vol 1(3), december 2011 3/jornamental 1(3)-f… · m. k. prithvi raje urs, k. rajashekar, a....

Matthiola incana Micropropagation Using Shoot Tips and Callus Induction Derived from

Lamina Explants and Rooting Capacity from Callus.................................................................129

A. Ahmadi Hesar, B. Kaviani, D. Hashemabadi, A.R. Tarang, S. Bohlooli Zanjani, M. H. Ansari.

Identification and Assessment of Fungal Diseases of Major Medicinal Plants

........................................................................................................................................................137

M. Nasr Esfahani, M. Monazzah.

An Easy and Simple Method for Estimating Total Shoot Length During Screening and Evaluation

of Mulberry (Morus spp.) Genotypes.........................................................................................147

M. Rekha, K. Kesavacharyulu, K. Rajashekar.

Marigold: The Possibilty Using Vermicompost as the Growth Medium................................153

F. Shadanpour, A. Mohammadi Torkashvand, K. Hashemi Majd.

The Role of Preservative Compounds on Number of Bacteria on the End of Stems and Vase

Solutions of Cut Gerbera.............................................................................................................161

T. Oraee, A. Asghar Zadeh, M. Kiani, A. Oraee.

Evaluation of Mulberry (Morus spp.) Genotypes for Tolerance to Major Abiotic Stresses........167

M. K. Prithvi Raje Urs, K. Rajashekar, A. Sarkar.

Synchronous Plantlet Formation by Using Banana Extract and In vitro Hardening in Orchid,

Dendrobium lituiflorum Lindl......................................................................................................175

S. Vyas, P. Kapoor-Pandey, S.Guha, I. Usha Rao.

Study on the Effect of Different Growing Media on the Growth and Yield of Gerbera

(Gerbera jamesonii L.)..................................................................................................................185

M. A. Khalaj, M. Amiri, S.S. Sindhu.

Vol 1(3), December 2011

Journal of

Ornamental and Horticultural Plants

It is approved publication of Journal of Ornamental and Horticultural Plants (JOHP) 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: Dr. Davood Hashemabadi

Executive Manager: Dr. Shahram Sedaghat Hoor

Editorial Board:

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

Associated Professor Naderi, R., University of Tehran, Iran

Professor Aytekin Polat, Ataturk University, Antakya, Turkey

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

Professor Peyvast G. University of Guilan, Iran

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

Assistant Professor YU,W. The Chinese University of Hongkong

Associated Professor Hokmabadi, H. Pistachio Research Institute, IranProfessor Salah El Deen M. Mahmoud, Al Azhr University, Egypt

Assistant Editor: Zahra Bagher Amiri

Abstracting/Indexing

Index Copernicous, Islamic World Science Citation Center (ISC), Open-J-Gate, Magiran, EBSCO

(under process).

Journal of Ornamental and Horticultural Plants (JOHP) is an international journal devoted to the

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Web Site: www. jornamental.com

Matthiola incana Micropropagation Using Shoot Tips and Callus Induction Derived from Lamina

Explants and Rooting Capacity from Callus....................................................................................129

Identification and Assessment of Fungal Diseases of Major Medicinal Plants................................137

An Easy and Simple Method for Estimating Total Shoot Length During Screening and Evaluation of

Mulberry (Morus spp.) Genotypes...................................................................................................147

Marigold: The Possibilty Using Vermicompost as the Growth Medium.........................................153

The Role of Preservative Compounds on Number of Bacteria on the End of Stems and Vase Solutions

of Cut Gerbera..................................................................................................................................161

Evaluation of Mulberry (Morus spp.) Genotypes for Tolerance to Major Abiotic Stresses...................167

Synchronous Plantlet Formation by Using Banana Extract and In vitro Hardening in Orchid,

Dendrobium lituiflorum Lindl.........................................................................................................175

Study on the Effect of Different Growing Media on the Growth and Yield of Gerbera

(Gerbera jamesonii L.)..................................................................................................................185

Content Page

www.jornamental.com

Journal of Ornamental and Horticultural Plants, 1(3): 129-136, December, 2011 129

Matthiola incana Micropropagation Using Shoot Tips

and Callus Induction Derived from Lamina Explants

and Rooting Capacity from Callus

Tissue culture is an attractive alternative for plant propagation.Micropropagation is a technique to ensure a constant and uniform source ofornamental plants. Matthiola incana is an important ornamental speciesmainly cultivate by seed. Matthiola incana seeds were germinated on solidMS medium without plant growth regulators. Shoot proliferation and rootformation are possible using kinetin (Kn) and naphthalene acetic acid (NAA).Shoot tips and leaf micro-cuttings derived from in vitro germinated seedlingswere subcultured on solid MS medium containing Kn (0, 0.5, 1 and 2 mg l-1)and NAA (0, 0.5, 1 and 2 mg l-1) for shoot tips explants and Kn (0, 0.5 and 1mg l-1) and NAA (0, 0.5 and 1 mg l-1) for leaf explants. Shoot tips mediasupplemented with 2 mg l-1 Kn without NAA and 2 mg l-1 NAA without Knresulted in the best shoot length (1.20 cm) and root number (1.90), respectively.The callus was induced from most leaf media after four weeks of culture. MSmediums containing 0.5 mg l-1 Kn and 0.5 mg l-1. The largest number (1.94)and the highest length (16.60 mm) of roots were obtained in MS mediumsupplemented with 1 mg l-1 Kn + 0.5 mg l-1 NAA. NAA prevented rootformation originated from callus with concentration of 1 mg l-1 + 0.5 and 1 mg l-1 Kn.

Keywords: Brassicaceae, Organogenesis, Ornamental Plants, Phytohormones.

Abstract

A. Ahmadi Hesar1, B. Kaviani2*, D. Hashemabadi2, A.R. Tarang3, S. Bohlooli Zanjani3 and M. H. Ansari4

1 M.Sc. Student, Rasht Branch, Islamic Azad University, Rasht, Iran2 Department of Horticultural Science, Rasht Branch, Islamic Azad University, Rasht, Iran3 North Biotechnology Institute, Rasht, Guilan, Iran4 Department of Agronomy, Rasht Branch, Islamic Azad University, Rasht, Iran

*Corresponding author’s email: [email protected]

Journal of Ornamental and Horticultural Plants, 1(3): 129-136, December, 2011130

INTRODUCTION

The ornamental species Matthiola incana, belonging to Brassicaceae, is a pot plant. TheBrassicaceae is a fairly large family with many economically important taxa, but from viewpointof tissue culture, it has been little studied. Natural propagation of Matthiola incana takes place byseed. The economic value of ornamental plants has increased significantly worldwide and isincreasing annually by 8-10% (Jain and Ochatt, 2010). The techniques for in vitro propagation ofornamental plants and tissue culture laboratory equipment are being continuously improved tomeet the demand of the floriculture breeding and industry (Rout et al., 2006). Tissue culture hasbecome a routine technique in agricultural and horticultural development which has revolutionizedthe ornamental industry and most popular application of this technique is micropropagation(Maira et al., 2010; Bhattacharya and Bhattacharyya, 2010). Micropropagation through tissueculture permits the regeneration of large numbers of disease free plants from small pieces(explants) of stock plants in a relatively short period and, crucially, without seasonal restrictions(Preil et al., 1988). In general, the number of publications on different aspects of the culture ofMatthiola incana is limited, with emphasis on micropropagation through somatic explants(Gautam et al., 1983). In the field of ornamental plants, tissue culture has allowed masspropagation of superior genotypes and plant improvement, thus enabling the commercializationof healthy and uniform planting material (Winkelmann et al., 2006; Nhut et al., 2006). Thesuccess of the micropropagation method depends on several factors like genotype, media, plantgrowth regulators and type of explants, which should be observed during the process (Pati et al.,2005; Nhut et al., 2010). In general, three modes of in vitro plant regeneration have been inpractice: organogenesis, embryogenesis and axillary proliferation. In tissue culture, cytokininsand auxins play a crucial role as promoters of cell division and act in the induction anddevelopment of meristematic centers leading to the formation of organs (Peeters et al., 1991). Themost frequently used growth regulators for micropropagation of ornamental plants by organogenesis,embryogenesis and axillary proliferation are naphthalenacetic acid (NAA), and benzyl adenine(BA) (Jain and Ochatt, 2010). Kn has been applied for micropropagation of many plants (Jain andOchatt, 2010). In this paper, potential of shoot tips and leaf explants of in vitro grown Matthiolaincana seedling to proliferation, and induction of callus and root by Kn and NAA has beendiscussed.

MATERIALS AND METHODS

Seeds of Matthiola incana were prepared from Mohaghegh-e-Ardabili University, Iran.The seeds were washed thoroughly under running tap water for 20 min and disinfected with a20% NaOCl aqueous solution and Tween-20 for 10 min then rinsed three times in steriledistilled water (10 min each). At the end, seeds were sterilized for 2 min in 70% ethanolfollowed by three times rinses with sterile distilled water (15 min each). Five seeds werecultivated in culture flasks on MS (Murashige and Skoog, 1962) basal medium without growthregulators. Micro-cuttings (shoot tips and leaves) were isolated from 4-week-old plants andcultivated on MS media supplemented with 0, 0.5, 1 and 2 mg l-1 Kn, and 0, 0.5, 1 and 2 mg l-

1 NAA for shoot tips, also, 0, 0.5 and 1 mg l-1 Kn, and 0, 0.5 and 1 mg l-1 NAA for leaves. Themedia were adjusted to pH 5.7-5.8 and solidified with 7 g/L Agar-agar. The media were pHadjusted before autoclaving at 121°C, 1 atm. for 20 min. The cultures were incubated in growthchamber whose environmental conditions were adjusted to 25±2°C and 75-80% relativehumidity, under a photosynthetic photon density flux 50 µmol/m2/s with a photoperiod of 14 hper day. Some characters such as callus, fresh weight, number of root, and root length werecalculated after 30 days. The experimental design was R.C.B.D. Each experiment was carriedout in three replicates and each replicate includes five specimens. Data were subjected toANOVA (analysis of variance) and significant differences between treatments means were de-termined by LSD test.


RESULTS AND DISCUSSION

The plant growth regulators are widely used for callus, rooting and shoot induction intissue culture studies. Therefore, we studied the effect of Kn and NAA on shoot proliferation,callus production and rooting of Matthiola incana, an ornamental plant. The medium supplementedwith 2 mg l-1 Kn without NAA resulted in the best shoot length (1.20 cm) (Table 1). Data analysisshowed that the effect of Kn, NAA and Kn × NAA were significant on the length of shoot and(p≤0.01) (Table 2). When the shoot tips were inoculated in the medium containing 2 mg l-1 NAAwithout Kn, the best result was observed for root number (1.90) (Table 1). Analysis of varianceshowed that the effect of Kn was no significant on the root number, while the effect of NAA andKn × NAA on the root number was significant (p≤0.05) (Table 2). Similar to our findings, manyresearchers showed that Kn induced multiple shoot formation (Sajina et al., 1997b; Mathai et al.,1997; Luo et al., 2009). Some studies showed the positive effect of NAA on rooting (Gautam etal., 1983; Hammaudeh et al., 1998; Lee-Epinosa et al., 2008). The results on leaf explantsrevealed that the largest number and highest length of root were obtained in MS basal mediumcontaining 0.5 mg l-1Kn + 1 mg l-1 NAA. Our data revealed that there are differences in the effectof the different concentrations of Kn and NAA on the root number and length. The most rootslength (16.60 mm) and the most number of roots (1.94) were found when we used 0.5 mg l-1 Kn+ 1 mg l-1 NAA (Table 3). This result was comparatively better than the growth of control. Dataanalysis showed that the effect of Kn and NAA was significant on the length and number of root(p≤0.01) (Table 4). Interaction effect of Kn and NAA was significant on the length and number ofroot (p≤0.01 and p≤0.05, respectively) (Table 4). The highest percent of callus induction (100%)was seen in explants grown in MS medium containing 0.5 mg l-1 NAA and 0.5 mg l-1 Kn + 0.5mg l-1 NAA (Table 3). Data analysis showed that the effect of Kn and NAA were significant onthe callus formation (p≤0.01) (Table 2). The effect of Kn + mg l-1 NAA was no significant on thecallus formation (Table 4). The most fresh weight between explants was obtained in explantsgrown in MS medium supplemented with 0.5 mg l-1 NAA (0.833 g) and 0.5 mg l-1 Kn + 1 NAA(0.817 g) (Table 3). Data analysis showed that the effect of Kn was significant on the fresh weight(p≤0.01) (Table 4). No the effect of NAA and Kn + NAA were significant on the fresh weight(Table 4).

In case of ornamental plants, leaf especially obtained from in vitro grown plantlets hasmore extensively been applied. We used from leaf explants taken from in vitro germinated seedsof Matthila incana. Many researchers applied leaves of ornamental plants as explants (Ibrahimand Debergh, 2000; Pati et al., 2004; Tyagi et al., 2010; Godo et al., 2010; Eeckant et al., 2010;Radice, 2010). Organogenesis takes place either directly or after callus formation. Studies onmany ornamental plants showed both kinds of organogenesis (Jain and Ochatt, 2010). There aremany reports on organogenesis via callus formation (Pati et al., 2010; Jain and Ochatt, 2010).Studies of Maira et al., (2010) on Anthurium andreanum Lind cv Rubrun revealed that the four-week-old in plants obtained from micro-cuttings, showed callus proliferation at the stem base.The development of plantlets was observed from callus tissue. In vitro leaf explants in Rosadamascena and some other ornamental plants were used for direct organogenesis (Leffering andKok, 1990; Ibrahim and Debergh, 2001; Dubios and de Vries, 1995). Nencheva (2010) showeddirect organogenesis from pedicel explants of Chrysanthemum. Cytokinins and auxins are usuallyknown to promote the formation of callus and root in many excited and in vitro cultured organs(Jain and Ochatt, 2010). Proper type and concentration of these hormones are different for eachspecies. We observed that callus was formed on the explants in many treatments. NAA did notstimulate much callus induction and root formation when it was applied alone (Table 3). Similarto our findings, many researchers showed that cytokinins and auxins induced callus induction androot formation in ornamental plants (Fuller and Fuller, 1995; Sangavai and Chellapandi, 2008;Hashemabadi and Kaviani, 2010; Dorion et al., 2010; Pati et al., 2010; Ochatt et al., 2010; Jainand Ochatt, 2010). Callus induction and root formation was performed for most Rhododendrongenotypes by indole-3-acetic acid (IAA), NAA, indole-3-butyric acid (IBA) and 2,4-Dichlorophenoxy


acetic acid (2,4-D) (Eeckaut et al., 2010). Rout et al., (1990) observed that the addition ofbenzylaminopurine (BAP) (2.0-3.0 mg l-1) as the only growth regulator in the culture mediumresulted in feeble callusing at the cut ends of the explants and the shoot elongation wasconsiderably slow.

Rooting is an important process in micropropagation. Without an effective root system,plant acclimatization will be difficult and the rate of plant propagation may be severely affected(Gomes et al., 2010). The ideal concentrations of cytokinins and auxins differ from species tospecies and need to be established accurately to achieve the effective rates of multiplication(Gomes et al., 2010). The most types of cytokinins and auxins applied for root formation oncallus or organs are BA, Kn and IAA, and NAA, IBA and 2,4-D, respectively. Some studiesshowed the positive effect of cytokinins on rooting (Gomes et al., 2010). A review of the literatureclearly points out to a negative effect of cytokinins on shoot rooting (Van Staden, 2008), althougha positive role has been occasionally referred (Nemeth, 1979; Bennett et al., 1994). Studies ofGodo et al., (2010) and Wong and Bhalla (2010) on Lysionotus pauciflorus Maxim. and Scaevola,respectively, showed that the regenerated shoots rooted easily on medium without any plantgrowth regulators. Current study showed the positive effect of Kn and NAA on root formation.Contrary to our findings, root formation was inhibited in the medium culture of Lilium longiflorumGeorgia containing BA (Han et al., 2004). Nayak et al., (2010) showed that the lowest rooting ofBambusa arundinacea was observed in medium without Kn. Fuller and Fuller (1995) demonstratedthat the least and most percentage of explants regeneration with root percent (5.0% and 65.0%) inBrassica spp. obtained in culture medium without IBA and Kn, and 2 mg l-1 IBA without Kn, re-spectively. The studies of Gautam et al., (1983) on in vitro regeneration of plantlets from somaticexplants of Matthiola incana showed only a few shoots developed on explants reared on MSmedium supplemented with 0.1 mg l-1 Kn. Also, NAA (1 and 4 mg l-1) induced profuse rooting inexplants. Nhut et al., (2010) demonstrated adventitious shoots of Begonia tuberous can be rootedon MS medium supplemented with 0.5 mg l-1 BA + 0.1 mg l-1 NAA. Root was induced on nodalsegments of Vanda teres on medium containing 2 mg l-1 Kn + 0.5 mg l-1 NAA (Alam et al., 2010).Tyagi et al., (2010) showed root induction at the cut ends of shoots obtained from leaf explants ofCrataeva adansonii on MS basal medium devoid of growth regulators. Shoot cuttings induceroots on MS medium with 1 mg l-1 NAA in 4-5 weeks, and in Dianthus caryophyllus L. with NAAand IBA (Casas et al., 2010). IAA (0.5-1 mg l-1) helped rooting in Pelargonium × hortorum(Dorion et al., 2010). Studies of Ruffoni et al., (2010) on Myrtus communis showed that rootingwas better in medium containing IAA than control, BA and BA + IAA. Ochatt et al., (2010)demonstrated that for rooting of Lathyrus odoratus L. micro-shoots, they are explanted ontomedium with 0.5-1 mg l-1 NAA for 3 weeks.

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Wong, C.E. and Bhalla, P.L. 2010. In vitro propagation of Australian native ornamental plant, Scaevola. In: Jain, S.M. and Ochatt, S.J. (eds) Protocols for In vitro Propagation of Ornamental Plants. Springer Protocols. Humana Press., pp 235-242.


Plant growth

Regulators (mg l-1)

Traits

Shoot length (mm) Root No.

0 Kn0.5 Kn1 Kn2 Kn0 NAA0.5 NAA1 NAA2 NAA0 Kn + 0 NAA0 Kn + 0.5 NAA0 Kn + 1 NAA0 Kn + 2 NAA0.5 Kn + 0 NAA0.5 Kn + 0.5 NAA0.5 Kn + 1 NAA0.5 Kn + 2 NAA1 Kn + 0 NAA1 Kn + 0.5 NAA1 Kn + 1 NAA1 Kn + 2 NAA2 Kn + 0 NAA2 Kn + 0.5 NAA2 Kn + 1 NAA2 Kn + 2 NAA

8.46a6.58b7.37ab8.58a9.26a7.25b5.76c8.72a6.95c7.65b9.45a9.48a9.20a7.50c3.65h6.50d8.92a5.85e4.75g10.00a12.00a8.55b5.28f8.92a

0.85a0.42a0.75a0.81a0.50b0.50b0.76ab1.05a0.36cd0.25d1.25ab1.90a0.56cd0.38cd0.45d0.75bc0.70cd1.60ab0.40d0.75bc0.45cd0.20d1.80a0.85bc

Table 1. Effect of different concentrations of Kn and NAA on the shoot length and root number of Matthiola inca.

Tables

In each column, means with the similar letters are not significantly different at 5% level of probability

using LSD test.

Source of variations dfM.S.

Shoot length Root No

KnNAAKn × NAAErrorc.v.

33964

0.174**0.477**0.175**0.0378225.18

0.770ns

1.120*2.470**0.4029.8

Table 2. Analysis of variance (ANOVA) for the effect of different concentrations of Kn and NAA on the shootlength and root number of Matthiola incana

**: Significant at α = 1%, *: Significant at α = 5%, ns=Not significant


Plant growth

Regulators (mg l-1)

Traits

Root length (mm) Root No. Callugenesis (%) Fresh weight (g)

0 Kn0.5 Kn1 Kn0 NAA0.5 NAA1 NAA0 Kn + 0 NAA0.5 Kn + 0 NAA1 Kn + 0 NAA0 Kn + 0.5 NAA0.5 Kn + 0.5 NAA1 Kn + 0.5 NAA0 Kn + 1 NAA0.5 Kn + 1 NAA1 Kn + 1 NAA

7.00a8.12a1.38b2.54b5.68a7.80a7.33d1.08g1.05g2.07f8.45c9.07b6.67e16.60a1.07g

1.28a1.00a0.17b0.22b0.73a1.33a0.81e0.26h0.30f1.15c1.10d0.21i1.30b1.94a0.32g

74.12a50.17b29.17c14.88c85.00a57.00b45.02e7.11g8.12g

100.00a100.00a55.71d80.23b55.93c33.79f

0.80a0.69a0.49b0.69a0.58a0.59a0.76b0.52e0.47f0.85a0.66c0.52d0.67c0.79a0.39f

Table 3. Effect of different concentrations of Kn and NAA on the root length and number, callugenesis percent andfresh weight of Matthiola incana.

In each column, means with the similar letters are not significantly different at 5% level of probability using

LSD test

**: Significant at α = 1%, *: Significant at α = 5%, ns=Not significant

Source of variations df

M.S.

Fresh weight Callus induction Root No. Root length

KnNAAKn × NAAErrorTotalc.v.

2241523

160.56**55.88**63.68**

7.68

50.40

3.22**1.40**0.70*0.20

59.04

4601.59**11139.37**

510.44ns258.44

30.04

0.17**0.03ns0.03ns0.01

22.46

Table 4. Analysis of variance (ANOVA) for the effect of different concentrations of Kn and NAA on the root lengthand number, callugenesis percent and fresh weight of Matthiola incana.


Identification and Assessment of Fungal Diseases of

Major Medicinal Plants

Medicinal plants are infected by fungal diseases. A four years surveyindicated that, there are various fungal infections in roots and shoots of severalmedicinal plants that grown in Isfahan. Lavender (Lavandula angustifolia),rosemarry (Rosmarinus officinalis) and viper’s bugloss (Borago officinalis) plantswere infected by Fusarium oxysporum and F. solani on roots, showing wilting andeventually plants death, though, F. culmorum was also isolated, but withoutpathogenicity on these hosts. The sage plants (Salvia officinalis) were also infectedby F. solani considerably. Burdock plants (Aractium lappal) were infected notonly by F.oxysporum and F.solani, but also Verticillium dahliae and V. albo- atrumand common balm (Melissa officinalisl) by F. solani. They were also simultaneouslyinfected by Rhizoctonia solani. The aerial infections were mainly powderymildews, downy mildews and rust. The naked seed pumpkin (Cucurbita pepo var.sterica) and common pumpkin (Cucurbita popo) were infected by Erysiphe cichoracearumand Sphaerotheca fuliginea., Johns-worth hypericum (Hypericum perforatum),by Leveillula guttiferatum and E. hypersici., estragon (Artemisia dracunculus) byE. artemisiae., bitter sweet (Solanum dulcamara) by E. beceleate., flixweld(Descurainia sophia) by E. communiis., marsh mallows (Althaea officinalis andMalva silvestris) by L. malvacearum., licoric (Glycyrrhiza globra) by L. leguminosar,dill (Anethum graveolens) and coriander (Coriandrum sativum) by L. umbelleferatum.,downy mildews were observed on spinach (Spinacia oleracea) Peronosporafarinosa, summer savory (Satureia hortensis) P. Lamii and waybread (Plantagomajor) P. alta. There was also rust diseases on medicinal plants including, estragon(Puccinia absinthi), wild thyme (Thymus serpllum) P. serpylli, P. mentha and alsoon pudding grass (Mentha pulegium) and peppermint (Mentha piperita) P. mentha.white rusts, Albugo candida, were also observed on some of the medicinal plantsincluding, flixweld and mother’s heart (Capsella bursa-pastoris). The two speciesof Verticillium dahha and V. albo- atrum with lower frequency on some of themedicinal plants especially on lavander, rosemarry, pudding gross, pepper mint,castor (Ricinus communis) was found and there were typical symptoms on peppermint. Rhizoctionia solani is an another fungal infecting agents, causing dry cankeron roots and underground stems of some of the medicinal plants in this area.

Keywords: Downy mildews, Medicinal plants, Powdery mildews, Rusts, Root rots.

M. Nasr Esfahani1*and M. Monazzah1

1 Agricultural and Natural Resources Research Center, Plant Pests and Diseases Research Institute,Isfahan, Iran

*Corresponding author,s email: [email protected]

Abstract


INTRODUCTION

Medicinal plants were planted in Iran for a long time. Avicenna wrote many books aboutmedicinal plants in Persian. As far as we know there are not comprehensive researches on wiltdiseases and root rot of medicinal plants in Iran. Only damping off by Phylophthora nicotianaewas reported from Castor (Ricinus communis) and Fusarium oxysporum was isolated from Cuminseed (Cuminum cyminuml) (Ershad, 1996). In current years, there are some reports on fungal dis-eases in medicinal plants, but they are not complementary. Powdery mildews, Erysiphe andLevillula genera, was reported from different region of Iran. E. sordido was reported from way-bread (Plantago major) and Indian plantago seed (Plantago psyllium). E. graminis, E. bicellata,E. artemisiae, E. communis, L.malvacearum, L. compositarum, Sphaerotheca fuliginea, L. tauricaand L. leguminosarum were reported from maidenhair fern (Adiantum capillus veneris), bittersweet (Solanum dulcamara), estragon (Artemisia dracunculus), flixweld (Descurainia sophia),marsh mallows (Althaea officinalis and Malva silvestris), yarrow (Achillea millefolium), dill (Ani-ethum graveolens), coriander (Coriandrum sativum) and licorice (Glycyrrhiza globra), respec-tively. Also, E. hyperici and L. guttiferarum was reported from johns-worth hypericum (Hypericumperforatum) (Ershad, 1996). Moreover, leaf spot diseases, Cercospora and Septoria genera, werereported in some medicinal plants. C. althaeina, C. ricinella, S. rubiae, S. rechingeri, S. sisymbriiwere isolated from marsh mallows, castor, madder (Rubia tinctorum), currant fraited rhubarb(Rheum ribes) and flixweld, respectively. Downy mildew caused by Pernospora genus, this genusincluding P. farinose, P. alta and P. lamii were isolated from spinach (Spinacia oleracea), way-bread and summer savory (Satureia hortensis), respectively (Ershad, 1996). Also, rust disease in-cluding Puccinia and Uromyces grnera were found in medicinal plants. P. menthae and P. serhylliwere found in wild thyme (Thymus serpllum). P. achilpeae, P. graminis, P. menthe, P. malvacearum,P. absinthi, P. dracunculine, U. glycyrrhizae were found in yarrow, maidenhair fern, peppermint(Mentha piperita) and pudding grass (Mentha pulegium), marsh mallows, estragon, madder,licoric, respectively. Loose smut, Ustilago nuda was reported in maidenhair fern, white rust, Albugocandidates was found in flixweld and mother’s heart (Capsella bursa-pastoris) (Ershad, 1996).

Fungal diseases also were reported on medicinal plants around the world. Rhizoctonia solaniwas identified as a leaf spot disease in malabar nut (Adhatoda vasica) in India (Verma et al., 2006).Pithomyces chartarum is known to cause leaf spot diseases of ashwagandha (Withania somnifera)in India (Verma et al., 2007). Wilt disease of cucumber (Cucumis sativus) caused by F. oxysporumf. sp. cucumerinum has been recorded in Turkey for a long time (Yıldız and Delen, 1977). Also F.oxysporum f. sp. radicis-cucumerinum causes wilting accompanied by root and stem rot has beenreported in this country (Karaca and Kahveci, 2009) and in British Columbia (Punja and Parker,2000). Macrophomina phaseolina was found to cause root rot in medicinal coleus (Coleusforskohlii) in India (Kamalakannan et al., 2005). Peronospora lamii causing damage to sage (Salviaofficinalis) and rosemary (R. officinalis) reported from the UK (Humphreys-Jones et al., 2006).Fusarium wilt caused by Fusarium solani on commercial field lavender was identified in China(Ren et al., 2007). Several species of powdery mildew fungi have been recorded on rosemary (Lev-eillula spp.) from Europe and Podosphaera fuliginea from USA (Farr and Rossman, 2009). Pow-dery mildew on rosemary associated with Golovinomyces biocellatus in Asia (Park et al., 2009).Podosphaera fusca (syn. Sphaerotheca fusca and S. fuliginea) has been recorded to infect Germanchamomile (Matricaria chamomilla) in Canada, Egypt, Germany, Switzerland, Russia (Farr andRossman, 2009). Golovinomyces cichoracearum (syn. Erysiphe cichoracearum) is a rather com-mon powdery mildew species infecting German chamomile in Europe (Farr and Rossman, 2009)and has been reported in Korea (Park et al., 2010).

There is no any report on vascular wilt disease, root rot and plant death on medicinal plantson the medicinal plants so far. Only damping-off disease caused by Phytophtora nicotianae andFusarium oxysporum were deducted from castor and cumin seed (Cuminum cyminum), respectively


(Ershad, 1996). This report is dealing with the fungal diseases, occurring on medicinal plants inIsfahan Province, Iran.


The surveys on fungal diseases were carried out as a national project in medicinal plantsfor 4 continues years (2005-2008). For this purpose, the fields of medicinal plants were surveyedthroughout early years and samples of plants that showed symptoms of wilting, damping off andleaf spots were collected.

The number of healthy and infected plants were recorded and then registered in tables, sep-arately. All the samples with the symptoms of disease were collected in the separate plastic bags.The name of plant, place and time of samplings were recorded accordingly. Samples were takeninto the lab as soon as possible in order to examination in due course time stored sample at refrig-erator were used to isolation of fungal agents, and keep in the fridge (Anon, 1985).

For isolation of root rot fungal agents, samples were washed thoroughly in running tapwater for 30 min to remove the debris adhered. Samples were carefully dissected into small piecesfrom border of healthy and infection regions of the roots. Then, surface-sterilized roots were placedon petri dishes containing potato dextrose agar medium (PDA) and incubated at 25°c±1 and 12hof illumination in incubator. Sub cultures were obtained from hyphal tips of the developing fungalcolonies then were placed in incubator again.

Spore suspensions of fungal isolates were cultured on WA medium as a single colony, inorder to obtain pure culture. Eventually, same single colonies were transferred randomly into thePDA medium in the same conditions as describe above. SNA medium was used for avoiding mu-tation in Fusarium spp. (Singleton et al., 1993).

Fusarium genus was identified by CLA medium or clove leaf agar and also identified baseon several keys and references (Barnett and Hunter, 1999). Petri dishes containing sterile and wetWhatMan paper were used to identify Verticillum genus. Hypha and spores of fungi were collectedfrom solid medium and then fungal isolates with a spore concentration of about 107 spores per mlwere propagated to prove pathogenicity tests. Inoculum of isolates were added to pots containingsterilized soil (soil: sand, 1:1ratio) (Booths, 1971).

The seedlings of the medicinal plants had been planted in sterilized pots and then weretransferred into pots with infested soils. Eventually, pots were placed in greenhouse conditions forthe further growth of tested medicinal plants (Joubert et al., 1970; Nasr Esfahani, 2004).

Some groups of plant pathogenic agents, namely downy mildews, powdery mildews, rustsand smuts could not grow on most commonly used nutrient media in lab conditions. So, thesefungi were identified as such, according to microscopic structures and classified on several relatedkeys and references (Ershad, 1996; Barnett and Hunter, 1999; Farr and Rossman, 2009).

Statistical Analyses

Variance analysis was summarized using statistical analysis system (SAS) version 8 (SASInstitute, Inc., Cary, NC). Duncan’s test (DMRT) in the level of 1% was performed to determinethe significance of differences in the means of infection percentage.

RESULTS

Results indicated that, there are several fungal diseases, which may infect the medicinalplants in Iran out of which, there are on aerial and soil parts of the infected plant, depending uponthe plant species and the fungal pathogens. Pathogenicity tests were carried out in greenhouse con-ditions and the fungi were re-isolated from these plants and identified as described below. The re-sults are also summaries in table 1.


Rosemary (Rosmarinus officinalis)

Wilting and die of the plant tissues in rosemary was occurred in Janatabad station. Infectedplants began to show wilting in leaves and grow poorly. Eventually, death and collapse was ob-served in these plants. The infection rate of root rot and wilting was of 40.81%, which a consider-able amount in comparison to other studied medicinal plants in these studies with a significanteffect (Table 1). F.oxysporum and F. solani was known to cause disease in rosemary (Table 1),while F. culmorum was not the cause of the disease. So, this genus was in association with F.oxys-porum and F. solani.

Lavander (Lavandula angustifolia)

Decline rate in lavander plants was also considerable in comparison with other medicinalplants. Even, root and crown rot was observed in lavander plants, which was planted in publicparks and landscape. Infection rate in these plants in Janatabad station was 20% with a significanteffect. It was categories in a signal statist of group along with sage as far as the disease severity isconcerned (Table 1). F. solani and F. oxysporm were isolated from this plant. These isolate wasobtained on PDA medium and maintain on SNA medium. CLA medium was used to identify.

Sage (Salvia officinalis)

Wilting and dryness also was found in sage plants like other medicinal plants. Infectedbushes were shown green death and stem became black. Roots begin to be infected and then stemand crowns were infected by fungi. F. solani was isolated from infected sage plants. Number ofinfected and health plants were counted and infection rate was evaluated accordingly (Table 1).

Viper's bugloss (Borago officinalis)

Wilt disease was the most prevalent disease in Janatabad station. All of perennial plantsdied after two years because of severe infection. When plants were replaced in this station in thesame year, infection was observed in Viper's bugloss again (Table 1). F. oxysporum and F. solaniwere isolated from PDA medium and maintained in SNA. CLA medium was used to identify them.

Burdock (Aractium lappal)Wilt and dryness diseases also were observed in burdock bushes. The symptom was green

dead in leaves, black rots in petiole and severe rots in stems and roots. Burdock plants had beeninfected not only by Fusarium species (F. oxysporum, F. solani) but also infected by Verticillumdahlia and V. albo-atrum. (fields have become contaminated with Verticillum). Wilt bushes werecounted in order to evaluate infection rate in 4 random blocks (Table 1).

Common balm (Melissa officinalis)

Dryness (chlorosis, necrosis) also was found in common balm plants in Janatabad station.Dry bushes were counted with 4 repeat to evaluate infection rate (Table 1). F. solani and Rhizoc-tonia solani were isolated and studied in lab and greenhouse.

Naked seed pumpkin (Cucurbita pepo var. sterica)

Naked seed pumpkin had been infected by powdery mildew. On the leaves and stems, dis-ease appears at first as small, white and powdery lesions that soon become covered with large,white patches of fungus growth. At the short time fungus cover both sides of leaves entirely. Even-tually leaves become necrotic and died. The pathogens that cause powdery mildew on vegetablesare introduced as Erysiphe cichoracearum and Sphaerotheca fuliginea. S. fuliginea is more prob-able to cause diseases in vegetables (is probably the most common and widespread diseases onvegetable). The infection rate of naked seed was estimated 100% (Table 1).


Johns-worth/hypericum (Hypericum perforatum)

This plant also was infected by powdery mildew. Symptoms on leaves were the same assymptoms on naked seed. Erysiphe hyperici and Levillula guttiferarum causes powdery mildewon Johns-worth/hypericum. White patches of fungus growth covered the entire surface of growingleaves and infection rate was assessed 100% (Table 1).

Other medicinal plants

Evidences show that powdery mildews are the most common diseases on medicinal plants.The late in the end of season, september, powdery mildews were characterized by the appearanceof spots on patches of a white, powdery, mildew growth on plant tissues and then entire leavescompletely were covered by the white powdery mildew. Sphaerotheca, Erysiphe and Levillulawere identified and introduce in references.

In blocks and fields powdery mildew diseases was observed on the vast number of medic-inal plants including estragon Artemisia dracunculus (Erysiphe artemisiae), bitter sweet Solanumdulcamara (E. bicellate), flixweld Descurainia sophia (E. communis), marsh mallows Althaea of-ficinalis and Malva silvestris (Levillula malvacearum), licoric Glycyrrhiza globra (L.) legumiu-osarum), naked seed pumpkin Cucurbita pepo var. sterica and common pumpkin Cucurbita popo(Sphaerotheca fuliginea), Vipers bugloss Borago officinalis (E. asperifoliarum), dill Anethumgraveolens and coriander Coriandrum sativum (L. umbelliferarum). Leaves of some medicinalplants were used for different purpose, so, powdery mildew is a significant disease on these me-dicinal plants.

The downy mildew of medicinal plants was the genus Pernospora. Downy mildew infectedthe spinach (Spinacia oleracea) very seriously. P. farinose was found as the disease agent. Thisdisease also was observed on summer savory (Satureia hortensis) in the field and P. lamii causedisease on them. P. alta was reported as disease agent on waybread (Plantago major).

Plant rusts are among the most destructive plant disease on medicinal plants. Puccinia andUromyces was identified and reported on medicinal plants in different references. Various speciesof Puccinia infected the medicinal plants and cause rust disease, including estragon Artemisia dra-cunculus (P. absinthi), Wild thyme Thymus serpllum (P. menthea and P. serpylli); causing severeinfection on leaves and stems, pudding grass Mentha pulegium and peppermint Mentha piperita(P. menthea).

White rust also was observed on some medicinal plants including: Flixweld and mother’sheart (Capsella bursa-pastoris). Albugo candida was introduced and identified on these plants.

Soil borne (some fungi are widespread in soils) disease are the common and significantdisease on medicinal plants. Verticillum dahliae was found on the vast number of plants. Thesefungi grow on the medium very slowly and isolation of them is very difficult. On the most time,other fungi like saprophyte grow on the media with these fungi and so develop on media very late.

Nevertheless, the abundance of V. dahliae and V. albo-atrum was very low in vascular tis-sues of roots and stems of some medicinal plants especially annual and perennial plants. Verticillumwas observed in some medicinal plants including lavander (Lavandula angustifolia), rosemary(Rosmarinus officinalis), pudding grass (Mentha pulegium), peppermint (Mentha piperita), Castor(Ricinus communis) and other plants, but the symptoms like wilt and death of the entire plant wasnot observed significantly and clearly in these plants. However, Verticillum infection results in de-scribing growth of plants. Also, the symptoms of Verticillum wilt and chlorotic in some brancheswas found significantly in peppermint.

Rhizoctonia disease causes losses on most medicinal plants in this study. The most commonsymptoms caused by Rhizoctonia on most plants was root rot and then there was the appearanceof stem canker on annual plants including castor (Ricinus communis), yarrow (Achillea mille-folium), common balm (Melissa officinalis) and perennial plants including lavender (Lavandula


angustifolia), rosemary (Rosmarinus officinalis), marsh mallows (Althaea officinalis and Malvasilvestris), viper's bugloss (Borago officinalis), burdock (Aractium lappal) and etc. Rhizoctoniaalso causes dry canker on underground stems (root stock, rhizome) and rotting of axillaries rootshair.

Physiological diseases also cause diseases in medicinal plants. Deficiency of elements wasobserved in most plants. Certain symptoms were chlorosis of leaves, scorching of the margins,parallel and symmetric discoloration of leaves. Common balm (Melissa officinalis) was sensitiveto deficiency of iron and upper leaves turn white and discolored.

DISCUSSION

Medicinal plants like other plants have special diseases. Data in this survey show that var-ious fungi disease appears on medicinal plants and infected aerial, foliage and underground partsof plants. Powdery mildews are airborne disease. This disease appeared on leaves and fresh stemson the late of the season and then covered entire surface of growing parts of plants. The symptomsof powdery mildew had been reported already by other researcher.

All data was collected by Ershad and was published in fungi books in 1996. In our study,powdery mildew was reported on some medicinal plants in the first time including: Naked seedpumpkin (Cucurbita pepo var. sterica) and common pumpkin (Cucurbita popo), Johns-worth hy-pericum (Hypericum perforatum), bitter sweet (Solanum dulcamara), flixweld (Descurainiasophia), marsh mallows (Althaea officinalis and Malva silvestris). This disease was already ob-served on estragon (Artemisia dracunculus), dill (Aniethum graveolens) and coriander (Coriandrumsativum) in Esfahan fields.

Another airborne disease is rust. This disease was observed on estragon (Artemisia dra-cunculus), wild thyme (Thymus serpllum), pudding grass (Mentha pulegium) and peppermint (Men-tha piperita). Also, other researchers have introduced rust disease on medicinal plants. In our study,these diseases were mentioned. White rust, airborne disease, was found on flixweld and mother’sheart (Capsella bursa-pastoris). White rust has been reported in other regions of Iran, recently.So, control of airborne disease is very important because aerial parts of these plants are used forsome medicinal purposes. So, every kind of control methods cannot be offered by researchers andor cannot be applied by farmers.

Therefore, it is necessary that other control methods will be offered by researchers like eu-genics and cultural methods, integrated pest management to reduce chemical controls, using fun-gicide with low residue and then these methods should be performed as comprehensive nationalproject. Control methods are very significant especially on medicinal plants which aerial parts ofthem and their leaves are used as medicinal purposes. Dryness, wilt and death of tissues were ob-served on medicinal plants by soil borne fungi. F. oxysporum and F. solani were two significantspecies and isolated from roots of medicinal plants. These two species was very common and wide-spread on rosemary (Rosmarinus officinalis) and lavender (Lavandula angustifolia). Also, theseplants were planted as ornamental plants on public parks and landscape. Damping off was so im-portant in these regions. F. oxysporum only was reported and isolated on cumin seed (Cuminumcyminuml) by other researchers (Ershad, 1996). Sage (Salvia officinalis), burdock (Aractium lap-pal), common balm (Melissa officinalisl) and other plants were infected to Fusarium especially F.solani but the symptoms was not so clear and severe disease and infection was not found on otherplants by Fusarium species.

Another soil borne fungi was Verticillium. V.dahlia and V. albo-atrum was identified andisolated from medicinal plants. The prominent species was V. dahlia. The frequency and patho-genicity of this species was higher other than Verticillium species. Also, V. dahlia produce mi-crosclerote on infected tissues in wet conditions and it was observed by binocular microscope,easily. Verticillum genus was isolated from vascular tissues of roots and underground stems of me-


dicinal plants especially on rosemary (Rosmarinus officinalis), lavender (Lavandula angustifolia),pudding grass (Mentha pulegium), peppermint (Mentha piperita), castor (Ricinus communis) andetc. This fungus penetrates on vascular tissues systemically and then it transfer from stem to aerialparts of plants, easily. When stem of plant was cut and grow on wet chamber, Verticillium growand develop on stem. It is necessary to mention that this fungus grow poorly and slowly on nutrientmedium and isolated difficulty. Nevertheless, research and survey about Verticillum has not beendone completely and significantly (Snowdon, 1991; Singh, 1992; Sutton, 1993). The symptomsof cancer, root rot, axillaries root rot of medicinal plants were caused by Rhizoctonia solani. Thisfungus cause canker on underground stems of medicinal plants, especially on thick undergroundstems. This disease cause scar and death of tissues and finally it cause medicinal plants to growpoorly.

This study show that root rot, vascular wilt, damping off of medicinal plant, which causeby soil borne fungi, are very significant disease in medicinal plants. Thus, it is probable medicinalplants will be planted in the vast number of fields. It is necessary that researcher consider and sur-vey different kind of control methods or prevention of soil borne fungi disease. Moreover, controlof pathogen agents of medicinal plants is different from other agricultural plants. In future, regard-ing to problems of chemical medicine, medicinal plants maybe will be used more than now andmedicinal plant will be very important plant in future. So, it is a nice suggestion that research proj-ect concentrate on nonchemical methods or integrated pest management in order to decrease con-suming of chemical and prevent adverse effects of them on human and nature (Walker, 1952;Candan and Suludere,1999; Amponsah et al., 2002; Merle shepard, 2003; Purohit and Vyas, 2004;).

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Karaca, G. and Kahveci, E. 2009. First report of Fusarium oxysporum f. sp. radicis-cucumerinum on cucumbers in Turkey. New Disease Reports 20, 9.

Merle shepard, B. 2003. Management of pests on Medicinal plants and influence of pest damage on active priciple (Final report). Costol Research and Education Center. Charlston, SC 29414.

Nasr Esfahani, M. 2001. Principles of diagnostic techniques in plant pathology. Jihad Daneshgahi Isfahan. Park, M.J., Choi, Y.J., Han, J.G. and Shin, H.D. 2010. First report in Korea of powdery mildew of

Matricaria chamomilla caused by Golovinomyces cichoracearum. New Disease Reports 20, 30.


Park, M.J., Han, J.G. and Shin, H.D. 2009. First Korean report of rosemary powdery mildew caused by Golovinomyces biocellatus . New Disease Reports 19, 60.

Punja Z.K. and Parker M. 2000. Development of Fusarium root and stem rot, a new disease on greenhouse cucumber in British Columbia, caused by Fusarium oxysporum f.sp. radicis-cucumerinum. Canadian Journal of Plant Pathology. 22:349-363.

Purohit, S.S. and Vyas S.P. 2004. Medicinal plant cultivation, a scientifical approach including processing and financial guidelines. Agrobios, India.

Ren, Y.Z., Tan, H., Li, Z.J., Du, J. and Li, H. 2007. First report of lavender wilt caused by Fusarium solani in China. New Disease Reports. 15, 55.

Singh, R.S. 1992. Diseases of vegetable crops. Oxford and IBM. Publishing Co. PVT. LTD. New Dehli, Bombay, Calcutta. p. 280-300.

Singleton, L.L., Mihail, J.D. and Rush, C.M. 1993. Methods for research on soil borne phythogenic fungi. APS Press.

Snowdon, A.L. 1991. Bulb diseases. p. 236-261 In: A colour atlas of post-harvest diseases and disorders of fruit and vegetables. Vol. 1 BPCC Hazell Book, England.

Sutton, A. 1993. Onions. Ciba Plant Protection Vegetables. Switzerland.Verma, O.P., Gupta, R.B.L. and Shivpuri A. 2007. A new host for Pithomyces chartarum, the cause

of a leaf spot disease on Withania somnifera. New Disease Reports. 15, 47.Verma, O.P., Singh, N. and Sharma, P. 2006. First report of Rhizoctonia solani causing leaf spot

of Adhatoda vasica. New Disease Reports. 14, 39. Walker, J.C. 1952. Diseases of vegetable crops, Mc Graw Hill Book Company Inc. New York.

Landon. p. 225-262.Yildiz M. and Delen, N. 1977. Studies on the occurrence of Fusarium wilt of cucumber in Ege

Region of Turkey. Journal of Turkish Phytopathology. 6:111-117.


Table 1. The infection of medicinal plants to fungal diseases.

Means followed by the same letter are not significantly different at P=0.01 level in Duncan's multiple range test

No Common name Scientific name Casual organisms

Mean No. of plantsDuncan's

groupTotal /Rep Healthy/Rep Infected/Rep Infection (%)

1

2

3

4

5

6

7

8

Rosemary

Lavender

Sage

Viper's bugloss

Burdock

Common balm

Naked seed pumpkin

Johns-worth/hypericum

Rosmarinusofficinalis

Lavandula angustifolia

Salvia officinalis

Borago officinalis

Aractium lappal

Melissa officinalis

Cucurbita pepovar. sterica

Hypericum perforatum

Fusarium oxysporumF. solani

F. culmorum

F.oxysporumF. solani

F. solani



Verticillium dahliaV. albo-atrum

F. solaniRhizoctonia solani

ErysiphecichoracearumSphaerotheca

fuliginea

E. hypericiLeveillula

guttiferarum

17.25

30.00

22.75

22.75

30.00

21.75

59.50

34.00

12.25

25.00

18.25

16.00

25.00

19.00

29.75

19.50

6.60

5.00

4.50

6.75

5.00

2.75

29.75

19.50

40.81

20.00

24.65

42.18

20.00

14.47

100.00

100.00

c

ab

ab

bc

ab

a

d

d

Tables

www.jornamental.com


An Easy and Simple Method for Estimating Total Shoot

Length During Screening and Evaluation of Mulberry

(Morus spp.) Genotypes

In mulberry (Morus sp.), grown for its foliage, which is the sole foodfor the silkworm (Bombyx mori L.), evolving high yielding varieties is a longdrawn and laborious process. One of the important selection parameter that hassignificant positive correlation with leaf yield is Total Shoot Length [TSL] ofthe mulberry plant. Measuring the length of all the shoots of the test genotypesto get the total shoot length during several stages of screening and evaluationrequires enormous skilled manpower and time. The enormity of the task itselfmost often than not leads to inaccuracies. Due to multi-collinearity among thecharacters such as number of shoots, length of the longest shoot and total shootlength, the expression of these characters as a single entity could be moreaccurate and time saving if appropriate statistical relationships are established.In view of this, a regression relationship was derived and a model developedfor estimating total shoot length by measuring only the length of longest shootand number of shoots per plant. The model was tested with four mulberryvarieties that are often used as checks in evaluation experiments and significantlyhigh coefficient of determination [R2] ranging from 0.81 to 0.91 were recorded.Further, evaluation of the models with two mulberry genotypes grown undertwo distinctly different growing environments also showed no significantdifference between the estimated and actual total shoot length. These testsconfirmed the efficacy of the models across varieties and growing environments,thus paving way for reduction in drudgery, savings in time and resources inmulberry breeding programmes.

Keywords: Evaluation, Models, Mulberry, Multi-collinearity, Total shoot length.

Abstract

M. Rekha1*, K. Kesavacharyulu2 and K. Rajashekar1

1 Central Sericultural Research and Training Institute, Mysore 57008, Karnataka, India2 Regional Sericultural Research Station, Chamarajanagar 571313, Karnataka, India



INTRODUCTION

Mulberry (Morus spp.) is an economically important crop plant grown for its foliage, which isthe sole food of the silkworm (Bombyx mori L.). Mulberry, which is a tree in nature, is trained as lowbushes and pruned about 5 times a year to harvest nutritious leaves for silkworm rearing. Generally,the number of shoots per plant varies between 5 and 20. Conventional plant breeding programme inmulberry involves evaluation of thousands of individual hybrids in the first phase of hybrid screening.The subsequent two stages are the Primary Yield Evaluation [PYE] and Final Yield Evaluation [FYE]of the short-listed hybrids in specific growing environments. The three-stage evaluation requires abouteleven years and involves eight years of data recording. Leaf yield and growth data are recorded fivetimes a year in all the three stages, rendering the process immensely laborious and time consuming.

Leaf yield in mulberry is determined by many growth parameters. It is reported by many workerssuch as Das and Krishnaswami (1969); Sarkar et al., (1987); Susheelamma et al., (1988); Bari et al.,(1989); Bindroo et al., (1990) and Singhvi et al., (2001) that the number of shoots per plant, length ofthe longest shoot and total shoot length are positively correlated with leaf yield. Hence, these charactersare considered important determinants of leaf yield and are measured in screening and evaluation oflarge heterogeneous populations and shortlisted promising hybrids for final yield evaluation as reportedby Sarkar et al., (1987). However, measuring all the shoots of each of the thousands of hybrids duringhybrid screening and short-listed ones during replicated trials at PYE and FYE stages, requires enormousskilled manpower and is a time consuming process. This is even more cumbersome in case of experi-ments conducted in rainfed conditions, where the measurements have to be recorded in a non-destructivemethod from standing crops. The enormity of the task and the drudgery involved, often leads to inac-curacies. Further, it appears that, while studying the association of morphological characters with leafyield, collinearity among number of shoots, length of the longest branch and total shoot length was notexploited for establishing a relation to reduce time, manpower and avoid human errors.

Keeping the above in view, the present study was undertaken to establish correlation among numberof shoots, length of longest shoot and total shoot length and to find out a way to estimate total shoot lengthbased on number of shoots and length of longest shoot without actually measuring all the shoots of a plant.


Data sets from primary yield evaluation trial (PYE) and final yield evaluation trial (FYE) underadvanced generation breeding programme were considered for the study. Data on four mulberry geno-types, V1, G4, RC1, and RC2, which were used as checks in primary yield evaluation trial, and evaluatedunder two distinct environments of optimum and sub-optimum irrigation were considered in the firstpart of the study for developing and validating the models. In the later part of the study aimed at con-firming the efficacy of the model across different growing environments, data on varieties V1 and RC1in the final yield evaluation stage, under optimum and sub-optimum levels of irrigation, were considered.The methodology consisted of model development and model validation and confirmation.

Development of Models

For developing the models, leaf yield and growth data of varieties V1, G4, RC1 and RC2grown under two environments of optimal and sub-optimal irrigated conditions, for primary yieldevaluation [PYE], were considered. The entire package of practices recommended for mulberry wasfollowed and leaf yield and growth data were recorded five times a year for two consecutive years.On 70th day after each pruning, data on number of shoots (NS), length of longest shoot (LLS) andtotal shoot length (TSL) were recorded from ten plants from each variety. A new variable NS × LLS(the product of NS and LLS) was obtained. Correlation of total shoot length (TSL) with number ofshoots, length of the longest shoot and total shoot length (NS × LLS) were calculated. Linear regres-sion relationships of total shoot length (TSL) to NS × LLS were developed for all the varieties underthe two growing environments, to develop two models specific to the environment. These modelsserved as original models for further validation. Further, the hypothesis (H0: b1 = b2) on the slopesof the two regression models under two conditions for each variety was tested using Z test.


Validation of Models

To confirm validity of the models across growing environments, an independent validationmethod was adopted, as they are statistically independent. The data from sub-optimum conditionsof irrigation was considered as validation set. The regression constants developed (original model)for the four varieties under optimum conditions of irrigation and the data on NS × LLS from thevalidation set were used to estimate total shoot length (TSL). The estimated total shoot length andthe actual total shoot length measured in the validation set were compared using paired t-test fol-lowing the method of Snedecor and Cochran (1976).

Confirmation

To confirm validity of the developed models across growing environments, data on three vari-ables viz., NS, LLS and actual TSL from two varieties V1 and RC1 grown under optimal and sub-optimal conditions, respectively, were collected for ten harvests over a period of two years. The newvariable NS × LLS was computed and used in the regression models obtained earlier from the dataof PYE, to get the estimated total shoot length. These estimated total shoot length data were comparedto the actual total shoot length using one-way ANOVA with Tukey’s multiple comparisons post-test.


Leaf yield in Mulberry is a complex character that is influenced by total shoot length and variousworkers such as Fotadar (2000); Masilamani et al., (1996) and Susheelamma (1988) have reported thedirect and indirect effect of number of shoots, length of the longest shoot and total shoot length on leafyield. It was also observed that number of shoots and length of the longest shoot in all the varietiestested are negatively correlated. This type of correlation between the component characters may leadto inappropriate evaluation. The current practice of measuring the length of all shoots [5-20/plant] ofeach plant several times during the three-stage screening and evaluation spread over eleven years, con-sumes enormous resources. The enormity of the task itself more often than not results in inaccuracies.

In an effort to develop a method that involves measurement of two variables per plant (num-ber of shoots and length of the longest shoot) and then find a new variable, which is the product ofthese two, to accurately estimate the total shoot length of a plant, consistent and positive correlationswere observed between total shoot length (TSL) and number of shoots (p<0.01) and also betweenTSL and NS × LLS (p<0.0001). However, no significant correlation was observed between TSLand length of the longest shoot (Table 1). The positive correlation of total shoot length [TSL] tonumber of shoots [NS] was relatively stronger in all genotypes in comparison to its correlation withlength of the longest shoot. Scatter plots and linear regression relation between total shoot lengthand NS × LLS for the varieties V1, G4, RC1 and RC2 under optimal and sub optimal conditions ofirrigation are shown in Fig.1. A linear function always best described the relationships. The regres-sion model, coefficient of determination and the Z statistic for the comparison of the slopes of themodels were also estimated (Table 2). High coefficient of determination (R2 = 0.81 to 0.91) wasobserved in all the genotypes, thus explaining the fitness of the model. The Z statistics for the com-parison of the slopes of the models for two growing conditions in all the genotypes were found tobe not significant at p<0.01. The model developed for optimal conditions was validated using thedata on NS × LLS with sub optimum conditions, which revealed the consistency of the newly de-fined variable, NS × LLS in explaining the total shoot length across varieties and growing environ-ments. The estimated total shoot length thus obtained was compared with the actual total shootlength measured using paired t-test (Table 3). The t-statistics were found to be not significant in allthe genotypes confirming the validity of the developed model. Confirmatory tests conducted withthe relevant data sets of two varieties, V1 and RC1, from the final yield evaluation (FYE) undertwo growing conditions to predict total shoot length showed that there is no statistical differencebetween the models’ mean estimates, or estimated and actual mean total shoot length (Table 4).

The new method developed, is less laborious, simple, faster and avoids inaccuracies, sinceit involves measuring two variables, which are relatively easier when compared to the enormity


of measuring all the shoots of all genotypes over several crops. Also the method is robust and con-sistent across different varieties and growth environments. Adopting the suggested method in mul-berry screening and evaluation would lead to reduction in drudgery and effective utilization ofresources without compromising on the accuracy of data.

Literature Cited

Bari, M.A., Qaiyyum, M.A. and Ahmed, S.U. 1989. Correlation studies in mulberry (Morus alba L.). Indian J. Seric., 28: 11-16.

Bindroo, B. B., Tiku, A. K. and Pandit, R. K. 1990. Variation of some metric traits in mulberry varieties. Indian Forester 116: 320-324.

Das, B.C. and Krishnaswami, S. 1969. Estimation of components of variation of leaf yield and its traits in mulberry. J. Sericult. Sci. Japan, 38: 242-248.

Fotadar, R.K. 2000. Correlation & path analysis in mulberry. National conference, CSRTI, Mysore., 16-18, pp:2

Masilamani, S., Thiagarajan, V., Susheelamma, B.N. and Datta, R.K. 1996. Path analysis for leaf yield components in mulberry (Morus spp.) grown under high altitude conditions. Seriocologia 36: 461-467.

Sarkar, A., Roy, B.N., Gupta, K.K. and Das, B.C. 1987. Character association in mulberry under close planting. Indian J. Seric., 26: 76-78.

Singhvi, N.R., Chakraborty, S., Singhal, B.K., Rekha, M., Sarkar, A. and Datta, R.K. 2001. Cause and effect relationship of leaf yield in mulberry as influenced by morphological and physiological traits in mulberry. Bull. Ind. Acad. Seri., 5:31-33.

Snedecor, G.W and Cochran, W. G. 1967. Statistical methods, sixth edition, Oxford & IBH publishingCo., New Delhi.

Susheelamma, B.N., Jolly, M.S., Kshama Giridhar, D.D, N.K. and Suryanarayana, N. 1988. Correlation and path analysis in mulberry under stress and non-stress conditions. Seriocologia 28: 239-243.


Character Genotypes Optimum Irrigation Sub-optimum Irrigation

Number of shoots [NS]

Length of the longest shoot(LLS)

NS × LLS

V1G4RC1RC2

V1G4RC1RC2

V1G4RC1RC2

0.687**0.674**0.585*0.676**

-0.0350.1530.105-0.004

0.911***0.953***0.902***0.936***

0.675**0.613**0.680**0.621*

0.1050.136-0.107-0.074

0.953***0.943***0.933***0.922***

Table 1. Correlation among various characters under optimal and sub optimal conditions of irrigation

** p<0.001 and *** p<0.0001

Mulberry

Variety

Original regression model

Z stat p<0.01

Optimum Sub optimum

irrigation R2 irrigation R2

Estimated Y=2.162+0.647xY=1.687+0.666xY=2.235+0.604xY=1.378+0.701x

0.8290.9080.8140.876

Y=1.110+0.729xY=1.538+0.669xY=1.095+0.715xY=2.232+0.637x

0.9090.8890.8690.849

1.220.061.530.94

NSNSNSNS

Table 2. Original models for four varieties grown under optimal and sub optimal conditions of irrigation

NS= Not significant

NS = Not significant

Tables

Mulberry variety

Original regression model

(Optimum irrigation) R2

Total shoot length (m) t stat

p<0.01Actual Estimated

V1G4RC1RC2

Y=2.162+0.647xY=1.687+0.666xY=2.235+0.604xY=1.378+0.701x

0.8290.9080.8140.876

12.109.798.9811.71

11.929.908.9011.81

NSNSNSNS

Table 3. Estimation of total shoot length and comparison with actual total shoot length as measured

Table 4. Comparison of Total shoot length estimated using the two models with the actual values as measured in the FYE trial

Mulberry variety

Estimated total shoot length (m)

Actual total shoot

length (m)

pa

(Tukeys)Regression model -1

(Optimum irrigation)

Regression model -2

(Sub-optimum irrigation)

V1RC1

11.219.37

11.309.54

10.908.99

0.8590.681


Figures

Fig.1. Relation between TSL and NS × LLS for four mulberry genotypes under optimal and sub-op-

timal conditions of irrigation.

Optimal Sub optimal


Marigold: The Possibilty Using Vermicompost as the

Growth Medium

In order to investigation of vermicompost effect on growth and yieldof marigold in pot medium, an experiment was done by a randomizedcompeletely block design in three raplications. Treatmnets were includedcontrol (30% v/v of soil plus 70% v/v of sand) and three levels ofvermicompost (20, 40, 60 % v/v of vermicompost + 30% v/v of sand andsoil) that applied in three lit. pots. Marigold seeds (Tagetes erecta cv.Tiashan)were planted in media. The shoots were cut and it was measured the bushheigh, the lateral branches, size and flower weight, dry weight of shoot, andthe concertretion of nutrient elements. The results showed that addedvermicompost to the growth media tend to improve the growth and yield ofmarigold than in the control. The Vermicompost (60%) had the highestweight, size and dry weight of shoot, but the maximum bush height wasobtained by 20% vermicompost. The most lateral branches was belong to40% vermicompost treatment. The results showed that the plants whichcultivated on 60% vermicompost medium had the most amount of nitrogen,phosphorus and calcium. The most amount of potassium was ralated to 40%vermicompost treatment.

Keywords: Growth medium, Marigold, Peat, Vermicompost.

Abstract

F. Shadanpour1*, A. Mohammadi Torkashvand2 and K. Hashemi Majd3

1 M.Sc. student, Department of Horticulture, Rasht Branch, Islamic Azad Univercity, Rasht, Iran2 Department of Horticulture, Rasht Branch, Islamic Azad University, Rasht, Iran3 Soil Science Department, Mohaghegh Ardebili University, Ardebil, Iran

*Corresponding author email: [email protected]


INTRODUCTION

For providing growth media of ornamental plants, some organic material are used in amend-ing some physical and chemical characteristics of growth media that can refer to peat, compost,grinded shells of trees, the hull of coconut and rough rice. Using manure, wood chips and paperwastes with volcanic mixture to produce croton, Cordyline, chrysanthemum media showed thatthese materials are very useful and appropriable as a growth medium (Cull, 1989). Broad leaf shell,conifer, the compost of mushrooms and municipal wastes compost can be used as a growth bed(Pool et al., 1998).

Today it has been proposed to use useful fauna of soil as the best natural way to keep bioticsystem in agriculture lands. Presentation of organic matter as the food of these useful fauna is oneof the advantageous of some fertilizer such as vermicompost (Saleh Rastin, 2001). Vermicompostis a bio-organic fertilizer which contains a biological active mixture of bacteria, enzymes, manure,earthworm capsule which led to decomposition of organic material and improvement of microbialactivity in growth media (Benitez, 1999).

Vermicompost had a positive effect on the germination rate in seeds by increasing the ca-pacity of water holding, nutrients and producing hormones (Tomati et al., 1998). The resultsshowed that the application of 10% vermicompost increased significantly dry weight in Magnoliavirginiana (Bachman, 2000). The produced vermicompost by animal wastes is quite monotonousand stable quality. In this material, primary contaminations have decreased, it is stable in long termwithout compaction of growth medium, and it provides some nutrient for plants. In the producingstage of vermicompost, even the drainage water discharge from the bed contain some nutrient andgrowth factors, which it has nutritional value. A study of Aglaonema and Dieffenbachia showedthat spraying by drainage water solution of bed; significantly increased the height, dry matter,diameter, fresh weight and nitrogen (Mahboub Khomami, 2005).

Atiyeh et al., (2000) showed that adding hog manure vermicompost to marigold and tomatoincreased the growth rate that vermicompost had more positive effect on germination rate in tomatothan marigold. Bachman (2008) had conduct some research to study the growth rate in some nurs-ery flora such as tomato, marigold and pepper in economical growth medium which contain ver-micompost. He observed that a mixture of 20% v/v hog manure vermicompost with growthmedium increased root and shoot dry matter, leave area, the rate of shoot to root in Tomato andmarigold, but it had a little effect on growth rate of pepper and santeria, so it had no effect on ger-mination rate in all plants.

Mal Yin (2010) reported that the mixture of sewage sludge vermicompost amounted 20%v/v with soil was the best rate. Atiyeh et al., (2002) studied the effect of hog vermicompost ongrowth rate and yield of Magnolia and concluded that the highest rate of growth was observed on30% and 40% rates of vermicompost and the lowest growth rate was observed on 90% and 100%proportions. They reported that the highest and the lowest rate of flower bud were in the proportionof 40% and 100%, respertively. In this study, the aim is to evaluate the possibility using vermi-compost as the growth medium of marigold.


An experiment was conducted by a completely randomized block design to study the effectof different rates of vermicompost in pot growth medium. Treatments include:1. Growth medium contains 30% v/v of soil + 70% v/v of sand (control)2. Growth medium contains 20% v/v of vermicompost + 30% v/v of sand + 50% v/v of soil3. Growth medium contains 40% v/v of vermicompost + 30% v/v of sand + 30% v/v of soil4. Growth medium contains 60% v/v of vermicompost + 30% v/v of sand + 10% v/v of soil

Some chemical characteristics of soil including pH and EC (in saturated extraction), texture(by hydrometric method), calcium carbonates (by titration method), available phosphorus (by spec-


trophotometry method), and available potassium (by flame photometry) were measured. Somechemical properties of vermicompost including pH and EC in extract 1:5 (vermicompost to distilledwater), CEC (by Chapman method), organic material, total nitrogen (Kjeldal method) were meas-ured (Jackson, 1967). In order to determination of nutrients in shoot dry matter, 1 g vermicompostpowder was turned to ashes in 550 0C and it was extracted by HCl 2N and in derived extraction,phosphorus by spectrophotometry, potassium and calcium by flame photometry were measured.

Growth media was poured on three lit. pots and then marigold seed was cultivated in thesepots. All necessary attention including the use of fertilizers, irrigation and poisoning were done.After four months, the plant shoots was cut to measure final height of plant, lateral branches, shootdry matter, size and weight of flower. The concentration of potassium, phosphorus, nitrogen, cal-cium and magnesium was measured in shoot dry matter. The results were analyzed by using LSDtest and SAS software.

RESULTS

Fig. 1 shows the results which related to the effect of different amount of vermicompost incultivation bed on the marigold height. Vermicompost increased the height of plant at 20 and 40%vermicompost, significantly, but the height decreased by 60% vermicompost that of course it wasnot significant than in the control. Adding vermicompost to medium increased the number of lateralbranches significantly. The number of lateral branches increased about 3 times in 60% vermicom-post treatment as compared with the control.

Vermicompost tend to increase in flower size than in the control, so that this increase was8.53 mm in 60% vermicompost treatment more than control. The flower weight has also increasedin vermicompost treatments which this increase was about 1.5 times more than control in all ver-micompost treatments. Shoot dry matter increased in the vermicompost treatments. Increasing drymatter was 4.73, 4.93 and 7.40 g more than control in 20, 40 and 60% vermicompost, respectively.

The impact of different vermicompost rates on nitrogen are observed in fig. 6. The signif-icant impact of vermicompost was not observed on the nitrogen of marigold shoot. Application ofvermicompost 20% decreased phosphorus concentration of plant shoot, significantly, but therewas not a significant difference at the 40 and 60% of vermicompost as compared with control.The use of vermicompost increased potassium of plant significantly in 40 and 60% vermicomposterates, but it had not significant difference at the 20% rate in compared to control. The increase inpotassium of shoot was 1.69 times more than control in 60% vermicompost. Thus, the calciumconcentration of shoot has increased in the vermicompost treatments which Ca of 60% vermicom-post treatment about 2 times more than control.

DISCUSSION

Vermicompost include some aerobic microorganisms such as Azotobacter and it lack ofnon aerobic bacteria, fungi and pathogenic microorganisms. Vermicompost like peat has beenformed of materials with pores, aerator capacity, drainage and high capacity for water holding, ithas a high surface area in exchanging nutrients. Vermicompost has little amount of solvable mineralthan primitive material, also it has more acid humic and more capacity for cations exchange (Atiyehet al., 2001). Vermicompost has many nutrients such as phosphorus, potassium, calcium, magne-sium as available for plants (Orozco et al., 2001).

Vermicompost contains plant growth hormones, several enzymes, more microbial popula-tions and it is free of harmful pathogens and small animals such as form colonies bacteria andSalmonella. The results of this experiment showed that the effect of vermicompost on plant heightwas similar to peat, while in the perlite bed, plant height was lowest. Atiyeh et al., (2002) studiedthe effect of pigs manure vermicompost on the growth and yield of french marigold. The mostvegetative growth had obtained at 30 and 40% v/v vermicompost and the least growth obtained of


90 and 100% values. The highest and the lowest number of flower buds were related to 40% and100%, respectively. The studies of Snagwan et al., (2010) showed that the adding 40% w/w ofsugar factor waste to vermicompost caused to increase in flower size than in 10-30% w/w rates.

Rani and Srivastava (1997) found that replacement of or of the nitrogen chemical fer-tilizers with vermicompost tend to increase in the rice height.Peyvast et al., (2008) investigated effect of soil and vermicompost composition in thegrowth bed of parsley (Petroselinum crispum) at different rations. Their results showed that theaddition of vermicompost to soil increased plant height. Walker and Bernal (2008) reported thatapplication of compost and organic fertilizer dramatically increased growth of best branch. Theeffect of vermicompost on plant growth is through effect on photosynthesis and various productsof plants such as leaves, stem and root to stimulate the material stored in the leaves and nutrientsuptake and water by roots. Bwamiki et al., (1998) showed that the use of organic amendmentssuch as compost can increase the production of yield.

Vermicompost contains some nutrients which are important in plant production. The nitro-gen, phosphorus and potassium levels of the earthworm wastes mostly 5 to 11 times is more thansoil and during the processing, the calcium, magnesium and micronutrients can also increase(Smith, 1998). There is much evidence that the activity of earthworms accelerates organic mattermineralization, decomposition of polysaccharides, increase the humus material, reducing the car-bon to nitrogen ratio and reducing availability of heavy elements (Domingues et al., 1997). Ver-micompost indirectly through the impact on soil micro flora will affect on plant growth. Forexample, adding vermicompost to the growth medium containing peat increases the colony for-mation of mycorrhiza (Cavender et al, 2003).

CONCLUSION

Vermicompost increased the plant growth and nutrient concentration in shoot. The best im-pact of vermicompost obtained at the 60% rate. Therefore, results showed that the vermicompostin an appropriate medium for growth of ornamental plant. It is proposed to study other researchesabout impact of vermicompost on ornamental plants growth in compared with other planting bedssuch as peat and perlite.

Literature Cited

Atiyeh, R.M., Arancon, N., Edwards, C.A. and Metzger, J.D. 2000. Influence of earthworm-processed pig manure on the growth and yield of greenhouse tomatoes. Bioresource Technology, 75 (3): 175-180.

Atiyeh, R.M., Arancon, N.Q., Edwards, C.A. and Metzger, J.D. 2002. The influence of earthworm-processedpig manure on the growth and productivity of marigolds. Bioresource technology, 81:(2) 103-108.

Atiyeh, R.M., Subler, S., Edwards, C.A. and Metzger, J.D. 2001. Pig manure vermicompost as a component of a horticultural bedding plant medium : Effects on physicochemical properties and plant growth. Bioresource Technology, 78: (1) 11-20.

Bachman, G. R., and Davis, W. E. 2000. Growth of Magnolia virginiana liners in vermicompost-amended media. PedoBiologia 43:579-590.

Bachman, G.R. and Metzger, J.D. 2008. Growth of bedding plants in potting substrate amended with vermicompost. Bioresource Technology, 99: 3755-3761.

Benitez, E., Nogales, R., Elvira, C., Masciandaro, G. and Ceccanti, B. 1999. Enzyme activities as indicators of the stabilization of sewage sludge composting with Eisenia foetida. Bioresorce technology, 67: 297-303.

Bwamiki, D.P., Zake, J.Y.K., Bekunda, M.A, Woomer, P.l., Bergstrom, L., Kirchman, H. 1998. Use of coffee husks as an organic amendment to improve soil fertility in Ugandan banana

13

14


production. Carbon and nitrogen dynamics in natural and agricultural tropical ecosystem, 113-127.

Cavender, N.D., Atiyeh, R.L. and Knee, M. 2003. Vermicompost stimulates mycorrhizal colonizationof roots of sorghum bicolor at the expense of plant growth. Pedobiologia, 47: 85-89.

Cull, D. C. 1989. Alternatives to peat as container media: Organic resources in UKActaHorticulturae 126: 69-81.

Domingues, J., Edwards, C.A. and Subler, S. 1997. A comparison of vermicomposting and composting. Biocycle, 38: 57-59. 58.

Jackson, M. L. 1967. Soil chemical analysis. Prentic-Hall of India private limited, New Delhi, India.Mahboub Khomami, A. 2005. Effect of liquid bio-fertilizer (vermiwash) in foliar application on

Dieffenbachia and Aglaonema nutrition and growth indices.Agricultural Sciences. 1(4): 175-187 (In Persian).

Mal Yin, X.Q., 2010. Effect of sewage vermicompost on the growth of Marigold.Ying Yanng Sheng TaiXuieBao, 21(5): 1346 – 1350. 83.

Orozco, F,h., Cegarra, G., Trujillo, L.M. and Roig, A. 1996. Vermicomposting of coffe pulpusing the earth worm Esientia fotida : Effects on C and N contents and availability of nutrients. Biology and Fertility of soil, 22: 162-166.

Peyvast, Gh., Olf, J.A., Madeni, S., forghani, A. and Samizadeh, H. 2008. Vermicompost as a soil supplement to improve growth and yield of parsley. International Journal of Vegetable Science, 14 (1): 82-92.

Pool, R. T., Conover, C. A. and Joiner, J. N. 1981. Soils and potting Mixes. SoilScience 132 (2): 179 - 202.

Rani, R. and Srivastava, O.p. 1997. Vermicompost: a potential supplement to nitrogenos fertilizer in rice nutrition. International Rice Research Notes, 22(3):30-31.

Saleh Rasti, N. 2001. Biological fertilizers anf its role at sustainable agriculture. The papers collections of industrial production of biological fertilizers in Iran, pp. 1-54 (In Persian).

Smith, K. 1998. Practical guide to raising earthworm (Basic vermiculture information) K & W rabbit and worm. Bioresource Technology, 84(2): 191-196.

Snagwan, P, Garg, V.K. and Kaushik, C.P. 2010. Grows and yeald response of Marigold to pottingmedia containing vermicompost produced from waster. The Envirmentalis 30(2):123 – 130.109.

Tomati, U., Grappelli, A., Galli, E. 1988. The hormone- like effect of earthworm casts on plant growth. Biology Soils, 5: 288-294. 17.

Walker, D.J. and Bernal, M.P. 2008. The effecfs of olive mill waste compost and poultry manure on the availability and plant uptake of nutrients in a highly saline soil. Bioresource Technology, 99: 396 - 403.


EC(dS/m)pHTextureCalcium CarbonatesAvailable phosphorus (mg/kg)Available potassium (mg/kg)

4.88.0Sandy Loam3.314.3656.8

Table 1. Some chemical characteristics of soil

EC(dS/m)pHCEC (me/100g)Organic carbon (%)Total nitrogen (%)Available phosphorous (mg/kg)Available potassium (mg/kg)Available Calcium (mg/kg)

23.47.743.465.40.0383.9750.085.0

Table 2. Some chemical characteristics of vermicompost

Table 3. The effect of different treatments on nitrogen, phosphorus, potassium, calcium and magnesiumconcentration of shoot organs of Marigold

Tables

Treatment Compound

N P K Ca

(%)

Control20% Vermicompost40% Vermicompost60% Vermicompost

30% soil + 70% sand 50% soil + 30% sand + 20% vermicompost 50% soil + 30% sand + 40% vermicompost 50% soil + 30% sand + 60% vermicompost

7.8 a5.7 abc6.5 ab7.2 a

0.20 b0.14 bc0.18 b0.21 b

2.50 bc2.06 cd4.16 a2.80 b

0.83 c1.56 a1.50 ab1.60 a


Figures

Fig. 1. The effect of treatments on the plant height

Fig. 2. The effect of treatments on the lateral branche number of plant

Fig. 3. The effect of treatments on the flower size of plant


Fig. 4. The effect of treatments on the flower weight of plant

Fig. 5. The effect of treatments on the shoot dry matter of plant


The Role of Preservative Compounds on Number of Bacteria

on the End of Stems and Vase Solutions of Cut Gerbera

This study was conducted to evaluate the effect of preservativesolutions on vase life, number of bacteria in the end of stem and in vasesolution of cut gerbera 'Double Dutch' and 'Red Explotion'. Cut flowerswere pulse-treated with nano-silver (2, 4, 6, 8 or 10 mg L-1) and thymol(12.5, 25, 50, 75 or 100 mg L-1) + 5 % sucrose. Experiment was conductedin completely randomized design with 5 replications and 1 flower in eachexperimental unit. Flower were harvested from a commercial greenhouseand transported to laboratory with 22±1ºC temperature and 60±5% relativehumidity. According to the results, these materials had positive effects onvase life of flowers. 6 mg L-1 nano-silver treatments in ‘Red Explotion’cultivar had highest longevity (14 days). All treatments were effective ondecreasing of bacteria in stem end and solution. In 4 and 6 mg L-1 SNPtreatments was not any bacteria in vase solutions of ‘Red Explotion’ cultivar.Resulty showed that nano-silver can be use for increasing the vase life ofcut gerbera ‘Double Dutch’ and ‘Red Explotion’.

Keywords: Anti microbial, Gerbera jamsonii, Nano-silver, Postharvaest, Thymol.

Abstract

T. Oraee1*, A. Asghar Zadeh2, M. Kiani3 and A. Oraee1

1 Former M.Sc. Student, Department of Horticulture, Shirvan Branch, Islamic Azad University,Shirvan, Iran2 Department of Horticulture, Islamic Azad University, Shirvan Branch, Islamic Azad University,Shirvan, Iran3 Research Center for Plant Sciences, Ferdowsi University of Mashhad, 91775-1491, Iran

*Corresponding author email: [email protected]


INTRODUCTION

The colours of cut gerbera are very variable and gerbera is a popular cut flower (Syros etal., 2004). Cut gerbera are sensitive to microbial contamination at the stem base (Balestra et al.,2005). Many agents have been used in cut flower vase solutions to extened vase life by reducedmicrobial contamination. The bactericides are the most important components in the preservativesolutions to control harmful bacteria and help to prevent bacterial embolism (Halevy and mayak,1981). Other material tested in preservative including sucrose (Kim and Lee. 2002) and thymol.Sucrose was used as substrate for respiration (Victoria et al.,2003) , it helps to maintain the osmoticpotential of the petal cells (Sujatha et al., 2003). Pre-treatment of cut roses with thymol have beenfound effective against some bacteria (Oraee et al., 2010). Solgi et al., (2009) reported thatpre-treatment of gerbera jamsonii ‘Dune’ with nano-silver particles (SNPs) is important as an an-tibacterial agent (Morones et al., 2005). Liu et al., (2009) reported vase life extention for cutgerbera jamsonii ‘Ruikou’ flowers following pulsing with 5 mg L-1 SNP solution for 24 h. Appli-cation of 10 mg L-1 SNP + 5% sucrose for 24 h extended the vase life of cut rosa hybrida ‘Dolcevita’ Flowers (Oraee et al., 2010). Aim of present study is increasing vase life of cut gerbera bynano silver and thymol .


Plant Material

Cut gerbera ‘Double Dutch’ and ‘Red Explotion’ were harvested from a hydroponic green-house in Mashhad, Iran. They were immediately stood upright in buckets partially filled with tapwater and transported to the laboratory.

SNP and Thymol Treatments

Flower stems were then re-cut under deionized water (DI) to 45 cm length. Cut gerberaswere pulse-treated for 24 hours at 22±1 °C with 2, 4, 6, 8 or 10 mg L-1 SNP (Nano cid company,Iran) and 12.5, 25, 50,75 or 100 mg L-1 thymol (Sigma company, USA( + 5% sucrose. Controlflowers were pulse-treated with DI water. The flowers were kept under controlled conditions: 12hours photoperiod at a photosynthetically activated radiation of 10 μmolm-2 s-1 provided by fluo-rescent lamps, 22±1 °C.

Assessments of Vase Life

The end of vase life was defined the time that flowers were showing symptoms of petalwilting or curling and stem bending (Geraspolus and Chebli, 1999).

Bacterial Counts

To determine the bacterial populations, vase water aliquots were tested during the experi-ment. Dilution carried out with 0.9% normal saline to achive 30-300 bacterial colonies in one petridish. 0.1 % ml of aliquots was spread on nutrient agar plate. They were incubated at 37° C for 24h before enumeration of bacteria (Bleeksma and Van Doorn, 2003).

To determine the stem bacteria numbers, 2 cm length segments were cut from the stemends. Explants were washed three times with sterile DI to reduce the surface loud of microbes.They were then grounded and diluted with 0.9% sterile normal saline. Liquid extract (0.1ml) wasspread on nutrient agar plates and bacterial colonies were enumerated after incubation for 24h at37˚ C (Balestra et al., 2005).

Statistical Analyses

Statistical significance between mean values was assessed using analysis of variance(ANOVA) and means were compared by Duncanُ s Multiple Range Test (DMRT; P≤ 0.05).



The results revealed that SNP and thymol at all concentrations increased vase life of allcultivars ('Double Dutch' and 'Red Explotion') compared to control. Our results were supportedthe by some previous researches. They suggested that vase life of cut gerbera is greatly improvedby chemical compounds and essential oils and there is a large difference among gerbera cultivars.Comparsion of two cultivars showed that 'Red Explotion' had longevity more than ‘Double Dutch’.There were significant differences between cultivars in response to SNP and thymol concentrations.Nano-silver at 6 mg L-1 was the most effective treatment for increasing the vase life of gerberajamsonii ‘ Double Dutch’ (14 days). However, the SNP pulse treatment at 8 and 10 mg L-1 causedvisible damage to flowers. In both cultivars with increasing of thymol concentration from 12.5 to100 mg L-1, the longevity of cut flowers increased (Table 1).

Our findings are similar to result of Solgi et al., (2009), they reported that gerbera flowersare sensitive to microbial contamination and vase life of cut gerbera was extended by essentialoils such as thymol and chemical compounds like SNP. Number of bacteria in the stem ends andvase solution tended to be increased throughout the gerbera vase life in the all treatments. Therewas significant difference in vase solution bacterial population between thymol and SNP pulsetreatments. SNP reduced the number of bacteria and delayed the petal wilting. Pre-treatment withSNP (2, 4, 6 mg L-1) in both cultivars was generally very effective against bacterial growth, but 8and 10 mg L-1 concentrations had no effect on the number of bacteria and have toxic effect on cutgerbera. The highest number of bacteria was recorded in control (P≤ 0.05). In 4 mg L-1 and 6 mgL-1 SNP in ‘Red Explotion’ was not any bacteria in the vase solution. In the other treatments, thenumber of bacteria was also less than control. There were significant differences in number of bac-teria in the stem ends between the 100 mg L-1 thymol and the control for the duration of assessment.In cut gerberas ‘Double Dutch’ and ‘Red Explotion’, we found that numbers of bacteria in stemend in third day was more than 106 CFU ml-1 and in vase solution was less than 104 CFU ml-1

(Table 1). Also there is relation between number of bacteria in vase solution and number of bacteriastem end (Oraee et al., 2010). We concluded that all compounds had antibacterial effects and vasesolution and stem ends microorganisms can decrease these preservative compounds. The mostconsistent antibacterial action was found in nano silver particles.

Literature Cited

Balestra, G.M., Agostini, R., Bellincontro, A., Mencarelli, F. and Varvaro, L. 2005. Bacterial populationsrelated to gerbera (Gerbera jamesonii L.) stem break. Phytopath. Mediterr. 44: 291-299.

Geraspolus, D. and Chebli, B. 1999. Effect of pre- and postharvest calcium applications on the vase life of cut gerberas. J. Hort. Sci. Bio. 74: 78-81.

Halevy, A.H. and Mayak, S. 1981. Senescence and postharvest physiology of cut-flowers. Part2. Hort. Rev. 3: 59-143.

Kim, Y. and Lee, J.S. 2002. Changes in bent neck, water balance and vase life of cut rose cultivarsaffected by preservative solution. J. Korean Soc. Hort. Sci. 43: 201-207.

Liu, J., He, S., Zhang, Z., Coa, J., Lv, P., He, S., Cheng, G. and Joyce, D.C. 2009. Nano-silver pulse treatments inhibit stem-end bacteria on cut gerbera cv. ‘Ruikou’ flowers. Postharvest Biol. Technol. 54: 59-62.

Morones, J.R., Elechiguerra, J.L., Camacho, A., Holt, K., Kouri, J.B., Ramirez, T.J. and Yacaman, M.J. 2005. The bacterial effect of silver nanoparticles. Nano. Tech. 16: 2346-2353.

Nair, S.A. and Sharma, T.V.R.S. 2003. Effect of chemical preservatives on enhancing vase-life of gerbera flowers. J. Trop. Agri. 41: 56-58.

Oraee, A. Kiani, M. and Ganji Moghadam, E. 2010. Effects of Nano-silver, Silver thiosulfate, 8-Hydroxy quinoline and some natural compounds on vase life of Rose. MSc thesis, Faculty Of Horticultural Sciences. Islamic Azad University Branch of Shirvan, Iran, PP.1-137.


Solgi, M., Kafi, M., Taghavi, T.S. and Naderi, R. 2009. Essential oils and silver nanoparticles (SNP) as novel agents to extend vase-life of gerbera (Gerbera jamsonii cv. ‘Dune’) flowers. Postharvest Biol. Technol. 53:155-158.

Sujatha, A., Singh, V. and Sharma, T.V.R.S. 2003. Effect of chemical preservatives on enhancingvase-life of gerbera flowers. J. Tropical Agri. 41: 56-58.

Syros, T., Yupsanis, T. and Omirou, M. 2004. Photosynthetic response and peroxidases in relation to water and nutrient deficiency in gerbera. Environ. Exp. Bot. 25:23-31.

Victoria, G.N., Marissen, N. and Van Meeteren, U. 2003. Effect of supplemental carbohydrates, In: A.V. Roberts, T. Debener and S. Gudin (eds). Encyclopedia of rose science, Elsevier Academic Press Oxford UK. 1:549-554.


Treatment

Vase life

(Days)

No. of stem end bacteria

in 3th day

(CFU ml-1)

No. of vase solution

bacteria in 3th day

(CFU ml-1)

Cultivars

Control

SNP(mg L-1)

Thymol(mg L-1)

246810

12.5255075100

6.2jkl*9.2ef12.2b14a11c

10.4cd

7.2ghij7.6ghi9.2ef

10.2cde11.2bc

4.4m6kl

9.6de10.6cd

8gh7hijk

5.8l6.4jkl6.8ijkl7.6ghi8.2fg

7.2×106d5.6×104 m7.6×103 t5.1×102u7.8×103 s4.1×104 p

8.05×105h8×105 i

5.6×104 l4.2×104 o7.9×103 r

8.9×106 a7.9×106c5.1×104 n4×104q

4.9×105 j8.4×105 g

8.2×106 b7.2×106 e7.1×106 f8×105 h

4.1×106 k

3.4×104 c8×103 d

30j <30j <78j

6.9×103 e

4.1×103 g3.3×103 h8×103 d

8.4×102 i7.1×102 j

8.4×104 a3.8×103g h5.6×103 f8.4×102 i3.2×103 h8.3×103 d

5.1×104 b3.3×103 h3.3×103 h3.3×103 h6.9×103 e

Table 1. Effect of different concentrations of nano silver particles and thymol on measured characteristies

*Mean in each column with the same letters are not significantly different at 5% level of probability using

DMRT.

Tables

www.jornamental.com


Evaluation of Mulberry (Morus spp.) Genotypes for

Tolerance to Major Abiotic Stresses

Sericulture has played a very important role in the socio-economicempowerment of rural and semi-urban population. The eco-friendly nature ofthe industry, in addition to its employment generation potential, low investmentsand frequent returns, has rendered the industry as one of the most suitableland based economic activity, particularly in the context of global movementagainst environmental degradation and global warming. The present thrust inincreasing silk production to meet the growing domestic and internationaldemand however, cannot rely upon horizontal expansion. In light of thecompetition to mulberry from other food and commercial crops, it has becomeimperative to utilize marginal, problematic soils for mulberry cultivation.Although soil amendment and management is one of the feasible means,genetic improvement of crops towards tolerance to stress is more effective,less costly, non-polluting and longer lasting. The present study was conductedwith six mulberry genotypes selected from a segregating population of 1152hybrids on the basis of their relative performance in two diverse environments- (i)Optimum growing conditions, wherein the recommended inputs were providedand (ii) Stress conditions, wherein all inputs including irrigation werewithdrawn. The six genotypes were further subjected evaluation under differentstress conditions like, soil moisture stress, alkalinity and salinity along withcontrol genotypes K-2 and V-1 maintained at optimal conditions. Significantvariability was recorded among the genotypes in respect of Leaf yield responseindex [LYRI], Stress resistance index [SRI] and Varietal score [VS]. Theresults indicated a high degree of plasticity in G-6, which is now christened asRC-2 [Resource Constraint-2], that would assure sustained leaf production insevere water stress, alkaline and saline conditions. The genotype is recommendedfor cultivation by the marginal and small farmers for economic utilization ofthe problematic soils.

Keywords: Alkalinity, Leaf yield response index, Plasticity, Salinity, Stress resistance index, Varietalscore, Water stress.

Abstract

M. K. Prithvi Raje Urs1*, K. Rajashekar1 and A. Sarkar1

1 Central Sericultural Research and Training Institute, Manandavadi Road, Srirampura, Mysore-570008India



INTRODUCTION

In view of the growing demand for silk in the domestic and international markets, it is mostappropriate for any agrarian economy, especially the ones that have a tradition in sericulture, toincrease their silk production. While increased productivity per unit area has significantly con-tributed towards production, it has now become imperative to develop strategies for utilizing prob-lematic soils, and tracts with limited water resources. Larger areas under the semi-irrigatedconditions exhibit alkalinity due to poor rainfall and scarcity of irrigation water. These soils containexcessive concentrations of exchangeable carbonates or bicarbonates of sodium that usually exceed30% of the soil’s cation exchangeable capacity and high pH, which affect plant survival and growthdue to physiological drought conditions and nutritional deficiencies. Utilization of problematicsoils is best achieved by growing tolerant plant species (Epstain, 1985; Epstain and Rains, 1987;Ashraf and Mc Neilly, 1988). Although soil amendment and management is one of the feasiblemeans, genetic improvement of crops towards tolerance to stress is more effective, less costly,non-polluting and longer lasting (Epstain et al., 1980 and Downtown, 1984).

Many studies have been conducted to identify salt tolerant mulberry genotypes by screeningunder coastal saline soils (Agastian and Vivekanandan, 1997; Chakraborty et al., 2000) and underinduced salinity conditions (Shaik and Vivekanandan, 1999; Mogili et al., 1998 and 2002; Prakashet al., 1998; Sarkar et al., 2000). Naidu et al., (1999) reported variability in reaction to alkalinitystress among tree species when screened under natural stress conditions. The studies on evolvingsoil moisture stress tolerant genotypes have led to the development of mulberry varieties like S-13 and S-34 (Susheelamma, 1987; Susheelamma and Jolly, 1986).

The present study subjected six mulberry genotypes selected from a segregating populationof 1152 hybrids on the basis of their relative performance in two diverse environments- (i) Opti-mum growing conditions, wherein the recommended inputs were provided and (ii) Stress condi-tions, wherein all inputs including irrigation were withdrawn. The test genotypes were subjectedto different stress conditions like soil moisture stress, alkalinity and salinity along with controlgenotypes K-2 and V-1 maintained at pH 7.0, with recommended dosage of NPK and irrigation.Significant variability was recorded among the genotypes in respect of leaf yield response index[LYRI], stress resistance index [SRI] and varietal score [VS].


The study was carried out under controlled conditions using pot culture technique. Six hy-brids short-listed for their superior performance under stress and non-stress environments wereconsidered for the study along with K-2, a variety popular in semi-irrigated conditions and V-1, avariety popular under optimum growing conditions, as checks. Well-rooted saplings of the geno-types were planted in pots lined with polyethylene sheets to provide full effect of the treatmentsunder simulated conditions to study the plasticity of genotypes under different conditions. The dif-ferent conditions were treated as environments.

The genotypes were put under soil moisture stress (irrigation once in 3 & 7 days), salinitystress (Ec- 4.0 & 7.0 mmho-cm) and alkalinity stress (pH 8.5 & 10.0). The controls were main-tained with recommended dosage of fertilizers i.e., N:P:K @ 300:120:120 kg/ha/yr in 5 equalsplits, pH 7.00 and Ec 0.8 mmho-cm

Seven different conditions comprising of optimal and six different conditions were simu-lated (Table 1). The stress levels were induced by adopting different frequencies of irrigation;adding sodium carbonate and calcium carbonate to induce alkalinity; adding sodium chloride andsodium carbonate to induce salinity. Soil samples were collected periodically from each pot, wheredifferent salinity and alkalinity levels were induced and analyzed after each harvest. The data in-dicated slight increase in pH and exchangeable sodium percentage (ESP) and sodium adsorptionRatio (SAR). However, pH, ESP and SAR were within normal range of variation and did not affect


the actual stress conditions induced. The experiment was conducted in randomized design withthree replications, considering individual pots as replications. Each ordered replicate was arrangedcompactly with a guard row around each replication. Pots were shifted at regular intervals anddata recorded each time during harvest.

The experiment was conducted for one year after establishment of saplings in pots. Leaveswere harvested at intervals of 70 days and the data of five leaf harvests were recorded. Averageleaf yield was considered for further analysis.

Plastic response of genotypes was measured by one-way and two-way ANOVA. Toleranceindices for different stress environments were determined following Rana (1986) and Maloo (1993)with slight modifications:

Leaf yield response index [LYRI]

LYRI = Leaf yield of a variety (average of all stress environments)

Mean yield of all varieties under stress environments

Stress resistance index [SRI]

SRI =Leaf yield of a variety (average of all stress environments)

Leaf yield of the variety under non-stress environment

Varietal score [VS] = LYRI x SRI


The mean values for leaf yield at different growth environments are presented in Table 2.Significant reduction was recorded in leaf yield of all genotypes in response to different stress fac-tors and also in the yield among the genotypes under a particular environment. Although the leafyield of the test genotypes significantly reduced in all the treatments (E2 to E7) compared to thecontrol (E1), the reduction in leaf yield was not similar in all the genotypes.

Under moderate stress conditions [E2], the leaf yield was found to vary from 56.65% inG-3 to 82.28% in G-6, in comparison to the yield in E1. The test genotype G-6 showed least re-duction under E2 and the mean leaf yield was almost equal to the superior check V-1. Under severesoil moisture stress [E3], the leaf yield was found to vary from 38.86% in V-1 to 60.10% in G-4,in comparison to the yield in E1. The test genotype G-4 showed least reduction under E3, but wasoutyielded by G-6 and G-2.

Under moderate stress conditions [E4], the leaf yield was found to vary from 46.21% in V-1 to 77.27% in G-6, in comparison to the yield in E1. The test genotype G-6 showed least reductionunder E4 and the mean leaf yield was also highest in the genotype. Under severe alkalinity stress[E5], the leaf yield was found to vary from 28.46% in V-1 to 47.60% in G-4, in comparison to theyield in E1. The test genotype G-4 showed least reduction under E5, followed by G-6, which outyielded all the test genotypes and both the checks.

Under moderate stress conditions [E6], the leaf yield was found to vary from 63.29% inG-3 to 86.69% in K-2, in comparison to the yield in E1. The check genotype K-2 showed least re-duction under E6 and the mean leaf yield was highest in the check genotype V-1, followed by G6,which did not show any significant difference in mean leaf yield with the check variety V-1. Undersevere salinity stress [E7], the leaf yield was found to vary from 47.99% in G-4 to 70.63% in G-6, in comparison to the yield in E1. The test genotype G-6 showed least reduction under E7, whichout yielded all the test genotypes and both the checks.

In all the treatments G-6, among the test genotypes, was found superior to K-2 in respect


of leaf yield, with the largest positive yield variance in case of mild soil moisture stress (87.60%),followed by mild alkalinity (51.87%) and severe salinity (50.24%). The variance was 47.96% incase of control maintained under non-stress conditions. However, the test genotype G-6 showedpositive yield variance against the superior check V-1 only in four of the six environments. Thehighest positive variance of 52.28% was recorded under mild alkalinity stress followed by severealkalinity stress [41.15%].

It is reported that due to effect of plasticity, not only a genotype behaves differently in dif-ferent environments but also; the different genotypes behave similarly in a particular environment(Bradshaw, 1965). In the present study, the maximum yield was obtained in the genotype V-1 underoptimal conditions and the minimum in K-2 under similar conditions. The yield of genotype K-2(95.16 g/plant) was only 61.50% of the yield of V-1 (154.80 g/plant) under optimal conditions,the yield difference being highly significant. But it is interesting to note that the leaf yield of K-2and V-1 was nearly similar in both alkaline stress treatments E4 and E5. Under severe soil moisturestress treatment [E3], the yield difference was not significant.

The leaf yield was dependent on both genotype and environments. Genotype x environmentinteraction was also found to be significant. Both environment and G x E terms were found to besignificant indicating the plastic response of the genotypes and differences in their response.Schlichting (1986) stated that comparing a large number of genotypes for plasticity couldn’t pro-vide sufficient information for the plastic response of a pair of genotypes. Hence the pair wisecomparison was made through two-way ANOVA. The result indicated how the genotypes variedin their plastic response when compared with the other selected genotypes (Table-3). The genotypepairs viz., G-1 Vs G-2, G-1 Vs G-6 did not differ significantly in respect of plasticity for yield.Similarly, G-2 Vs G-6, G-5 and G-3 did not differ significantly in respect of plastic response toyield. All other pairs were found to be highly significant.

The tolerance indices measured by using leaf yield under stress and control conditions in-dicated clear differences between the genotypes (Table 4). The test genotype G-6 recorded highestvalues of the two indices, Leaf yield response index (LYRI) and Varietal score (VS) in all the treat-ments. The genotype G-6 ranked a very close second in respect of Stress resistance Index (SRI).Rana (1986) and Singh (1991) critically analyzed the selection criteria for salt tolerance in cropplants and concluded that the tolerant genotype shows higher rankings for leaf yield response,stress resistance indices and varietal scores.

The results indicated a high degree of plasticity in G-6 that would assure sustained leaf pro-duction in alkaline and semi-irrigated tracts. The genotype could be recommended for cultivationby the marginal and small farmers in semi-arid and alkali-affected tracts for economic utilizationof the soils through sericulture. This will not only increase the silk production but also will improvethe economy of the poor farmers in those areas.

Literature Cited

Agastian, S.T.P., Vivekanandan, M. 1997. Rooting potential of mulberry genotypes in costal saline areas. Sericologia, 37:521-523.

Ashraf, M. and McNeilly, T. 1988. Variability in salt tolerance of nine spring wheat cultivars. J.Agron. Crop Sci., 160:14-21.

Bradshaw, A.D. 1965. Evolutionary significance of phenotypic plasticity in plants. Adv. Genet., 13:115-155.

Chakraborty, S.P., Biswas, C.R., Vijayan, K., Roy, B.N. and Sarathchandra, B. 2000. Evaluation of mulberry varieties for coastal saline soils of West Bengal. Bull. Ind. Acad. Seri., 4:41-45.

Downtown, W.J.S. 1984. Salt tolerance in food crops: Perspectives for improvements. CRC Critical Reviews in plant sciences, 1:183-201.

Epstain, E. 1985. Salt tolerant crops: origin, development and prospects of the concept. Plant and


Soil, 89:187-198. Epstain, E., Norlyn, J.D., Rush, D.W., Kingsbury, R.W., Kelly, D.B., Cunningham, G.A., Wrona,

A.F. 1980. Saline culture of crops: a genetic approach. Science, 210:399-404.Epstain, E. and Rains, D.W. 1987. Advances in salt tolerance. Plant and Soil, 99:17-19. Maloo, S.R. 1993. Breeding and screening techniques for salt tolerance in crop plants. In management

of salt-affected soils and waters. (eds.) Somani, L.L., Totawat, K.L.), Agrotech Publishing Academy, Udaipur, pp. 321-357.

Mogili, T., Sarkar, A. and Munirathnam R.M. 2002. Effect of salinity stress on some improved varieties of mulberry, Morus spp. Sericologia, 42:149-163.

Mogili, T., Sarkar, A., Suzuki, M. and Munirathnam R.M. 1998. Evaluation of salt tolerance in promising mulberry genotypes under simulated conditions. Current technology seminar on mulberry and silkworm genetics & molecular biology and agronomy, Central Sericultural Research and Training Institute, Mysore, India, Abstract No. 6.

Naidu, C.V., Srinivasa Sastry, P.S. and Srivasuki, K.P. 1999. Performance of some tree species inalkali soils. Indian Forester 125:508-512.

Prakash, B.G., Bongale, U.D. and Dandin, S.B. 1998. Screening of mulberry germplasm accessions for salt tolerance. Sericologia, 38:367-372.

Rana, R.S. 1986. Evaluation and utilization of traditionally grown cereal cultivars of salt affected areas in India. Indian J. Genet., 46 (Suppl.):121-135.

Sarkar, A., Mogili, T., Sathyanarayana, K., Reddy, M.M. and Umadevi, K. 2000. Identification of mulberry genotypes (Morus spp.) for tolerance to alkalinity. Seminar on Sericulture Technologies- an Appraisal, Central Sericultural Research and Training Institute, Mysore, India. pp. 1

Schlichting, D. 1986. The evolution of phenotypic plasticity in plants. A. Rev. Ecol. Syst., 17:667-693.Shaik M.A.S. and Vivekanandan, M. 1999. Evaluation of salinity tolerance in mulberry varieties

by exploring rooting potential. Sericologia, 34:311-321.Singh, K.N. 1991. Recent approaches to breeding for salt tolerance in crop plants. Golden Jubilee

Symposium on Genet. Research and Education: Current Trends and the Next Fifty years. 1:197-198.

Susheelamma, B.N. 1987. Evaluation and evolution of drought resistant mulberry varieties for sericulture. Ph. D. Thesis, University of Mysore, India.

Susheelamma, B.N. and Jolly, M.S. 1986. Evaluation of morpho-physiological parameters associated with drought resistance in mulberry. Ind. J. Seric., 25:6-14.


Sl.

No.Experiment No. Treatment details

1234567

E-1E-2E-3E-4E-5E-6E-7

Optimal- water daily (7/7), full dose of fertilizers (F)Water once in 3 days (1/3), full dose of fertilizers Water once a week (1/7), full dose of fertilizers Water daily, full dose of fertilizers, pH 8.50Water daily, full dose of fertilizers, pH 10.0Water daily, full dose of fertilizers, Ec 4.0 mmho-cm

Water daily, full dose of fertilizers, Ec 7.0 mmho-cm

Table 1. Details on different experiments and their treatment information.

Tables

Table 2. Mean values for leaf yield (g/plant) under different growing environments*

Sl.

No Environment G-1 G-2 G-3

Mulberry genotype

G-4 G-5 G-6 K-2 V-1

1

2

3

4

5

E1- Non- stressIndexFertilizer stress

E2- Mild (50% dose)Index

E3- Severe (25% dose)IndexSoil moisture stress

E4- Mild (once in 3 days)Index

E5- Severe (once in 7 days)IndexAlkalinity stress

E6- Mild (pH- 8.5)Index

E7- Severe (pH-10)IndexSalinity stress

E8- Mild (Ec-4.0 mmho-cm )Index

E9- Severe (Ec-7.0 mmho-cm)

IndexC. D. at 5%

125.73100.00

86.7368.9872.8357.92

93.4774.3467.4553.64

86.4268.7347.1637.50

94.3075.0070.1355.7714.90

134.50100.00

88.0365.4474.3355.26

94.1670.0068.4950.92

93.2969.3658.3843.40

92.0068.4086.6064.3810.95

135.09100.00

73.0954.1057.4342.51

76.5356.6555.9041.37

76.8256.8650.7137.53

85.5063.2972.2353.4608.96

107.73100.00

62.2657.7951.9648.23

76.9371.4164.7560.10

67.1662.3451.3647.60

83.7377.7251.7047.9910.97

126.06100.00

80.8964.1665.1051.64

72.7557.7152.9642.01

97.5177.3555.1043.70

87.6369.5172.6657.6312.08

140.80100.00

100.9071.6689.2663.39

115.8682.2867.9648.26

108.9477.3762.1944.16

111.2378.9999.4670.6314.44

95.16100.00

75.9679.8265.2668.57

61.7664.9050.4052.96

71.7375.3741.9144.04

82.5086.6966.2069.5610.32

154.80100.00

108.0969.8297.7363.16

117.5675.9460.1638.86

71.5446.2144.0628.46

124.8080.6294.0060.7214.82

* Results of one-way ANOVA


Table 4. Indices for measuring tolerance in mulberry genotypes under different stress conditions

Table 3. Significance between genotype pair grown in different environments as calculated by two-way ANOVA(Mean square)

LYRI – Leaf yield response index; SRI -Stress resistance index; VS – varietal score.

G- Genotype; E-Environment (treatment); *- significant at 5% level;

**- significant at 1% level; NS- Non-significant.

STRESS INDEX G-1 G-2 G-3 G-4 G-5 G-6 K-2 V-1

Soil Moisture

stress

Alkalinitystress

Salinitystress

LYRISRIVS

LYRISRIVS

LYRISRIVS

1.080.640.690.990.530.520.970.650.63

1.090.600.661.120.560.631.050.660.70

0.890.490.430.940.470.440.930.580.54

0.950.660.620.870.550.480.800.630.50

0.840.500.421.130.610.680.950.640.60

1.230.650.801.260.610.771.240.750.93

0.750.590.440.840.600.500.880.780.69

1.190.570.680.850.370.321.170.640.75

Genotype

Source of

variation G-2 G-4 G-6

Genotype

G-5 G-3 G-1 V-1

G-1

G-2

G-4

G-6

G-5

G-3

K-2

GE

G X EGE

G X EGE

G X EGE

G X EGE

G X EGE

G X EGE

G X E

6.67*53.67**1.09 NS

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

52.06**45.87**2.54**

134.11**60.85**4.66**

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

56.65**45.70**1.37 NS35.35**56.92**1.48 NS243.43**46.16**6.16**

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

3.11 NS43.36**2.63*

22.75**62.38**1.85 NS31.89**51.19**5.95**92.53**50.58**2.70*

---------

12.87**66.55**2.75*

56.32**95.46**1.66 NS21.81**82.15**5.75**

151.67**68.82**3.83**

2.97 NS79.09 NS

2.64*------

59.38**41.59**3.10**

150.66**56.52**4.44**

0.21 NS44.86**6.85**

262.37**44.62**4.23**37.85**49.33**3.87**27.69**75.05**8.77**

--

38.99**64.46**6.51**20.45**77.93**10.10**195.23**67.64**14.83**1.31 NS67.39**5.84**67.81**66.42**12.79**114.41**92.91**10.91**211.11**66.79**13.16**

www.jornamental.com


Synchronous Plantlet Formation by Using Banana Extract

and In vitro Hardening in Orchid, Dendrobium lituiflorumLindl.

The present study was undertaken to investigate the role of banana extract(BE) on synchronous plantlet formation from seeds of Dendrobium lituiflorumLindl. The seedlings formed on modified Knudson C (KC) medium supplementedwith 20% (v/v) BE had significantly high fresh and dry weights. Morphologicallyuniform plantlets with elongated leaves and well developed roots were formedon KC medium with 20% (v/v) BE in comparison to KC medium without BE(control) after 30 d of fourth subculture. The plantlets so formed weresubjected to in vitro hardening on agar-agar gelled medium, Luffa sponge andcocopeat: perlite (9:1) as support matrices, each containing one-half strengthKC major salts and were successfully acclimatized under greenhouse conditions.Banana extract helps in synchronous plantlet formation in vitro which is notonly beneficial for conservation purposes but also to the biotech industries asa large number of uniform plantlets can be obtained for transplantation,thereby, reducing the cost of production.

Keywords: Orchids, Propagation, Support Matrices, Synchrony.

Abstract

S. Vyas1, P. Kapoor-Pandey1, S.Guha1 and I. Usha Rao1*1 Department of Botany, University of Delhi, Delhi, India



INTRODUCTION

Synchrony in the present report refers to the uniformity in plantlet growth and is exclusivelya phenotypic parameter. In other instances, synchrony has been extensively used in cell culture(Sharma, 1999), as in a cell population growing at a logarithmic rate, all cells should be in a ho-mogeneous state in order to analyse the chemical and physical setup at each phase. Synchronizedsystem, essential to study cell cycle related events, is induced by blocking the cell cycle at S (syn-thetic) or M (mitotic) phases. Several chemicals used in inducing synchrony are colchicine, am-phidicolin, hydroxyurea, etc. Sharma (1999) elaborated that synchronization in plant cells is alsoimportant for the study of cell cycle related events and to study specific enzyme activity and in-crease in production of useful metabolites. The success of tissue culture on a commercial scalelies in the fact that a large number of similar sized propagules at a given stage can be produced ina given time period. Asynchrony is considered disadvantageous in vitro as it hampers seedlingmaintenance and transplanting (Thompson et al., 2006). This may further lead to an increase inthe cost of production. Synchrony with respect to germination of seeds of the orchid, Cypripediumcalceolus var. pubescens was also reported by Chu and Mudge (1994) where it referred to the oc-currence of majority of protocorms in a treatment at a given developmental stage at approximatelysame time. Synchrony in other aspects of tissue culture such as secondary somatic embryogenesishas been reported by Mondal et al., (2001) in Camellia sinensis. In tissue culture, induction ofmass somatic embryogenesis and successful somatic hybrid production can be achieved by syn-chrony in cell behavior (Sharma, 1999).

Commercial propagation of orchids has many bottlenecks such as low multiplication rate,vitrification, poor rooting and high mortality during acclimatization being the major limitations(Hew, 1994). At this juncture, rapid production of synchronous plantlets in vitro with well devel-oped leaves and established root system is extremely desirable as it will provide ample materialfor acclimatization in the greenhouse and finally to the field conditions. Dendrobium lituiflorumLindl. is an extremely rare and threatened sympodial epiphyte of North-Eastern states of India(Chowdhery, 2001). The ultimate success of tissue culture protocol on a commercial scale dependson the production of plants raised by tissue culture at low cost and with high survival rates (Haz-arika, 2006). Mass scale production of plantlets can be achieved by many methods, involving re-duction of the cost of medium ingredients and use of a natural additive that leads to fasterproduction of synchronous plantlets, thus, reducing the time period of plantlet formation. Even iflow cost in vitro propagation can be achieved, the transplantation stage continues to be a majorbottleneck in the micropropagation (Hazarika, 2003). After ex vitro transfer, these plantlets mighteasily be impaired by sudden changes in environmental conditions, and need a period of acclima-tization to correct the abnormalities (Pospíšilová et al., 1999). It is, therefore necessary, that a pro-tocol is devised wherein the in vitro grown plantlets can gradually adapt to the new environmentalconditions. It is at this juncture that the process of in vitro hardening, where the process of ac-climatization can begin while the plantlets are still under in vitro conditions, becomes important(Hazarika, 2006). For mass propagation of D. lituiflorum orchid for conservation, in vitro methodneeds to be devised that would lead to the formation of synchronous plantlets (so that a large num-ber of morphologically uniform plantlets can be obtained at the same time) which can be trans-planted to the greenhouse or field conditions on a large scale. BE is responsible for higherpercentage germination and rapid progress to advanced stages of development and root formation(Vyas et al., 2009). In this communication, we report that besides the above mentioned roles ofBE, it plays a significant role in mass scale synchronous plantlet formation which is a prerequisitefor a commercial venture. The in vitro raised plantlets can be used for commercial purposes onlywhen these can be successfully acclimatized. After ex vitro transfer, these plantlets might easilybe impaired by sudden changes in environmental conditions, and need a period of acclimatizationto correct the abnormalities (Pospíšilová et al., 1999). It is, therefore necessary, that a protocol is


devised wherein the in vitro grown plantlets can gradually adapt to the new environmental condi-tions. It is at this juncture that the process of in vitro hardening, where the process of acclimatizationcan begin while the plantlets are still under in vitro conditions, becomes important (Hazarika,2006). The study also compares the suitability of inexpensive support matrices in in vitro hardeningof the plantlets.


The capsules of Dendrobium lituiflorum Lindl. were procured from Assam, India. Bananaextract was prepared according to Vyas et al., (2009) by cutting banana into thin circular slicesand blending these with distilled water in the ratio of 4:1 (w/v). The modified KC medium (Knud-son, 1946) was prepared by adding BE in the required volume to KC medium. BE was used at 1-20% (v/v). Agar–agar (Qualigens, India) and sucrose (Daurala, Meerut, India) were used at 0.8%and 2% (w/v), respectively, in the medium and the pH was adjusted to 5.8. 50 ml medium wasdispensed in culture bottle (500 ml) and was autoclaved at 121ºC at 1.06 kg cm-2 for 30 min. Thecultures, inoculated under aseptic conditions, were then incubated under 12 h photoperiod by coolwhite fluorescent tubes (30 μmol m-2 s-1, Philips, India) at 25±2ºC. The subculture of propaguleswas carried out after every 30 d on fresh medium of similar composition.

The fresh weight of the propagules was taken directly by removing cultures from themedium and gently separating the agar-agar sticking to these propagules with a soft brush. Thecultures whose fresh weight was recorded were then wrapped in Aluminium foil and kept in ovenat 60ºC and the weight was periodically recorded after every 24 h and three consecutive readingswere taken until the weight stabilized.

The plantlets were subjected to in vitro hardening on agar-agar and Luffa sponge or coco-peat: perlite (9:1) in liquid medium. Each of these contained one-half strength KC major salts onlyexcept Ca(NO3)2.4H2O with each culture bottle containing 25 ml medium. The Luffa sponge (de-rived from dried fruits of Luffa aegyptica) was used as a matrix in liquid media (Gangopadhyayet al., 2009) was cut transversely. The plantlets were further transferred to the greenhouse in plastictrays containing cocopeat: perlite (9:1) (potting mix) and maintained under controlled conditionsof light, 25 2º C and 85-90% relative humidity.

Twelve replicates were carried out for each treatment and the experiments were repeatedtwice. Fifteen replicates were taken for recording the data on in vitro hardening. The data was sub-jected to univariate analysis of variance and least significant difference (LSD) at p ≤ 0.05 was ap-plied to test statistical significance by using SPSS, Sigmastat, Chicago, IL, USA.

RESULTS

The seeds inoculated on KC medium supplemented with BE [1-20% (v/v)] germinated andthe propagules on BE supplemented media showed early rooting and rapid progress to advancedstages of development leading to rapid seedling formation in comparison to control (Vyas et al.,2009). After 30 d of third subculture, the most optimum response was observed on 20% (v/v) BEsupplemented KC medium. The plantlets showed significantly higher shoot and root lengths incomparison to control (Fig. 1A). There was no significant difference in the fresh weight of propag-ules in comparison to control upon incorporation up to 10% (v/v) BE in the KC medium but it wassignificantly high on KC medium supplemented with 20% (v/v) BE (Fig. 1B). When dry weightwas recorded, no significant difference was observed in comparison to control up to 5% (v/v) BEsupplemented KC medium. But with further higher percentages of BE [10% and 20% (v/v)], itwas significantly higher than the control (Fig. 1B).

After 30 d of fourth subculture, shoot, leaf and root length and root number were signifi-cantly higher on 20% (v/v) BE supplemented KC medium in comparison to control (Figs. 1C;2A). The plantlets on 20% (v/v) BE supplemented KC medium exhibited elongated intense green

+-


leaves and well developed velamenous roots in comparison to control which were with tiny leavesand roots (Fig. 2A). Synchronous growth pattern was observed in cultures on 20% (v/v) BE sup-plemented KC medium (Fig. 2B-D). The plantlets were morphologically uniform. The shoot androot lengths of these plantlets were nearly similar and these exhibited well formed root system andwere produced on a mass scale.

These synchronously growing plantlets were then subjected to in vitro hardening on plainagar-agar medium and Luffa sponge and cocopeat: perlite as support matrices. The plantlets hard-ened in vitro on Luffa sponge as support matrix exhibited elongated intense green leaves withbroad lamina and long velamenous and light green roots some of which intertwined with fibres ofLuffa sponge (Fig. 3A). The plantlets on cocopeat: perlite as support matrix formed long leaveswith broad lamina, but many basal leaves senesced (Fig. 3B). The plantlets on agar-agar mediumdeveloped a cluster of numerous green and velamenous roots but the leaves had narrow laminaand some of the leaves also turned chlorotic (Fig. 3C). The plantlets immediately before transferexhibited well formed leaves and roots (Fig. 3D). The number of shoots and roots of the plantletson 0 d of inoculation on Luffa sponge, cocopeat: perlite and agar-agar was approximately similar(Fig. 4A). The maximum increase in the number of shoots of plantlets was observed on Luffasponge as support matrix followed by agar-agar and cocopeat: perlite after 30 d of in vitro hard-ening in comparison to that on 0 d of inoculation (Fig. 4B). The number of roots also increased inplantlets on medium with Luffa sponge as support matrix (Fig. 4B). These plantlets also showedmaximum shoot and root length (Fig. 4C&D). Some of the roots were smaller than the other roots.So the root length was clubbed into two categories- those lesser than or equal to two cm were re-ferred to as short roots while those above two cm as long roots. The maximum length of shortroots was observed in plantlets on Luffa sponge followed by those on agar-agar and cocopeat: per-lite while that of long roots was observed in plantlets on Luffa sponge followed by cocopeat: perliteand agar-agar (Fig. 4D).

A marked feature of leaf senescence was observed during in vitro hardening. The plantletson cocopeat: perlite recorded the maximum percentage of leaf senescence followed by those onagar-agar. The minimum percentage of senescing leaves was observed in plantlets on Luffa spongeas support matrix (Fig. 4E). The plantlets with shoots showing pseudobulbs and well developedintense green leaves and long light green roots with velamen, growing in the greenhouse conditionsexhibited prolific growth after six weeks of transfer (Fig. 3 E-F).

DISCUSSION

Synchronous formation of plantlets (morphological uniformity) of Dendrobium lituiflorumon 20% (v/v) BE supplemented KC medium with elongated intense green leaves and well devel-oped roots was observed after 30 d of fourth subculture. This synchronous growth and developmentis desirable as it helps in providing a large number of plantlets at a given stage of developmentafter a particular time period. In Dendrobium lituiflorum, BE was not only promotory in causinghigher percentage germination, early rooting and faster growth and development in comparison tocontrol (Vyas et al., 2009), it was also effective in achieving a large number of plantlets with higherfresh and dry weights indicating increased biomass and exhibiting the same stage of developmentwhen it was supplemented at 20% (v/v) to KC medium as reported in this investigation. Sudeepet al., (1997) also observed significant increase in shoot length and leaf number of Dendrobiumnobile in Vacin and Went medium (Vacin and Went, 1949) supplemented with 10% banana pulp.Pierik et al., (1988) observed that although banana homogenate was inhibitory in seed germinationof Paphiopedilum ciliolare but it promoted the growth of seedlings. The growth promoting effectof banana can be speculatively attributed to any of the following reasons in the present study: (a)buffering of media, (b) chelation of iron rendering it more readily available to the plants, (c) mineralnutrients in concentration and form that is appropriate for the plants and (d) growth substances


(Arditti, 1968) and needs to be confirmed with further experimentation. Banana contains IAA(Khalifah, 1966b), GA7 and GAx (Khalifah, 1966a) and zeatin, zeatin riboside and 2-iP (Van Stadenand Stewart, 1975). The growth-enhancing effect of banana might occur due to the individualaction of any of these substances or a synergistic effect of two or more of them (Arditti, 1968).Synchronous plantlet formation is advantageous as it not only reduces the time frame for completeplantlet formation (which is advantageous for commercial growers), it also eases the task of trans-planting as a large number of plantlets can be removed from the culture vessel without an additionalstep of subculture for the remaining smaller plantlets (Vyas, 2010). In the present study, a largenumber of uniform plantlets with well developed root system and shoot growth can be transferredfor in vitro hardening and finally to the greenhouse conditions which will be beneficial for theconservation of this threatened orchid. Synchronous rapid plantlet regeneration, thus, have a majorimpact at a commercial level as an efficient time saving strategy.

Synchrony in secondary somatic embryogenesis was also achieved by Mondal et al., (2001)in Camellia sinensis by using nitrate salts of potassium (9.39 mM) and ammonium (10.3 mM)with potassium sulphate (1.5 mM) and also on MS medium containing the above mentioned saltswith BAP (8.88 μM), IBA (0.98 μM) and glutamine (10 mM). Our report differs from previousstudies as in this case a natural additive (BE) has been used as a supplement to KC medium forthe study instead of synthetic plant growth regulators. Banana grows in tropical areas which formthe native habitat of this orchid, reduces the input cost considerably at a commercial scale (NayarGopalan, 1962; Vyas, 2010).

The plantlets growing in the medium enriched with minerals of the basal medium (here,KC), natural additives and sucrose depend mainly on the exogenous supply of nutrients for theirgrowth and development. In the present study, the plantlets of Dendrobium lituiflorum were sub-jected to in vitro hardening by using various support matrices (Luffa sponge and cocopeat: perlite)in culture vessels and on agar-agar medium without sucrose containing one-half strength of KCmajor salts. Sucrose was eliminated from the medium in the present study for two reasons, firstly,although it promotes growth, but it also depresses photosynthesis and reduces in vitro hardening(Deng and Donnelly, 1993) and secondly, to minimize contamination (see De Faria et al., 2004).Besides, the use of growth-regulating substances, vitamins and other organic substances can beminimized because some of these will be produced endogenously in sufficient quantities by theplantlets growing autotrophically (Kozai, 1991). This view is also supported by Deb and Imchen(2010) who reported the use of 1/10th liquid MS basal medium without sucrose for the in vitrohardening of three orchid species, Arachnis labrosa, Cleisostoma racemiferum and Malaxiskhasiana on support matrices such as charcoal pieces, brick chips, mosses and decayed wood.Therefore, in the present study, only minimal amount of nutrients were provided to the plantlets.

Senescence of leaves was observed in the cultures hardened in vitro on the two support ma-trices, viz., Luffa sponge and cocopeat: perlite and agar-agar gelled medium. Pic et al., (2002),while working on pea, speculated that acceleration of leaf senescence is an adaptation in plantssubjected to water shortage, firstly, as it reduces the water demand accumulated over the wholeplant cycle and secondly, as it allows recycling of scarce resources to the reproductive sinks. Mer-rien et al., (1981) emphasized that it is hastened by water or nitrogen deficits. In the present find-ings, since plantlets on the support matrices viz., cocopeat: perlite, Luffa sponge and agar-agarmedium (containing liquid KC medium without sucrose with one-half strength major salts) showedsenescing leaves, it was possible that both water and nitrogen deficits were causing it. However,the percentage of senescing leaves varied in the support matrices. If nitrogen deficit was presumedto be the major factor, the percentage senescence would have been nearly same in plantlets in allthe three treatments as same amount of KC salts were present under all the three conditions. There-fore, possibly water deficit was the main reason for the leaf senescence which was the highest inthe plantlets on cocopeat: perlite which was drier than agar-agar gelled medium and Luffa sponge


(all containing one-half strength KC major salts without sucrose). The above results clearly pointtowards the efficacy of Luffa sponge as support matrix for the in vitro hardening of Dendrobiumlituiflorum. The roots received ample space to grow and entangled the fibrous network of the Luffasponge which aided in plantlet growth. Gangopadhyay et al., (2004, 2005) successfully micro-propagated Philodendron and pineapple by using Luffa sponge as a matrix instead of agar-agar.Gangopadhyay et al., (2004) reported that for the in vitro propagation of Philodendron, Luffasponge was better than coir. The plantlets of Dendrobium lituiflorum exhibited successful survivalin the greenhouse conditions after six weeks of transfer.

CONCLUSIONS

For conservation of threatened orchids, synchronous plantlet formation is important as thesecan then be transferred to the greenhouse and ultimately to the field conditions. Secondly, syn-chronous plantlet formation is a big boost to the biotech industries where a large number of plantletsat a particular stage of development at a given time period are required. Besides, incorporation ofbanana extract, an inexpensive and readily available natural additive, can bring down the cost ofproduction considerably without compromising with the quality of plantlets produced. In vitrohardening by using inexpensive support matrices also serves as a cost effective measure aiding insuccessful acclimatization of these plantlets produced at a mass scale in vitro (Vyas, 2010).

ACKNOWLEDGEMENTS

The authors thank Department of Biotechnology for providing financial assistance to carryout this research work. S.V. is thankful to University Grants Commission, New Delhi, India, forthe award of Senior Research Fellowship.

Literature Cited

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Chowdhery, H.J. 2001. Orchid diversity in North-East India. J. Orchid Soc. India 15:1-17.Chu, C.C. and Mudge, K.W. 1994. Effects of pre-chilling and liquid suspension culture on seed

germination of the yellow Lady’s slipper orchid (Cypripedium calceolus var. pubescens). Lindleyana 9:153-159.

Deb, C.R. and Imchen, T. 2010. An efficient in vitro hardening technique of tissue culture raised plants. Biotechnology 9:79-83.

De Faria, R., Rodrigues, F.N., Oliviera, L.D.V.R. and Müller, C. 2004. In vitro Dendrobium nobile plant growth and rooting in different sucrose concentrations. Hort. Brasileira 22:780-783.

Deng, R. and Donnelly, D.J. 1993. In vitro hardening of new raspberry by CO2 enrichment and reduced medium sucrose concentration. Hort. Sci. 281: 1048-1051.

Gangopadhyay, G., Bandyopadhyay, T., Basu Gangopadhyay, S. and Mukherjee, K.K. 2004. Luffasponge - a unique matrix for tissue culture of Philodendron. Curr. Sci. 86: 315-319.

Gangopadhyay, G., Bandyopadhyay, T., Poddar, R., Basu Gangopadhyay, S. and Mukherjee, K.K. 2005. Encapsulation of pineapple microshoots in alginate beads of temporary storage. Curr. Sci. 88: 972-977.

Hazarika, B.N. 2003. Acclimatization of tissue-cultured plants. Curr. Sci. 85:1704-1712.Hazarika, B.N. 2006. Morpho-physiological disorders in in vitro culture of plants. Scientia Hort.

108:105-120.Hew, C.S. 1994. Orchid cut-flower production in ASEAN countries. p. 363-401. In: J. Arditti (ed.), Orchid Biology: Reviews and Perspectives. Vol. 6. John Wiley & Sons Inc., New York, USA.Khalifah, R.A. 1966a. Gibberellin-like substances from the developing banana fruit. Z. Pflanzenphysiol.

76:280–283.


Khalifah, R.A. 1966b. Indolyl-3-acetic acid from the developing banana. Nature 212:1471–1472.Knudson, L. 1946. A new nutrient solution for germination of orchid seed. Am. Orchid Soc. Bull.

15:214–217.Kozai, T. 1991. Photoautotrophic micropropagation. In vitro Cell. Devel. Biol. Plant 27:47-51.Merrien, A., Blanchet, R. and Gelfi, N. 1981. Relationships between water supply, leaf area development

and survival, and production in sunflower (Helianthus annuus L.). Agronomie 1:917-922.Mondal, T.K., Bhattacharya, A. and Ahuja, P.S. 2001. Induction of synchronous secondary somatic

embryogenesis in Camellia sinensis (L.) O. Kuntze. J. Plant Physiol. 158:945-951.Nayar Gopalan, T. 1962. Bananas in India. The fact technical society, Udyogmandal.Pic, E., Teyssendier de la Serve, B., Tardieu, F. and Turc, O. 2002. Leaf senescence induced by mild

water deficit follows the same sequence of macroscopic, biochemical, and molecular eventsas monocarpic senescence in pea. Plant Physiol. 128:236-246.

Pierik, R.L.M., Sprenkels, P.A., Van der Harst, B. and Van der Mays, Q.G. 1988. Seed germinationand further development of plantlets of Paphiopedilum ciliolare Pfitz. In vitro. Sci. Hort. 34:139-153.


Figures

Fig. 1A

Fig. 1B

Fig. 1C

Fig. 1. Effect of BE on growth and fresh and dry weights of plantlets of Dendrobium lituiflorum.

A. The plantlets growing on 20% (v/v) BE supplemented KC medium show maximum growth in com-parison to control after 30 d of third subculture.B. The fresh and dry weights of plantlets on 20% (v/v) BE supplemented KC medium are significantlyhigher than those on control.C. The cultures on KC medium supplemented with 20% (v/v) BE show maximum growth in comparisonto control and other treatments after 30 d of fourth subculture. (X_

= 12 replicates, *, L.S.D., p<0.05)


Fig. 2. Effect of BE on growth of plantlets on KC medium after 30 d of fourth subculture.A. The cultures growing on 20% (v/v) BE supplemented KC medium (lower row) show significant shoot androot growth in comparison to control (upper row).B. Synchronous mass scale production of culture on KC medium with 20% (v/v) BE.C&D. Synchronously developed plantlets on KC medium supplemented with 20% (v/v) BE taken out of culturebottles to show shoot and root growth.

Fig. 3. In vitro hardening and acclimatization of plantlets of Dendrobium lituiflorum.A. Plantlets on plain agar-agar medium showing a few chlorotic leaves.B. Plantlets on Luffa sponge showing well expanded leaves growing prolifically.C. Plantlets on cocopeat: perlite (9:1) showing senesced leaves.D. Prolifically growing plantlets after in vitro hardening immediately before transfer tothe greenhouse conditions.E. Plantlets growing vigorously in the greenhouse on potting mix [cocopeat : perlite (9:1)]after six weeks of transfer.F. Plantlets taken out of potting mix showing well expanded intense green leaves andlight green long roots with velamen.


Fig. 4A

Fig. 4A

Fig. 4. Effect of various support matrices on in vitro hardening of plantlets of Dendrobium lituiflorum on liquid KC medium(one-half strength major salts only) after 30 d.

A. The number of shoots and roots on zero day of inoculation on various support matrices viz., Luffa sponge, cocopeat:perlite (9:1) and agar-agar medium.B. Among the three matrices, maximum increase in the number of shoots and roots is observed on Luffa sponge as supportmatrix (cf A). After that the maximum number of shoots are on cocopeat: perlite (9:1) and roots on agar-agar medium, re-spectively. C. The shoot length is higher in cultures on Luffa sponge followed by cocopeat: perlite and agar-agar medium. D. The length of both short and long roots is maximum on Luffa sponge. E. The percentage of senescing leaves is maximum in plantlets on cocopeat: perlite followed by agar-agar and minimumin plantlets on Luffa sponge.

Fig. 4B Fig. 4C

Fig. 4D Fig. 4E


Study on the Effect of Different Growing Media on the

Growth and Yield of Gerbera (Gerbera jamesonii L.)

To study the effect of different substrates on growth and yield ofgerbera, this experiment was carried out as randomized completely blockdesign with 14 treatments and 3 replications. Treatments were as fallowing:fine sand, peat + fine sand (25%+ 75%), peat + fine sand (50% + 50%),perlite + peat (75% + 25%), perlite + peat (50% + 50%), perlite + peat (25%+ 75%), perlite + peat + expanded clay (25% + 70% + 5%), perlite + peat +expanded clay (50 %+ 25% + 25%), perlite + peat + expanded clay(25%+ 50% + 25%), perlite + expanded clay (50%+ 50%), cocopeat, cocopeat +perlite (75% + 25%), cocopeat + perlite (50% + 50%), coco peat + perlite +expanded clay (50% + 25% + 25%), plants were fertilized with a samenutrient solution. Results showed that the growing medium containing perlite +peat + expanded clay (25% + 70% + 5%) was the best treatment. In thissubstrate, flower number, flower diameter, shoot diameter, stem neck diameter,flower height and vase life showed significant difference among growing media.

Keywords: Gerbera, Expanded Clay, Growth, Peat, Perlite, Yield.

Abstract

M. A. Khalaj1, M. Amiri 2 and S.S. Sindhu3

1 Scientific Board Member of the National Ornamental Plant Research Station (Mahallat), Iran.2 M.Sc. Student, Plant Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran. 3 Division of Floriculture and Landscaping, Indian Agricultural Research Institute, New Delhi-110012.



INTRODUCTION

Gerbera (Gerbera jamesonii L.) is one of the herbaceous plants with colorful and beautifulflowers that are used as cut, pot and garden flower. Various planting beds around the world is usedfor growing gerbera such as perlite, rock wool, vermiculite, sand, coconut fiber (cocopeat), ex-panded clay, organic substrates, compost cow, zeolite, pumice, sand etc. that reported by Khalaj,2007 and Fakhri, et al., 1995.

Soil-less cultures have been successfully used for several decades with the aim to intensifyproduction and reduce cost (Maloupa et al., 1993). Peat is the most widely used substrate for pottedplant production in the nurseries and accounts for a significant portion of the materials used togrow potted plants (Marfa` et al., 2002; Ribeiro et al., 2007). Since the last few years, cocopeat,also known as coir dust or coconut mesocarp has been considered as a renewable sphagnum peatsubstitute for the use in horticulture (Yau and Murphy, 2000; Pickering, 1997).

Perlite has been widely used in soil-less cultures too. Perlite, an aluminosilicate of volcanicorigin, is rather inert (low buffering and cation exchange capacities of 0–1 mg/L). In general, ithas a closed cellular structure, with the majority of water being retained superficially and releasedslowly at a relatively low tension, providing excellent drainage of the medium and aeration of rhi-zosphere (Maloupa et al., 1993). The objective of this study was to determine the effect of differentsubstrates on growth and yield of gerbera under an open soil-less production system.


This experiment was carried out as Randomized Completely Block Design (RCBD) with14 treatments and 3 replications for study on the effect of different substrate on growth and yieldof gerbera (Gerbera jamesonii L.) over a period of 6 months along with their physical and chemicalproperties (Verdonck and Gabriels, 1992) are mentioned at table 1.

Plants were fertilized with a same nutrient solution. In this experiment, sand, perlite andexpanded clay were used with 0.5-1, 1-2 and 3-5 mm in diameter range respectively. The green-house temperature and relative humidity were 18-28 °C and 50-70% and also the amount of lightwas 23000-25000 (Lumen/m2). Gerbera transplanted in 4 liters size pots. They were irrigated 3-4 times per day.

Electrical conductivity and pH of nutrient solution was 5.5-6.5 and 1.5-2 ds/m respectively.In a period of six months, some quality and quantity characteristics of flowers were measured suchas flower number, flower stem height, flower disc diameter, stem diameter, stem neck diameterand vase life. Standard procedures were followed to collect the data for growth and flowering pa-rameters. The statistical analysis of the treatments was tested using analysis of variance and meanswere compared by Duncan's Multiple Range Test (Steel et al., 1996).


The selecting media is based on many factors such as availability, high quality and lowprice for producers. The different types of media can be used as peat and recently cocopeat (coconutfiber), rock wool, vermiculite, perlite, expanded clay, pumice and sand. In this experiment, basedon various sources of external and internal reviews, common media used in various gerbera cultureswere evaluated (Sindahu et al., 2010; Khalaj, 2007; Venezia et al., 1997; Mascecarini, 1998; Pisanuet al., 1994).

There are significance differences (P≤1%) between the flower numbers of treatments (table2). The results of analysis (table 3) showed that 7th treatment, which includes a mixture of perlite+ peat + expanded clay (25% + 70% + 5%), produced maximum flower numbers against otherswith 31 numbers and sand bed alone produced 11.3 flowers that have lowest production. The flowernumbers of gerbera in 7th treatment could be the results of faster plant development due to goodroot system and better physicochemical properties of mixes. Growth medium is known to have a


large effect on value of potted ornamental plants (Vendrame et al., 2005). Cation exchange ca-pacity (CEC) of Bed No.7 is 80 Cmol charge (table 1). According to different researches, or-ganic materials and high cation exchange capacity (CEC) increase the absorption and storageof nutrient, water and also by creating of suitable conditions for plant root growth, can increasequalitative and quantitative characteristics of flowers. If peat has been used alone, it because ofpressing and decreasing ventilation and so sand or perlite due to little or no good propertieswould not be useful (Khalaj, 2007).

Among the physical characteristics, aeration and water holding capacity are probably themost important factors while, among the chemical characteristics, nutritional status, and salinitylevel have a crucial role on plant development (Dewayne et al., 2003). Nowak and Strojny (2004)reported that the total porosity, bulk density, shrinkage water capacity and air capacity of the grow-ing substrates had significant effects on the number and weight of fresh flowers in gerbera. Datashowed that flower disc diameter influenced significantly (P≤1%) by the different media (table 2)and the largest flower diameter, 11.6 cm in 7th treatment and the lowest flower diameter 10.9 cmfrom 1st (sand alone) is derived (table 3). In Fakhri et al., (1995) experimental design reportedthat the largest flower diameter obtained from mixes of peat and perlite. They have been notedthat media physicochemical characteristics improving because of the organic matter existence wasthe main reason of differences.

Results showed significant difference (P≤5%) in the stem and stem neck diameter (table2).Significantly greatest mean stem and stem neck diameter were produced in medium 7 with 0.79and 0.58 cm respectively (table 3). Similar results were reported by Aswath and Padmanabha,2004. There was significant difference (P≤5%) in the flower height (table 2), significantly greatermean flower height were produced in medium 7 with 54.5 cm, the highest of growing media (table3). Greater flower height and more yields produced by plants grown in medium 7 suggest that thistreatment is best suited for growing gerbera flower in among these media. Medium 7, by 0.39 ds/m has the least salinity than other media, so good root medium has provided for nutrient absorptionand growth for plants.

Survey such as that conducted by Papadopoulos (1996) has shown that mixture of per-lite and peat with equal volume produced the maximum flower height with 69 cm. Aswath andPadmanabha (2004) reported that in gerbera electrical conductivity in medium had significant in-fluence on stalk length, stalk thickness and flower diameter. Ozcelik et al., (1997) studied duringthe 1994-95 years on the effects of different planting media as the alone or the combination onquality and quantity of gerbera, they observed that the most appropriate mixture for gerbera yieldin 15-month period. A strong relationship between substrate physicochemical properties and ger-bera quantity and quality characteristics has been reported in this survey.

Data showed that significant differences (P<5%) in the gerbera vase life grown on mediawith varying substrate (table 2). In medium 7, has the longest gerbera vase life as 13.6 days (table3). The vase life is directly related to dry matter production as well as size of flowers. This findingis in agreement with Manins et al., (1995) findings which showed significant differences betweendifferent substrates on gerbera vase life. De Jong (1978) found that gerbera flowers with strongstem were less likely to fold in the vase due to turgor pressure maintained. As the vegetative growthwas found to be better in coco peat combinations, the flower set was early, producing high qualitycut flowers.

The recent study confirms the fact that selection of the appropriate medium of growth forcut flowers (in this case gerbera jamesonii L.) was very important from yield and quality point ofview. The medium must ensure the production of plants of the required quality on cost effectivebasis. In the present study, perlite + peat + expanded clay mix (%25 + %70 + %5) produced sig-nificantly the maximum number of flowers per plant and other quality characteristics among dif-ferent media.


Literature cited

Aswath, C. and Padmanabha P. 2004. Effect of cocopeat medium and electrical conductivity on production of gerbera. J. of Orna. Hort. 7(1):15-22.

De Jong, J. 1978. Dry storage and subsequent recovery of cut gerbera flowers as an aid in selectionfor longevity. Sci. Hort. 9: 389-397.

Dewayne, L.I., Richard, W.H. and Thomas, H.Y. 2003. Growth media for container grown ornamental plants. The Environmental Horticulture Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida, BUL241.

Fakhri, M., Maloupa, E. and Gerasopoulos, D. 1995. Effect of substrate and frequency of irrigation in yield and quality of three Gerbera jamesonii cultivars, Acta Hort. (ISHS), 408: 41-45.

Khalaj, M. 2007. Gerbera cultivation guide. National Research Station of Flowers and Ornamental Plants Publications, Bulletin No. 86.394. Markazi, Iran.

Maloupa, E. I., Mitsios, P., Martinez, F., and Bladenopoulou, S.1993. Study of substrates used in Gerbera culture in plastic greenhouse, Acta Hort. (ISHS), 323: 139-144.

Manins, V.I., Papadimitriou, M.D. and Kefakis, M.D. 1995. Hydroponics culture of tomato andgerbera in different substrates. Acta Hort. (ISHS), 408: 11-16.

Marfa`, O., Lemaire, F., Ca´ceres, R., Giuffrida, F. and Gue´rin, V. 2002. Relationships between growing media fertility percolate composition and fertigation strategy in peat-substitute substrate used for growing ornamental shrubs. Sci. Hort. 94, 309–321.

Mascecarini, L. 1998. Gerbera cultivation in growing media. Horticulture International. 6:19, 86-88. Nowak, J. S. and Strojny, Z. 2004. The effect of physical properties of organic growing medium

on cut flower yield of gerbera. Folia Universitatis Agriculturae Stetinensis, Agricultura. 94: 133-138.

Özçelik, A., Besroglu, A., Özaltin, A.S. and Özgümüs, A.1997. The use of different media for greenhouse gerbera cut flower production, Acta Hort. (ISHS), 491:425-432.

Papadopoulos , E., Gerasopoulos, D. and Maloupa, E. 1996. Effect of substrate and frequency of irrigation on growth, yield and quality of Gerbera jamesonii Bolus cultivated in pots. Agriculturamediterranea. 126:3, 297-302.

Pickering, J.S. 1997. An alternative to peat. The Garden 122: 428-429.Pisanu, B., Carletti, M. and Leoni, S. 1994. Gerbera jamesonii cultivation with different inert

substrates. Acta Hort. (ISHS), 361: 590-602. Ribeiro, H.M., Romero, A.M., Pereira, H., Borges, P., Cabral, F. and Vaconcelos, E. 2007. Evaluation

of a compost obtained from forestry wastes and solid phase of pig slurry as a substrate for seedlings production. Bioresour. Techno. 98, 3294–3297.

Sindhu S.S., Gholap D.B., Singh, M.C. and Dhiman, M.R. 2010. Effect of medium amendments on growth and flowering in gerbera. Indian J. Hort. 67(Special Issue) : 391-394.

Steel, R.G., Dickey, D.A. and Torrie, J.H., 1996. Principles and procedures of statistics: A biometrical approach. McGraw-Hill College. pp. 672.

Vendrame, A.W., Maguire, I. and Moore, K.K. 2005. Growth of selected bedding plants as affected by different compost percentages. Proc. Fla. State Hort. Soc. 118: 368-371.

Venezia, A., Martignon, G., Schiavi, M. and Cassarotti, D. 1997. Soil-less culture of gerbera, open and closed systems. Gerbera fuori suolo: sistema aperto e chiuso. Culture Protette. 26(9): pp, 129-135.

Verdonck, O. and Gabriels, R.1992. Reference method for the determination of physical properties of plant substrates. Acta Hort. (ISHS), 302: 169-179.

Yau, P.Y. and Murphy, R.J. 2000. Biodegraded cocopeat as a horticultural substrate. Acta Hort. (ISHS), 517: 275-278.


Tables

substrates Porosity (%) CEC (Cmol(+)/ kg) EC (dS/m) pH (1:2)

1) Fine sand

2) Peat + Fine Sand (25%+75%)

3) Peat + Fine Sand (50% +50%)

4) Perlite + Peat (75% + 25% )

5) Perlite + Peat (50% + 50% )

6) Perlite + Peat (25% + 75%)

7) Perlite + Peat + E.C. (25% + 70% + 5% )

8) Perlite + Peat + E.C. (50 %+ 25%+ 25% )

9) Perlite + Peat + E.C. (25%+ 50% + 25% )

10) Perlite + E.C. (50% + 50% ),

11) Coco peat

12)Coco peat + Perlite (75 %+ 25% )

13) Coco peat + Perlite (50% + 50%)

14) Coco peat + Perlite + E.C. (50% +25%+25%)

40

41.1

42.7

73.7

79.4

86.3

80.7

62.7

66.2

59

90

84.1

78.6

66.3

0.75

3.5

7.7

26.5

57.2

94.9

80.3

22.4

43.5

35.3

75

54

34.5

27.6

1.04

1.02

0.99

0.84

0.65

0.41

0.34

0.49

0.39

0.18

0.5

0.64

0.77

0.45

6.91

6.87

6.82

6.54

6.15

6.65

6.17

7.75

6.51

8.29

5.29

5.75

6.17

7.48

Table 1. Physical and chemical properties of substrates used in this experiment

(Treatments mix by (v/v) of substrates), E.C.: Expanded Clay

ns, * and ** indicate no significant difference, significant at 5% and 1%, respectively

S.O.V. df Flower

number

Flower

diameter

Stem

diameter

Stem neck

diameter

Flower

heightVase life

ReplicationSubstratesError

CV

21326

101.3 ns1211.2 **

489.3

20.7

0.32 ns5.47 **

2.27

2.6

0.001 ns0.004 *0.001

5.25

0.001 ns0.001 *0.001

4.93

30.24 ns369.2**123.05

4.29

0.104 ns2.23*0.823

8.22

Table 2. Analysis of variance of gerbera quality and quantity characteristics

Table 3. Effect of different substrates on the yield and growth of gerbera

Flower number Flower disc.

diameter (cm)

Stem diameter

(cm)

Stem neck

diameter (cm)

Flower height

(cm)Vase life (days)

T1T2T3T4T5T6T7T8T9T10T11T12T13T14

11.3311.6717.0023.6722.3323.3331.0023.6727.6723.6716.6720.0018.0023.33

ddcdabcbcabcaabcababccdbccdabc

10.8811.6311.0711.0211.1311.4712.3511.0211.1811.1811.1211.2811.2510.94

dbbcdcdbcdbcacdbcdbcdbcdbcdbcdcd

0.660.690.660.650.640.670.790.680.680.690.690.690.690.70

bbbbbbabbbbbbb

0.490.520.520.490.510.500.580.490.510.510.510.510.500.49

bbbbbbabbbbbbb

48.451.350.444.951.653.054.548.448.246.051.054.354.253.2

bcababcabaabcbccabaaa

10.611.410.711.610.810.313.611.311.111.310.610.710.310.1

bbbbbbabbbbbbb

Treatment

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