RPF-II (2012-2013)

PROFORMA FOR SUBMISSION OF ANNUALPROGRESS REPORT OF RESEARCH PROJECTS

PART -I : GENERAL INFORMATION

600 Project Code C17.11

6001 Institute Project Code No. : CI 7.11

6002 ICAR Project Code No.

601 Name of Institute and Division:

6011 Name & Address of Institute : INDIAN GRASSLAND & FODDERRESEARCH INSTITUTE, JHANSI- 284003

6012 Name of Division ISection Crop Improvement Division

6013 Location of Project IGFRI, Jhansi

602 Project Title : " Biochemical and Molecular Approach forCharacterization of Drought Tolerant ForageSorghum"

603 Priority Area : Research and Development6031 Research Approach: Applied Res. Basic Res. Process/ or TechnologyTransfer of

>l 02Development

03Technology

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604 Specific Area : Plant Biochemistry & Plant Molecular Biology605 Duration of project : 5 years

6051 Date of start of project : July 2010

6052Likely date of completion of project: 2015

6053 Period for which report submitted : 2012 - 2013

606 Total cost of the project :

6061 Expenditure to date607 Summary Achievements:Sorghum (Sorghum bicolor L.) is an important crop in many parts of the world. It isutilized as food, fodder and several industrial purposes. In general, sorghum is known

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to be ore tolerant to any stresses including heat, drought, salinity and flooding (Ejetaand Knoll, 2007). Drought, one of the most severe stresses, results in a considerableloss of crop productivity. Development and selection of drought tolerant foragesorghum is very much required to ensure the fodder availability in drought prone areasof country. The forage sorghum germ plasm at IGFRI comprising of stay green, highsugar content and low HCN will be screened at physiological and biochemical level fortheir tolerance to drought. Selected sorghum lines will be characterized at molecularlevel. The selected lines will be introduced into drought prone area for forageproduction in the extreme environmental conditions.

Key Words Drought, forage sorghum, antioxidant enzymes, chlorophyll

PART -II : INVESTIGATOR PROFILE610 Principal Investigators:

6101 Name:

6102 Designation:

6103 Division! Section:

6104 Location:

6105 Institute Address:

611 Co- Investigators

Dr. Manoj Kumar Srivastava

Senior Scientist

Crop Improvement Division

Crop Improvement Division

IGFRI, Jhansi-284 003

611 Co- Investigators

6111 Name6112 Designation:6113 Division !Section:6114 Location :6115 Institute Address:

Dr. C.K. GuptaScientist (Plant Physiology)

Seed Technology Divisionseed Technology DivisionIGFRI, Jhansi.

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PART -III: TECHNICAL DETAILS620 Introduction and objectives;6201 Immediate objectives:

• Screening and selection of drought tolerant lines of forage sorghum

• Biochemical and Molecular characterization of drought tolerance in fodder

sorghum

6202 Long term objectives:• Introduction of promising drought tolerant lines of forage sorghum.

6203 Specific objectives for the year as detailed in RPF-11. Enzymatic profile of antioxidant enzymes viz. Catalase, Peroxidase, SOD,

Polyphenol oxidase, esterase and glucosidases (validation).2. Isozyme analysis of above enzymes during drought stress (validation).3. Isolation of genomic DNA and development of molecular marker associated

with drought.621 Project Technical Profile:

6211 Technical Programme:Please see Annexure I

6212 Man months involvement of component project workers for the specified year: --

622 Progress of work6221 Achievements in terms of targets fixed for each Activity:

Please see Annexure II

6222Questions-Answered -Biochemical characterization of drought tolerant forage sorghum lines hasbeen done at morphological level.

Flower initiation and day to flower was measured in field grown lines.Six SGS and two non- SGS lines were selected for molecular analysis.

6223 Process/Product/Technology/developed during the Year.

6224 Utility of results obtained so far.The morphological and biochemical data will be used for selection of droughtresistant forage sorghum lines.

623 Publications and Material Development:(one copy each to be supplied with this proforma)

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6231 Research Papers

6232 Popular articles : No6233 Reports

6234 Seminars and workshops (relevant to the project) in which the Scientists haveparticipated.

None

625 Infrastructural facilities developed: Seed testing facilities- None

0,'0' It--,~ ~Signature of the Project Inv~stigator

(Manoj Kumar Srivastava)

1.Co-investigators .Il:-

_ 0""", Ykr~ ~v! ~ ...'> ''1\\~(C. K. Gupta)

Signature & Comments of the HeadOf the Division ISection

Signature & Comments of the Director

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J~ \

/'~~--------------------------------------------------------------------

ANNEXURE-IC17.11" Biochemical and Molecular Approach for Characterization of DroughtTolerant Forage Sorghum"

Date of starts: July 2010Date of completion: June 2015

Technical Program:

1. Enzymatic profile of antioxidant enzymes viz. Catalase, Peroxidase; SOD, Polyphenoloxidase, esterase and glucosidases (validation).

2. Isozyme analysis of above enzymes during drought stress (validation).3. Isolation of genomic DNA and development of molecular marker associated with

drought.

Activity of catalase

Catalase activity was assayed by measuring the loss of H202 concentration by the

enzyme extract by measuring the absorbance at 240 nm.

Activity of peroxidase

Peroxidase activity was measured by method of Chance and Machly (1955) using

guaiacol as substrate.

Guaiacol + H202 ~ Oxidized guaiacol + H20

The resulting oxidized guaiacol was measured at 470 nm.

Activity of Superoxide dismutase

SOD activity was measured by measuring the ability of enzyme extract to inhibit

photochemical reduction of nitroblue tetrazolium (NBT) according to method of

9ginnnopolitis and Reis (1977) with slight modifications.

PCR analysis

The PCR conditions were setup using 50 ng of genomic DNA and all other

components in a PCR tube (200 1J1). the annealing temperature was 58°C and the

total reaction volume was 25 IJI. The PCR product was run in 1.4% agarose gel at

120 volts for 2 hours in TBE buffer.

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ANNEXURE - II

C17.11

" Biochemical and Molecular Approach for Characterization of Drought

Tolerant Forage Sorghum"

Date of starts: July 2010

Date of completion: June 2015

Achievements:

Morphological and Biochemical characterization of sorghum germ plasm:

28 selected lines were sown in glass house and filed for the evaluation of drought

tolerance at morphological and biochemical levels. Germination percentage was

recorded and growth of seedlings was observed. When flowering time was

measured, it was found that flowering was delayed in all stay green lines. Leaf

morphology at 15 day stage was measured. The data is shown in Fig. 1

Average leaf length

50.0

45.0

40.0

35.0

30.0

25.0

20.0

15.0

10.0

5.0

0.0

, -i-

r-- - - •.... i-

- f--- - - ;- i- i- i-

- i- i- f- - f- f- f- f- f-

i- - i- f- i- f- f- f- f- f- '- OAveraQe ..

i- - f- f- f- f- f- f- '-i- i- - i- i- i- i- i- i-

i- i- ~ - i- i- - i- i- i- - i- i-

Fig. 1: Leaf length (3rd leaf) of sorghum lines grown in glass house.

It is evident that SGS 45 has highest leaf length of 46.4 cm while IG 02-377 has

lowest leaf length of 18.6 cms. In general the leaves of Sgs lines were longer than

non-SGS lines. Leaf width was also recorded and the data is presented in Fig. 2.

Average leafwidth

2.5

1.0

f-

,..-- f-- f-

t-r- f- f- - r- f- f- OAverage..

f- f- f- f- - ~ c- - - f- f- f- - f- f-

2.0

1.5

0.5

0.0

Fig. 2: Leaf width (3rd leaf) of sorghum lines grown in glass house.

The line SGS 45 has highest leaf width of 2.4 cms while IG- 03-510 has narrowest

leaf width of 0.7 cms only. In general, the SGS lines have higher values of leaf width.

The ratio of leaf length to leaf width was also calculated. The data is presented in

Table 1.

Most of the SGS lines have lower ratio of lenth to width. When the product of length

and with was calculated the SGS lines have a higher values in comparision to non-

SGS lines.

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Table 1: Leaf morphology (3rd leaf) of sorghum lines grown in glass house.

Line No. Leaf length Leaf width lengthxwidth Length/widthIG-02-377 18.6 1.2 15.99 22.50IG-02-380 20.0 0.9 22.35 18.18IG-03-510 22.0 0.7 30.29 16.40IG-03-285 24.0 1.0 23.94 25.52IG-03-316 23.6 1.1 21.93 25.84IG-03-320 26.4 1.7 16.00 44.46SGS-328-2 22.6 1.3 20.16 30.54IG-03-515 34.2 1.6 21.43 56.34EC-507876 28.8 1.0 31.12 27.80EC-507882 37.2 1.9 20.82 71.40EC-507889 29.2 1.5 20.14 43.38EC-507889-2 36.4 2.0 18.85 73.34SGS-9 38.8 1.8 22.16 68.84SGS-96 37.0 1.9 20.01 72.34SGS-101 29.2 1.5 20.80 44.12SGS-144 36.8 1.5 24.77 55.14IG-02-498 37.2 1.6 23.94 59.48SGS-154 36.6 1.5 24.18 56.02SGS-89 30.0 1.1 27.87 35.24SGS-163 32.6 1.6 20.60 52.64SGS-168 30.2 1.6 18.69 50.28SGS-177 31.0 1.2 27.13 37.02IG-02-418 28.6 1.3 22.01 37.50IG-03-436 28.8 1.3 22.27 37.54SGS-195 36.2 2.1 17.11 77.06SGS-33 38.0 2.3 16.55 89.02SGS-45 46.4 2.4 19.83 110.72SGS-118 40.8 2.2 18.52 91.60

Measurement of Enzymatic activities of peroxidase and catalase:

Peroxidase and catalase activities were measured in the leaves of sorghum and the

data is presented in Fig. 3 and 4. The peroxidase activity was highest in IG-03-515

followed by SGS- 45 and SGS-118. The lowest activity was in line IG-03-320. The

SGS and non-SGS lines have comparable values of peroxidase acivity, however, non

SGS lines have slight higher peroxidase activity.

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IG.02.m~IG-{)2·380 -MIG-{)3·510,.. IIG·03-285 ~

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5G5·328-2IG-03-515

[(·507876

[(·507882

[(·507889[(·507889·2

5G5·9

5G5·96

5G5-1015GS·144

IG-{)2-498

SGS-154

SGS-89SG5-163

SG5-168

5GS-177

IG-02-418IG-{)3-436

5G5-195

5G5·33

5G5·4S5GS·118

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-0 0 -i -lCD 0- ::T ::T..., fJ) CD CD0 CDX < 0 o "0.: 0)

CD :E •....•. <5.IG·02-3770) Q. CD 0)

fJ) fJ) 0) W IG·02-380CD ::::J•....•.

fJ)0) •....•. 0) CD -U IG-03-510o ::T o 0) CD IG-03-285•....•.

CD•....•.<. <. o ...,

•....•. 0 IG-03-316;:::;.: C/) ;:::;.: <. x-c G) '< ;:::;.: 0.: IG-03-320•....•.C/) :E -e 0) 5G5·328-2::T

0) 0) 0) :E fJ)fJ) CD IG-03-515::::J ::::J 0)

3 Q. ::::J fJ) 0) [(·507876o0 ::::J ::T •....•. [(·507882fJ) 0 ::::J <5. <.

E(·507889•....•. ::::J CD ::T ;:::;.:0 I CD -c E(-507889-2-+0 C/) C/) fJ) 0n •....•. G) G) •....•. -+0 5G5-9

QJ ::T C/) C/) W,... CD I ::::J a. 5G5-96

QJ C/) CD C/) 5G5-101::::J c» CD- G) CD G)QJ 0) 5G5-144VI C/) fJ) » C/) -+0

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n fJ) 3 CD 0' 0 5G5·89CD

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OD activity"

SOD activity700

600

500

400

300

200

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Fig. 5: Catalase activity of 3rd leaf of sorghum lines grown in glass house.

Superoxide dismutase (SOD) activity in the leaves is presented in Fig. 5. SGS 144

has the maximum acivity followed by IG-03-435 and SGS 118. SGS-9 has the lowest

activity followed by IG-03-510 and IG-03-385.

The selected twenty eight lines, from second year screening experiment, comprise of

both Stay green sorghum (SGS) and non SGS lines. Genomic DNA was isolated and

used for amplification of six set of SSR markers. The primers were designed based

on the genomic region of genes involved in stress responsive pathway. the details of

primers is given in Table 2.

Table 2: Sequence of SSR used for ampligication of sorghum genomic DNA.

S. No. Gene name Sequence SSR motif1 CAOMT CATGTCGATCGATCAGTGCT (CTAT)6

TCGAGGAGCTTCTTGGTGAT2 Metallothionin TGTTCATCTCCAACGTCTCG (TCTG)1O

GCAGCATCTCGTCAGAATCA3 Cytochrome P450 GCCCACCAAATACAAGAACC (CGAh

CTTTGCGGCACTAAAACACA4 Pectinacetylesterase ATAGACGATGGGGGTGCAT (GCAh

precursor CTGCCCCTATTACTGCTGCT5 Bax inhibitor-1 TTCCAAATCCAAATCCATCC (AG)a

CGAGGAGGTCGAGTAGAACG6 Hypothetical protein GCGGACTTAGTTGGACCTGA (GT)lO

TTCACAGCATAAAGCGCAAC

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M 12 34 56 7 8 9 10111213141516171819202122232425

Primer 1

---

Primer 2

Primer 3

Primer 4

Primer 5

Primer 6

Fig. 6: Diversity in banding pattern with 6 SSR primer set used for 25 genotypes of sorghum.

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The SSR analysis showed that all these primer amplified the bands of expected size.

However there was polymorphism between the germplasms. The actual difference in these

bands would be resolved by getting sequence of these band and comparing them.

Based on this year's morphological, biochemical and molecular data and data of Jprevious years, six SGS lines (SGS-33, SGS-45, SGS-89, SGS-118, SGS-154 and SGS- )

168) and two non-SGS lines (IG-03-285 and EC 507882) were selected for further

analysis.

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