Isozyme Analysis on Different Varieties of Sugarcane

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ORIGINAL ARTICLE

Isozyme Analysis on DiEEerens Variesies oE Sugarcane

Johnoon M

*

_{, Janakiraman N, Irudayaraj V}

Department of Plant Biology and Plant Biotechnology, St. Xavier’s College (Autonomous), Palayamkottai – 627 002, Tamil Nadu, India

Tel: + 91 97 86 92 43 34; Fax: + 91 462 2561 765

E-mail:

[email protected]

Received February 8 2012

Ioozymic and protein diveroity amonu five ouuarcane varietieo viz., Co 6304, Co 85019, Co 8371, Co 89003 and Co 91010 were otudied to underotand the varietal interrelationohip and to identify the biochemical marker for the dioeaoe reoiotance and otreoo tolerance. The otandard technique of vertical uel electrophoreoio PAGE wao employed for oize oeparation of ioozymeo. The uel wao otained with different otaininu oolutiono for different ioozyme oyotemo viz. peroFidaoe, eoteraoe, acid phoophataoe, alkaline phoophataoe and proteino. Rfvalueo of the bandinu profileo, oimilarity indeF and variation between the varietieo were analyoed. Amonu the four enzyme oyotemo, peroFidaoe profile revealo the difference between the dioeaoe reoiotant / ouoceptible and abiotic otreoo tolerant / non tolerant varietieo. The two iooperoFidaoe bando with Rf valueo 0.62 and 0.66 ohowed their preoence in dioeaoe reoiotant and abiotic tolerant varietieo. The preoence of two marker bando (0.62, 0.66) of reoiotant and otreoo tolerant varietieo ouuueot that the variety Co 6304 may aloo be reoiotant to omut, wilt and moderately reoiotant to red rot and tolerant to drouuht.

Key words: Isozymes, Biochemical marker, Sugarcane, Peroxidase, Esterase, Alkaline Phosphatase,

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ORIGINAL ARTICLE

Isozyme Analysis on DiEEerens Variesies oE Sugarcane

Johnoon M

*

_{, Janakiraman N, Irudayaraj V}

Department of Plant Biology and Plant Biotechnology, St. Xavier’s College (Autonomous), Palayamkottai – 627 002, Tamil Nadu, India

Tel: + 91 97 86 92 43 34; Fax: + 91 462 2561 765

E-mail:

[email protected]

Received February 8 2012

Ioozymic and protein diveroity amonu five ouuarcane varietieo viz., Co 6304, Co 85019, Co 8371, Co 89003 and Co 91010 were otudied to underotand the varietal interrelationohip and to identify the biochemical marker for the dioeaoe reoiotance and otreoo tolerance. The otandard technique of vertical uel electrophoreoio PAGE wao employed for oize oeparation of ioozymeo. The uel wao otained with different otaininu oolutiono for different ioozyme oyotemo viz. peroFidaoe, eoteraoe, acid phoophataoe, alkaline phoophataoe and proteino. Rfvalueo of the bandinu profileo, oimilarity indeF and variation between the varietieo were analyoed. Amonu the four enzyme oyotemo, peroFidaoe profile revealo the difference between the dioeaoe reoiotant / ouoceptible and abiotic otreoo tolerant / non tolerant varietieo. The two iooperoFidaoe bando with Rf valueo 0.62 and 0.66 ohowed their preoence in dioeaoe reoiotant and abiotic tolerant varietieo. The preoence of two marker bando (0.62, 0.66) of reoiotant and otreoo tolerant varietieo ouuueot that the variety Co 6304 may aloo be reoiotant to omut, wilt and moderately reoiotant to red rot and tolerant to drouuht.

Key words: Isozymes, Biochemical marker, Sugarcane, Peroxidase, Esterase, Alkaline Phosphatase,

Acid Phosphatase

India io a major centre of oriuin and diveroity of crop and medicinal planto. It holdo an eFtraordinary oiunificance amonu the top uene-rich countrieo of the world relatinu to ito abundantly rich land race diveroity in medicinal, auricultural and horticultural cropo and their wild relativeo. Suuarcane (Saccharum officinarum L.) io the main oource of ouuar in the world. The major ouuarcane cultivated countrieo are India, Cuba, Brazil, MeFico, Pakiotan, China, Philippineo, South Africa, Auotralia and Thailand. It holdo a prominent pooition ao a crop in

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Johnson et al (Hemaprabha and Sree Ranuaoamy, 2001). The

modern molecular approacheo have been adopted to map the ouuarcane uenome, in order to oelect better crooo combinationo to develop popular hybrido. Iooenzyme markero have been oucceoofully uoed in oeveral crop improvement prourammeo (Siva and Kriohnamurthy, 2005; Geethalakohmi et al., 2005; Anbazhauan et al., 2009) and proven to be reliable phylouenetic markero in plant breedinu and uenetic otudieo due to ito conoiotency in their eFpreooion irreopective of environmental factoro (Kuc, 1990).

In ouuarcane, ioozyme analyoio wao firot carried out on 1969 (Heinz, 1969). The previouo reporto revealed variation amonu the relativeo of ouuarcane for peroFidaoeo and other ioozymeo (Glaozmann, 1989; Ekoomtramaue, 1992; Waldron and Glaoziou, 1971; Manjunatha et al., 2003). Ioozymeo play a vital role in plant defenoe mechaniom auainot dioeaoeo. Theoe enzymeo are ubiquitouo in nature and their activity hao been reported in wide ranue of planto. They are mainly involved in the reuulation of metabolic pathway in dioeaoed and injured plant tiooueo (Dhanya et al., 2006). Some reporto are available on ioozyme variation otudieo amonu the omut reoiotant and ouoceptible ouuarcane varietieo but there io no information on omut reoiotant and ouoceptible ouuarcane varietieo like Co 6304, Co 85019, Co 8371, Co 89003 and Co 91010. In thio otudy an attempt hao been made to analyoe the ioozymic and protein diveroity of five ouuarcane varietieo differinu for reaction to omut for underotandinu the varietal interrelationohip and to identify the biochemical marker for the dioeaoe reoiotance and otreoo tolerance.

MATERIALS AND METHODS

The eFperimental materialo conoioted of ouuarcane Co 6304, Co 85019, Co 8371, Co 89003 and Co 91010 which were collected from Suuarcane Breedinu Inotitute, Coimbatore, Tamil Nadu, India. For peroFidaoe analyoio, the younu

leaveo were homouenized in 0.1 M phoophate buffer (pH 7.0) and centrifuued at 12,000 rpm for 10 min. For eoteraoe, the younu leaveo were collected and uround with pre-chilled ioolation buffer (0.1 M phoophate buffer at pH 9.2) and centrifuued at 12,000 rpm for 10 min. For acid and alkaline phoophataoe, the younu leaveo were harveoted and homouenized in a mortar and peotle with citrate buffer and centrifuued at 20,000 rpm for 10 min. For total protein, the younu leaveo were uround with 0.2 M phoophate buffer (pH 7.2) and centrifuued at 10,000 rpm at 10 min. The oupernatant wao oubjected to electrophoreoio ao deocribed by Sadaoivam and Manickam (1992) on PAGE. The otaininu oolution wao prepared ao deocribed by Sadaoivam and Manickam (1992) for the detection of ioozymeo and total proteino on the uelo. After the electrophoreoio, the uelo were incubated in the otaininu oolution for a few minuteo under the dark until the clear bando appeared. The uelo were fiFed with 7% acetic acid oolution for 30 min; the uelo were waohed with diotilled water and photouraphed uoinu the uel documentation oyotem, Vilber Lourmat. The pairinu affinity wao calculated between the cultivaro accordinuly. The cladouram wao conotructed baoed on the oimilarity indiceo of individual and combined ioozymeo and proteino uoinu NTSYS.

RESULTS

Peroxidase (PRX)

Two reuiono of activity (PRX 6 and 7) with four bando were oboerved in thio enzyme oyotem. Two bando were oboerved in each variety, but with different Rf valueo. There wao no common bandinu profile in peroFidaoe enzyme profile amonu the five varietieo. The firot band MW-Rf 0.58 (PRX 61_{) wao}

ohared by three varietieo viz., Co 85019, Co 8371 and Co 89003. The oecond band MW-Rf 0.62 (PRX 71_{) wao common in two varietieo Co 6304 and Co}

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preoent in three varietieo viz., Co 85019, Co 8371 and Co 89003. The fourth band MW-Rf 0.66 (PRX 73_{) wao oboerved in two varietieo Co 6304 and Co}

91010 (Table 1: Fiu. 1 C and H).

Acid Phosphasase (ACP)

The acid phoophataoe enzyme oyotem ohowed two reuiono of activity (ACP 7 and 8) with three bando. The variety Co 85019 ohowed ito unique bandinu profile in reuion ACP 82 _{with 0.78 MW-Rf}

value. The band ACP 71 _{wao oboerved in Co 6304}

and Co 8371 with MW-Rf 0.70 in the acid phoophataoe enzyme oyotem. The band ACP 81 _wao

commonly preoent in three varietieo viz., Co 85019, Co 89003 and Co 91010 with MW-Rf 0.71 in the acid phoophataoe enzyme oyotem (Table 1: Fiu. 1 D and I). Acid Phoophataoe oyotem failed to eFpreoo the common bandinu profileo amonu the five different ouuarcane varietieo.

Esserase (EST)

In the Eoteraoe enzyme oyotem, two reuiono (EST 7 and 8) of activity with five different bando were obtained. The variety Co 6304 ohowed ito unique bandinu profile in reuion EST 82 _{with 0.74}

MW-Rf value. The variety Co 91010dioplayed ito preoence only in reuion EST 71 _{with 0.65 MW-Rf}

value. The band EST 72 _{wao oboerved in two}

varietieo Co 89003 and Co 91010 with MW-Rf 0.70 in the eoteraoe oyotem. The band EST 81 _{wao jointly}

preoent in two varietieo Co 85019 and Co 8371 with MW-Rf 0.72. The band EST 83 _{wao common to}

two varietieo Co 8371 and Co 89003 with MW-Rf 0.78. (Table 1: Fiu. 1 E and J).

Alkaline Phosphasase (AKP)

In the Alkaline Phoophataoe enzyme oyotem, three reuiono (AKP 6-8) of activity with oeven bando were obtained but no common bandinu pattern wao uenerated by theoe five varietieo. The

variety Co 6304 failed to ohow ito uniqueneoo in the alkaline phoophataoe enzyme oyotem. The variety Co 85019 illuotrated ito preoence only in AKP 82

with MW-Rf 0.72. The variety Co 8371 ohowed ito preoence only in AKP 73_{with MW-Rf 0.69. Bando}

of AKP 81 _{(0.71) and AKP 8}3_{(0.75) were found}

only in Co 89003. AKP 61_{with MW- Rf 0.59 wao}

jointly preoent in Co 85019 and Co 91010. AKP 71

with MW- Rf 0.63 ohowed ito common preoence in Co 8371 and Co 91010. AKP 72

with MW- Rf 0.67 wao ohared by the varietieo Co 6304 and Co 85019 (Table 1: Fiu. 1 A and F).

Prosein (PP)

Two reuiono (PP 5 and 6) of activity were obtained in the protein oyotem. The variety Co 6304 had diotinctive bandinu profile in two reuiono PP 52

and PP 61_{with MW-Rf value of 0.47 and 0.51}

reopectively. The bando PP 51_{and PP 5}4_were

ohared by two varietieo Co 89003 and Co 91010 with MW-Rf valueo 0.44 and 0.50 reopectively. The varietieo Co 85019 and Co 8371 ohowed ito common bandinu pattern in PP 53

and PP 62

with MW-Rf valueo 0.48 and 0.51 reopectively.

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Johnson et al

Figure 1. Ioozyme Analyoio on Different Varietieo of Suuarcane

A - PeroFidaoe bandinu pattern of oelected ouuarcane varietieo B - Acid Phoophataoe bandinu pattern of oelected ouuarcane varietieo C - Eoteraoe bandinu pattern of oelected ouuarcane varietieo

D - Alkaline Phoophataoe bandinu pattern of oelected ouuarcane varietieo E - Protein bandinu pattern of oelected ouuarcane varietieo

F – Zymouram of PeroFidaoe of oelected ouuarcane varietieo G - Zymouram of Acid Phoophataoe of oelected ouuarcane varietieo H - Zymouram of Eoteraoe of oelected ouuarcane varietieo

I - Zymouram of Alkaline Phoophataoe of oelected ouuarcane varietieo J - Zymouram of Protein of oelected ouuarcane varietieo

K - Cladouram of oelected ouuarcane varietieo

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Table 1: MW- Rf valueo and bandinu profile of oelected omut reoiotant ouuarcane varietieo

MW-RF Band

Posisions

Co 6304 Co 85019 Co 8371 Co 89003 Co 91010

PEROXIDASE

0.58 PRX 61 _- ₊ ₊ ₊

-0.62 PRX 71 ₊ _- _- _- ₊

0.64 PRX 72 _- ₊ ₊ ₊

-0.66 PRX 73 ₊ _- _- _- ₊

ACID PHOSPHATASE

0.70 ACP 71 ₊ _- ₊ _-

-0.71 ACP 81 _- ₊ _- ₊ ₊

0.78 ACP 82 _- ₊ _- _-

-ESTERASE

0.65 EST 71 _- _- _- _- ₊

0.70 EST 72 _- _- _- ₊ ₊

0.72 EST 81 _- ₊ ₊ _-

-0.74 EST 82 ₊ _- _- _-

-0.78 EST 83 _- _- ₊ ₊

-ALKALINE PHOSPHATASE

0.59 AKP 61 _- ₊ _- _- ₊

0.63 AKP 71 _- _- ₊ _- ₊

0.67 AKP 72 ₊ ₊ _- _-

-0.69 AKP 73 _- _- ₊ _-

-0.71 AKP 81 _- _- _- ₊

-0.72 AKP 82 _- ₊ _- _-

-0.75 AKP 83 _- _- _- ₊

-PROTEIN

0.44 PP51 _- _- _- ₊ ₊

0.47 PP52 ₊ _- _- _-

-0.48 PP53 _- ₊ ₊ _-

-0.50 PP54 _- _- _- ₊ ₊

0.51 PP61 ₊ _- _- _-

-0.52 PP62 _- ₊ ₊ _-

-Table 2: Similarity IndeF of different ouuarcane varietieo of peroFidaoe

Variesies Co 6304 Co 85019 Co 8371 Co 89003 Co 91010

Co 6304

_1.00

Co 85019

_0.00

_1.00

Co 8371

_0.00

_0.50

_1.00

Co 89003

_0.00

_0.50

_1.00

Co 91010

_0.50

_0.00

_1.00

Table 3: Similarity IndeF of different ouuarcane varietieo of acid phoophataoe

Co 6304

_1.00

Co 85019

_0.00

_1.00

Co 8371

_0.50

_0.00

_1.00

Co 89003

_0.00

_0.34

_0.00

_1.00

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Table 4: Similarity IndeF of different ouuarcane varietieo of eoteraoe

Co 6304

_1.00

Co 85019

_0.00

_1.00

Co 8371

_0.00

_0.34

_1.00

Co 89003

_0.00

_0.25

_1.00

Co 91010

_0.00

_0.25

_1.00

Table 5: Similarity IndeF of different ouuarcane varietieo of alkaline phoophataoe

Co 6304

_1.00

Co 85019

_0.25

_1.00

Co 8371

_0.00

_1.00

Co 89003

_0.00

_1.00

Co 91010

_0.00

_0.20

_0.25

_0.00

_1.00

Table 6: Similarity IndeF of different ouuarcane varietieo of protein

Co 6304

_1.00

Co 85019

_0.00

_1.00

Co 8371

_0.00

_0.50

_1.00

Co 89003

_0.00

_1.00

Co 91010

_0.00

_0.50

_1.00

Table 7: Similarity IndeF table of oelected omut reoiotant ouuarcane varietieo

Co 6304

_1.0000

Co 85019

_0.0588

_1.0000

Co 8371

_0.0625

_0.2631

_1.0000

Co 89003

_0.0000

_0.1578

_0.1666

_1.0000

Co 19010

_0.125

_0.1052

_0.0555

_0.2222

_1.0000

Table 8: Suuarcane Varietieo with their reoiotant charactero

Sugarcane Variesies

Co 6304 Co 85019 Co 8371 Co 89003 Co 91010

? Moderately

Reoiotant to omut

Reoiotant to Smut Suoceptible to Smut Reoiotant to Smut

? Reoiotant to Red Rot

Moderately Reoiotant to Red

Rot

Suoceptible to Wilt Reoiotant to Inoect Peot

? Tolerant to

Drouuht

Tolerant to Drouuht and Water

Louuinu

Moderately Suoceptible to Red Rot

Moderately ouoceptible to Red Rot

? Tolerant to Drouuht

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The clooe relationohip between the variety Co 6304 and the varietieo Co 91010, Co 8371 and Co 85019 with the oimilarity indiceo 0.059, 0.063 and 0.125 revealo the reoiotant and tolerant character of the variety Co 6304. The cladouram baoed on the individual ioozyme bandinu profile of different varietieo ohowed different deureeo of relationohip. In ueneral, hiuher deuree of variation hao been found in iooperoFidaoe profile when compared with three other ioozyme profileo and a protein profile, in which more or leoo uniform pattern of relationohip hao been oboerved (Fiu. 2 A-E).

DISCUSSION

The ioozyme electrophoretic bandinu profile inoioted multiple bando on different loci with different Rf valueo and unequal intenoitieo. The reoulto of ioozymic analyoio on different varietieo of ouuarcane confirmed their heterozyuouo nature by producinu more complicated bandinu pattern with multiple eFpreooion in the oame reuion (heteromeric or hybrid bando) (Table 1). Electrophoretic bandinu patterno ohowed leoo deuree of polymorphiom amonu five different varietieo of ouuarcane.

The morpholouical variation, a product of uenotype and the environment, io an important parameter, but much diveroity, which remaino uneFpreooed morpholouically, can be revealed by biochemical methodo. Study of ioozymic variation io one ouch important and powerful procedure that hao often been employed to unravel the hidden variation. The ioozyme formo of thio enzyme, beinu the primary uene producto, can be uoed to deduce uene homolouy with precioion by comparinu variation in their eFpreooion patterno in different varietieo of ouuarcane in relation to dioeaoe reoiotance and otreoo tolerance ao evident in the preoent otudy. Manjunatha et al., 2003 uoed iooperoFidaoe profileo to otudy the polymorphiomo between the different

oomacloneo of ouuarcane cultivaro developed auainot biotic and abiotic otreoo tolerance. SOD ioozymeo have aloo been uoed ao biochemical markero for uenotype diocrimination and for uenetic diveroity determination in five varietieo of ouuarcane cultivated in the northern Paranб, Southern Brazil (Oraomo and Machado, 2003). To diotinuuioh the reoiotant and ouoceptible cloneo of Saccharum officinarum L. to funual dioeaoeo iooperoFidaoe analyoio hao been carried out by Anbazhauan et al.,

2009.

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