Volume 20(2), 118- 125, 2016 JOURNAL of Horticulture, Forestry and Biotechnology www.journal-hfb.usab-tm.ro

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The Effects of Taxus Baccata L. Aqueous Extracts on Germination, Seedling Growth and Physiological Parameters of Test Species Lobiuc A. 1,2, Cuibari Roxana 1, Frunzete Mădălina 1,*, Naela C.1, Marian Burducea1, Ardelean Mirela 3, Zamfirache Maria-Magdalena 1

1Departament of Biology, Faculty of Biology, “Alexandru Ioan Cuza” University of Iași, Bd. Carol I, No. 20 A,

code 700505, Iași, Romania; 2 “Universitatea Ștefan cel Mare”, Universitatii str. 13, Suceava, România

3“Vasile Goldiș” University, Revolutiei str. 94, Arad, Romania;

*Corresponding author. Email: f.madalinaelena@yahoo.com;

Abstract The present paper aimed to assess the phytotoxic potential of Taxus baccata L. (yew) aqueous extracts on Raphanus sativus L. and Cucumis sativus L. germination, growth, chlorophyll content and chlorophyll fluorescence (ɸPSII) of the seedling.

In the case of Raphanus sativus the germination indices were negatively affected by the leaves and aril extracts, with the 5% leaves extract having the most pronounced effect on the germination percentage, energy and speed of germination and vitality index. Leaves and aril extracts significantly reduced the fresh mass (with up to 41%) and radicle length (with up to 71%) of the seedlings, while the length of the hypocotyl was reduced especially by the bark extracts. Inhibitory effects on chlorophyll contents were exerted by 5% aril and 1% leaves extracts while chlorophyll fluorescence (ϕPSII) was decreased, however not significantly, by leaves extracts.

In the case of Cucumis sativus, inhibitory effects on germination were recorded for leaves 1% and aril 5% extracts. Morphometric inhibition was especially observed for 5% aril extract, which reduced the mass, hypocotyl and radicle lengths and also chlorophyll content and fluorescence parameters. The registered effects can be associated with the high contents of phenolic in the extracts as determined by UV-Vis spectrometry and also to physico-chemical characteristics of extracts. Taxus baccata is a well-known species for the toxic effect of its constituent, taxol, on animal cells. The tested extracts displayed a certain degree of toxicity also on plants which may add to the uses of this specie. Further research may include optimization of the extraction process and more thorough identification of compounds present in extracts.

Key words yew, phytotoxicity, morphometry, chlorophyll, fluorescence

Taxus baccata L. (yew) is a glaciary relict coniferous

specie, belonging to the Taxaceae family. It is a specie

with a long standing use by humans, especially for its

wood, from which furniture is produced but also for the

dyeing properties of the bark [27]. Some extracts form

Taxus are used in cosmetics products, as well as in

traditional medicine, for treating headache, cold or

nervous system disordes [15]. More recently Taxus

baccata is used for extraction of the anti-proliferative

compound taxol, an alkaloid with potent activity [22].

These are the main reasons for over exploitation of this

specie in many countries [8]. More over the specie is

considered endangered in many countries being

included in several red lists, including in Romania [25,

26], while habitats with yew are listed in the Habitats

Annex II [3].

The main factors affecting the Taxus

population establishment and trends are considered the

available amount of light in relation with other species’

competition, deforestation, browsing and bark stripping

by herbivores [8]. The requirements of yew towards

abiotic ecological factors are relatively well know, the

specie growing well in higher humidity, on calcareous

soils, altitude between 100-400 m in regions with mean

temperature between 8-21° C [41,27].

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However, significantly less data are available

concerning the ecology of Taxus in interaction with

individuals of other species. Also, it considered that

Taxus seedling are not competitive in establishing new

populations, on one hand due to grazing and on the

other hand, due to inhibition of growth induced by

shade [31,3]. The possibility of self-inhibition of

seedling growth by other Taxus individuals was,

however, not studied previously, while it is known that

this phenomenon occurs in several forest species [21].

Autotoxicity is reported to occur in both manipulated

and natural ecosystems, in both herbaceous and tree

species [18]. Toxic effects of leachates or exudates

involve the presence of chemical substances, such as

phenolics [35,46], which affect surrounding plants by

interference with various metabolic pathways [4].

Few studies have documented the allelopathic

effects of Taxus on other species, showing a certain

degree of toxicity towards other [47]. Moreover,

various Taxus tissues contain relatively high amounts

of phenolic substances [6,7], and alkaloids [28], which

may have phytotoxic activity [2,30,33]. Therefore, the

current study aims to test whether aqueous extracts of

Taxus organs may induce growth inhibition, by

subjecting seeds and individuals of fast-growing test

species to extracts treatments and assessing

morphological and physiological growth parameters.

Materials and Methods

Germination and growth conditions

The effects of yew extracts on germination

and initial growth were assessed using two test species,

frequently used for their fast germination and growth

and sensitivity towards external factors, Raphanus

sativus L. and Cucumis sativus L.

Petri dishes, sterilized, lined with sterile filter

paper were used for placing 16 radish and 10 cucumber

seeds per plate, subsequently wetting the paper with

distilled water for control plates or aqueous extracts

obtained from different yew organs. Plates were treated

initially with 3 ml of solution, then the filter paper was

kept moist when needed. The plates were placed in a

controlled environment growth chamber (Snijders

Scientific), with a 14:10 h photoperiod, 22-26°C

temperature and 70% relative humidity. Three plates

were used for each treatment variant, placed in a

random fashion in the growth chamber. The total

duration of the experiment was 120 hours.

Extract preparation

Aqueous extracts were prepared from arils,

leaves and bark of yew. From each organ, 1 g or 5 g of

tissue was grinded and suspended in 99 or 95 ml of

distilled water, in order to obtain 1% and 5% extracts

from each organ. The mixtures were placed on a heated

water bath and kept 150 minutes at 50° C to facilitate

compound extraction. Subsequently, the mixtures were

filtered and stored in refrigerated conditions before use.

Measurements of germination

For assessing the influence of extracts on the

germination process, four germination-related indices

were determined, namely the germination percentage

(GP), the speed of germination (SG), the energy of

germination (EG) and the vitality index (VI). These

two indices are among the most widely used for

assessing germination performance [5,23] and

calculated as follows.

GP =

EG =

VI =

Morphometric assessments

Effects of external factors on plant growth are

frequently evaluated by measurements of plant organs,

as growth stimulation or inhibition directly are directly

reflected in the length of these organs. Therefore, the

length of the hypocotyl, of the radicle and the mass of

fresh plants were measured after 120 hours from the

beginning of the experiment. A number of 8 radish

plantlets or 5 cucumber plantlets per dish were

measured, or the maximum available number of

plantlets per dish.

Evaluation of chlorophyll content

The content of assimilatory pigments in leaves

was estimated using a portable chlorophyll meter,

CCM-200 plus, Opti-Sciences Inc., UK. The device

measures the chlorophyll content by relating the

amount of light transmitted through leaf tissue to the

amount of pigments. A number of 8 radish plants or 5

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cucumber plants or the maximum available number of

plants per dish were measured.

Chlorophyll fluorescence measurements

The influence of the extracts on the yield of

the photosynthetic apparatus were evaluated by

measuring the PSII quantum yield parameter using a

portable chlorophyll fluorescence meter, FMS2,

HansaTech Ltd., UK. Measurements were done on 5

leaves per dish per test specie, and the Fm’, Fs and

ɸPSII indices were recorded.

Total phenolic contents of extracts

The content of phenolic compounds in Taxus

extracts was evaluated using the spectrophotometric

method described in [10]. Briefly, ethanolic (50%)

extracts were prepared, at a concentration of 5 g fresh

material (leaves, bark and aril) per 100 ml ethanol

solution. An aliquot of 0.1 ml of each extracts was

incubated with 0.1 ml of Folin reagent and 1 ml of

distilled water. After 5 minutes 0.8 ml of 7.5%

Na2CO3 solution was added and incubated for further

90 min. The absorbances of the mixture were red at

760 nm using an UVmini-1240 Shimadzu

spectrophotometer. Results were expressed as gallic

acid equivalent per gram of fresh material, calculating

the means and standard errors of three replications per

each type of extract.

Results and Discussion

The germination process, in terms of germination

percentage, was influenced especially in the case of

radish seeds compared to the cucumber seeds (Fig. 1).

The germination percentage was significantly reduced,

for radish seeds, by most extracts, except for the 5%

bark extract. The most significant inhibition was

observed for the leaves 5% extract, followed by the

bark 1%, aril 1%, aril 5% and leaves 1% extracts. The

same germination index, in the case of cucumber seeds,

was mainly influenced by the aril 5%, leaves 5% and

bark 5% extracts, followed by aril 1%, leaves 1% and

bark 1% extracts, in this order.

Fig. 1. Germination percentage of seeds (A Cucumis sativus, B Raphanus sativus)

Speed of germination was similarly, more affected in

the case of radish seeds, where significant inhibition

was observed for the leaves 5%, aril 1%, bark 1%,

leaves 1% and aril 5% extracts (Fig. 2). The speed of

germination of cucumber seeds, although not

significantly, was reduced by all extracts, more so by

the leaves 5% and aril 5% ones.

Fig. 2. Speed of germination of seeds (A Cucumis sativus, B Raphanus sativus)

The energy of germination was reduced in radish compared to controls, mainly by leaves extracts, followed by aril and

bark extracts (Fig. 3).

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Fig. 3. Energy of germination of seeds (A Cucumis sativus, B Raphanus sativus)

Less pronounced inhibition was observed for this index

in cucumber, however reduction of values were

observed for most extracts. The vitality index was

reduce, for both species, by leaves extracts (Fig. 4).

Fig. 4. Vitality index of seeds (A Cucumis sativus, B Raphanus sativus)

The radicle length at 120 h was reduced in treated

plants compared to control ones (Fig. 5) in both

cucumber and radish. The most significant reduction

was recorded in leaves 5% and 1% and aril 5%

treatments.

Fig. 5. Radicle length of plantlets (A Cucumis sativus, B Raphanus sativus)

The hypocotyl length recorded more heterogeneous

values than radicle length (Fig. 6). Cucumber plantlets

recorded a reduction of this parameter for aril

treatments, while other treatments determined similar

or higher values comparing to controls, suggesting

possible hypocotyl growth stimulation. For radish

plantlets, growth of hypocotyl was significantly

reduced by bark extracts, but no stimulation was

observed.

Fig. 6. Hypocotyl length of plantlets (A Cucumis sativus, B Raphanus sativus)

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A similar trend in fresh mass of plants with hypocotyl

elongation was observed (Fig. 7), as the values of

treated cucumber plantlets were lower than of controls

for aril 1% and 5% and leaves 1% extracts and similar

or higher in other treatments. Radish plantlets recorded

lower fresh mass values compared to controls in all

treatments, especially for aril 5% and leaves 1%

extracts.

Fig. 7. Fresh mass of plantlets (A Cucumis sativus, B Raphanus sativus)

Chlorophyll contents, in cotyledons of cucumber

plantlets, were increased by most of the extracts, with

the exception of aril 5% one. In the same time, radish

cotyledons recorded lowered chlorophyll contents

under all treatments, especially under aril 5% and

leaves 1% extracts (Fig. 8).

Fig. 8. Chlorophyll contents of cotyledons (A Cucumis sativus, B Raphanus sativus)

Chlorophyll fluorescence, however, recorded little

influence from extracts treatments, the only significant

reduction of ϕPSII parameters recorded for cucumber

cotyledons under leaves 1% extracts (Fig. 9).

Fig. 9. Chlorophyll fluorescence ϕPSII index of cotyledons (A Cucumis sativus, B Raphanus sativus)

Total phenolic content of extracts showed higher

amounts in leaves extracts, followed by bark and aril

extracts, with an increase in contents according to

extract concentration (Fig. 10).

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Fig. 10. Total phenolic content of yew extracts

The yew extracts affected both germination

and growth of test species. Generally, the effects were

more visible in the case of Rapahanus sativus seeds,

compared to Cucumis sativus seeds, possibly as a

consequence of different histo-anatomical structures

and chemical composition of the two seed types, as

differences are known to occur between species,

leading to different sensitivity towards exogenous

factors [45,36].

The stronger effects on germination, recorded

in the case of leaves extracts, may be attributed to the

elevated amounts of phenolic compounds in these

organs, compared to other extracts. The effects of

phenolics on germination have been investigated, with

inhibition of the process reported for various species,

including radish and cucumber [42,14,17]. However,

since germination inhibition occurred also in aril

extracts and bark 5% extract, the inhibitory effects may

be owed to other compounds as well, as these extracts

showed small amounts of phenolic compounds. The

composition of Taxus organs is reported to be rich in

alkaloids, reason for which yew is exploited in the

pharmaceutical industry [12,11]. The inhibitory effects

of alkaloids on germination are known [43,1,19],

which may explain the observed effects on radish and

cucumber.

Growth of plants was also influenced by the

treatments, especially concerning radicle elongation,

but also regarding fresh mass. Reduction in radicle

growth by aril and leaves extracts and in hypocotyl

length by bark extracts indicate once more that the

effects are the result of the activity of several classes of

compounds, not phenolics alone. Synergism between

different types of compounds is known to occur in

phytotoxicity, increasing the effects of substances [34].

A less significant influence was observed on

plantlets photosynthetic apparatus, with mixed

response concerning pigment content, stimulatory in

the case of cucumber and inhibitory in the case of

radish, once again underlining the sensitivity of radish

towards exogenous application of compounds.

However, the functioning of the photosystem II

appeared relatively unaffected, considering the

chlorophyll fluorescence values, comparable under

most treatments with those of control plants.

Toxicity of aqueous leachates from various vegetal

organs of both herbaceous [32] and tree species

[46,39,40] are reported, the water soluble nature of

involved compounds indicating the occurrence of such

effects under natural conditions. Both alkaloids

[30,37,19] and phenolics [33,2] may influence

germination and growth of plants, possible

mechanisms of action including altered nutrient uptake,

inhibition of cell division, effects on enzymes and

proteins [17] or DNA and RNA interactions, modified

protein biosynthesis [44,4]. Autotoxicity of these

compounds was observed for several tree species,

including conifers [13,24,35]. In the case of Taxus

baccata, autotoxicity was proposed [38,29] but, as far

as the available literature is concerned, effective testing

was not performed. Furthermore, there are few reports

on the toxicity of Taxus extracts on other species

[9,47], which, however, indicate the allelopathic effects

of yew. The obtained results confirm the previous

findings, and suggest an opportunity for autoxicity tests

of Taxus leachates.

Conclusions

Taking in consideration all parameters

analyzed, the Taxus extracts appear to exert a certain

phytotoxic activity on test species used. Phenolic

compounds and alkaloids may be responsible for

displayed effects, further identification of compounds

and corresponding toxicity testing being required.

Furthermore, toxicity assessment of Taxus extracts

towards Taxus seedlings could be useful for

autotoxicity hypothesis testing.

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