journal of plant science plantica2)-2017.pdf · of paper and synthesis of pharmaceuticals and...

ISSN: 2456 – 9259

PLANTICA: 1(2), October, 2017

PLANTICA

Journal of Plant Science

The Official Publication of

Association of Plant Science Researchers

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AN

TIC

A

PLANTICA Journal of Plant Science

ISSN: 2456 – 9259

Vol. – 1, No. – 2 October, 2017

(A Quarterly Journal)

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Official Publication of

Association of Plant Science Researchers Dehradun, Uttarakhand, India

_________________________________________________________________________________________ PLANTICA - Journal of Plant Science, Volume 1 (2) October, 2017 The Official Publication of Association of Plant Science Researchers - APSR ____________________________________________________________________________________________________________________________________________

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__________________________________________________________________________________________ PLANTICA - Journal of Plant Science, Volume 1 (2) October, 2017 The Official Publication of Association of Plant Science Researchers - APSR ____________________________________________________________________________________________________________________________________________

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__________________________________________________________________________________________ PLANTICA - Journal of Plant Science Volume 1 (2),October, 2017 ____________________________________________________________________________________________________________________________________________

Article Index

Research Article: 1. A REVIEW ON SCREENING OF EXTRACELLULAR HYDROLYTIC ENZYME PRODUCING MICROORGANISMS Arun Kumar Sharma, Vaishali Srivastava and Vinay Sharma* Department of Bioscience and Biotechnology, Banasthali University, Rajasthan, India *Corresponding Author: [email protected] 2. EFFECT OF MICRONUTRIENTS ON DIFFERENT YIELD PARAMETERS OF SUGARCANE Harsh Vardhan Chauhan*, Rekha Balodi and R.K. Sahu Centre of Advance Faculty Training in Plant Pathology G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand – 263145, India *Corresponding author: [email protected] 3. EFFECT OF NITROUS ACID TREATMENT ON LIPASE PRODUCTION BY LOCAL SOIL FUNGAL ISOLATE Arun Kumar Sharma, Sapna Kumari and Vinay Sharma* Department of Bioscience and Biotechnology, Banasthali University, Rajasthan, India *Corresponding Author: [email protected] 4. EFFICACY OF ORGANIC FERTILIZER- AMIRTHAKARAISAL ON THE GROWTH OF CHILLI PLANT Anjali, Tanveer Hassan, Muralitharan R, Murugalatha N.*, Rinkey Arya,Vijay Kumar and Naveen Chandra Department of Agriculture, Quantum School of Graduate Studies,Quantum Global Campus, Roorkee, Uttarakhand – 247667, India *Corresponding author –[email protected] 5. PARTIAL PURIFICATION OF CUNNINGHAMELLA LIPASE Arun Kumar Sharma, Sapna Kumari and Vinay Sharma* Department of Bioscience and Biotechnology, Banasthali University, Rajasthan, India *Corresponding Author: [email protected] 6. RESPONSE OF PACLOBUTRAZOL AS SPRAY & DRENCH ON POT GROWN FUCHSIA CV. ‘PINK GALORE’ Yachna Gupta and Pooja Kaintura* Department of Floriculture and Landscaping, Dr. Y. S. Parmar University of Horticulture & Forestry, Solan (H.P.), India *Corresponding author:[email protected] 7. ISOLATION AND CHARACTERIZATION OF DESIRABLE COLOUR MUTANT OF CARNATION Pooja Kaintura Department of Horticulture, VCSG Uttarakhand University of Horticulture and Forestry, Bharsar , Uttarakhand, 263145 *Corresponding author:[email protected] 8. EFFECTS OF SALINITY STRESS CONDITION IN SEED GERMINATION AND VIGOUR OF Aegle marmelos Rakesh Singh, V.P Nautiyal, J.S. Chauhan and Ganga Dutt Department of Seed Science and Technology, Hemwati Nandan Bahuguna Garhwal University (Central University), Srinagar, Uttarakhand, India, Corresponding author:[email protected]









PLANTICA – Journal of Plant Science ISSN: 2456 - 9259 (The Official Publication of Association of Plant Science Researchers) Plantica, Vol. 1 (2), 2017: 37 – 48

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Sharma et. al. | A REVIEW ON SCREENING OF EXTRACELLULAR HYDROLYTIC ENZYME 37

Review Article

A REVIEW ON SCREENING OF EXTRACELLULAR HYDROLYTIC ENZYME

PRODUCING MICROORGANISMS

Arun Kumar Sharma, Vaishali Srivastava and Vinay Sharma*

Department of Bioscience and Biotechnology, Banasthali University, Rajasthan, India

*Corresponding Author: [email protected]

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Abstract In past few years the novel potential of utilizing microbes as biotechnological sources of

enzymes of industrially important has encouraged improved attention in the investigation of

extracellular enzymatic activity in numerous microbes. Commercial enzymes are generally

extracted from microorganisms because of their biochemical and physiological properties,

easy cultivation at large scale and easy genetic manipulation for improved productivity.

Starch degrading enzymes such as amylase have received huge consideration due to their

noticeable technological importance and economic advantages. Celluloses are observed as the

most important renewable resource for bioconversion and cellulose is usually hydrolyzed

cellulase enzyme. Proteases are the imperative industrial enzymes and cover about 25% of

global commercial enzymes. Lipases are utilized in the modification of oils and fats,

detergents, food processing, synthesis of pharmaceuticals and fine chemicals, manufacturing

of paper and synthesis of pharmaceuticals and cosmetics, brewing, bakery, biofuels and

processing of leather. These extracellular enzymes are manufactured by numerous

microorganisms, usually by bacteria and fungi. Therefore present review deals with the

screening of hydrolytic enzyme producers.

Key words: Amylase, cellulase, extracellular enzymes, lipase, microorganisms, protease.

Introduction:

ceans act as a seed bank hosting a

rare “biosphere” of thousands of

distinct microbes which turn into

active in response to seasonal or ecological

alterations. Proteases are produced and

secreted by all creatures. Proteases are also

known as proteinases and protein

degrading enzymes are universal proteins

that stimulate the cleavage of peptide

bonds present in other proteins and are

necessary for cellular growth and

differentiation (Rao et al. 1998). Proteases

are considered imperative category of

industrial enzymes occupying a main share

of about 60% of the total global enzyme

market (Gupta et al. 2005). In nature,

proteases are produced and secreted by

animal, plant and microorganisms but

microbes are the favorite source for getting

proteases because of their faster growth

rate, easy genetic manipulation for

enhanced productivity of stable enzyme

and ability of protease production in very

small time period and easy purification

(North, 1982; Rao et al. 1998).

Cellulase are considered an

important category of enzymes produced

mainly by bacteria, fungi and protozoans

that stimulate hydrolysis of cellulose into

monomers (glucose). The main industrial

applications of cellulases are in fabric

industry for ‘bio-polishing’ of clothes and

producing better look of garments as well

O



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as in domestic laundry detergents for

increasing textile softness and clearness.

Cellulase is utilized in the fermentation of

biomass into biofuels, fibre alteration and

they are also utilized for pharmaceutical

applications (Sadhu and Maity, 2013).

Cellulases are produced by numerous

microorganisms such as bacteria, yeast and

fungi. There is rising attention in cellulase

production by bacteria since bacteria

possess elevated growth rate than fungi

and has excellent possibility to be utilized

in cellulase production (Maki et al. 2011).

Lipases are a main category of

biocatalysts that stimulate cleavage of

ester bonds present in triacylglycerol into

fatty acids and glycerol. Lipases are

produced and secreted by microbes

(bacteria, fungi and yeast), plants and

animals but lipases purified from bacteria

and fungi are more cost-effective and

stable (Snellman et al. 2002). Bacterial

lipases are broadly utilized in food and

dairy industry, ripening of cheese, flavour

enrichment, detergent industry, fabric

industries, for production of compounds or

polymers (biodegradable in nature),

diverse transesterification reactions,

beauty industry, in paper and pulp

industry, in production of biodiesel and in

pharmaceutical applications (Jaeger et al.

1994; Benjamin and Pandey, 1996; Sagar

et al. 2013).

Amylase is synthesized and

secreted into saliva by human salivary

glands, where it starts the chemical

digestion of food. Amylases (alpha

amylase) are also synthesized by the

pancreas to cleave food starch into

disaccharides (maltose) which are further

acted by additional enzymes to simplest

sugar (glucose) to provide the body with

principal energy source. Plants and

numerous bacteria also manufacture and

secret amylase. Like diastase, amylase was

the first enzyme to be discovered and

separated by Anselme Payen in 1833 (Hill

and Needham, 1970). Amylases are

valuable enzymes which are mostly

engaged in the starch utilizing industries

for the conversion of polysaccharides

(starch) into simple sugar components

(Akpan et al. 1999). All amylases belong

to the class hydrolase and show biological

activity on α-1,4-glycosidic bonds.

Amylases cover approximately 30% of the

global enzyme market (Singhania et al.

2009). Considering the ever rising demand

of these hydrolytic enzymes we need to

understand the methods to isolate the

novel microorganisms producing and

secreting these enzymes. Therefore, the

present review is focused on different

screening methods used for isolation and

identification of enzyme producers.

Lipases

Lipase producing microbes have

been reported from a wide range of

environments such as industrial wastes,

compost heaps, oilseeds, deteriorated food

vegetable oils processing factories and

dairy products (Sharma et al. 2016).

Mucor, Penicillium, Aspergillus, Rhizopus

and Geotrichum have been established as

the most luxuriant sources of enzyme

lipase (Singh and Mukhopadhayay 2012;

Gopinath et al. 2013). Lipases have shown

their tremendous applications in industries

such as oleochemistry, organic synthesis,

detergent formulation, nutrition, dairy,

textiles, tea and paper (Ghosh et al. 1996).

Realizing the demand of lipases for

different industrial uses, which are

expected to further increase in the near

future, a massive screening programme

was initiated in the search of potential

extracellular lipase releasing fungi for

which samples were collected from a

variety of habitats and screened on

different media (Pandey et al. 2015).

Isolation of microorganisms from soil

sample


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Generally serial dilution agar plate

method is utilized for isolation of

microorganisms (bacteria and fungi). In

this technique, the soil sample is serially

diluted to obtain the 10-1

to 10-8

dilutions

and inoculum from each dilution is poured

on to nutrient agar plates and potato

dextrose agar plates for the isolation of

bacteria and fungi, respectively. Nutrient

agar medium (NAM) plates are incubated

at 37ºC and PDA plates are kept at 28 ºC

for 3 days and 6 days, respectively.

Colonies from NAM plates and PDA

plates are then maintained in their

respective media slants at 4ºC till their use

for screening (Sharma et al. 2015a & b:

Sharma et al. 2016a & b).

Screening of lipase producers

Lipases in today’s time utilized in

various industrial processes as well as

products and new areas are continuously

being added, which comprise the

manufacturing of single cell protein,

cosmetics pulping, lubricants etc. Lipid

degrading microorganisms have been

isolated from different areas such as

wastes released from vegetable oil

processing factories dairies, other

industries, oil contaminated soil and

decomposing food, compost piles,

petroleum tips and hot springs.

Oil contaminated soil samples were

taken by Veerapagu et al. (2013) from

diverse oil mills of Dharmapuri and

serially diluted upto 10-8

dilution followed

by plating on NAM plates. At the end of

incubation colonies of bacteria were

detected and out of total colonies, 200

colonies were isolated and screened for

lipolytic activity on tributyrin agar

medium (TBA).

Qualitative screening

The bacteria appeared on NAM

plates and fungi appeared on PDA plates

are screened for lipolytic activity in TBA

medium. Lipolytic activity is detected

directly by transformations noticed in the

appearance of substrate like tributyrin and

triolein. These substrates are added into

agar medium at 2% w/v concentration

during preparation. TBA medium is

prepared, poured on Petri plates, allowed

to solidify followed by incoculation of

bacterial or fungal colonies. Inoculated

plates are kept at respective temperature

for appropriate days. Production of

extracellular lipase in these plates is

indicated by appearance of zone of

hydrolysis around colonies. Clear zone

develops due to hydrolysis of tributyrin by

lipase enzyme, therefore opacity of

medium is not retained. Negative colonies

retains the opacity of the medium around

themselves (Cihangir and Sarikaya, 2004;

Griebeler et al. 2009; Xia et al. 2011).

Qualitative screening for Colorimetric

assay using copper soap method

Fatty acids released through

hydrolysis of substrate (olive oil) by lipase

can be estimated by a cupric

acetate/pyridine reagent. Fatty acids

complex along with Cu++

to form cupric

salts/ soaps that absorb light in the visible

range (λmax 715nm), provides a blue color.

Concentration of fatty acid librated by

lipase is estimated using a reference curve

of oleic acid. Olive oil is utilized as a

substrate. The reaction cocktail constitute

of 1ml of crude lipase, 2.5ml of olive oil is

kept for 5min. After that the enzyme

catalyzed reaction is terminated by the

addition of 1.0ml of 6N HCl and 5ml

benzene. The top layer 4ml is transferred

into a test tube and 1.0ml of cupric acetate

pyridine is added. The Free fatty acids

(FFA) solubilized in benzene are estimated

by measuring the absorption at 715nm.

Lipase activity is estimated by measuring

the quantity of FFA from the reference

curve of oleic acid. One unit of lipase

activity is defined as the quantity of

enzyme, required to release 1μmol FFA at

37 °C in 1min (Namboodiri and


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Chattopadhyaya, 2000: Veerapagu et al.

2013).

Olive oil PVA emulsion method

Lipase activity is determined

tritrimetrically using olive oil-Poly vinyl

alcohol (PVA) method according to

processes of Horiuti et al. (1976). PVA is

used for emulsification of olive oil in

water. One unit of lipase is defined as the

quantity, required to release 1 µM fatty

acid per minute at 30ºC at pH 7.

Spectrophotometric method

Pandey et al. (2015) described

production and estimation of P.

aurantiogriseum lipase activity. For

production of lipase, P. aurantiogriseum

was grown in modified Czapek-Dox broth

containing 1.5% olive oil. Cultivation of

fungi was done at 30ºC for 96 h in 250ml

shake flasks each having 50ml of Czapek-

Dox medium. Filtration of fungal culture

was done through 4 or 5 layers of muslin

cloth. The filtrate obtained was subjected

to centrifugation at 10,000 rpm at 4ºC for

10min to acquire supernatant, which was

considered as crude lipase. The

extracellular lipase activity in the

supernatant was detected by

spectrophotometric procedure using p-

nitrophenyl palmitate (p-NPP) (Winkler

and Stuckmann 1979). Freshly prepared

1.2ml of p-NPP solution was incubated in

a shaker water bath at 37ºC for 10min.

After 10min, 0.5ml of crude enzyme

sample was added and the reaction

cocktail was further kept at 37ºC in a water

bath for 30min. Formation of yellow color

due to release of p-nitrophenol was

indicative of lipase activity. To terminate

the reaction, 0.1ml of 100mM CaCl2.2H2O

was added to the solution. The absorbance

of yellow color was evaluated at 410nm

against a control (enzyme free). One

International Unit (IU) of enzyme was

defined as the quantity of enzyme, which

released 1 μM of p-nitrophenol ml-1

min-1

under standard assay conditions. Other

investigators have utilized

spectrophotometric assay using p-NPP as a

substrate (Joshi et al. 2006; Pera et al.

2006; Karanam and Medicherla, 2008).

Proteases

Proteases are important category of

hydrolytic enzymes and produced by

several microorganisms. Among all

proteolytic microbes, the Bacillus genus

assumes significance due to its potential

for production in huge quantities.

Furthermore numerous medium

components like carbon and nitrogen

sources, physiological factors for eg. pH,

incubation temperature and incubation

period and biological factors for eg. the

genetic nature of the producer organism

affects the biochemical behavior of the

microbial strain and subsequent pattern of

metabolite production (Swamy et al.

2014).

Proteases carry out diverse

activities in pharmaceutical, detergent,

laundry, food, leather, food processing etc.

These enzymes are broadly utilized in

dairy industry as milk coagulating agent

and meat tenderizing agent in food

industry, decrease of tissue inflammation

(medical) application (Swamy et al. 2014).

Proteases produced from marine water

bacteria are used for various

pharmaceutical applications for eg.

digestive drugs, anti-inflammatory drugs

and anti-tumour agents (Gonzalez and

Isaacs, 1999). The manufacture or

processing of enzymes is an imperative

fact of today’s pharmaceutical industries

and these investigations chiefly center on

the cytotoxic, haemolytic and

antimicrobial activity of protease enzyme

extracted from screened bacterial strains

(Vijayasurya et al. 2014). A proteolytic

enzyme exhibits the body’s chief

protection against tumour so these

enzymes are probably beneficial as

anticancer agents. A complete suppression


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of cancer is probable by the bacterial

protease. Proteases exhibit antitumor

activity if given to the cancer patient at the

site of tumor occurrence at very low dose

(Gupta et al. 2002). Fifteen bacterium

strains were isolated and characterized

from soil samples that were collected at

different regions of Ravulapalem, Andhra

Pradesh. One of which had the highest

proteolytic activity, was selected and its

proteolytic activity was further checked by

spectrophotometric analysis (Ramalakshmi

et al. 2012).

Qualitative screening for protease

producers

Skim milk agar plate, gelatin agar

plate and casein agar plate are used for

screening of extracellular protease

producers. The ingredients of casein agar

plate (g/L) are yeast extract 2.5, dry milk

50, glucose 1, pancreatic digest of casein 5

and agar 12.5. Skim milk agar medium

(g/L) contains skim milk powder 28,

dextrose 1, yeast extract 2.5, casein 5, agar

15, pH 7.0 (Sevinc and Demirkan, 2011)

whereas gelatin agar medium (g/L)

contains peptone 1, NaCl 5, gelatin 100

and agar 20 (Josephine et al. 2012).

Microbial isolates are inoculated in these

agar plates followed by incubation at

respective temperature for appropriate

time duration. Protease producers are

identified by looking zone of protein

hydrolysis around their colonies. In case of

gelatin agar medium, plates are flooded

with HgCl2 solution. Mercury chloride

reacts with undegraded gelatin to create

opacity making the clear zones easier to

observe (Abdel Galil 1992; Choudhary and

Jain, 2012; Mohanasrinivasan et al. 2012).

In case of negative results, no clearing is

observed around colonies.

Quantitative screening

Proteolytic isolates obtained from

primary screening are further confirmed

for their protein degrading capabilities in

submerged fermentation. These isolates

are allowed to grow in fermentation broth

containing inducer of protease expression

and protease activity from the enzyme

supernatant is determined using casein as

substrate as described earlier by Carrie

Cupp-Enyard (2008). Two test tubes are

marked as test (T) and blank (B). Five ml

of 0.65% casein solution is added in both

the tubes followed by incubation at 37˚C

for 5min. One ml of protease supernatant

is added in T-test tube, mixed correctly

and placed at 37˚C in a water bath for

30min for allowing the proteolytic reaction

to take place. At the end of incubation,

5ml of trichloroacetic acid (TCA) reagent

is added in both the tubes to stop the

catalytic reaction. One ml of protease

supernatant is added in blank tube to

match its final volume with T-tube. Then,

it is kept for 15min at room temperature.

Mixture from both tubes is filtered by

Whatmann’s No 1 filter paper. Two ml of

filtrate from test and blank tubes were

transferred in two new tubes and marked

as test (T) and blank (B). Five ml of

Na2CO3 is added in both tubes followed by

addition of 1ml of Follin’s reagent. The

resultant mixtures in both tubes are kept in

dark for 30min at room temperature for the

apparance of blue colored complex. The

absorbance of the blue colored complex is

determined at 660nm against a reagent

blank with tyrosine reference curve. One

protease unit is defined as the quantity of

enzyme that liberates 1 μM of tyrosine per

min at at 37 ºC, pH 7.5 (Mohapatra et al.

2003; Alnahdi, 2012; Sharma et al.

2015b).

Cellulases

Plant biomass contains cellulose as

the main ingredient. Cellulose contribute

for 50% of the total dry weight of plant

biomass and about 50% of the dry weight

of secondary sources of biomass for eg.

wastes from agricultural fields (Haruta et

al. 2003). Cellulose has pulled global


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consideration as a renewable resource that

can be transformed into biological

products and bioenergy. It has been

become the economic interest to develop

an effective method to hydrolyze the

cellulosic biomass. Cellulase is an

imperative and important category of

enzyme for hydrolysis of cellulosic

biomass into simple sugars. Cellulase is

utilized for depolymerization of cellulose

into fermentable sugar (glucose)

(sacharification) (Xing-hua et al. 2009).

Cellulolytic microbes can transform

cellulose polymer into soluble simple

sugars either by acid or enzymatic

hydrolysis. Hence, microbial cellulose

exploitation is accountable for one of the

major material flows in the biosphere

(Lynd et al. 2002). Despite a global and

massive exploitation of usual cellulosic

sources, there are still plentiful amount of

cellulosic sources, cellulose containing

unprocessed materials and waste products

that are not utilized or which could be

exploited more proficiently (Sonia et al.

2013). Celluloytic potential of certain

bacterial genera for eg. Cellulomonas

species, Pseudomonas species, Bacillus

species and Micrococcus species has been

reported. Quantity of product of cellulase

catalyzed reaction depend on a range of

factors such as size of inoculum, pH,

temperature, presence or absence of

inducers, medium additives, aeration,

agitation and incubation period

(Shanmugapriya et al. 2012). Cellulases

are being utilized in the fabric industry for

softening of cotton and denim finishing, in

laundry detergents for colour care,

cleaning, mash formation in the food

industry, drainage improvement and fibre

modification in the pulp and paper

industry and they are still utilized for

pharmaceutical applications (Cherry et al.

2003).

Qualitative screening for cellulase

producers

Nutrient agar medium containing

1% carboxymethylcellulose (CMC) is

utilized for screening of cellulase

producing bacteria. CMC agar plates are

prepared and inoculated with bacterial

culture followed by incubation at 37ºC for

2-3 days. The incubated CMC agar plates

are flooded with 1% congo red and kept

for 15min at room temperature. Then

plates are thoroughly counterstained with

1M NaCl. Appearance of clear zone

around bacterial colonies indicates

hydrolysis of cellulose by extracellular

cellulase enzyme. These positive bacterial

isolates are selected for further production

of cellulase in submerged fermentation

(Sethi et al. 2013; Patagundi et al. 2014).


Cellulolytic isolates obtained from

primary screening on CMC agar plate are

further confirmed for their cellulose

degrading potential in submerged

fermentation. Isolates are allowed to grow

in production media containing inducer of

cellulase expression and cellulase activity

from the enzyme supernatant is determined

by Dinitrosalisic acid (DNS) method

(Miller, 1959), in which reducing sugars

generated by the activity of cellulase on

CMC are estimated. Enzyme supernatant

is added to 0.5ml of 1% CMC in 0.05M

phosphate buffer and placed at 50ºC for

30min. At the end of incubation, the

reaction is terminated by the addition of

1.5ml of DNS reagent and heated at 100ºC

in water bath for 10min. Sugars released

by enzymatic reaction are estimated by

taking absorbance of coloured complex at

540nm. Production of cellulase is

determined using reference curve of

glucose (Shoham et al. 1999). One unit (U)

of enzyme activity is defined as the

quantity of enzyme, which is needed to

liberate 1 mol of glucose in 1min under

standard assay conditions (Muhammad et

al. 2012).


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Amylases

In the food industry starch

degrading enzymes have a broad spectrum

of applications, for eg. manufacturing of

high fructose corn syrups, glucose syrups,

maltose syrup, decrease in the viscosity of

sugar syrups, decrease of turbidity to form

clarified juice of fruits for increased shelf-

life, solubilisation (mixing of starch in

water) and saccharification (conversion of

starch into monomers) of starch in the

beverages industry. Amylases delay the

staling of bread and some other baked

products, therefore they are used in baking

industry. The paper industry utilizes

amylases for decreasing the viscosity of

starch to attain the proper covering of

paper. Amylases are utilized in the fabric

industry for warp sizing of cloth fibers as

well as utilized as a digestive supplement

in the medicine industry. Amylases

contribute approximately 30% of the

global enzyme production (Singhania et al.

2009). Amylase enzyme excreted by

Pseudomonas fluorescens bacteria is

utilized for removal of biofilm and

biodegradation of extracellular polymeric

substances (EPS) (Molobela et al. 2010).

Fungal species belonging to the genera

Aspergillus are most commonly utilized

for alpha amylase production. Solid-state

fermentation (SSF) is a cost effective

method of production of alpha amylase

using fungal species because low cost

substrate is used. Other microbes produce

and secret large quantity of alpha amylase

are Proteus sp., Pseudomonas sp.,

Escherichia sp., Serratia sp., Micrococcus

sp., Penicillium, Cephalosporium, Mucor,

Neurospora and Candida (Karnwal and

Nigam, 2013).

Qualitative screening for amylase

producers

For testing the secretion of amylase

enzyme by microorganisms, bacterial and

fungal isolates are inoculated in the

medium contacting (g/L): peptone 5; beef

extract 3; soluble starch 2 and agar 15

(Pillai et al. 2010). Inoculated plates are

incubated at respective temperature to

allow the growth of microorganisms and

production of extracellular alpha-amylase

in their vicinity. At the end of incubation

starch degrading isolates are recognized by

incubating the plates with Gram’s iodine

(1 g of iodine crystals and 2.0 g of KI are

solubilized in 100ml of distilled water).

Starch reacts with iodine to produce a deep

blue iodine-starch complex that covers the

whole agar. Amylolytic colonies are

surrounded by a clear zone, which

develops due to hydrolysis of starch by

alpha amylase. There is no clear zone

around the negative colonies, indicates that

they did not produce any extracellular

alpha amylase, hence starch in their

vicinity is not degraded and blue colour of

starch-iodine complex is maintained

(Alfred, 2007; Khokhar et al. 2011;

Alariya et al. 2013; Patel et al. 2014;

Sharma et al. 2015a).


Cultures of amylolytic isolates

selected from primary screening on starch

agar plate are further confirmed for their

starch degrading capabilities in submerged

fermentation. Isolates are allowed to grow

in production medium and amylase

activity from the enzyme supernatant is

estimated by estimation of reducing sugar

(maltose), which is released by the

catalytic activity of alpha amylase on

soluble starch. Liberated reducing sugars

are determined by dinitrosalicylic acid

(DNS) technique (Ogundero, 1979, 1982

a, b). Two test tubes are marked as test (T)

and a blank (B). One ml of 1% starch

reagent (prepared in 20 mM sodium

phosphate buffer, pH 6.9) is added in both

tubes followed by incubation at 37ºC for

5min. One ml of enzyme supernatant is

added in T-test tube and enzymatic

reaction is allowed to occur at 37ºC for

30min. One ml of coloring reagent


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(Dinitrosalicylic acid solution) is added in

both tubes to stop the reaction. One ml of

enzyme supernatant is added to the blank

tube to bring the final volume to 3ml,

which is equivalent to final volume of T-

test tube. Both test tubes are placed at

100ºC for 15min for the formation of

brown color. Test tubes are cooled and

9ml of distilled water is added in both the

tubes followed by measurement of

absorbance of brown colored complex at

540nm against the reagent blank using

reference curve of maltose (Pillai et al.

2010; Khan and Priya, 2011; Kumar et al.

2012; Saini et al. 2014).

Conclusion:

icrobial enzymes are more

beneficial than enzymes

obtained from plants or animals

due to the huge variety of catalytic

activities, large quantity of enzyme, easy

genetic modification for enhanced

productivity, constant supply because of

lack of seasonal variations and fast growth

of microbes on low-cost media. Search for

microorganisms capable of biodegradation

is one of the extensive areas of research.

Enzymes produced from microbes that can

tolerate extreme pH might be mainly

beneficial for industrial applications under

greatly alkaline situations. e.g. in the

manufacturing of detergents. These

hydrolytic enzymes also found promising

applications in various industries such as

food, pharmaceutical, cosmetic, oil,

chemical, medical diagnostics etc.

Qualitative and quantitative screening is

used for identification and isolation of

extracellular enzyme producers.

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Chauhan et. al. | EFFECT OF MICRONUTRIENTS ON DIFFERENT YIELD 49

Research Article

EFFECT OF MICRONUTRIENTS ON DIFFERENT YIELD PARAMETERS OF

SUGARCANE

Harsh Vardhan Chauhan*, Rekha Balodi and R.K. Sahu

Centre of Advance Faculty Training in Plant Pathology

G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand - 263145, India

*Corresponding author: [email protected]

______________________________________________________________________________

Abstract Micronutrients are those nutrient elements, which are required in very small quantities for plant

growth and development. The micronutrients essential for green plants are iron, copper, manganese,

zinc, boron, molybdenum and chloride. Most of the micronutrients are important constituents of

enzymes or co-enzymes produced by an organism for performing various physiological processes.

When the availability of these elements is very low, it produces characteristic deficiency symptoms

and the plant growth is affected. On the other hand, excess availability and uptake of these elements

may cause phytotoxic symptoms resulting in lower yields. Hence, it is always essential to maintain

their availability at optimum levels and in correct proportions for realizing the highest productivity.

Micronutrients play a very vital role for growth and development of sugarcane. Micronutrients

reduce the severity of disease. The results indicate that there was no disease incidence in Zinc

sulphate, Borax, Zinc sulphate+Borax, Zinc sulphate+Copper sulphate+Borax and

Borax+Elemental sulphur. Zn (zinc sulphate) + Cu (copper sulphate) + B (borax) not only

reduced the disease but also enhance the yield and growth parameters i.e. clumps, tillers, cane

girth, cane height, cane weight, nodes, internodes, brix, sucrops % juice purity, CCS

(commercial cane sugar), cane yield and CCS yield. A very high amount of biomass is produced

by sugarcane, which results in greater removal of micronutrients.

Key Words: Micronutrients, sugarcane and yield

Introduction:

n general, a severely nutrient stressed

plant is more vulnerable to disease than

the one at a nutritional optimum.

Specific nutrients are known to reduce

disease severity by increasing tolerance to

disease through compensation of pathogenic

damage, facilitating disease escape,

enhancing physiologic resistance of the

plant and reducing pathogen virulence

(Huber, 1981). Sugarcane is one of the

important cash crop next to cotton and

cultivated in most of the states of India

except hilly tracks. During 2012-13 the total

area coverage 5.06 mh with a production of

336.15 mt, productivity of 66.7 M t/ha,

sugar production is 24.8mt and percentage

of sugar recovery was 9.99 per cent (Anon,

2013 b). India is next only to Brazil in

production. In India, UP having maximum

area under sugarcane 2.21 mh, production

13.05 mt, average cane yield was 59 mt/ha,

whereas, the productivity was highest in

I




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Tamil Nadu 101 mt/ha (Anon, 2013 b).

While in Uttarakhand during 2012-13 area

under sugarcane was 1.10 mt with

production of 6.71mt and productivity 61.07

mt/ha (Anon, 2013 b) which was lower than

the national average 71.7 t/ha (Anon, 2013

c). Similar to yield, sugar recovery 8.89 per

cent and sugar production 3.37 lakh tonnes

both were lower in Uttarakhand when

compare with national average (Anon, 2013

b).

Sugarcane crop is infected by both

abiotic and biotic stress. In abiotic viz.

drought, waterlogging, salinity/alkalinity

and variations of temperatures, etc., whereas

in biotic different groups of organisms and

others associated which include fungi,

bacteria, phytoplasma, nematodes,

phanerogamic plants and viruses etc. Among

them red rot, wilt, smut, pokkah boeng,

ratoon stunting, wilt, mosaic, leaf spots,

YLD and grassy shoot are of great concern

and causing losses in yield every year in

varying quantiy (Singh et al., 1991). Pokkah

boeng disease of sugarcane is one of them

which were reported as minor foliar disease

caused by Fusarium moniliformae in early

1930s. It has been noticed that the diseased

plant become deficient of trace elements and

few major elements simultaneously. The pol

per cent and sucrose percentage in juice also

declined. Since this disease is spreading fast

in wider areas, it appears essential to study

the deterioration due to this disease in

sugarcane (Singh et al., 2006).

Pokkah boeng is an emerging minor

disease not only in central Uttar Pradesh but

also in the whole of the Southern and

Northern sugarcane growing zone of India

causing reduction in the yield of sugarcane.

Approximately 40.8 - 64.5% sugars can be

reduced from sugarcanes infected by

Fusarium moniliforme var. subglutinans,

depending upon the cultivars (Dohare, et al.,

2003). In India the incidence and severity of

Pokkah boeng disease has been reported

from major sugarcane growing states like

Uttar Pradesh, Uttarakhand, Maharashtra,

Karnatka, Andra Pradesh, Punjab, Haryana,

Rajsthan,Assam, Tamil Nadu and Bihar etc.

(Anon, 2013 a).

Keeping in view the importance of

sugarcane and its economic value and

visualizing the emergence of disease in

northern sugarcane growing areas

Materials and Methods:

reparation of field: Field preparations were begun in the

first week of April 2013. The soil

was well pulverized by one disc

ploughing followed by 3 to 4 harrowing

with disc harrow. The field was then

levelled by tractor drawn leveller and

applied irrigation for moisture.

Fertilizer schedule: The fertilizer were applied @ 150 kg N/ha,

60 kg P2O5/ha and 40 kg K2O/ha, in the

form of urea, di-ammonium phosphate

(DAP) respectively. Nitrogen was applied in

two split doses. Half of nitrogen was mixed

thoroughly with full dose of phosphorus and

potash and applied as basal dose just before

leveling of field whereas the second half of

nitrogen was applied as top dressing along

the rows at the time of tillering prior to onset

of monsoon.

Planting: The planting was done on 5 April, 2013 at

optimum soil moisture for germination

following the standard method of planting of

sugarcane at a spacing of 75 cm. Sugarcane

variety Co-1148 has been taken as planting

material. Seed pieces have been made by

cutting them into three bud sett from the

upper 2/3 portion of cane. The

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micronutrients were applied at the rate of 25

kg/ha Zn (zinc sulphate), 25 kg/ha Mn

(mangnese sulphate), 5 kg/ha Cu (copper

sulphate), 40 kg/ha S (elemental sulphur), 10

kg/ha B (borax), 750 g/ha Mo (ammonium

molybdate) , 25 kg/ha Zn (zinc sulphate) +

25 kg/ha Mn (mangnese sulphate), 25 kg/ha

Zn (zinc sulphate) + 5 kg/ha Cu (copper

sulphate), 25 kg/ha Zn (zinc sulphate) + 40

kg/ha S (elemental sulphur), 25 kg/ha Zn

(zinc sulphate) + 10 kg/ha B (borax), 25

kg/ha Zn (zinc sulphate) + 750 g/ha Mo

(ammonium molybdate), 25 kg/ha Zn (zinc

sulphate) + 5 kg/ha Cu (copper sulphate) +

10 kg/ha B (borax), 10 kg/ha B (borax) +25

kg/ha Mn (mangnese sulphate, 10 kg/ha B

(borax) + 5 kg/ha Cu (copper Sulphate), 10

kg/ha B (borax) + 40 kg/ha S (elemental

sulphur , 10 kg/ha B (borax) + 750 g/ha Mo

(ammonium molybdate) as soil application

in the furrows at the time of planting and

Ferrous Sulphate was applied as foliar spray

at the rate of 0.1 % at the time of tillering

in the respective treatment plots are advised

by the expert of Department of Soil

Science. Then the setts were sown at a depth

of 6 to 8 cm in furrow and after planting the

setts, furrow were covered with the soil and

the experimental area was rolled later.

Yield parameters:

Clumps: Group of side shoots growing from

the base at ground level.

Tiller(s): Side shoot growing from the base

of the stem (at a ground level).

Cane girth: Cane girth is measured by

Berniar calipers

Cane height: Cane height is measured by

graduated tape

Cane weight: Cane weight is taken by using

simple balance

Nodes: The stalk is broken up in segments

called joints or nodes.

Internodes: The area between two nodes is

called internode.

Brix: The percentage of total solids in

sugarcane juice, read from Brix hydrometer.

Sucrose per cent: Sucrose per cent is

recorded by Polarimeter

Juice Purity %: Sucrose /Brix × 100

CCS (Commercial Cane Sugar): The

convertible sucrose or cane sugar content of

sugarcane.

CCS % (Commercial Cane Sugar %): 1.022

× Sucrose – 0.292 × Brix, where, 1.022 and

0.292 are Constant

Cane Yield: Number of milliable cane ×

Cane weight (kg)

CCS Yield: Cane Yield × CCS %

Note: - Cane yield and CCS yield is

converted in tonnes per hectare by using

following formula:

kg/plotin yield Cane areaPlot

10 tons/hayield Cane

kg/plotin yield CCS areaPlot

10 tons/hayield CCS

Statistical analysis:

The data were analysed statically at the

Computer Centre of G.B. Pant University of

Agriculture and Technology, Pantnagar,

using Randomized Block Design (RBD).

The treatments were compared by the means

of critical differences (CD) at 5% level of

significance.


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Results and Discussion:

s in the case of other crops, sugarcane

also requires all the micronutrients for

optimum growth and yield. These

elements are equally important to achieve

quality canes. Sugarcane produces very high

amount of biomass, which resulted in greater

removal of the micronutrients creating the

deficiencies.

a) Per cent incidence of disease:

The results are presented in table 1. The

results indicate that there was no disease

incidence in Zinc sulphate, Borax, Zinc

sulphate + Borax, Zinc sulphate + Copper

sulphate + Borax and Borax + Elemental

sulphur. Whereas, less disease incidence

was recorded in case of Zinc sulphate +

Elemental sulphur applied plot i.e. 0.79 %

followed by Zinc sulphate +Ammonium

molybdate, Zinc sulphate + Manganese

sulphate, Zinc sulphate + Copper sulphate

(0.8%, 1.27% and 1.56% respectively. The

maximum per cent incidence i.e. 4.58% was

shown by Ammonium molybdate followed

by Manganese sulphate (2.39%) and Copper

sulphate (2.23%). Though all the treatments

were significantly different however

incidence due to Ammonium molybdate was

found different in comparison to others i.e.

4.58 %.

b) Number of clumps:

Table 1, shows the effect of different

treatments on the no. of clumps. Maximum

no. of clumps were recorded in Zinc

sulphate + Copper sulphate + Borax (15.66)

followed by Zinc sulphate + Borax (14.66),

Zinc sulphate + Manganese sulphate and

Borax + Manganese sulphate (14.33)

however minimum no. of clumps were

observed in Ammonium molybdate (10)

followed by Elemental Sulphur (10.33),

Ferrous sulphate (11). Effect of Zinc

sulphate + Copper sulphate + Borax, Zinc

sulphate + Borax, Zinc sulphate +

Manganese sulphate and Borax +

Manganese sulphate different while others

were at par. were significantly. Zinc

sulphate + Copper sulphate + Borax not only

reduced the pokkah boeng incidence but also

enhanced the number of clumps in

sugarcane.

c) Number of tillers:

Table 1, reveals the effect of different

treatments on the no. of tillers. Maximum

no. of tillers were recorded in Zinc sulphate

+Copper sulphate + Borax i.e. 52.33 as

compared to check (39) followed by Zinc

sulphate + Elemental Sulphur (50.66) and

were signifiantcly different. While minimum

no. of tillers was observed in Ammonium

molybdate i.e. 38.33. Zinc sulphate +Copper

sulphate + Borax not only reduced the

pokkah boeng incidence but also enhanced

the number of tillers in sugarcane.

However, these results are in agreement

with the finding of Nayyar,

et al. (1984), Shinde, et al. (1986) and

Banger, et al. (1991) who have reported an

increase in tillers production with the

application of Zn and Fe. Kumaresan, et al.

(1987) had reported that with the application

of Zn, Fe and Cu increase the tillers.

d) Number of milliable cane:

The effect of different treatments on the

number of milliable cane is shown in Table

1, Zinc sulphate + Copper sulphate + Borax

show the maximum no of milliable cane i.e.

39 followed by Zinc sulphate + Manganese

sulphate and Zinc sulphate + Borax i.e.

38.66 in both which were significantly

different. However minimum no of milliable

cane was observed in Elemental Sulphur i.e.

28.66 followed by Ammonium molybdate

(29). Zinc sulphate + Copper sulphate +

A


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Borax not only reduced the pokkah boeng

incidence but also enhanced the number of

milliable cane in sugarcane. The results of

the present studies are in accordance with

the findings of Maribo and Albuquerque,

(1981), Anon, (1983) and Nayyar, et al.,

(1984) who have recorded an increase in the

number of millable cane with the application

of minimum levels of zinc, Copper, Boron

Iron and manganese.

e) Cane height:

Table 1, shows that in all treatments, the

maximum cane height has been recorded for

Zinc sulphate + Copper sulphate + Borax i.e

1.92 m followed by Zinc sulphate + Borax

i.e. 1.9 m, Zinc sulphate + Manganese

sulphate i.e. 1.88m and Zinc sulphate +

Elemental Sulphur i.e. 1.87 m, while

minimum was observed in Copper sulphate

i.e.1.79m as compared to control i.e. 1.69m.

The treatment doesn’t show any significant

difference. Zinc sulphate + Copper sulphate

+ Borax not only reduced the pokkah boeng

incidence but also enhanced the cane height

of sugarcane. These findings are in

accordance with the earlier findings of

Jamro, et al. (2002) who found that among

the micronutrients the lowest rates of Zn, Cu

and B were found more effective in

increasing the plant height. Oad, et al. 2002

had also got similar findings as earlier.

However, these results confirm the

finding of Nayyar, et al. (1984), Kumaresan,

et al. (1987) and Banger, et al. (1991) who

have also reported an increase in plant

height with the application of Zn, Cu and Fe.

However, Willcox, (1981) and Tonapy, et

al., (1965) have observed negative

correlation of Cu with plant height.

f) Cane girth:

In the Table 1, has shown that, the

maximum cane girth is recorded in for Zinc

sulphate+Copper sulphate+Borax (2.67cm)

followed by B Zinc sulphate+ Borax

(2.66cm) and Zinc sulphate+Elemental

sulphur (2.62cm) while minimum in Sulphur

i.e. Ammonium molybdate (1.82cm) and

Elemental sulphur (1.84cm) as compared to

control (1.81 cm) and were signifiantcly

different. Zinc sulphate+Copper

sulphate+Borax not only reduced the pokkah

boeng incidence but also enhanced the cane

girth of sugarcane.

However, these results are in

agreement with the findings of Ahmad,

(1977); Nayyar, et al. (1984); Sen, et al.,

(1985); Shinde, et al., (1986) and

Kumaresan, et al. (1987) who had reported

the highest value of stalk diameter with the

application of Zn, Cu, Fe and Mn.

g) Cane weight:


treatments on the cane weight. Positive

result was observed in all the treatments.

Maximum cane weight was observed in Zinc

sulphate, Zinc sulphate + Borax and Zinc

sulphate + Copper sulphate + Borax (0.8)

whereas, minimum cane weight was

observed in Elemental sulphur and Borax +

Ammonium molybdate (0.73). The

treatments don’t showed any significant

difference. Zinc sulphate, Zinc sulphate +

Borax and Zinc sulphate + Copper sulphate

+ Borax enhanced the cane weight of

sugarcane. These findings are in accordance

with the earlier findings of Jamro, et al.,

2002 who had found that among the

micronutrients the lowest dose of Zn, Cu

and B were more effective in increasing the

cane weight.Oad, et al., 2002 had also got

similar results.

However, Kanwar and Randhawa,

(1967), Singh and Singh, (1973), Bowen,

(1975) and Willcox, (1981) had reported

that the essentiality of Zn, Cu, and Mn for

the production of growth regulating

hormones for the vegetative growth of plant.


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h) Number of internodes:


treatments on the no. of internodes. All

treatment shows the positive results as

compared to check. Maximum number of

inter nodes were recorded in Zinc

sulphate+Borax and Zinc sulphate+Copper

sulphate+Borax (17) in both cases while

minimum number of inter nodes in

Ammonium molybdate (15.9) as compared

to check (15.33). The treatments don’t

showed any significant difference. Zinc


sulphate+Borax enhanced the no. of

internodes in sugarcane. These findings are

in accordance with the earlier findings of

Jamro, et al. (2002) who found that among

the micronutrients the lowest rates of Zn, Cu

and B were found more effective in

increasing the number of internodes. Prior to

this Oad, et al. (2002) had also got similar

findings. Singh and Singh, (1973); Bowen,

(1975) and Nayyar, et al. (1984) showed

that the application of micronutrients (Fe,

Cu, Zn, Mg, Mn, B and Mo) improved the

number of internodes over check treatments.

Kumaresan, et al. (1987) had also reported

an increase in the number of internodes with

the application of Zn, Fe and Cu.

i) Internodal length:


treatments on the internodal length. All

treatment shows the positive results as

compared to check. Maximum inter nodal

length was noted in Zinc sulphate+Borax

and Zinc sulphate+Copper sulphate+Borax

(11.36cm) in both cases while minimum

internodal length was recorded in Copper

sulphate (10.86 cm) followed by Mangenese

sulphate (10.93 cm) as compared to check

(10.93 cm). The treatments don’t showed

any significant difference. Zinc


sulphate+Borax enhanced the intermodal

length in sugarcane. These findings are in

accordance with the earlier findings of

Jamro, et al. (2002) who had found that

among the micronutrients the lowest dose of

Zn, Cu and B were more effective in

increasing the internodal length. Oad, et al.

(2002) had also got similar findings as

earlier.

However, the results of the present

studies are in the line with the results

reported by Anon, (1983); Sen, et al. (1985)

and Banger, et al. (1991) who had reported

an increase in length of internodes with the

application of Zn, Fe, Cu and B. Kumaresan,

et al. (1987) had reported that with the

application of Zn, Fe and Cu.

j) Brix value:


treatments on the Brix per cent. Maximum

Brix per cent was observed in Zinc sulphate,

Copper sulphate, Zinc sulphate + Borax,

Zinc sulphate + Copper sulphate + Borax,

Borax + Mangense sulphate and Borax +

Elemental sulphur (18.66%) whereas

minimum Brix per cent was recorded in

Ammonium molybdate i.e. 17.33% as

compared to check (17.33%). The

treatments don’t showed any significant

difference. Zinc sulphate, Copper sulphate,

Zinc sulphate + Borax, Zinc sulphate +

Copper sulphate + Borax, Borax +

Mangense sulphate and Borax + Elemental

sulphur enhanced the brix per cent in

sugarcane.

The data manifested that the lowest

rate of zinc, copper and boron were more

effective in increasing the cane brix. These

results are in agreement with the findings of

Li, (1985); Kumaresan, et al. (1987); Patel, et

al. (1991) and Banger and Sharma, (1992)

who reported an improvement in juice quality

with the application of Zn, Cu, Fe and Mn.


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k) Sucrose per cent:


treatments on the Sucrose per cent.

Maximum Sucrose per cent was observed in

Zinc sulphate + Copper sulphate + Borax

(15.59%) followed by Zinc sulphate + Borax

(15.53%) however least sucrose per cent

was observed in Ammonium molybdate

(14.51%) as compared to check (14.1%) and

were signifiantcly different. Zinc

sulphate+Copper sulphate+Borax and Zinc

sulphate + Borax not only reduced the

incidence of pokkah boeng but also

enhanced the sucrose per cent in sugarcane.

Yadav, et al. (1987) and Kumaresan,

et al. (1987) had reported an increase in

sucrose percentage with the foliar

application of Zn, Fe, and Cu. As Zn play an

important role in the photosynthesis

activities of the plant (Tsui, 1984), therefore,

an increase in the sucrose per cent may be

due to more absorption of Zn in the cane

plant.

l) Juice purity Per cent:


treatments on the Juice purity per cent. Zinc

sulphate + Copper sulphate + Borax shows

the maximum juice purity per cent i.e.

86.59% followed by Borax + Ammonium

molybdate (84.76%) and Zinc sulphate +

Copper sulphate (84.46%) while minimum

juice purity was recorded in Ammonium

molybdate (81.66%) as compared to check

(81.45%) and were significantly different.

Zinc sulphate + Copper sulphate + Borax

not only reduced the pokkah boeng

incidence but also enhanced the juice purity

in sugarcane

m) CCS per cent:


treatments on the CCS per cent. Maximum

CCS per cent was recorded in Zinc sulphate

+ Copper sulphate + Borax (10.51%)

followed by Zinc sulphate + Borax (10.47%)

while minimum CCS per cent was recorded

in Ammonium molybdate and Mangenese

sulphate (9.76%) as compared to check

(9.34). However, the treatments don’t

showed any significant difference. Zinc

sulphate + Copper sulphate + Borax and

Zinc sulphate + Borax not only reduced the

pokkah boeng incidence but also enhanced

the CCS per cent in sugarcane.

An increase in the CCS% with the

application of Zn, Cu and Boron may be

attributed to their significant effect on the

brix and sucrose per cent which ultimately

resulted in high CCS per cent.

However, Sen, et al. (1985) and

Yadav, et al. (1988) have reported an

increase in CCS % with the help of Zn, Cu,

B, Fe, and Mn. n) Cane yield: Table 1,

shows the effect of different treatments on

the Cane yield. Here maximum cane yield

was recorded in Zinc sulphate + Copper

sulphate + Borax (31.36 kg/plot or 69.60

t/ha) followed by Zinc sulphate + Borax

(30.93 kg/plot or 68.66 t/ha) and Zinc

sulphate + Mangenese sulphate (30.92

kg/plot or 68.62 t/ha) as compared to control

(21.13 kg/plot or 46.91 t/ha and were

significantly different. Zinc sulphate +

Copper sulphate + Borax not only reduced

the pokkah boeng incidence but also

enhanced the cane yield of sugarcane.

However, these results are in line

with the findings of Nayyar, et al. (1984);

Kumaresan, et al. (1987); Yadav, et al.

(1988); Velu, (1989) and Cambria, et al.

(1989) all these workers have reported an

increase in cane yield with an adequate but

not excessive application of Zn, Cu, Mg and

Mn.


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Table 1: Effect of different treatments on the pokkah boeng and other growth / yield parameters

Sl.

No.

Treatments No. of

clumps

Per plot

No. of tillers

Per plot

No. of milliable

Cane Per plot

Cane

height

(m)

Cane girth

(cm)

Cane weight

(kg/plot)

No. of

internodes

1 Zinc sulphate 14.00 44 35.33 1.85 2.58 0.8 16.4

2 Manganese sulphate 12.33 41 31 1.81 2.31 0.76 16.26

3 Copper sulphate 11.66 40.66 30 1.79 2.25 0.77 16.2

4 Elemental sulphur 10.33 40 28.66 1.82 1.84 0.73 16.03

5 Borax 13.66 43 33.33 1.84 2.55 0.79 16.36

6 Ammonium molybdate 10.00 38.33 29 1.80 1.82 0.76 15.9

7 Ferrous sulphate 11.00 40.33 31 1.82 1.86 0.78 16.3

Zinc sulphate + Manganese

sulphate 14.33 50 38.66 1.88 2.50 0.79 16.9

9 Zinc sulphate + Copper

sulphate 13.66 49.33 36.33 1.84 2.4 0.76 16.63

10 Zinc sulphate + Elemental

sulphur 14.00 50.66 38 1.87 2.62 0.75 16.76

11 Zinc sulphate + Borax 14.66 52 38.66 1.9 2.66 0.80 17.03

12 Zinc sulphate + Ammonium

molybdate 13.66 48.33 30.66 1.86 2.50 0.77 16.66

13 Zinc sulphate + Copper

sulphate + Borax 15.66 52.33 39 1.92 2.67 0.8 17.06

14 Borax + Manganese sulphate 14.33 50.33 35 1.85 2.48 0.79 16.66

15 Borax + Copper sulphate 14.00 45.66 34.33 1.83 2.58 0.76 16.46

16 Borax + Elemental sulphur 13.66 48.33 33.66 1.84 2.53 0.73 16.6

17 Borax + Ammonium

molybdate 3.33 44 32 1.83 2.55 0.76 16.36

18 Control 10.00 39 29 1.69 1.81 0.73 15.33

CD at 5% 2.34 5.34 6.17 0.14 0.21 0.11 0.95

CV 10.86 7.08 11.09 4.59 5.57 8.99 3.48

.


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Table 1: Effect of different treatments on the pokkah boeng and other growth / yield parameters

Sl.

No.

Treatments Internodal

length (cm)

Brix % Sucrose

%

Juice

purity %

CCS % Cane yield CCS yield % Disease

incidence Kg/plot t/ha Kg/plot t/ha

1 Zinc sulphate 11.2 18.66 15.31 82.26 10.18 28.26 62.75 2.88 6.38 0

2 Manganese sulphate 10.93 18 14.70 81.83 9.76 23.67 52.55 2.30 5.09 2.39

3 Copper sulphate 10.86 18.66 15.23 82.01 10.10 23.11 51.31 2.33 5.17 2.23

4 Elemental sulphur 11.1 18.33 14.92 83.66 9.85 21.13 46.89 2.07 4.57 1.633333

5 Borax 11.16 18.5 15.24 82.52 10.17 26.55 58.94 2.69 5.98 0

6 Ammonium molybdate 11.03 17.33 14.51 81.66 9.763 22.13 49.12 2.16 4.78 4.58

7 Ferrous sulphate 11.13 17.66 14.73 83.49 9.89 24.3 53.5 2.39 5.27 1.733333

8 Zinc sulphate + Manganese

sulphate 11.3 17.66 14.89 84.28 10.05 30.92 68.62 3.11 6.83 1.27

9 Zinc sulphate + Copper sulphate 11.13 17.83 15.04 84.46 10.16 27.43 60.88 2.78 6.14 1.566667

10 Zinc sulphate + Elemental sulphur 11.26 17.66 15.29 83.76 10.46 28.45 63.15 2.97 6.56 0.79

11 Zinc sulphate + Borax 11.36 18.66 15.53 81.86 10.47 30.93 68.66 3.24 7.17 0

12 Zinc sulphate + Ammonium

molybdate 11.16 17.66 14.81 83.93 9.97 23.79 52.81 2.38 5.27 0.8

13 Zinc sulphate + Copper sulphate +

Borax 11.36 18.66 15.59 86.59 10.51 31.36 69.60 3.25 7.19 0

14 Borax + Manganese sulphate 11.06 18.66 15.37 82.40 10.25 27.7 61.48 2.85 6.32 1.493333

15 Borax + Copper sulphate 11.03 18.33 15.15 82.73 10.13 26.26 58.30 2.61 5.79 1.02

16 Borax + Elemental sulphur 11.06 18.66 15.15 81.74 10.03 24.66 54.75 2.49 5.50 0

17 Borax + Ammonium molybdate 11.1 17.5 14.82 84.76 10.03 24.4 54.15 2.43 5.40 1.363333

18 Control 10.93 17.33 14.1 81.45 9.34 21.13 46.91 1.97 4.37 18.66

CD at 5 % .76* 1.44* .605** 7.62** .76* 5.00** 11.06** .55** 1.19** 2.41**

CV 4.12 4.81 2.42 5.53 4.58 11.64 11.60 12.70 12.49 66.36


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o) Commercial cane sugar (CCS) yield:


treatments on the CCS yield. Here,

maximum CCS yield was recorded for Zinc

sulphate + Copper sulphate + Borax (3.25

kg/plot or 7.19 t/ha followed by Zinc

sulphate + Borax (3.24 kg/plot or 7.71 t/ha)

and Zins sulphate + Mangenese sulphate

(3.11 kg/plot or 6.83 t/ha) as compared to

control 1.97 kg/plot or 4.37 t/ha). The effect

of treatment were significantly different

while that of Zinc sulphate + Copper

sulphate + Borax and Zinc sulphate + Borax

were at par. Zinc sulphate + Copper sulphate

+ Borax not only reduced the pokkah boeng

incidence but also enhanced the CCS yield

of sugarcane.

Increase in cane yield, CCS % and

sugar production with the application of Zn,

Cu, Mo, B and Mn has also been reported by

Ahmad (1977); Maribo and Albuquerque

(1981); Kumaresan, et al. (1987); Yadav, et

al. (1988) and Cambria, et al. (1989).

Conclusion:

nder field conditions results indicate

that there was a considerable

reduction of pokkah boeng

incidence in all treatments. Variation in

pokkah boeng incidence among different

treatments showed significant difference.

There was no incidence of pokkah boeng in

the treatments of Zn (zinc sulphate), Boron (borax), Zn (zinc sulphate)+ Boron (borax), Zn

(zinc sulphate)+Cu (copper sulphate)+Boron

(borax) and Boron (borax)+S (elemental

sulphur). In application of Zn (zinc

sulphate)+S(elemental sulphur) applied plot i.e.,

0.79 per cent show less disease incidence

whereas, Mo (ammonium molybdate) showed

the maximum per cent incidence i.e., 4.58 per

cent.

Zn (zinc sulphate)+Cu (copper

sulphate)+Boron (Borax) not only reduced

the incidence of pokkah boeng. At the same

time, they also enhanced the growth

parameters of sugarcane crop such as

number of clumps & number of tiller,

number of milliable cane, cane height, cane

girth, cane weight, number of internodes,

intermodal length, brix per cent sucrose per

cent, juice per cent, CCS per cent, cane yield

and CCS yield.

Reference:

Ahmad, M. (1977). Effect of secondary and

micronutrient application on the

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Anonymous (2013 c). Sugarcane

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on yield and quality of sugarcane.

Indian Sugar. XLI: 403-404.

Bowen, J.E. (1975). Recognizing and

satisfying the micronutrients

U


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Jamro, G.H., Kazi, B.R., Oad, F.C. Jamali,

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P., Manickam, T.S. and

Kothandaraman. (1987). Need for

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increasing yield and quality of

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Li, S. L. (1985). Combined application of

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Soil Sci. Turang Tongbao. 16: 156-

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and Patil, J.R. (1986). Response of

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Tonapy, G. K. (1965). Preliminary trials

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(1987). Response of sugarcane to

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Sugarcane Res., Lucknow. 226. 002

India


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Sharma et. al. | EFFECT OF NITROUS ACID TREATMENT ON LIPASE PRODUCTION 61

Research Article

EFFECT OF NITROUS ACID TREATMENT ON LIPASE PRODUCTION BY LOCAL

SOIL FUNGAL ISOLATE

Arun Kumar Sharma, Sapna Kumari and Vinay Sharma*



___________________________________________________________________________

Abstract The present investigation explains identification and strain improvement of Cunninghamella

sp. for enhanced production of lipase in submerged fermentation. Four hyperproducer fungal

isolates previously isolated from 4 different soil samples were identified belonging to genera

Rhizopus, Cunninghamella and Aspergillus luchuensis by looking for macroscopic (Agar

plate culture) and microscopic features (lactophenol cotton blue staining). Lipolytic potential

of Cunninghamella sp. was increased by strain improvement using random (induced)

mutagenesis technique with chemical mutagen (nitrous acid). Lipase activity of

Cunninghamella sp. was increased up to 11 % (156.79 ± 3.19 U/ml) as compared to wild

strain (141.23 ± 1.73 U/ml) after 90 minutes incubation with nitrous acid. This hyperproducer

mutagenic strain was assigned the code name Cunninghamella sp. NA90. Lipase activity was

decreased after short time exposure with nitrous acid whereas long time treatment increased

lipase activity. Nitrous acid was found potent mutagenic agent for enhancing lipolytic

potential of Cunninghamella sp.

Keywords: Lipase, strain improvement, Cunninghamella sp., hyperproducer, nitrous acid.

Introduction:

ipases are capable of hydrolyzing

ester linkage within fat/oil

(triacylglycerols) into free fatty

acids, monoacylglycerols,

diacylglycerols and glycerol under

aqueous condition and also capable of

catalyzing reverse reactions such as

transesterification and esterification under

non aqueous conditions (Sharma et al.

2001; Fernandes et al. 2007). In past few

years, attention for microbial lipases has

enhanced due to their properties and

stability. These enzymes are being utilized

in various industrial sectors such as

detergent, food, pulp and paper,

pharmaceutical, perfume and cosmetic,

oleochemical, biofuel production

(Ranganathan et al. 2008; Tamalampudi et

al. 2008) and modification of fat quality

due to their versality of chemical reactions

they catalyze (Fernandes et al. 2007).

Lipases can be extracted from

plants (Villeneuve, 2003), animals

(Shimokawa et al. 2005) and microbes

(Ionita et al. 1997; Mahadik et al. 2002;

Burkert et al. 2004). Enzymatic

transformation of lipidic substances has

several advantages (high quality of derived

product, less energy consumption etc) than

chemical transformation. Microbial lipases

can be obtained from bacteria and fungi

(Teng and Xu, 2008). Most of the bacterial

lipases reported so far are non specific in

their specificity to substrate, few are

thermostable (Pogaku et al. 2010), and

therefore fungal lipases may be a good

L



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choice for industries since these enzymes

are produced extracellularly and has

specificity to their substrate (Rani and

Panneerselvam, 2009). Among the fungi,

filamentous fungi belonging to genera

Aspergillus (Sharma et al. 2016a),

Rhizopus (Bapiraju et al. 2005),

Penicillium (Chahinian et al. 2000) and

Trichoderma (Kashmiri et al. 2006) have

been reported as potent producer of

lipases.

In general microbes cannot

synthesize large quantity of enzyme

because of regulatory mechanisms

therefore quantity of microbial enzyme can

be raised by suppression of these

regulatory mechanisms (strain

improvement) and by optimization studies

(Sharma et al. 2016b). A number of

publications deal with optimization of

culture conditions for increased lipase

production (Hasan et al. 2006; Teng and

Xu, 2008) but very few papers deals with

strain improvement for enhanced lipase

production (Ellaiah et al. 2002; Bapiraju

et al. 2005; Sharma et al. 2016a).

In view of this background, the

present investigation was undertaken to

identify the four lipolytic soil fungal

isolates and to increase the productivity of

lipase from selected isolate through

induced mutagenesis with nitrous acid.


icroorganisms and lipase

production

Twenty one soil fungi

(isolated from wheat field,

mustard field, petrol pump of Newai Town

and medicinal plant garden of Banasthali

University) were utilized for lipase

production in submerged fermentation

according to the method and production

medium used by Sharma et al. (2017).

Thereafter lipase activity was determined

according to the method of Winkler and

Stuckmann (1979) (Results are not shown

here).

Identification of hyperproducer isolate

Hyper producer fungal isolate from

each of the soil sample was identified by

observation of Petri plate culture and

microscopic examination of the fungal

slide. Isolate was point inoculated at the

centre of PDA plate for visual

observations while its slide was made in

lacto phenol cotton blue stain for its

microscopic examination by the procedure

as previously explained by Aneja (1996).

Mutagenesis of hyperproducer isolate

Hyperproducer isolate from

mustard field identified as

Cunninghamella sp. was used for strain

improvement (chemical mutagenesis)

using nitrous acid. Fungal spore

suspension was prepared by adding 2 ml of

sterile distilled water in 5 days old Petri

plate culture and 0.9 ml of spore

suspension was mixed 0.1 ml sodium

nitrite solution (0.01M) in eppendorf vial

and placed in an incubator for 15, 30, 45,

60, 75 and 90 minutes. Epppendorf vial

were taken outside after respective

incubation time and centrifuged for 10000

rpm for 10 minutes for removal of

supernatant of nitrous acid. Pellet of

treated spores was washed two times by

adding 1 ml of distilled water followed by

centrifugation. Finally 0.5 ml of sterile

distilled water was added in pellet and

used for inoculation in PDA plates (Mala

et al. 2001; Vanama et al. 2014). 0.1 ml of

each of the treated spore suspension was

aseptically transferred in PDA plates.

Inoculated PDA plates were incubated in

at 28 0C, 150 rpm for 5 days.

Identification of hyperproducer

mutagenic isolate

Spore suspension from each of the

nitrous acid mutagenic culture from

respective PDA plates was transferred in

M


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SmF. Thereafter lipase activity was

measured after 3 days of incubation and

compared with untreated culture.


dentification of lipolytic soil fungal

isolates Figure 1 presents that

hyperproducer fungus from wheat

field soil sample was identified as

Rhizopus sp. based on its macroscopic and

microscopic features. Macroscopic feature:

the growth of fungus was rapid on PDA

plates and filled the entire plate with

fluffy, cottony like growth within 5 days.

Initially, surface colour of the colony was

whitish (Fig.1a) which turned brown (Fig.

1b) with age due to the maturation of the

sporangiospores within the sporangium

while reverse colour of colony was white.

Texture of colony was cotton-candy.

Microscopic features: root like

extremely branching non-septate hyphae

(rhizoid) was observed. Rhizoid was found

at the contact point of sporangiophore and

stolon. Sporangiophores were unbranced,

non-septate, brown coloured, smooth

walled, originated from stolon (aerial

hyphae), solitary or developed in group of

2 to 3 and were up to 1500 µm in length

and 19 µm in width. A hemispherical

structure (columella) (Figs. 1c, 1f) was

observed at the top of each

sporangiophore. A globular sac like spores

bearing structure (sporangium) was

observed on the top of columella (Fig. 1e)

(Guimarães et al. 2006).

Figure-1: Macroscopic and microscopic images of Rhizopus sp.; (a): 4 days old culture; (b):

8 days old culture; (c), (d), (e), (f): microscopic images magnified to 40 X.

I

a b c

d e f


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Figure-2: Macroscopic and microscopic images of Cunninghamella sp. (Isolated from petrol

pump soil); (a): 4 days old culture; (b): 8 days old culture; (c), (d), (e), (f): microscopic

images magnified to 40 X.

Figure 2 and 3 represents that

hyperproducer fungus from petrol pump

and mustard field soil samples was

identified as Cunninghamella sp. based on

its macroscopic and microscopic features.

Macroscopic feature: fungus was growing

very rapidly and filled the entire plate

within 5 days. Initially, surface colour of

the colony was white (Fig. 2a, Fig. 3a)

which turned dark brown (Fig. 2b, Fig. 3b)

with the age while reverse colour was

whitish. Texture was powdery.

Microscopic features: hyphae were

non-septate. Sporangiophores were long,

non-septate, brown coloured, smooth

walled structure (Fig. 2e) which ended in a

swollen vesicle (Fig. 2f). Vesicles were

pyriform or subglobose in shape and up to

40 µm in diameter. Sporangiola or spores

were globose (10 µm in diameter), hyaline

singly but brownish in cluster (Chung-

Wen et al. 2005) (Figs. 3d, 3f).

Figure 4 shows that hyperproducer

fungus from medicinal plant garden soil

sample was identified as Aspergillus

luchuensis based on its macroscopic and

microscopic features. Macroscopic feature:

Circular colony was surrounded by a white

ring at the periphery. Surface colour of the

colony was brownish. Velvety texture was

observed (Fig. 4a).

Microscopic features: Septate

hyaline hyphae were seen. Conidiophore

stipes were long, hyaline, unbranched,

brown coloured and non-septate. Globose

vesicles (60 µm diameter) were seen at the

apex of conidiophores stipes (Figs. 4d, 4e).

Large circular black conidial head was

seen at apex of conidiophores stipes (Figs.

4b, 4c). Biseriate nature of conidial head

was observed in which vesicles were

attached to metulae, which were further

attached to phialides (conidia producing

cells). Chain of conidia was seen at the top

of phialides (Fig. 4c). Conidia were

globose, brown coloured and up to 4 µm in

diameter (Madavasamy and

Panneerselvam, 2012; Gandipilli et al.

2013).

a b c

d e f


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Figure-3: Macroscopic and microscopic images of Cunninghamella sp. (Isolated from

mustard field soil); (a): 4 days old culture; (b): 8 days old culture; (c), (d), (e), (f):

microscopic images magnified to 40 X.

Figure - 4: Macroscopic and microscopic images of Aspergillus luchuensis; (a): 4 days old

culture; (b), (c), (d), (e), (f): microscopic images magnified to 40 X.

Chemical mutagenesis

Table 1 depicts that best nitrous

acid mutagenic strain (Cunninghamella sp.

NA90) demonstrated high level of lipase

activity (156.79 ± 3.19 U/ml) as compared

to lipase activity (141.23 ± 1.73 U/ml) of

wild strain (Cunninghamella sp.). The best

mutant strain (Cunninghamella sp. NA90)

was obtained after 90 minutes treatment of

fungal spores with nitrous acid. Short time

incubation (15 min, 30 min) with nitrous

acid decreased lipase activity (133.23 ±

f

a b c

d e

a b c

d e f


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3.23 U/ml for Cunninghamella sp. NA15

and 122.02 ± 0.30 U/ml for

Cunninghamella sp. NA30) while long time

treatment (45, 60, 75, 90 min) increased

lipase activity as compared to wild strain.

Lipase activity was gradually increased

with the rise of incubation time with

nitrous acid and reached to maximum after

90 min treatment time. Specific activity of

Cunninghamella sp. NA90 mutant strain

was 8.17 ± 0.47 U/mg as compared to 6.87

± 0.34 U/mg of wild strain. Our results

suggest that nitrous acid can increase

lipolytic activity from Cunninghamella sp.

Table-1: Lipase activity and specific activity from wild and nitrous acid mutagenic

strains of Cunninghamella sp after 3 days of incubation.

Similar to our study, Mala et al.

(2001) increased lipolytic potential of A.

niger by UV rays and nitrous acid

mutagenesis. Nitrous acid was found more

potent mutagenic agent that UV rays. Best

UV mutant demonstrated 14.9 % more

lipase activity after 4 min exposure

whereas best nitrous acid mutant showed

33.9 % more lipase activity after 60 min

treatment as compared to parent strain.

Toscano et al. (2011) reported that

chemical mutagenesis (N-methyl-N-nitro-

N-nitrosoguanidine) (NMG) was found

more effective than physical mutagenesis

(UV rays) for strain improvement of

Aspergillus niger for increased lipase

production. Rajeshkumar and Ilyas (2011)

increased lipolytic activity of A. fumigates,

A. niger, and Penicillium sp. by induced

mutagenesis using sodium azide, UV and

ethyl methane sulphonate. Ellaiah et al.

(2002) isolated mutant strains of

Aspergillus by treatment of the wild strain

using UV rays and NMG. Bapiraju et al.

(2004) increased lipase activity of

Rhizopus sp. by induced mutagenesis

using UV rays and NMG.

Conclusion: Laboratory study was carried out

to identify the four lipolytic soil

fungal isolates (by examination of

macroscopic and microscopic

features) and to enhance the lipase activity

of selected isolate (by strain improvement

using nitrous acid as mutagen). Out of

four, one isolate was identified as

Rhizopus sp., two were belonging to the

genera Cunninghamella and one was

identified as Aspergillus luchuensis.

Lipase activity of wild strain of

Cunninghamella sp. was increased up to

11 % by incubation of its spores with

nitrous acid for 90 min. In our study

treatment of spores with nitrous acid for

long duration (60 min, 90 min) increased

lipase activity from Cunninghamella sp.

whereas short duration exposure (15 min,

30 min) decreased lipase activity. It is

hoped that Cunninghamella sp. can be

used further for bulk production of lipase

in submerged fermentation.

Incubation time of

spores with HNO3

(min)

Mutant strains of

Cunninghamella sp.

Lipase

activity

(U/ml/min)

Protein

content

(mg /ml)

Specific

activity

(U/mg)

Wild strain (0 min) Cunninghamella sp. 141.23±1.73 20.56 6.87±0.34

15 min Cunninghamella sp. NA15 133.23±3.23 20.75 6.42±0.33






A


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Acknowledgements:

We are thankful to Professor

Aditya Shastri, Vice-Chancellor,

Banasthali University for providing

research facilities in the Department.

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Anjali et. al. | EFFICACY OF ORGANIC FERTILIZER- AMIRTHAKARAISAL 70

Research Article

EFFICACY OF ORGANIC FERTILIZER- AMIRTHAKARAISAL ON THE GROWTH OF

CHILLI PLANT

Anjali, Tanveer Hassan, Muralitharan R, Murugalatha N.*, Rinkey Arya,

Vijay Kumar and Naveen Chandra

Department of Agriculture, Quantum School of Graduate Studies,

Quantum Global Campus, Roorkee, Uttarakhand – 247667, India

*Corresponding author - [email protected]

___________________________________________________________________________

Abstract Chilli plant belonging to Solonaceae family is one of the major cash crop grown in India.

Chilli being a good source of vitamin is used in wide range of medicines against tonsillitis,

diphtheria, loss of appetite etc., Application of inorganic fertiliser in soil for the growth of

plants has deteriorated the quality of soil and reduced the plant growth and yield. The paper

examines the effect of Amirthakaraisal, an organic fertiliser on the growth of chilli plant. The

organic fertiliser Amirthakaraisal is prepared by using only the cow ingredients. The seeds of

chilli plant were soaked in various concentration of organic manure (1%, 3%, 5% & 7%)

prior germination. Organic fertiliser at the rate of 1, 3, 5 , 7 % concentration was sprayed on

all plants. Plant height and root length were measured. Maximum shoot length and root

length were observed in the plants treated with 7% organic manure. Effective results were

obtained after application of organic fertilisers as compared to water as control.

Key Words: Chilli plant, organic fertiliser- Amirthakaraisal, soil fertility, growth

Introduction:

ndia is the world’s most important

country in agriculture. Most people in

India depend on agriculture because of

its economic and ecological importance.

The nature of soil has supported many of

the staple crops. In order to increase the

yield, people believe that chemicals above

could give high yield. Though they may

stimulate the growth of the crops and

provide a good yield, they may contain

ingredients that may be toxic to the skin or

respiratory system. These chemicals when

applied more in the field can affect the

crop growth, build up in the soil causing

day term imbalance in the soil pH and

fertility. These conditions may affect the

nutrients in the soil and the friendly

microorganisms. Chemical fertilizer

reduces the protein content of the crops

and also degrades the carbohydrate quality

of crop (Marzouk and Kassem, 2011).

Vegetable and fruit grown on chemically

over fertilised soil are also prone to attacks

by insects and disease (Karunji et al.

2006). Natural products as fertilizer have

now made its way to overcome these

problems. Organic farming originated

early in the 20th

century in an alternative

agricultural system rapidly changing

farming practices. Organic cultures are

developed in combination of tradition.

Innovation and science to benefit the

shared environment and promote fair

relationship and a good quality of life.

Health awareness and environment issues

in agriculture have demanded organic food

production. The International Federation

I



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of Organic Agriculture Movement

(IFOAM) started the establishment of

organic farming on 5th

Nov, 1972 in

France. Amirthakaraisal is an organic

formation termed from 4 ingredients like

cow dung, cow urine, country sugar and

water. Amirthakaraisal is one of the

effective organic manure which is used as

a growth stimulator, growth promoter and

immunity booster. Amrithakaraisal proved

its value by providing strength and great

resistance to the crop (Rajukkahhu,

Ramadas and JecithaKudumba, 2007).

Amirthakaraisal is mixed with irrigation

water acts as an toxic for the soil and

makes it rich in nutrients. Earth worms

which live deep under the soil surface

come to the top to feed on this solution.

The two direct constituents from, cow used

in Amirthakaraisal are cow dung, urine

mixed in a proper ratio and then allowed it

to ferment by using jaggery as a

fermented. Cow urine provides nitrogen

which is essential for plant growth. Cow

dung act as a medium foe growth of

beneficial microbes (Saritha and

Vijaykumari, 2013). Organically and

chemically manage farming system have

high soil organic matter and total nitrogen

with an increase in soil pH, increase in

concentration of nutrients and microbial

population under natural organic

management (Alvarez et al, 1998;

Drinkwater et al. 1995;Reganold, 1998;

Clark et al. 1998; Dinesh et al. 2000; Lee,

2010) . In our present study the efficiency

of Amirthakaraisal was assessed in Chilli

plant to achieve maximum yield and

quality.


oil and Seed:

Soil was collected from the

Agricultural Research Block of

Quantum Global Campus, Roorkee. Seeds

were collected from the commercial seed

supplier from the Roorkee.

Preparation of Amirthakaraisal:

Ingredients are follows

Cow dunk - 1Kg

Cow urine - 1 litre

Jaggery - a hand full

Water - 10 litre

The cow dung and urine were taken

in a wide mouthed pot and thoroughly

mixed. 10 litre was added to the mixture

and a handful of country sugar (jaggery)

was also added and stirred well until the

sugar gets dissolved. The mixture was

allowed to ferment for 24 hours and stored

in the shade by covering it with plastic

mosquito net to prevent house flies.

The Amirthakaraisal solution was

diluted to 1%, 3%, 5% , 7% solution and

were sprayed on 3rd

,7th

,11th

,16th

, 20th

,and

24th

day in morning to all the plants. The

plants were irrigated twice a day (Gayathri

et al. 2015).

Biochemical Observation:

Germination percentage of seeds, shoot

length and root length were observed and

measured respectively. The percentage of

germination was calculated three days

after sowing the seeds, root length and

shoot length are taken on 25th

day.

The percentage germination was

calculated by using the following formula:

Germination percentage

=


he effect of organic manure with

Amirthakaraisal on chilli plant has

shown marvellous result on

different concentrations of 1%, 3%, 5%,

and 7% solution. The seeds were soaked in

different concentrations of organic manure

before germination process and the seeds

S T


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soaked in water alone were taken as

control.

Germination rate was found to be

maximum in the seeds soaked in 7% when

compared to other concentrations. 82% of

germination was obtained in the seeds

soaked in 7% organic manure. Seeds

soaked in 1%, 3% and 5% organic manure

showed germination of 66%, 68% and

73% respectively. Seeds soaked in water

provided only 50% of germination (Fig.

1). Gayathri et al. (2015) has stated that

seeds soaked in organic manure

Panchagavya have provided 83.33%

germination in Tomato plant, 77.8 % in

French beans and 66.67% in Lady’s finger.

Shoot length and root length of the

plant were measured on 25th

day after the

application of varying concentrations of

Amirthakaraisal to the plants. The shoot

length and root length were found to be

higher in plants treated with 7%

Amirthakaraisal solution. The root length

was found to be 5.08 cm and the shoot

length was 14.25 cm (Fig. 2). 1% treated

seeds showed minimum root length of 4.10

cm and shoot length of 13.09 cm. The

water treated seeds acting as control

showed minimum shoot length of 12.08

cm and root length of 4.02 cm. The current

trend of using organic practices has

improved yields in crops of rainfedares in

Indai (Singh et al., 2001; Ramesh et al.,

2005). Organic practices has increased the

yield in crops like Chilli (Subhashini et al.

2001), moringa (Beaulah et al, 2002),

green gram (Somasundaram et al. 2003)

and French beans (Selvaraj, 2003). The

organic manure has well developed the

roots which had allowed them to spread

wider and deeper inside the soil. This has

aided them in absorbing the nutrients,

minerals and water. The tap root system in

chilli plant can store more nutrients and

water which is an advantage in regions

with minimal water. The shoots were

sturdy and capable of transporting

nutrients and provided the plant a physical

support.

Figure 1 Germination % of the chilli seeds with varying concentrations of Amirthakaraisal


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Fig. 2 Shoot length and Root Length of the chilli plants treated with varying

concentrations of Amirthakaraisal

Application of organic manure

Amirthakaraisal has improved the structure

of the soil and increased its ability to hold

water and nutrients. Microorganisms

present in the organic manure breaks down

and releases nutrients into the soil. These

microorganisms obtain energy from

decaying plants and animal matter which

provided a complete package of nutrients

for the soil. Few other organic manure

such as Panchagavya has improved the

sustainability in agriculture (Tharmaraj et

al. 2011). In conclusion Amirthakaraisal

can be used as an organic growth promoter

for vegetable growers and cost

benefitable.

References:

Marzouk, H.A and Kassem H.A.

(2011).Improving fruit quality,

nutritional value and yield of

Zaghloul dates by the application

of organic and or mineral

fertilisers.Scientia

Horticulturae.127: 249-245.

Karunjgi, J. Ekborn, B and Kyamanywa,

S. (2006). Effects of organic versus

and conventional fertilisers on

insect pests, natural enemies and

yield of Phaseolus

vulgaris.Agriculture.Ecosystems

and environment. 115:51-55.

Saritha, M. And Vijaykumari. (2013).

Influence of selected organic

manures on the seed germination

and seedling growth of cluster

bean. Journal of Wollega

University, Ethiopa, pp:2226-7522.

Alvarez, C.E., Garcia, C. and Carracedo,

A.E. (1988). Soil fertility and

mineral nutrition of organic banana

plantation in Tenerife. Biological

Agriculture and Horticulture,

5:313-323.


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Drinkwater, L.E., Letourneau, D.K.,

Workneh, F., Bruggen, A.H.C. &

Shennan, C. (1995). Fundamental

difference between conventional

and organic tomato agroecosystems

in California. Ecological

Applications. 5: 1098–1112.

Reganold, J.P. (1988). Comparison of soil

properties as influenced by organic

and conventional farming systems.

American Journal of Alternative

Agriculture. 3: 144–155.

Clark, M.S., Horwath, W.R., Shennan, C.

& Scow, K.M. (1998). Changes in

soil chemical properties resulting

from organic and low-input

farming practices. Agronomy

Journal. 90: 662–671.

Dinesh, R., Dubey, R.P., Ganeshamurthy,

A.N. & Prasad, G.S. (2000).

Organic manuring in ricebased

cropping system: effects on soil

microbial biomass and selected

enzyme activities. Current Science.

79: 1716–1720.

Lee, J. (2010). Effect of application

methods of organic fertilizer on

growth, soil chemical properties

and microbial densities in organic

bulb onion production. Scientia

Horticulturae. 124: 299– 305.

Gayathri, V., Nesiriya, M., Karthika, A.

And Jisha Sebastian. (2015).

Styudy on the growth of vegetable

crops using panchagavya. Int.

Journal of Current

Research.7(10):21093-21096.

Singh, G.R., Chaure, N.K., and Parihar, S.

S. (2001). Organic Farming for

sustainable agriculture Indian

Farmer, 52, 12-17.

Ramesh, P, Singh, M, & Subbarao, A.

(2005). Organic Farming: Its

relevance to the Indian context.

Current Science. 88(4):561-568.

Subhasini, S.; Arumugasamy, A.;

Vijaylalakshmi, K. and

Balasubramanian, A.V. (2001).

Vrkshayurveda- Ayurveda and

plants. Centre of Indian Knowledge

system, Chennai, India, pp: 33.

Beaulah, A. (2001). Growth and

development of moringa (Moringa

oleifera Lam.) under organic and

inorganic systems of culture.

Ph.D.Thesis, Tamil Nadu

Agric.Univ., Coimbatore.

Somasundaram, E., Sankaran, N., Meena,

S., Thiyagarajan, T. M.,

Chandaragiri, K and

Panneerselvam, S. (2003).

Response of green gram to varied

levels of panchagavya (organic

nutrition) foliar spray. Madras

Agric. J., 90: 169-172.

Selvaraj, N. (2003). Report on the work

done on organic farming at

Horticultural Research Station.

TNAU, Ooty, Indian, pp: 2-5.

Tharmaraj, K., Ganesh, P., Suresh Kumar,

R., Anandan, A. And Loanjinathan,

K. (2011). A Critical reciew on

panchagavya – A boon plant

growth. International Journal of

Pharmaceutical and Biological

Archives. 2(6): 1611-1164.


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Sharma et. al. | PARTIAL PURIFICATION OF CUNNINGHAMELLA LIPASE

75

Research Article

PARTIAL PURIFICATION OF CUNNINGHAMELLA LIPASE

Arun Kumar Sharma, Sapna Kumari and Vinay Sharma*



______________________________________________________________________________

Abstract Lipases are considered only after carbohydrases and proteases in global enzyme market and

contribute to nearly 5% of enzyme market. They are found in animals, plants and microbes hence

classified as animal, plant and microbial lipases. In most of the industries, they prefer

exploitation of crude enzyme instead of purified preparation to reduce the cost and time of

purification. Hence, the present study is focused on salt (ammonium sulfate) purification of

extracellular lipase obtained from submerged fermentation of lipolytic Cunninghamella sp.

Among all precipitates, highest level of lipase activity (101.18 ± 4.3 U/ml) and specific activity

(7.36 ± 1.4 U/mg) was observed in 70% precipitate which suggest that lipase was maximally

precipitated at 70% ammonium sulfate concentration. Hence, 70% salt concentration was found

optimum for maximum precipitation of Cunninghamella lipase.

Keywords: Cunninghamella, ammonium sulfate, animal, lipase, precipitates, specific activity.

Introduction:

ipases (EC 3.1.1.3) are water soluble

enzymes cleave ester bonds of long

chain water immiscible

triacylglycerols into fatty acids,

monoacylglycerols and diacylglycerols. On

the other hand lipases also catalyze reverse

reactions such as esterification and

transesterification. True lipases exhibit

interfacial kinds of activation in which

lipase are active only when they are

absorbed at interface of oil and water. This

property of lipases makes them different

from esterases (Yamamoto and Fujiwara,

1995). Lipases have a broad range of potent

applications in the hydrolysis (aqueous

condition), esterification (Sharma et al.

2002), and transesterification (non-aqueous

condition) of triacylglycerols, in the chiral

selective synthesis of esters (Ray, 2012), oil

spillage degradation (Naz and Jadhav,

2013), biodiesel production (Siva et al.

2015), textile industry (Hasan et al. 2006)

and oil effluent treatment (Basheer et al.

2011). The capability of lipases to carry out

very precise chemical transformation

(biotransformation) has made them more

and more popular in the chemical, food,

pharmaceutical and detergent industries

(Grbavcic et al. 2007).

Lipase extracted from pig pancreatic

is one of the earlier recognized sources of

lipase. Plant lipases are not utilized

commercially due to poor yield and complex

process of their production. The present

commercial sources of lipases are

microorganisms because their production is

more convenient, safe and simple (Jaeger et

al. 1994; Gombert et al. 1999; Maia et al.

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76

2001). Among microbial lipases, fungal

lipases are produced extracellularly and

broadly utilized in applications of industries,

especially in the food industry (Sharma et al.

2001). However, the synthesis of fungal

lipases depends upon culture media

ingredients (carbon, nitrogen and mineral

sources) and incubation conditions (pH,

temperature, shaking speed, oxygen content

etc). Major commercially significant lipase

producing fungi are: Aspergillus terries

(Gulati et al. 1999), Aspergillus niger (Jamil

and Omar, 1992), Rhizopus japonicus

(Suzuki et al. 1986), Rhizopus arrhizus

(Elibol et al. 2000), Rhizopus niveus

(Tweddell et al. 1998), Candida rugosa

(Benjamin and Pandey, 1998), Humicola

lanuginose (Martinelle et al. 1995),

Trichoderma harzianum (Ulker et al. 2011),

Fusarium oxysporum (Hoshino et al. 1992)

and Mucor miehei (Patti et al. 2000) whereas

commercially imperative lipase producing

bacteria are: Pseudomonas sp. (Borkar et al.

2009), Bacillus subtilis (Devi et al. 2012),

Bacillus alcalophilus (Ghanem et al. 2000)

and Acinetobacter sp. (Kasana et al. 2008).

Lipolytic fungi are well known for their

existence in various habitats such as oil

contaminated soil, effluents from dairy

industries, wastes of vegetable oil and

decomposed food (Iftikhar et al. 2010).

Microbial lipases are generally

produced by submerged fermentation (SmF)

at industrial scale (Ito et al. 2001), however

solid state fermentation (SSF) can also be

utilized (Mohseni et al. 2012). SmF offer

several advantages such as less space is

required, process parameters can be easily

controlled, chances of contamination are

very less, quantity of enzyme is higher and

separation of fungal mycelium is easier.

Lipases from a great number of fungal,

bacterial and a certain animal and plant

sources have been purified to homogeneity.

Various approaches (ammonium sulfate

precipitation, ion exchange chromatography

followed by gel filtration) are being

currently utilized for the purification of

different fungal lipases (Saxena et al. 2003).

In view of the regular demand for

fungal lipases in different industrial sectors,

the current study was undertaken with the

objective of partial purification of

Cunninghamella lipase using solid

ammonium sulfate.


roduction of extracellular lipase

Cunninghamella species previously

isolated from mustard field soil

sample and identified by us. 100 ml

of fermentation broth was prepared and

inoculated aseptically with 1 ml of spore

suspension made from 6 days old sporulated

culture of Cunninghamella sp. Inoculated

flasks were kept in appropriate cultivation

conditions. The composition of fermentation

broth in g/100ml was as follows: KH2PO4,

0.1; olive oil, 1 ml; MgSO4.7H2O, 0.1;

bacteriological peptone, 4; sucrose, 0.5 and

(NH4)2SO4, 0.1 (Xia et al. 2011). For

extraction of mycelium free crude protein

lysate, culture broth was filtered at the end

of 3 days incubation and any residual

mycelium and impurities were further

separated by centrifugation of filtrate. This

centrifugation separated impurities in the

pellet and crude protein lysate in the

supernatant, later was utilized as source of

extracellular lipase for partial purification.

Partial purification

Five ml of crude protein extract was

used for partial purification using solid

ammonium sulfate. Different fractions

(30%, 70% and 80%) of ammonium sulfate

were prepared. Initially 0.87g of solid

P


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77

(NH4)2SO4 was added in 5 ml of crude

enzyme lysate to obtain 0 to 30% salt

saturation. (NH4)2SO4 was dissolved in

protein lysate by keeping the beaker on

magnetic stirrer for 30 min. Then beaker

was transferred in refrigerator at 4 0C for

overnight for insolubilization of proteins.

Next day Insoluble proteins were separated

by centrifugation of the mixture at 10000

rpm for 10 min at 4 0C. This resulted in the

formation of pellet of precipitated proteins

and supernatant of dissolved proteins.

Supernatant was taken in another tube. Two

ml of Tris HCl buffer (0.05 M, pH-8.0) was

added in pellet and use for estimation of

lipase activity and protein content. Now

0.141g of solid (NH4)2SO4 was added in

supernatant for achieving the salt saturation

from 30% to 70%. Ammonium sulfate was

solubilized on magnetic stirrer for 30 min

followed by overnight incubation at 4 0C.

Thereafter it was centrifuged to obtain the

pellet and supernatant. Pellet was again

dissolved in 2 ml of buffer followed by

estimation of protein content by the

procedure of Lowry et al. (1951) and

extracellular lipase activity by the procedure

of Winkler and Stuckmann (1979) in which

p-nitrophenyl palmitate was utilized as

substrate. Salt saturation of supernatant was

further increased from 70% to 80% by

adding 0.35g of ammonium sulfate followed

by solubilization on magnetic stirrer,

overnight incubation, centrifugation to

obtain the pellet of 80% fractionate. Pellet

was again solubilized in buffer followed by

estimation of protein content and lipase

activity. Grams of ammonium sulfate are

calculated by the following formula:

where S1 is initial percent salt saturation and

S2 is percent salt saturation we want to

obtain. This equation is based on assumption

that 100% saturation is equal to 4.05 M

(NH4)2SO4.

Specific lipase activity was

determined by dividing lipase activity with

protein content.

Statistical analysis:

All the spectrophotometric

estimations were done in triplicates and

mean values of them along with standard

deviations are presented in this paper.


able 1 represents that at 30%

saturation lipase activity was 34.46 ±

2.3 U/ml with specific activity of

4.37 ± 0.7 U/mg. Lipase activity and

specific activity were increased and reached

to maximum (101.18 ± 4.3 U/ml and 7.36 ±

1.4 U/mg) at 70% fractionate. Thereafter

lipase activity and specific activity were

reduced and reached to lowest (12.98 ± 2.4

U/ml and 1.82 ± 0.4 U/mg) at 80%

fractionate. Specific activity is indicator of

the purity of enzyme. Among other

fractions, 70% fraction represents highly

purified lipase, which indicates that lipase

was maximally precipitated at 70% salt

saturation. Therefore 70% salt saturation

was found optimum concentration for

maximum precipitation of present lipase.

The order of lipase precipitation in our study

was 70% > 30% > 80%. Very little amount

of lipase was precipitated at 80% saturation

therefore lipase activity and specific activity

both are lowest at this concentration.

T

Grams of (NH4)2SO4

533 × (S2-S1)

100-0.3 S2 =

)


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78

Table-1: Lipase activity and specific activity of different fractions of solid (NH4)2SO4.

The present results suggest that 30%

salt concentration was not sufficient for

optimum precipitation of lipase because this

concentration was not sufficient to break the

interaction of enzyme with water molecules

(solvated layer). As the concentration of

ammonium sulfate was increased to 70%

then most of the H-bonds (solvated layer)

were broken down hence maximum

molecules of enzyme were precipitated at

this concentration. When salt percentage

was further increased to 80% then remaining

H- bonds were cleaved and very few

molecules of lipases were precipitated.

Similar to present results,

Jayaprakash and Ebenezer (2012) reported

optimum specific activity (367.14 U/mg) at

70% salt saturation as compared to crude

lysate (139.35 U/mg). Similarly Tipre et al.

(2014) also observed maximum activity of

Staphylococcus sp. lipase (33.67 U/ml) in

70% salt fraction as compared to crude

lysate (12.5 U/ml). Das et al. (2016)

reported purification of Aspergillus

tamarii lipase by 80% ammonium sulfate

saturation. Specific activity was 86,877.14

U/mg at 80% fraction as compared to crude

lysate (32,938.06 U/mg). Borkar et al.

(2009) reported that specific activity for

Pseudomonas aeruginosa lipase was

2618.07 U/mg at 30% fraction as compared

to crude lysate (125.59 U/mg) and other

fractions (40% to 90%). Islam et al. (2008)

reported stepwise purification strategy for

Liza parsia lipase in which lipase

demonstrated higher activity at 85%

saturation further purified by

chromatographic methods. Souza et al.

(2014) documented higher specific activity

for Aspergillus japonicus lipase in 60% salt

precipitate. Çorbacı et al. (2014) have

reported partial purification of Candida

odintsovae lipase by ammonium sulfate

(25%-75%). Shu et al. (2006) reported

highest specific activity (12.7 U/mg) for

Antrodia cinnamomea lipase at 70% salt

saturation as compared to crude lysate (10.9

U/mg) whereas Kashmiri et al. (2006)

reported purification of Trichoderma Viride

lipase at 35-60% salt concentration.

Several investigators (Palekar et al.

2000; Sharma et al. 2001; Saxena et al.

2003) have reviewed purification strategies

from time to time for microbial lipases.

Various methods of purification of lipase

have been recently explained by Singh and

Mukhopadhyay (2012). Ghosh et al. (1996)

summarized the strategies of microbial

lipase purification. Kumarevel et al. (2005)

reported purification of Cunninghamella

verticillata lipase by acetone precipitation

(5% to 50% saturation). Using the acetone

precipitation many lipases have been

purified and their molecular weight have

been reported by Muderhwa et al. (1986) for

Rhodotorula pilimena, by Sugihara et al.

(1995) for Pichia burtonii, Hofelmann et al.

(1985) for Aspergillus niger, Das et al.

(2016) for Aspergillus tamari, Hiol et al.

(1999) for Mucor hiemalis, Gopinath et al.

(2002) for Cunninghamella verticillata and

Gopinath et al. (2003) for Geotrichum

candidum.

Ammonium sulfate

fractions

Lipase activity

(U/ml)

Protein content

(mg/ml)

Specific activity

(U/mg)

30% 34.46 ± 2.3 7.87 4.37 ± 0.7

70% 101.18 ± 4.3 13.86 7.36 ± 1.4

80% 12.98 ± 2.4 7.21 1.82 ± 0.4


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79

Conclusions:

ipases are hydrolytic enzymes of

industrial importance because of the

chemical reactions catalyzed by

them. By keeping increasing demand

of lipases in mind and their applications, the

aim of present investigation was partial

purification of Cunninghamella lipase using

solid ammonium sulfate. The present results

suggest that 70% salt fraction demonstrated

higher lipase activity and specific activity as

compared to 30% and 80% fractions.

Further chromatographic purification of

lipase can be carried out for investigation of

properties of purified lipase.

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Sukumaran, K., Elyas, K.K. and

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Annadurai, G. (2002). Purification of

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80

Gopinath, S.C.B., Hilda, A., Priya, T.L.,

Annadurai, G. and Anbu, P. (2003).

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Bezbradica, D.I., Siler-Marinkovic,

S.S. and Knezevic, Z.D. (2007).

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Hasan, F., Shah, A.A. and Hameed, A.

(2006). Industrial applications of

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Gupta and Kaintura | RESPONSE OF PACLOBUTRAZOL AS SPRAY & DRENCH 83

Research Article

RESPONSE OF PACLOBUTRAZOL AS SPRAY & DRENCH ON POT GROWN FUCHSIA

CV. ‘PINK GALORE’

Yachna Gupta and Pooja Kaintura*

Department of Floriculture and Landscaping,

Dr. Y. S. Parmar University of Horticulture & Forestry, Solan (H.P.), India


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Abstract Present investigations were carried out at the experimental farm of Department of Floriculture

and Landscaping, Dr. Y. S. Parmar University of Horticulture & Forestry, Solan (HP) with the

objective to study the effect of paclobutrazol as drench, spray or combination of both on growth

and flowering of Fuchsia cv. ‘Pink Galore’. The experiment was laid out in a completely

randomized design consisting of 16 treatments, replicated thrice and each having 5 pots. When

the cuttings were 8 to 10 cm in length, drench treatments of PP333 @ 10, 20 & 30 ppm were

given per pot. After 30 days of drenching, PP333 @ 10, 20 & 30 ppm was applied as foliar

spray. All the paclobutrazol treated plants resulted in reduced plant height, plant spread,

internodal length, flower stalk length and flower size.Days to flower bud formation and

flowering were advanced in drench and combination treatments, whereas, spray treatments

delayed flower bud formation and flowering. The number of flowers open at a time and duration

of flowering were increased in all paclobutrazol treated plants. The presentability of plants

increased in all the paclobutrazol treated plants as compared to control. However, the most

presentable pots were obtained when the plants were drenched with 10 ppm paclobutrazol.

Key words: Paclobutrazol (PP333), Fuchsia, presentability

Introduction:

ot plants are generally valued for their

foliage and flowers, with Fuchsia

being an important flowering pot

plant, requires strong, straight trunk, dense

crown, prolific and early flowering for its

presentability. To achieve the desired shape

of the plants Davis and Anderson (1989)

advocated the use of growth retardants in

manipulating shape, size and form of pot

plants. Paclobutrazol, a recently developed

triazole, is quite effective in checking

growth and manipulating flowering of

Fuchsia’s, yet its consistency remains to be

evaluated for the cultivar under study.

Application techniques also have a huge

impact on the effectiveness of a growth

retardant on the crop. The way a retardant is

applied can greatly influence the growth

pattern of the crop thereby, modifying its

presentability.

P



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ooted cuttings of cultivar ‘Pink

Galore’ were planted (one cutting

per pot) in pots of six inch size

containing a sterilized potting mixture of

sand : soil : vermicompost in equal ratio.

Simultaneously, slow release fertilizer @ 2g

per pot was added. After 45 days of

planting, plants were pinched to encourage

laterals and regular hoeing and weeding was

performed at weekly intervals. When plants

became 8-10 cm in length, drench

treatments of PP333 @ 10, 20 & 30 ppm

were given per pot. Before drenching, the

pots were irrigated and PP333 solution of

desired concentration was added @

250ml/pot. Spray treatments were applied @

10, 20, 30 ppm after 30 days of drenching.

Application of NPK (19: 19: 19) @

2g/litre/pot was done fortnightly through

water soluble fertilizers. The pots were kept

under 75% shade net till the light intensity

was upto 40,000 lux, there after the pots

were shifted to poly house as the light

intensity decreased in the net house than the

required light intensity of Fuchsia which is

approximately 60,000 lux.


eight reduction (table1) was

achieved in plants treated with

paclobutrazol as drench, spray and

their combinations but maximum percent

reduction over control (33.21%) was

achieved when a drench of 30ppm

paclobutrazol was given. This reduction in

height is due to inhibition of GA

biosynthesis by the activity of paclobutrazol.

Advance flowering (table2) by 24.46% and

7.03% over control by drench (10 ppm) and

combination (10 ppm drench + 20 ppm

spray) treatments, respectively, was

achieved. This earliness in flowering is

attributed to general property of retardants to

reduce the indigenous level of GA to a

permissible concentration required for

flowering (Armitage, 1984). On the

contrary, spray of paclobutrazol @ 10ppm

delayed flowering (table 2) by 12.14 days.

This delay in flowering may be attributed to

the reduced GA synthesis and prolonged

vegetative phase by paclobutrazol

application. Paclobutrazol delayed flowering

by 7 days in Fuchsia cv. Beacon (Gad et al.,

1997). Schekel and Blau (1986) also

reported delayed flowering with the

application of PP333 @ 2-16ppm.

The number of flowers (table3)open

at a time per plants increased significantly in

all the paclobutrazol treatments. However,

maximum percent increase (65.78%) over

control was achieved when the plants were

drenched with 10ppm paclobutrazol.

Bazzocchi (2001) also reported higher

flower production in Fuchsia cv. Beacon

when enriched the medium with

paclobutrazol @ 0.5 mg/plant. Similar

results were reported by Andrasek (1989) in

Pelargonium x hortorum cv. Springtime and

Mertens (1992) in Rhododendron hybrids

with paclobutrazol.

Flower length (table4) was reduced

in all the treatments of paclobutrazol, but

maximum percent reduction (44.79%) over

control was obtained with 30 ppm drench

and 30 ppm spray of paclobutrazol. The

decrease in flower size of Fuchsia when

treated with paclobutrazol was also reported

by Bazzocchi (2001). The decrease in

inflorescence length was also reported by

Gianfagna and Wulster (1986) in Freesia,

Horn and Wischer (1987) in Elatior

begonias and Heusiel and Witt (1985) in

Azalea.

R

H


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As far as duration of flowering (table5) is

concerned, Andrade et al. (1991) reported

that paclobutrazol causes increase in the

duration of flowering. Similar results were

obtained in all

Table 1: Effect of paclobutrazol on plant height (cm) of Fuchsia cv. ‘Pink Galore’

Treatments PBZ Spray Mean

PBZ Drench 0 ppm

(S0)

10 ppm

(S1)

20 ppm

(S2)

30 ppm

(S3)

0 ppm (D0) 24.33 19.77 20.77 21.03 21.47

10 ppm (D1) 16.50 16.77 16.87 18.60 17.18

20 ppm (D2) 17.30 17.27 16.73 18.52 17.45

30 ppm (D3) 16.25 17.52 18.94 19.03 17.93

Mean 18.60 17.83 18.33 19.30

CD0.05 D - 0.73 S - 0.73 D* S - 1.46

Table 2: Effect of paclobutrazol on days to first flowering on Fuchsia cv. ‘Pink Galore’


PBZ Drench 0 ppm

(S0)

10 ppm

(S1)

20 ppm

(S2)

30 ppm

(S3)

0 ppm (D0) 101.30 148.10 140.00 118.50 127.00

10 ppm (D1) 96.10 99.12 94.18 98.47 96.97

20 ppm (D2) 96.27 95.20 96.72 95.58 95.94

30 ppm (D3) 95.82 95.55 96.69 96.77 96.21

Mean 97.36 109.50 106.90 102.30

CD0.05

D - 3.27 S - 3.27 D*S - 6.54

Table 3: Effect of paclobutrazol on number of flowers open at a time of Fuchsia cv. ‘Pink Galore’


PBZ Drench 0 ppm

(S0)

10 ppm

(S1)

20 ppm

(S2)

30 ppm

(S3)

0 ppm (D0) 6.92 5.73 9.38 15.87 9.48

10 ppm (D1) 20.22 18.53 17.93 16.13 18.20

20 ppm (D2) 17.40 14.95 19.68 17.13 17.29

30 ppm (D3) 15.47 15.87 15.13 15.14 15.40

Mean 15.00 13.77 15.53 16.07

CD0.05

D - 1.38 S - 1.38 D*S - 2.76


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Table 4: Effect of paclobutrazol on flower length (cm) of Fuchsia cv. ‘Pink Galore’


PBZ Drench 0 ppm

(S0)

10 ppm

(S1)

20 ppm

(S2)

30 ppm

(S3)

0 ppm (D0) 8.64 9.23 7.71 6.50 8.02

10 ppm (D1) 5.05 5.65 6.79 6.02 5.88

20 ppm (D2) 6.50 6.66 5.56 6.21 6.23

30 ppm (D3) 5.15 5.29 5.43 4.77 5.16

Mean 6.34 6.71 6.37 5.87

CD0.05

D - 0.32 S - 0.32 D*S - 0.64

Table 5: Effect of paclobutrazol on duration (days) of flowering of Fuchsia cv. ‘Pink Galore’


PBZ Drench 0 ppm

(S0)

10 ppm

(S1)

20 ppm

(S2)

30 ppm

(S3)

0 ppm (D0) 15.19 17.73 20.15 18.53 17.90

10 ppm (D1) 19.25 21.92 18.10 20.47 19.93

20 ppm (D2) 19.27 19.97 18.39 20.47 19.52

30 ppm (D3) 20.13 25.53 24.00 22.30 22.99

Mean 18.46 21.29 20.16 20.44

CD0.05

D - 1.05 S - 1.05 D*S - 2.10

Table 6: Effect of paclobutrazol on presentability of Fuchsia cv. ‘Pink Galore’


PBZ Drench 0 ppm

(S0)

10 ppm

(S1)

20 ppm

(S2)

30 ppm

(S3)

0 ppm (D0) 65.07 78.47 84.60 80.80 77.23

10 ppm (D1) 86.07 82.98 84.07 83.83 84.24

20 ppm (D2) 85.53 84.60 82.94 78.38 82.86

30 ppm (D3) 84.70 85.60 82.43 80.35 83.27

Mean 80.34 82.91 83.51 80.84

CD0.05

D - 1.54 S - 1.54 D*S - 3.08

treatments with paclobutrazol (table 5). But

maximum percent increase (40.50%)

duration of flowering over control was

achieved with 30ppm drenches along with

10ppm foliar application of paclobutrazol.

The increased duration of flowering by

paclobutrazol may be attributed to more

number of flowers produced by

paclobutrazol application.

The presentability (table6) of plants

enhanced in all the plants treated with

paclobutrazol as compared to control.

Presentability, which primarily depends

upon the compactness and the ratio of plant

with the pot, was found to be maximum


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(86.07) in plants drenched with 10 ppm

paclobutrazol. Increase in the presentability

score was due to the overall enhancement of

the presentability attributes, which resulted

in production of dwarf, compact plants with

shorter stalks, balanced growth and

flowering. Similar boost in the presentability

in other ornamentals was reported by Bharti

and Rajesh (2001) in Geranium and by

Pathak and Sharma (1996) in Begonia x

tuberhybrida and Primula obconica.

References:

Andrade-Rodrguez-M, Colinas Leon MT

and Lozoya-Saldana H. (1991).

Chemical treatment for growth control in

impatiens (I.walleriana S.). Revista

Chapingo. 15: 73-74.

Andrasek K. (1989). Increasing the

ornamental value of Hibiscus rosa-

sinensis and P.hortorum cv. Springtime

by using gibberellin inhibitor growth

regulator. Acta Horticulture. 251: 329-

333

Armitage AM. (1984). Chlormequat induced

early flowering of hybrid geranium: the

influence of gibbrellic acid. Hort

Science21 (1): 116-118

Bazzocchi R, Giorgioni ME and Bedonni B.

(2001). Morphological alterations

induced in Fuchsia X hybrida by

paclobutazol and uniconazole. Italus-

Hortus. 8 (1): 26-33

Bharti S and Bhalla Rajesh. (2001).

Paclobutrazol and azotobacter enhance

the presentability of potted geraniums.

Journal of Ornamental Horticulture. 5:

63-64

Davis TD and Anderson AS. (1989). Growth

retardants as aid in adapting new

floricultural crops to pot culture. Acta

Horticulturae 252:77-84

Gad M, Schmidt G and Gerzson L. (1997).

Comparison of application methods of

growth retardants on the growth and

flowering of Fuchsia magellanica Lan.

Horticultural Science. 29(1/2): 70-73

Gianfagna TJ and Wolster BG. (1986).

Growth retardants as an aid to adapting

freesia to pot culture. Hort Science.

21(2): 263-264

Horn W and Wischer M. (1987). Bonzi, a

new growth retardant for Elatior

begonias. Gb+ -G – Gartnerborse –

und- Gartenwelt. 87(51): 1894-1895

Mertens M. (1992). Large-flowered

rhododendrons as pot plants.

Verbondsnieuws-voor-de-Belgische-

Sierteelt. 36(2): 67-69

Narender, Pathak and Sharma Y.D. (1996).

Effect of plant bioregulators and potting

mixture on Primula obconica. Journal of

Ornamental Horticulture. 4(1/2): 30-32

Schekel A and Blau S. (1986). Influence of

paclobutrazol and gibberellic acid on

flowering of geranium. Hort Science. 22:

1155 (Abst.)


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Kaintura | ISOLATION AND CHARACTERIZATION OF DESIRABLE COLOUR 88

Short Communication

ISOLATION AND CHARACTERIZATION OF DESIRABLE COLOUR MUTANT OF

CARNATION

Pooja Kaintura

Department of Horticulture, VCSG Uttarakhand University of Horticulture and Forestry,

Bharsar , Uttarakhand, 263145


______________________________________________________________________________

Abstract Development of new cultivars through conventional or modern techniques has been a great

demand in commercial floriculture. New colour, earliness, stem length, number of flowers, plant

architecture, resistance to abiotic and biotic stress, productivity and vase life are the main

attributes required in new cultivars. Mutation breeding is an established method for crop

improvement and has played a major role in the development of many new flower colour/shape

mutant varieties in ornamentals. A spontaneous mutant was observed in a carnation at

Floriculture Center of VCSG Uttarakhand University of Horticulture and Forestry, Bharsar,

Uttarakhand during 2013.The mother plant of the mutant was variety ‘Tempo’. Initially plant

showed sectoral chimera on flower but latter on mutant plant developed flower having change in

whole colour of flower from creamish white to deep pink on few branches.

Key words: Carnation, mutant and isolation

Introduction:

arnation (Dianthus caryophyllus L.)

is a leading cut flower in

International flower market having

great commercial value as a cut flower due

to its excellent keeping quality, wide array

of colour and forms. It belongs to the family

Caryophyllaceae having diploid

chromosome number 2n=30. Carnation,

apart from producing cut flowers can also

become useful in gardening for bedding,

edging, borders, pots and rock gardens. Due

to its popularity it has been subjected to

intense breeding efforts for the past few

hundred years resulting in development of

almost all possible colour through different

technique. Development of new cultivars

through conventional or modern techniques

has always been in demand in commercial

floriculture. Mutation breeding is an

established method for crop improvement

and has played a major role in the

development of many new flower

colour/shape mutant varieties in ornamentals

New colour, earliness, stem length, number

of flowers, plant architecture, resistance to

abiotic and biotic stress, productivity and

vase life are the main attributes required in

new cultivars.

Inductions of variation through

mutation has been found beneficial in many

crops. Any change in the dominant genes is

easily expressed in the first generation and

thus the selection of mutant of directly

perceptible characters like flower colour,

shape, size etc is generally very easy and

can directly be put to commercial

use.(Gustafsson, 19601 and Broertjes, 1969

2

and 19723).

C



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Observation and Discussion:

A spontaneous mutant of

Carnation has been observed at at

Floriculture Center of College of

Horticulture, VCSG Uttarakhand University

of Horticulture and Forestry, Bharsar

,Uttarakhand located at a latitudeof

30°03’35"N and longitude of 78°59’42"E and at an

altitudeof 2000 m above mean sea level

during 2012.. The area falls under wet

temperate type of climate in central

Himalayan region.

The mother plant of the mutant

was variety ‘Tempo’ having cream colour

flower base having red colour on margins of

petal. The mother plants of the mutant were

initially planted at naturally ventilated

polyhouse having Fan and pad system.

Initially muntant plant showed sectoral

chimera on flower showing change in half

of petal colour (plate 1 and plate 2) but latter

on mutant plant developed flower having

change in whole colour of flower from

creamish white to deep pink on few

branches (plate 3).

Plate – 1

Plate – 2

Plate – 3

Momose M et al (2013) studied the

molecular mechanisms underlying

spontaneous bud mutationsin carnation, and

described a new active hAT type

transposable element, designated Tdic101,

the movement of which caused a bud

http://www.ncbi.nlm.nih.gov/pubmed/?term=Momose%20M%5BAuthor%5D&cauthor=true&cauthor_uid=23543146


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mutation in carnation that led to a change of

flower color from purple to deep pink. The

color change was attributed to Tdic101

insertion into the second intron of F3'H, the

gene for flavonoid 3'-hydroxylase

responsible for purple pigment production.

Regions on the deep pink flowers of the

mutant can revert to purple, a visible

phenotype of, as we show, excision of the

transposable element. Sequence analysis

revealed that Tdic101 has the characteristics

of an autonomous element encoding a

transposase. A related, but non-autonomous

element dTdic102 was found to move in the

genome of the bud mutant as well. Its

mobilization might be the result of

transposase activities provided by other

elements such as Tdic101. In carnation,

therefore, the movement of transposable

elements plays an important role in the

emergence of a bud mutation.

Iizuka K., 1979 treated Rooted cuttings

of 30 leading varieties of carnation in Japan

were used as the materials for induction of

flower color changes by .gamma.-rays. The

total dose of 3 kR was given at the exposure

rate 120 R/min and 52 R/min, respectively.

To stimulate the growth of axillary buds and

to get complete variants, the growth points

of the primary and secondary stems were cut

back 3 times, once before and twice after

irradiation. Flower color changes occurred

with a high frequency of 3.3-30% including

complete and partial changes, of which 43%

was complete. Flower color changes

occurred in various directions, frequently

from genetically recessive to dominant

colors. Flavonoid analyses were performed

on flower petals on both original and

changed colors separately by paper

chromatography. All anthocyanins were

identified, but the flavonoids except

anthocyanins were judged simply by

comparison of the number and position of

the spots on the chromatograms. In 'Clear

Pink', anthocyanidin changed from cyanidin

to pelargonidin. This is the only case caused

by gene mutation. Changes in flavonoid

patterns, except anthocyanins, followed the

changes of anthocyanins. Several spots

observed in the chromatogram of the

original color disappeared and new spots

appeared simultaneously in that of the

changed color. The above-mentioned

phenomenon together with the high

frequency of variability, especially from

recessive to dominant character, indicate as

the cause of this variability, the significance

of the uncovering or rearrangement of

chimeral layers rather than mutation. True

mutations and some chromosomal

aberrations also may be involved.

Scovel G(2000) identified two

mutants which were heterozygous for a

mutation in a gene termed evergreen. In

these mutants, spike-like clusters of

bracteoles subtend each flower. Genetic

analysis of the mutants confirmed the semi

dominant nature of this nuclear mutation and

that the two original mutants were allelic at

the evergreen locus. In homozygous mutant

plants, a more severe phenotype was

observed. Flower formation was completely

blocked and spike like clusters of bracteoles

did not subtend any flowers. Morphological

characterization of mutant plants revealed

that vegetative growth and inflorescence

structure are not affected by the mutant

allele. In plants heterozygous for the

evergreen mutation, fertility, petal and pistil

length, calyx diameter, and stamen number

were not affected.

References:

Broertjes, C. 1969. Induced mutation and

breeding methods in vegatively

propagated species. In: Induced mutations

in plants. IAEA, Vienna, pp 325-329.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Scovel%20G%5BAuthor%5D&cauthor=true&cauthor_uid=11218088


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Broertjes, C. 1972. Improvement of

vegatively Propagated crops by ionizing

radiations. In: Induced mutation and plant

improvement. IAEA, Vienna, pp 393-

299.

Gustafsson, A. 1960. Polyploidy and

mutagenesis in forest tree breeding. In:

Proceedingof 5th world Cong. II.

Genetics and Tree Improvement, 793-

806.

Iizuka K., 1979: Induction of flower color

changes in carnation dianthus

caryophyllus by gamma irradiation.

Bulletin Of The Faculty Of Agriculture

Tamagawa University: 9-42

Momose M

, Nakayama M, Itoh Y,

Umemoto N, Toguri T, Ozeki Y. 2013

An active hAT transposable element

causing bud mutation of carnation by

insertion into the flavonoid 3'-

hydroxylase gene.Mol Genet Genomics.

Apr; 288(3-4):175-84.

Scovel G, Altshuler T, Liu Z, Vainstein A.

2000 The Evergreen gene is essential for

flower initiation in carnation J Hered.

2000 Nov-Dec;91(6):487-91.

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http://www.ncbi.nlm.nih.gov/pubmed/23543146

http://www.ncbi.nlm.nih.gov/pubmed/?term=Scovel%20G%5BAuthor%5D&cauthor=true&cauthor_uid=11218088

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http://www.ncbi.nlm.nih.gov/pubmed/11218088

PLANTICA – Journal of Plant Science ISSN: 2456 - 9259 (The Official Publication of Association of Plant Science Researchers) Plantica, Vol. 1 (2), 2017: 92 –98

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Singh et. al. | EFFECTS OF SALINITY STRESS CONDITION IN SEED GERMINATION AND VIGOUR 92

Research Article

EFFECTS OF SALINITY STRESS CONDITION IN SEED GERMINATION AND VIGOUR

OF Aegle marmelos

Rakesh Singh, V.P Nautiyal, J.S. Chauhan and Ganga Dutt

.

Department of Seed Science and Technology

Hemwati Nandan Bahuguna Garhwal University (Central University), Srinagar, Uttarakhand,

India

Corresponding author: [email protected]

_____________________________________________________________________________

Abstract In cultivation of medicinal trees, stress conditions are very important problem. Salinity stress is

major problem of increasing production in crop growing areas throughout the world. The

objective of this research was to evaluate effect of salinity stress (NaCl) on seed germination and

vigour of bael (Aegle marmelos). The present study was conducted to examine the effects of

Salinity stress (seeds were soaked in solution with concentration of 5% 10% and 15% NaCl for 3

hours) treatment along with control (Without any treatment) on seed germination and seedling

quality character of bael (Aegle marmelos). The results showed that the effect of salinity was

significantly decrease seed germination percentage; seedling length, seedling vigour and dry

matter production than control. Mean comparison showed that control and stress conditions

treatments those after 25 days and the maximum germination (76.67), seedling length (14.4 cm),

dry matter production (0.19gm), vigour index -I ( 1073.79), and vigour index 2 (14.22).were

achieved in control (Without any treatment) of bael seeds but in stress condition failed to

improve germination. Hence bael seeds can be showed lower germiniability and vigour on

Salinity stress condition.

Key words: Salinity stress (NaCl), germination, seedling, vigour and bael.

Introduction:

egle marmelos (L.) Corr.,is a popular

medicinal tree belonging to the

family Rutaceae and its various parts

are used in Ayurvedic and Siddha medicines

to treat a variety of ailments. The tree grows

wild in dry forests of hills and plains of

tropical and subtropical region of Central

and Southern India, Burma, Pakistan,

Bangladesh, Sri Lanka, Northern Malaya,

and Java Islands (Islam et al., 1995). Almost

all parts of the tree are used in preparing

herbal medicine (Kala, 2006) .The roots are

useful for treating diarrhea, dysentery and

dyspepsia. The tree is rich in alkaloids,

among which aegline, marmesin, marmin

and marmelosin are the major ones. The

compounds luvangetin and pyranocoumarin,

isolated from seeds showed significant

antiulcer activity (Goel et al., 1997).

Essential oil isolated from the leaf has

antifungal activity (Rana et al., 1997).

The foundation for revitalization of local

health traditions (FRLHT), Bangalore, India

assessed threat status of bael (A. marmelos)

A



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tree as rare, endangered and threatened

(RET) species, especially endangered

species and importance is being given for

mass multiplication through various

propagation techniques. The tree is normally

grown with seeds (Nayak and Sen, 1999).

Many medicinal plants need to be cultivated

in order to satisfy their increasing demand,

but salinity and other forms of abiotic stress

represent serious threats to the growth and

crop yields (Qureshi et al., 2005). In fact,

salinity is a major environmental problem to

crop productivity throughout the arid and

semi-arid regions of the world (Foolad &

Lin, 1997). So, salt tolerance at germination

stage is an important factor and has

detrimental effects on germination of seeds

(Sharma et al., 2004). Seed germination

seedling growth characters are very

important factors for determining seedling

establishment. Seed vigour index and shoot

length are among the most sensitive to

Salinity stress, followed by root length and

coleoptiles length. Environmental stress

during seed production period can has

negative impact on seeds quality (Sediyma

et al., 1972).

The purpose of this experiment is

considering salinity stress impacts which are

results of NaCl on germination of Aegle

marmelos seeds. In the present study,

different concentration of NaCl was used

for salinity stress induction, respectively in

seed germination and vigour of Aegle

marmelos, an ethanobotanically highly

medicinal plant.


he freshly ripped fruits of Aegle

marmelos were collected from

healthy well growing tree from

chauras campus of H.N.B.G. University

Srinagar in the month of May-June, 2014.

Then the collected fruits were breakdown to

remove the outer shell of fruits and to get

the seeds. Removed seeds were macerated

with water for 48 hours, to remove the pulp

and after that seeds were dried properly at

room temperature (25 ±10C) for 3-4 days.

Treatment Preparation:

The study was performed in a randomized

factorial method with 100 seeds of 4

replicates prepared as per International Seed

Testing Association (ISTA) and were

sterilized with 1% HgCl2 solution for five

minutes followed by three times wash with

double distilled water and were kept in

germinator maintained at 25±2°C 25 days,

for control 100 seed per replicate were put in

to the petri dishes and water was used as a

wetting agent. For salinity stress, seeds were

soaked in solution with concentration of 5%

10% and 15% NaCl for 3 hours separately

and were transferred to petri dishes for

salinity stress.

During the process of germination, the seeds

were observed for days to first germination

and based on the germination observations

taken on every day germination. After 25

days of germination period, seedlings were

evaluated for germination based on normal

seedling characters and the results were

reported in percentage. Ten randomly

selected normal seedlings were measured for

the vigour parameters, After the final day of

testing (the 25th

day), mean daily

germination, percentage of germination,

mean germination time, germination rate,

germination energy, allometric trait, seedling

strength index were calculated using the

following formulas:

Final Germination Percentage (FGP)

The following formula was used to calculate

the percentage of germination (Nicols,

1968).

T


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S: Number of germinated seeds.

T: Total number of seeds.

Seedling length (cm):

Ten normal seedlings were selected

randomly and measured the shoot & root

length of them. The root and shoot length

was measured from the tip of primary root to

base of the hypostyle, and from the base of

primary leaf to the base of hypostyle,

respectively. Total length of seedling

obtained by adding roots and shoots length.

Root length (cm):

Final count was observed on 25th day and

normal seedlings were selected randomly

and measured the root length of them. The

root length was measured from the tip of

primary root to base of the hypocotyls and

the mean root length was expressed in

centimetres.

Shoot length (cm):

Final count was observed on 25th day and

normal seedlings were selected randomly

and measured the shoot length of them. The

shoot length was measured from the base of

primary leaf to the base of hypocotyls and

the mean shoot length was expressed in

centimetres.

Seedling fresh and dry weight (mg):

Ten normal seedlings used for measuring the

seedling length, initially fresh weight was

taken and put in the butter paper bag and

dried in a hot air oven, maintained at 70 ±

1ºC temperature for 24 hours. Then the

seedlings were removed and allowed to cool

in a desiccators for 30 minutes, the weighing

was done in an electronic balance. The

weights of dried samples were recorded and

average of ten seedling dry weight in

milligrams was recorded. Fresh weight – Dry weight

Dry weight (%) = -------------------------------------- X 100

Fresh weight

Vigor Index of Seedling:

The vigour index of seedling was calculated

by adopting the method suggested by (Abdul

Baki & Anderson 1973) and expressed as

whole number for each treatment by using

the below formula.

A). Vigour Index-I Vigour index-I was

computed by using the following formula

and expressed as whole number (Abdul &

Baker, 1973).

Vigour Index-I = Germination (%) X

Seedling length (cm)

Where, seedling length = Root length +

Shoot length

B). Vigour Index-II

The Vigour index - II of seedling was

calculated by adopting the method suggested

by (Abdul-Baki & Anderson 1973), and

expressed as whole number for each

treatment by using the formula mentioned

below.

Vigour index-II = Germination (%) X

Seedling dry weight (mg)

Statistical analysis

Data recorded during the course of

investigations were subjected to statistical

analysis under Factorial Completely random

block design by Snedecor and Cochram

(1968). Valid conciliations were drawn after

the determination of significance difference

between the treatments, at 5 and 1 per cent

level of probability. Critical difference was

calculated in order to compare the treatment

means.


ffect of seed salinity stres showed

lower germination than untreated

seeds. Seeds salinity stress for 5%

10% and 15% NaCl for 3 h resulted in later

E


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emergence than control (without any

treatment) 25 days after final count. Total

germination percentage showed significantly

lower germination in salinity treated seeds

than control (untreated) seeds. Therefore,

optimal yield could be achieved by fast

germination and uniform emergence on the

nursery. This implies that salinity is the

important factor to decrease germination.

However, total percentage in non treated

seeds was higher compared with those

derived from the salinity treated seeds for all

salinity stress levels. The effects of salinity

stress on seedlingsl length have been

showed in table -1.comparison of seedlings

length means in different salinity stress

levels showed that when salinity level

increase, seedlings length decrease. Effect of

seed salinity stress on the germination

percentage of bael seedling (Table 1)

showed significant effect of salinity stress

on the germination of bael seeds with seed

treatment for 5% NaCl for 3 h which

recorded significantly lower germination

percentage ( 70.33%) than control (76.67%).

But above 5% concentration, the salt had an

inhibitor action and the length of seed lings

are being shortened in depending on the

concentration of NaCl.

Seedling growth and vigour index:

Effect of salinity stress on seedlings growth

of the bael seedlings showed significant

effect of salinity stress on the seedlings

length, dry matter production and vigour

index (Table 1 and 2) Significantlylower

seedling length (2.05cm) were recorded in

seeds that were treated NaCl 5% than

control seeds(14.4). Similarly, dry matter

production (40mg) and vigour index –I

(143.86), vigour index-II (3.09) that was

salinity treated were inferior to the control.

When salinity level increase, seedlings

length and vigour index decrease. The most

reduction in seedlings length related to

control. But, for all salinity levels, reduction

was significantly higher in salinity treated

seeds than control seeds. This could be due

to Salinity affects plant growth, activity of

major cytosolic enzymes by disturbing

intracellular potassium homeostasis, causing

oxidative stress and programmed cell death,

reduced nutrient uptake, metabolic toxicity,

inhibition of photosynthesis, reduced CO2

assimilation and reduced root respiration

(Sairam & Srivastava, 2002; Cuin &

Shabala, 2007; Chen et al., 2007; Shabala,

2009; Abogadallah, 2010; Demirkiran et al.,

2013 and Liu et al., 2014). Yildirim et al.,

2008 & Mori et al., 2011 .Earlier studies

have shown that NaCl treatment decreased

the some growth parameters such as fresh

weight of shoot and root of plants Sivritepe

et al., 2003 & Jamil et al., (2006) A negative

effect of salinity on germination and

emergence has been reported for several

vegetables species. Botia et al., (1998).

Had studied that Salt also delays

germination and slows its speed. This delay

could be due to the alteration of some

enzymes and hormones which are present in

the Seed. Gill et al., (2003) added that the

germination delay can be due to a problem

of seed hydration following a high osmotic

potential which cause the inhibition of some

mechanisms leading to radicle emergence.

Jaber Arves et. al,(2013) have been studied

similar the effects of salinity stress (made by

sodium chloride and calcium chloride) and

temperature interaction on germination

characteristics of hyssopus officinalis.

Exposure to environmental stress due to

salinity has been reported to result in

adverse effects on the growth of plants.


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Table 1: Effect of salinity stress on germination percentage, seedling length and dry matter

production in bael (Aegle marmelos)

Treatment Germination % Seedling length(cm) Dry matter production

(gm seedlings-10

)

Mean S.D Mean S.D Mean S.D

Control 76.67 4.163 14.04 .115 .19 .011

NaCl 5% 70.33 1.528 2.05 .495 .04 .005

NaCl 10% 30.33 1.528 1.51 .190 .02 .004

NaCl 15% 12.67 3.055 .10 .023 .02 .009

Table 2: Effect of salinity stress on Vigour Index- I and Vigour Index- II in bael

(Aegle marmelos)

Treatment Vigour index I Vigour index II

Mean S.D. Mean S.D.

Control 1073.79 40.250 14.22 1.251

NaCl 5% 143.86 32.616 3.09 .317

NaCl 10% 47.53 8.008 3.09 .317

NaCl 15% 1.31 .354 .25 .021

Conclusion:

he final conclusion was found that

the Based on the information

obtained in this research work was

found that the salinity stress treatments for

5%,10%,and 15% for 3 h was significantly

decrease the germination percentage,

seedling weight & vigour indexes than

control in Aegle marmelos. Exposure to

environmental stress due to salinity has been

reported to result in adverse effects on the

growth of plants.

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