Dhanasekar & Reddy : Genotypic variation for raffinose family oligosaccharides in cowpea

25
Genotypic variation for
oligosaccharides in cowpea

raffinose

family

P Dhanasekar and KS Reddy

Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre,
Trombay, Mumbai 400 085, Maharashtra, India
Email: sekar@barc.gov.in

Abstract
Cowpea (Vigna unguiculata) is a protein rich arid legume crop. The presence of anti-nutritional
factors in cowpea diminishes its value and adoptability. Among the low molecular weight antinutritional elements, raffinose family oligosaccharides (RFOs) pose a challenge as they remain
recalcitrant to normal physical processing methods. Breeding of varieties with low RFOs offers a
tenable solution to contain this flatulence contributing factor. With a view to discerning the
genotypic variation for RFOs, 92 cowpea genotypes including mutants, varieties, germplasm and
advanced breeding lines were analyzed following the enzymatic method (Megazyme Kit). Glucose
and sucrose contents were also analyzed simultaneously. Cowpea exhibited significant genotypic
variation for glucose, sucrose and total RFOs content. The range of RFOs content along with that
of glucose and sucrose, genotypic and phenotypic coefficient of variations, heritability, genetic
advance and putative classification of genotypes based on the RFOs content are discussed.
Correlations among the various contents as well between seed coat colour and RFOs content
along with selection criterion for genetic improvement of these anti-nutritional factors have been
deliberated.
Key words: Anti-nutritional factors, Cowpea, Genitic variation, RFOs content

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Pulses Challenges & Opportunities under Changing Climatic Scenario

Cowpea (Vigna unguiculata) is a protein rich legume crop that is widely grown in
the arid and semi-arid regions of the world including India. The ability of cowpea to
tide over adverse conditions like high temperature and drought enhances its climate
change resilience potential. The role of cowpea in soil nutrient amelioration through
biological nitrogen fixation could be realized from its ability to grow under poor soil
conditions. Its inherent shade tolerance attribute makes it suitable for intercropping
and its dual purpose nature adds additional revenue to the cultivators. In spite of its
multifariousness, the presence of anti-nutritional factors (ANF) in cowpea diminishes
its value and adoptability. The ANFs range from low molecular weight secondary
metabolites like glucosinolates, saponins, polyphenols, tannins, saponins, flatulence
causing oligosaccharides, cyanogens or alkaloids to high molecular weight proteins
like -amylase, lipase or protease inhibitors, lectins and other toxins (Khokhar and
Apenten 2003). These secondary metabolites though have imminent physiological role
in plants to tide over adverse growing conditions, may turn anti-nutritional in humans
upon consumption. Among the low molecular weight anti-nutritional elements, raffinose
family oligosaccharides (RFOs) pose a challenge as they are heat stable (Rao and
Belavady 1978) and remain recalcitrant to normal physical processing methods like
heating and boiling. RFO represent a class of soluble but nonreducing and nonstructural
oligosaccharides having (16) linkage between sucrose and galactosyl subunits (Luthi
et al. 2004). Therefore, these sugars are indigestible in human and monogastric animals
as they lack -galactosidase, hydrolyzing enzyme responsible for RFO breakdown
(Kumar et al. 2010). Consequently, RFO escape digestion and absorption in the small
intestine, but large intestinal microflora metabolize RFO and produce carbon dioxide,
hydrogen, and small quantities of methane, causing flatulence, diarrhea, and stomach
discomfort in humans. As RFO act as substrate for intestinal bacteria, they are also
considered as prebiotics (Swennen et al. 2006). These oligosaccharides also participate
in important plant processes such as desiccation during seed maturation, carbon sourcing
in the early stages of germination, translocation of photoassimilates, abiotic stress
tolerance (Martinez et al. 2008) and also support the growth of root nodulating bacteria
in the rhizosphere of legume plants (Gage and Long 1998). Therefore, to increase the
acceptability of cowpea in human diets, RFO concentration needs to be reduced without
affecting their physiological role in plants and beneficial effect onhuman health.
Breeding of varieties with low RFOs offers a tenable solution to contain this
flatulence contributing factor. With a view to discerning the genotypic variation for
RFOs the present investigation was conceived involving 92 cowpea genotypes.
Ninety-two diverse cowpea genotypes including mutants, varieties, germplasm
and advanced breeding lines were analyzed in duplicate for RFOs content following
the enzymatic method using raffinose assay kit (Megazyme, Ireland) according to the
manufacturers instruction. Simultaneously, the genotypes were also assayed for glucose
and sucrose contents. Briefly, 500 mg of powdered seed meal was extracted in sodium
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Dhanasekar & Reddy : Genotypic variation for raffinose family oligosaccharides in cowpea

acetate buffer following ethanol inactivation of endogenous galactosidase enzymes.

The extracts after chloroform purification were apportioned into three tubes to which;
buffer, invertase and -galactosidase + invertase were added respectively to hydrolyze
the complex RFOs into D-galactose, D-fructose, and D-glucose molecules. The resulting
D-glucose concentration was determined using glucose oxidase/peroxidase reagent
(GOPOD) that produced a red quinoneimine, the concentration of which was
determined at A510 nm using a spectrophotometer (Jasco, USA). This method
determined alloligosaccharides including raffinose, stachyose, and verbascose
concentration as a group. Total RFO concentration was calculated on a molar basis as
1 mol of each oligosaccharide contains 1 mol of D-glucose. The anova, genotypic and
phenotypic coefficient of variation, genetic advance and broadsense heritability were
calculated using SAS 9.1.3 software. Spearmans pairwise rank correlation was also
worked out between each of glucose, sucrose and RFOs content and also between the
RFOs content and seed coat colour.
Cowpea exhibited significant genotypic variation for glucose, sucrose and total
RFOs content (Table 1). The RFOs content ranged from 4.27 (TC501 ) to 9.67
(Bhagyalakshmi) mmoles/100gm with a mean value of 7.77 mmoles/100gm, while
glucose and sucrose contents ranged from 0 to 0.83 mmoles/100gm and 1.69 to 8.78
mmoles/100gm respectively. Among the 92 genotypes, 16 genotypes contained low
RFOs (<7 mmoles/100gm), while 72 genotypes contained medium (7-9 mmoles/100gm)
and 4 genotypes contained very high (>9 mmoles/100gm) RFOs. The CV (%) was high
for glucose (11.3) followed by sucrose and RFOs (9.3). The GCV (%) was high for sucrose
(28.80) while it was low in case of RFOs (8.9). Likewise, the broadsense heritability of
sucrose was very high (95.06) in comparison to RFOs (64.99). The genetic advance (%
mean) for sucrose (57.85) was more as against that of RFOs (14.83). No significant
correlation was observed between glucose and RFOs content, though sucrose and RFOs
showed a strong positive association (r=0.39). Seed coat color and RFOs content were
also found to be uncorrelated even though green coloured genotype showed numerically
high RFOs content.
Natural variability exists in cowpea for RFOs and sucrose, which are responsible
for causing flatulence. The total RFOs content as estimated by enzymatic assay has
been confirmed to be accurate and precise as that of the HPAEC-PAD (Gangola et al.
2013). The total RFOs content in cowpea (4.27 to 9.67 mmol/100gm) is slightly higher
than that reported by Gangola et al. (2013) in chickpea (1.58 to 5.83mmol/100gm) and
comparable to that of soybean (3.5 to 9.22 mmol/100gm) as reported by Kumar et al.
(2010). It has been stated that even though cowpea contains higher or comparable
flatulent sugars than that of soybean, it contains low raffinose (most flatulent sugar)
than soybean (Lambot 2002) and coupled with its low phytate, has the potential to
replace soybean as an industrial crop. The sucrose content was comparatively less in
cowpea in comparison to soybean, while more than that of chickpea. Strong correlation
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Pulses Challenges & Opportunities under Changing Climatic Scenario

Table 1:
Sr. No.
1

Genotypic variation for glucose (glu), sucrose (Suc) and raffinose family
oligosaccharides (RFOs) content (mmol/100gm) in cowpea
Genotype

Glu

Suc

RFO

BLRC1

0.08

3.28

7.07

Sr. No. Genotype

54

Bhagyalakshmi

Glu

Suc

RFO

0.17

7.31

9.67

GC3SKN

0.08

4.78

7.66

55

Kanakamony

0.11

2.88

8.67

IT86F2K14

0.38

3.26

5.58

56

KM5

0.07

3.58

7.35

Anaswara

0.02

4.35

8.37

57

C152

0.08

4.17

8.19

PGCP11

0.00

4.16

6.81

58

KBC2

0.10

4.33

7.60

TC601

0.00

3.38

6.92

59

DCS47-1

0.12

4.17

7.62

BLRC2

0.02

3.65

7.53

60

TC501

0.07

3.56

4.27

BLRC3

0.02

3.23

6.97

61

CPD103

0.09

2.54

7.35

BLRC4

0.03

3.88

6.36

62

CPD115

0.07

4.43

8.58

10

BLRC5

0.03

4.35

7.55

63

CPD118

0.04

2.78

8.59

11

BLRC6

0.02

4.45

7.98

64

JOB129

0.06

4.90

7.89

12

BLRC7

0.04

3.87

7.92

65

ArkaSamrudhi

0.09

3.67

8.00

13

BLRC8

0.01

3.12

7.78

66

V585

0.07

3.46

8.03

14

BLRC9

0.02

2.76

7.40

67

CO2

0.06

2.99

8.42

15

BLRC10

0.03

4.20

7.97

68

CO4

0.04

3.43

8.85

16

BLRC11

0.07

2.12

8.97

69

CO6

0.08

3.51

6.90

17

BLRC12

0.02

3.00

6.85

70

TVx944

0.09

4.13

7.62

18

BLRC13

0.04

3.13

7.68

71

COCP7

0.08

4.92

7.40

19

BLRC14

0.02

3.03

6.81

72

ARC-1

0.09

2.92

7.31

20

BLRC15

0.00

2.70

7.34

73

TC503l

0.17

2.71

7.40

21

BLRC16

0.01

2.43

7.74

74

TCM418SDT

0.07

4.17

8.39

22

BLRC17

0.01

3.13

7.74

75

TC501-4

0.07

5.16

8.68

23

BLRC18

0.02

4.80

6.95

76

TC1-6-10

0.08

4.77

9.15

24

BLRC19

0.07

3.86

7.94

77

TC1-6-9E

0.09

4.47

8.34

25

BLRC20

0.01

4.24

8.10

78

TC605

0.07

4.51

7.52

26

BLRC21

0.02

3.67

7.34

79

TC603

0.29

4.75

7.87

27

BLRC22

0.12

3.36

6.69

80

TC1-26-E

0.15

6.03

8.98

28

BLRC23

0.01

4.49

8.66

81

TC201

0.03

3.61

7.98

29

PGCP3

0.02

6.45

7.58

82

TCM503w

0.09

3.95

8.13

30

PGCP6

0.10

3.85

7.72

83

ALSANDO

0.09

8.78

9.10

31

PGCP5

0.12

6.68

8.18

84

CPD91

0.11

5.52

7.56

32

PGCP4

0.74

2.73

6.00

85

PGCP1

0.09

4.26

7.63

33

PGCP13

0.02

5.64

8.97

86

GC510

0.10

4.37

8.68

34

GC3JOD

0.02

3.71

8.12

87

NBC3

0.04

2.92

7.07

35

GC502

0.03

3.32

6.16

88

NBC2

0.83

3.85

7.71

36

GC521

0.07

2.31

7.66

89

PGCP12

0.13

6.25

7.78

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Dhanasekar & Reddy : Genotypic variation for raffinose family oligosaccharides in cowpea
Sr. No.
37

Genotype

Glu

Suc

RFO

GC4

0.05

6.72

8.46

Sr. No. Genotype

90

PGCP14

Glu

Suc

RFO

0.25

4.01

6.69

38

GC5

0.45

3.39

6.82

91

Lola

0.23

6.21

9.51

39

IT389561

0.02

5.23

8.19

92

Sarika

0.14

3.96

7.84

40

IC402175

0.01

3.61

8.41

mean

0.09

4.02

7.77

41

IC402172

0.02

4.79

8.62

min

0.00

1.69

4.27

42

RC101

0.01

4.19

8.38

max

0.83

8.78

9.67

43

IT86F20141

0.24

3.29

6.46

CV

11.28

9.28

9.27

44

IT00K1197

0.14

2.91

7.10

SE

0.01

0.26

0.51

45

IT86F20895

0.03

4.13

8.16

CD(5%)

0.02

0.74

1.43

46

IC521495

0.41

2.62

7.21

h2

95.06

64.99

47

IC202797

0.07

4.46

7.22

GA (%mean)

57.85

14.83

48

IC202784

0.00

1.69

8.35

GCV(%)

28.80

8.93

49

EC517140

0.00

4.57

7.90

PCV(%)

29.54

11.08

50

EC536635

0.09

2.73

8.40

51

IC366776

0.04

4.74

8.77

52

NBC1

0.04

4.30

8.05

53

Anaswara

0.06

3.19

8.93

between sucrose and total RFOs (r=0.39*) even though was significant, the coefficient
of determination (r2=0.15) was less suggesting that the observed correlation may not be
actual. Nevertheless, the strong association could be made use while making selections
in breeding programme. Low GCV in comparison to PCV in both the cases showed the
involvement of genetic and environmental components. Similar trend was observed in
chickpea also (Gangola et al. 2013). The environmental influence was more evident in
case of RFOs content necessitating the need for multi-location screening. High GCV
coupled with high heritability and genetic advance for sucrose revealed the involvement
of additive gene action while non-additive genes contributed to RFOs content. Selection
for sucrose content could be possible in early segregating generations, while delayed
selection would be desirable for RFOs. Simultaneously, selection could be done in
different locations for RFOs and sucrose content that showed strong correlation as well
as high heritability and genetic advance.
Thus, it could be concluded that even though cowpea is gaining importance in the
present context of climate change, its adoptability could be enhanced by breeding
varieties with low difficult to process anti-nutritional factors such as flatulence causing
oligosaccharides. The genotypes identified with low RFOs content (TC501, IT86F2K14),
after due confirmation, could be utilized in breeding programme.

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