4.43 BA is used. For root formation, the use of WPM medium (pH adjusted to 7.0) supplemented with 23.62 μM indole-3-butyric acid (IBA) and 4.7gl−1 activated charcoal is recommended. For acelimatization, subjecting rooted plantlets to 70%, 50%, and 30% mesh screen, each successively for a period of 7 d, has resulted in 97% plantlet survival.

Structural equation modelling: an alternative for assessing causal relationships in threatened plant populations

Structural equation modelling (SEM) is a powerful tool to explore and contrast hypotheses on causal relationships among variables using observational data. It constitutes an alternative to experimental approaches that is especially useful in the conservation of small populations where the implementation of treatments may have a negative effect on population viability. We are presently applying SEM to study the factors that condition reproductive success, seed emergence and plantlet survival in several plant species. We are also using model comparisons through multi-sample analysis to assess the implications of different microhabitats on the viability of a population. The most outstanding advantages of this tool are the global perspective used in the study of complex problems, the ability to discern the essential from the accessory, and the possibility of evaluating one's own hypotheses. The basic procedure, the limitations of this method and further applications in conservation and management are also discussed.

Micropropagation of Thapsia garganica--a medicinal plant.

The micropropagation of T. garganica, a medicinally important plant, was investigated as an option for conservation purposes as wild populations are becoming sparse. A Murashige and Skoog agar medium supplemented with 0.5 mg l(-1) alpha-naphthaleneacetic acid and 1.5 mg l(-1) benzyl-6-adenine significantly improved the production of multiple shoots directly from petiole and leaflet explants compared to other plant growth regulator (PGR) combinations. Medium free of PGRs promoted rooting at a low incidence. Acclimatisation was low--52% plantlet survival--as micropropagated plantlets were highly susceptible to fungal rot once removed from culture. Application of antifungal agents to in vitro-derived plantlets as pre- and post-acclimatisation treatments during transplanting significantly reduced ex vitro mortality.

In vitro flowering of Bambusa edulis and subsequent plantlet survival

Bamboo shoots could be induced to flower in vitro, but there is very little information on the effect of growth components on flowering. In this study, multiple shoots grown from in vitro, spikelet-derived, somatic embryos of Bambusa edulis were used for in vitro flowering. Multiple shoots flowered on Murashige and Skoog medium (MS) with 0.5 mM thidiazuron (TDZ) and 30 g l sucrose. Different

In vitro and ex vitro growth of grapevine rootstock '5BB' as influenced by number of air exchanges and the presence or absence of sucrose in culture media

Single node cuttings of grapevine rootstock `5BB' were cultured for 5 weeks under different numbers of air exchange (0, 0.8, 1.5, 2.5 and 4.4 h −1) with or without sucrose in the medium. To determine the effect of number of air exchanges related to presence or absence of sucrose, in vitro growth, net photosynthetic rate, stomata characteristics and ex vitro growth were investigated. Increasing numbers of air exchanges accelerated plantlet growth regardless of the presence or absence of sucrose in the medium; under the same number of air exchanges, plantlet growth and net photosynthesis were greater in sucrose-containing medium compared with sucrose-free medium. However, a high number of air exchanges (4.4 h−1) inhibited the growth and photosynthesis in sucrose-containing medium compared to sucrose-free medium. A higher number of stomata was observed in sucrose-containing medium, and larger size stomata was observed in sucrose-free medium. These results emphasize the importance of ventilation (increased number of air exchanges) during in vitro culture, for ex vitro plantlet survival was greatly affected by with or without air exchanges. Without air exchanges ex vitro plantlet survival was less than 70%, while air exchanges increased ex vitro survival by more than 90% with greater growth.

Optimisation of the tissue culture protocol for the endangered Aloe polyphylla

An optimised, rapid tissue culture protocol was established for the highly endangered Aloe polyphylla (Schonland ex Pillans). Shoot cultures of Aloe polyphylla were initiated from young shoot explants of in vitro grown plants. The basal medium was MS medium (Murashige and Skoog 1962), supplemented with 100mgl - 1 myo-inositol, and 30gl - 1 sucrose. Different cytokinins (kinetin, zeatin and BA) singly or in combination with auxins (IBA and NAA), were tested for shoot proliferation activity. All the cytokinins gave good shoot proliferation. The optimal concentrations for shoot proliferation of each of the cytokinins tested were: zeatin (0.5mgl - 1 ), kinetin (1.5mgl - 1 ) and BA (1.5mgl - 1 ). In combination with auxins, the optimal combinations were kinetin/NAA (2.0/0.1mgl - 1 ), kinetin/IBA (1.5/1.0mgl - 1 ), zeatin/IBA (1.0/0.5mgl - 1 ), zeatin/NAA (1.0/1.0mgl - 1 ), BA/IBA (1.0/1.0mgl - 1 ) and BA/NAA (1.5/0.1 mgl - 1 ). Although it gave the highest number of shoots per explant, BA (1.0–3.0mgl - 1 ) induced hyperhydricity. Temperature and sucrose also influenced shoot proliferation. The optimal temperature was 25 °C, while 30gl - 1 was the optimal concentration of sucrose. Plants rooted well in plant growth regulator-free MS medium. Amongst the potting mixtures tested, soil:sand:vermiculite (1:1:1 v/v/v) was the best, with a 98% plantlet survival.

Cultivo de embriões de tomate in vitro visando a introgressão de genes de Lycopersicon peruvianum em L. esculentum

Estudou-se diferentes protocolos de germinação e manutenção de híbridos interespecíficos (Lycopersicon esculentum X L. peruvianum) pela melhoria das condições de cultura in vitro bem como da aclimatação das plantas germinadas. Quanto à regeneração dos embriões, três formulações de meios de cultura para resgate de embrião foram avaliadas, em combinação com distintos períodos de tempo. A recuperação de híbridos interespecíficos foi influenciada pelo meio de cultura utilizado para o plaqueamento das sementes e pelo intervalo entre a polinização artificial e o plaqueamento. O meio HLH proporcionou os melhores resultados. O intervalo entre 25 e 35 dias após a polinização foi determinado como sendo o período ideal para recuperação. Para aclimatização, foram avaliadas plantas de L. esculentum, L. peruvianum, híbridos interespecíficos (F1) e das gerações RC1 e RC2. O processo de aclimatização foi influenciado pelo tamanho das gemas utilizadas na repicagem anterior à aclimatização, pelos materiais genéticos utilizados e pelo tempo de manutencão das plântulas em tubo de ensaio. A geracão F1 apresentou a melhor capacidade de aclimatização. O período ideal para aclimatização variou entre 26 e 35 dias após a repicagem em tubo de ensaio. As aclimatizações realizadas sob condições ambientais amenas, alcançaram êxito total quanto à sobrevivência das plântulas.

The Effect of Hexazinone, Sulfometuron, Metsulfuron, and Atrazine on the Germination Success of Selected Ceanothus and Rubus Species

Abstract This study documents the impact pre-emergent forestry herbicides have on germination of some selected seral woody competitors in the Pacific Northwest. Four commonly used pre-emergent soil-active herbicides (hexazinone, sulfometuron, metsulfuron, and atrazine) typically used for herbaceous weed control were applied at six rates over stratified seed of Ceanothus velutinus (CEVE) and Ceanothus integerrimus (CEIN) in a greenhouse efficacy trial. In addition, hexazinone and sulfometuron were applied over stratified Rubus ursinus (RUUR) and Rubus parviflorus (RUPA) seed and sulfometuron over stratified seed of Rubus spectabilis (RUSP) at the same six rates. Numbers of seed to successfully germinate and develop true leaves were counted over a 9 wk period immediately following herbicide application. The hexazinone treatments reduced germination and growth of CEVE, CEIN, and RUPA. The RUUR species was tolerant of the hexazinone herbicide at low rates but at higher rates was strongly affected. The sulfometuron treatments had less effect on survival probability than hexazinone but strongly reduced the average dry weight of plantlets of all species. Seedling dry weight decreased with increasing rate of both metsulfuron and atrazine. Increasing the metsulfuron rate reduced the probability for CEVE seedlings to survive but not CEIN. Finally, atrazine sharply reduced the plantlet survival and reduced dry weight of both CEVE and CEIN even at low rates.West. J. Appl. For. 17(4):194–201.

Culture method and photosynthetic photon flux affect photosynthesis, growth and survival of Limonium ‘Misty Blue’ in vitro

Microcuttings (shoots each with two leaves) of Limonium ‘Misty Blue’ were cultivated in vitro for 28 days under photoautotrophic (sucrose-free culture medium; CO 2 and photosynthetic photon flux (PPF) enriched conditions), photomixotrophic (medium with 30 g l −1 sucrose; CO 2 and PPF enriched conditions) and heterotrophic (medium with 30 g l −1 sucrose; CO 2 non-enriched conditions) methods. Several growth variables were measured during and at the end of cultivation: shoot fresh and dry weight, percentage of shoot dry matter, root fresh weight, number of leaves, leaf area, chlorophyll and sugar content of leaves, stomatal density and size, net photosynthetic rate (NPR) and percent survival of plantlets ex vitro. Plantlets grown in photoautotrophic and photomixotrophic methods had more leaves, high chlorophyll and sugar contents, high NPR, and showed high percent survival. However, these plantlets possessed less number of stomata per square millimeter. In contrast, the plantlets grown by the heterotrophic method showed decreased values of these growth variables except for the number of stomata per square millimeter. These results indicate that CO 2 enrichment for plantlets in vitro at a relatively high PPF would promote photosynthesis and hence growth of chlorophyllous explants/plantlets in vitro. The resulting plantlets were acclimatized better and sooner on ex vitro transplantation.

The effect of inoculating endophytic N2-fixing bacteria on micropropagated sugarcane plants

The aim of this work was to evaluate the effect of the inoculation of endophytic N2-fixing bacteria on the development of micropropagated sugarcane plants. The endophytic population of each inoculated species was monitored during the growth period, and biological nitrogen fixation (BNF) contribution of each inoculation treatment was assessed using the 15N-isotope dilution technique. Seven different combinations of inoculum were used, using five endophytic diazotrophic species (Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Azospirillum amazonense and Burkholderia sp.), originally isolated from sugarcane plants. The results showed a clear physiological effect on the development of the inoculated plants, resulting in alteration of the dry matter-partitioning pattern and increase on root dry matter as compared to uninoculated plants. Indeed, all inoculated diazotrophic species could be reisolated in high numbers from the rhizomes of the inoculated plants, even 400 days after inoculation (DAI), suggesting the establishment of the inoculated bacteria. However, a negative effect of the mixture of all five species on the survival of plantlets was observed 45 days after inoculation, just after acclimatization. The analysis of the BNF contribution using the 15N-isotope dilution technique showed that inoculation promoted some increase in the BNF contribution to the plant tissues. The best treatment was the mixture of all five strains, followed by the treatment with a mixture of Herbaspirillum spp. The contribution was much lower when the plants were inoculated with a mixture of G. diazotrophicus with A. amazonense and Burkholderia sp. A BNF contribution around 30% of total nitrogen accumulated was observed in micropropagated plants inoculated with the mixture of strains, suggesting that the combination of species in the inocula is the best strategy to improve sugarcane crops dependent on the biological nitrogen fixation process.

In vitro propagation of Opuntia ellisiana Griff. and acclimatization to field conditions

The genus Opuntia is a valuable forage resource in arid and semiarid lands during periods of drought and shortage of herbaceous plants. However, absolute minimum temperatures in the plains of Mendoza represent a limiting factor to cultivate several species. Opuntia ellisiana is a cold hardy species, so the goals of this study were to massively propagate is using in vitro culture techniques, and then to acclimatize plantlets obtained to field conditions. Different sterilization protocols were tested. Areoles were isolated in laminar airflow cabinet, and cultured on Murashige-Skoog medium, supplemented with sucrose and different BAP and IBA combinations. Explants were grown at 27 +/- 2 degrees C, under a 16-h photoperiod. The shoots produced were used in the rooting assay using different auxin combinations. In the most efficient growth treatment, plantlets reached 100% shooting after 35 days of culture, and a mean length of 10.2 mm after 49 days of culture. A 100% rooted plantlets was obtained on a medium containing 5 mg L-1 IBA, after 12 days of culture. Acclimatization was achieved under greenhouse conditions, showing 100% plantlet survival. This study suggests that O. ellisiana can be successfully micropropagated by areoles, and easily acclimatizated to field conditions.

CULTURE OF VERY YOUNG PHASEOLUS VULGARIS L. PODS AND PLANTLET REGENERATION

Introduction First results on the development of an in vitro pod culture technique for young pods of P. vulgaris (PV) are reported by Geerts et al (2000). Different culture techniques for 2-dlay old Phaseolus pods were described using a modified Phillips ^i al (1982) medium for maturation. The authors showed that the application of high and variable osmolality conditions, similar to those observed in vivo, during pod culture and before extracting the embryos, gave the best results in terms of ovule and embryo development (Geerts et a/. 1999). However, the plantlet survival remained very low and no regeneration was described. In this paper, we report the method used to obtain the first regeneration of plantlets from 2 days-old Phaseolus embryos using an in vitro pod culture technique. Material and methods A P. vulgaris genotype (NI 637) was grown in a growth chamber under the following controlled conditions: day/night temperature of 24/20°C, light intensity of 580 |imol,m*^s^ and a day length of 11 h 30 min. Pod culture technique was adapted fi-om Geerts et al (2000). Basal medium contains Phillips et al. (1982) mineral salts, 80 g.F^ sucrose, 1 mg.F^ thiamin HCl, 5 mg.F^ nicotinic acid, 0.5 mg.r' pyridoxme, lOOmg.r' myoinositol, 1,000 mg.I'^ L-glutamine, 1,000 mg.F^ casein hydrolysate, 0.1 jLiM NAA, 10 \xM adenine and 1.0 |iM Benzylaminopurine. Seven pod culture methods were compared using three different osmoticum agents (Table I): four solid media (SI, S2, S3 and S4) and three liquid media (LI, L2 and L3). For each of the solid cultures, a sequence of three media with a decreasing osmotic pressure were used. Pods were cultured on 580 mosm of osmolality during I day, transferred to 450 mosm for 2 days more, and lastly transferred for 4 days more to basal medium (350 mosm). For S4 technique, pods were directly transferred to basal medium for 1 week. Table L Type and quantity of osmoticum added to the basal medium. Techniques Osmoticum added Osmolalities of media 580 mosm ^*^ 450 mosm 350 mosm SV''' LI Sucrose 63 g.r^ 22 g.r' S2 L2 Mannitol 36 g.r' 16 g.r ^ S3 L3 Polyethylene glycol 8000 128 g.r' 55 g.r' _ S4 ^*^ at this osmolality, vitamins, amino acids and growth regulators of basal medium are modified : 0.25 mg.f' thiamin HGl, 1.25 mg.T^ nicotinic acid, 0.125 mg.r' pyridoxine, 25 mg.r' myoinositol, 250 mg.r' Lglutamine, 250 mg.r' casein hydrolysate, 0.095 ^M abscisic acid, 5.5 |iM Tryptophan and 0.1 ^M NAA. ^**^ 7 g.r'Select Agar-Gihco BRL.

Flooding tolerance and cell wall alterations in maize mesocotyl during hypoxia

This research aimed to characterize the tolerance to flooding and alterations in pectic and hemicellulose fractions from mesocotyl of maize tolerant to flooding when submitted to hypoxia. In order to characterize tolerance seeds from maize cultivars Saracura BRS-4154 and BR 107 tolerant and sensitive to low oxygen levels, respectively, were set to germinate. Plantlet survival was evaluated during five days after having been submitted to hypoxia. After fractionation with ammonium oxalate 0.5% (w/v) and KOH 2M and 4M, Saracura BRS-4154 cell wall was obtained from mesocotyl segments with different damage intensities caused by oxygen deficiency exposure. The cell wall fractions were analyzed by gel filtration and gas chromatography, and also by Infrared Spectrum with Fourrier Transformation (FTIR). The hypoxia period lasting three days or longer caused cell lysis and in advanced stages plant death. The gelic profile from pectic, hemicellulose 2M and 4M fractions from samples with translucid and constriction zone showed the appearance of low molecular weight compounds, similar to glucose. The main neutral sugars in pectic and hemicellulose fractions were arabinose, xilose and mannose. The FTIR spectrum showed a gradual decrease in pectic substances from mesocotyl with normal to translucid and constriction appearance respectively.

In Vitro Colonization of Micropropagated Pieris floribunda by Ericoid Mycorrhizae. II. Effects on Acclimatization and Growth

Inoculation of microshoots of Pieris floribunda (Pursh ex Sims) Benth. and Hook. (mountain andromeda) with isolates of Hymenoscyphus ericae (Read) Korf and Kernan ericoid mycorrhizal fungi stimulated growth during 1 month in vitro. However, no benefits were apparent after 3 months in a greenhouse. Acclimatization of plantlets of P. floribunda to greenhouse conditions following in vitro inoculation improved survival (42% vs. 16% for controls). The protocol reported herein is similar to procedures utilized currently for micropropagation of various ericaceous species and has potential to improve plantlet survival during acclimatization.

In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.

Physiological changes and growth of micropropagated chile ancho pepper plantlets during acclimatization and post-acclimatization

Little is known about physiological changes that occur with micropropagated chile ancho pepper (Capsicum annuum L. cv. San Luis) plantlets during acclimatization. Plantlets were transferred to ex vitro conditions to study selected physiological changes and growth performance during acclimatization and post-acclimatization. The physiology of the plantlets was characterized by measuring leaf gas exchange and water status. Plant growth was determined by assessing plant height, leaf number, total leaf area, relative growth rate (RGR), and leaf, root, and stem dry matter (DM). Chile pepper plantlets became acclimatized within 6 days after transplantation. During this period, physiological adjustments occurred, which were critical for plantlet survival. After initial ex vitro transplanting, plantlets experienced water deficit [leaf wilting and reduced relative water content (RWC)], which corresponded with reduced stomatal conductance (gs) and transpiration (E), and an increase in stomatal resistance (rs). Thus, leaf stomata that developed in vitro were functional ex vitro. Because of this stomatal control, plantlets minimized transplant shock, recovered and survived. Prior to transplanting, plantlets were photomixotrophic, as indicated by low photosynthetic rates (A). During acclimatization, RWC, gs, E, and A were significantly lower two days after transplanting. However, within 6 days after transplanting, plantlets recovered and became photoautotrophic – attaining high A, gs, and E. Water use efficiency was initially low during the first days after transplanting, but increased dramatically at the end of the acclimatization period in part due to increased A. The stabilization and improvement of plantlet water status and gas exchange during acclimatization and post-acclimatization closely correlated with increased plantlet growth.

Micropropagation of Camptotheca acuminata decaisne from axillary buds, shoot tips, and seed embryos in a tissue culture system

Micropropagation of the anti-cancer plant Camptotheca acuminata Decaisne from axillary buds and seed embryos was investigated. Axillary buds from greenhouse seedlings required a period of culture in media free of N6-benzyladenine (BA) before multiple shoot induction began. Direct induction of multiple shoots on BA-containing medium resulted in high mortality of the axillary buds. Multiple shoot induction from the greenhouse axillary buds was best achieved on B5 with 4.4 μM BA+0.5μM α-naphthaleneacetic acid, forming an average of three 2-mm tall shoots per bud in 8 wk. Elongation of these multiple shoots was successful at a lower BA level (0.22 μM) on B5 medium. Both in vitro and ex vitro rooting of the microcuttings was feasible with indole-3-butyric acid in the culture media, but ex vitro rooting led to high plantlet survival. Seed embryos were not ideal explants for multiple shoot induction. Shoot tips and axillary buds of in vitro-germinated seedlings showed an optimal multiple shoot formation on B5 with 8.9 μM BA, double the optimal BA level for greenhouse axillary buds. Using axillary buds to propagate C. acuminata plants in vitro is feasible for mass propagation of desired clonal lines high in camptothecin concentrations.

In vitro propagation of Eucomis L'Hérit species - plants with medicinal and horticultural potential

Eucomis (Family Hyacinthaceae) are deciduousgeophytes with long, narrow leaves and erect, densely packed flower spikes. Thebulbs are greatly valued in traditional medicine for the treatment of a variety ofailments, and are thus heavily harvested for trade in South Africa's traditional herbal markets. Eucomis species propagaterelatively slowly from offsets and seed, and this, together with theirover-utilisation ethnopharmacologically, has led to their threatened status. Thein vitro propagation of the genusEucomis was investigated to optimise this technique for thebulk production of plants for commercial and conservation purposes. Multipleshoot production was initiated from leaf explants, in all species studied. AMurashige and Skoog (MS) medium, supplemented with 100mg amp;ell;−1 myo-inositol, 20g amp;ell;−1 sucrose, and solidified with 2g amp;ell;−1 Gelrite® was used. Theoptimal hormone combination for shoot initiation in the majority of species was1 mg amp;ell;−1 NAA and 1mg amp;ell;−1 BA. Optimal root initiation wasdemonstrated on media supplemented with 1mg amp;ell;−1 IAA, IBA or NAA, depending onspecies. A continuous culture system using this protocol produced 25–30plantlets per culture bottle, with 10–25 specimens per bottle availablefor acclimatisation. To maximise plantlet survival, different support media usedduring the acclimatisation process were necessary. Certain species respondedbest on a vermiculite medium, while perlite (which holds less water) wasnecessary for the optimal survival rate of other species. Acclimatised plantletswere repotted in a sand: soil mix (1:1).