Journal of Ornamental Plants4... · Journal of Ornamental Plants It is approved publication of...

An Efficient In Vitro Propagation, Antioxidant and Antimicrobial Activities of AphyllorchisMontana (Reichenb.f.)............................................................................................................189-204

Ganesan Mahendran

Encapsulation of Protocorm of Cymbidium bicolor Lindl. for Short-Term Storage and

Germplasm Exchange..............................................................................................................205-215

G. Mahendran, N. Parimala Devi and V. Narmatha Bai

Comparison of Different Pot Mixtures Containing Perlite on Growth and Morphological

Characteristics of Pothos (Scindapsus Aureum L.)...............................................................217-226

Fatemeh Bidarnamani and Hossein Zarei

Combined Effect of Humic Acid and NPK on Growth and Flower Development of Tulipa gesnerianain Faisalabad, Pakistan..........................................................................................................227-236

A. Ali, S.U. Rehman, S. Raza and S.U. Allah

The Regulating Effect of the Growth of Indole Butyric Acid and the Time of Stem Cutting

Preparation on Peroliferation of Damask Rose Ornamental Shrub..................................237-243

Mahsa Kashefi, Hossein Zarei and Farzaneh Bahadori

Study on Effects of Ascorbic Acid and Citric Acid on Vase Life of Cut Lisianthus (Eustomagrandiflorum ‘Mariachi Blue’...............................................................................................245-252

Farnaz Sheikh, Seyed Hossein Neamati, Navid Vahdati and Ali Dolatkhahi

Pollen Germinability and Cross-Pollination Success in Persian Cyclamen (Cyclamen persicumMill.).........................................................................................................................................253-261

Mohammad Kermanshahani, Roohangiz Naderi, Reza Fattahi and Ahmad Khalighi

Volume 4, Number 4

December 2014

Journal of Ornamental Plants


It is approved publication of Journal of Ornamental Plants (based on approbation of 61st session

of "Survey and Confirmation Commission for Scientific Journals" at Islamic Azad University dated

on 01/25/2010.

Publisher: Islamic Azad University, Rasht, Iran.

Executive Director: Dr. Ali Mohammadi Torkashvand

Editor-in-Chief: Professor Roohangiz Naderi

Executive Manager: Dr. Shahram Sedaghat Hoor

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Professor Ramin, A., Isfahan University of Technology, Iran

Professor Abdollah Hatamzadeh, University of Guilan, Iran

Professor Honarnejad, R., Islamic Azad University-Varamin Branch, Iran

Associate Professor Shahram Sedaghathoor, Islamic Azad University, Rasht Branch, Iran

Dr. Davood Hashemabadi, Islamic Azad University, Rasht Branch, Iran

Associate Professor Moazzam Hassanpour Asil, University of Guilan, Iran

Assistant Professor Behzad Kaviani, Islamic Azad University, Rasht Branch, Iran

Professor Nagar, P.K., Institute of Himalayan Bio-Resource Technology, India

Professor Salah El Deen, M.M., Al Azhr University, Egypt

Assistant Editor: Zahra Bagheramiri

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Web Site: www. jornamental.com

An Efficient In Vitro Propagation, Antioxidant and Antimicrobial Activities of Aphyllorchis Montana(Reichenb.f.)...............................................................................................................................189-204Ganesan Mahendran

Encapsulation of Protocorm of Cymbidium bicolor Lindl. for Short-Term Storage and Germplasm

Exchange....................................................................................................................................205-215 G. Mahendran, N. Parimala Devi and V. Narmatha Bai

Comparison of Different Pot Mixtures Containing Perlite on Growth and Morphological Characteristics

of Pothos (Scindapsus Aureum L.).............................................................................................217-226Fatemeh Bidarnamani and Hossein Zarei

Combined Effect of Humic Acid and NPK on Growth and Flower Development of Tulipa gesneriana in

Faisalabad, Pakistan....................................................................................................................227-236A. Ali, S.U. Rehman, S. Raza and S.U. Allah

The Regulating Effect of the Growth of Indole Butyric Acid and the Time of Stem Cutting Preparation

on Peroliferation of Damask Rose Ornamental Shrub...............................................................237-243Mahsa Kashefi, Hossein Zarei and Farzaneh Bahadori

Study on Effects of Ascorbic Acid and Citric Acid on Vase Life of Cut Lisianthus (Eustoma grandiflorum‘Mariachi Blue’...........................................................................................................................245-252 Farnaz Sheikh, Seyed Hossein Neamati, Navid Vahdati and Ali Dolatkhahi

Pollen Germinability and Cross-Pollination Success in Persian Cyclamen (Cyclamen persicumMill.).............................................................................................................................................253-261Mohammad Kermanshahani, Roohangiz Naderi, Reza Fattahi and Ahmad Khalighi

Content Page

www.jornamental.com

189Journal of Ornamental Plants, Volume 4, Number 4: 189-204, December, 2014

An Efficient In Vitro Propagation, Antioxidant and Antimicrobial

Activities of Aphyllorchis montana (Reichenb. F.)

Keywords: Antimicrobial activity, Antioxidant activity, Aphyllorchis montana, Asymbiotic seed ger-

mination, In vitro propagation, Plant growth regulators, 2,2-Diphenyl-1- picrylhydrazyl.

Abbreviations: IAA (Indol-3-acetic acid), BM-TM (BM-1Terrestrial orchid medium), IBA (Indole-

3-butyric acid), NAA (α- Naphthaleneacetic acid), TDZ (Thidiazuron), BA (6-Benzyl adenine), KIN

(6-Furfurylaminopurine), 2-iP (2-Isopentenyladenine), GA3 (Gibberellic acid), Zt (Zeatin), DPPH (2,

2-diphenyl-1-picrylhydrazyl), ABTS (2, 2’azinobis (3-ethylbenzothiozoline-6-sulfonic acid) diammonium

salt), TPTZ (2, 4, 6-tripyridyl-S-triazine), EDTA (Ethylenediamine tetraacetic acid).

Ganesan Mahendran

Research scholar, Department of Botany, Bharathiar University, Coimbatore- 641046, India.

*Corresponding author,s email: [email protected]

Abstract

An in vitro plant regeneration protocol was successfully established inAphyllorchis montana, a saprophytic achlorophyllous orchid by culturingimmature seeds. Among the six basal media evaluated for seed germination,BM-TM medium was found to be the best followed by KC medium. After 40days, all the media turned brown and the growths of the protocorms werearrested. Activated charcoal, 1 g/l in half strength BM-TM was found to besuitable for further development of protocorms. Half strength BM-TM mediumwas supplemented with different growth regulators either individually or incombinations for multiplication of shoots. Of the five cytokinins tested, TDZ at6.8 μM was found to be most effective for multiple shoot induction yielding17.24 ± 0.27 shoots after 10 weeks of culture. Addition of low concentration ofNAA (1.3 μM) in MS medium supplemented with the cytokinin TDZ (6.8 μM)favoured shoot multiplication. A mean number of 27.56 ± 0.54 shoots with3.92 ± 0.11 number of roots were produced per explant. The response of theseed derived protocorm to the different types of organic additives viz., peptoneand yeast extract and coconut water was also evaluated. The addition of theseorganic additives to the medium containing TDZ enhanced the number ofshoot regeneration. The plantlets were acclimatized in plastic pots containingsterilized vermiculite. The survival rate was 100 % when maintained in theculture room condition (25 ± 2 °C). Screening of the antibacterial, antioxidantactivity and estimation of total phenolics and flavonoid content of methanolicextracts of micropropagated plants were also carried out and compared withthat of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolicsand flavonoid content than in vitro propagated plants.

Journal of Ornamental Plants, Volume 4, Number 4: 189-204, December, 2014190

INTRODUCTIONOrchids belong to the most diverse plant family known to man. They have complex lifecy-

cle, mycorrhizal association (seed germination) and specific pollination syndrome. It is a family

of considerable economic importance particularly in horticulture and floristry. Apart from the hor-

ticultural value, orchids are used in traditional herbal medicine. Many orchid species are threatened

globally by over collection from the natural habitat for horticultural purpose. Plant tissue culture

and micropropagation techniquesplay an important role in conservation programs and management

of botanical collection.

Aphyllorchis montana (family Orchidaceae) is native to the region and rare in natural habitat

(Sinu et al., 2012) has drawn much attention in recent years. The extract of it has been in use since

olden times to cure cough, cold, anemia and for its vitality strengthening properties (Prajapati etal., 2003). A. montanais used in traditional Indian systems for diabetic activity (Bhavani et al.,2012; Sreenu et al., 2013). The species is also one of the important components of ‘Astvarga’ and

used in the preparation of ‘Chyavanprash’, a highly popular Ayurvedic tonic. The medicinal prop-

erties are possibly due to production of secondary metabolites, including phenolic compounds.

The role of antioxidants due to the phenolic compounds in this species cannot be ignored. To fulfill

the high demand of pharmaceutical industries, at present raw material is largely being drawn from

the wild; this has severely affected its availability in natural habitat and the species has been con-

sidered rare in the Western Ghats (Sinu et al., 2012). In spite of this, there has been hardly any

effort to commercially cultivate this species.

The vegetative growth of the terrestrial mycohetrrotrophic orchid is absolutely an under-

ground mechanism; it challenges to locate them in vegetative condition, species-specific fungi for

seed germination, poor seed viability and low rate of germination in natural condition and poor

rooting ability of vegetative cuttings of rhizomes. In spite of this, there has been hardly any effort

to commercially cultivate this species. Inevitably, therefore, rapid multiplication of this important

drug yielding genotype is imperative. Alternatively, in vitro micropropagation would be beneficial

in accelerating large scale multiplication and conservation of this important plant species. In vitroseed germination has been suggested as a suitable propagation method for conservation of orchids

(Kauth et al., 2006; Stewart and Kane, 2006).Therefore, development of rapid protocol for high

frequency in vitro plant regeneration in this important medicinal herb became necessary in order

to reduce the existing pressure on natural populations and continuous supply of plant materials for

the pharmaceutical industry. As tissue culture technique has now become a well–established

method for large scale plants developed for commercial utilization of several endangered medicinal

plants. The major goal of the present investigation was to standardize the best media for seed ger-

mination, growth regulators combinations for high frequency plantlet production from protocorm

explants of A. montana species. In order to enhance the shoot bud multiplication rate, various

growth regulators were examined to identify the best growth hormone combinations for maximum

number of shoot buds production and to evaluate the antioxidant, antimicrobial activity of both

wild-grown and in vitro regenerated plants.

MATERIALS AND METHODSPlant material, explant preparation and surface sterilization

Immature seeds of Aphyllorchis Montana (Reichenb.f.) were collected from Vellingiri Hills

(longitude 60-40' and 70-10' E and latitude 10o-55 and 11o-10' N 1200) at an attitude of 1000-

1250 msl. Tamilnadu, India. Freshly collected green capsules were washed thoroughly under run-

ning tap water. The pods were then immersed in Teepol3 - 5%(v/v) for 2 - 5 minutes by continuous

shaking and then rinsed with sterile distilled water thrice and the pod were pretreated with 0.1%

(W/V) of Bavistin, a fungicide for 5 minutes and rinsed in double distilled water. Then the explants

were surface sterilized in 0.01% mercuric chloride solution for 5 minutes and rinsed thoroughly

with sterile distilled water (5-7 times). The capsules were dipped in 70% ethanol for 30 seconds

Journal of Ornamental Plants, Volume 4, Number 4: 189-204, December, 2014 191

and flamed. The surface sterilized pods were cut opened with sterile blade and seeds were extracted

using sterileforceps and spread as thin film in test tubes containing 20 ml of culture media. Cultures

were maintained at 25 ± 2 ºC under cool white fluorescent tubes at a light intensity of 50 µmol m-2 s-1

with 16/8-h L/D photoperiod.

Seed viability testSeed viability was tested according to Vellupillai et al. (1997). For enumerating seed via-

bility percentage, seeds from the fresh capsules were treated with 1% (w/v) 2, 3, 5-triphenyl tetra-

zolium chloride (pH 7.0) in the dark overnight. Treated seeds were observed with a light

microscope and scored as either viable (red embryo) or nonviable (white embryo).

Optimization of culture medium for asymbiotic seed cultureImmature seeds of A. Montana were inoculated on Knudson C modified Morel (Morel1965;

KCM), Lindemann (Lindemann et al., 1970; LM), Mitra medium (Mitra et al., 1976; M), Knudson

C medium (Knudson, 1946; KC), Murashige and Skoog medium (Murashige and Skoog, 1962, MS)

and BM-1-Terrestrial Orchid media (Van Waes and Debergh,1986 procured from Hi-Media Labora-

tories Mumbai, India) initially to find out the suitable medium for maximum seed germination. The

best medium for seed germination was selected for further studies. All media contained 2% sucrose

and were solidified with 0.8% agar (Hi Media Laboratories, India). Activated charcoal (Hi-Media

Laboratories Mumbai, India) was added at 0.5, 1.0 or 2.0 g/l to half strength basal BM-TM medium

to arrest phenolic exudation and further development of the protocorms. The pH of the media was

adjusted to 5.6–5.8 with 1 N NaOH or HCl before autoclaving at 121ºC, 105 kPa for 20 min.

Multiplication of protocormsFor the multiplication of protocorms, BM-TM medium was supplemented with cytokinins

such as BA (1.10 to 8.80 µM), Kn (1.15 to 9.20 µM), 2-iP (1.01 to 8.12), Zt (1.1 to 9.1 µM) and

TDZ (1.1 to 9.0 µM) either individually or in combination with NAA (1.3 µM). All media con-

tained 20 gl-1 sucrose and were solidified with 0.8% agar (Hi Media Laboratories, India). The cul-

tures were maintained at 25 ± 2ºC temperature with 75–80% relative humidity and a 16/8 h

(light/dark) photoperiod provided with diffuse light (50 µmol m-2 s-1). Final observation on the

number of multiple shoot and the shoot length, root number and root length were recorded after

70 days of culture.

Ex vitro plant establishmentFor ex vitro establishment, well-developed plantlets were rinsed thoroughly with tap water

to remove residual nutrients and agar from the plant body and transplanted to plastic pot containing

vermiculite. The plastic pots were covered initially with polyethylene bags and maintained for two

months inside the culture room for acclimatization under cool white tubular fluorescent lights (40

W, 220 V, Philips Electronics India Ltd.) at 50 µmol−l m−2 s−1 with a 16 h photoperiod at 25±2°C.

The transplants were transferred to National Orchidarium, Yearcaud, Tamil Nadu.

Preparation of methanol extractsThe biomass from in vitro propagated plants (8 week old culture, BM-TM medium supple-

mented with TDZ (6.8μM) and NAA (1.3μM) shoots were collected) and wild grown plants (whole

plants) were washed under tap water and dried in oven at 60ºC for two days. The material was

powdered by using electric blender and stored in clean labelled airtight bottles. The powder (100g)

was extracted by maceration in 300 ml of methanol (100%) for 3 days with frequent agitation. The

mixture was filtered through Whatman No. 1 filter paper and the filtrate was concentrated and

dried in petridishes at 60ºC in the oven.


Estimation of total phenolics (TPC) and flavonoids content (TFC)The total phenolics content of the extracts was determined and calculated as gallic acid

equivalent (GAE) in mg/g DW from the calibration curve according to method described by Sid-

dhuraju and Becker (2003). The total flavonoids content of sample extract was determined fol-

lowing a colorimetric method and values were expressed as mg/g rutin equivalent (RE) of extract

according to the method described by Zhishen et al.(1999).

In vitro antioxidant activityThe free radical scavenging activity of the A. Montana methanol extracts of wild-grown

plants and in vitro propagated plants were evaluated by using DPPH• (Blois, 1958), ABTS•+ cation

radical (Re et al., 1999) and ferric reducing antioxidant power (FRAP) activity (Pulido et al., 2000)

were measured using standard methods.

Antimicrobial activity Test bacteria

The antibacterial activity of isolated compounds 1-6 were evaluated against 6 pathogenic bac-

teria such as Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphylo-coccus aureus (ATCC 29213), Streptococcus pnumoniae (ATCC 33400) Klebsiella pnumoniae(ATCC 10031) and Bacillus subtilis (ATCC 6633) procured from the Institute of Microbial Technol-

ogy (IMTECH) Chandigarh, India. All the strains were stored in the appropriate medium before use.

Disc diffusion methodDisc diffusion method (Joshi et al., 2010) was used for the evaluation of antibacterial ac-

tivity of A. Montana methanol extracts of wild-grown plants and in vitro propagated plantsusing

100 μl of suspension containing 108 CFU/ml of bacteria spread on the inoculated agar. A sterile

cotton swab was dipped into the inoculums suspension to remove the excess of fluid. Whatmann

filter paper discs (6 mm diameter) were prepared at the concentration of 25 μg/disc for wild-grown

plants and in vitro propagated plants extracts and 10 μg/disc reference antibiotic (Ciprofloxacin).

A disc prepared with only the corresponding volume of DMSO was used as negative control. The

petriplates were then incubated and antimicrobial activity was evaluated by measuring the diameter

of the zones of inhibition around the disc. The experiments were repeated in triplicate and the

result was expressed as average value.

Minimum inhibitory concentrationThe minimum inhibitory concentration (MIC) of wild-grown plants and in -vitro propa-

gated plants of A. montanawas determined using the micro-dilution assay in 96-well micro-

plates (Siddiqi et al., 2011). Briefly, 500 µl of each re-suspended sample (1.0 mg/ml) in DMSO

(2%). Serial two-fold dilutions were prepared from the stock solution to give concentrations

ranging from 500μg to 3.90 μg/ml of the wild-grown plants and in vitro propagated plants ex-

tracts. The highest concentration of DMSO remaining after dilution (5%, v/v) caused no inhibi-

tion to bacterial growth. DMSO served as negative control. Streptomycin and Ciprofloxacin

were served as positive controls. An aliquot of 100 μl standardized suspension of the test bacteria

(108 CFU/ ml) was transferred to a well of 96 plates. Then, 100 μl diluted samples were also

added to each well and incubated at 37°C for 24h. The MIC was defined as the lowest concen-

tration of samples which inhibited the visible growth of tested microorganisms. For further re-

confirmation, 20 μl of MTT reagent (1mg/ml) was added as an indicator for microbial growth

to each well of the microtitre plates, followed by 20 min incubation at 37°C. The reagent-sus-

pension colour will remain clear or yellowish indicating complete inhibition activity as opposed

to dark blue for growth (Eloff, 1998). The MIC was recorded as the most repeatable minimum

concentration of triplicate.


Experimental design and statistical analysis The percent of seed germination was recorded

after 10, 20 and 30 days of culture. Percent germination

was calculated by dividing the number of germinated

seeds by the total number of seeds inoculated. Num-

ber of multiple shoots, height, root number and root

length was recorded after 70 days of culture. The exper-

iments were repeated thrice and each set had five repli-

cates. The significance of differences among means was

assessed out using Duncan’s multiple range test at P <

0.05 (ANOVA). Data of the antioxidant, total

phenolic and flavonoid assays were expressed as

the mean ± standard deviation (SD) of three in-

dependent measurements. Correlation analysis

was performed between phenolics and

flavonoids with antioxidant activity using Pear-

son correlation two-tailed. The results were an-

alyzed statistically using SPSS Version 17 (SPSS

Inc., Chicago, USA).

RESULTSIn vitro seed germination

Tetrazolium (TZ) viability test indi-

cated a mean embryo viability of 90%. Seeds

germinated on all the media tested, however,

the percentage of seed germination and proto-

corm developmentwas not consistent and var-

ied in different media. The embryos enlarged

and occupied the entire seed coat within 10

days after sowing. Germination as evidenced

by enlargement of the embryo was first ob-

served in BM-TM medium, followed by both

KC and KCM medium. The seed germination

was low in MS, Mitra and Lindmann orchid

medium (Fig. 1).

After 30 days, the embryos by repeated

cell divisions emerged and rupturing the testa in

BM-TM medium (Fig. 2a). The percentage seed

germination was 79% in BM-TM basal medium

(Fig. 2a), 52% in KC medium and 42.50 % in

KCM medium. After 40 days, all the media turned

brown possibly indicating phenolic exudation and

further development of protocorms were arrested

(Fig. 2b).The above medium was reduced half

strength and supplemented with activated char-

coal at various concentrations (0.5 to 3 g/l) to pre-

Fig.1. Effect of various culture media on seedgermination of A. montana

Error bars indicates ± S.E

Fig.2. Asymbiotic seed germination and multipleshoot development of A. montana (Bar-1cm)

a. Seed derived protocorms developed on BM-TMmedium after 30 days of cultureb. Phenolic exudation on BM-TM medium c. Development of protocorms on half strength BM-TMmedium with activated charcoal 1 g/l d. Development of multiple protocorm on half strengthBM-TM medium supplemented with TDZ at 9.8 μMe. Multiple protocorm development on half strengthBM-TM medium fortified with TDZ (6.8μM) and 20%CW f. Regenerated plantlet g. Hardened plantleth. Hardened plantlet after 2 months under ex vitro condition


vent the phenolic exudation and induce further development of protocorms (Fig. 2c). Among the dif-

ferent concentrations of activated charcoal tested, 1g/l completely arrested phenolic exudation and it

was found to bethe optimum concentration for the development of protocorms.

Effect of cytokininson development of multiple shootsThe seed derived protocorm explants cultured on half strength BM-TM medium without

any growth regulators formed a single shoot. In the presence of cytokinin, the explants responded

positively in producing multiple shoots (multiple seedlings). Of the five cyokinins tested, TDZ

was found to be more efficient than other cytokinins with respect to initiation and subsequent pro-

liferation of shoots (Table 1). Among the different levels TDZ tested, the maximum number of

shoots was observed on the half strength BM-TM medium containing 6.8 μM of TDZ (17.24 ±

0.27) (Table 1, Fig. 2d).The shoot buds first appeared as small white protuberances at basal surface

at the protocorm, which eventually developed into multiple shoots within 30-35 days.The number

of shoot buds increased with increasing concentration of TDZ up to an optimal level of 6.8 μM.

Among the various concentrations of Zt and 2-ip tested, maximum number of the multiple shoots

was recorded in half strength BM-TM medium supplemented with 9.1 μM Z and 8.12 μM 2-iP

(12.65 shoots/explant). NAA (1.3 μM) in combination with cytokinins also favored shoot forma-

tion. However, the response for multiple shoot induction varied (Table 2). Among the different

combinations, maximum number of shoots were produced in half strength BM-TM medium sup-

plemented with TDZ (6.8 μM) and NAA (1.3 µM).

Effect of TDZ and growthadjuvants on multiple shoot inductionMultiple shoots were also formed when seed derived protocorms were cultured on BM-

TM medium supplemented with TDZ at 6.8 μM in combination with growth adjuvants like pep-

BA (µM/l) Kn (µM/l) Z (µM/l) TDZ (µM/l) 2ip (µM/l) No. of

hoots/explant

Shoot

length (cm)

No. of

Root/explant

Length of

root (cm)

1.102.203.304.406.608.80

------------------------

------

1.152.323.454.646.909.20

------------------

------------

1.12.23.34.56.89.1------------

------------------

1.12.23.34.56.89.0------

------------------------

1.012.033.044.066.098.12

1.20±0.71 g

2.56±0.54 f

5.12±0.33 d

9.51±0.49 c

8.51±0.12 c

5.76±0.21 d

1.10±0.73 g

1.32±0.26 g

2.65±0.12 f

3.97±0.75 de

5.14±0.65 d

7.50±0.30 cd

4.60±0.16 de

1.25±0.50 g

2.53±0.11 f

5.54±0.64 d

7.20±0.81 cd

12.65±0.16 b

2.30±0.83 f

3.65±0.76 e

5.65±0.76 d

8.97±0.75 c

17.24±0.27 a

12.30±0.65 b

2.15±0.58 f

2.87±0.98 f

3.87±0.70 e

5.82±0.23 d

9.70±0.65 c

12.65±0.45 b

2.60±0.66 bc

2.00±0.12 d

2.41±0.34 c

2.01±0.52 d

1.16±0.22 f

1.00±0.40 f

1.00±0.56 f

2.11±0.77 d

2.19±0.14 d

2.52±0.40 bc

2.82±0.49 b

2.42±0.43 c

2.63±0.87 bc

2.61±0.54 bc

3.00±0.20 a

2.81±0.28 b

3.00±0.79 a

2.84±0.65 b

3.16±0.66 a

3.00±0.79 a

2.71±0.54 b

2.01±0.02 d

1.46±0.02 e

1.00±0.40 f

2.11±0.57 d

2.41±0.54 c

3.00±0.69 a

3.00±0.10 a

2.42±0.40 c

2.84±0.92 b

3.76±0.23 b

3.25±0.45 c

2.30±0.76 de

2.38±0.25 de

2.13±0.671.84±0.12 f

1.90±0.71 f

2.18±0.29 e

2.64±0.21 d

1.89±0.78 f

3.65±0.62 bc

3.12±0.62 cd

1.34±0.52 g

2.00±0.70 e

3.12±0.41 cd

3.89±0.63 b

3.60±0.29 bc

2.72±0.56 d

2.29±0.12 de

2.69±0.34 d

2.93±0.18 de

2.31±0.51 de

2.12±0.11 e

4.94±0.22 a

2.12±0.34 e

3.81±0.70 b

3.61±0.07 bc

3.53±0.75 bc

3.50±0.65 bc

3.12±0.29 c

1.53±0.22 d

1.09±0.46 de

1.22±0.34 de

2.19±0.63 c

2.72±0.62 b

3.30±0.20 a

3.09±0.90 a

3.12±0.17 a

2.16±0.24 c

2.06±0.87 c

1.72±0.89 d

1.16±0.51 de

1.71±0.41 d

1.54±0.26 d

2.73±0.31 b

2.39±0.32 bc

3.66±0.12 a

2.53±0.34 bc

1.75±0.34 d

2.00±0.00 c

2.19±0.11 c

2.30±0.13 bc

2.70±0.71 b

2.00±0.92 c

1.09±0.92 de

1.48±0.44 d

2.00±0.23 c

2.50±0.32 bc

2.43±0.43 c

2.87±0.69 b

Values represent mean ± S.E. of twice repeated experiments each with 5 replications. Means in a column with thedifferent letter (superscript) are significantly different according to DMRT (P < 0.05).

Table 1. Effect of cytokinins on multiple shoot induction from seed derived protocorm of A. montana.


tone, yeast extract and coconut water (0.5 g- 3 g/l or 5-20%). The mean number of multiple shoots

varied among the treatments (Table 3). A significantly higher number of multiple shoots (28.33 ±

0.27)were obtained on medium supplemented with TDZ (6.8 μM) and 20% CW (Fig. 2e). Other

organic additives were also effective in inducing multiple shoots, however their number remained

low when compared to CW (20%). The mean length of the shoot per explant was in the range of

0.46 ± 0.14 - 1.76 ± 0.23 cm which is lower when compared to the medium supplemented with

TDZ (6.8 μM) individually (Table 1).

BA

(µM)

Kn

(µM)

Z

(µM)

TDZ

(µM)

2-ip

(µM)

NAA

(µM)

No. of shoot

/explant

Shoot length

(cm)

No. of Root/ex-

plant

Root length

(cm)

1.102.203.304.406.608.80

------------------------

------

1.152.323.454.646.909.20

------------------

------------

1.12.23.34.56.89.1------------

------------------

1.12.23.34.56.89.0------

------------------------

1.012.033.044.066.098.12

1.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.31.3

1.48±0.61 h

3.76±0.54 fg

7.62±0.53 e

12.47±0.34 d

10.51±0.54 d

6.76±0.63 e

1.76±0.21 h

2.11±0.46 g

3.54±0.14 fg

4.32±0.80 f

5.64±0.55 f

7.50±0.37 e

1.60±0.56 h

2.62±0.84 g

2.70±0.65 g

4.47±0.07 f

5.32±0.66 f

7.33±0.87 e

4.81±0.33 f

7.12±0.87 e

12.57±0.61 d

19.78±0.81 bc

27.56±0.54 a

22.65±0.11 b

2.98±0.12 g

1.41±0.17 h

3.25±0.24 fg

5.98±0.35 f

10.70±0.89 d

16.43±0.54 c

2.06±0.12 bc

2.34±0.87 b

2.65±0.66 b

2.70±0.33 b

3.11±0.44 b

1.00±0.31 c

1.22±0.31 c

2.23±0.54 bc

2.43±0.54 b

2.65±0.10 b

3.13±0.23 a

3.61±0.55 a

2.97±0.16 ab

3.20±0.19 a

3.61±0.89 a

3.44±0.29 a

3.87±0.64 a

3.98±0.16 a

3.16±0.62 a

3.45±0.69 a

2.67±0.21 b

2.35±0.98 b

1.56±0.33 c

1.34±0.54 c

2.43±0.67 b

2.78±0.88 b

3.22±0.23 a

3.54±0.82 a

2.17±0.10 bc

1.65±0.92 c

4.56±0.11 bc

5.12±0.34 bc

6.74±0.54 b

10.12±0.61 a

7.09±0.12 b

4.17±0.44 bc

1.30±0.41 e

2.32±0.18 d

3.11±0.65 c

4.50±0.23 bc

4.21±0.11 bc

4.82±0.62 bc

1.02±0.11 e

2.34±0.45 d

2.23±0.41 d

3.89±0.63 c

3.60±0.29 c

2.72±0.56 d

2.29±0.12 d

2.69±0.34 d

2.93±0.18 cd

3.31±0.51 c

3.92±0.11 bc

4.23±0.32 bc

2.12±0.34 d

3.78±0.32 c

3.24±0.34 c

3.76±0.87 c

3.87±0.76 c

3.15±0.33 c

1.00±0.11 d

1.22±0.54 d

1.76±0.76 d

1.00±0.12 d

1.22±0.62 d

2.00±0.43 c

1.56±0.23 d

1.12±0.21 d

2.16±0.32 c

2.06±0.43 c

1.10±0.54 d

1.21±0.11 d

1.65±0.20 d

1.23±0.43 d

2.73±0.31 b

2.39±0.32 c

3.66±0.12 a

2.53±0.34 b

1.75±0.34 d

2.00±0.00 c

2.19±0.11 c

2.30±0.13 c

2.70±0.71 b

3.41±0.12 a

1.32±0.14 d

1.98±0.35 cd

2.23±0.34 c

2.17±0.41 c

2.21±0.91 c

2.11±0.43 c

Values represent mean ± S.E. of twice repeated experiments each with 5 replications. Means in a column with thedifferent letter (superscript) are significantly different according to DMRT (P < 0.05).

Table 2. Effect of cytokinins and auxin on multiple shoot developed from seed derived protocorm of A. montana.

TDZ

(µM)

Peptone

(g/l)

Coconut

water (%)

Yeast extract

(g/l)

No. of

shootsShoot length (cm) Root number Root length (cm)

6.86.86.8 6.86.86.86.86.86.8 6.86.86.86.8

Control 17.24±0.27 e

18.66±0.11 e

20.21±0.26 de

23.82±0.32 c

20.89±0.44 de

21.71±0.54 d

23.61±0.11 c

27.24±0.76 b

28.33±0.27 a

17.89±0.61 e

14.32±0.79 f

12.18±0.43 g

9.29±0.99 h

1.46±0.02 c

1.11±0.31 e

1.16±0.82 e

0.97±0.12 f

0.46±0.14 g

1.76±0.23 b

1.49±0.62 c

1.17±0.39 e

1.98±0.42 a

1.21±0.26 d

1.11±0.13 e

1.00±0.43 f

1.00±0.54 f

2.12±0.11 d

2.19±0.32 d

1.98±0.18 e

1.50±0.12 f

1.00±0.43 g

2.12±0.22 d

3.92±0.31 c

5.54±0.52 a

7.21±0.29 a

1.00±0.32 g

1.00±0.11 g

0.91±0.11 g

0.00±0.00 h

2.70±0.71 a

1.92±0.22 b

1.70±0.43 b

1.40±0.65 c

0.98±0.36 e

1.10±0.12 d

1.87±0.11 b

1.72±0.18 b

1.45±0.13 c

0.50±0.17 f

0.40±0.11 f

0.10±0.03 g

0.00±0.00 h

0.51.02.03.0--------

----5

101520----

----

0.51.02.03.0

Values represent mean ± S.E. of twice repeated experiments each with 5 replications. Means in a column with the different letter(superscript) are significantly different according to DMRT (P < 0.05).

Table 3. Effect of growth adjuvants on multiple shoot induction from seed derived protocorm of A. montana.


Ex vitro establishment of plantletsThe plantlets (Fig. 2f) were transferred to the potting medium containing vermiculite and

covered with polythene bag. After 2 months, the cover was gradually loosened, thus dropping the

humidity (65–70%). This procedure subsequently resulted in in vitro hardening of the plants. The

survival rate was 100% when maintained in culture room condition (25±2 °C). New shoot and

root were formed after two months of in vitro hardening (Fig. 2g & h).The hardened plantlets are

maintained in the National Orchidarium, Tamil Nadu, India with 80% field establishment rate.

Total phenolics and flavonoid content of the samplesThe results of the phenolics and flavonoid content of both methanol extract of the wild

plant and in vitro regenerated plants are shown in Table 4. The results revealed that the phenolics

content of the wild grown plant of A. montana (121.34 ±1.23 mg/1g) was significantly (P < 0.05)

higher than that of the in vitro regenerated plant of A. montana (61.89 ±3.19 mg/1g). Also, the

flavonoid content of the methanol extracts as revealed in Table 4 indicated that wild grown plant

A. montana (93.62 ± 1.17 mg/1g) had significantly (p< 0.05) higher flavonoid content than invitro regenerated plants (34.59 ±1.14 mg/1g).

Antioxidant activityIn the DPPH assay, the wild-grown plants of A. montanashowed higher scavenging activity

(IC50 = 26.87mg/ml) compared to the positive standard (BHT) (IC50 = 34.59mg/ml) and in vitropropagated plant (IC50 = 41.45mg/ml) (Table 4). To determine the relationship between the levels

of the total phenolics and the antioxidant capacity of the extracts, correlation and regeneration

analysis was performed. Total phenolic content of wild grown plant methanolic extract correlated

with radical scavenging activity against DPPH (r2 = -0.987, P˂0.01) and flavonoid (r2 = -0.971,

P˂0.01) are presented in Table 5 whereas, in in vitro regenerated plant methanolic extract, the sig-

nificant correlation was not observed.

Sample Total pheno-

lics content

(TPC) (mg/ga)

Total flavonoids

content (TFC)

(mg/gb)

DPPH

IC50

(µg/ml)

FRAP (mmol

Fe(II)/g

extractc)

ABTS

(μmoltrolox/g

extractd)

Wild grown plant extractIn vitro propagated plantsBHA

121.34 ±1.2361.89 ±3.19

93.62 ±1.1734.59 ±1.14

26.87 a

41.45 c

34.59 b

2987.10±19.33 a

1034.23±67.44 c

1213.89±80.86 b

38912.19± 456.12 a

20246.53± 291.24 c

21682.08± 359.47 b

Values are mean of three replicate determinations standard deviation. Mean values followed by different superscript in a col-umn are significantly different (P< 0.05). a gallic acid equivalents (GAE)b rutin equivalent (RE) c mmol of ferrous equivalents / g extract; d µmol of trolox equivalents/ g extract; BHA- Butylated hydroxyl anisole

Table 4. Comparison of total phenol, flavonoids content and antioxidant activity of in vitro propagated plantand wild grown plant of A. montana

Parameters

Phenolics Flavonoids

Wild grown

plant extract

In vitro propa-

gated plants

Wild grown

plant extract

In vitro propagated

plants

DPPHABTSFRAP

-0.987**0.980**0.992**

0.4780.898-0.672

-0.971**0.973**0.966*

0.6690.732

0.980**

Table 5. Correlation between phenolics, flavonoids and different antioxidant parameters of in vitro regen-erated and wild grown plant methanol extract of A. Montana.


The Trolox equivalent antioxidant capacity (TEAC) was measured using the improved

ABTSradical cation decolorization assay. The decolorization of ABTS•+ cation radical is an un-

ambiguous way to measure the total equivalent antioxidant capacity of test compounds or plant

samples. Since, TEAC is a measurement of the effective antioxidant activity of the extract. A higher

TEAC value would imply greater antioxidant activity of the sample. Similarly to DPPH assay, the

wild grown plant showed the highest amount of ABTS•+ radical quenching ability and then BHA

and in-vitro plant material (Table 4). From the correlation analysis, it is conceived that phenolics

(r2 = 0.980, P˂0.01) and flavonoids (0.973, P˂0.01) of A. montana wild grown plantmethanol ex-

tract wasthe main contributors for their reducing activity (Table 5).

Antioxidative activity has been proposed to be related to reducing power. Therefore, the

antioxidant potential of A. montana wild grown plant and in vitro propagated methanolic extract

was estimated for their ability to reduce TPTZ–Fe (III) complex to TPTZ–Fe (II) (Table 4). The

FRAP activities of wild-grown plant of A. montana (2987.10 ± 19.33 mmol Fe (II)/g extract) and

in vitro propagated plant (1034.23 ± 67.44 mmol Fe (II)/g extract) are presented in Table 4. Similar

to DPPH and ABTS radical scavenging activity, the reducing power of wild-grown plant was sig-

nificantly higher than in vitro samples. From the correlation analysis, it is conceived that phenolics

(r2 =0.992, P<0.01) and flavonoids (r2 =0.966, P<0.05) of wild grown plant methanolic extract of

A. montana were the main contributors for their reducing activity (Table 5).

Antimicrobial activityAntibacterial activity was compared between in vitro propagated and wild-grown plants.

The results of antibacterial assay are presented in Table 6. The result from the disc diffusion method

measured in inhibition zone (IZ in mm) of in vitro propagated and wild-grown plants methanol

extracts against bacterial strains ranged from 4.54 ± 0.31 to 22.67 ± 0.11mm (Table 6). Methanol

extract of wild-grown plants of S. corymbosa showed high antimicrobial activity against S. aureus,B. subtilis and K. pnumoniae at 25 μg/ml. The antimicrobial activity of in vitro propagated and

wild-grown plants methanol extracts of A. montana was quantitatively assessed by MIC against

the six bacterial strains at various concentration range from 500 to 3.90 μg/ml. The wild-grown

plants methanol extract exhibited potent growth inhibitory activity against K. pnumoniae, B. subtilisand S. aureus with MIC value of 7.81 and 15.62 μg/ml respectively (Table 6).

Micro-organ

Inhibition zone diameters of the test

compounds (mm)

Minimum inhibitory concentration (MIC,

μg/ml)

Wild grown

plant extract

25μg/disc

In vitropropagated

plants

25μg/disc

Ciprofloxacin

10 μg/disc

Wild

grown

plant

extract

In vitropropa-

gated

plants

Ciprofl

oxacin

Streptomycin

E. coli (ATCC25922)P. aeruginosa(ATCC 27853)S. aureus(ATCC 29213)S. pnumoniae( ATCC 33400)K. pnumoniae(ATCC 10031)B. subtilis(ATCC 6633)

8.11±0.91

7.11±0.34

11.87±0.13

6.55±0.62

10.21±0.91

10.17±0.23

4.61±0.23

5.54±0.21

6.42±0.98

4.54±0.31

8.12±0.67

9.97±0.11

21.77±0.11

20.47±0.17

20.77±0.11

21.07±0.05

19.17±0.11

22.67±0.11

31.25

62.5

15.62

62.5

7.81

15.62

125

125

31.25

125

15.62

31.25

3.90

1.90

7.81

3.90

1.90

3.90

1.90

3.90

1.90

3.90

3.90

7.81

Table 6. Evaluation of antimicrobial activity of methanolic extract of A. montana by agar disc diffusionmethod and microdilution method.


DISCUSSIONTetrazolium staining indicated that A. montana seed viability was higher than that of observed

during germination experiments. However, many studies on the Orchidaceae indicate that the viability

testing often is not a good indicator of germinability (Johnson et al., 2007; Mahendran and Narmatha

Bai, 2009; Mahendran et al., 2013). Because of this, the viability estimates should be confirmed by

germination tests. Mass propagation of orchids through asymbiotic seed germination is a tool for

conservation of the declining orchid propagation in nature (Kauth et al., 2006; Stewart and Kane,

2006; Mahendran and Narmatha Bai, 2009; Roy et al., 2011; Mahendran et al., 2013). The success

of asymbiotic seed germination is dependent upon identification of suitable medium and abiotic con-

ditions. The nutritional requirements of most orchids vary due to their enormous diversity and com-

plex mycorrhizal interactions (Arditti et al., 1990). Burgeff (1959) has reported that under culture

condition the seedling of saprophytic species do not develop beyond the first leaf primordia and the

first root. However in the present study fully developed seedlings were regenerated on BM-TM

medium supplemented with growth regulators. In A. montana, among the six basal media evaluated

for asymbiotic seed germination, BM-TM medium was found to be the best for seed germination

followed by KC medium. Though all the media favoured seed germination, the growth of the proto-

corm was arrested beyond protocorm stage due to browning. Browning, a problem in in vitro culture

of orchids, results from the accumulation of phenolic compounds and causes the loss of growth ca-

pacity and tissue death during culture (Rittirat et al., 2012), but can be induced by different factors.

In Grammatophyllum speciosum, as much as 70% PLB browning occurred after 8% glucose was

added as the carbon source to MS medium (Pimsen and Kanchanapoom, 2011) while in Phalaenopsiscornu-cervi, New Dogashima medium induced up to 30% PLB browning (Rittirat et al., 2012). MS

medium containing 0.5 mg/l NAA was the optimal medium for Cymbidium faberi, 74% of PLBs turn-

ing green and 26% of PLBs undergoing browning (Tao et al., 2011). The application of TDZ (3 mg/l)

in half-strength MS medium with 60-day subculture interval stimulated browning in as many as 45%

of Phalaenopsis somatic embryos (Gow et al., 2009a). In a separate study, Gow et al., (2009b) found

that incubation of Phalaenopsis leaf explants in the dark for 15 days followed by 45 days under light

induced browning in as many as 90% of embryos after 60 days. In Eria bambusifolia, MS medium

reduced PLB browning more effectively than Knudson C medium (52% and 64%, respectively) with

subsequently higher percentage germination (48% and 36%, respectively) (Basker and Narmatha Bai,

2010). However, in this study, protocorms browning was presumably caused by the high salt concen-

tration of full-strength medium. Since terrestrial orchids usually require the medium with lower salt

concentrations for seed germination (Rasmussen, 1995), the diluted macro-elements of MS medium

(1/2, 1/4, or 1/8 MS) was preferable in several orchid species (Lee, 1998). In the present study, half

strength BM-TM medium supplemented with activated charcoal was necessary for further growth of

the protocorm in A. montana. This promotive effect has been attributed to the ability of activated char-

coal to absorb phenolic compounds released by the plantlet into the media (Pan and van Staden, 1998).

The varied responses in different media might be due to the composition of the media. All

the media used in the present study have different mineral composition. BM-TM medium is en-

riched with organic nitrogen sources such as casin hydrolysate, amino acids and vitamins, thus

conditions are suspected to be responsible for enhancing seed germination which is in agreement

with many workers (Kauth et al., 2006; Stewart and Kane, 2006; Roy et al., 2011; Mahendran etal., 2013). These findings indicate the existence of species–medium specificity. Such species –

specific medium for seed germination have also been reported by many workers (Arditti and Ernst,

1993; Roy et al., 2011; Mahendran et al., 2013).

Orchids needauxin or cytokinin for the formation of new protocorms and plantlets develop-

ment. The type and concentrations of growth regulators play an important role during in vitro mul-

tiplication of many orchid species (Arditti and Ernst,1993). In A. montana, the protocorm developed

into multiple shoots directly on half strength BM-TM medium fortified with cytokinins or cytokinin

in combination with NAA. Among the cytokinins, TDZ was more effective for inducing maximum


number of multiple shoots (17.24 ± 0.27). TDZ also found to be suitable for protocorm proliferation

for Dendrobium hybrids, Dendrobium candidum, Satyrium nepalense and Phalaenopsis gigantean(Martin and Madassery, 2006; Zhao et al., 2007; Mahendran and Narmatha Bai, 2009; Niknejad etal., 2011). In A. montana TDZ in combination with NAA enhanced the number of secondary pro-

tocorms/multiple shoots. Combined effects of cytokinin (BA or TDZ) and auxin (NAA) proved to

be useful in induction of protocorm and seedling in many orchid species like Phalaenopsis and

Doritaenopsis (Park et al., 2002; 2003) and Dendrobium candidum (Zhao et al., 2008). The ratio of

auxin and cytokinin for the initiation of shoot buds or PLB formation varies from species to species

(Teng et al., 1997). Cytokinin and auxin in the ratio of 2:1produced shoot buds in Rhynchostylis gi-gantea (Van Le et al., 1999), Vanda spathulata (Decruse et al., 2003), Phalaenopsis and Renantheraimschootiana and Vanda coerulea (Seeni and Latha, 2000). An auxin in combination with cytokinin

has been well documented for multiplication in various species (Javed et al., 2013) as observed in

our study. However, the concentration of auxin required was found to be very low which indicate a

high endogenous level of auxin in the explants. This may be due to reinforced by high requirement

of exogenous cytokinin as the auxin has been shown to inhibit the transcription of cytokinin biosyn-

thesis geneas suggested by Tanaka et al.(2006) and Javed et al.(2013).

The organic supplements have been shown to stimulate seed germination and seedling

growth of many orchids (Arditti and Ernst, 1993; Zeng et al., 2012; Zhang et al., 2013). However,

their effects are complex and may vary depending on species, the types of explants or the devel-

opmental stages. In the present study the combined effect of organic additives with TDZ (6.8 µM),

various organic additives (peptone, yeast extract and coconut water) at different concentrations (0

.5, 1.0, 2.0, 3.0 g/l or 5, 10, 15 and 20%) were used together with the optimal TDZ concentration

(Table 3). This study was needed so as to familiarize their ability to affect the shoot induction and

multiplication rate and to optimize the medium composition for maximum plantlet regeneration.

Addition of organic additives (peptone, yeast extract and coconut water) at various concentrations

(0 .5, 1.0, 2.0, 3.0 g/l or 5, 10, 15and 20%) in combination with the optimum concentrations of cy-

tokinins was found to be superior for shoot initiation and multiplication. Among the different levels

of organic additives tested, TDZ (6.8 µM) in combination with coconut water (20%) was found to

be the best for multiplication of shoots (28.33±0.27) with a mean number of 1.98 ± 0.42 shoot

length per explant (Table 3). This increase in the number of multiple shoots could be attributed to

the biochemical compounds of the tender coconut milk such as amino acids, organic acids, inor-

ganic ions, vitamins, sugars, lipids, nitrogenous compounds and hormones. Similarly, the effec-

tiveness of coconut water for promoting shoots differentiation has also been reported in

Phalaenopsis gigantean (Murdad et al., 2006), Gastrochilus calceolaris (Pathak et al., 2011),

Vanda Kasem’s Delight (Gnasekara et al., 2012) and Dendrobium AlyaPink (Nambiar et al., 2012).

In the present study, wild–grown plant gave higher phenolic compounds and total flavonoid

content probably due to a higher stress associated to their growth conditions. Otherwise, in vitro cul-

tured seem to be under less stress producing lower amounts of phenolics and total flavonoids com-

pounds (secondary metabolites). In fact, in vitro cultured plant grown with controlled light, temperature

and nutrients. Similarly early researchers found that lower amounts of phenolic compounds in vitrocultured samples in Poliomintha glabrescens (Garcia-Perez et al., 2012), Hypericum spp (Danova etal., 2012), Cichorium pumilum (Khateeb et al., 2012), Hypericum undulatum (Rainha et al., 2013),

Melissa officinalis (Barros et al., 2013) and Swertia corymbosa (Mahendran and Narmatha Bai, 2014).

Free-radical scavenging activity (FRSA) was determined to evaluate the antioxidant potential

of in vitro propagated plants and these were compared with levels in wild-grown plants. Considering

the fact that the mechanisms of antioxidant processes are complex, therefore, an approach with mul-

tiple assays in screening work is highly advisable (Matkowski, 2008; Amoo et al., 2012; Mahendran

and Narmatha Bai, 2014). In the present study, various methods of in vitro assays were performed to

determine the antioxidant activity of in vitro propagated plants and wild grown plants of A. montana.

The DPPH free radical scavenging assay has been widely used to evaluate antioxidant capac-


ities. Antioxidants react with DPPH, reducing a number of DPPH molecules equal to the number of

available hydroxyl groups (Matthaus, 2002). The degree of discoloration indicates that the samples to

scavenge DPPH radical due to its ability to donate hydrogen proton. Similar to DPPH, the decoloriza-

tion of ABTS●+ radical reflects the capacity of an antioxidant species to donate electrons or hydrogen

atoms to inactivate this radical species. The ABTS●+ radical cation is generated from the reaction of

ABTS with potassium persulfate overnight in water (Re et al., 1999). In the present study, the methano-

lic extract of plants growing wild seemed to exhibit a higher antioxidant potential compared to plants

growing in vitro under controlled conditions. The differences between the responses of methanolic

extracts prepared from wild and in vitro growing explants could be attributed to the differences in the

chemical constituents of the plant cells that are likely to develop in response to the surrounding envi-

ronmental conditions and different components are accumulated during different growth phases or

due to mutations (Parsaeimehr et al., 2010; Mohanty et al., 2011). The radical scavenging activity of

extracts can be credited to the presence of its major phenolic compounds (Guimaraes et al., 2010).

The antioxidant activity of phenolic compounds is related to the hydroxyl groups linked to the aromatic

ring, which are capable of donating hydrogen atoms with electrons and stabilizing free radicals (Dor-

man et al., 2003; Yanishlieva et al., 2006). In P. glabrescens, C. pumilum, M. officinalis and S. corym-bosa higher antioxidant activity correlated with higher total phenolic contents (Garcia-Perez et al.,2012; Khateeb et al., 2012; Barros et al., 2013; Mahendran and Narmatha Bai, 2014).

CONCLUSIONIn conclusion, the present study is the first to report seed derived protocorm explants and

the successful plant regeneration in A. montana. The protocol described here could be applied in

a propagation program for genetic resource conservation and commercial purposes. The total phe-

nolics, flavonoid content and antioxidant activity of in vitro propagated plants methanol extract

were lower than that of wild-grown plants’ methanol extract. In addition, we provide evidence that

anti-bacterial activity of A. montana extracts revealed that wild-grown plants’ methanol extract at

25 μg/ml exhibited strong antibacterial activity against S. aureus, B. subtilis and K. pnumoniaewith MIC values of 7.81 and 15.62 μg/ml respectably. Further work on the type of phytocon-

stituents and purification of individual groups of bioactive components can reveal the exact po-

tential of the plant to inhibit pathogenic microbes and induce the antioxidation. Tissue culturing

and the in vitro propagation of Aphyllorchis explants could be a possible method for the large scale

commercial production of biologically active components.

ACKNOWLEDGEMENTSThe financial support by University Grants Commission (UGC-F.No-37-97/2009(SR)),

New Delhi is gratefully acknowledged.

Literature CitedAmoo, S.O., Aremu, A.O. and Van Staden, J. 2012. In vitro plant regeneration, secondary metabolite

production and antioxidant activity of micropropagated Aloe arborescens Mill. Plant Cell,

Tissue and Organ Culture,111: 345–358.

Arditti, J. and Ernst, R. 1993. Micropropagation of orchids. John Wiley and Sons. New York.

Arditti, J., Ernst, R., Yam, T.W. and Glabe, C. 1990. The contribution to orchid mycorrhizal fungi

to seed germination: A speculative review. Lindleyana, 5: 249-255.

Barros, L., Duenas, M., Dias, M.I., Sousa, M.J., Santos-Buelga, C. and Ferreira, I.C.F.R. 2013.

Phenolic profiles of cultivated, in vitro cultured and commercial samples of Melissa officinalis L.

infusions. Food Chemistry, 136: 1–8.

Basker, S., and Narmatha Bai V. 2010. In vitro propagation of an epiphytic and rare orchid Eria bambusifolia Lindl. Research in Biotechnology,1: 15–20.


Bhavani, P., Sreenu, T., Tharangini, K., Geethanjali, J., Govinda Reddy, T. and Venkata Lakshmi,

D. 2012. Hypoglycemic activity of ethanolic extract of Aphyllorchis montanaon alloxan

induce diabetes in rats. International Journal of Chemistry and Pharmaceutical Sciences,

3(3): 27-29.

Blois, M.S. 1958. Antioxidant determinations by the use of a stable free radical. Nature 26: 1199-1200.

Burgeff, H. 1959. Mycorrhiza of orchids, pp. 361-395. In: Withner C.L. (ed), The Orchids: A Scientific

Survey, The Ronald Press Company, New York.

Danova, K., Nikolova-Damianova, B., Denev, R., and Dimitrov, D. 2012. Influence of vitamins

on polyphenolic content, morphological development, and stress response in shoot cultures

of Hypericum spp. Plant Cell, Tissue and Organ Culture,110: 383–393.

Decruse, S.W., Gangaprasad, A., Seeni S. and Menon V.S. 2003. Micropropagation and ecorestoration

of Vanda spathulata, an exquisite orchid. Plant Cell, Tissue and Organ Culture,72: 199-202.

Dorman, H.J.D., Peltoketo, A., Hiltunen, R. and Tikkanen, M.J. 2003. Characterisation of the antioxidant

properties of deodorized aqueous extracts from selected lamiaceae herbs. Food Chemistry,

83: 255–262.

Eloff, J.N. 1998. A sensitive and quick microplate method to determine the minimal inhibitory

concentration of plant extracts for bacteria. Planta Medica, 64: 711–713.

Garcia-Perez, E., Gutierrez-Uribe, J.A. and Garcia-Lara, S. 2012. Luteolin content and antioxidant

activity in micropropagated plants of Poliomintha glabrescens (Gray). Plant Cell, Tissue

and Organ Culture,108: 521–527.

Gnasekaran, R., Poobathy, R., Mahmood, M., Samian, M.R. and Subramaniam S. 2012. Effects

of complex organic additives on improving the growth of PLBs of Vanda Kasem’s Delight.

Australian Journal of Crop Science, 6: 1245-1248.

Gow, W.P., Chen, J.T. and Chang, W.C. 2009a. Enhancement of direct somatic embryo-genesis

and plantlet growth from leaf explants of Phalaenopsis by adjusting culture period and explant

length. Acta Physiologiae Plantarum, 32: 621–627.

Gow, W.P., Chen J.T. and Chang, W.C. 2009b. Effects of genotype, light regime, explants position

and orientation on direct somatic embryogenesis from leaf explants of Phalaenopsis orchids.

Acta Physiologiae Plantarum, 31: 363–369.

Guimaraes, R., Sousa, M.J. and Ferreira, I.C.F.R. 2010. Contribution of essential oils and phenolics

to the antioxidant properties of aromatic plants. Industrial Crops and Products, 32: 152–156.

Javed, S.B., Anis, M., Khan P.R. and Aref, I.M. 2013. In vitro regeneration and multiplication for

mass propagation of Acacia ehrenbergiana Hayne: a potential reclaiment of denude arid

lands. Agroforestry Systems,87: 621–629.

Johnson, T.R., Stewart, S.L., Daniela, D., Kane, M.E. and Richardson, L. 2007. A symbiotic and

symbiotic seed germination of Eulophiaalta (orchidaceae) - Preliminary evidence for the

symbiotic culture advantage. Plant Cell, Tissue and Organ Culture, 90: 313-323.

Joshi, S.C., Verma, A.R. and Mathela, C.S. 2010. Antioxidant and antibacterial activities of the

leaf essential oilsof himalayan lauraceae species. Food and Chemical Toxicology, 48: 37–40.

Kauth, P.J., Vendrame, W.A. and Kane, M.E. 2006. In vitro seed culture and seedling development

of Calopogon tuberosus. Plant Cell, Tissue and Organ Culture, 85: 91–102.

Khateeb, W.A., Hussein, E., Qouta L., Aludatt, M., Al-Shara, B. and Abu-zaiton, A. 2012. In vitropropagation and characterization of phenolic content along with antioxidant and antimicrobial

activities of Cichori umpumilum Jacq. Plant Cell, Tissue and Organ Culture,110: 103–110.

Knudson, L. 1946. A new nutrient solution for the germination of orchid seed. American Orchid

Society Bulletin, 15: 214–217.

Lee, Y.I. 1998. The study of embryo development and seed germination in vitro in slipper orchids.

M.Sc thesis. National Taiwan University, Taiwan.

Lindemann, E.G.P., Gunckel, J.E. and Davidson, O.W. 1970. Meristem culture of Cattleya. American

Orchid Society Bulletin, 39: 1002-1004.


Mahendran, G., Muniappan, V., Ashwini, M., Muthukumar, T. and Narmatha Bai, V. 2013. Asymbiotic

seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal

fungus on seedling development. Acta Physiologiae Plantarum, 35: 829-840.

Mahendran, G. and Narmatha Bai, V. 2009. Mass propagation of Satyrium nepalense D. Don- A

medicinal orchid via seed germination. Scientia Horticulturae,119: 203-207.

Mahendran, G. and Narmatha Bai, V. 2014. Micropropagation, antioxidant properties and phytochemical

assessment of Swertia corymbosa (Griseb.) Wight ex C. B. Clarke: a medicinal plant .Acta

PhysiologiaePlantarum, 36: 589–603.

Martin, K.P. and Madassery, J. 2006. Rapid in vitro propagation of Dendrobium hybrids through

direct shoot formation from foliar explants, and protocorm-like bodies. Scientia Horticulturae,

108: 95–99.

Matkowski, A. 2008. Plant in vitro culture for the production of antioxidants – A review. Biotechnology

Advances, 26: 548–560.

Matthaus, B. 2002. Antioxidant activity of extracts obtained from residues of different oil seeds.

Journal of Agricultural and Food Chemistry,50: 3444–3452.

Mitra, G.C., Prasad, R.N. and Chowdhury, A.R. 1976. Inorganic salts and differentiation of protocorms

in seed callus of an orchid and correlated changes in its free amino acid content. Indian

Journal of Experimental Biology,14: 350–351.

Mohanty, S., Parida, R., Singh, S., Joshi, R., Subudhi, E. and Nayak, S. 2011. Biochemical and

molecular profiling of micropropagated and conventionally grown Kaempferia galanga.

Plant Cell, Tissue and Organ Culture,106: 39–46.

Morel, G.M. 1965. Clonal propagation of orchids by meristem culture. Cymbidium Society News,

20: 3–11.

Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio-assays with tobacco

tissue cultures. Physiologia Plantarum,15: 473–497.

Murdad, R., Hwa, K.S., Seng, C.K., Latip, M.A., Aziz, Z.A. and Ripin, R. 2006. High frequency

multiplication of Phalaenopsis gigantean using trimmed bases protocorms technique. Scientia

Horticulturae,111: 73-79.

Nambiar, N., Tee, C.S. and Maziah, M. 2012. Effects of organic additives and different carbohydrate

sources on proliferation of protocorm like bodies in Dendrobium Alya Pink. Plant Omics

Journal, 5: 10-18.

Niknejad, A., Kadir, M.A. and Kadzimin, S.B. 2011. In vitro plant regeneration from protocorms-like

bodies (PLBs) and callus of Phalaenopsis gigantea (Epidendroideae: Orchidaceae). African

Journal of Biotechnology, 10: 11808-11816.

Pan, M.J. and van Staden, J. 1998. The use of charcoal in in vitro culture – A review. Plant Growth

Regulation, 26: 155-163.

Park, S.Y., Murthy, H.N. and Paek, K.Y. 2002. Rapid propagation of Phalaenopsis from floral

stalk-derived leaves. In vitro Cellular & Developmental Biology – Plant,38: 168-172.

Park, S.Y., Murthy, H.N. and Paek, K.Y. 2003. Protocorm-like body induction and subsequent plant

regeneration from root tip cultures of Doritaenopsis. Plant Science, 164: 919-923.

Parsaeimehr, A., Sargsyan, E. and Javidnia, K. 2010. A comparative study of the antibacterial, antifungal

and antioxidant activity and total content of phenolic compounds of cell cultures and wild

plants of three endemic species of Ephedra. Molecules 15: 1668–1678.

Pathak, P., Piri, H., Vij, S.P., Mahant, K.C. and Chauhan, S. 2011. In vitro propagation and mass

scale multiplication of a critically endangered epiphytic orchid, Gastrochilus calceolaris(Bhch.-Ham ex J.E.Sm) D. Don using immature seeds. Indian Journal of Experimental Biology,

49: 711-716.

Pimsen, M., and Kanchanapoom, K. 2011. Effect of basal media and sugar types on in vitro regeneration

of Grammatophyllum speciosum Blume. Notulae Scientia Biologicae,3(3): 101–104.


Prajapati, N.S., Purohit, S.S., Sharma, A.K. and Kumar, T. 2003. A handbook of medicinal plants. A

complete source book. Agrobios.

Pulido, R., Bravo, L. and Sauro-Calixto, F. 2000. Antioxidant activity of dietary polyphenols as determined

by a modified ferric reducing/antioxidant power assay. Journal of Agricultural and Food Chemistry,

48: 3396-3402.

Rainha, N., Koci, K., Coelho, A.V., Lima, E., Baptista, J. and Ferreira, F. 2013. HPLC–UV–ESI-MS

analysis of phenolic compounds and antioxidant properties of Hypericum undulatum shoot cultures

and wild-growing plants. Phytochemistry, 86: 83–91.

Rasmussen, H.N. 1995. Terrestrial orchids: from seed to mycotrophic plant. Cambridge University

Press., Cambridge.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M. and Rice- Evans, C. 1999. Antioxidant

activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology

and Medicine, 26: 1231-1237.

Rittirat, S., Thammasiri, K. and Te-chato, S. 2012. Effect of media and sucrose concentrations with

or without activated charcoal on the plantlet growth of Phalaenopsis cornu-cervi (Breda) Blume

& Rchb. f. International Journal of Agricultural Technology, 8(6): 2075–2085.

Roy, A.R., Patel, R.S., Patel, V.V., Sajeev, S. and Deka, B.C. 2011. Asymbiotic seed germination, mass

propagation and seedling development of Vanda coerulea Griff ex. Lindl. (BlueVanda): An in vitroprotocol for an endangered orchid. Scientia Horticulturae,128: 325-331.

Seeni, S. and Latha, P.G. 2000. In vitro multiplication and eco rehabilitation of the endangered Blue

Vanda. Plant Cell, Tissue and Organ Culture, 61: 1–8.

Siddhuraju, P. and Becker, K. 2003. Studies on antioxidant activities of Mucuna seed (Mucunapruriens var. utilis) extracts and certain non-protein amino/imino acids through in vitro models. Journal

of the Science of Food and Agriculture, 83: 1517-1524.

Siddiqi, R., Naz, S., Ahmad, S. and Sayeed, S.A. 2011. Antimicrobial activity of the polyphenolic

fractions derived from Grewia asiatica, Eugenia jambolana and Carissa carandas. Journal of

Food Science and Technology, 46: 250–256.

Sinu, P.A., Sinu, N. and Chandrashekara, K. 2012. Ecology and population structure of a terrestrial

mycoheterotrophic orchid, Aphyllorchis montana Rchb.f. (Orchidaceae) in soppinabetta forests

of the western ghats. Journal of Threatened Taxa, 4(9): 2915–2919.

Sreenu, T., VenkataRamana, K., Jyothibasu, T. and Delhiraj, N. 2013. Hypoglycemic activity of ethanolic

extract of Aphyllorchis montana induced by streptozocin in rats. International Journal of Pharmacy

Technology Research, 5: 320-323.

Stewart, S.L. and Kane, M.E. 2006. Symbiotic germination of Habenariama croceratitis (Orchidaceae),

a rare Florida terrestrial orchid. Plant Cell, Tissue and Organ Culture, 86: 159–167.

Tanaka, M., Takei, K., Kojima, M., Sakakibara, H. and Mori, H. 2006. Auxin controls local cytokinin

biosynthesis in the nodal stem in apical dominance. The Plant Journal, 45(6): 1028–1036.

Tao, J., Yu, L., Kong, F. and Zhao, D. 2011. Effects of plant growth regulators on in vitro propagation

of Cymbidium faberi Rolfe. African Journal of Biotechnology, 10(69): 15639–15646.

Teng, W.L., Nicholson, L. and Teng, M.C. 1997. Micropropagation of Spathoglottis plicata. Plant Cell

Reports, 16:831-835.

Van Le, B., Hang Phuong, N.T., Anh Hong, L.T. and Tran Thanh Van, K. 1999. High frequency shoot

regeneration from Rhynchostylis gigantea (Orchidaceae) using thin cell layers. Plant Growth

Regulation, 28: 179-185.

Van Waes, J.M. and Debergh, P.C. 1986. In vitro germination of some western european orchids. Physiologia

Plantarum, 67: 253-261.

Vellupillai, M., Swaru, P.S. and Goh, C.J. 1997. Histological and protein changes during early stage

of seed germination in the orchid Dendrobium curmenatum. Journal of Horticultural Sciences,

76: 941-948.

Yanishlieva, N.V., Marinova, E. and Pokorny, J. 2006. Natural antioxidants from herbs and spices.


European Journal of Lipid Science and Technology,108: 776–793.

Zeng, S., Wu, K., Teixeira da Silva, J.A., Zhang, J., Chen, Z., Xia, N. and Duan, J. 2012. Asymbiotic

seed germination, seedling development and reintroduction of Paphiopedilum wardii Sumerh.,

an endangered terrestrial orchid. Scientia Horticulturae,138: 198–209.

Zhang, Y., Lee, Y.L., Deng, L. and Zhao, S. 2013.Asymbiotic germination of immature seeds and the

seedling development of Cypripedium macranthos Sw., an endangered lady’sslipper orchid.

ScientiaHorticulturae, 164: 130–136.

Zhao, P., Wang, W., Feng, F.S., Wu, F., Yang, Z.Q. and Wang, W.J. 2007. High frequency shoot regeneration

through transverse thin cell layer culture in Dendrobium candidum Wall. Ex. Lindl. Plant Cell,

Tissue and Organ Culture, 90: 131-139.

Zhao, P., Wu, F., Feng, F.S. and Wang, W.J. 2008. Protocorm-like body (PLB) formation and plant regeneration

from the callus culture of Dendrobium candidum Wall ex Lindl. In Vitro Cellular & Developmental

Biology – Plant, 44: 178-185.

Zhishen, J., Mengcheng, T. and Jianming, W. 1999. The determination of flavonoid contents in mulberry

and their scavenging effects on superoxide radicals. Food Chemistry, 64: 555-559.


Encapsulation of Protocorm of Cymbidium bicolor Lindl.

for Short-Term Storage and Germplasm Exchange

Keywords: Acclimatization, Encapsulation, Epiphytic orchid, Germplasm preservation, Protocorms,

Sodium alginate, Synthetic seeds.

Abbreviations: ANOVA (Analysis of variance), SPSS (Statistical package for the social sciences),

CaCl2.2H2O (Calcium chloride), DDW (Double distilled water), TDZ (Thidiazuron), BA (6-Benzyl

adenine), Z (Zeatin), Kn (6-Furfurylaminopurine).

G. Mahendran, N. Parimala Devi and V. Narmatha Bai

Research scholar, Department of Botany, Bharathiar University, Coimbatore- 641046, India.


The present study describes the encapsulation of protocorm of

Cymbidium bicolor Lindl. from 60 days-old in seed cultures for short-

term conservation and propagation. Various concentrations and combinations

of gelling matrix (sodium alginate) and complexing agents (calcium

chloride) were tested to prepare uniform beads. The ideal beads were ob-

tained through a combination of 3% sodium alginate and 100 mM calcium

chloride. Encapsulated protocorms exhibited the best re-growth and con-

version frequency on MS medium supplemented with BA (4.42 μM). En-

capsulated protocorms stored at 25°C were green and retained the viability

with potential for conversion (52%) and germination even after 360

days. The encapsulated protocorms stored at 4°C remained viable up to

30 days beyond which the conversion rate decreased drastically. Well-de-

veloped plantlets were transplanted into plastic pots containing vermiculite

and maintained for 60 days in the culture room for acclimatization. The

90% of the recovered plantlets were hardened off and established suc-

cessfully in the soil. The present study could be useful for large scale

propagation as well as short term storage of this commercial orchid.

Abstract


INTRODUCTIONThe encapsulation technique is an important application of micropropagation that offers

the potential of easy handling, exchange of germplasm between laboratories, efficient short- or

long-term storage and improves delivery of in vitro regenerated plantlets to the field or to the green

house (Piccioni and Standardi, 1995; Chand and Singh, 2004; Rai et al., 2009). Synseed technology

provides a means for the transportation of propagules to distant places as well as to different lab-

oratories without a loss in vigor for shoot organogenesis in micropropagation programs (Rihan etal., 2011; Hung and Trueman, 2012a,b; Lata et al., 2012; Reddy et al., 2012). Therefore, appro-

priate storage conditions and a definite storage period are prerequisites to maintain synseed viability

during transportation that leads to successful commercialization of synseed technology (Sharma

and Shahzad, 2012; Sharma et al., 2013).

Synseedare prepared using unipolar structures such as hairy roots (Uozumi et al., 1992;

Nakashimada et al., 1995), apical shoot tips (Rai et al., 2008; Singh et al., 2009), axillary buds (Ahmad

and Anis, 2010; Singh et al., 2010) and protocorm-like bodies of orchids (Sarmah et al., 2010). Orchids

are a group of economically important plants valued for cut flowers. Synthetic seed technology has

been employed for the mass multiplication and for the storage of number of the orchids (Saiprasad

and Polisetty, 2003; Nhut et al., 2005; Sarmah et al., 2010). The exploitation of the encapsulation tech-

nology has proven to be successful specifically for a number of Orchidaceae species, such as Dendro-bium wardianum (Sharma et al., 1992), Cymbidium gianteum (Corrie and Tandon, 1993), Geodorumdensiflorum (Datta et al., 1999), Spathoglottis plicata (Khor et al., 1998), Dendrobium densiflorum(Vij et al., 2001), Dendrobium, Oncidium, and Cattleya (Saiprasad and Polisetty, 2003), Ipsea mal-abarica (Martin, 2003), Vanilla planifolia (Divakaran et al., 2006), Vanda coerulea (Sarmah et al.,2010), Coelogyne breviscapa (Mohanraj et al., 2009), Aranda × Vanda (Gantait et al., 2012), Cym-bidium devonianum (Das et al., 2011), Flickingeria nodosa (Nagananda et al., 2011), Dendrobiumcandidum (Zhang and Yan, 2011), Phalaenopsis bellina (Khoddamzadeh et al., 2011), Dendrobiumnobile (Mohanty et al., 2012) and Dendrobium Shavin White (Bustam et al., 2013).

Cymbidium or ‘‘boat orchid’’ is a popular orchid grown commercially worldwide (Chugh etal., 2009). Today, orchids such as Cymbidium, Dendrobium, Oncidium and Phalaenopsis are marketed

globally and the orchid industry contributes substantially to the economy of many the South East

Asian countries. Cymbidium bicolor Lindl. is an important horticultural orchid known for its beautiful

flowers (Chugh et al., 2009). In Cymbidium, plantlets were regenerated in in vitro using shoot tips

(Morel, 1964), mature and immature seeds (Chung et al., 1985; Shimasaki and Uemoto, 1990), green

capsules (Hossain et al., 2010; Deb and Pongener, 2011), flower stalks (Wang, 1988), pseudo bulbs

(Shimasaki and Uemoto, 1990), shoot segments (Nayak et al., 1997), flower buds (Shimasaki and

Uemoto, 1990), protocorm-like bodies (PLBs) (Begum et al., 1994; Huan and Tanaka, 2004; Teixeira

da Silva et al., 2007), thin cell layers of PLBs (Malabadi et al., 2008), artificial seeds (Nhut et al.,2005) and through somatic embryogenesis (Chang and Chang, 1998; Huan and Tanaka, 2004; Ma-

hendran and Narmatha Bai, 2012). Hoque et al. (1994) reported that Phytomax medium was the best

among the five different media (Phytomax, Modified Vacin and Went, KC, KCM and LO medium)

tested for large scale multiplication of C. bicolor. The present study was carried out with the aim to

optimize the methods for the production of synseeds using seed derived protocorm for propagation

and in vitro short-term storage. Broadly, three experiments were conducted (i) to assess the effect of

encapsulation matrix on the formation of synthetic seeds (ii) to test the efficiency of growth regulators

for in vitro conversion of synthetic seeds under aseptic conditions and (iii) to study the effect of stor-

age (4°C and 25°C) temperature on the conversion of encapsulated protocorms.

MATERIALS AND METHODSExplant source

Green pods of Cymbidium bicolour collected from National Yercaud Orchidarium Salem,

India, were surface sterilized through the procedure adopted by Mahendran et al. (2013) and used


to raise aseptic protocorms in seed germination medium that comprised Lindemann orchid (LO)

(Lindemann et al.,1970) under controlled conditions of light, temperature and humidity (Mahen-

dran et al., 2013). The protocorms were taken from in vitro seed cultures of C. bicolor and used

as the source of explants for the preparation of synseeds.

Encapsulation matrix and complexing agentSodium alginate (Qualigens, India) was used as gelling agent and prepared in double dis-

tilled water (DDW) and liquid B5 medium (with 3% sucrose) at different concentrations, i.e., 1,

2, 3, 4 and 5% (w/v). For complexion, 25, 50, 75, 100 and 200 mM calcium chloride (CaCl2.2H2O)

solution was prepared in liquid B5 medium. The pH of the gel matrix and the complexing agent

was adjusted to 5.8 prior to autoclaving at 121°C for 20 min.

Encapsulation of explantsEncapsulation was accomplished by mixing the 60-day-oldprotocorms with sodium alginate

solution and dropping them in CaCl2.2H2O solution using a pipette. The droplets containing the

explants were held at least for 25–30 min to achieve polymerization. The alginate beads containing

the protocorms were retrieved from the solution and rinsed twice with sterilized DDW to remove

the traces of CaCl2.2H2O and transferred to sterile filter paper in Petri dishes for 5 min under the

laminar airflow cabinet to eliminate the excess of water and thereafter transferred to culture vials

containing nutrient medium.

Planting media and culture conditionsThe encapsulated protocorms (alginate beads) were transferred to test tube (Borosil, India)

containing B5 basal medium without plant growth regulator (PGR) and B5 medium supplemented

with cytokinins like BA (1.10, 2.21, 4.42, 8.84 or 13.26 µM), Kn (1.16, 2.32, 4. 64, 9.28 or 13.92

µM), TDZ (1.13, 2.26, 4.52, 9.24 or 13.76 µM) or Zt (1.12, 2.24, 3.36, 4.58 or 6.90 µM), individ-

ually. The culture medium was gelled with 0.8 % (w/v) agar and pH was adjusted to 5.8 prior to

autoclaving at 121°C for 20 min. Cultures were maintained at 25 ± 2°C under 16/8 h light–dark

conditions with a photosynthetic photon flux density (PPFD) of 50 μmol m-2 s-1 provided by cool

white fluorescent tubes (40 W, Philips, India).

Short-term storage of encapsulatedprotocormTwo sets of 150 encapsulated protocorms were kept in two sterile wide mouth culture flask

(Borosil, India) properly covered with aluminium foil and stored in refrigerator at 4°C and culture

room at 25 ± 2°C. Six different exposure times (0, 30, 60, 90, 180 and 360 days) were evaluated for

conversion of synseeds into plantlets. After each storage period, 15 encapsulated were transferred

to B5 medium containing optimal concentration of PGRs for conversion into plantlets. During stor-

age period the beads were sprayed with sterile DDW after every storage period to ensure the moist

conditions so that the beads may not shrink by losing water. The number of shoot regeneration, root,

shoot and root length of encapsulated protocorm was recorded after 8 weeks of culture.

Hardening and acclimatizationPlantlets with well-developed root and shoot system were removed from the culture medium

and washed gently under running tap water to remove any adherent gel from the roots and transferred

to plastic cups containing sterile vermiculite. These were kept under similar culture conditions as

mentioned earlier and covered with transparent polythene bags to ensure high humidity. These were

irrigated after every 10 days with one-fourth strength B5 salt solution (without vitamins) for 2 month.

Polythene bags were removed gradually after 1 month in order to acclimatize the plantlets and after

8 weeks they were transferred transplanted into plastic pot (10 x 8 cm; height x diameter) containing

sterilized vermiculite (50 g per pot) and maintained in greenhouse under normal day light conditions.


Statistical analysisThe results were expressed as mean ± SE of three independent replicates of independent

experiments. Data were subjected to analysis of variance (one way ANOVA) and Duncan’s Mul-

tiple Range Test (DMRT) using SPSS version 17.0.

RESULTS AND DISCUSSIONThe formation of beads and the subsequent success of the encapsulation depend on the con-

centration of alginate and calcium chloride used and it may vary with different propagules as well

as with the different plant species (Sharma and Shahzad, 2012). Hence, the concentrations of these

two solutions and complexion time must be optimized for the formation of an ideal bead. In most

of the reports, 3% (w/v) sodium alginate and100 mM calcium chloride for 20–30 min has proved

to be the best combination for the formation of an ideal synseed (Tabassum et al., 2010; Ahmad

and Anis, 2010; Ozudogru et al., 2011; Alatar and Faisal, 2012; Hung and Trueman, 2011, 2012a,

b; Gantait et al., 2012). However, 3% sodium alginate upon complexion with 75 mM calcium

chloride for 20–30 min was found to be optimum combination for proper hardening of beads or-

chids such as of Dendrobium, Oncidium and Cattleya orchids (Saiprasad and Polisetty, 2003). In

contrast, for the encapsulation of nodal segments of medicinal plant such as Pogostemon cablin(Swamy et al., 2009), Spilanthes acmella (Sharma et al., 2009b) and the microshoots of Zingiberofficinale (Sundararaj et al., 2010), 4% sodium alginate with 100 mM calcium chloride was opti-

mum. This variation in sodium alginate concentration for synseed formation in different plant

species might be due to the variation of the source from which the chemicals were purchased as

suggested by Ghosh and Sen (1994) and Mandal et al. (2000). In the present study, alginate-beads

containing seed derived protocorm (beads) showed different morphology (clearness, form, and

consistency) based on the concentrations of sodium alginate and calcium chloride used. Ideal beads

were obtained with 3% sodium alginate in 100 mM CaCl2 solution (Fig. 1c). At lower concentra-

tions (1–2%), sodium alginate became unsuitable for encapsulation because of a reduction in its

gelling ability following exposure to high temperature during autoclaving (Larkin et al., 1998).

On the contrary, high concentrations of sodium alginate (5–6%), beads were diametric but too

hard, causing considerable delay in sprouting of shoots (Ahmad and Anis, 2010; Sharma et al.,2009 a,b; Gantait and Sinniah, 2012) which was also observed in our study (Fig.1a-d).

The most desirable property of the encapsulated explants is their ability to retain viability

in terms of regrowth and conversion abilities after encapsulation (Adriani et al., 2000; Micheli etal., 2007). In the present study, the ideal beads produced by encapsulating protocorm in 3% sodium

alginate and 100 mM CaCl2.2H2O were cultured on B5 basal medium without any PGR as well as

with various concentrations of BA, Kn, Zt and TDZ (Fig.1e). Synseeds cultured on B5 basal

medium exhibited 90% regeneration response and this occurred after 10 weeks of culture. Addition

of higher concentration of cytokinins enhanced the regeneration potential of the beads and the

shoots emerged out within 10-15 days of inoculation onto the regeneration medium. An average

of 30.25±0.11 shoots/bead was produced in the medium containing 4.42 µM of BA with 87% re-

generation response after 10 weeks of culture (Table 1; Fig. 1f). The shoot buds first appeared as

small white protuberances over the surface of the protocorm which eventually developed into mul-

tiple shoots within 45–60 days. The number of shoot buds increased with increasing concentration

of BA up to an optimal level of 4.42 µM. Among the various concentrations of Kn and TDZ tested,

maximum number of the multiple shoots were recorded in B5 medium supplemented with 13.92

µM Kn (23.89±0.54 shoots/encapsulated explant), 13.76 µM TDZ (6.41±0.50 shoots/bead) and

6.90 µM zeatin (14.56±0.23 shoots/bead). Emergence of single or multiple shoots from the en-

capsulated explants has also been reported earlier in other medicinal plants (Mandal et al., 2000;

Lata et al., 2009; Shrivastava et al., 2009). Mishra et al. (2011) also described that encapsulated

explants of Picrorhiza kurroa exhibited simultaneous production of shoots and roots while rest of


the non-rooted shoots were transferred to root induction medium for the development of roots.

Supplementation of PGRs to the regeneration medium has been found to eliminate the requirement

of an additional in vitro root induction step prior to acclimatization (Sharma and Shahzad, 2012).

In the present study, encapsulated explants of C. bicolor exhibited simultaneous production of

shoots and roots without any specific root induction medium (Fig.1g). B5 medium supplemented

with BA (4.43 µM) significantly increased the number of protocorms as well as induced roots

(Table 1). Similar result observed in Cassava (Danso and Ford-Lloyd, 2003) and Zingiber officinale(Sundararaj et al., 2010). In contrast, Gangopadhyay et al. (2005) devised a two-step method to

achieve maximum bead conversion in A. comosus (pineapple). In the first step, shoots were re-

trieved from encapsulated beads and in the second step, these microshoots were rooted in liquid

medium (supplemented with 0.01 mM IBA and 0.002 mM Kn) supported with luffa-sponge.

Values represent mean±S.E. Each treatment was repeated twice and each treatment counted

of 5 replicate culture tubes, each containing five encapsulated protocorms. Means in a column

with the different letter (superscript) are significantly different according to DMRT (p < 0.05).

In C. bicolor the encapsulated protocorms stored at 25°C gave promising results for ger-

mination and regeneration. Table 2 presents the germination and conversion competency of syn-

thetic seeds stored at 4°C and 25°C for 0, 30, 60, 90, 180 and 360 days. Regeneration percent of

the encapsulated protocorms decreased gradually with an increase in storage duration at 25°C with

being significantly more (p≤0.05) compared to that stored in 4°C. Encapsulated protocorms stored

at 4°C up to 30 days retained their viability (10.23%) and with the advancement of storage duration

(60 days) the synthetic seed turned necrotic, shrunken and brown, resulting in complete death,

whereas, beads stored at 25°C were green, with potential for conversion and with 52% germination

even at 360 days. Storage of encapsulated protocorm/PLBs is greatly influenced by the tempera-

ture. However, the response of synthetic seeds to storage temperature appears to be species specific

(Bustam et al., 2012 and Gantait and Sinniah, 2012). The failure of prolonged storage in 4°C in

the present study corresponds to the earlier reports where, in low temperature (4°C) storage, the

storage life of synthetic seed was rather short (Redenbaugh et al., 1987; Gantait et al., 2012).Sim-

BA

(µM/l)

Kn

(µM/l)

TDZ

(µM/l)

Zt

(µM/l)

Regeneration

response (%)

No. of

shoots/bead

Shoot length

(cm)/bead

No. of

Roots

Root length

(cm)

-

1.10

2.21

4.42

8.84

13.26

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1.16

2.32

4.64

9.28

13.92

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1.13

2.26

4.52

9.24

13.76

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

1.12

2.24

3.36

4.58

6.90

90

92

97

87

98

79

89

90

76

98

100

90

89

100

90

90

87

90

96

97

100

2.43±0.12 i

10.23±0.34 e

16.65±0.98 c

30.25±0.11 a

20.17±0.13 b

17.12±0.34 c

3.56±0.78 g

7.87±0.91 f

12.43±0.19 d

17.81±0.71 c

23.89±0.54 b

3.54±0.43 h

3.76±0.11 g

4.12±0.61 g

5.45±0.99 g

6.41±0.50 f

2.45±0.81 i

4.76±0.11 g

5.10±0.89 g

10.010.21 e

14.56± 0.23 c

3.21±0.23 d

2.11±0.14 g

2.54±0.62 e

2.58±0.74 e

2.33±0.54 f

3.32±0.12 d

2.32±0.65 f

2.12±0 .76 g

3.98±0.17 b

3.30±0.22 d

2.23±0.21 f

4.54±0.76 a

4.32±0.54 a

3.80±0.27 c

3.76±0.76 c

2.34±0.41 f

3.50±0.16 e

2.54±0.58 e

2.20±0.43 f

3.20±0.03 d

3.60±0.36 c

1.87±0.89 c

2.12±0.43 b

2.11±0.98 b

1.11±0.43 f

1.06±0.11 g

0.80±0.21 h

2.61±0.12 b

3.19±0.34 a

1.43±0.19 e

1.67±0.20 d

1.21±0.21 f

1.11±0.56 f

1.00±0.22 g

1.12±0.31 f

0.95±0.93 h

0.81±0.21 h

2.52±0.75 ab

3.84±0.84 a

1.66±0.61 d

1.00±0.39 g

1.41±0.14 e

2.87±0.43 a

2.56 ±0.11 a

2.11±0.65 b

1.15±0.13 f

1.00±0.55 g

0.59±0.63 h

1.29±0.63 de

1.10±0.23 f

1.49±0.49 d

1.38±0.53 d

1.22±0.88 e

1.21±0.56 e

1.19±0.77 ef

1.87±0.60 c

1.69±0.93 c

1.11±0.09 f

1.13±0.99 f

1.00±0.21 g

1.08±0.33 g

1.32±0.52 d

1.12±0.79 f

Values represent mean±S.E. Each treatment was repeated twice and each treatment counted of 5 replicate culturetubes, each containing five encapsulated protocorms. Means in a column with the different letter (superscript) aresignificantly different according to DMRT (p < 0.05).

Table 1. Effect of different plant growth regulators on regeneration of encapsulated protocorm of C. bicolor.


ilarly, the germination of encapsulated PLBs of Aranda x Vanda coerulea also showed marked de-

cline, following storage at low temperature (Gantait et al., 2012). Storage at room temperature

(25°C) implemented in this study was effective for short-term storage and handling without re-

frigerated containers and beads stored up to 360 days gave considerable conversion rate (52%).

Similarly, in Aranda x Vanda coerulea beads stored at 4°C showed rapid deterioration and faced

complete death within 160 days while those stored for 200 days at 25°C showed relatively high

conversion/ regeneration (71.6%)(Gantait and Sinniah, 2012). Encapsulated PLBs of Dendrobiumwere successfully stored up to 135 days with 52% survival at 25±2°C (Bustam et al., 2012). In

Vanda coerulea encapsulated PLBs stored at 4°C retaining their viability up to 100 days (Sharmah

et al., 2010) and in Phalaenopsis bellina beads stored at 25°C up to 60 days did not show any re-

sponse (Khoddamzadeh et al., 2011). The decline in the viability and germination rate of stored

encapsulated protocorms (4°C) may be related to oxygen deficiency in the gel bead as reported by

earlier researchers (Danso and Ford-Lloyd, 2003; Gantait et al., 2012).

The regeneration efficiency and the number of seedling/shoots and shoots length declined

with increase in storage duration at both 4°C and 25°C of plant markedly reduced with an increase

in storage time. Till 30 days of storage at both 25°C and 4°C, number of shoots reduced from

30.25±0.11 to 19. 12±0.71 while the shoot length reduced from 2.58±0.74 to 2.29±0.97. At 360

days of storage, capsules stored at 25°C showed 18.87±0.44 shoots/bead, whereas, capsules stored

at 4°C lost their viability completely after 30 days. Similarly, in relation to morphological changes,

the numbers of shoots, root numbers, shoot and rootlength of thestored synthetic seed on optimized

conversion medium decreased in a linear manner with increase in storage duration till 360 days.

Root numbers, on the other hand, was unaffected by the storage duration under refrigerated condi-

tions (4°C), as each encapsulated protocorms produced one root after 30 days of storage time. While

storage at 25°C had a different trend with an increase in storage time, a linear reduction in root num-

ber was observed (1.00 to 0.53) up to 180 days of storage (Table 2). According to Danso and Ford-

Lloyd (2003) and Gantait et al. (2012) the decline in conversion or morphogenesis i.e.shoot forming

capacity as the result of prolonged storage could be due to inhibited respiration of tissues.

The plantlets were transferred to the potting medium containing vermiculite and covered

with polythene bag and maintained at 25±2°C. After 2 months, the cover was gradually loosened,

thus dropping the humidity (65–70%) (Fig.1h and i) this procedure subsequently resulted in invitro hardening of the plants. Acclimatization of plants grown in in vitro to ex vitro conditions is

a critical step for many species, requiring time and expensive installation that restrict the commer-

cial application of micropropagation process. About 90% survival rate in the present study was

obtained when plantlets were transferred to field.

Storage

temperature (°C)

Storage

duration (days)

Regeneration

response (%)

No of

shoots/bead

Shoot length

(cm)/bead

No of

Roots

Root Length

(cm)

4°C

0

30

80

10.23

30.25±0.11a

19.12±0.71e

2.58±0.74 a

2.40±0.56 a

1.11±0.43 a

1.31 ±0.50 a

1.15±0.13 a

1.00±0.90 b

60 Complete death

25°C 30

60

90

180

360

80

75

70

60

52

30.12±0.45 a

27.65±0.98 b

25.83±0.71 c

20.59±0.60 d

18.87±0.44 e

2.29±0.97 a

1.95±0.62 b

1.14±0.74 b

0.83±0.54 d

0.32±0.12 e

1.00±0.11 a

0.95±0.98 b

0.94±0.43 b

0.53±0.81 b

0.45±0.77 c

1.00±0.11 a

0.95±0.98 b

0.94±0.43 b

0.53±0.81 b

0.45±0.77 c

Values represent mean±SE. Each treatment was repeated twice and each treatment counted of 5 replicate culture tubes,each containing five encapsulated protocorms. Means in a column with the different letter (superscript) are significantlydifferent according to DMRT (p< 0.05).

Table 2. Effect of storage at 4°C and 25 ± 2°C for different time periods on conversion of encapsulated protocormof C. bicolor on B5 medium containing BA (4.42 µM).


CONCLUSIONIn conclusion, this study developed highly effective techniquesfor synthetic seed production,

short-term conservation and regeneration of plantlets. The synthetic seed development protocol

illustrated here offers a substitute scheme for mass propagation and germplasm distribution of this

important orchid to laboratories and extension centers in distant places. Exultant plant recovery

from encapsulated shoot tips following room temperature storage for more than 360 days indicates

that the technique described in this report can be potentially exploited for short-term storage with

the retention of genetic uniformity.

Fig. 1. Alginate-encapsulation, short-term storage and regeneration of C. bicolor usingseed derived protocorm.

a. Beads prepared with 2% sodium alginate in75 mM calcium chloride solution. (Bar = 2 cm) b. Beads prepared with 3% alginate in 75mM calcium chloride solution. (Bar = 2 cm) c. Firm beads at 3 % sodium alginate in 100 mM CaCl2 solution (Bar = 2 cm) d. Hard brads 5 % at sodium alginate in 100 mM CaCl2 solution. (Bar = 2 cm) e. Conversion of synthetic seed & formation of multiple shoot/ seedling on B5 medium con-taining BA (4.42 μM). (Bar = 2 cm) f. Conversion of synthetic seed & formation of multiple protocorm/ seedling on B5 mediumcontaining Kn (13.92 μM). (Bar = 2 cm) g. plantlets (Bar = 1cm) h. In vitro hardening. (Bar = 2 cm) i. Plantlet transferred to pot containing vermiculite cori peat (1:1). (Bar = 2 cm)


ACKNOWLEDGEMENTSThe financial support by University Grants Commission (UGC), New Delhi is gratefully

acknowledged.

Literature CitedAdriani, M., Piccioni, E. and Standardi, A. 2000. Effect of different treatments on the conversion

of ‘Hayward’ kiwifruit synthetic seeds to whole plants following encapsulation of in vitroderived buds. New Zeal. Journal of Crop and Horticultural Science, 28:59–67.

Ahmad, N. and Anis, M. 2010. Direct plant regeneration from encapsulated nodal segments of

Vitex negundo. Biologia Plantarum, 54:748–752.

Alatar, A. and Faisal, M. 2012. Encapsulation of Rauvolfia tetraphylla microshoots as artificial

seeds and evaluation of genetic fidelity using RAPD and ISSR markers. Journal of Medicinal

Plants Research, 6:1367–1374.

Begum, A.A., Tamaki, M. and Kako, S. 1994. Formation of protocorm-like bodies (PLBs) and

shoot development through in vitro culture of outer tissue of Cymbidium PLB. Journal of

the Japanese Society for Horticultural Science, 63:663–673.

Bustam, S., Sinniah, U.R., Kadir, M.A., Zaman, F.Q. and Subramaniam, S. 2013. Selection of optimal

stage for protocorm-like bodies and production of artificial seeds for direct regeneration

on different media and short term storage of Dendrobium Shavin White. Plant Growth Regulators,

69: 215- 224.

Chand, S. and Singh, A. K. 2004. Plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb., a timber yielding leguminous tree species. Journal of Plant Physiology,

161:237–243.

Chang, C. and Chang, W. C. 1998. Plant regeneration from callus of Cymbidium ensifolium var.

misericors. Plant Cell Reports, 17:251-255.

Chugh, S., Guha, S. and Usha Rao, I. 2009. Micropropagation of orchids: A review on the potential

of different explants. Scientia Horticulturae, 22:507–520.

Chung, J.D., Chun, C.K., and Choi, S.O. 1985. Asymbiotic germination of Cymbidium ensifolium II. Effects of several supplements to the medium, pH values and light and/or dark culture

periods on growth of rhizome and organogenesis for rhizome. Journal of the Korean Society

for Horticultural Science, 26:86–92.

Corrie, S. and Tandon, P. 1993. Propagation of Cymbidium gianteum wall through high frequency

conversion of encapsulated protocorms under in vivo and in vitro conditions. Indian Journal

of Experimental Biology, 31:61–64.

Danso, K.E. and Ford-Lyoyd, B.V. 2003. Encapsulation of nodal cuttings and shoot tips for storage

and exchange of cassava germplasm. Plant Cell Reports, 21:718–725.

Das, M.C., Kumaria, S. and Tandon, P. 2011. Storage and high conversion frequency of encapsulated

protocorm-like bodies of Cymbidium devonianum (orchid). Journal of Horticultural Science

and Biotechnology, 86: 611-615.

Datta, K.B., Kanjilal, B. and Sarker, D. 1999. Artificial seed technology. Development of a protocol

in Geodorum densiflorum (Lam.) Schltr. An endangered orchid. Current Science, 76:1142-1145.

Deb, C.R. and Pongener, A. 2011. Asymbiotic seed germination and in vitro seedling development

of Cymbidium aloifolium (L.) Sw.: a multipurpose orchid. Journal of Plant Biochemistry

and Biotechnology, 20:90–95.

Divakaran, M., NirmalBabu, K. and Peter, K.V. 2006. Conservation of Vanilla species, in vitro.

Scientia Horticulturae, 110:175–180.

Gangopadhyay, G., Bandyopadhyay, T., Ramit, P., Gangopadhyay, S.B. and Mukherjee, K.K. 2005.

Encapsulation of pineapple microshoots in alginate beads for temporary storage. Current

Science, 88:972–977.


Gantait, S., Bustam, S. and Sinniah, U.R. 2012. Alginate-encapsulation, short-term storage and

plant regeneration from protocorm-like bodies of Aranda Wan Chark Kuan ‘Blue’× Vanda coerulea Grifft. ex. Lindl. (Orchidaceae). Plant Growth Regulation, 67:257–270.

Gantait, S. and Sinniah, U.R. 2012. Storability, post-storage conversion and genetic stability assessment

of alginate-encapsulated shoot tips of monopodial orchid hybrid Aranda Wan CharkKuan

‘Blue’ 3 Vanda coerulea Grifft.ex. Lindl. Plant Biotechnology Reports, 7: 257- 266.

Ghosh, B. and Sen, S. 1994. Plant regeneration from alginate encapsulated somatic embryos of Asparagus cooperi Baker. Plant Cell Reports, 13:381–385.

Hoque, M.I., Roy, A.R., Sarker, R.H. and Haque, M. M. 1994. Micropropagation of Cymbidium bicolor through in vitro culture. Plant Tissue Culture, 4:45–51.

Hossain, M.M., Sharma, M., Teixeira Da Silva, J.A. and Pathak, P. 2010. Seed germination and

tissue culture of Cymbidium giganteum Wall. ex Lindl. Scientia Horticulturae,123:479–487.

Huan, L.V.T. and Tanaka, M. 2004. Callus induction from protocorm-like body segments and plant

regeneration in Cymbidium (Orchidaceae). Journal of Horticultural Science and Biotechnology,

79:406–410.

Hung, C.D. and Trueman, S.J. 2011. Encapsulation technology for short term preservation and

germplasm distribution of the African mahogany Khaya senegalensis. Plant Cell, Tissue

and Organ Culture, 107:397–405.

Hung, C.D. and Trueman, S.J. 2012a. Alginate encapsulation of shoot tips and nodal segments for

short-term storage and distribution of the eucalypt Corymbia torelliana × C. citriodora. Acta

Physiologiae Plantarum, 34:117–128.

Hung, C.D. and Trueman, S.J.P. 2012b. Reservation of encapsulated shoot tips and nodes of the

tropical hardwoods Corymbiatorelliana × C. citriodora and Khaya senegalensis. Plant Cell,

Tissue and Organ Culture, 109:341–352.

Khoddamzadeh, A.A., Sinniah, U.R., Kadir, M.A., Kadzimin, S.B., Mahmood, M. and Subramaniam,

S. 2011. Establishment of a short-term storage method via encapsulation of protocorm-like

bodies in Phalaenopsis bellina (Rchb.f.) Christenson. Seed Science and Technology, 39:697-702.

Khor, E., Ng, W.F. and Loh, C.S. 1998. Two-coat system for encapsulation of Spathoglottis plicata (Orchidaceae) seeds and protocorms. Biotechnology and Bioengineering, 59:635-639.

Larkin, P.J., Davies, P.A. and Tanner, G.J. 1998. Nurse culture of low number of Medicago and

Nicotiana protoplasts using calcium alginate beads. Plant Science, 58:203–210.

Lata, H., Chandra, S., Khan, I.A. and El-Sohly, M.A. 2009. Propagation through alginate encapsulation

of axillary buds of Cannabis sativa L. an important medicinal plant. Physiology and Molecular

Biology of Plants, 15:79–86.

Lata, H., Chandra, S., Mehmedic, Z., Khan, I.A. and El-Sohly, M. A. 2012. In vitro germplasm

conservation of high D9-tetrahydrocannabinol yielding elite clones of Cannabis sativa L.

under slow growth conditions. Acta Physiologiae Plantarum, 34:743–750.

Lindemann, E.G.P., Gunckel, J.E. and Davidson, O.W. 1970. Meristem culture of Cattleya. American

Orchid Society Bulletin, 39:1002-1004.

Mahendran, G., Muniappan, V., Ashwini, M., Muthukumar, T. and Narmatha Bai, V. 2013. Asymbiotic

seed germination of Cymbidium bicolor Lindl. (Orchidaceae) and the influence of mycorrhizal

fungus on seedling development. Acta Physiologiae Plantarum, 35:829–840.

Mahendran, G. and Narmatha Bai, V. 2012. Direct somatic embryogenesis and plant regeneration

from seed derived protocorms of Cymbidium bicolor Lindl. Scientia Horticulturae, 135:40–44.

Malabadi, R.B., Teixeira da Silva, J.A., Nataraj, K. and Mulgund, G.S. 2008. Shoot tip transverse

thin cell layers and 2, 4-epibrassinolide in the micropropagation of Cymbidium bicolor Lindl. Floriculture and Ornamental Biotechnology, 2: 44–48.

Mandal, J., Pattnaik, S. and Chand, P.K. 2000. Alginate encapsulation of axillary buds of Ocimum americanum L. (Hoary basil), O. basilicum (sweet basil), O. gratissium (shrubby basil) and


O. sanctum (sacred basil). In Vitro Cellular and Developmental Biology- Plant, 36:287–92.

Martin, K.P. 2003. Clonal propagation, encapsulation and reintroduction of Ipsea malabarica (Reichb.f)

Hook JD, An endangered orchid. In Vitro Cellular and Developmental Biology- Plant,

39:322-326

Micheli, M., Hafiz, I.A. and Standardi, A. 2007. Encapsulation of in vitro derived explants of olive

(Oleaeuropaea L. cv. Moraiolo) II. Effects of storage on capsule and derived shoots performance.

Scientia Horticulturae, 113:286–292.

Mishra, J., Singh, M., Palni, L.M.S. and Nandi, S.K. 2011. Assessment of genetic fidelity of encapsulated

microshoots of Picrorhiza kurrooa. Plant Cell Tissue Organ Culture, 104:181–186.

Mohanraj, R., Ananthan, R. and Narmatha Bai, V. 2009. Production and storage of synthetic seeds

in Coelogyne breviscapa Lindl. Asian Journal of Biotechnology, 1:124–128.

Mohanty, P., Nongkling, P., Das, M.C., Kumaria, S. and Tandon, P. 2012. Short-term storage of

alginate-encapsulated protocorm-like bodies of Dendrobium nobile Lindl.: an endangered

medicinal orchid from North-east India. 3 Biotech, 3 (3): 235- 239.

Morel, G. 1964. Tissue culture—a new means of clonal propagation of orchids. American Orchid

Society Bulletin, 33:437–478.

Nagananda, G.S., Nalini, S. and Rajath, S. 2011. Regeneration of encapsulated protocorm like bodies

of medicinally important vulnerable orchid Flickingeria nodosa (Dalz.) Seidenf. International

Journal of Botany, 7:301-313.

Nakashimada, Y., Uozumi, N. and Kobayashi, T. 1995. Production of plantlets for use as artificial

seeds from horse radish hairy roots fragmented in a blender. Journal of Fermentation and

Bioengineering, 79:458–464.

Nayak, N.R., Rath, S.P. and Patnaik, S. 1997. In vitro propagation of three epiphytic orchids, Cymbidium aloifolium (L.) Sw., Dendrobium aphyllum (Roxb.) Fisch. And Dendrobium moschatum (Buch-Ham) Sw. through thidiazuron-induced high frequency shoot proliferation. Scientia

Horticulturae, 71:243–250.

Nhut, D.T., Tien, T.N.T., Huong, M.T.N., Hien, N.T.H., Huyen, P.X., Luan, V.Q., Le, B.V. and Teixeira

da Silva, J.A. 2005. Artificial seeds for preservation and propagation of Cymbidium spp.

Propagation of Ornamental Plants, 5: 67–73.

Ozudogru, E.A., Kirdok, E., Kaya, E., Capuana, M., De Carlo, A. and Engelmann, F. 2011.Medium-term

conservation of redwood [Sequoia sempervirens (D. Don) Endl.] in vitro shoot cultures and

encapsulated buds. Scientia Horticulturae, 127:431–435.

Piccioni, E. and Standardi, A. 1995. Encapsulation of micropropagated buds of six woody species.

Plant Cell Tissue Organ Culture, 42:221–226.

Rai, M.K., Asthana, P., Singh, S.K., Jaiswal, V.S. and Jaiswal, U. 2009. The encapsulation technology

in fruit plants a review. Biotechnology Advance, 27:671–679.

Rai, M.K., Jaiswal, V.S. and Jaiswal, U. 2008. Encapsulation of shoot tips of guava (Psidium guajava L.) for short-term storage and germplasm exchange. Scientia Horticulturae, 118:33–38.

Reddy, M.C., Murthy, K.S.R. and Pullaiah, T. 2012. Synthetic seeds: A review in agriculture and

forestry. African Journal of Biotechnology, 11:14254–14275.

Redenbaugh, K., Slade, D., Viss, P. and Fujii, J.A.A. 1987. Synthetic seed technology for mass

cloning of crop plants: problems and perspectives. Horticultural Science, 22:796–814.

Rihan, H.Z., Al-Issawi, M., Burchett, S. and Fuller, M.P. 2011. Encapsulation of cauliflower (Brassica oleracea var. botrytis) microshoots as artificial seeds and their conversion and growth in

commercial substrates. Plant Cell Tissue Organ Culture, 107:243–250.

Saiprasad, G.V.S. and Polisetty, R. 2003. Propagation of three orchid genera using encapsulated

protocorm-like bodies. In vitro Cellular and Developmental Biology- Plant, 39: 42–48.

Sarmah, D.K., Borthakur, M. and Borua, P.K. 2010. Artificial seed production from encapsulated

PLBs regenerated from leaf base of Vanda coerulea Grifft. ex. Lindl. – An endangered orchid.


Current Science, 98:686–690.

Sharma, S. and Shahzad, A. 2012. Encapsulation technology for short-term storage and conservation

of a woody climber, Decalepis hamiltonii Wight and Arn. Plant Cell Tissue Organ Culture,

111:191–198.

Sharma, S., Shahzad, A., Jan, N. and Sahai, A. 2009a. In vitro studies on shoot regeneration through

various explants and alginate-encapsulated nodal segments of Spilanthes mauritiana DC.,

an endangered medicinal herb. International Journal of Plant Developmental Biology, 3:56–61.

Sharma, S., Shahzad, A. and Sahai, A. 2009b. Artificial seeds for propagation and preservation of

Spilanthes acmella (L.) Murr., a threatened pesticidal plant species. International Journal

of Plant Developmental Biology, 3:62–64.

Sharma, S., Shahzad, A. and Teixeira da Silva, J.A. 2013.Synseed technology a complete synthesis.

Biotechnology Advance, 31:186–207.

Sharma, A., Tandon, P. and Kumar, A. 1992. Regeneration of Dendrobium wardianum Warner

(Orchidaceae) from synthetic seeds. Indian Journal of Experimental Biology, 30: 747-748.

Shimasaki, K. and Uemoto, S. 1990. Micropropagation of a terrestrial Cymbidium species using

rhizomes developed from seeds and pseudo bulbs. Plant Cell Tissue Organ Culture, 22:237–244.

Shrivastava, V., Khan, S.A. and Banerjee, S. 2009. An evaluation of genetic fidelity of encapsulated

microshoots of the medicinal plant: Cineraria maritima following 6 months of storage. Plant

Cell Tissue Organ Culture, 99:193–198.

Singh, S.K., Rai, M.K., Asthana, P., Pandey, S., Jaiswal, V.S. and Jaiswal, U. 2009. Plant regeneration

from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr, a medicinally important

and herbal pesticidal plant species. Acta Physiologiae Plantarum, 31:649–653.

Singh, S.K., Rai, M.K., Asthana, P. and Sahoo, L. 2010. Alginate-encapsulation of nodal segments

for propagation, short-term conservation and germplasm exchange and distribution of Eclipta alba (L.). Acta Physiologiae Plantarum, 32: 607–610.

Sundararaj, S.G., Agrawal, A. and Tyagi, R.K. 2010. Encapsulation for in vitro short term storage

and exchange of ginger (Zingiber officinale Rosc.) germplasm. Scientia Horticulturae,

125:761–766.

Swamy, M.K., Balasubramanya, S. and Anuradha, M. 2009. Germplasm conservation of patchouli

(Pogostemon cablin Benth.) by encapsulation of in vitro derived nodal segments. International

Journal of Biodiversity and Conservation, 1:224–230.

Tabassum, B., Nasir, I.A., Farooq, A.M., Rehman, Z., Latif, Z. and Husain, T. 2010. Viability assessment

of in vitro produced synthetic seeds of cucumber. African Journal of Biotechnology, 9:7026–7032.

Teixeira da Silva, J.A., Giang, D.T.T., Chan, M.T., SanjayaNorikane, A., Chai, M.L., Chico-Ruız,

J., Penna, S., Granstrom, T. and Tanaka, M. 2007. The influence of different carbon sources,

photohetero-, photoauto- and photo mixotrophic conditions on protocorm-like body organogenesis

and callus formation in thin cell layer culture of hybrid Cymbidium (Orchidaceae). Orchid

Science and Biotechnology, 1:15–23.

Uozumi, N., Nakashimada, K.Y. and Kobayashi, T. 1992. Production of artificial seed from horseradish

hairy root. Journal of Fermentation and Bioengineering, 74:21–26.

Vij, S.P., Kaur, P. and Gupta, A. 2001. Synseeds and their utility in orchids: Dendrobium densiflorumLindl. Phytomorphology, 51:159–165.

Wang, X. 1988. Tissue culture of Cymbidium: plant and flower induction in vitro. Lindleyana

3:184–189.

Zhang, Y.F. and Yan, S. 2011. Factors affecting germination and propagators of artificial seeds of

Dendrobium candidum. International Conference on Agricultural and Biosystems Engineering.

Advances in Biomedical Engineering 1-2: 404-410.

www.jornamental.com


Comparison of Different Pot Mixtures Containing Perlite

on Growth and Morphological Characteristics of Pothos

(Scindapsus aureum L.)

Keywords: Compost, Perlite, Pothos, Pot mixture.

Fatemeh Bidarnamani1 and Hossein Zarei2

1 Horticultural Department in Institute of Agricultural Research, University of Zabol, Zabol, Iran.2 Assistant Professor of Horticulture Department, Gorgan University of Agricultural Sciences and Natural

Resources.


To Select an appropriate medium for the growth of plant is one of the

problems of most greenhouse owners in production of pot ornamentals. So,

current research was conducted to evaluate effect of some available media in

mixtured by perlite on the growth of pothos. Study was based on a completely

randomized design with 5 treatments, 8 measuring times and 6 replications

in a fiberglass greenhouse of Gorgan University of Agricultural Sciences

and Natural Resources during 2009-2010. The treatments include ratios of

perlite+ leaf compost, perlite+ rice husk, perlite+ cocopeat, perlite+ composted

forest trees and perlite+ mushroom compost. Parameters such as plant height,

stem diameter, leaf number, leaf fresh and dry weight and chlorophyll

content were measured. Moreover, plants were compared according to their

overall shape and appearance. The results of data analysis showed that the

effect of medium, measuring time and their interaction were significant in all

traits. The marketing value of pothos including plant height, leaf number and

chlorophyll content, had a better response in perlite+ leaf compost and

perlite+ mushroom compost media.

Abstract


INTRODUCTIONThe production of foliage plants is an important agricultural industry. There is a demand

for tropical foliage plants in homes, hotels, business offices, airports and other public building as

result of human being form natural environments (Dole and Wilkins, 2005).

Pothos (Scindapsus aureum or Epipremnum aureum L.) from Araceae is one of well-known

foliage plants. It requires medium indirect light; tolerated bright light, but the lengthy and direct

light of sun will scorch the leaves (Dole and Wilkins, 2005).

The various substrates are mixed to produce a better medium in cutting production of or-

namentals (Altman and Freudenberg, 1983). However, considerable differences are observed be-

tween the qualities of cuttings grown on various media combinations (Moursy, 2001; Wilson,

1983), depends on the plant species and environmental conditions of the nursery. Although, effects

of different pot mixtures on growth and development of some ornamentals are previously investi-

gated (Verdonck and Gabriels, 1992), there are not such reports for ornamental pothos (Cottenie

et al., 1982).

An appropriate growth medium would provide sufficient anchorage or support to the plant,

serves as reservoir for nutrients and water, allow oxygen diffusion to the roots and gaseous ex-

change between the roots and atmosphere (Abad et al., 2002; Richards and Beardsell, 1986). The

major growth media currently used in Kuwait are perlite and peat moss. There is an interest to

subtitute current media by relatively inexpensive substrates that have a great importance to growers

of the country. In order to reduce cost of imported expensive organic materials to be used in growth

media, it is recommended to extend to a wide range of plant species grown in the growth media

containing higher ratio of sand (Abo-Rezq et al., 2009).

Perlite is recognized to have a unique importance as a superior growing media for hydro-

ponic cultures (Robins and Evans, 2004). It is very useful for increasing aeration and drainage

within the container due to its uniformity and lightness. In addition, because of the physical shape

of perlite particles, it provides a suitable balance between moisture retention and aeration. Adding

cocopeat into perlite enhanced the growth and productivity of gerbera (Paradiso and Pascale, 2008).

In an experiment on pothos plant, it was reported that Leaf number was higher in the media

containing 3:1 leaf-mold/cocopeat mixture. It is concluded that these differences represent a direct

effect on the rooting process and the substrates characteristics have the high importance in the

quality of rooted cuttings (Khayyat et al., 2007).

Awang et al. (2009) indicated that certain chemical and physical properties of cocopeat can

be improved through incorporation of burnt rice hull and its positive effect was clearly reflected

in the growth and development of Celosia cristata.

The shell of almond is also seems to be a good substrate for growing ornamental plants.

Ficus benjamina in mixture of %20 almond shells and %80 peat had the high height, dry and fresh

weight of shoot and root and nitrogen content of foliar (Lao and Jimenez, 2004). Younis et al.(2010) expressed the medium containing equal ratio of sand, silt, leaf compost and spent compost,

showed the best result for the production of croton plants.

Jackson et al. (2005) reported that roots can grow effectively and vigorously in substrates

containing cotton gin compost. For growing Cypress, maximum height was achieved in plants

grown in mixtures containing spent mushroom compost (Benito et al., 2005). So in the present re-

search, a combination of major mentioned mediums were investigated to grow ornamental pothos

in the condition of north of Iran.

MATERIAL AND METHODThis study was carried out at a Quonset fiberglass greenhouse in Gorgan University of Agri-

cultural Sciences and Natural Resources during 2009-2010. The experiment was based on a com-


pletely randomized design with five treatments, eight measuring times and six replications. Treat-

ments werecontained equal volume ratio of substrates, m1: perlite+leaf compost, m2: perlite+rice

husk, m3: perlite+ cocopeat, m4: perlite+ spent mushroom compost and m5: perlite+ composted

bark of forest trees. Measurements were performed in eight times from December 2009 till July

2010 (a measurement of factors in each month).

The substrates were provided in volume ratio and three uniform pothos plantlets were trans-

planted into each pot with mouth diameter of 21 cm). Parameters such as plant height, stem diam-

eter, leaf number, leaf fresh and dry weight and chlorophyll content were measured; and finally

the plants were compared according to their overall visual appearance by a score of 1 to 10. During

the study were used no fertilizer or nutrition materials. Irrigation and mist were carried out handy

and uniformly for all treatments. The plants were irrigated each 10 days in cool months and each

4-7 days in warm months the pH and EC of was irrigation water 6.55 and 642 μs/cm, respectively).

A sunshade of 30 percent was used for protecting plants from sunburn in warm months. All

achieved data were analyzed using SPSS software.

The purpose of this study was to investigate the influence of growing substrates composition

on growth and development of a popular indoor plant, golden pothos, and determination the effect

of pot mixtures on the morphological characteristics of pothos.

RESULT AND DISCUSSIONThe measurement of temperature showed the minimum temperature of the inside of green-

house was about 12-14 ºC during cold season and 17 ºC and 25 ºC in spring and early summer, re-

Media N (%) Mg (mg/kg) K (mg/kg) P (mg/kg) Fe (mg/kg)

PerliteLeaf MoldRice HuskCocopeatMushroom CompostComposted Bark of Forest Trees

0.0391.692.043.041.113.34

140360160880680

1060

182528671917880784

1121

19021

25032

4.416.423.042.82.6

20.3

Table 1: The amount of nutrients in the used substrates

Fig. 1. Height of pothos during the time in different pot mixtures


spectively. The humidity was retained 30-40% in greenhouse in all months comparatively. table 1

showed the amount of nutrients in the composition of the used substrates.

Analysis of potted medium reflected that a low nutrients medium, with low water-holding

capacities, can be amended with different organic materials with at different rates (Younis et al.,2010).

Plant heightData analysis showed that the effect of media, time and their interaction on plants height

were significant.

Interaction diagram shows that m1 substrate caused the longest plants height in all meas-

uring months. The highest values of plant length (373 cm) had been seen in m1 treatment. Accord-

ing to Figure 1; the m1 medium had 250 cm growth only in final stage of measurement (July 2010).

Current research showed that nitrogen content of substrate is important for increase in the plant

growth. Substrate analysis showed that there is the most amount of N in cocopeat and composted

forest trees, respectively, whiles the mixture of them is not caused to favorable condition in the

growth of plant.

Figure 2 showed that the lowest height obtained at m5 medium containing forest trees bark

compost+ perlite. It seems that the fresh organic material in forest trees compost caused to decrease

Fig. 3. The change of height at different times of year

Fig. 2. Effect of different media on height of pothos plant


the growth in this treatment. The plants cultivated in this substrate had no the desirable growth.

Effect of time on pothos growth showed that the height increased during December to July

(Figure 3.) The growth increased slowly from December to March, but the plant height increase

quickly after gradual warming of weather and increasing light level in April. The reason of this

theorem is clear, because the pothos is a tropical and native plant in southeastern Asia (Malaysia

and Indonesia) and New Guinea (Abd-El-Hadi and Shanan, 2010). It has the slight growth in cool

and cloudy days of winter, but after April, the length plants of increases quickly due to air warming

and higher light level into the greenhouse area; so the height of plants increased up to 126 cm just

in July month. The difference of pothos height increases in December to March wasn’t significant

but in three final month, they had significant variations.

Although pothos plant had the better growth in warm seasons, but in addition to warm tem-

perature, air ventilation is necessary for optimal growth and lack of ventilation may damage to fo-

liage plants: moreover, because of increase in respiration the plant receives sufficient water

(GhasemiGhehsareh and Kafi, 2009).

Leaf numberThe presence of leaves on the cuttings may reflect earlier growth of root system, but the

other environmental factors can also be involved (Khayyat et al., 2007). Data analysis showed that

the effect of media, time and their interaction on leaf number were significant. Results of figure 4

showed that substrate m1 was better than other pot media on leaf number, and July month caused

the maximum new leaf number.

According to Figure 5; among various pot media, m1 and m2 had the maximum and mini-

Fig. 4. Effect of different media and measuring time on newleaf number in pothos

Fig. 5. Effect of different pot mixture on new leaf number inpothos


mum of new leaf number, respectively. There wasn’t significant difference between m2 and m4;

also between m3 and m5 mixtures. But there were significant differences between m1 and m2 and

m4.

The effect of measuring time on new leaf number showed that new leaf number in each

measurement stages were increased from December until July. It was due to gradual change of

warm and light condition in greenhouse. In March month because of sudden cold and cloudy at-

mosphere, new leaf number and height was lower than previous month. According to the Figure

6; total new leaf number from December till May was the same of June (similar to height). Thus

economic times for pothos production are after May month. Appearance of new leaf on the plant

was due to more photosynthesis to produce new leaf.

Stem diameterThe result of data analysis showed that the effect of media, time and their interaction on

stem diameter were significant.

Figures 7 and 8 show the effect of time and treatments on stem diameter of pothos. Effect

of different pot mixtures onstem diameter factor (Figure 8) showed that m4 and m5 caused the

thicker stem diameter, while these media had no appropriate effect on height and new leaf number.

Benito et al. (2005) represented no differences in stem diameter in cypress affected by the growing

media, which is in controversy with the finding of current experiment on pothos.

According to Figure 9, increase in stem diameter was ascending from December until

March, whiles there is not an obvious change from April to July. In primary months of measuring,

because of undesirable weather for plants growing (due to cool air and low light), the plants gen-

Fig. 6. The change of new leaf number at different times ofyear

Fig. 7. Effect of different pot mixture and measuring time onstem diameter of pothos


erated little leaf, but it increases the stem diameter. The plants increase their diameter for resistance

against unfavorable situations (temperature decline, low light and etc.) but during weather warm-

ing, the plants produced new leaf and increase the height instead of adding diameter.

ChlorophyllThe result of Figure 10 showed that medium m1 had the heighest chlorophyll, but it had

not significant difference with medium m4. The results of Ebrahimi et al. (2012) showed that co-

copeat + perlite substrates had the most effect on chlorophyll a, chlorophyll b, total chlorophyll,

and carotenoid in the old and young leaves of strawberry.

One of the physiological reasons of decreasing growth may be due to a disorder in the plant

photosynthetic system. One of the ways to study disorders in photosynthesis is chlorophyll fluo-

rescence (Soltani, 2004).

For synthesis of chlorophyll molecules, nitrogen and magnesium are necessary. Cocopeat

and forest trees bark compost had the highest nitrogen and magnesium in compared with other

substrates, but perlite+ cocopeat and perlite+ forest trees bark compost substrates had the minimum

level of chlorophyll content.

Also the result of Hasanpur et al. (2009) on Lilium’s flower showed substrate had not the

significant effect on chlorophyll index, that it accordance with result of current experiment.

Leaf areaThe result of data analysis showed that the effect of media, time and their interaction on

leaf area, leaf fresh and dry weight were significant. Figure 11 showed the media containing leaf

mold and spent mushroom compost had the most leaf area because of the most leaf number. But

the media containing perlite + composted bark offorest trees and perlite+ cocopeat which had min-

Fig. 8. Effect of different pot mixture on stem diameter ofpothos

Fig. 9. Effect of different pot mixture and measuringtime on stem diameter of pothos


imum leaf number; they had the minimum leaf area too.

Matsiak and Nowak (1998) on Ficus benjamina and Khayyat et al. (2007) on Pothos plants

found that the greatest leaf area was obtained in peat moss medium mixed with different media.

Treder et al. (1999) mentioned that the greatest leaf area of different species of ficus plants obtained

in peat moss media.

In Khayyat et al. (2007) report, higher leaf area in pothos was observed in the medium of

peat moss/cocopeat (1:3 ratio). However, no significant differences were shown between recent

media and cocopeat/peat moss (1:1 ratio) and cocopeat media.

Morphological evaluationAt the end of study, it was computed a numerical value to each plant according to its overall

morphological appearance by using Amerin method (Amerin et al. 1965). For this evaluation, four

characteristics were used: plant length, leaf color, leaf size and uniformity in growing leaves. Each

factor contained maximum grade of 2.5 and all four factors may cause the maximum score level

of 10. These numbers were then used for the comparison and determination of the best and worst

media for pothos in this trial.

Finally the comparison between various substrates showed that media m1 and m4 were the

best media in this trial. They caused similar results for higher new leaf number and higher length.

While the media m3 and m5 were worst media because of little growth even after 8 months. The

Fig. 10. Effect of different media on pothos leaf chloro-phyll content

Fig. 11. Effect of different media and measuring time dur-ing the year on pothos leaf area


media containing perlite with 5 composted bark of forest trees or cocopeat were not caused to a

suitable growth of pothos, because the plants could not uptake necessary N, Mg and Fe elements,

due to insolubility or unavailability of elements in the final pot mixture. Accordingly, it was ob-

served the decrease in height, leaf number and chlorophyll. So the plants with suitable growth had

better appearance and sale price.

Literature CitedAbad, M., Noguera, P., Puchades, R., Maquieira, A. and Noguera, V. 2002. Physico-chemical and

chemical properties of some coconut dusts for use as a peat substitute for containerized ornamental

plants. Bioresource Technollogy, 82: 241-245.

Abd-El-Hadi, M. and Shanan, N. 2010. Enhancement growth characters of Pothos plants (Epipremnum aureum Lindl.) grown in different improved pot media. International Journal of Academic

Research. 2(2): 89-97.

Fig. 12. Effect of different media on morphologicalevaluation of pothos

Fig. 13. Pothos plants in perlite + forest trees compost as worst media (in left side), perlite +leaf mold as best media (in right side)


Abo-Rezq, H., Albano, M. and Thomas, B. 2009. The effect of sand in growing media on selected

plant species. European Journal of Scientific Research. 26(4):618-623.

Altman, A. and Freudenberg, D. 1983. Quality of Pelargonium graveolens cutting as affected by the

rooting medium. Science Horticulturae, 19: 379-385.

Amerin, M. A, Pangborn, R. and Rossler, E.B. 1965. Principles of sensory evaluation of food. Academic

press, Inc. New York. 602p.

Awang, Y., ShazmiShaharom, A., RosliB.Selamat, M. 2009. Chemical and physical characteristics

of cocopeat-based media mixtures and their effects on the growth and development of

Celosia cristata. American Journal of Agricultural and Biological Sciences. 4 (1): 63-71.

Benito, M., Masaguer, A., De Antonio, R. and Moliner, A. 2004. Use of pruning waste compost as

a component in soilless growing media. Bioresource Technology. 95(5): 597-603.

Cottenie, A., Verloo, M., Velghe, G. and Aerlynk, R. C.O. 1982.Chemical analysis of plant and

soil. Laboratory of Analytical and Agro-Chemistry State Univ., Ghent, Belgium.

Dole, J.M. and Wilkins, H.F. 2005. Floriculture principles and species. (2nded.). Person Education,

Inc., Upper Saddle River, New Jersey.

Ebrahimi, R., Ebrahimi, F. and Ahmadizadeh, M. 2012. Effect of different substrates on herbaceous

pigments and chlorophyll amount of strawberry in hydroponic cultivation system. American-

Eurasian journal of Agriculture & Environment Science. 12(2): 154-158.

Jackson, B.E., Wright, A.N., Cole, D.M. and Sibley, J.L. 2005. Cotton gin compost as a substrate

component in container production of nursery crops. Journal of Environmental Horticulture,

23(3):118–122.

Khayyat, M., Nazari, F. and Salehi, H. 2007. Effects of different pot mixtures on pothos (Epipremnum aureum Lindl. and Andre ‘Golden Pothos’) growth and development. American-Eurasian

Journal of Agriculture and Environmental Science, 2 (4):341-348.

Lao, M.T. and Jimenez, S. 2004. Evaluation of almond shell as a culture substrate for ornamental

plants Ficus benjamina. International Journal of Experimental Botany.79-84.

Matsiak, B. and J. Nowak, 1998.Acclimatization and the growth of Ficus benjamina micro cuttings

as affected by carbon dioxide concentration. Journal of Horticultural Science and Biotechnology,

73:2, 185-188.

Moursy, K.S. 2001. Effect of some prepared organic media on plant growth in newly reclaimed

soils. Ph.D. Thesis Agric. Fac. Cairo Univ. Egypt.

Richards, D.M.L. and Beardsell, D.V. 1986. The influence of particle-size distribution in pinebark:

sand: Brown coal potting mixes on water supply, aeration and plant growth. Science Horticulturae,

29: 1-14.

Robbins, J.A. and Evans, M.R. 2004. Growing media for container production in a greenhouse or

nurseries. Part 1: Components and mixes. agriculture and natural resources. Division of

Agriculture: University of Arkansas, Fayetteville.

Soltani, A. 2004. Chlorophyll fluorescence and its application.Internal Press.University of Agricultural

Science and Natural Resource, Gorgan, Iran.

Treder, J., Matysiak, B., Nowak, J. and Papadopoulos, A.P. 1999. The effects of potting media and

concentration of nutrient solution on growth and nutrient content of three Ficus species cultivated

on ebb-and flow benches. Acta Horticulturae, 481: 433-439.

Verdonck, O. and Gabriels, R. 1992. I. Reference method for the determination of physical properties

of plant substrates. II. Reference method for the determination of chemical properties of

plant substrates. Acta Horticulturae, 302: 169-179.

Wilson, G.C.S. 1983. The physico-chemical and physical properties of horticultural substrates.

Acta Horticulturea, 150: 19-32.

Yonis, A., RiazAtif, R., Waseem, M., Asif Khan, M. and Nadeem, M. 2010. Production of Quality

Croton (Codiaeum variegatum) plants by using different growing media. American-Eurasian

Journal of Agricltural and Environmental Science, 7(2): 232-237.


Combined Effect of Humic Acid and NPK on Growth

and Flower Development of Tulipa gesneriana in Faisalabad,

Pakistan

Keywords: Flower quality, Humic acid, Nutrient uptake, Plant growth, Tulip cut flower.

A. Ali1*, S.U. Rehman2, S. Raza3 and S.J. Butt4

1 Institute of Horticultural Sciences, University of Agriculture, Faisalabad-Pakistan2 Organic Plant Production and Agroecosystems Research in the Tropics and Subtropics, University of

Kassel, Steinstr. 19, D-37213 Witzenhausen-Germany3 Institute of Soil & Environmental Sciences, University of Agriculture, Faisalabad–Pakistan4 Department of Horticulture, PMAS-Arid Agriculture University, Rawalpindi-Pakistan


An experiment was conducted in green house during 2011-2012 to

elucidate the impact of application of NPK alone and in combination with

different levels of humic acid (HA) and also to identify which combination is

optimum to improve growth and floral attributes of Tulip. Five treatments

comprising T0: (control), T1: 10g/m2 NPK (17:17:17), T2: HA 0.75ml (8%) +

10g/m2 NPK (17:17:17), T3: HA 1.00 ml (8%) + 10g/m2 NPK (17:17:17) and

T4: HA 1.25ml (8%) + 10g/m2 NPK (17:17:17) were replicated thrice and

arranged following Randomized Completely Block Design (RCBD). All the

vegetative and reproductive parameters were significantly enhanced by the

addition of humic aid and NPK. The obtained results revealed that treatment

T4 was the most effective compared with the other treatments and gave the

inimitable outcomes concerning earliest sprouting and flowering, plant height

increment, leaf area expansion, stem diameter, leaf chlorophyll contents, stalk

length, vase life, fresh and dry flower biomass. T4 also showed the maximum

nutrient contents followed by T3 and T2 when compared with control. The

plants grown without HA and NPK application (control) followed by a single

application of NPK (T1) exhibited poor growth with reduced yield of inferior

quality. Combined application of HA and NPK is recommended to get

improved and uniformed crop stand, plant growth and flower quality.

Abstract


INTRODUCTIONTulips are one of the most popular springs of all time and ranked third most popular flowers

world-wide next to Rose and Chrysanthemum in the cut flower trade (Van, 1999). The Netherland

has prominent status by largest bulb grower and exporter in the world with about 65% of the total

sales of flowers and plants (Schneider, 1991). Tulips are the top most liked flowering geophytes’

of the Netherland and occupies the largest area under any true bulb crop in the world followed by

Narcissus, Iris, Hyacinthus and Lilium (Khan et al., 2006).

Production of cut flowers in Pakistan is estimated at about 10-12 thousand tons per annum

(Khan, 2011) and Pakistan has tremendous potential for cultivation of floral crops on commercial

scale due to availability of favorable soil climate and geographic location (Manzoor et al., 2001).

The unique natural advantage to this country is the seasonal diversity in producing and supplying

the best quality of cut tulip and other flowers during the winter months’ time of the European coun-

tries. Europe experiences extreme shortage of cut flowers in winter because of frost, fog and snow

causing reduction in flower yield, quality and production and depend largely on import to meet

their domestic demands for spiritual and traditional festivities like Christmas, Valentine, Mother’s

Day and Happy New Year celebrations. Pakistan has strong potential to earn foreign exchange

with minimum cost of production by raising and improving the standards of our floricultural pro-

duce (Bukhari, 2005).

Humic based fertilizers and mineral nutrients are the excellent combination which provides

the ideal environment for plant growth and development (Mawgoud et al., 2007; Sara et al., 2010).

The use of humic acid (HA) is a promising natural resource to be utilized as an alternative for in-

creasing crop production. It makes important contributions to improve soil stability and fertility,

also improves flower quality that lead to exceptional plant growth and nutrient uptake (Knicker etal., 1993). Humic acid is an effective agent to be used as a complement to fertilizer and is mostly

recommended for soil reclamation and reduces the harmful effects of synthetic fertilizers. It also

has the potential for the economization of water and fertilizers. Approximately 65-70% of organic

matter in the soil is mainly derived from humic and fulvic acid substances (Khristeva, 1953). Za-

ghloul et al. (2009) examined the effect of potassium humate on vegetative growth and chemical

constituents of ornamental plant Thuja orientalis L. and concluded that plant height, stem diameter,

root length, fresh and dry weights of shoots and roots and NPK contents were significantly im-

proved by application of K-humate. Nikbakht et al. (2008) revealed that exogenous application of

humic acid improved NPK and chlorophyll contents in leaves as well as flower quality, flower di-

ameter, flower longevity, plant height, nutrient uptake and prolonged the vase life of Gerbera

flower. Evans and Li (2003) confirmed positive effect of humic acid on the growth of annual or-

namental seedling like Pansy, Marigold, Geranium, Vinca and Impatiens. All vegetative and floral

parameters were significantly improved by increasing the HA concentration. Dore and Peacock

(1997) also concluded that humic substances act as a soil conditioner for the growth of turf grass

and improved root growth. As a consequence, the use of humic substances has often been proposed

as a method to improve crop production.

Keeping in view the importance of Tulip in global cut flower industry, a research project

was arranged to determine the potential of varying levels of HA in combination with NPK on plant

growth, nutrient contents, flower development and post-harvest life of Tulip.

MATERIALS AND METHODSResearch work was conducted in the green house, Rose Research Area, Institute of Horti-

culture Sciences, University of Agriculture, Faisalabad during 2011-2012 (latitude 31°30 N, lon-

gitude 73°10 E and altitude 213 m) in order to evaluate the effect of application of NPK and humic

acid (HA) at varying levels on growth, yield and quality of cut flower Tulipa gesneriana L. viz.

Triumph. Five treatment plan with three replication was designed in this experiment.


TreatmentsControl T0

10 g/m2 NPK (17:17:17) (2 Applications) T1

0.75 ml of 8% HUMIC ACID + 10 g/m2 NPK (17:17:17) (2 Applications) T2



The treatments were laid out following Randomized Completely Block Design (RCBD).

Before the start of the experiment, soil samples from various blocks of experimental field were

collected and analyzed for various physico-chemical properties (Table 1). The soil was thoroughly

tilled, levelled and all necessary cultural practices (weeding, plant protection measures, earthing

up etc.) were done before plantation. Treatment materials (HA and NPK) were obtained from the

Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad, Pakistan ex-

tracted from organic residues and macronutrient combinations.

The planting material (corms) was obtained from “Green works Pvt. Limited, Lahore” an

importer of “Stoop Flower Bulb Company, Holland”. The corms were stored at 4±1°C and were

taken out seven days before planting and kept in a laboratory at ambient temperature. Corms were

planted during the end week of November, 2012 according to the layout of the experiment with

10 cm plant to plant distance on 60 cm spaced ridges. Seven corms were planted in each treatment

and each treatment was replicated thrice with a total of 105 corms used in the study. The first ap-

plication of humic acid and NPK fertilizers were applied at the time of planting bulbs while the

second dose was applied at 3-leaf stage. All other cultural practices like weeding, plant protection

measures, staking, earthing up etc. were kept same for all treatments during entire period of study.

Plants were allowed to grow and data regarding growth, flowering and corm indices were collected

by using standard procedures.

STATISTICAL ANALYSISThe data pertaining to various parameters was analyzed by ANOVA technique using Sta-

tistics 8.1® computer based software and significance of means was tested according to least sig-

nificant difference test (LSD) at 5 % probability (Steel et al., 1997).

RESULTSThe data collected revealed that all the growth and yield parameters exhibited similar trend

throughout towards the application of humic acid (HA) and NPK and responded positively with

improved plants growth when compared with control (T0). Among all the treatments T4 was the

most effective giving plants with maximum growth and development while the plants grown with-

out HA and NPK application (control) followed by a single application of NPK (T1) exhibited poor

growth with reduced yield of inferior quality.

The data regarding number of days to sprouting and sprouting percentage (Table 2) showed

Parameter Unit Value

pHsECeOrganic MatterNitrogenPhosphorusPotassiumZinc

---dS m-1

%mg kg-1

mg kg-1

mg kg-1

mg kg-1

8.792.470.6611.579.62041.9

Table 1. Physical and chemical properties of soil used forexperiment.


significant outcome towards application of HA and NPK. Earliest sprouting (47.14 days) and max-

imum sprouting percentage (97.29%) was recorded in plants which received highest concentration

of HA along with NPK (T4) while application of NPK alone (T1) took maximum number of days

(55.19 days) and gave minimum sprouting percentage (88.41%) compared with control. Similar

pattern was observed in case of plant height. Maximum (40.27cm) and minimum (33.61cm) plant

height was recorded in plant receiving treatments T4 and T1 respectively (Table 2).

Mean comparison of treatments for leaf area and stem diameter (Table 2) depicted the su-

periority of T4 over all the other treatments by giving maximum leaf area (149.57cm2) and pro-

ducing highest stem diameter (10.06mm). In contrast, the least leaf area (135.84cm2) and stem

diameter (5.04mm) was observed in T1. These results revealed that treatments are highly significant

with respect to leaf area and stem diameter of tulip statistically and these attributes increased with

increase in humic acid application that advocated the importance of HA for stem diameter.

Data pertaining to stalk length and days to flower emergence (DAP) presented in Table 3

also indicated the highly significant dominance of T4. Maximum (38.15cm) and minimum

(33.36cm) stalk length was examined in the treatments T4 and T1 respectively as compared to con-

trol T0 (31.54cm). Minimum number of days for flower emergence (123.66 days) were taken by

the plants of treatment T4 followed by T3 (125.42 days), T2 (127.42 days) and T1 (133.64 days) re-

spectively compared with control T0 (136.73 days). Significant progress was also examined in case

of vase life and findings clearly confirmed the supremacy of T4 over other treatments. Maximum

vase life was produced by T4 (8.66 days) plants followed by T3 (6.42 days), T2 (5.71 days) and

least vase life was recorded in T1 (4.90 days) compared with control T0 (3.00 days).

Mean values obtained for chlorophyll contents (SPAD value) of leaves are presented in

Table 3. The result indicated that soil application of humic acid along with macronutrient gave

highly significant (p <0.05) results on leaf chlorophyll contents of Tulip. The highest value was

achieved in T4 (45.62 SPAD) with rest of other treatments giving lower values. Data regarding

tepal diameter also exhibited highly significant results with highest tepal diameter recorded in T4

(10.51mm) while minimum in case of T1 (6.64mm) with reference to control T0 (6.28mm).

The results in Fig. 3 showed clear positive significant trend in fresh and dry biomass with

Treatment Sprouting

days

Sprouting

percentage

Plant

height (cm)Leaf area

(cm2)

Stem diameter

(mm)

T0

T1

T2

T3

T4

58.43 a

55.19 b

52.67 c

49.81 d

47.14 e

85.59 e

88.41 d

91.53 c

94.67 b

97.29 a

29.39 e

33.61 d

36.61 c

38.15 b

40.27 a

130.14 e

135.84 d

140.86 c

145.28 b

149.57 a

4.53 e

5.04 d

6.20 c

8.28 b

10.06 a

Means in columns followed by the same letters are not significantly different at α≤0.05 level according to LSD test.

Table 2. Effects of different humic acid levels on days to sprouting, sprouting percentage, plant height,leaf area, stem diameter of tulip flower ‘Triumph’.

Treatment Stalk length

(cm)

Days to flower

emergence (DAP)

Vase life

(days)Leaf chlorophyll

contents (SPAD)

Tepal diameter

(mm)

T0

T1

T2

T3

T4

31.54 e

33.36 d

35.69 c

36.11 b

38.15 a

136.73 a

133.64 b

127.42 c

125.42 d

123.66 e

3.00 e

4.90 d

5.71 c

6.42 b

8.66 a

39.02 e

36.34 d

40.76 c

42.68 b

45.62 a

6.28 e

6.64 d

7.33 c

8.03 b

10.51 a

Means in columns followed by the same letters are not significantly different at α≤0.05 level according to LSD test.

Table 3. Effects of different humic acid levels on stalk length, days to flower emergence, vase life, leafchlorophyll contents, tepal diameter of tulip flower ‘Triumph’.


increase in the concentration of humic acid along with NPK. Treatment T4 demonstrated the highest

fresh weight (34.67g) and dry weight (6.88g) of flower which was statistically superior to other

treatments. The lowest observation for both measured traits was recorded in T1 and control T0.

It is evident from the data (Table 4) that all minerals contents (N, P and K %) under inves-

tigation were gradually increased by increasing humic acid concentrations. The observation re-

garding the nutrient uptake was statistically analyzed and noted that T4 has got the maximum

nitrogen (3.11%), highest phosphorus contents (2.95%) and abundant available potassium contents

(4.24%) followed by T3, T2 and T1 compared with control T0 which had 0.97% N, 0.21% P and

2.32% K respectively.

DISCUSSIONThis study focused on the response of tulip flower for different humic acid concentrations

Fig. 1. The fresh and dry biomass of flowers of Tulipa gesneriana L.‘Triumph’ as influenced by humic acid application.

Treatment Dry matter

(%)

Leaf N contents

(%)

Leaf P contents

(%)Leaf K contents

(%)

T0

T1

T2

T3

T4

12.30 c

10.45 d

12.14 c

15.40 b

19.84 a

3.23 e

3.70 d

4.40 c

5.23 b

5.97 a

0.21 d

0.84 c

1.15 c

2.01 b

2.95 a

2.32 d

2.52 c

2.76 c

3.00 b

4.24 a

Means in columns followed by the same letters are not significantly different at α≤0.05 level according toLSD test.

Table 4. Effects of different humic acid levels on dry matter, N, P and K contents in leaves oftulip flower ‘Triumph’.


along with NPK to recognize the best treatment for maximum plant growth, nutrient uptake and

vase life of flower. Significant role of humic acid and NPK was determined in greenhouse exper-

iment under ecological Faisalabad’s soil condition. The absolute findings of the experiment are in

line with and verified by the findings of multiple researchers and is given as under categorically.

Vegetative growth responseThe ingredients for combined treatment of humic acid and NPK used in this research played

a crucial role in plant growth and development of Tulip. Seadh et al., 2012 in their findings con-

firmed the luminous efficacy of the humic acid and NPK on vegetative growth and flower devel-

opment. Early growth and maximum germination percentage have been observed in our study with

increased concentration of humic acid and NPK. These results are in line with the findings of Dixit

and Kishore (1967) who reported that humic acid is growth promoter that makes the seeds possible

to sprout earlier and reduce the mean germination time in a number of species including ornamental

seeds. Miroslava (1962) findings are also in line with our results that sodium humate at a concen-

tration of 100 mg l-1 accelerates the uptake of water by the swelling seed during the initial stage of

imbibition. The fact that the seed takes up a sufficient amount of water sooner make it possible for

the activation of enzyme systems which ensure normal germination to take place. So it may be the

reason for the increased sprouting percentage.

In the lights of results of this study, a gradual increment in plant height, leaf area and stem

diameter was observed with an increase in the concentration of humic acid application. These ob-

servations are in accordance with the findings of Turkmen et al., 2004 who reported significant

effect of different levels of humic acid (0, 500, 1000 and 2000 mg kg-1) on plant height and vege-

tative growth for stem elongation. Basbag (2008) stated that plants provided with the highest dose

of HA @ 150 ppm produce maximum leaf expansion. It was also reported by Atiyeh et al., 2002

that there is improvement of nutrient uptake especially of nitrogen, phosphorous and sulfur and

also some hormones by the activity of HA. These results may be due to the combined application

of HA and macronutrients which helps to transport vital sugars through plant membranes and pro-

motes proper cell division, cell wall formation and also acts as an enzyme activator in protein and

hormones (Mahgoub et al., 2010).

Stalk length is a well-known attribute in bulbous cut flowers. Our results revealed increment

in stalk length under increased application of humic acid along with macronutrients (NPK). The

results suggested that application of HA and NPK in combination produced more effect than their

single applications. This may be due to improvement of plant growth in terms of stem elongation

by hormone like activity of HA. These results are in harmony with those of Turkmen et al. (2004)

who reported the significant effect of different levels of HA on stem length improvement. This is

well documented by many other researchers (Arancon et al., 2003; Atiyeh et al., 2002). Padem

and Alan, 1995 observed that combined NPK application may elevate the nitrogen, potassium and

phosphorus contents. The increase in stalk length might be due to elevated levels of macronutrients

and humic acid which have a positive effect on floral characteristics.

Leaf chlorophyll contents (SPAD) responseIt was concluded that among HA treatments, the treatment containing highest concentration

of HA along with NPK had more leaf chlorophyll contents as compared to rest of the treatments.

The increase in chlorophyll contents might be due to improvement in nitrogen assimilation from

NPK (Minotti et al., 1994). The beneficial effect of HA on photosynthetic pigments may be due

to its role in increasing the rates of photochemical reduction (Coco and Agnolla, 1988). These re-

sults are in agreement with Visser (1986) who reported that photosynthesis efficiency and chloro-

phyll contents were significantly increased in humus treated plants. These humic substances were

able to support electron transport so as to increase photosynthesis efficiency.


Reproductive growth responseOur findings concluded that minimum number of days to flower initiation were taken by

the plants which received increased application of HA along with NPK. The results showed that

the combination of HA and NPK induced earlier flowering. Balanced nutrition is one of the best

practices to get early flowering in plants (Kumar and Haripriya, 2010). Ricardo et al., 1993 ob-

served that the application of HA may lead to improved production of marigold bedding plants

and earlier flowering. The application of the HA and nutrients increase the root portion of the

plants which enhance rooting and growth performance of the bedding plants.

The vase life of tulip plants increased significantly under increasing dose of humic acid

treatments. Similar results were painted by Kulikova et al., (2005) who reported that HAs have

auxin-like activity that enhance the nutrient uptake which may be responsible for the good floral

growth. Khenizy et al., 2013 also reported that HA plays vital role for maximum water uptake and

ultimately prolong the vase life in Gerbera cut flowers.

Tepal diameter also responded with similar trend upon application of treatments and max-

imum diameter was recorded at highest dose of HA along with NPK (T4). Combined effect of

humic substances along with macro-nutrients has a vital role in the growth and development of

strawberry plant described by Pilanali et al., 2003.

Fresh and dry weight responseFresh and dry weights of flower are important indicators with reference to plant growth.

The application of HA and NPK contents increases vegetative growth which in turn resulted in

higher assimilate production (carbohydrates and proteins) which might have been utilized for better

development of plants (Shanmugam and Veeraputhran, 2001). The results revealed that treatments

are highly significant and are statistically at par. Fresh and dry weight of the flower (g) increased

with an increase in the HA application showed the importance of HA for yield. The results of the

present study are harmonious with those of Arnacon et al., 2003; Kauser et al., 1985; Azarmi etal., 2008; Kumar et al., 2003. They all reported that plants treated with HA and NPK showed more

fresh and dry weights for different crops and also increased the total carbohydrates which may

also be due to more availability of nitrogen and phosphorus which increase leaf photosynthetic

rates and protein synthesis.

Nutrient uptake responseHumic acids promote the conversion of mineral nutrients into available forms for plants.

Our findings claim that nutrient absorption by plants became significantly higher when HA and

NPK was applied together. Maximum nutrient accumulation was recorded in the leaves of plants

which received maximum concentration of HA with NPK.

Nitrogen is primarily responsible for vegetative growth. Nitrogen assimilates into amino

acids and protein and is also a component of chlorophyll and is required for several enzyme reac-

tions. Magda, (2003) elaborated that nitrogen contents increased with increase in HA and NPK

application in squash plant. These results are also in line with the findings of Chen and Aviad.

(1990) who reported significant differences in Nitrogen uptake by leaves.

Phosphorus is a major component in plant DNA and RNA. Phosphorus is also critical in

root development, crop maturity and seed production. Increase in the accumulation of phosphorus

under HA and NPK application was also reported by Shehata et al., 2011. They observed that HA

plays vital role in nutrient accumulation to roots and hence phosphorous absorption rate increased

by HA application at critical growth stages.

Potassium increases water use efficiency and transforms sugars to starch in the grain-filling

process. It's important for a plant's ability to withstand extreme cold and hot temperatures, drought

and pests. The accumulation of K in plant leaves become higher by increasing HA and NPK ap-


plication. Homogenous results showing the positive co-relationship between the doses of HA and

the potassium contents of the leaves were observed by Nikbakht et al. (2008) and Chen et al.(2004).

CONCLUSIONThe combined effect of application of varying levels of HA along with NPK evaluated on

tulip cultivar viz. Triumph revealed that addition of humic acid along with macro nutrient mixes

(NPK) had a positive effect on the growth of Tulipa gesneriana L. The magnificent role of HA in

this study has been observed as a greatest growth enhancement tonic especially for the cut flower

industry. The scientific findings related with this particular field are very rare; however a conclusive

clue has emerged in this experiment for further research. Plant growth gradually increased with

increasing the concentration of HA with NPK. All the vegetative and floral growth production was

vigorous without soil and environmental degradation. The potential exists for growers to use HA

along with macro-nutrients on a commercial scale in order to achieve better growth and flower

quality in tulip.

Literature CitedAlbuzio, A.G., Concheri, S., Nardi, G. and Agnola, D. 1994. Effect of humic fraction of on the development

of oat seedlings grown in varied nutritional condition. In: Senesi, N. & Mianom, T. M (eds),

Humic substances in the global environment and implications on human health. Elsevier

Science, Amsterdam. 7: 199-204.

Arancon, N.Q., Lee, S.C., Edwards, A. and Atiyeh, R. 2003. Effect of humic acids derived from

cattle, food and paper-waste vermicompost on growth of green house plants. Pedobiologia.

47: 741-744.

Atiyeh, R. M., Edwards, C.A., Metzger, J.D., Lee, S. and Arancon, N.Q. 2002. The influence of

humic acids derived from earth worm processed organic wastes on plant growth. Bioresource

Technollogy, 84: 7–14.

Azarmi, R., Ziveh, P. S. and Starri, M. R. 2008. Effect of humic acid derived from vermicompost

on growth, yield and nutrition status of tomato (Lycopersicon esculentum L.). African Journal

of Biotechnology, 7: 2397-2401.

Basbag, S. 2008. Effects of humic acid application on yield and quality of cotton (Gossybium hrisutum L.). Asian Journal of Chemistry, 20: 1961- 1966.

Bukhari, R.A.S. 2005. Development of sustainable commercial floriculture in Pakistan. Proceedings

of the National Seminar on Streamlining: "Production and Export" of Cut-Flowers and

House Plants, March 2nd to 4th, 2005. Horticultural Foundation of Pakistan, Islamabad,

Pakistan.p 105.

Chen, Y. and Aviad, T. 1990. Effects of humic substances on plant growth. In: humic substances

in soils and crop science (Ed. P.R. Malcom and P.R. Bloom), Sci. Soc. of Amer. Madison.

p. 161-186.

Chen, Y., Clapp, C. E. and Magen, H. 2004. Mechanisms of plant growth stimulation by humic

substances: The role of organo-iron complexes. Soil Science and Plant Nutrition, 50: 1089–1095.

Coco, G. and Agnella, G.D. 1988. Plant growth regulator activity of soluble humic complexes.

Canadian Journal of Soil Science, 64: 225-228.

Dixit, V. K, and Kishore, N. 1997. Effect of humic acid and fulvic acid fraction of soil organic

matter on seed germination. Indian Journal of Science, 1: 202-206.

Dore, S. P. and Peacock, C.H. 1997. The effect of humate and organic fertilizers on establishment

and nutrition of creeping bent grasses. International Turfgrass Society, 8: 437-444.

Evans, M. R. and Li, G. 2003. Effect of humic acids on growth of annual ornamental seedling

plugs. Hort Technology, 13: 4-9.


Kauser, S., Mali, A. and Azam, F. 1985. Effect of humic acid on corn seedling growth. Environmental

and Experimental Botany, 25: 245-252.

Khan, M. J. 2011. Horticulture in Pakistan: planning perspective. Presentation at International

Seminar on Horticulture and Agribusiness in Developing Countries at UAF. p 1-17.

Khan, F.U., Jhon, A.Q., Khan, F.A and Mir, M.M. 2006. Effect of NPK and Zn on growth, flowering

and bulb production in tulip under polyhouse conditions in Kashmir. Journal of Horticultural

Science, 1(2): 129-134.

Khenizy, S. A., Amal, M. A. and Yasser, M. E. 2013. Effect of humic acid on vase life of gerbera

flowers after cutting. Journal of Horticultural Science and Ornamental Plants, 5: 127-136.

Khristeva, L. A. 1953. The participation of humic acids and other organic substances in nutrition

of higher plants. Pochvovedenie. 10: 46-59.

Knicker, H., Frund, R. and Ludemann, H. D. 1993. The chemical nature of nitrogen in native soil

organic matter. Naturwissenschaften. 80: 219-221.

Kulikova, N. A., Perminova, I.V., Badun, L.G.A., Chernysheva, M.G., Koroleva, O.V. and Tsvetkova,

E.A. 2005. Estimation of uptake of humic substances from different sources by Escherichiacoli cells under optimum and salt stress conditions by use of tritium-labeled humic materials.

Applied and Environmental Microbiology, 76: 223-230.

Kulikova, N. A., Stapanoava, E.V. and Koroleva, O.V. 2005. Mitigating activity of humic substances:

Direct influence on Biota. In: Use of humic substances to remediate polluted environment:

from theory to practices: Earth and environmental series, Permenova, IV. (eds). Kluwer

Acedemic Publisher, USA : 285-309.

Kumar, J., Amin, M. and Singh, P.V. 2003. Effect of humic acid and NPK sprays on Apricot. Journal

of Plant Nutrition. 21: 63: 73.

Kumar, S. and Haripriya, K. 2010. Effect of foliar application of iron and zinc on growth, flowering

and yield of nerium (Nerium Odorum L.). Plant Archives. 2: 637-640.

Lee, Y. S. and Bartlette, R. J. 1976. Stimulation of plant growth by humic substances as a substitute

of fertilizers. Soil Science Socity of America Journal, 40: 876-879.

Magda, H.M. 2003. Effect of some sources of nitrogen fertilizer and concentration of humic acid

on the productivity of squash plant. Egyptian Journal of Applied Science, 19: 293-309.

Mahgoub, M.H., Quesni, E. l., Fatima, E.M. and Kandil, M. M. 2010. Response of vegetative

growth and chemical constituents of Schefflera arboricola L. plant to foliar application of

inorganic fertilizer (grow-more) and ammonium nitrate at Nubaria. Ozean Journal of Applied

Science, 3: 177-184.

Manzoor, R., Shaukat, A.S. and Mazahar, H. B. 2001. Economics of floriculture in Pakistan: A case

study of Lahore market. Pakistan Economic and Social Review, 2: 87-102.

Mawgoud, A.M.R., Greadly, N.H.M., Helmy, Y.I. and Singer, S. M. 2007. Responses of tomato

plants to different rates of humic based fertilizers and NPK fertilization. Journal Applied

Science Reserch, 3:169-174.

Minotti, P. L., Halseth, D. E. and Sieczka, J. B. 1994. Field chlorophyll measurement to assess the

nitrogen status of potato varieties. Hort Science. 29:1497-1500.

Nikbakht, A., Mohsen, K., Mesbah, B., Ping, X.Y., Ancheng, L. and Nemat-Allah, E. 2008. Effect

of humic acid on plant growth, nutrient uptake and postharvest life of gerbera. Journal of

Plant Nutrition. 31: 2155-2167.

Padem, H. and Alan, R. 1995. The effect of foliar fertilizers on yield, chlorophyll and chemical

content of lettuce (Lactuca sativa L.). Atatürk University Journal Agriculture Faculty, 26:

21–34.

Pilanali, N. and Kaplan, M. 2003. Investigation of effects on nutrient uptake of humic acid application

of different forms to strawberry plant. Journal of Plant Nutrition, 26: 835–843.

Ricardo, R., Raymond, P. and Graeme, P. B. 1993.The use of a commercial organic biostimulant


for improved production of marigold cultivars. Journal of Home & Consumer Horticulture.

1: 83-93.

Sara, T., Omella, F., Silvia, Q. and Serenella, N. 2010. Humic substances biological activity at the

plant-soil interface from environmental aspects to molecular factors. Plant Signaling and

Behavior, 5(6): 635–643.

Schneider, V. B. 1991. The comparison between Italian and Dutch floriculture, technical and economical

aspects. Acta Horticulturae, 295: 121-138.

Seadh, S.E., El-Hendi, M. H., Abd El-Aal, H. A. and El-Sayed, S. O. 2012. Effect of NPK rates

and humic acid applications on growth of Egyptian cotton. Journal of Plant Production. 3:

2287-2299.

Shanmugam, P. M. and Veeraputhran, R. 2001. Effect of organic and inorganic nitrogen and zinc

application on soil fertility and nutrient uptake of rabi rice (Oryza sativa). Madras Agricultural

Journal, 88(7-9): 514-517.

Shehata, S.A., Gharib, A. A., El-Mogy, M., Gawad, M. A. and Shalaby, E. A. 2011. Influence of

compost, amino and humic acids on the growth, and yield and chemical parameters of strawberries.

Journal of Medicinal Plants Research, 5: 2304-2308.

Steel, R.G.D., Torrie, J.H. and Dicky, D. A. 1997. Principles and procedures of statistics. A Biometric

Approach, 3rd ed. Mc Graw Hill Book Co., New York.

Turkmen, O., Dursun A., T. and Erdinc, M. C. 2004. Calcium and humic acid effect seed germination,

growth and nutrient content of tomato (Lycopersicon esculantum L.) seedling under saline

soil condition. Acta Agriculturæ Scandinavica, Section,54: 168-170.

Van, L.G. 1999. The world cut flower industry: Trends and prospects. Acta Horticulturae. 4: 135- 138.

Visser, S. A. 1986. Effect of humic substances on plant growth, in humic substances effect on soil

and plants, Italy, Reda. 4: 89-135.

Zaghloul, S.M., Fatima, E.M. and Mazhar, A.M. 2009. Influence of potassium humate on growth

and chemical constituents of Thuja orientalis L. seedlings. Ozean Journal of Applied Sciences,

2: 1-9.


The Effect of Indole Butyric Acid and the Time of Stem

Cutting Preparation on Propagation of Damask Rose

Ornamental Shrub

Keywords: Indole butyric acid, Morphological characteristics, Rosa damascena.

Mahsa Kashefi1*, Hossein Zarei2 and Farzaneh Bahadori3

1 Graduated student, Department of Horticultural Science, Agricultural Sciences and Natural Resources

University of Gorgan, Gorgan, Iran.2 Assistant Professor, Department of Horticultural Science, Agricultural Sciences and Natural Resources

University of Gorgan, Gorgan, Iran.3 Assistant Professor, Agriculture and Natural Resources Research Center of Semnan, Semnan, Iran.


In order to investigate the morphological reactions of cutting of

damask rose to IBA (indole butyric acid) in different times, an experiment

was done in a factorial experiment based on RCD and three times with three

replications and 10 observations per each replication. In this experiment, the

simple effects of IBA, time and their interaction effects were measured

against morphological properties of damask rose rooting. Important measured

factors were the root length, the percentage of rooting, the percentage of

callus and the dry root weight. After immersing the cutting in IBA quickly

for 5 seconds, the cutting were placed in the medium in a research greenhouse

under mist system. According to the findings of the present research, in the

simple effect of IBA, their time and interaction, the maximum increase in

the average root length was obtained in 4000 mg/L-1 IBA and in cutting

taken in winter. Similarly, the maximum rooting percentage was achieved in

cutting treated with 2000 and 4000 mg/L IBA in March. The highest root dry

weight was gained in March and in a concentration of 4000 mg/L of IBA.

Abstract


INTRODUCTIONRegarding the necessity to reduce the dependence on oil revenue in the country, paying

attention to the role of agriculture and natural resources in establishing a proper setting for export

development becomes highlighted in order to develop nonoil exports. The damask rose (Rosadamascena Mill.) belongs to the Rosaceae family. It is cultivated in many parts of the world

thanks to enjoying an extraordinary aroma and an immense diversity. This family consists of

2000 cultivars and about 100 genera and the chromosome basis of most of them is 2n=14. Iran

is among the oldest countries producing flower and rose water in the world, with a 2500-year

background.

Damask rose is a deciduous shrub and evergreen samples can hardly be found among

them (Rout et al., 1999; Nybom et al., 2005). The propagation of this plant is usually asexual

and is done through cutting. This method of propagation is not fast, but is the easiest and best

method to producing new plants and its greatest advantage is the production of plants similar

to the parent (Ruchala et al., 2002). Cutting is the best method for propagation of deciduous

ornamental shrubs and evergreen broad-leaved and conifer trees (Khosh-khui, 1997). Research

showed that the soft wood cutting of the maple tree is rooted from the terminal parts of young

shoots in late spring under mist system and treating them with IBA enhanced rooting precent-

age. The results obtained from the study on the effect of cutting length and different concen-

trations of IBA (3000, 4000, and 5000 mg/L) on rooting of the cuttings of ornamental camellia

revealed that, 5000 mg L-1 IBA, had the highest rooting precentage (Hashemabadi and

Sedaghathoor, 2005).

The aim of this study is determination of best IBA concentration and time of cutting on

rooting of damask rose.

MATERIALS AND METHODSIn order to investigate on different IBA concentrations and time of cutting on rooting of

damask rose, an experiment was conducted in the greenhouse of Agricultural Research and Nat-

ural Resources Center of Semnan province under a intermittent mist system. The cuttings carried

out during March, June, and October after application of IBA through the quick immersion

method and planting in a light soil bed. The study was replicated three times in a factorial ex-

periment based on RCD with 10 cutting in each plot. The best time of cutting is when the first

cold results in abscission of leaves. In cold climates, cutting is sometimes done in late winter or

early spring, while in warm climates it is from February to mid April (Khosh-khui et al., 2007).

If enhanced vegetative growth of plants is required, soft wood cuttings should not be used, how-

ever, usage of hard wood cuttings harvested in late fall and early winter is recommended (Ha-

jiyan, 1996). Mist system provided relative humidity above the cultivation bed. In the current

research, a hydrometer measured the relative humidity of the greenhouse, where it was variable

between 60 to 80 percent in all of the three times. This system has been very effective in en-

hancing rooting of difficult rooting, soft wood cutting of large-leaved cultivars and broad-leaved

evergreen ones unlike conifers.

Cuttings were planted in the rooting bed after 5ʺ immersing in the iprodione + carbendazim

fungicide solution (2/1000) and IBA (0, 500, 1000, 2000, and 4000 mg/L). After 60 days, the cut-

tings were taken out of the bed and the results were recorded.

In this research, the simple effects of IBA and the time of cutting and their interaction on

the mean length of the root, rooting percentage, the percentage of callus and the dry weight of the

root were measured. Ruler was used in order to measurement the mean length of the root. After

transferred to laboratory, the root was separated completely from the cutting and washed to remove

any external materials, then exposed 70 °C for 48 hours. After taking out the dried roots related to

each sample from the oven, every sample was weighed separately on a digital balance (P<0.01).


Statistical analysis was done by SPSS software and the means were compared using the Tukey

test at a 5% probability.

RESULTSIn the results of this research, according to ANOVA (Table 1) and F-test, it can be stated

that the simple effects of IBA and the time of cutting length of the root, rooting percentage, cal-

lusing percentage, and dry weight of the root were significant at 5% level, interaction effect of

IBA and time was not significant at the 5% probability level in these traits except average root

longth (Table 1).

Fig. 1 and 2 showed that the highest mean length of the root was seen in 4000 mg L-1 IBA,

while the lowest one was obtained in the control treatment (Fig.1). Furthermore, the highest root

mean length was obtained in cuttings obtained in March, (Fig. 2).

According to results, in all three times, March, June and October, the cutting pre-

pared in March were better in all properties than those obtained in June and October. 4000

mgL-1 IBA in cutting obtained in March, had the highest root length among all treatments

(Fig. 3).

Rooting percentageThe IBA at 4000 mg L-1 enhanced rooting percentage in cuttings with a significant differ-

S. O. V df

MS

Average root

length

Rooting

percentage

Callus

percentage

Root dry

weight

IBA (H) Time of cutting (T)H*TError TotalCV

428

3045

7.079*23.214*1.790*0.102

16.63

1147.778*5446.667*182.778ns

157.778

19.15

1214.444*3795.556*167.778ns

126.667

16.08

8.314*30.132*2.426ns

2.023

71.84

*, n,s : Significant at 5% probability level and not significant, respectively.

Table 1. ANOVA table of effect of IBA and time on morphological characteristics of Rosa damascene Mill.

Fig. 1. The effect of IBA on average root length ofRosa damascena Mill.

Fig. 2. Times of cutting on average root length ofRosa damascena Mill.


ence of 5% compared to 0 and 500 mgL-1 (Table 1, Fig. 4). Similarly, the maximum percentage of

rooting was obtained in cuttings obtained in March (Fig. 5).

Callus percentageThe comparison of March, June, and October revealed that callus percentage were the best

in March than other times (Fig. 8). The 4000 mg L-1 IBA showed a positive impact in terms of

callus percentage compared to other treatments and increased callus percentage at 5% probability

(Fig. 7). Interaction effect of IBA and time of cutting had not significant effect on callus percentage

(Table 1).

Root dry weightBased on the ANOVA (Table 1), it can be observed that the difference among different

levels of IBA and various times of cutting are significant at 5% probability level, their interaction

effect on root dry weight was not significant (Table 1). The maximum root dry weight was obtained

in 4000 mg L-1 IBA and the minimum one to the control treatment (Fig. 9).

Fig. 3. Interaction of IBA concentrations and times of cuttingon average root length of Rosa damascena Mill.

Fig. 5. The effect of imes of cutting on rooting per-centage of Rosa damascena Mill.

Fig. 4. The effect of IBA concentrations on rootingpercentage of Rosa damascena Mill.


DISCUSSIONAccording to the results of this study and evaluation of measured traits in the cuttings of

ornamental shrub of damask rose in the greenhouse at different concentrations of IBA and various

times of cutting preparation, it can be said that the March improvied all of the traits including the

Fig. 6. The comparison of average root length in control treatment(a) and 4000 mg L-1(b).

Fig. 8. Times of cutting on callus percentage ofRosa damascena Mill.

Fig. 7. The effect of IBA concentrations on calluspercentage of Rosa damascena Mill.

Fig. 10. Times of cutting on root dry weight of Rosa damascena Mill.

Fig. 9. The effect of IBA concentrations on root dryweight of Rosa damascena Mill.


root mean length, rooting percentage, callus percentage, and root dry weight. Regarding the IBA

concentrations, it should be noted that application IBA showed a clear impact on rooting in damask

rose. Overall, effect of March with 4000 mg L-1 IBA are the best treatment to improve rooting in

cutting of damask rose flower.

The results obtained from the effect of different levels of IBA on the diameter of stalks ob-

tained from apple cutting showed that there is no significant difference between various treatments

and the control. This can imply that IBA, the applied growth regulator, affects most of the properties

related to the root (Delargy and Wright, 2006).

The results of the investigation of two methods of quick immersion end of cutting and

spraying IBA and NAA on cutting showed that method of hormone application had significant in-

fluence in some species. For example, Aglaonema, Gardenia, and ivy cuttings with quick immer-

sion of end cuttings in high concentrations of IBA has been known to be effective compared with

spraying on Chrysanthemum, Begonia, and Dieffenbachia (Blythe et al., 2004).

In another study, it has been shown that semi-hardwood cutting prepared during the first

week of February, immersed quickly in 4000 mg L-1 IBA, had the highest callus, rooting percent-

ages and root dry weight as much as 91.8% compared with the first week of November (Zarin Ball

et al., 2005). In addition, rooting percentage of cuttings in quick immersion was better tha other

methods (Eftekhari and Moalemi, 2003; Zarin Ball et al., 2005).

IBA resulted in significant increase of rooting percentage of Conocarpus compared

Dombeya natalensis, Polygonella polygamy, and Thunbergia grandiflora (Gupta and Kher, 1989;

Edward and Watson, 2001; Heather et al., 2010).

With regard to the effect of treatments on cutting callus percentages, the results manigfest

that application of IBA is more effective than NAA, in Bougainvillea spectabillis, Hibiscus rosa-sinensis, Thunbergia grandiflora, and Polygonella polygama (Widiastoety and Soebijanto, 1988;

Moalemi and Chehrazi, 2003; Gupta and Kher, 1989; Heather et al., 2010), However, it is not con-

sistent with the results obtained by other researchers on Callistemon citrinus and Nerium oleanderL. This incompatibility might be due to the difference of plant types.

According to literatures, external usage of IBA increased IBA mechanism is developing

IAA (indole acetic acid) and prepared amino acids required for proteins that are involved in the

formation of root primordia (Ryugo and Breen, 2003).

The research on Callistemon citrinus demonstrated that the root length and the number of

roots are directly correlated with increased root weight. It was also reported that the highest per-

centage of rooting, maximum number and length of roots, the highest root dry weight, and the

maximum leaf number were obtained in cuttings treated with IBA at 4000 mg L-1 concentration

that obtained in February (Zarin Ball et al., 2005).

By stimulating rooting, auxin makes carbohydrates and nitrogenous substances transfer

from the leaves to the root, and increased dry and fresh weight of roots (Hashemabadi and

Sedaghathoor, 2005).

Hussein (2008) reported that in his study on Thunbergia grandiflora maximum rooting per-

centage and the longest root were obtained in 4000 mg L-1 and the highest number of roots was

achieved in 6000 mg L-1.

Literature CitedAbdulsalam, K.S. and Siraj, M. S. 1994. Effect of some biological agents and naphthalene acetic

acid (NAA) on rooting response of some ornamental shrubs. Journal of King Saud University.

6: 135-141.

Anonymous. 1976. The biology and ecology of rosa x hybrid (rose). Australian Government, Department

of Health and Ageing Office of the Gene Technology Ragulator.

Batuli, H. 2005. Damask rose (Rosa damascena Mill.). Agricultural and Natural Resources Research


Center’s report of Isfahan. Isfahan. 3: 37-46.

Blythe, E. K., Sibley, J. L., Ruter, J. M. and Tilt, K. M. 2004. Cutting propagation of foliage crops

using a foliar application of auxin. Scientia Horticulturae. 103: 31-37.

Delargy, J .A. and Wright, C. E. 2006. Root formation in cuttings of apple in relation to auxin application

and to etiolation. Department of Agricultural Botany, The Queens University of Belfast,

Newforge Lane, Belfast BT9 5PX. 82: 341-347.

Edward, F. G. and Watson. D. G. 2001. Conocarpus erectus. University of Florida. Fact Sheet ST. 179.

Gresbach, J . 2007. Growing Temperate Fruit Trees in Kenya. World Agroforestry Center (ICRAF).

P: 138.

Eftekhari, S. A. and Moalemi, N. 2003. Compare the effect of the growth regulator IBA on rooting

cuttings Citrus decumana Murry in plastic tunnels and outdoors in spring and autumn. Journal

of Agriculture and Natural Resources. 10(3): 107-199.

Gupta, V. N. and Kher, M. A. 1989. Studies on the rooting of Dombeya natalensis by semi-hard

wood cutting under intermittent mist with the aid of auxins. C. A. B International. P: 3.

Hajiyan, S. 1996. Increasing sexual and vegetative damask rose (Rosa damascena Mill). Ms. Thesis,

Shiraz University, Shiraz.

Hashemabadi, D. and Sedaghathoor, Sh. 2005. Effects of artificial auxin (IBA, NAA) on rooting

cuttings of ornamental shrub camellia (Camellia Japonica). Fourth Congress of Horticultural

Sciences. Ferdowsi University. 536-538.

Heather, A., Hector, W. S. Thetford, M. and Thetford, P. 2010. Vegatative propagation of two florida

native wild flower species: Polygonella polygama and Polygonella robusta. University of

Florida, Department of Environmental Horticulture, Gainesville, Florida. P: 9.

Hussein, M .M. M. 2008. Studies on the rooting and the consequent plant growth on the stem cutting

of Thunbergia grandiflora, (Roxb. ex RottL.) Rox. 1. Effect of different planting dates.

World Journal of Agricultural Sciences. 4 (2): 125-132.

Khosh-khui, M. 1997. Plant propagation principles and practices sixth edition. Prentice Hall, N.J.

USA. P: 770.

Khosh-khui, M., Sheybani, B., Ruhani, A. and Tafazoli, A. 2007. The principles of Horticulture.

Shiraz University Press. Shiraz. Iran. P: 139-310.

Moalemi, N. and Chehrazi, M. 2003. The effect of auxin on rooting cuttings, leaf and leafless

rosette (Bougainvillea spectabillis) in plastic tunnel. Proceedings of the Third Congress of Horticulture.

p: 110.

Nybom, H., Werlemark, G., Esselink, D. G. and Vosman, B. 2005. Sexual preferences linked to rose

taxonomy and cytology. Acta Horticulturae. 690: 21-27.

Rout, G. R., Samantaray, S., Mottley, J. and Das, P. 1999. Biotechnology of the rose: a review of recent

progress. Scientia Horticulturae. 81: 201-228.

Ruchala, S. L., Zhang, D., Mitchell. W. and Jianhau, L. 2002. Improving vegetative propagation

techniques of sweet fern (Comptonia peregrina). Proceedings of the International Plant

Propagator’s Society. 52: 381-387.

Ryugo, K. and Breen. P. J. 2003. Indole acetic acid metaholism in cuttings of plum (Prunus cerasifera × P. munsoniana cv. Mariana 2624). American Society of Horticultural Science, 99: 247.

Widiastoety, D. and Soebijanto, M. 1988. Rooting of stem cuttings of Hibiscus rosa-sinensis. Buletin Penelition Horticulturae. 16: 73-83.

Zarin Ball, M., Moalemi, N. and Daneshvar, M. H. 2005. The effect of different concentrations of

auxin, time and environmental conditions on rooting cuttings semi-hardwood cuttings of

Citrus decumana Murry. Journal of Agricultural Science. Vol. XV. 2: 13-26.

www.jornamental.com


Study on Effects of Ascorbic Acid and Citric Acid on Vase Life

of Cut Lisianthus (Eustoma grandiflorum) ‘Mariachi Blue’

Keywords: Cut flowers, Organic Acids, Postharvest life, Preservative solution.

Farnaz Sheikh1, Seyed Hossein Neamati2, Navid Vahdati3* and Ali Dolatkhahi3

1 MSc, Horticulture Department, Ferdowsi University of Mashhad.2 Assistant Professor, Horticulture Department, Ferdowsi University of Mashhad.3 PhD Student, Horticulture Department, Ferdowsi University of Mashhad.


The postharvest life of cut Eustoma grandiflorum flowers is limited

in open flowers. Therefore a factorial experiment based on a completely

randomized design with ascorbic acid (AsA) at 4 levels (0, 100, 200, 300

mg L-1) and citric acid (CA) at 3 levels (0, 100, 200 mg L-1) with 3

replications and 3 samples for each replications, was conducted for this

purpose. Results indicated that a significant increase with applying ascorbic

and citric acid nearly in all traits both individually and in combination,

with higher concentrations imposing greater effects (p≤0.05 and p≤0.01).

The highest vase life (17.6 days) and petal water content (68.9%) was

observed for the interaction of ascorbic acid (300 mg L-1) and citric acid

(100 mg L-1) and ascorbic acid (300 mg L-1) and citric acid (200 mg L-1), re-

spectively, which shows a 94 and 252% increase compared to control (9.1

days and 27.3%). Along with this, relative water content and petal water

content raised with AsA and CA increase. Water content also showed a

similar manner. Fresh weight decreased in all treatments during experiment,

but this reduction was much less in AsA (300 mg L-1) alone and in

interactions with CA levels. According to the results of this experiment,

ascorbic acid and/or citric acid as cheap, safe and biodegradable compounds

are suitable alternatives for chemical treatments in order to prolong vase

life of cut flowers of Eustoma. Commercialization of these compounds for

optimum formulations needs further experiments.

Abstract

246 Journal of Ornamental Plants, Volume 4, Number 4: 245-252, December, 2014

INTRODUCTIONCut flowers are precious products in horticulture. Maintaining good quality of cut flowers

and extending the vase life, is considered important and practical for having acceptable products

for the markets especially for sensitive species. In general, short vase life of cut flowers is attributed

to several factors e.g. carbohydrate depletion (Ketsa, 1989); water stress (Sankat and Mujaffar,

1994), microorganisms (especially bacteria) (van Doorn and Witte, 1991) and ethylene (Wu et al.,1991). In addition, a major cause of deterioration in cut flowers is blockage of xylem vessels by

microorganisms that accumulate in the vase solution or in the vessels themselves (Danaeeet al,

2011). For many years, floral preservatives have been acidified and have usually included biocides

to inhibit bacterial proliferation (Nowak and Rudnicki, 1990).

Lisianthus is one of the newly introduced cut flowers and is ranked as one of the top ten

economically important cut flower in the world (Dole and Wilkins, 1999). Mean vase life of

lisianthus cut flowers is considered to be 2 weeks; however it varies greatly among its cultivars

(Dole and Wilkins, 1999).

Ascorbic acid (AsA) is a bacteria growth inhibitor commercialized recently, which has been

extensively evaluated for protecting cut flowers from physical plugging (Jin et al., 2006, Hatami

et al., 2010). AsA is synthesized in higher plants and affects plant growth and development. It

plays an important role in the electron transport system (El-Kobisy et al., 2005). Robinson (1973)

reported that AsA acts as a co-enzymatic reaction by which carbohydrates; proteins are metabolized

and involved in photosynthesis and respiration processes. Bolkhina et al. (2003) stated that AsA

is the most abundant antioxidant which protects plant cells, and it is currently considered to be a

regulator on cell division and differentiation and added to that, it is involved in a wide range of

important functions such as antioxidant defense, photoprotection, regulation of photosynthesis and

growth. Pretreatment of cut rose with AsA for 12 h prolonged vase life of plant exposed to water

deficit stress (Jin et al., 2006).

Besides ascorbic acid, citric acid is also known as a potential component of vase solution

in order to enhance cut flowers vase life (Vahdati et al., 2011). Citric acid (CA), is as well a wide

spread organic acid in plant kingdom and makes a weak acid when dissolved in water. CA is used

to adjust water pH and to control the growth of microorganisms. CA is commercially advised for

a number of cut flowers like chrysanthemum (Dole and Wilkins, 1999). Also, CA reduces the risk

of vascular blockage in cut flowers through its anti-embolism habit (Bhattacharjee et al., 1993).

Adding CA to vase solution decreased pH and microbial activity and hence increased vase life of

cut carnation flowers by 30-40% over control (Ebrahimzadeh et al., 2003). An enhancing effect

of CA was also observed in ‘Bougati’ and ‘Rollet’ cultivars of cut rose flowers (Lechinani, 2006).

The main purpose of this work was to determine the response of cut lisianthus flowers to

ascorbic and citric acid preservatives and their effects on vase life and qualitative characteristics

of cut flowers.

MATERIALS AND METHODSPlant materials

Lisianthus (Eustoma grandiflorum. Cv. Mariachi Blue) flowers grown in a standard green-

house were supplied from a local grower. Cut flowers were trimmed to 30 cm after arrival to the

laboratory and were placed in prepared solutions. Experimental conditions were carefully moni-

tored at 20 ± 2 °C mean temperature, 60 ± 10 % RH and 15 µmol m-2s-1 with a 12 hour photoperiod

using florescent lamps.

TreatmentsAsA and CA are soluble in water and are easily dissolved in optimum concentrations. Four

levels of AsA as follow; 0, 100, 200, 300 mg L-1 and three levels of CA at 0, 100, 200 mg L-1 were


used alone and in combination. A 300 ml preservative solution was used for each replication and

cut flowers were placed in the solutions after cutting to 30 cm long. Sucrose at 4% was added in

all treatments as a base solution.

Vase life The postharvest life of cut Eustoma grandiflorum flower is limited by poor bud opening

and bent neck in open flowers. The vase life of the inflorescence was considered terminated when

50% of the open florets had wilted (Cho et al., 2001).

Relative water content (RWC)Relative water content (RWC) from each sample was determined according to Barros and

Virtly method (1962). For this purpose, two excised leaves per plant were weighed (fresh weight,

FW) and placed in water in the dark with their petioles plunged in distilled water for 6 hours to

allow them to reach full turgidity and, hence, to determine their turgid weight (TW). These leaves

were then dried at 70 ºC for 24 h and their dry weight (DW) was recorded. Finally, RWC was cal-

culated using the below equation:

% RWC= (FW-DW)/ (TW-DW) × 100

Petal water contentFor this trait, on the 12th day from the start of the experiment, 1g of petals from all replica-

tions was sampled and each sample were taken as FW and then dried at 70 ºC for 24 h and their

DW was recorded. Petal water content (% WP) was then determined with the below equation

(Kalate Jari et al., 2008):

%WP= FW-DW/DW × 100

Fresh weight loss Weight reduction and relative fresh weight of cut flowers were measured in one day inter-

vals through the experiment from day 1 to 10 (Karimi et al., 2008). Weight decrease compared to

day one (initial) was then calculated using equation below:

Weight decrease compared to initial (%) = (Wt-W0/W0) × 100

Which in the equation:

Wt = fresh weight in day ten

W0 = initial fresh weight (day one)

The opening of each vase was covered in order to limit vase solution evaporative loss and

to allow determination of the uptake by stems of the different treatments. Solution uptake volume

was calculated by subtracting the volume of evaporated solution from flasks of the same volume

without cut flowers.

Water content of cut flowerAt the end of vase life period of the examined flowers, they were oven dried for 48 hr at 72 ºC.

Dry weight was measured after 2 days and water content was calculated using equation below:

WC = (FW-DW/FW) × 100

Total water content was determined by subtracting the dry weight of whole flower from

their corresponding fresh weight. Results were divided to dry weight using the below formula

(Kalate Jari et al., 2008).

Water content = (FW-DW)/DW

Statistical analysisThis experiment was conducted as a factorial experiment based on completely randomized


design with 3 replications and 3 samples (individual flowers) for each replication. Data were an-

alyzed as factorial ANOVAs using JMP4.

RESULTS Vase life

According to the results shown in table 1, using AsA and/or CA and their interactions as

preservatives, significantly increased the vase life of lisianthus cut flowers, over control (p≤0.01

and p≤0.05 respectively). Highest vase life (16.81 and 15.77 days) was observed in solutions con-

taining AsA (300 mg L-1) and CA (200 mg L-1), respectively. Highest vase life of interaction treat-

ments (17.6 days) was observed in AsA 300 and CA 100 mg L-1 which shows a 94% increase

compared to control (Table 2).

Relative water content (RWC)According to the results, effects of AsA and CA treatments alone and in interaction on RWC

are shown significant (p≤0.05) (Table 1). RWC increases up to (74.22%) in 200 mg L-1AsA but

decreases to (61.26%) again in 300 mg L-1 AsA. Highest interaction value (81.7%) is observed in

200 mg L-1 AsA and 200 mg L-1 CA and shows a 39% enhancement over control (Table 2).

Petal water contentResults from petal water content measurement shows a significant difference in AsA and

CA treatments over control (p≤0.05) (Table 1). Highest measured petal water content is shown

Vase life Relative water

content

Petal water

contentFresh

weight loss

Water

content

Ascorbic acid (AsA)Citric acid (CA)(AsA) × (CA)ErrorCV (%)

32.24**31.17**12.71*4.29

26.75

717.96*410.23*321.87*210.5122.12

861.62*782.25*208.1 ns278.0725.79

270.21*254.95*72.69 ns

81.0129.37

41.39 *71.2*

64.92*23.7827.27

*, ** and ns represent significance at 0.05 , 0.01 and non-significant.

Table 1. Analysis of variance for measured traits in this experiment.

AsA

(mg L-1)

CA

(mg L-1)

Vase Life

(day)

Relative water

content (%)

Petal water

content (%)

Fresh weight

loss (g)

Water

content (%)

0100200300

000000

100 100100 200 200200 300300300

00000

100200

0100200

0100200

0100200

0100200

12.51 b

13.33 b

13.55 b

16.81 a

12.58 b

13.8 b

15.77 a

9.1 f

10.9 ef

17.6 a

12.3 def

13 cde

15.3 abcd

12.9 cde

13.8 bcde

13.3 cde

16 abc

17.6 a

16.9 ab

52.55 b

64.14 ab

74.22 a

61.26 ab

59.74 b

59.87 b

69.79 a

58.9 cd

44.3 d

64.6 bc

59.6 c

70.1 abc

77.5 ab

66.1 bc

78.5 ab

81.7 a

75.7 ab

68.5 abc

58.3 cd

37.89 b

40.93 ab

57.3 a

54.99 a

39.01 b

49.42 ab

54.91 a

27.3 abcde

38.7 abcd

47.7 abc

37.8 abcd

36.2 abcd

48.8 abc

55.2 ab

62.4 a

54.3 ab

35.7 abcd

60.4 a

68.9 a

41.86 a

37.96 ab

33.07 b

29.36 b

39.57 a

36.6 ab

30.53 b

42.4 ab

48.7 a

34.5 abc

41.7 ab

40.1 ab

32 abc

40.4 ab

34.6 abc

24.2 abcd

33.8 abc

23 abcd

31.4 abc

77.02 b

77.5 b

77.46 b

78.65 a

74.85 b

78.91 ab

79.21 a

76 abc

75.1 bc

80 abc

79.9 ab

78.2 ab

74.4 bc

68.9 c

79.9 ab

83.6 a

74.7 bc

82.5 ab

78.8 ab

*In each column, means with the similar letters are not significantly different at 5% level.

Table 2.Mean comparison of different ascorbic and citric acid treatments on measured traits.


AsA and CA 200 mg L-1. Interaction treatments were not significant in any statistical level, but a

considerable increase was observed compared to control (Table 2).

Fresh weight loss Effects of AsA and CA on initial weight variations (day 10 to day 0) were significant

(p≤0.05), but however, interaction effects were not significant (Table 1). The least weight decrease

(23%) of cut flowers during the experiment was observed in interaction of AsA 300 mg L-1 and

CA 100 mg L-1 (Table 2). Fig. 1 shows the weight decrease of cut flowers in different treatments

used for this experiment. According to them an increase in acid concentration results in a lower

weight decrease.

Water content of cut flowerAccording to the results, AsA showed no significant difference with control but CA and in-

teraction treatments on WC are significant (p≤0.05) (Table 1). Highest WC measured (83.6%) of

interaction treatments was observed in AsA 200 mg L-1 and CA 200 mg L-1 (Table 2).

DISCUSSIONIn the present study, it was demonstrated that AsA and CA treatments significantly extends

vase life in cut lisianthus flowers. Many researches have revealed that the presence of microor-

ganisms in water can cause vascular blockage of cut flower stems (Van Doorn and Witte,

1991).AsA has been shown to have an improving role in postharvest vase life of cut flowers (Jin

et al., 2006; Sujata et al., 2003).

AsA is the most abundant antioxidant which protects plant cells and involved in a wide range

of important functions as antioxidant defense, photoprotection, regulationof photosynthesis and growth

(Bolkhina et al., 2003; El-Kobisky et al., 2005). It was reported that AsA pretreatment increased an-

a

b

Fig. 1. Change in fresh weight of cut flowers during the first six days ofthe experiment in AsA (a) and CA (b) treatments.


tioxidant activity of some enzymes (superoxide dismutase and ascorbate peroxidase), which leads to

improved vase life of cut rose (cv. Samantha) exposed to water deficit stress (Jin et al., 2006).

In addition, it is well known that acidic solutions inhibits bacterial growth and proliferation

(Raskin, 1992b), thus considering AsA and CA as weak acids, extended vase life of lisianthus cut

flower in this experiment can be attributed to inhibit the growth of microorganisms in the treatments

mentioned above. In addition, CA can alleviate water uptake and extend vase life due to its anti-

embolism trait which is due to a decrease in microorganisms and low vascular blockage risk with

it (Bhattacharjee et al., 1993). Other investigators also suggested that reduced pH generally has

been considered to improve flower vase life and most flower preservatives contain an acidifier to

reduce the pH of the vase solution (Halvey and Mayak, 1980).

Petals of a cut flower are the main ornamental parts and turgidity of this part is important

for a good looking and marketable product. Petal turgidity depends largely on water uptake and

maintenance in treatments suggested (Vahdati et al, 2011). Results of this experiment show a sig-

nificantly higher water uptake and turgid petals which subsequently increases the cut flower fresh

weight. The increases in water uptake and subsequently cut flower fresh weight, is apparently con-

sidered due to the acidifying and stress alleviating properties of AsA (Hatami et al., 2010). Ac-

cording to our results, it can generally be discussed that, the major part of the water uptake is

gathered in the petals which in fact helps to have a better visual quality in treated cut flowers. CA

in another study showed to be effective on decreasing water potential in cut rose cv. Cara Mia pe-

duncle and a higher turgidity was observed (Durkin, 1979). AsA alleviates water stress in plants

and prevents cell membrane from induced damages and cut flower deterioration by the stress (Jin

et al., 2006). Similar to Jin et al. (2006), a very small decrease in cut flowers fresh weight over

control in this experiment is observed in AsA treatments. AsA prevents senescence and chlorophyll

degradation which agree with our results.

Relative water content (RWC) is an index representing the amount of water in the plant or-

gans and shows the ability of a plant in maintaining water under stress conditions (Abbaszadeh etal., 2008). Hence, in a controlled environment for an experiment, the measured RWC shows the

response of a plant and the higher the measured amount, the greater the ability of a treatment for

keeping water (Abbaszadeh et al., 2008). Therefore, according to these results, it seems that at day

10 of the experiment (end of control vase life duration), samples placed in control treatments were

under severe stress and could not take up and keep water properly, whereas AsA treatments in

comparison, at the same time were in normal non stress conditions which can be the main reason

for better looking flowers.

CONCLUSIONIn conclusion, adding AsA and CA to cut flower preservation solutions, increased vase life

and preserved cut flowers for a longer period due to an acidic and anti-stress properties. Acidic

solution, on the other hand, prevented bacterial growth and proliferation (vascular blockage) and

facilitated water uptake in cut flowers. AsA showed a greater effect rather than CA treatments and

a significant difference in vase life of lisianthus cut flowers was observed with higher concentra-

tions performing better. According to the results it can generally be mentioned that AsA and CA

as cheap, safe and biodegradable compounds can be suitable alternative chemical treatments in

order to prolong vase life of cut lisianthus (Eustoma grandiflorum cv. Mariachi Blue (flowers

which is a fact that would be much appreciated by the growers and handlers of cut flowers. Com-

mercialization of these compounds for optimum formulations of course needs further experiments.

Literature CitedAbbaszadeh1, B., Ashourabadi, Sharifi, E., Lebaschi, M. H., Hajibagher Kandy, M. N. and Moghadami,

F. 2008. The effect of drought stress on proline contents, soluble sugars, chlorophyll and


relative water contents of balm (Melissa officinalis L.). Iranian Journal of Medicine and

Aromatic Plants. 23: 504-513.

Barrs, H.D. and Weaterley, P.E. 1962. A re-examination of the relative turgidity techniques for the

estimating water deficit in leaves. Australian Journal of Biological Sciences. 15: 413-428.

Bhattacharjee, S. K., Singh, V. and Saxena, N. K. 1993. Studies on vegetative growth, flowering,

flower quality and vase life of roses. Journal of Primary Industries.21 (2): 67-71.

Blokhina, O., Virolainen, E. and Fagerstedt, K.V. 2003. Antioxidant, oxidative damage and oxygen

deprivations stress: A review. Annual Botany. 91:179-194.

Cho, M.S., Celikel, F., Dodge, L. and Reid, M.S. 2001. Sucrose enhances the postharvest quality

of cut flowers of Eustoma grandiflorum (Raf.) Shinn. Proceeding. VII International Symposium

on Postharvest. Acta Horticulture. 543.

Danaee, E., Mostofi, Y. and Moradi, P. 2011. Effect of GA3 and BA on postharvest quality and vase

life of gerbera (Gerbera jamesonii. cv. Good Timing) cut flowers.Horticulture, Environment,

and Biotechnology. 52 (2), 140-144.

Dole, J. M. and Wilkins, P. 1999. Floriculture principles and species. Prentice Hall Inc. Upper Saddle

River, New Jersey.

Durkin, D. J. 1979. Effect of millipore filtration, citric acid and sucrose on peduncle water potential

of cut rose flowers. Journal of American Society of Horticultural Sciences. 104 (6):860-863.

Ebrahimzadeh, A., Masiha, C., Nazemieh, A. and Valizade, M. 2003.Studying the effects of preserving

solutions on longevity and some other qualitative traits of Dianthus caryophyllus cut flower.

Iranian Journal of Horticultural Science and Technology, (4): 31-42.

El-Kobisy, D.S., Kady, K.A., Medani, R.A. and Agamy, R.A. 2005. Response of pea plant (Pisum sativum L.) to treatment with ascorbic acid. Egyptian Journal of Applied Science, 20:36-50.

Halevy, A. H. and Mayak, S. 1980. Senescence and postharvest physiology of cut flowers.Part 2.

In. J. Janick (ed). Horticultural Reviews 2. AVΙ Pub. Westport, Conn.

Hatami, M., Ghasemnezhad, M., Hatamzadeh, A. and Omran, S. G. 2010. Effect of ascorbic acid on

antioxidant capacity during flower development in 'Royal Class' rose cut flowers. Acta

Horticulturae. 877.

Jin, J., Shan A, N., Maa, N., Bai, B.J. and Gaoa, J. 2006. Regulation of ascorbate peroxidase at the

transcript level is involved in tolerance to postharvest water deficit stress in the cut rose

(Rosa hybrida L.) cv. Samantha. Postharvest Biology and Technology, 40:236–243

Kalate Jari S., Khalighi A., Moradi F. and Fatahi Moghadam M. R. 2008. The effect of calcium

chloride and calcium nitrate on quality and vase life of rose flowers cv. Red Giant. Iranian

Journal of Horticultural Science and Technology. No. 3: 163-179.

Karimi., M., Asil., M.H. Lahiji., H. S. and Sasani., S.T. 2008. Effects of temperature and different

chemical treatments for extending Lilium cut flower shelf life. Iranian Journal of Horticultural

Science and Technology. No. 43: 1-9.

Ketsa, S. 1989.Vase life characteristics of inflorescences of dendrobium ‘Pompadour’. Hort Science,

64: 611–615.

Lechinani, A. 2006. Effects of different treatments on vase life of two rose cut flower cultivars.

Proceeding of Fifth Iranian Horticultural Congress, Shiraz.

Nowak, J. and Rudnicki, R.M. 1990. Postharvest handling and storage of cut flowers, florist greens

and potted plants, p: 210. Timber Press, Porthand, Oregan.

Raskin, I. 1992b. Salicylate, a new plant hormone. Plant Physiology, 99: 799–803.

Robinson, F.A. 1973. Vitamins. In Phytochemistry. Vol. III: 195-220. Lawrence, Miller, P., (Ed)

Van- Reinhold Co., New York.

Sankat, C.K. and Mujaffar, S. 1994. Water balance in cut anthurium flowers in storage and its

effect on quality. Acta Horticulturae. 368: 723–32.

Sujata, A. N., Vijai Singh, T.V. and Sharma. R.S. 2003. Effect of chemical preservatives on enhancing


vase-life of gerbera flowers. Journal of Tropical Agriculture. 41: 56-58.

Vahdati, N., Tehranifar, A., Bayat, H. and Selahvarzi, Y. 2012. Salicylic and citric acid treatments improve

the vase life of cut Chrysanthemum flowers. Journal of Agriculture and Science Technology.

Vol. 14, 879-887.

Van Doorn, W.G. and De Witte, Y. 1991. The mode of action of bacteria inthe vascular occlusion of cut rose

flowers. Acta Horticulturae. 298: 165–7.

Wu, M.J., Van Doorn, W.G. and Reid, M.S. 1991.Variation in the senescence of carnation (Dianthus caryophyllus L.) cultivars. II.Comparison of sensitivity to exogenous ethylene and of ethylene

binding. Scientia Horticulturae. 48: 109–16.


Pollen Germinability and Cross-Pollination Success in

Persian Cyclamen (Cyclamen persicum Mill.)

Keywords: Cross-pollination success, Cyclamen, Medium composition, Pollen germination.

Mohammad Kermanshahani*, Roohangiz Naderi, Reza Fattahi and Ahmad Khalighi

Department of Horticultural Sciences, Faculty of Agriculture and Natural Resources, University of

Tehran, Karaj, Iran.


Low seed yield is a limiting factor for cross breeding and hybrid

seed production in cyclamen. This study was performed to investigate

pollen germination and its relation to cross-pollination success and fruit

set in this plant. In order to achieve a high level of pollen germination, the

effect of different concentrations of chemical compounds were examined

on in vitro pollen germination of cyclamen in modified Brewbaker and

Kwack medium, containing sucrose (10 and 20%), calcium nitrate (0, 200

and 300 mg l-1), and boric acid (0, 100 and 200mg l-1) at two pH levels (5.5

and 6.5). Maximum pollen germination was obtained in media containing

higher concentration of calcium and boron regardless of sucrose concentration

and pH level. Pollen germination percentage was genotype-dependent.

Cross-pollination was performed among four different genotypes characterized

by various pollen germination percentages. There was a direct correlation

between cross-pollination success and pollen germination percentage.

Genotypes with 30% higher pollen germination led to 10% increase in

fruit set.

Abstract


INTRODUCTIONCyclamen is a commercially valuable potted flowering plant (Tanaka et al., 2013b), which

is typically propagated by seed. Cyclamen seeds are expensive [up to 0.20 € per seed] (Schwenkel,

2001) that are usually sold as single seeds (Dole and Wilkins, 2005). The high price is primarily

because of the laborious work for emasculation and hand pollination, which are essential for hybrid

seeds production (Takamura, 2007). Cross-pollination success is extremely important not only for

F1 seed production, but also for cyclamen breeding. However, in controlled crosses, there are some

problems such as low pollen viability and germination (Ewald and Schwenkel, 1997; Nan, 2008)

and inflorescence abortion (Ewald and Schwenkel, 1997), leading to loss of a large number of

crosses, thereby reducing fruit and seed yields.

Emasculation and artificial pollination are required for producing cyclamen hybrid seeds.

The stigma dries 3–5 days after anthesis (Reinhardt et al., 2007); hence, the best time for emascu-

lation is a few days before and after anthesis. Takamura (2007) has suggested flower bud emascu-

lation to be done seven days before anthesis, and then the emasculated flower be pollinated at the

expected time of anthesis. This would increase seed set percentage. Temperatures above 30°C have

detrimental effects on cyclamen pollen germination; 15–25°C has been reported to be the optimal

range (Takamura et al., 1996). Higher relative humidity (RH) allows pollens to absorb water easily

(Ferrari et al., 1981). Naturally, petals abscise without drying after fertilization, which is catalyzed

by ethylene (Halvey et al., 1984), and ovaries will continue to grow for creating seed capsules

after 3 months (Naderi, 2000).

High pollen germination percentage and rapid growth of the pollen tube are very important

for sufficient seed yield. One of the methods to assess pollen viability is in vitro germination. In

in vitro condition, beside the genetic factor, medium elements and compounds, pH, and incubation

temperature affect pollen germination and pollen tube growth (Abdul-Baki, 1992; Sawidis and

Reiss, 1995; Taylor and Hepler, 1997). Once the stored nutrients in pollen grain run out, external

nutrients are required for further growth (Cruzan, 1986; Mulcahy and Mulcahy, 1982; Herrero and

Hormaza, 1996). For example, sugars are required in medium. Pollen consumes sugars (especially

sucrose) as respiration substrates for rapid synthesis of cell wall material and elongation (Baker

and Baker, 1979; Mascarenhas, 1993; Schlupmann et al., 1994; Derksen et al., 1995; Okusaka and

Hiratsuka, 2009). Sucrose level in medium depends on plant species and nutrient reserves of the

pollens (Vasil, 1960; Sahar and Spiegel, 1984; Golan-Goldhirh et al., 1991; Mortazavi et al., 2007).

Plant metabolic processes are cytoplasmic-pH-dependent (Tupy and Rhova, 1984). Some studies

have reported proton and ion gradients roles for polarized pollen tube growth) Malho et al., 1994;

Pierson et al., 1994; Malho and Trewavas, 1996; Feijó et al., 1999; Hepler et al., 2006; Michard

et al., 2008).

Boron and calcium are required for growth and development of vascular plants (Cakmak etal., 1995; Stangoulis et al., 2001; Zhang et al., 2007). The essential and supplementary roles of these

elements in pollen germination have been observed in both in vitro and in vivo conditions (Brewbaker

and Kwack, 1963; Robbertse et al., 1990; Loomis and Durst, 1992; Feijó et al., 1995; Nyomora etal., 2000; Wang et al., 2003; Mortazavi et al., 2007; Vaknin et al., 2008; Lee et al., 2009).

Supplemental calcium contributes to pollen tube guidance and enters the tube via the tip

(Vaknin et al., 2008; Steinhorst and Kudla, 2013). Furthermore, it affects mechanical properties of

the pollen tube wall (Steer and Steer, 1989) and pollen tube sensitivity to tropic trigger (Bou Daher

and Geitmann, 2011). In many plants, besides calcium, boron plays critical role in sexual reproduc-

tion (Brown et al., 2002). Boron is required for normal pollen germination and tube growth. Some

of genes related to B transporter are especially expressed in pollens (Tanaka et al., 2013a). Boron

deficiency affects growing points, such as pollen tube tips (Loomis and Durst, 1992).

Improved hybrid cyclamen seed production is beneficial for cross breeding and hybrid seed

production, for which, higher cross-pollination success is required, however. For this reason, we


conducted an experiment to investigate the relationship between pollen germination and cross-

pollination success. Moreover, to assess pollen viability, the effects of different concentrations of

chemical compounds were examined on in vitro pollen germination.

MATERIALS AND METHODSIn vitro germination test

In order to avoid possible discrepancies, the pollen grains were collected from a single

flower four days after anthesis in the morning. The pollen grains were cultured in modified Brew-

baker and Kwack liquid medium (Brewbaker and Kwack, 1963) containing sucrose (10 and 20%),

Ca(NO3)2 (0, 200 and 300 mg l-1) and H3BO3 (0, 100 and 200 mg l-1). pH of the media was adjusted

to 5.5 or 6.5 using 0.1 M HCl/NaOH. Considering these factors, 36 types of medium were prepared.

The cultures were maintained in germinator at 15°C and in dark. Germinated pollens were counted

after 24 h. A pollen grain was considered to be germinated if the length of the pollen tube was

equal to or longer than the diameter of the grain (Abdul-Baki, 1992). For each germination test,

800-1200 grains from four microscopic fields were counted. The best medium in this experiment

was used for determining pollen germination percentage of other genotypes. Tube length was meas-

ured on 30 randomly chosen pollen tubes per genotype after 48 h.

Cross-pollinationFlowering cyclamens from four inbred diploid genotypes (G1, G2, G3 and G4) were used,

which had been growing in greenhouse conditions. Cross-pollination was done among these geno-

types as shown in Fig. 1 (approximately 100 times for each direct cross). By removing stamens

together with petals, the flowers were emasculated. They were then covered by a paper bags. Next,

fresh pollens that were gathered from flowers of the male parent were placed on the stigma 5–7

days after emasculation. Finally, the flowers were covered again. Temperature and relative humidity

of the greenhouse were 15–20°C and 60–70%.

Data of medium composition were analyzed in a factorial design using SAS software 9.3,

and the means were compared using Duncan’s multiple range test. For pollen germination per-

centage in different genotypes and cross-pollination success in crosses among genotypes, data

were analyzed in a completely randomized design using SAS software 9.3, and Duncan’s multiple

range test was used to compare the means.

RESULTS AND DISCUSSIONIn vitro germination

Variation in pollen germination among the treatments was high. Sucrose 10% was better

than sucrose 20% (Fig. 2a). Takamura (1996) already reported low concentrations of sucrose (be-

tween 5-15%) to be proper for pollen germination in cyclamen. High concentration of sucrose can

a b c d e

Fig. 1. Different stages of emasculation and cross-pollination in cyclamen. a: Appropriate time for re-moving stamens (bud stage); b: Emasculated female parent; c: Releasing pollens of male parents on

ear cleaner or nail, d: Placing pollens on stigma of an emasculated flower; e: Packing.


decrease water absorption and pollen germination, which depends on the species, nevertheless

(Stone, 2003).

Media pH when was 5.5 increased pollen germination in comparison to when it was 6.5

(Fig. 2b). Acidification results in cell membrane loosening (Franklin-Tong, 1999), which is nec-

essary for pollen germination and pollen tube growth. Proton efflux and proton-sugar co-transport

has been seen in growing pollen tubes. It shows H+ exchanges with the medium for cytoplasmic

pH regulation of pollen tube (Tupy and Rhova, 1984). Cyclamen is an acidophilic plant (Dole and

Wilkins, 2005). Therefore, the better pollen germination in low pH could be a response to its aci-

dophilic nature.

Calcium nitrate in medium had a positive effect on pollen germination; 200 mg l-1 was op-

timum (Fig. 3). Many researchers have confirmed the essential role of Ca2+ for pollen germination

(Steinhorst and Kudla, 2013). Cell structure is directly and indirectly affected by Ca2+ concentration

(Zhang et al., 2007). The tip growth of pollen is correlated with Ca2+ gradient. Extracellular Ca2+

ions establish this gradient in combination with internal stores (Steinhorst and Kudla, 2013). Cal-

cium also has signal transduction role in pollen germination (Zhang et al., 2007). Proper concen-

tration of Ca2+ for pollen germination varies with different species. Similarly, many other studies

reported that high or low Ca concentrations had decreased pollen germination (Voyiatzis and

Paraskevopoulou-Paroussi, 2002; Lee et al., 2009). With high Ca concentration, the unbalanced

Ca gradient in the pollen tube tip could suppress pollen tube growth (Lee et al., 2009).

Without calcium, boric acid decreased pollen germination percentage. Reduction in pollen

Fig. 2. Pollen germination percentage of cyclamen. (a) In two sucrose concentrations. (b) Attwo pH levels. Values with different letter are significantly (P<0.01) different.

Fig. 3. Pollen germination percentage of cyclamen indifferent concentrations of calcium. Values with differ-

ent letter are significantly (P<0.01) different.


germination may be attributed to the higherB combination with rhamnogalacturonan II in pollen

tube wall, which results in pollen cell wall stability (Hu and Brown, 1994; Kaneko et al., 1997).

The reduction of pollen germination by boron in absence of calcium was already reported (Bruyn,

1966; Pfahler, 1967).

There are interactions between calcium and boron in plant tissues (Brewbaker and Kwack,

1963). The highest pollen germination percentage was obtained in media containing the highest

concentration of boron and calcium (Fig. 4). Some researchers have demonstrated that boric acid

is effective on pollen germination and pollen tube elongation in medium (Nyomora et al., 2000;

Wang et al., 2003). Pollen tube precursors form strong complexes with B (Loomis and Durst,

1992). Some authors suggested B to improve pollen germination by promoting H+-ATPase activity

(Feijó et al., 1995; Obermeyer and Blatt, 1995). Boron is needed also for cell wall expanding

(Fleischer et al., 1998). In our study, B had positive effect on pollen germination provided that Ca

was present. Synergistic interaction between Ca and B in increasing pollen germination has already

been reported (Bruyn, 1966; Pfahler, 1968).

Maximum pollen germination percentage (84%) was obtained in medium containing 10%

sucrose, 300 mg l-1 Ca and 200 mg l-1 B having pH of 5.5. The second medium by 80% germina-

tion was containing 20% sucrose, 300 mg l-1 Ca and 200 mg l-1 B with pH of 6.5. Medium con-

taining 10% sucrose, 200 mg l-1 Ca and 200 mg l-1 B with pH of 5.5 and by 76% germination was

the third.

Fig. 4. Pollen germination percentage of cyclamen in differentconcentrations of calcium and boron. Values with different

letter(s) are significantly (P<0.01) different.

Fig. 4. Pollen germination percentage of cyclamen in different concen-trations of calcium and boron. Values with different letter(s) are signifi-

cantly (P<0.01) different.


Pollen germination in different genotypesPollen germination percentage depended on the genotype. As shown in Figs. 5 and 6, there

were significant differences among the genotypes. Variation in pollen germination percentage of

different cyclamen genotypes has already been reported by Ewald and Schwenkel (1997) and Nan

(2008).

Some genotypes showed low germination rate (Fig. 7). Rapid pollen tube growth might be

beneficial during the fertilization process. Stigma receptivity in cyclamen is 16 days approximately

(Schwartz-Tzachor et al., 2006). However, pollens with low germination capability may be fol-

lowed by low seed yields, especially if the pollination is done late.

Cross-pollination successAveragely, 24% of crosses led to fruit set (Table 1). Naturally, a few ovaries are able to pro-

duce seeds. The main reason for the low fruit yield in cyclamen is the high rate of inflorescences

abortion (Ewald and Schwenkel, 1997); furthermore, most (approximately 80%) ovules fail to be

fertilized. Callose inclusion is also essential for fertilization in cyclamen (Reinhardt et al., 2008).

According to Ewald and Schwenkel (1997), the number of pollen tubes in styles varied be-

tween 0 and 100, and a few of them were capable to reach the ovules. In our study, pollen germi-

nation percentage was 44–84% (Fig. 5); it was reported to be 15–51% in other experiments (Ewald

and Schwenkel, 1997; Nan, 2008). In vitro condition seems to be more favorable for cyclamen

pollen germination than in vivo condition. Cross success was higher when male parent showed

higher pollen germination. Furthermore, female parents had different fruit set when crossed with

a specific male parent (Table 1), which shows ovules quality the quality depends on genotype

(Reinhardt et al., 2008) to be important for fruit set. These studies suggest that the pollens ger-

minability and ovules quality determine final seed yield in cyclamen.

With respect to our result, genotypes with high germination percentage are useful for use

a b

Fig. 6. pollen germination in two cyclamen genotypes: G1(a) and G4 (b).

Table 1. Cross-pollination success (%) in crosses among four cyclamen genotypes with

different pollen germination percentage.

♀ ♂ G1 G2 G3 G4

G1

G2

G3

G4

–

35 a

32 ab

24 bcde

34 a

–

30 abc

22 cde

25 bcd

20 de

–

19 de

19 de

15 e

17 de

–

Values with different letter are significantly (P<0.01) different. ♂ = male parents, ♀ = femaleparents.


as male parents in cross breeding. Two genotypes with higher pollen germination led to 10% higher

cross-pollination success. Considering the fact that fruit set in cyclamen is low, the 10% higher

cross-pollination success would be beneficial in large scale forimproving F1 seed production.

Literature CitedAbdul-Baki, A.A. 1992. Determination of pollen viability in tomatoes. Journal of American Society

of Horticulture Science, 117(3):473-476.

Baker, H.B. and Baker, I. 1979. Starch in angiosperm pollen grains and; its evolutionary significance.

American Journal of Botany, 66(5):591-600.

Bou Daher, F. and Geitmann, A. 2011. Actin is involved in pollen tube tropism throughredefining

the spatial targeting of secretory vesicles. Traffic, 12(11): 1537–1551.

Brewbaker, J.L. and Kwack, B.H. 1963. The essential role of calcium ion in pollen germination

and pollen tube growth. American Journal of Botany, 50(9): 859-865.

Brown, P.H., Bellaloui,N., Wimmer, M.A., Bassil, E.S., Ruiz, J., Hu, H., Pfeffer, H., Dannel, F.

and Romheld, V. 2002. Boron in plant biology. Plant Biology, 4(2): 205–223.

Bruyn, J.D. 1966. The in vitro germination of pollen of Setaria sphacelata 2. Relationships between

boron and certain cations. Physiolgia Plantarum, 19(2): 322-327.

Cakmak, I., Kurze, H. and Marschner, H. 1995. Short-term effects ofboron, germanium and high

light intensity on membrane permeabilityin boron deficient leaves of sunflower. Physiolgia

Plantarum, 95(1): 11–18.

Cruzan, M.B. 1986. Pollen tube distributions in Nicotiana glauca: evidence for density dependent

growth. American Journal of Botany, 73(6): 902–907.

Derksen, J., Rutten, T., Van Amstel, T., de Win, A., Doris, F. and Steer, M. 1995. Regulation of pollen

tube growth. Acta Botanica Neerlandica, 44(2): 93–119.

Dole, J.M. and Wilkins, H.F. 2005. Floriculture: principles and spicies. Prentice-Hall, New Jersey.

Ewald, A. and Schwenkel, H.G. 1997. Fertilization and seed formation of Cyclamen persicum Mill. Pp 113-118. In: R.H. Ellis, M. Black, A.J. Murdoch and T.D. Hong (eds.), Basic and

Applied Aspects of Seed Biology. Springer, Netherlands.

Feijó, J.A., Malhó, R. and Obermeyer, G. 1995. Ion dynamics and its possible role during in vitropollen germination and tube growth. Protoplasma, 187(1-4): 155–167.

Feijó, J.A., Sainhas, J., Hackett, G.R., Kunkel, J.G. and Hepler, P.K. 1999. Growing pollen tubes

possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip.

The Journal of Cell Biology, 144(3): 483–496.

Ferrari, T.E, Lee, S.S. and Wallace, D.H. 1981. Biochemistry and physiology of recognition in

pollen-stigma interactions. Phytopathology, 71(7): 752-755.

Fleischer, A., Titel, C. and Ehwald, R. 1998. The boron requirement and cell wall properties of

growing and stationary suspension-cultured Chenopodium album L. cells. Plant Physiology,

117(4): 1401–1410.

Franklin-Tong, V.E. 1999. Signaling and the modulation of pollen tube growth. The Plant Cell,

11(4): 727-738.

Golan-Goldhirsh, A., Schmidhalter, U., Müller, M. and Oertli, J.J. 1991. Germination of Pistacia vera L. pollen in liquid medium. Sexual Plant Reproduction,4(3): 182-187.

Halevy, A. H., Whitehead, C.S. and Kofranek, A.M. 1984. Does pollination induce corolla abscission

of cyclamen flowers by promoting ethylene production?. Plant Physiology, 75(4):1090-1093.

Herrero, M. and Hormaza, J.I. 1996. Pistil strategies controlling pollen tube growth. Sexual Plant

Reproduction, 9(6): 343–347.

Hepler, P.K., Lovy-Wheeler, A., McKenna, S.T. and Kunkel, J.G. 2006. Ions and pollen tube growth.

Pp 47-69. In: R. Malho (ed.),The Pollen Tube. Springer, Berlin, Heidelberg.

Hu, H. and Brown, P.H. 1994. Localization of boron in cell walls of squash and tobacco and its association


with pectin: evidence for a structural role of boron in the cell wall. Plant Physiology, 105(2):

681–689.

Kaneko, S.T., Ishii, T. and Matsunaga, T. 1997. A boron rhamnogalacturonan II complex from

bamboo shoot cell walls. Phytochemistry, 44(2): 243–248.

Lee, S.H., Kim,W.S. and Han, T.H. 2009. Effects of post-harvest foliar boron and calcium applications

on subsequent season's pollen germination and pollen tube growth of pear (Pyrus pyrifolia).

Scientia Horticulturae, 122(1): 77-82.

Loomis, W.D. and Durst, R.W. 1992. Chemistry and biology of boron. Bio Factors, 3(4): 229–239.

Malho, R., Read, N. D., Pais, M.S. and Trewavas, A.J. 1994. Role of cytosolic-free calcium in the reorientation

of pollen-tube growth. The Plant Journal,5(3): 331–341.

Malho, R. and Trewavas, A.J. 1996. Localized apical increases of cytosolic free calcium control pollen

tube orientation. The Plant Cell, 8(11): 1935–1949.

Mascarenhas, J.P. 1993. Molecular mechanisms of pollen tube growth and differentiation. The Plant

Cell, 5(10): 1303–1314.

Michard, E., Dias, P. and Feijó, J.A. 2008. Tobacco pollen tubes as cellular models for ion dynamics:

improved spatial and temporal resolution of extracellular flux and free cytosolic concentration

of calcium and protons using pHluorin and YC3.1 Ca Meleon. Sexual Plant Reproduction,

21(3): 169-181.

Mortazavi, M.H., Arzani, K. and Moeini, A. 2007. The effect of different concentrations of several

chemical compounds on in vitro germination of three cultivar of date palm (in Persian).

Scientific Journal of Agriculture, 30(4): 1-8.

Mulcahy, G.B. and Mulcahy, D.L. 1982. The two phases of growth of Petunia hybrida (Hort. Vilm-Andz.)

pollen tubes through compatible styles. Journal of Palynology,18: 61–64.

Naderi, R. 2000. The effects of hormonal and nutritional treatments on quantitative and qualitative

characteristics of four genotypes of cyclamen. Ph.D. Thesis. University of Tehran, Iran.

Nan, W. 2008. Determination of vitality and germination rate in cyclamen pollen. Journal of Shanxi

Agriculture University (Natural Science Edition), 2: 23.

Nyomora, A.M.S., Brown, P.H., Pinney, K. and Polito, V.S. 2000. Foliar application of boron to

almond trees affects pollen quality. Journal of the American Society for Horticultural

Science,125(2): 265–270.

Obermeyer, G. and Blatt, M.R. 1995. Electrical properties of intact pollen grains of Lilium longiflorum:

characteristics of the non-germination grain. Journal of Exprimental Botany,46(7): 803–813.

Okusaka, K. and Hiratsuka, S. 2009. Fructose inhibits pear pollen germination on agar medium

without loss of viability. Scientia Horticulturae, 122(1): 51-55.

Pfahler, P.L. 1967. In vitro germination and pollen tube growth of maize (Zea mays L.) Pollen. I. Calcium

and boron effects. Canadian Journal of Botany,45(6): 839-845.

Pfahler, P.L.1968. In vitro germination and pollen tube growth of maize (Zea mays) pollen. II. Pollen

source, calcium, and boron interactions. Canadian Journal of Botany, 46(3): 235-240.

Pierson, E.S., Miller, D.D., Callaham, D.A., Shipley, A.M., Rivers, B.A., Cresti, M. and Hepler,

P.K. 1994. Pollen-tube growth is coupled to the extracellular calcium-ion flux and the intracellular

calcium gradient: effect of BAPTA-typebuffers and hypertonic media. The Plant Cell, 6(12):

1815–1828.

Reinhardt, S., Ewald, A. and Hellwig, F. 2007. The Anatomy of the stigma and style from Cyclamen persicum (Mill.) cv. “pure white” and its relation to pollination success. Plant Biology, 9(1):

158-162.

Reinhardt, S., Ewald, A. and Hellwig, F. 2008. The correlation between ovule quality parameters

and the seed yield at Cyclamen persicum Mill. Journal of Applied Botany and Food Quality,

82(1): 76-82.

Robbertse, P.J., Lock, J.J., Stoffberg, E. and Coetzer, L.A. 1990. Effect of boron on directionality


of pollen-tube growth in Petunia and Agapanthus. South African Journal of Botany, 56(4):

487–492.

Sahar, N. and Spiegel-Roy, P. 1984. In vitro pollen germination of avocado pollen. Hort Science, 19(6):

886-888.

Sawidis, T. and Reiss, H.D. 1995. Effects of heavy metals on pollen tube growth and ultrastructure.

Protoplasma, 185(3-4): 113-122.

Schlupmann, H., Bacic, A. and Read, S.M. 1994. Uridine diphosphate glucose metabolism and callose

synthesis in cultured pollen tubes of Nicotiana alata Link et Otto. Plant Physiology, 105(2):

659-670.

Schwartz-Tzachor, R., Dafni, A., Potts, S.G. and Eisikowitch, D. 2006. An ancient pollinator of a contemporary

plant (Cyclamen persicum): When pollination syndromes break down. Flora, 201(5): 370-373.

Schwenkel, H.G. 2001. Introduction: Botany, Economic Importance, Cultivars, Micropropagation of

C. persicum. Pp 3–7. In: H.G. Schwenkel (ed.), Reproduction of Cyclamen persicum Mill.

through somatic embryogenesis using suspension culture systems. European Communities.

Stangoulis, J.C.R., Reid, R.J., Brown, P.H. and Graham, R.D.2001. Kinetic analysis of boron transport

in Chara. Planta, 213(1): 142–146.

Steer, M.W.and Steer, J.M. 1989. Pollen tube tip growth. New Phytologist, 111(3): 323–358.

Steinhorst, L. and Kudla, J. 2013. Calcium-A central regulator of pollen germination and tube growth.

Biochimica et Biophysica Acta, 1833(7): 1573-1581.

Stone, L.M. 2003. Floral biology and propagation of blue-flowered Conospermum spp. Ph.D. Thesis.

University of Murdoch, Australia.

Takamura, T. 2007. Cyclamen. Pp 459-478. In: N. O. Anderson (ed.), Flower Breeding and Genetics.

Springer, Netherlands.

Takamura, T., Nakao, T. and Tanaka, M. 1996. Effects of light and temperature on the in vitro germination

of cyclamen (Cyclamen persicum) pollen grains. Technical Bulletin of Faculty of Agriculture-Kagawa

University, 48(1): 39-45.

Tanaka, Y., Oshima, Y., Yamamura, T. 2013b. Multi-petal cyclamen flowers produced by AGAMOUS

chimeric repressor expression. Scientific Reports, 3: 2641.

Tanaka, N., Uraguchi, S., Saito, A. 2013a. Roles of Pollen-Specific Boron Efflux Transporter, OsBOR4,

in the rice fertilization process. Plant and Cell Physiology, 54(12): 2011-2019.

Taylor, L.P. and Hepler, P.K. 1997. Pollen germination and tube growth. Plant Physiology and Plant

Molecular Biology, 48(1): 461–491.

Tupy, J.and Rhova,L.1984. Changes and growth effect of pH in pollen tube culture. Journal of Plant

Physiology, 115(1): 1-10.

Vaknin, Y., Barr, N. and Saranga, Y. 2008. Preliminary investigations into the significance of floral

applications of calcium, boron and polyphenols for increased seed set in confection sunflowers

(Helianthus annuus L.). Field Crops Research, 107(2): 155-160.

Vasil, I.K. 1960. Studies on pollen germination of certain Cucurbitaceae. American Journal of Botany,47(4):

239-248.

Voyiatzis, D.G. and Paraskevopoulou-Paroussi, G. 2002. Factors affecting the quality and in vitro germination

capacity of strawberry pollen. Journal of Horticultural Science and Biotechnology, 77(2): 200–203.

Wang, Q., Lu, L., Wu, X., Li, Y. and Lin, J. 2003. Boron influences pollen germination and pollen

tube growth in Picea meyeri. Tree Physiology, 23(5): 345-351.

Zhang, J., Liu, J., Chen, Z. and Lin, J. 2007. In vitro germination and growth of lily pollen tubes is affected

by calcium inhibitor with reference to calcium distribution. Flora, 202(7): 581-588.

مجله گیاهان زینتىwww.jornamental.com قابل دسترس در سایتشماره استاندارد بین المللى چاپ: 6433-2251 شماره استاندارد بین المللى آنالین: 2251-6441

قــدرت جوانه زنــى دانــه ى گــرده و موفقیــت دورگ گیــرى در گل (Cyclamen persicum Mill.) ایرانــى ســیکالمن

محمد کرمانشاهانى*، روح انگیز نادرى، رضا فتاحى و احمد خلیقى گروه علوم باغبانى پردیس کشاورزى و منابع طبیعى دانشگاه تهران، کرج

تاریخ تایید: 4 دى 1393 تاریخ دریافت: 5 آبان 1393 [email protected] :ایمیل نویسنده مسئول *

کلیــد واژگــان: موفقیت دورگ گیرى، سیکالمن، ترکیب محیط کشت، قدرت جوانه زنى.

7 مجله گیاهان زینتى، سال چهارم، شماره 4، (1393)

ــذر دورگ در ــد ب ــالح و تولی ــده در اص ــل محدودکنن ــک عام ــذر ی ــم ب ــرد ک عملک

ــاه ســیکالمن می باشــد. مطالعــه حــارض بــه منظــور بررســی جوانه زنــی دانــه ی گــرده گی

و رابطــه ی آن بــا موفقیــت دورگ گیــری و تشــکیل میــوه در ایــن گیــاه انجــام شــد. جهــت

دســتیابی بــه ســطح باالیــی از جوانه زنــی گــرده، تأثیــر غلظت هــای مختلــف مــواد

ــاکاروز (۱۰ و ــامل س ــواک ش ــر و ک ــه ی بروبیک ــر یافت ــت تغیی ــط کش ــیمیایی در محی ش

ــید (۰، ــک اس ــرت) و بوری ــر لی ــرم ب ــیم (۰، ۲۰۰ و ۳۰۰ میلی گ ــرتات کلس ــد)، نی ۲۰ درص

۱۰۰ و ۲۰۰ میلی گــرم بــر لیــرت) در دو ســطح pH (۵/۵ و ۶/۵) بــر جوانه زنــی دورن

شیشــه ای دانه هــای گــرده ی ســیکالمن بررســی گردیــد. بیشــرتین جوانه زنــی دانــه ی گــرده

در محیــط کشــت های حــاوی بیشــرتین غلظــت از کلســیم و بــور، رصف نظــر از غلظــت

ســاکاروز و ســطح pH به دســت آمــد. جوانه زنــی دانــه ی گــرده وابســته بــه ژنوتیــپ بــود.

ــز ــرده متامی ــه ی گ ــی دان ــد جوانه زن ــر درص ــه از نظ ــپ ک ــار ژنوتی ــن چه ــری بی دورگ گی

بودنــد، انجــام شــد. همبســتگی مســتقیم بیــن موفقیــت دورگ گیــری و درصــد جوانه زنــی

ــرده ــرت گ ــی بیش ــد جوانه زن ــای دارای ۳۰ درص ــد. ژنوتیپ ه ــاهده ش ــرده مش ــه ی گ دان

منجــر بــه ۱۰ درصــد افزایــش در تولیــد میــوه شــدند.

دهــیـکـ چ

عمر گلجایی گل های بریدنی لیزیانتوس محدود است، بنابراین به منظور افزایش

با تصادفی کامال طرح پایه بر فاکتوریل آزمایش لیزیانتوس، بریدنی گل گلجایی عمر

آسکوربیک اسید در ۴ سطح (۰، ۱۰۰، ۲۰۰ و ۳۰۰ میلی گرم در لیرت) و سیرتیک اسید در ۳

سطح (۰، ۱۰۰ و ۲۰۰ میلی گرم در لیرت) و ۳ تکرار و ۳ منونه در هر تکرار طراحی و اجرا

گردید. نتایج افزایش قابل مالحظه ای در سطح احتامل ۵ و ۱ درصد در کاربرد آسکوربیک

یا متقابل اثر ساده اندازه گیری شده به صورت و سیرتیک اسید تقریبا در متام صفات

نشان داد. باالترین عمر گلجایی (۱۷/۶ روز) و محتوی آب گلربگ (۶۸/۹٪) به ترتیب در

تیامر برهمکنش آسکوربیک اسید (۳۰۰ میلی گرم در لیرت) و سیرتیک اسید (۱۰۰ میلی گرم

لیرت) و برهمکنش آسکوربیک اسید (۳۰۰ میلی گرم در لیرت) و سیرتیک اسید (۲۰۰ در

میلی گرم در لیرت) مشاهده شد، که نشان دهنده یک افزایش ۹۴ و ۲۵۲ درصدی نسبت به

شاهد (۹/۱ روز و ۲۷/۳٪) در این دو صفت است. در همین راستا، محتوی نسبی آب برگ

و محتوی آب گلربگ با افزایش سطوح آسکوربیک و سیرتیک اسید افزایش یافت. محتوای

آب گیاه نیز روند مشابهی را بروز داد. وزن تر گل ها در متامی تیامرها در طی آزمایش

کاهش یافت اما این کاهش در تیامر باالی آسکوربیک اسید (۳۰۰ میلی گرم در لیرت) به

تنهایی و در برهمکنش به مراتب کمرت بود. با توجه به نتایج این آزمایش، آسکوربیک

و سیرتیک اسید ترکیباتی با منشا طبيعي ایمن و ارزان هستند که می توان آن ها را به

عنوان جایگزین های مناسبی برای مواد شیمیایی متداول در افزایش عمر گلجایي گل های

بریدنی در نظر گرفت. تجاری سازی این محلول ها نیازمند آزمایش های بیشرت است.

دهــیـکـ چ

اسـتفاده از اسید آسـکوربیک و اسـید سـیتریک به منظور افزایش ماندگارى گل(Eustoma grandiflorum ‘Mariachi Blue’) شـاخه بریده ى لیسیانتوس

فرناز شیخى1، سید حسین نعمتى2، نوید وحدتى3* و على دولتخواهى3 1 کارشناسى ارشد، گروه باغبانى، دانشگاه فردوسى مشهد، مشهد، ایران

2 استادیار گروه باغبانى، دانشگاه فردوسى مشهد، مشهد، ایران3 دانشجوى دکترى، گروه باغبانى، دانشگاه فردوسى مشهد، مشهد، ایران

تاریخ تایید: 19 آذر 1393 تاریخ دریافت: 24 تیر 1393 [email protected] :ایمیل نویسنده مسئول *

کلیــد واژگــان: گل هاى بریدنى، اسیدهاى آلى، عمر گلجایى، محلول نگهدارنده.

مجله گیاهان زینتىwww.jornamental.com قابل دسترس در سایت

شماره استاندارد بین المللى چاپ: 6433-2251 شماره استاندارد بین المللى آنالین: 2251-6441

مجله گیاهان زینتى، سال چهارم، شماره 4، (1393)6

ــدی ــاقه گل محم ــای س ــی قلمه ه ــای مورفولوژیک ــی واکنش ه ــور بررس به منظ

بــه اینــدول بوتیریــک اســید در زمان هــای مختلــف، آزمایشــی به صــورت فاکتوریــل در

قالــب طــرح بلوک هــای کامــال تصادفــی در ســه زمــان و در ســه تکــرار و ١٠ مشــاهده

در هــر تکــرار به اجــرا در آمــد. در ایــن آزمایــش اثــرات ســاده اینــدول بوتیریــک اســید،

ــدی ــه دهی گل محم ــی ریش ــات مورفولوژیک ــر صف ــا ب ــل آن ه ــرات متقاب ــان و اث زم

اندازه گیــری شــد. از فاکتورهــای مهــم مــورد اندازه گیــری می تــوان بــه میانگیــن طــول

ریشــه، درصــد ریشــه زایی، درصــد کالــوس زایــی و وزن خشــک ریشــه اشــاره کــرد. پــس

از فروبــری رسیــع قلمه هــا در IBA بــه مــدت ٥ ثانیــه، قلمه هــا در گلخانــه پژوهشــی

تحــت سیســتم مه پــاش در بســرت کشــت قــرار گرفتنــد. باتوجــه بــه یافته هــای پژوهــش

ــن ــش میانگی ــا؛ بیشــرتین افزای ــل آن ه ــرات متقاب ــان و اث ــر ســاده IBA، زم حــارض، اث

طــول ریشــه در تیــامر بــا ٤٠٠٠ میلی گــرم در لیــرت IBA و در قلمه هایــی کــه زمســتان

ــه ــی ک ــز در قلمه های ــن بیشــرتین درصــد ریشــه زایی نی ــد، همچنی ــه شــده بودن گرفت

ــا ٢٠٠٠ و ٤٠٠٠ میلی گــرم در لیــرت IBA در اســفند مــاه تیــامر شــدند، حاصــل شــد. ب

بیشــرتین میــزان وزن خشــک ریشــه نیــز، در اســفند مــاه و در غلظــت ٤٠٠٠ میلی گــرم

در لیــرت اینــدول بوتیریــک اســید بدســت آمــد.

دهــیـکـ چ

اثر تنظیم کننده هاى رشد ایندول بوتیریک اسید و زمان تهیه قلمه بر افزونش درختچه زینتى گل محمدىمهسا کاشفى1*، حسین زارعى2 و فرزانه بهادرى3

1 فارغ التحصیل گروه علوم باغبانى، دانشگاه منابع طبیعى و علوم کشاورزى دانشگاه گرگان، گرگان، ایران

2 استادیار گروه علوم باغبانى، دانشگاه منابع طبیعى و علوم کشاورزى دانشگاه گرگان، گرگان، ایران

3 استادیار مرکز تحقیقات منابع طبیعى و کشاورزى سمنان، سمنان، ایران

تاریخ تایید: 7 دى 1393 تاریخ دریافت: 21 بهمن 1392 [email protected] :ایمیل نویسنده مسئول *

کلیــد واژگــان: ایندول بوتیریک اسید، صفات مورفولوژیک، گل محمدى.



ــرات اســید هیومیــک (HA) و NPK در ســال ۲۰۱۲- ــن مطالعــه به منظــور بررســی اث ای

۲۰۱۱ بــا هــدف تعییــن بهرتیــن نــرخ رشــد و گلدهــی روی گل اللــه رقــم ’تریومــف‘ انجــام

:T۲ ،(۱۷:۱۷:۱۷) NPK ۱۰ گــرم در مــرت مربــع :T۱ ،(شــاهد) T۰ شــد. ۵ تیــامر عبارتنــد از

:T۳ ،(۱۷:۱۷:۱۷) NPK ــع ــرت مرب ــرم در م ــک (۸٪) + ۱۰ گ ــید هیومی ــرت اس ۰/۷۵ میلی لی

۱/۲۵ :T۴ ( ۱۷:۱۷:۱۷) و NPK ۱ میلی لیــرت اســید هیومیــک (۸٪) + ۱۰ گــرم درمرتمربــع

ــب ــه در قال ــع NPK (۱۷:۱۷:۱۷) ک ــرم در مرتمرب ــیدهیومیک (۸٪)+ ۱۰ گ ــرت اس میلی لی

ــی ــات رویش ــه خصوصی ــد. هم ــرار گرفتن ــی ق ــورد بررس ــرار م ــه تک ــا س ــرح RCBD ب ط

ــد ــرار گرفتن ــک و NPK ق ــید هیومی ــر اس ــت تاثی ــی داری تح ــورت معن ــی به ص و زایش

و نتایــج حاصلــه نشــان داد کــه تیــامر T۴ نســبت بــه ســایر تیامرهــا موثرتــر بــود. ایــن

تیــامر نتایــج بی نظیــری از نظــر زود گلدهــی، افزایــش ارتفــاع گیــاه، توســعه ســطح بــرگ،

قطــر ســاقه، مقــدار کلروفیــل بــرگ، طــول ســاقه گلدهنــده، عمــر گلجایــی و وزن تــر و

T۲ خشــک گل به همــراه داشــت. ایــن تیــامر مقــدار مــواد غذایــی را نیــز در مقایســه بــا

و T۳ افزایــش داد. گیاهــان شــاهد و بعــد از آن گیاهــان تیــامر T۱ رشــد ضعیفــی داشــته

و عملکــرد و کیفیــت آن هــا کاهــش یافــت. نتایــج حاصــل از ایــن مطالعــه نشــان داد کــه

ــه ــع) ب ــرم در مرتمرب ــا NPK (۱۰ گ ــراه ب ــک ۸٪) هم ــرت اســید هیومی HA (۱/۲۵ میلی لی

بهبــود یکنواختــی محصــول، رشــد گیــاه و کیفیــت گل اللــه کمــک می کنــد.

دهــیـکـ چ

Tulipa gesneriana روى رشد و نمو گل NPK تاثیر تلفیق اسید هیومیک ودر فیصل آباد پاکستان

آ. على1، اس.یو. رحمان2، اس. رازا3 و اس.ج. بات41 موسسه علوم باغبانى، دانشگاه کشاورزى، فیصل آباد، پاکستان

2 موسسه تحقیقات اکوسیستم کشاورزى و تولید گیاهان ارگانیک مناطق گرمسیرى و نیمه گرمسیرى، دانشگاه کاسل، استینستر، ویتزنهاسن، آلمان

3 موسسه علوم محیطى و خاك، دانشگاه کشاورزى، فیصل آباد، پاکستان

4 گروه باغبانى، دانشگاه کشاورزى، راوالپیندى، پاکستان

تاریخ تایید: 9 دى 1393 تاریخ دریافت: 5 آبان 1392 [email protected] :ایمیل نویسنده مسئول *

کلیــد واژگــان: کیفیت گل، اسید هیومیک، جذب عناصر غذایى، رشد گیاه، شاخه برید ى الله.




انتخـاب بسـرت کاشـت مناسـب بـرای رشـد گیاه یکـی از معضـالت بیشـرت گلکاران

در تولیـد گیاهـان زینتـی گلدانـی اسـت. بنابرایـن این تحقیـق برای بررسـی تاثیر بعضی

بسـرتهای کاشـت قابل دسـرتس در مخلوط با پرلیت روی رشـد و منو پوتوس اسـت. این

مطالعـه بـر اسـاس طـرح کامـال تصادفـی با ٥ تیـامر، ٨ زمـان اندازه گیـری و ٦ تکـرار در

یـک گلخانـه بـا پوشـش فایـربگالس در دانشـگاه کشـاورزی و منابـع طبیعـی گـرگان طی

سـال های ٢٠١٠-٢٠٠٩ انجـام شـد. تیامرهـا شـامل حجم مسـاوی از پرلیت + کمپوسـت

بـرگ، پرلیـت + شـلتوک برنـج، پرلیـت + کوکوپیت، پرلیت + کمپوسـت درختـان جنگلی

و پرلیـت + کمپوسـت قـارچ بودنـد. صفاتـی مثـل ارتفـاع گیاه، قطر سـاقه، تعـداد برگ،

وزن تـر و خشـک بـرگ و مقـدار کلروفیل بـرگ اندازه گیری شـدند. همچنیـن گیاهان از

نظـر انـدازه و شـکل ظاهـری نیـز با هم مقایسـه شـدند. نتایـج آنالیز داده ها نشـان داد

کـه اثـر بسـرت کاشـت، زمـان اندازه گیـری و اثـر متقابـل آن هـا در متـام صفـات معنی دار

بودنـد. خصوصیـات رشـد مثـل ارتفـاع بوته، تعـداد برگ و مقـدار کلروفیل، کـه با ارزش

بازاریابـی ایـن گیـاه مرتبـط اسـت، در بسـرتهای پرلیـت + کمپوسـت بـرگ و پرلیـت +

کمپوسـت قـارچ بهـرت از تیامرهـای دیگـر بودنـد. بنابرایـن، ایـن بسـرتها می توانـد بـرای

تولیـد پوتـوس بـا کیفیـت در گلخانه ها اسـتفاده شـوند.

دهــیـکـ چ

مقایسـه مخلوط مختلف بستر حاوى پرلیت روى رشـد و خصوصیات مورفولوژیکى (Scindapsus Aureum L.) پوتوس

فاطمه بیدارنمانى 1 و حسین زارعى 2 گروه باغبانى موسسه تحقیقات کشاورزى، دانشگاه زابل، زابل، ایران

استادیار گروه باغبانى، دانشگاه منابع طبیعى و علوم کشاورزى گرگان، گرگان، ایرانتاریخ تایید: 9 دى 1393 تاریخ دریافت: 5 آبان 1393

[email protected] :ایمیل نویسنده مسئول *

کلیــد واژگــان: کمپوست، پرلیت، پوتوس، مخلوط گلدان.



Cymbidium bicolor Lindl. ایـن مطالعـه کپسـوله کـردن پیـش پداژه هـای گیـاه

۶۰ روزه ی حاصـل از کشـت بـذر را بـرای نگهـداری کوتـاه مـدت و ازدیـاد این گیـاه، ترشیح

می کنـد. غلظت هـا و ترکیبـات ژل زمینـه (آلژینـات سـدیم) و ترکیبـات کمپلکـس (کلریـد

کلسـیم) بـرای تهیـه تیله های یکنواخت آزمایش شـدند. تیله های ایـده آل از ترکیب ۳ درصد

آلژینـات سـدیم و ۱۰۰ میلی مـول کلریـد کلسـیم بدسـت آمـد. پیـش پداژ ه هـای کپسـوله

۴/۴۲) BA تکمیـل شـده بـا MS شـده بهرتیـن بـاز رشـد و نگهـداری را روی محیـط کشـت

میکرومـول) نشـان دادنـد. پیـش پداژه هـای کپسـوله شـده کـه در ۲۵ درجـه سـانتی گراد

نگهـداری شـده بودنـد، سـبز بودنـد و پـس از ۳۶۰ روز ۵۲ درصـد قـوه نامیه داشـتند. پیش

پداژ هـای کپسـوله شـده کـه در ۴ درجه سـانتی گراد نگهـداری شـده بودنـد، تـا ۳۰ روز زنده

ماندنـد و سـپس رسیعـا قدرت انبارمانـی آن ها کاهش یافـت. گیاهچه های کامال رشـد کرده

بـه گلدان هـای پالسـتیکی حـاوی ورمیکولیـت منتقـل و ۶۰ روز در رشایـط اتـاق نگهـداری

شـدند تـا بومی سـازی انجـام شـود. ۹۰ درصـد گیاهچه هـای ریـکاوری شـده مقاوم سـازی

شـده و به صـورت موفقیت آمیـزی بـه خـاک منتقل شـدند. این مطالعـه می توانـد در ازدیاد

مقیـاس وسـیع و انبـارداری کوتـاه مـدت ایـن گیاه تجاری سـودمند باشـد.

دهــیـکـ چ

کپسـوله کردن پیش پداژه هـاى گیـاه .Cymbidium bicolor Lindl براى انبار کوتاه مدت و تبادل ژرم پالسـم

جى. ماهندران*، ان. پاریماال دوى و وى. نارماتها باى محقق گروه گیاهشناسى دانشگاه بهاراتیار، هندوستان

تاریخ تایید: 2 دى 1393 تاریخ دریافت: 21 خرداد 1393 [email protected] :ایمیل نویسنده مسئول *

کلیــــد واژگــــان: بومى ســازى، کپســوله کــردن، ارکیــد دارزى، نگهــدارى از ژرم پالســم، پیــش پــداژه، آلژینــات ســدیم، بذرهــاى مصنوعــى.




Aphyllorchis Montana (Reichenb.f .). یک دستورالعمل باززایی درون شیشه ای برای گیاهکـه یـک ارکیـد بـدون کلروفیل اسـپورفیت اسـت، از طریق کشـت بذرهای نابالـغ به صورت موفقیت آمیـز انجـام شـد. در بیـن ۶ محیـط کشـت پایه کـه بـرای جوانه زنی بذرهـا ارزیابی شـد، محیط کشـت BM-TM و سـپس KC بهرتیـن بودند. ۴۰ روز بعـد، همه ی محیط های کشـت قهـوه ای شـده و رشـد پیـش پداژه ها متوقف شـد. زغال فعـال به مقدار یـک گرم در لیـرت در محیـط کشـت BM-TM ٪۵۰ بـرای منـو بهـرت پیـش پداژه هـا بهـرت بـود. ایـن محیط کشـت بـا تنظیـم کننده هـای رشـد مختلـف بـه تنهایـی یـا در ترکیـب بـا هـم بـرای تکثیـر شاخسـاره ها تکمیل شـد. از بین ۵ سیتوکنین آزمایش شده، TDZ در غلظت ۶/۸ میکرومول بـرای القـای تکثیـر شاخسـاره موثرترین بود و پـس از ۱۰ روز ۰/۲۷±۱۷/۲۴ شاخسـاره تولید شـد. افـزودن مقـدار کمـی NAA (۱/۳ میکرومـول) بـه محیـط کشـت MS تکمیل شـده با سـیتوکنین TDZ (۶/۸ میکرومـول) بـرای شـاخه زایی مطلـوب بود. میانگیـن ۰/۵۴ ± ۲۷/۵۶ شاخسـاره بـا ۰/۱۱ ± ۳/۹۲ ریشـه در هـر منونه گیاهی بدسـت آمد. واکنـش بذرهای حاصل از پیش پداژه هـا بـه انـواع مختلـف ترکیبـات آلـی اضافـه شـده مثل پپتـون، عصـاره مخمر و آب نارگیـل نیـز ارزیابـی شـد. افـزودن ایـن ترکیبـات بـه محیط کشـت حـاوی TDZ تعداد شاخسـاره را افزایـش داد. گیاهچه هـا در گلدان هـای پالسـتیکی حـاوی ورمی کولیت اسـرتیل شـده سـازگار شـدند. میـزان بقـاء گیاهچه هـا در اتاق کشـت ۲۵ درجـه سـانتی گراد، صد در صـد بـود. ارزیابـی فعالیت ضد باکرتیایی و آنتی اکسـیدانی و تخمین مـواد فنولیک و مقدار فالونوئیـد عصـاره متانولـی گیاهـان ریز ازدیاد شـده نیز انجـام و با گیاهان خودرو مقایسـه شـد. در همـه آزمایش هـا، عصـاره متانولی گیاهان خـودرو مقادیر بیشـرتی از ترکیبات فوق

نسـبت به گیاهان درون شیشـه ای داشـتند.

دهــیـکـ چ

یک تکثیر درون شیشـه اى کارآمد همراه با فعالیت هاى آنتى اکسـیدانى یک تکثیر درون شیشـه اى کارآمد همراه با فعالیت هاى آنتى اکسـیدانى Aphyllorchis Montana (Reichenb.f.) و ضدمیکروبى گیاه و ضدمیکروبى گیاه

گانسان ماهندرانمحقق گروه گیاهشناسى دانشگاه بهاراتیار، هندوستان

تاریخ تایید: 29 آبان 1393 تاریخ دریافت: 21 خرداد 1393 [email protected] :ایمیل نویسنده مسئول *

.Aphyllorchis MontanaAphyllorchis Montana (Reichenb.f (Reichenb.f.)، ، جوانه زنى جوانه زنى ،.). کلیــد واژگــان: کلیــد واژگــان: فعالیت ضد باکتریایى، فعالیت آنتى اکسیدانىفعالیت ضد باکتریایى، فعالیت آنتى اکسیدانى، غیرهمزیستى بذر، تکثیر درون شیشه اى، تنظیم کننده هاى رشد، غیرهمزیستى بذر، تکثیر درون شیشه اى، تنظیم کننده هاى رشد، 2،22،2- دى فنیل-- دى فنیل-1- پیکریل هیدرازیل. - پیکریل هیدرازیل.



www.jornamental.comThe Journal of Ornamental Plants, is an open access journal that provides rapid publication of manuscripts

on Ornamental plants, Floriculture and Landscape. Journal of Ornamental Plants is published in English,

as a printed journal and in electronic form.

All articles published in Journal of Ornamental Plants are peer-reviewed. All manuscripts should convey im-

portant results that have not been published, nor under consideration anywhere else. Journal of Orna-

mental Plants will be available online around the world free of charge at http://www.jornamental.com.

In addition, no page charge are required from the author(s). The Journal of Ornamental Plants is pub-

lished quarterly by Islamic Azad University, Rasht Branch, Rasht, Iran.

Manuscript Submission

Please read the “Instructions to Authors” before submitting your manuscript. Submit manuscripts as e-

mail attachment to Dr. Ali Mohammadi Torkashvand, Executive Director of Journal of Ornamental Plants,

at [email protected]. Electronic submission of manuscripts is strongly encouraged, provided that

the text, tables, and figures are included in a single Microsoft Word 2003 file. A manuscript acknowledg-

ment including manuscript number will be emailed to the corresponding author within 72 hours.

Please do not hesitate to contact meif you have any questions about the journal. We look forward to

your participation in the Journal of Ornamental Plants.

Address: Islamic azad University, Rasht Branch

Horticultural Department,

Agriculture Faculty,

Rasht,

Iran.

P.O.Box 41335-3516

Email: [email protected]

URL: http:// www.jornamental.com

Topics and Types of PaperJournal of Ornamental Plants is an international journal to the publication of original papers and reviews

in the Ornamental plants, Floriculture and Landscape fields. Articles in the journal deal with Ornamental

plants, Floriculture and Landscape. The scope of JOP includes all Ornamental plants, Floriculture and

Landscape. The journal is concerned with Ornamental plants, Floriculture and Landscape and covers

all aspects of physiology, molecular biology, biotechnology, protected cultivation, and environmental areas

of plants. The journal welcomes the submission of manuscripts that meet the general criteria of signif-

icance and scientific excellence, and will publish:

● Research articles

● Short Communications

● Review

Papers are welcome reporting studies in all aspects of Ornamental plants, Floriculture and Landscape

including:

Any Novel Approaches in Plant Science

Biotechnology

Environmental Stress Physiology

Genetices and Breeding

Photosynthesis, Sources-Sink Physiology

Postharvest Biology

Seed Physiology

Soil-Plant-Water Relationships

Modelling

Published by:Islamic Azad University, Rasht Branch, Iran


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