Phenotypic characterization and stability analysis for biomass and essential oil yields of fifteen genotypes of five Ocimum species

Industrial Crops and Products 77 (2015) 21–29

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Phenotypic characterization and stability analysis for biomass and essential oil yields of fifteen genotypes of five Ocimum species Rajendra P. Patel a , Rakshapal Singh a , Shilpi K. Saikia b , B.R. Rajeswara Rao c , K.P. Sastry c , M. Zaim d , Raj K. Lal e,∗ a

Biology Central Facility, CSIR—Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, U.P. 226015, India Microbial Technology and Nematology, CSIR—Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, U.P. 226015, India c CSIR–CIMAP, Research Centre Hyderabad, 500039, India d Virology, CSIR—Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, U.P. 226015, India e Genetics and Plant Breeding, CSIR—Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, U.P. 226015, India b

a r t i c l e

i n f o

Article history: Received 23 February 2015 Received in revised form 1 June 2015 Accepted 20 August 2015 Keywords: Biomass Environmental indices Essential oil yield Phenotypic Stability parameters

a b s t r a c t In order to characterize the germplasm of 15 genotypes of 5 Ocimum species collected from different regions of India. Based on significant variations and distinct morphotypes were identified in the germplasm. PCA analysis of 11 quantitative traits revealed that the first three components accounted for 82.51% of the total variance. The leaf length, width and leaf area, primary branches, inflorescence length, oil content and 1000 test seed weight were the most important traits accounting for 46.61% of the total variability in the first PC variable. Hierachical cluster analysis produced two distinct clusters of the Basilicum and the Sanctum group. The Basilicum group contained the genotypes of O. basilicum (OCB-7 to OCB-11), O. americanum (OCA-12) and O. kilimandscharicum (OCK-15). The Sanctum group comprised of O. sanctum (OCS-1 to OCS-6) and O. gratissimum (OCG-13) genotypes. Five genotypes viz. OCB-9, OCB-8, OCB-7, OCB-11 and OC-A12 were identified as high-yielding and stable genotypes. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The cultivation of therapeutically important Ocimum species is increasing globally due to their immense pharmaceutical and nutraceutical significance and wide ranges of adaptability to different soils and climatic conditions. The genus has more than 150 species (Simon et al., 1999) and several varieties or cultivars amongst which the two species O. tenuiflorum L.f. (syn. O. sanctum L.) and O. basilicum L. are the most prevalent for commercial cultivation in Indian subcontinent. The leaves and inflorescence of Ocimum are rich source of essential oil consisting of a huge variety of terpenes as its constituents. The criteria for characterization of taxa are very complex. Phenotypic traits such as leaf shape, leaf colour, flower colour etc. have traditionally being used for taxonomic classifications. Due to extensive cultivation practices and inter and intra-specific hybridization, Ocimum species display enormous morphogenetic variability among their species, including phenology (colour, shape and size of flowers, leaves, stems) growth characteristics,

∗ Corresponding author. Fax: +91 522 2342666. E-mail addresses: [email protected], [email protected] (R.K. Lal). http://dx.doi.org/10.1016/j.indcrop.2015.08.043 0926-6690/© 2015 Elsevier B.V. All rights reserved.

reproductive behaviour and chemical composition (Svecova and Neugebauerov, 2010). More than 60 variants in Ocimum are identified with green, red and violet leaves (Ibrahim et al., 2011). ´ Carovic-Stanko et al. (2011a,b) classified the O. basilicum cultivars in six morphotypes. Several investigations on genetic diversity, morphological variability, taxonomic and phylogenetic relationships, as well as essential oil composition and bioactivity of Ocimum species have been reported earlier (Patel et al., 2015; Verma et al., 2013; Rao ´ et al., 2011; Carovic-Stanko et al., 2010; Masi et al., 2006; Vieira et al., 2003) but reports on genotypic stability in Ocimum are very meagre. The interaction of different genotype with environment has great interest in many aspects of genomic and breeding research. The inconsistent performance of the genotypes under varying environments creates problem in the adaptation of a crop for large scale cultivation. In Ocimum, productivity performance is represented mainly by biomass and essential oil yield of better quality. Plant breeders always look for the genotypes that express stability for high yield across the years/environmental or locations. In general, a genotype is considered stable when its performance across the environments does not significantly fluctuate from the mean performance of a group of selected genotypes.

In order to evaluate the stability of genotypes, field experiments were conducted for two years (2007–08 and 2008–09) in three locations of India namely, (1) CSIR–CIMAP research farm Lucknow [(north India, humid subtropical climate, located at 128 m above MSL, latitude 26 8 N and longitude 80.9 E); mean annual rainfall as 800 mm (80–85% of which is received between July and September)], (2) CSIR, CIMAP, Research centre Hyderabad [(south India, semi-arid tropical climate, located at 542 m above MSL, latitude 17◦ 25 N and longitude 78◦ 33 E); mean annual rainfall as 764 mm (80% of which is received between June and September

235 98 215 12.4 21.2 9.5 250 234 220 0.60 0.23 0.45 7.8 7.3 5.7 Loamy sand Alfic ustochrept Red sandy loam 85 59 67 13.9 16.9 22.2 7–22.8 16–18.5 17.4–28.8 77 72 65 24.7 25.2 23.2

Available P (kg ha−1 ) Available N (kg ha−1 ) Organic C (%) pH Soil texture Avg. RH (%) Avg.

Temp: temperature; Min: minimum; Max:maximum; Avg: average; R.H: relative humidity. C: carbon; N: nitrogen; P: phosphorus; K: potassium. a Humid Subtropical, Latitude 26◦ 8 N; longitude 80◦ 9 E, altitude 128 m above mean sea level; annual rainfall 800 mm, 80–85% from July to September and winter season characterized by cold and fog. b Tropical wet, dry to semi-arid Latitude 17◦ –25 N; longitude 78◦ –33 E, altitude 542 m above mean sea level annual rainfall 764 mm, 80% between June and September and winter season characterized by mild cold and dry plenty sunlight) c Semiarid tropical, Latitude 13◦ 05 N; longitude 77◦ 35 E, altitude 930 m above mean sea level; annual rainfall 870 mm, 80% from May to October and winter season characterized by mild cold with plenty of sunlight.

2.2. Experimental location

18.8–31.7 19.7–30.7 19.8–28.2

The plant material used in the present study comprised of a total of 15 accessions of Ocimum placed within five species. Among the 15 genotypes, six accessions were from O. tenuiflorum L.f. (syn. O. sanctum L.), five accessions were of O. basilicum L., two accessions were from O. gratissimum L. and a single accession each was from O. kilimandscharicum Baker ex. Guerke and Ocimum canam Sim. syn. O. amricanam were selected for the study. The detail of planting material is described in (Table 1).

Lucknowa Hydearbad b Bangalore c

2.1. Plant material

Temp. (◦ C) Min–Max

2. Material and methods

Avg. RH (%)

Various methods have been proposed to assess genotypic stability in crops (Lin et al., 1986; Becker and Léon, 1988; Piepho, 1998). Univariate and multivariate are the widely accepted methods of stability (Lin et al., 1986; Lal, 2007). Joint regression analysis (univariate method) is the most documented and relevant method (Becker and Léon, 1988). Finlay and Wilkinson (1963) proposed that a genotype with regression coefficient equal to zero (ˇi = 0) as stable, while Eberhart and Russell (1966) emphasized the need of considering both linear (ˇi ) as well as non-linear (S2 di ) components of genotype × environment interactions in judging the stability of a genotype. Studies of genotypic stability for most economic traits viz. biomass and yield essential oil yields have been reported very scanty on Ocimum crop. Lal, (2014) reported the stability of essential oil yield in 40 Ocimum genotypes grown in multiyear under northern region (Lucknow) of India. However, no stability studies have been performed together in a multilocation and multiyear for biomass as well as essential oil yield for Ocimum crop. Considering the significance of this important crop, the present study aims to characterize and determine the stability and adaptability of two most desirable traits for recommendation for cultivation of Ocimum across the years/locations.

Avg.

CSIR–CIMAP, Lucknow (UP) CSIR–CIMAP, RC, Hyderabad (AP) CSIR–CIMAP, RC, Hyderabad (AP) CSIR–CIMAP, Lucknow (UP) CSIR–CIMAP, Lucknow (UP) Nasik (Maharastra) CSIR–CIMAP, RC, Hyderabad (AP) CSIR–CIMAP, RC, Hyderabad (AP) CSIR–CIMAP, RC, Hyderabad (AP) CSIR–CIMAP, RC, Hyderabad (AP) CSIR–CIMAP, Lucknow (UP) CSIR–CIMAP, RC, Hyderabad (AP) CSIR–CIMAP, RC, Hyderabad (AP) CSIR–CIMAP, RC, Hyderabad (AP) CSIR–CIMAP, RC, Hyderabad (AP)

Temp. (◦ C) Min–Max

Origin/Source of planting material

Ocimum tenuiflorum O. tenuiflorum O. tenuiflorum O. tenuiflorum O. tenuiflorum O. tenuiflorum O. basilicum O. basilicum O. basilicum O. basilicum O. basilicum O. americanum O. gratissimum O. gratissimum O. kilimandscharicum

Soil characteristics

Botanical name

OCS-1 OCS-2 OCS-3 OCS-4 OCS-5 OCS-6 OCB-7 OCB-8 OCB-9 OCB-10 OCB-11 OCA-12 OCG-13 OCG-14 OCK-15

During harvesting

Genotypes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Growth period

S. No.

Experimental Location

Table 1 Description of planting materials employed in the present study.

Exchangeable K (kg ha−1 )

R.P. Patel et al. / Industrial Crops and Products 77 (2015) 21–29 Table 2 Description of edapho-climatic conditions and initial soil characteristics (0–15 cm), the concentration of extractable nutrients in the soil, and the average minimum and maximum temperatures of the three locations (Lucknow, Hyderabad and Bangalore) during two cropping years (2007–2009).

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Table 3 Phenotypic variability in Ocimum genotypes. Germplasm

Plant height

Plant habit

Stem colour

Leaf colour

Leaf shape

Flower colour

Spike colour

Seed colour

OCS-1 OCS-2 OCS-3 OCS-4 OCS-5 OCS-6 OCB-7 OCB-8 OCB-9 OCB-10 OCB-11 OCA-12 OCG-13 OCG-14 OCK-15

Semi tall Semi tall Tall Medium Semi tall Tall Tall Tall Tall Short Tall Tall Tall Tall Tall

Erect Erect Erect Erect Erect Erect Erect Erect Erect Bushy Erect Erect Erect Erect Intermediate

Green purple green Purple Purple Purple Purple green green purple Green Green Green Green Dark green Green

Light green Purple green Deep purple Deep Purple Deep Purple Deep Purple Pale green Dark green Dark green Pale green Light green Light Green Pale green Dark green Pale green

ovate ovate ovate ovate ovate ovate Narrowly ovate Narrowly ovate Elliptic Narrowly ovate Narrowly ovate Narrowly ovate Broadly ovate Ovate Elliptic

Greenish white Purplish white Purple Purple Purple Purple White Pinkish-white Pinkish-white Milky white Pinkish-white Lemon-white Greenish white Greenish white White

Green Purple green Purple Purple Purple Purple Green Light purple Dark purple Pale green Green Green Green Green Greenish gray

Brown Brown Brown Brown Brown Brown Black Black Black Black Black Black Reddish brown Reddish brown Black

Note: >80 cm (tall), 70–80 cm (semi tall), 60–70 cm (medium), <60 cm (short).

and winter season is characterized by mild, cool, dry weather)] and (3) CSIR–CIMAP, Research Centre Bangalore [(south India, semi-arid tropical climate, located at 930 m above MSL, latitude 13◦ 05 N and longitude 77◦ 55 E, 870 mm annual precipitation received between May to October)]. The edapho-climatic conditions of the three studied locations during the experimental period (2007–08 and 2008–09) are detailed in (Table 2). 2.3. Designing of experiments All experiments were laid-out in randomized block design with three replications. The soil samples were analysed for available nutrients (Jackson, 1973). The details of the soil characteristics of the experimental fields are presented in Tables 1 and 2 . All the fifteen genotypes were transplanted in the respective locations for two consecutive cropping years (2007–08 and 2008–09). Individual plot size was 2.5 m × 3 m (7.5 m2 ) in both the cropping years. Each plot received vermi-compost (1.5 t ha−1 ), single superphosphate (P2 O5 40 kg ha−1 ) and muriate of potash (K2 O 40 kg ha−1 ) prior to planting. The vermicompost was produced for a period of 12 weeks from de-oiled waste of Cymbopogon grasses (lemongrass and citronella) using Eudrilus eugineae an epigeic species of earthworms. Vermicompost had a total N content of 1.1%, Olsen’s P content of 0.49%, exchangeable K content of 0.57%, total organic carbon of 19.17% and moisture content 45–50% on dry weight basis (Singh et al., 2012). Seeds of all the 15 Ocimum genotypes were sown in nursery beds in the last week of May at all the experimental loca-

tions. Healthy and uniformly grown seedlings were transplanted into planting holes having 10 cm depth and 5.0 cm diameter at 45 × 30 cm spacing during the first week of July at all three experimental locations. Transplanting of Ocimum genotypes was done in a similar manner in the second cropping year in all the locations. The fields were irrigated after planting and thereafter at 10–15-day intervals. Nitrogen (as urea) was supplied @ 50 kg ha−1 in 2 splits doses at 15 days intervals. Weeds were manually removed 25 and 45 days after transplanting seedlings. 2.4. Observations on phenotypic and quantitative agromorphological traits The genotypes were characterized for different qualitative phenotypic traits viz. plant habit, shape of leaf blade, leaf margin, leaf colour, stem colour, flower colour and seed colour.The data for quantitative morphological characters viz., plant height (cm), plant canopy (cm2 ), No. of primary branches, leaf length (cm), leaf width (cm), leaf area (cm2 ), fresh biomass/plant (g), essential oil content (% w/v), test seeds weight (g), spike length (cm) and number of whorls/spike were recorded on 10 randomly selected plants from all the blocks. All the traits were measured at full flowering stage of crop. The genotypic stability analysis was performed for fresh biomass and essential oil yield in the field conditions across two consecutive years (2007–09) and three locations (Lucknow, Hyderabad and Bangalore). Crop was harvested manually 15–20 cm above the ground level during each growing season.

Table 4 Variation in morphological traits of Ocimum genotypes. Genotypes

PH (cm)

PC (cm)

PB (No)

B/P (gm)

LL (cm)

LW (cm)

LA (cm2 )

LS (cm)

W/In. (No.)

EOC (%)

TSW (gm)

OCS-1 OCS-2 OCS-3 OCS-4 OCS-5 OCS-6 OCB-7 OCS-8 OCB-9 OCB-10 OCB-11 OCA-12 OCG-13 OCG-14 OCK-15

79.66 70.9 88.73 69.46 79.6 85.26 83.33 90.6 90.2 59.73 91.33 88.2 84.26 80.53 93.06

57.53 55.86 65.06 66.66 71.86 70.13 59.93 62.26 60.6 54.33 67.33 58.86 65.00 45.80 62.93

18.41 17.91 15.85 9.35 13.56 14.30 17.23 18.09 17.01 17.18 19.90 15.03 17.86 23.55 16.40

110 95 121 84 81 96 254 295 218 140 202 156 112 98 163

5.39 5.00 4.83 4.70 4.20 4.53 7.73 8.50 5.53 4.85 5.02 5.30 9.80 12.00 4.60

3.13 2.75 2.83 2.73 2.26 2.30 4.50 4.50 2.84 2.20 3.20 2.86 6.40 6.13 2.53

8.32 10.35 10.38 8.90 7.93 8.86 21.88 19.25 12.64 4.37 7.63 6.75 33.61 43.17 9.08

10.00 8.00 8.45 6.66 8.52 6.33 16.80 20.65 16.33 6.45 14.75 19.93 15.00 15.00 23.70

10.25 10.66 10.82 14.66 11.30 12.66 17.00 18.00 13.66 8.00 14.00 16.33 14.66 16.00 19.65

0.25 0.39 0.27 0.12 0.10 0.25 0.28 0.28 0.35 0.27 0.27 0.44 0.25 0.48 0.27

0.28 0.31 0.35 0.28 0.29 0.28 1.53 1.94 1.35 1.80 1.38 1.37 1.15 1.09 1.08

PH: plant height, PC: plant canopy, PB: No of primary branches, B/P: biomass per plant, LL: leaf length, LW: leaf width, LA: leaf area, LS: length of spike, W/in: weight of inflorescence, EOC: essential oil content, TSW: test seed weight.

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Fig. 1. Distribution of Ocimum germplasm accessions in Euclidean distance model in relation to morphological traits.

2.5. Statistical analysis 2.5.1. Principal component analysis of agro-morphological traits Data recorded for Hyderabad experimental location were subjected for phenotyping and used to calculate Euclidean distance (Ed) for all pairs of accessions. Euclidean distance obtained from morphological traits was used to construct hierarchical dendogram with the help of SPSS statistical software ver.17. Principal component analysis (PCA) was performed to demonstrate the relationship among Ocimum germplasm accessions in terms of their position relative to two coordinate axes. 2.5.2. Analysis of variance for stability The Pooled mean data over three locations and two years were analyzed through CSIR–CIMAP ver.1 software available at the Department of Genetics and Plant Breeding i.e., based on the Singh and Chaudhary (1979). The stability parameters (mean yield, regression coefficient (ˇi ) and deviation from the regression (S2 di ) were used to report the comparative performance and selection of suitable genotypes (Eberhart and Russell, 1966).

while other reports defines five forms (Mondello et al., 2002). The organ specific qualitative differences, viz. the purple small leaf and inflorescence of O. tenuiflorum (OCS-3 to OCS-6), green leaf and thin inflorescence with clove note of OCS-1, thick and large leaf with clove essence of O. gratissimum (OCG-14), green and wrinkled leaf (OCB-7, OCB-8) with sweet aroma of O. basilicum, tall plant, large gray–green inflorescence with camphor’s smell of O. kilimandscharicum (OCK-15) allowed clear and unambiguous identification of all investigated species. The present study in good agreement with those of; (Chhaya et al., 2013; Verma et al., 2013; ´ Nurzynska-Wierdak, 2007; Labra et al., 2004) who demonstrated the considerable variation in their morphology. These morphological variations can be accounted by the genetic background, natural inter and/or intra-specific hybridization and interaction of genotypes with environments are potent factors for determining the expression of phenotypic characters in plants. However, variations allowed distinguishing some of the accessions among studied germplasm depending on quantitative agro-morphological parameters. 3.1. Analysis of quantitative morphological traits

3. Results and discussion The studied genotypes were characterized on the basis of plant type, leaf, flower, inflorescence and seed morphology as described in the (Table 3). Plant growth habit classified the accessions into erect, bushy and intermediate type. In the genus Ocimum morohological traits viz. pigmentation in the leaf, stem, flower and inflorescence have been used for long time for proper identification of cultivars (Chhaya et al., 2013; Svecova and Neugebauerov, 2010). O. tenuiflorum accessions have been differentiated into three morphological forms (green, purple and purple-green) on the basis of leaf foliage colour. Lawrence (1989) and Maheshwari et al. (1987) classified O. tenuiflorum as green, purple and mixed type (perhaps natural hybrid between green and purple type)

Agro-morphological data showed enormous morphological variation in studied genotypes. The quantitative characters such as plant height, plant canopy, leaf area, leaf stem ratio, essential oil content etc. were considered as yield contributing traits; therefore, picked as agronomic, as well as morphological descriptors for the studied genetic stocks. The agricultural practices and morphological observations under uniform conditions (CSIR–CIMAP, Resource centre, Hyderabad) allowed us to identify three morphotypes among the accessions, based mainly on plant canopy, leaf area and inflorescence format. On the basis of variability in plant height (59.73–93.06 cm) four types of plants were identified (Table 3). Masi et al. (2006) grouped the basil accessions into three types based on plant height and leaf size. Our results are slightly differ-

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Table 5 Eigen values and loading of variables in Principal Component axis. Eigen values of correlation matrix PC 1

PC 2

PC 3

Eigen value Explained proportion of variance (%) Cumulative proportion of variation (%)

5.127 46.61% 46.61%

2.510 22.81% 69.42%

1.439 13% 82.51%

Loading variables Plant height (cm) Plant canopy (cm) No. of primary branches Fresh biomass yield/plant (g) Leaf length (cm) Leaf width (cm) Leaf area (cm2 ) Length of inflorescence (cm) Whorls/inflorescence (No) Essential oil content (%) Test seed weight (g)

Eigen vector .411 −.561 .745 .527 .858 .812 .783 .735 .632 .652 .663

.684 .590 −.337 .620 −.354 −.286 −.392 .590 .571 −.268 .298

.217 .479 −.248 −.334 .322 .448 .460 −.067 .273 −.465 −.423 Fig. 2. Hierachical clustering of Ocimum germplasm accessions in relation to morphological characters.

ent from earlier reports of other workers who have reported plant height variation from 30 to 69.6 cm. Omer et al. (2008) and Kritikar and Basu (1984) reported 60 to 69.6 cm plant height in O. tenuiflorum. Omer et al. (2008), Labra et al. (2004) and Simon et al. (1999) reported 20 to 57.21 cm heights in O. basilicum plants. Verma et al. (2013), Omer et al. (2008) and Simon et al. (1999) reported height variation from 30 to 63.3 cm in the plant of O. americanum. Shadia et al. (2007) and Simon et al. (1999) reported narrow spreading of O. basilicum plants (30–48 cm) in comparison to our observations (54.33–67.33 cm). No such type of grouping is reported so far, however variation among the accessions has earlier been reported (Chhaya et al., 2013; Rao et al., 2011; Erum et al., 2011; Javanmardi et al., 2002) for this quantitative trait. In the present investigation, leaf size variations (4.37–43.17 cm2 ) were in accordance to the reports by Ahmad and Khaliq (2002); Vieira et al. (2001). Genetic variability in Ocimum genotypes explored promising results for future breeding research (Patel et al., 2015). The highest average biomass yield per plant was exhibited by that of O. basilicum followed by O. kilimandscharicum and O. americanum in comparison to O. tenuiflorum and O. gratissimum accessions which reflect the different genetic backgrounds of the accessions (Rao et al., 2011; Zheljazkov et al., 2008; Telci et al., 2006). The essen-

tial oil content (0.1–0.48%) in the present study was comparable to the reports of Verma et al. (2013) and Rao et al. (2011). Contrarily, in O. basilicum cv. purple and O. basilicum cv. green from Iran, the essential oil yields were found to be 0.20% and 0.50%, respectively (Sajjadi, 2006). Kitchlu et al. (2013) reported the essential oils varied from 0.16 to 0.55% in the plants of O. tenuiflorum collected from different geographical locations of India. Such variations in the essential oil content of Ocimum across different locations might be attributed to the varied agroclimatic conditions of the regions. The high average essential oil yield in O. basilicum accessions than O. tenuiflorum is a subject of previous studies (Bowes and Zheljazkov, 2004). Javanmardi et al. (2002), Lachowicz et al. (1997) and Putievsky (1993) reported that essential oil content and yield may be influenced by individual genotypes and phenological stage of the plants. Moreover, the selection of highly discriminated variables is important for morphological characterization. Multivariate techniques and cluster analysis are used in the characterization of germplasm to explore the genetic variation (Vieira et al., 2001).

Fig. 3. Scattargram showing relationship of genotype adaptation (regression coefficient) and fresh biomass yield.

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Fig. 4. Scattargram showing relationship of genotype adaptation (regression coefficient) and essential oil yield.

Table 6 Analysis of variance (ANOVA) for stability (Eberhart and Russell, 1966) in Ocimum germplasm accession for ten characters under three locations/environments. Characters

DF

Plant height (cm)

Plant canopy (cm2 )

Primary branches (nos)

Leaf length (cm)

Leaf width (cm)

Leaf area (cm2 )

Leaf stem ratio

Herb yield (t/ha)

E. oil content (% w/v)

E. oil yield (lit/ha)

14 2 28

255.37** 2548.95** 35.93**

139.83** 2932.8** 30.25**

24.74** 6.11** 5.69**

12.25** 0.94** 0.32**

4.35** 1.74** 0.11**

202.82** 30.11** 9.41**

0.49** 1.10** 0.22**

27.29** 127.79** 5.83**

0.03** 0.0002 0.0001

654.52** 724.57** 89.41**

30 1 14

203.46** 5097.89** 37.68

231.22** 5865.60** 24.49

5.72** 12.22** 3.74

0.36** 1.88** 0.29

0.22** 3.48** 0.13

10.79** 60.22** 10.94

0.28** 2.19** 0.20

13.96** 255.57** 4.05

0.0010 0.0077** 0.00125

131.75** 1449.13** 104.87

15 90

31.90 5.48

48.53 6.32

7.14 0.82

0.34 0.04

0.08 0.05

7.36 0.66

0.22 0.0044

7.11 0.29

0.000487 0.000191

69.01 5.32

Source

Genotype (G) Locations (Y) Genotype (G) × Location (Y) Locations (Y) + [GX Y] ion (Linear) year Genotype × Location (Lin.) (G×E) Pooled Dev. Pooled Error **

MSS

P < 0.01.

3.2. Principal component analysis and cluster formation The present investigation showed clear morphological differences among the fifteen genotypes for all characters studied. Indeed, many contrasts in foliage colour, productivity and oil composition were recognized between the different species. To get better insight into inter and intra- and inter-specific taxonomy, a variety of different approaches based on geographic origin, morphology, chemical composition and molecular marker have been used earlier (Bernhardt et al., 2014; Moghaddam et al., 2011; ´ Carovic-Stanko et al., 2011a,b). Morphological data (Table 4) were plotted in two-dimensional space, two distinct groups of genotypes were identified (Fig. 1). The observation summarized in the PCA analysis showed that accessions of O. tenuiflorum and O. gratissimum were quantitatively similar to each other, but distinct from the accessions of all other species. Further, the variation between accessions of the other three species was lower, as revealed by the first three variables of PC (Table 5). The correlation coefficients derived from three PCA axis cumulatively contributed to 82.51% of the total variance, first PC accounted 46.6% of the total multivariate variation, while the second accounted 22.8% and the third for 13%. The high degree of variation in the first three PC indicates a high extent of diversity for these morphological traits. The leaf length, leaf width, leaf area, No. of primary branches, length of inflorescence, essential oil content and 1000 test seed weight were the

Table 7 Environmental indices of 10 characters of Ocimum genotypes. Agromorphic variables

Plant height Plant canopy Primary branches Leaf length Leaf width Leaf area Leaf stem ratio Fresh biomass yield Essential oil content Essential oil yield

Growing locations Lucknow

Hyderabad

Bangalore

4.36 11.07 −0.55 −0.22 −0.39 0.24 −0.19 −0.25 −0.015 −2.64

10.3 4.7 0.67 0.27 0.23 1.28 −0.11 3.03 −0.001 7.88

−14.65 −15.71 −0.14 −0.047 0.15 −1.52 0.3 −2.78 0.01 −5.23

most important traits accounting for 46.61% of the total variability in the first PC variable. In order to describe relationships among the genotypes based on quantitative morphological data, present study highlighted the clustering of 15 accessions which bring out the separation between different species or between reproductively isolated groups viz., the Basilicum group and the Sanctum group (Khosla, 1995). Dendogram produced grouping defined two distinct clusters of the basilicum and the sanctum group (Figs. 2–4). The closest morphological profiles were those of accessions comprises three species (O.

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Table 8 Mean yield, stability parameters (Eberhart and Russell, 1966) of the traits fresh herb and essential oil yield of fifteen Ocimum genotypes grown at three locations. Genotypes

OCS-1 OCS-2 OCS-3 OCS-4 OCS-5 OCS-6 OCB-7 OCB-8 OCB-9 OCB-10 OCB-11 OCA-12 OCG-13 OCG-14 OCK-15 Mean

Fresh herb yield (t ha−1 )

Essential oil yield (l ha−1 ) 2

Mean (Rank)

ˇi

S di

Mean (rank)

ˇi

S2 di

14.16 (5) 11.7(8) 10.63(12) 10.60(13) 11.11(9) 10.90 (10) 16.08(3) 16.48(2) 19.05(1) 13.06(7) 16.05 (4) 15.85(11) 8.50(15) 10.05(14) 13.85(6) 13.20

0.94 1.30 0.56 0.37 0.48 0.32 1.37 0.86 1.07 0.88 1.26 1.26 1.78 1.91 0.58 1.00

12.90 45.71 0.57 3.55 14.24 1.01 -0.24 0.17 1.72 13.68 0.97 1.15 0.22 0.04 6.55

33.87 (9) 45.03 (5) 27.81(11) 12.23 (15) 13.64 (14) 26.41 (12) 45.5 (4) 47.37(3) 57.74 (2) 36.14 (8) 43.52 (6) 62.9 (1) 21.65 (13) 41.96 (7) 30.14 (10) 36.39

1.34 2.03 0.09 0.04 −0.18 0.17 1.35 1.02 2.01 0.36 0.61 1.00 1.9 3.45 −0.26 1.00

−5.01 543.25 9.54 −5.28 64.09 37.90 25.30 22.64 73.37 119.06 24.60 31.61 −3.571 5.93 21.80

Fig. 5. Most stable genotypes OCB-9 for high biomass and OCA-12 for high essential oil yield.

americanum, O. kilimandscharicum and O. basilicum) with tall plant height, large spike, higher plant and seed weight along with high to medium essential oil yield grouped together in major cluster A (Basilicum group) while, other 7 accessions comprising two species (O. tenuiflorum and O. gratissimum) with tall to medium plants height, less spread canopy, and small plant weight and spike clustered together in major cluster B (Sanctum group). Due to highest branching and largest leaf size OCG-14 accession out grouped from the rest. D2 analysis of 15 genotypes revealed highest divergence in OCG-14 genotype (Patel et al., 2015). Morphological trait analysis provided an inexpensive and reliable categorization of accessions and it appears to be a sound basis for further discrimination and authentication of genotypes by essential oil data and molecular markers. 3.3. Stability analysis for fresh biomass and essential oil yields The analysis of variance for stability (Table 6) revealed that all the genotypes differed significantly for desirable traits representing the existence of genetic variation. G × E interaction was significant for fresh biomass and essential oil yields. Environment (linear) was significantly pronounced for all the characters whereas, G × E interaction (linear) were significant for number of primary branches, fresh herbage yield and dry herbage yield/plant. The most desirable traits ie. fresh biomass and essential oil yield having significant

G × E interaction were considered for analysis of stability parameters. Data of environmental indices in relation to climatic conditions of experimental locations (Table 7 and Fig. 6) also revealed that the environment of Hyderabad (semi arid tropics) was most favourable for the phenotypic traits of Ocimum genotypes studied, followed by Lucknow agro-climate, while Bangalore was least favourable for important traits. Stability for desirable economic traits should be considered an important feature of yield trials experiments across the years/environments. Based on Eberhart and Russell (1966) stability statistics, the different genotypes of Ocimum should have a high mean yield, regression coefficient (ˇi ) = 1 or close to 1.0 and deviation from regression (S2 di ) = 0 or close to 0 can be classified as stable genotype. The genotypes OCB-9 (Fig. 5) followed by OCB-8, OCB7, OCB-11, OCS-1, OCK-15 and OCA-12 gave the high yield over the grand mean yield (13.2 t/ha) with the non significant regression value from 1 with small deviation from regression, thereby revealing stable performance across the environments (Table 8). The genotypes; OCS-2, OCS-3, OCS-4, OCS-5, OCS-6, OCB-10 OCG-13 and OCG-14 was poor and produced below average biomass yield. All these genotypes (except OCS-2) had non significant regression coefficient but significant high deviation from regression (S2 di ) indicating sensitivity to environmental changes. The genotype OCS-1 produced above average herb yield and had non significant regression coefficient and high deviation from regression

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R.P. Patel et al. / Industrial Crops and Products 77 (2015) 21–29

Environmental indices of ten traits of Ocimum genotypes over three locaons 15 10.3

11.07

10

5

7.88 4.7

4.36

3.03 0.67

0 Plant height

Plant canopy

0.27

0.23 0.15

1.28 0.24

-0.55 -0.14 -0.22-0.047 -0.39 Primary Leaf length Leaf width branches

-5

0.3

0.01 -0.015 -0.25 -0.19 -0.11 -0.001 oil Essenal oil Fresh Leaf area Leaf stem Essenal -1.52 rao biomass content yield -2.64 -2.78 yield

Lucknow Hyderabad Bangalore

-5.23 -10

-15

-14.65 -15.71

-20 Fig. 6. Environmental indices of ten characters of Ocimum genotypes over three locations (Lucknow; Hyderabad and Bangalore).

revealing sensitivity to environmental changes and gave higher yield when the environmental conditions were conductive. With respect to essential oil yield performance, the genotypes OCA-12 (Figs. 5 and 6), OCB-8, OCB-7, OCB-11 and gave the high essential oil yield over the grand mean with the regression coefficient value close to unity (ˇi = 1) and small deviation from regression (S2 di ), indicating the stability across the locations. Stability of OCA-12 (O. canum) genotype (Figs. 5 and 6) for essential oil yield in our study is in agreement with the results of Lal, (2014). Nevertheless, the observations from the present study defined substantial variations in phenology among all fifteen genotypes. Phylogenetic relationship based on morphological traits among the accessions defined two major clusters/groups viz. Basilicum (OCB7- OCB-11, OCA-12 and OCK-15) and Sanctum (OCS1–OCS6, OCG-13). Based on the Eberhart and Russell’s model, five genotypes namely, OCB-9, OCB-8, OCB-7, OCB-11 and OCA-12 were identified as high-yielding and stable due to their ability to adopt in wide environmental conditions across the locations and years and these specific genotypes were identified for further commercial exploitation. Of these, two genotypes OCB-9 and OCA-12 were found superior for biomass and essential oil yields across the years and locations.

4. Conclusions The genotypes OCS-2 and OCB-9 with high essential oil yield and high values of S2 di are performed under favourable environmental conditions. Genotype OCG-14, which also produced more essential oil yield than average yield, had low deviation from regression, thereby exhibiting less sensitivity to environmental changes. The genotype OCS1 produced below average essential oil yield and had regression coefficient close to 1.0 and low deviation from regression revealing stability for poor yield. The genotypes; OCS3, OCS-4, OCS-5, OCS-6, OCB-10, OCG-13 and OCK-15 produced below average herb yield and had significant regression coefficient from unity or high value for deviation from regression, hence considered as unstable. These genotypes are suitable and recommended for cultivation for obtaining high biomass and essential oil yields across the 3 studied locations covering north and south India.

Acknowledgements The authors are grateful to the Director, CSIR-CIMAP, Lucknow and Scientist-In-Charge, CRC, Hyderabad (India) for providing all necessary facilities for this study.

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