- Email: [email protected]
Floral biology of wild marigold (Tagetes minuta L.) and its relation to essential oil composition
Industrial Crops & Products xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Floral biology of wild marigold (Tagetes minuta L.) and its relation to essential oil composition Ajay Kumar, Rahul Dev Gautam, Ashok Kumar*, Sanatsujat Singh Agro technology of Medicinal, Aromatic and Commercially Important Plants Division CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176 061, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Essential oil Floral development Light microscopy Pollen viability Seed set Wild marigold
Wild marigold (Tagetes minuta L.) is being commercially cultivated due to high demand of its essential oil in flavor and perfumery industry. Present study investigates pollination behavior, mechanism of pollination and floral developmental of wild marigold in to 11 stages each having distinct characteristic feature as observed by visual and light microscopy. Pollen production starts from stage-2, shape of pollen is spherical tricolpate and size varies from 0.025 - 0.031 mm. However, highest pollen availability and viability is observed at stage-6. Bilobed stigma protrude from the flowers at stage-7 indicating protandry. High bee activity is evident under open field conditions suggesting entomophilous mechanism of pollination. Based on viable seed formation and per cent germination, open field conditions are most suitable for good seed set as these conditions favor cross pollination. Despite poor seed set in self-pollinated condition germination percentage was not affected significantly. Floral biology of wild marigold suggests often cross pollinated breeding behaviour. Gas Chromatography - Mass spectrometry results showed significant variation in essential oil composition at foliage and inflorescence. The (Z)-β-ocimene (52.01 %) content was highest in inflorescence while dihydrotagetone (84.85 %) content was highest in foliage. Knowledge of pollination behavior of wild marigold is critical to decide the breeding strategies required for its improvement.
1. Introduction Wild marigold (Tagetes minuta L.) is a plant native to South America (subtropical and tropical America). It belongs to Asteraceae family and has spread throughout the world as a weed (Singh et al., 2003). It is an erect, annual, hermaphrodite herb possessing green stem up to 1−2 m in height, compound leaves with narrow serrated leaflets. Wild marigold is an annual crop suitable for cultivation in the plain and hilly areas, as a monocrop or intercrop. In India, wild marigold is found naturally in the western Himalayas between altitudes range of 1000–2500 m (Thappa et al., 1993). Himachal Pradesh and the hills of Uttarakhand and Uttar Pradesh are the regions where wild marigold occurs in its natural habitat. The wild growth of this plant in these regions of India forms the source of essential oil commonly known as “Tagetes oil” (Singh et al., 2003). Wild marigold oil is used as raw material in flavour and perfumery (Bahare et al., 2018). Various studies on wild marigold suggest that there are variations in the essential oil composition based on harvesting stages (Zygadlo et al., 1993; Kéïta et al., 2000), parts of the plant used (Weaver et al., 1994) and different chemotypes within the species (Gil et al., 2000). The main chemical
⁎
components identified in essential oil of wild marigold are β-ocimene, dihydrotagetone, tagetone, β-phellandrene, limonene and tagetenone (Ester et al., 2008). Volatile oil of wild marigold is used in perfumery and as a flavor component in food products, and have suppressive biological activity against different pathogens and insects (Vasudevan et al., 1997). The essential oil has been reported to have antimicrobial (Shahzadi et al., 2010; Muyima et al., 2013), insecticidal (Wanzala, 2009), repellent (Wanzala, 2009; Wanzala and Ogoma, 2013), antioxidant (Kyarimpa et al., 2015; Mahmoud, 2013), antifungal (Grange and Ahmed, 1988), nematicidal (Adekunle et al., 2007) and allelopathic (Scrivanti et al., 2003; Arora et al., 2015). Due to high demand of its essential oil, there has been increasing interest in the cultivation of this plant for commercial production (Singh et al., 2003). Knowledge of pollination behavior of a plant species is critical to decide the breeding strategies required for its improvement. Morphological study of different stages of floral development are useful in understanding the mechanism of pollination in a crop. In the present study, various floral developmental stages and pollination behaviour of wild marigold were identified based on their distinct morphological characteristics like size of bud including corolla growth, anthesis,
Corresponding author. E-mail address: [email protected] (A. Kumar).
https://doi.org/10.1016/j.indcrop.2019.111996 Received 17 July 2019; Received in revised form 13 November 2019; Accepted 18 November 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Ajay Kumar, et al., Industrial Crops & Products, https://doi.org/10.1016/j.indcrop.2019.111996
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
2.4. Light microscopy
length of pistil, pollen availability, pollen viability and stigma receptivity. Seed set was also observed in open field and controlled (polyhouse) conditions. Chemical characterization of essential oil was also studied.
Estimates on pollen availability, viability, stigma development and receptivity were done in accordance to various developmental stages. Anthers from different floral development stages were crushed on a glass slide, stained with acetocarmine and analyzed under light microscope (Olympus Binocular Research Microscope Model CX21i). Images were captured at 10X and 40 X magnifications with Magnus CMOS10 MP C-Mount camera attached to the microscope and software used was NIS Element F 2.20.
2. Materials and methods 2.1. Plant material and growth conditions Flower development was studied in open pollinated wild marigold plants of variety ‘Himgold’ which is being maintained at Experimental Farm of AMACIP division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh (India) located at 32 °N, 76 °E and 1300 m above mean sea level. Agro-climatically, the location represents the mid-hill zone of Himachal Pradesh (Zone-II). This zone is characterized by humid sub-temperate climate and high mean annual rainfall (∼2500 mm). Observations were made on plants during flowering stage. Floral buds were observed daily during the forenoon.
2.5. Pollen production and viability Pollen production and viability was determined at different floral developmental stages. Florets of identified developmental stages were taken in three replicates from selected plants. During dissection all the five anthers from each floret were removed very gently and stained with acetocarmine. The number of both mature and immature pollen grains were recorded under microscope (Fig. 2). Pollen size of mature pollen was measured by using ocular and stage micrometer under Olympus Binocular Research Microscope Model CX21i light microscope and recorded in micrometers. Anther dehiscence in wild marigold takes place from very early stages of floral development. Anthers of various stages of florets were crushed on a glass slide, stained and analyzed under microscope. The staining was performed with the help of acetocarmine dye. Data was recorded and analyzed to assess pollen viability.
2.2. Floral characterization Observations were made with respect to number and type of florets per flower head, structural modifications of corolla and calyx, number and position of anthers, dehiscence of anthers, position of ovary and number of achenes formed. 2.3. Floral developmental stages The flower developmental stages follow event of flower bud initiation and flower differentiation. The whole developmental process was divided into distinct stages (Fig. 1) each having characteristic feature starting from formation of immature buds in the heads, further increase in size of florets, corresponding development of the anthers and stigma, anthesis, opening of the petals, senescence of petals and seed formation stages. These identified floral development stages were photographed with Nikon –DSLR camera. The floret length and floret diameter at various stages were recorded to determine full floral development events. Both pistil and anthers were dissected out from these stages of floret development. Immature bud stages (Stages 1–3) were dissected under stereo microscope (10 X magnification) as the bud size was very small (Kamenetsky, 1994). Data of flower head length, head diameter, pistil length at different developmental stages was also recorded.
2.6. Pollination and seed set Seed set of wild marigold both in open field conditions (Cross pollination) and protected conditions (self-pollination under polyhouse) was evaluated. Self-pollination was observed using selfing bags and keeping individual plant in isolation under polyhouse conditions. During flowering season, individual wild marigold plants were selected randomly and bagged. All the open flowers were removed before bagging. Data was recorded in triplicate by selecting five individual plants (50 heads each) both from open field and protected conditions. Florets were dissected using forceps and the seeds were taken out and counted. Seed test weight (1000 seeds) was also measured with the help of analytical weighing balance.
Fig. 1. Flower development stages (1–11) of Tagetes minuta. 2
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
Fig. 2. Pollen grains of Tagetes minuta.
Corp., Tokyo, Japan) having an AOC 5000 Auto injector equipped with ZB-5 MS capillary column (SGE International, Ringwood, Australia) with 30 m length ×0.25 mm i.d. and 0.25-μm film thickness. Temperature was set at 70 °C for 3 min and then to 220 °C for five minutes at the rate of 4 °C minute−1. MS source temperature was 240 °C and interface temperature was 250 °C. Sample injection volume was 2 μL of pure oil. Split ratio was 10.0 and mass scan was 40–800 amu. The carrier gas was nitrogen with constant velocity of 1.05 mL / minute
2.7. Viability and seed germination test Seed viability test was performed by tetrazolium method. Both open and self-pollinated seeds were preconditioned (soaked) with distilled water for overnight. Seed coat was removed gently so as to protect it from any mechanical injury. Seeds were then soaked in 0.5 % tetrazolium stain and kept in dark for 2−3 hours at 30 °C. Seeds which retain stain were viable and which do not were non-viable. Viability percentage was calculated as number of seed stained / Total number of seed × 100. Seed of both self-pollinated and open pollinated plants was further evaluated for its germination potential. Seeds were sown in pots containing mixture of sand, soil and FYM (1:1:1) ensuring proper water, and temperature (20−25 °C) conditions maintained at room conditions. Seed germination was recorded at periodic intervals till 8th day of final count.
2.9.2. Identification of components A homologous series of n-alkanes C9– C24 (SUPELCO SigmaAldrich) was used for calculating the retention index (RI). Identification of components was performed by calculating RI value for every peak in GCMS spectra and then comparing it with Adams tabulated indexes (not exceeding ± 10) (Adams, 1995) stored in New York mass spectral (MS) library, National Institute of Standards and Technology (NIST) (Stein, 2005). After this Mass spectral fragmentation pattern of the components was compared with those reported in literature.
2.8. Essential oil extraction Essential oil extraction was performed with 1 kg sample of wild marigold each from different floral development stages. Samples were also collected from inflorescence and foliage. Samples were subjected to hydro distillation for 4 h in glass Clevenger-type apparatus. The essential oil was extracted and moisture content in the essential oil was removed with anhydrous sodium sulfate. It was stored in a dark glass bottle at 4 °C for further chemical characterization.
2.10. Statistical analysis All the data were recorded in triplicates. Values were then used to perform statistical analysis and create graphics in Microsoft Excel 2016. Means ± standard deviation, Standard error of the mean were calculated for all measurements 3. Results and discussion
2.9. Gas chromatography analysis 3.1. Floral characterization GC analysis was carried out with the help of Shimadzu GC 2010 gas chromatograph having flame ionization detector (FID) equipped with ZB-5 MS capillary column (SGE International, Ringwood, Australia) with 30 m length ×0.25 mm i.d. and 0.25-μm film thickness. Auto injection split mode was performed with 2 μL injection volume from (10 μL oil in 2 mL of dichloromethane). The carrier gas was nitrogen with constant velocity of 1.05 mL / minute; the oven temperature was set at 70 °C for three minute and then to 220 °C for five minutes at the rate of 4 °C minute−1; injector and detector temperatures were 220 °C and 250 °C, respectively.
Each flower head consists of ray and disc florets held inside. Ray florets are ligulate, three in number (white colour) having inferior ovary below. Calyx (group of sepals) is represented by awn like scales above ovary, through which bilobed stigma stick out. Single achene develops per floret that are yellowish brown to dark brown in colour. Disc florets are bisexual with inferior ovary bearing a single ovule. Corolla (group of petals) tube consists of five fused petals with epipetalous five stamens and bicarpellary syncarpous inferior ovary with single ovule bearing bilobed stigma constitutes the gynoecium. Anthers are linear, dehiscing longitudinally and enclosing style. Bilobed stigma extrose with anthers positioned below, releasing their pollen on the outside of the flower.
2.9.1. Gas chromatography–mass spectrometry GC–MS analysis was carried out with the help of QP2010 (Shimadzu 3
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
Fig. 3. Florets of wild marigold (A) Ray Floret (B) Disc Floret.
morphological description has been studied for plants like Arabidopsis thaliana (L.) Heynh (Schneit et al., 1995) and Stevia rebaudiana (Bertoni) (Yadav et al., 2014). Floral developmental stages were studied for use as visual reference for understanding the pollination behavior of the plant and distinctions among different developmental stages (Fig. 5) are as under: Stage-1: Flower head was found to be immature, small and elongated in this stage and bract tightly enclosing 5–8 immature florets. All the floral parts are green including stigma and anthers. Average size of the Flower head in this stage was 3.5 mm and diameter was 0.91 mm, while stigma and anthers were indistinct in this stage. Stage-2: Flower head size increased almost double as compared with stage-1 (6.5 mm) and average diameter of bud was 1.41 mm. Florets are inside the closed flower head and average size of pistil at this stage was observed to be 2.5 mm. Pollen production starts in stage-2. Pollen grains are shed in the cavity between anthers and stigma as stigma is also surrounded by anthers at this early stage. As the stigma grows it takes out pollen grains out of this cavity along with its slow movement. Pollen viability test suggested 22.6 per cent viability pointing towards
3.2. Floral induction and development The flower head is initially greenish in colour and turns yellowish at maturity. Flower heads are characterized by the presence of glands which contributes to essential oil production. The arrangement of flowers in wild marigold is panicle-like. Plants produce many clusters of 45–80 cylindrical flower heads. Each head is 10−15 mm long surrounded by 4–5 involucre bracts. Each head generally contains about 3 ray florets and 3–5 disc florets (Fig. 3A and B). Color of corolla is white, Achenes are brown in colour 09−12 mm long and the pappus consisting of 1–4 tiny scales and awns 1−3 mm long. Test weight of 1000 achenes varies from 0.58 g to 0.68 g. Anthers are small, elongated, five in number and are fused (Fig. 4A). Stigma is bi-lobed (Fig. 4B) and style is surrounded by anthers. Similar type of observations was also made for head, ray florets and disc florets (Wang and Chen, 2006), essential oil (Singh et al., 2003) and floral examination (Bandana, 2017). Wild marigold floral development stages can be differentiated into 11 distinct stages, each having distinct morphological features which are described stage wise accordingly. Earlier such type of
Fig. 4. Floral development stages of Tagetes minuta (A) anthers (B) pistil. 4
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
Fig. 5. Variations at different flower developmental stages in (A) Bud/Flower head length (including stigma) (B) Bud/Flower head diameter, (C) Pistil length. Vertical bars represent standard error.
Fig. 7. Variations at different developmental stages in (A) Pollen count/anther (B) Pollen viability, Vertical bars represent standard error.
immature stage of pollen grains production. Pollen shape was spherical tricolpate and reticulately ornamental on the surface. Pollen size ranged from 0.025 mm to 0.031 mm. Stage-3: Average length and diameter of the flower head during this stage was 8.38 mm and 1.76 mm, respectively. Pistil length was observed to be 3.37 mm and pollen viability was 32.65 per cent at this particular stage. Stage-4: In this stage, average flower head length was found to be 9.27 mm and diameter was 2.04 mm, whereas average length of pistil was 3.52 mm. Pollen viability is 33.47 per cent at this stage. Oil glands were observed to be present on the flower head from this stage (Fig. 6A). These specialized structures contribute to essential oil production. Oil contains different biomolecules and have sweet smell. This helps to attract insects for ensuring cross pollination and seed set. Plants species have evolved this mechanism as strategies to strengthen fitness of the population. Stage-5: Flower head become ready to open at this stage and florets
still remains inside. Average length and diameter of flower head was observed to be 11.28 mm and 2.21 mm, respectively. Pistil length at this stage was 3.65 mm. pollen viability was observed to be 45.05 per cent at this stage. A curvation in stigma lobes was observed during this stage. This stage represents the initiation of stigma receptivity. Stage-6: Flower head distinguished by one prominent white petal (one petal stage) emerging from the flower head. Average flower head length at this stage was 12.12 mm and diameter was 2.34 mm. Stigma length enlarge to 4.57 mm at this stage. Stage-6 recorded highest pollen viability (67.19 per cent) as well as highest stage for pollen availability (Fig. 7A and B). Stage-7: This stage was distinguished by two prominent white petals emerging from flower head (two petal stage). The average length of flower head was 12.98 mm and diameter was 2.34 mm. At stage-6 the stigma becomes “curved” and continues till last stage. Pistil growth is highest at stage-7 with average pistil length 5.22 mm (Figs. 5C and 4 B)
Fig. 6. Oil glands (A) Immature flower head (Stage-4) (B) Mature flower heads. 5
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
At this stage bilobed stigma is clearly visible outside the floret, which is curved and average length of floret including stigma is 12.98 mm. It is the stage of highest stigma receptivity which is very important stage for pollination. Pollen availability further declines and the pollen viability is reduced to 40.67 per cent. Stage-8: Flower head is distinguished by three prominent petals (three petal stage) and is characterized by maximum growth in size of floral bud and diameter. The average length of flower bud is 13.20 mm and bud diameter is 2.36 mm. Pistil length enlarge to 5.18 mm at this stage. Pollen viability is 37.94 per cent at this stage. Ovary starts turning light black at this stage. This indicates the stage of initiation of seed development process. Stage-9: Corolla and stigma show senescence and dry up. Stigma turns to pale brown colour and corolla gets fully dried up and turn dark brown but calyx is still light green. The average length of flower head was 12.18 mm and diameter reduce to was 1.85 mm. Pistil length was 4.77 mm at this stage. Stage-10: Whole flower head starts drying; corolla and stigma turn dark brown and seeds attain maturity. The average length /of flower head was 10.92 mm and diameter was reduced to 1.71 mm. stigma length is 4.98 mm at this stage. Stage-11: At this stage seed is ready for dispersal. During this stage fragrance in form of sweet smell is still prevalent which attracts insects. Insects in turn help in seed dispersal.
were considerably variable in the different parts of the plant. The (Z)-βocimene was significantly higher (46.42 %) in inflorescence compared with its content in foliage. On the contrary, dihydrotagetone content was significantly higher (73.91 %) in foliage as compared to its content in inflorescence. The limonene content was also recorded to be higher (1.83 %) in foliage while lower in inflorescence. These observations suggest that dihydrotagetone content decreases and (Z)-β-ocimene content increases when plant enters reproductive phase. Prakasa rao et al., 1999 showed similar findings that essential oil in leaf contains more dihydrotagetone and less (Z)-β-ocimene as compared to oil in inflorescence. Similar to (Z)-β-ocimene, (Z)-tagetone, (Z)-ocimenone and (E)-ocimenone contents were higher in inflorescence (6.43 %, 4.67 % and 17.17 %, respectively). 3.6. Variation of essential oil composition at each floral development stages Analysis of essential oil components at each floral development stages (Table 2) revealed considerable variations. The dihydrotagetone content was highest at stages 1–5 (vegetative phases) while (E)-ocimenone content was significantly lower in all these stages. The (Z)-βocimene content was significantly higher at satges 6–10 (reproductive phases) while limonene content was significantly lower in these stages. 4. Conclusion Eleven floral developmental stages of wild marigold each having distinct characteristic features were identified on the basis of size of bud development including corolla growth, anthesis, length of stigma, pollen production and viability which gave an insight regarding the breeding behavior of wild marigold. Pollen availability starts from stage-2 and continues till stage-8. Stage-5 represents the initiation of stigma receptivity. Stigma growth is highest in stage-7 indicating highest receptivity during this stage. Total seed production was higher in open field conditions as these conditions favors cross pollination. Seed weight was also higher in cross pollinated condition as compare to self-pollinated condition. Despite poor seed set in self-pollinated condition germination percentage was not effected much. Significant variations in essential oil composition of inflorescence and foliage were recorded. (Z)-β-ocimene content was highest in inflorescence, while Dihydrotagetone content was highest in foliage. These variations in essential oil composition are possibly due to change of crop from vegetative phase to reproductive phase. Self-pollination in a species results in homozygous genotypes and maintains homogeneity of populations, while cross pollination leads to heterozygous individuals and promotes heterogeneity in population. The floral biology of wild marigold suggests often cross pollinated breeding behaviour.
3.3. Seed set At maturity when wild marigold seeds are ready for dispersal, total seed formation was manually counted by dissecting out the seeds from floret. Seed set percentage was higher (94.43 %) in open field condition than that of self -pollinated conditions (45.57 %) (Fig. 8). Open field conditions favors cross pollination resulting in higher seed set percentage than the self-pollinated conditions. Results suggest open field conditions favor good seed set in wild marigold. 3.4. Seed viability and germination test Seed viability test suggest 86 % viabilty in open pollinated and 83 % viabilty in self pollinated condition. There was no significant difference between seed germination percentage for both open pollinated and self pollinated conditions and it was comparable, 81.40 ± 2.06 for open pollinated and 79.80 ± 1.46 for self-pollination under polyhouse conditions. 3.5. Variation of essential oil composition in inflorescence and foliage Essential oil composition of wild marigold analysed by GC and GC–MS led to the identification of six major compounds which contributed maximum to the total volume (Table 1). In the present study, the major components of Tagetes essential oil
Authors contribution All authors contributed equally. Disclosure statements No potential conflict of interest was reported by the authors. Declaration of Competing Interest Authors declare that they don’t have any conflict of interest Acknowledgement We thankfully acknowledge the support of Dr. Sanjay Kumar, Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur (HP, India) for providing the facilities for the study and Council of Scientific and Industrial Research, New Delhi (India) for providing financial assistance under the Project HCP-0007 entitled
Fig. 8. Percent seed set in open field and self pollinated conditions. 6
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
Table 1 Variations of essential oil composition of wild marigold in foliage and inflorescence. Chemical compounds (%) Plant Part
(Z)-β-ocimene
Dihydrotagetone
Limonene
(Z)-tagetone
(Z)- ocimenone
(E)-ocimenone
Foliage Inflorescence RIa RI Lit.b
5.59 ± 3.50 52.01 ± 3.23 1041 1050
84.85 ± 5.04 10.94 ± 0.62 1057 1054
3.30 ± 0.11 1.47 ± 0.29 1034 1031
1.87 ± 0.62 8.30 ± 0.36 1157 1153
0.09 ± 0.09 4.76 ± 0.69 1234 1231
0.14 ± 0.14 17.31 ± 3.97 1243 1239
RIa (retention indices), RI Lit.b (retention indices from literature). Table 2 Variations of essential oil composition of wild marigold at different floral development stages. Chemical compounds (%) Stages
(Z)-β-ocimene
Dihydrotagetone
Limonene
(Z)-tagetone
(Z)- ocimenone
(E)-ocimenone
Stage-1 Stage-2 Stage-3 Stage-4 Stage-5 Stage-6 Stage-7 Stage-8 Stage-9 Stage-10
0.29 ± 0.15 0.56 ± 0.23 0.95 ± 0.47 0.65 ± 0.12 2.66 ± 0.42 24.27 ± 2.23 53.70 ± 0.25 52.88 ± 0.55 46.18 ± 1.87 38.7 ± 0.52
53.92 ± 1.06 51.26 ± 2.16 60.56 ± 2.56 72.97 ± 2.22 82.07 ± 1.22 27.60 ± 1.14 7.20 ± 0.01 10.65 ± 0.88 7.08 ± 0.78 7.31 ± 0.84
4.36 ± 0.32 5.03 ± 0.97 6.72 ± 0.38 5.24 ± 0.59 3.13 ± 0.18 3.83 ± 0.19 2.44 ± 0.01 1.70 ± 0.33 1.61 ± 0.11 1.98 ± 0.06
1.13 ± 0.58 2.58 ± 0.56 5.06 ± 1.04 1.02 ± 0.57 1.83 ± 0.36 13.10 ± 2.05 6.67 ± 0.02 8.74 ± 0.18 7.77 ± 0.08 7.94 ± 1.43
2.71 ± 0.29 3.41 ± 1.09 4.30 ± 0.74 1.25 ± 0.43 0.13 ± 0.06 7.38 ± 0.88 5.45 ± 0.27 4.49 ± 0.55 9.0 ± 1.64 7.94 ± 1.43
0.75 ± 0.40 0.81 ± 0.44 1.18 ± 0.16 0.25 ± 0.25 0.20 ± 0.10 9.97 ± 0.60 11.73 ± 0.66 15.87 ± 1.74 20.33 ± 0.87 18.86 ± 0.06
Data are arithmetic means ± S.E., n = 3.
“CSIR Aroma-Mission”
activities of Tagetes minuta, Lippia javanica and Foeniculum vulgare essential oils from the Eastern Cape Province of South Africa. J. Essent. Oil. Bear Pl. 7 (1), 68–78. Prakasa Rao, E.V.S., Syamasundar, K.V., Gopinath, C.T., Ramesh, S., 1999. Agronomical and chemical studies on Tagetes minuta grown in a red soil of a semiarid tropical region in India. J. Essent. Oil Res. 11, 259–261. Schneit, K., Hülskamp, M., Pruitt, R.E., 1995. Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. Plant J. 7, 731–749. Scrivanti, L.R., Zunino, M.P., Zygadlo, J.A., 2003. Tagetes minuta and Schinusareira essential oils as allelopathic agents. Biochem. Syst. Ecol. 31 (6), 563–572. Shahzadi, I., Hassan, A., Khan, U.W., Shah, M.M., 2010. Evaluating biological activities of the seed extracts from Tagetes minuta L. found in Northern Pakistan. J. Med. Plants Res. 4 (20), 2108–2112. Singh, V., Singh, B., Kaul, V.K., 2003. Domestication of wild marigold (Tagetes minuta L.) as a potential economic crop in western Himalaya and north Indian plains. Econ. Bot. 57, 535–544. Stein, S.E., 2005. Mass Spectral Database and Software, Version3.02. National Institute of Standards and Technology (NIST), Gaithersburg, MD. Thappa, R., Agarwal, S., Kalia, N., Kapoor, R., 1993. Changes in chemical composition of Tagetes minuta oil at various stages of flowering and fruiting. J. Essent. Oil Res. 5, 375–379. Vasudevan, P., Suman, K., Satyawati, S., 1997. Tagetes: a multipurpose plant. Bioresour. Technol. 62 (29), 33. Wang, C.M., Chen, C.H., 2006. Tagetes minuta L. (Asteraceae), a newly naturalized plant in Taiwan. Taiwania. 51 (1), 32–35. Wanzala, W., 2009. Ethnobotanicals for Management of the Brown Ear Tick, Rhipicephalus Appendiculatus in Western Kenya. Ponsen & Looijen, Wageningen, pp. 231. Wanzala, W., Ogoma, S.B., 2013. Chemical composition and mosquito repellency of essential oil of Tagetes minuta from the Southern slopes of Mount Elgon in Western Kenya. J. Essent. Oil. Bear Pl. 16 (2), 216–232. Weaver, D.K., Wells, C.D., Dankel, F.V., Bertsch, W., Sing, S.E., Sirharan, S., 1994. Insecticidal activity of floral, foliar and root extracts of Tagetes minuta (Asterales: asteraceae) against adult Mexican bean weavils (Coleoptera: bruchidae). J. Econ. Entomo. 87, 1718–1725. Yadav, A.K., Singh, S., Rajeev, 2014. Self-incompatibility evidenced through scanning electron microscopy and pollination behaviour in Stevia rebaudiana. Indian J. Agr. Sci. 84 (1), 93–100. Zygadlo, J.A., Maestr, D.M., Ariza Espinar, L., 1993. The volatile oil of Tagetes Argentina Cabrera. J. Essent. Oil Res. 5 (1), 85–86.
References Adams, P.R., 1995. Identification of essential oil components by Gas Chromatography/ Mass Spectroscopy, 4th ed. Allured Publishing Corporation, Suite A Carol Stream, IL, USA, pp. 60188–62403 336 Gundersen Drive. Adekunle, O.K., Acharya, R., Singh, B., 2007. Toxicity of pure compounds isolated from Tagetes minuta oil to Meloidogyne incognita, Australas. Plant Dis. Notes. 2, 101–104. Arora, K., Batish, D.R., Singh, P.H., Kohli, R.K., 2015. Allelopathic potential of the essential oil of wild marigold (Tagetes minuta L.) against some invasive weeds. J. Agri. Environ. Sci. 3, 56–60. Bahare, S., Marco, V., Maria, F., Bezerra, M.B., Joara, N.P.C., Antonio Linkoln, A.B.L., Henrique Douglas, M.C., Sara, V., Dorota, K., Hubert, A., Mehdi, S., Nathália, C.C.L., Zubaida, Y., Miquel, M., Marcello, I., Simone, C., Javad, S.R., 2018. Tagetes spp. essential oils and other extracts: chemical characterization and biological activity. Molecules 23, 2847. Bandana, K., 2017. Studies on morphological, essential oil and molecular diversity in Tagetes minuta L. in Himachal Pradesh. PhD Thesis. YSPUHF Nauni, Solan (H.P) India. Ester, R.C., Griselda, B., Alfredo, F.S., Gustavo, A.V., María, F.Z., 2008. Chemical composition of essential oil from Tagetes minuta L. leaves and flower. J. Arg. Chem. Soc. 96, 1–2. Gil, A., Ghersa, C.M., Leicach, S., 2000. Essential oil yield and composition of Tagetes minuta accessions from Argentina. Biochem. Syst. Ecol. 28, 261–274. Grange, M., Ahmed, S., 1988. Handbook of Plants With Pest-control Properties. John Wiley & Sons (Wiley-Interscience), Inc., New York, pp. 470. Kamenetsky, R., 1994. Life cycle, flower initiation and propagation of the desert geophyte Allium rothii. Int. J. Plant Sc. 155, 597–605. Kéïta, S., Vincent, C., Schmit, J.P., Ramaswamy, S., Bélanger, A., 2000. Effect of various essential oils on Callosobruchus maculatus (F.) (Coleoptera: bruchidae). J. Stored Prod. Res. 36, 355–364. Kyarimpa, C., Omolo, I.N., Kabasa, J.D., Nagawa, C.B., Wasswa, J., Kikawa, C.R., 2015. Evaluation of anti-oxidant properties in essential oil and solvent extracts from Tagetes minuta. Afr. J. Pure Appl. Chem. 9 (5), 98–104. Mahmoud, G.L., 2013. Biological effects, antioxidant and anticancer activities of marigold and basil essential oils. J. Med. Plants Res. 7 (10), 561–572. Muyima, O.N., Nziweni, S., Mabinya, L.V., 2013. Antimicrobial and antioxidative
7
Contents lists available at ScienceDirect
Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop
Floral biology of wild marigold (Tagetes minuta L.) and its relation to essential oil composition Ajay Kumar, Rahul Dev Gautam, Ashok Kumar*, Sanatsujat Singh Agro technology of Medicinal, Aromatic and Commercially Important Plants Division CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh 176 061, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Essential oil Floral development Light microscopy Pollen viability Seed set Wild marigold
Wild marigold (Tagetes minuta L.) is being commercially cultivated due to high demand of its essential oil in flavor and perfumery industry. Present study investigates pollination behavior, mechanism of pollination and floral developmental of wild marigold in to 11 stages each having distinct characteristic feature as observed by visual and light microscopy. Pollen production starts from stage-2, shape of pollen is spherical tricolpate and size varies from 0.025 - 0.031 mm. However, highest pollen availability and viability is observed at stage-6. Bilobed stigma protrude from the flowers at stage-7 indicating protandry. High bee activity is evident under open field conditions suggesting entomophilous mechanism of pollination. Based on viable seed formation and per cent germination, open field conditions are most suitable for good seed set as these conditions favor cross pollination. Despite poor seed set in self-pollinated condition germination percentage was not affected significantly. Floral biology of wild marigold suggests often cross pollinated breeding behaviour. Gas Chromatography - Mass spectrometry results showed significant variation in essential oil composition at foliage and inflorescence. The (Z)-β-ocimene (52.01 %) content was highest in inflorescence while dihydrotagetone (84.85 %) content was highest in foliage. Knowledge of pollination behavior of wild marigold is critical to decide the breeding strategies required for its improvement.
1. Introduction Wild marigold (Tagetes minuta L.) is a plant native to South America (subtropical and tropical America). It belongs to Asteraceae family and has spread throughout the world as a weed (Singh et al., 2003). It is an erect, annual, hermaphrodite herb possessing green stem up to 1−2 m in height, compound leaves with narrow serrated leaflets. Wild marigold is an annual crop suitable for cultivation in the plain and hilly areas, as a monocrop or intercrop. In India, wild marigold is found naturally in the western Himalayas between altitudes range of 1000–2500 m (Thappa et al., 1993). Himachal Pradesh and the hills of Uttarakhand and Uttar Pradesh are the regions where wild marigold occurs in its natural habitat. The wild growth of this plant in these regions of India forms the source of essential oil commonly known as “Tagetes oil” (Singh et al., 2003). Wild marigold oil is used as raw material in flavour and perfumery (Bahare et al., 2018). Various studies on wild marigold suggest that there are variations in the essential oil composition based on harvesting stages (Zygadlo et al., 1993; Kéïta et al., 2000), parts of the plant used (Weaver et al., 1994) and different chemotypes within the species (Gil et al., 2000). The main chemical
⁎
components identified in essential oil of wild marigold are β-ocimene, dihydrotagetone, tagetone, β-phellandrene, limonene and tagetenone (Ester et al., 2008). Volatile oil of wild marigold is used in perfumery and as a flavor component in food products, and have suppressive biological activity against different pathogens and insects (Vasudevan et al., 1997). The essential oil has been reported to have antimicrobial (Shahzadi et al., 2010; Muyima et al., 2013), insecticidal (Wanzala, 2009), repellent (Wanzala, 2009; Wanzala and Ogoma, 2013), antioxidant (Kyarimpa et al., 2015; Mahmoud, 2013), antifungal (Grange and Ahmed, 1988), nematicidal (Adekunle et al., 2007) and allelopathic (Scrivanti et al., 2003; Arora et al., 2015). Due to high demand of its essential oil, there has been increasing interest in the cultivation of this plant for commercial production (Singh et al., 2003). Knowledge of pollination behavior of a plant species is critical to decide the breeding strategies required for its improvement. Morphological study of different stages of floral development are useful in understanding the mechanism of pollination in a crop. In the present study, various floral developmental stages and pollination behaviour of wild marigold were identified based on their distinct morphological characteristics like size of bud including corolla growth, anthesis,
Corresponding author. E-mail address: [email protected] (A. Kumar).
https://doi.org/10.1016/j.indcrop.2019.111996 Received 17 July 2019; Received in revised form 13 November 2019; Accepted 18 November 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Ajay Kumar, et al., Industrial Crops & Products, https://doi.org/10.1016/j.indcrop.2019.111996
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
2.4. Light microscopy
length of pistil, pollen availability, pollen viability and stigma receptivity. Seed set was also observed in open field and controlled (polyhouse) conditions. Chemical characterization of essential oil was also studied.
Estimates on pollen availability, viability, stigma development and receptivity were done in accordance to various developmental stages. Anthers from different floral development stages were crushed on a glass slide, stained with acetocarmine and analyzed under light microscope (Olympus Binocular Research Microscope Model CX21i). Images were captured at 10X and 40 X magnifications with Magnus CMOS10 MP C-Mount camera attached to the microscope and software used was NIS Element F 2.20.
2. Materials and methods 2.1. Plant material and growth conditions Flower development was studied in open pollinated wild marigold plants of variety ‘Himgold’ which is being maintained at Experimental Farm of AMACIP division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh (India) located at 32 °N, 76 °E and 1300 m above mean sea level. Agro-climatically, the location represents the mid-hill zone of Himachal Pradesh (Zone-II). This zone is characterized by humid sub-temperate climate and high mean annual rainfall (∼2500 mm). Observations were made on plants during flowering stage. Floral buds were observed daily during the forenoon.
2.5. Pollen production and viability Pollen production and viability was determined at different floral developmental stages. Florets of identified developmental stages were taken in three replicates from selected plants. During dissection all the five anthers from each floret were removed very gently and stained with acetocarmine. The number of both mature and immature pollen grains were recorded under microscope (Fig. 2). Pollen size of mature pollen was measured by using ocular and stage micrometer under Olympus Binocular Research Microscope Model CX21i light microscope and recorded in micrometers. Anther dehiscence in wild marigold takes place from very early stages of floral development. Anthers of various stages of florets were crushed on a glass slide, stained and analyzed under microscope. The staining was performed with the help of acetocarmine dye. Data was recorded and analyzed to assess pollen viability.
2.2. Floral characterization Observations were made with respect to number and type of florets per flower head, structural modifications of corolla and calyx, number and position of anthers, dehiscence of anthers, position of ovary and number of achenes formed. 2.3. Floral developmental stages The flower developmental stages follow event of flower bud initiation and flower differentiation. The whole developmental process was divided into distinct stages (Fig. 1) each having characteristic feature starting from formation of immature buds in the heads, further increase in size of florets, corresponding development of the anthers and stigma, anthesis, opening of the petals, senescence of petals and seed formation stages. These identified floral development stages were photographed with Nikon –DSLR camera. The floret length and floret diameter at various stages were recorded to determine full floral development events. Both pistil and anthers were dissected out from these stages of floret development. Immature bud stages (Stages 1–3) were dissected under stereo microscope (10 X magnification) as the bud size was very small (Kamenetsky, 1994). Data of flower head length, head diameter, pistil length at different developmental stages was also recorded.
2.6. Pollination and seed set Seed set of wild marigold both in open field conditions (Cross pollination) and protected conditions (self-pollination under polyhouse) was evaluated. Self-pollination was observed using selfing bags and keeping individual plant in isolation under polyhouse conditions. During flowering season, individual wild marigold plants were selected randomly and bagged. All the open flowers were removed before bagging. Data was recorded in triplicate by selecting five individual plants (50 heads each) both from open field and protected conditions. Florets were dissected using forceps and the seeds were taken out and counted. Seed test weight (1000 seeds) was also measured with the help of analytical weighing balance.
Fig. 1. Flower development stages (1–11) of Tagetes minuta. 2
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
Fig. 2. Pollen grains of Tagetes minuta.
Corp., Tokyo, Japan) having an AOC 5000 Auto injector equipped with ZB-5 MS capillary column (SGE International, Ringwood, Australia) with 30 m length ×0.25 mm i.d. and 0.25-μm film thickness. Temperature was set at 70 °C for 3 min and then to 220 °C for five minutes at the rate of 4 °C minute−1. MS source temperature was 240 °C and interface temperature was 250 °C. Sample injection volume was 2 μL of pure oil. Split ratio was 10.0 and mass scan was 40–800 amu. The carrier gas was nitrogen with constant velocity of 1.05 mL / minute
2.7. Viability and seed germination test Seed viability test was performed by tetrazolium method. Both open and self-pollinated seeds were preconditioned (soaked) with distilled water for overnight. Seed coat was removed gently so as to protect it from any mechanical injury. Seeds were then soaked in 0.5 % tetrazolium stain and kept in dark for 2−3 hours at 30 °C. Seeds which retain stain were viable and which do not were non-viable. Viability percentage was calculated as number of seed stained / Total number of seed × 100. Seed of both self-pollinated and open pollinated plants was further evaluated for its germination potential. Seeds were sown in pots containing mixture of sand, soil and FYM (1:1:1) ensuring proper water, and temperature (20−25 °C) conditions maintained at room conditions. Seed germination was recorded at periodic intervals till 8th day of final count.
2.9.2. Identification of components A homologous series of n-alkanes C9– C24 (SUPELCO SigmaAldrich) was used for calculating the retention index (RI). Identification of components was performed by calculating RI value for every peak in GCMS spectra and then comparing it with Adams tabulated indexes (not exceeding ± 10) (Adams, 1995) stored in New York mass spectral (MS) library, National Institute of Standards and Technology (NIST) (Stein, 2005). After this Mass spectral fragmentation pattern of the components was compared with those reported in literature.
2.8. Essential oil extraction Essential oil extraction was performed with 1 kg sample of wild marigold each from different floral development stages. Samples were also collected from inflorescence and foliage. Samples were subjected to hydro distillation for 4 h in glass Clevenger-type apparatus. The essential oil was extracted and moisture content in the essential oil was removed with anhydrous sodium sulfate. It was stored in a dark glass bottle at 4 °C for further chemical characterization.
2.10. Statistical analysis All the data were recorded in triplicates. Values were then used to perform statistical analysis and create graphics in Microsoft Excel 2016. Means ± standard deviation, Standard error of the mean were calculated for all measurements 3. Results and discussion
2.9. Gas chromatography analysis 3.1. Floral characterization GC analysis was carried out with the help of Shimadzu GC 2010 gas chromatograph having flame ionization detector (FID) equipped with ZB-5 MS capillary column (SGE International, Ringwood, Australia) with 30 m length ×0.25 mm i.d. and 0.25-μm film thickness. Auto injection split mode was performed with 2 μL injection volume from (10 μL oil in 2 mL of dichloromethane). The carrier gas was nitrogen with constant velocity of 1.05 mL / minute; the oven temperature was set at 70 °C for three minute and then to 220 °C for five minutes at the rate of 4 °C minute−1; injector and detector temperatures were 220 °C and 250 °C, respectively.
Each flower head consists of ray and disc florets held inside. Ray florets are ligulate, three in number (white colour) having inferior ovary below. Calyx (group of sepals) is represented by awn like scales above ovary, through which bilobed stigma stick out. Single achene develops per floret that are yellowish brown to dark brown in colour. Disc florets are bisexual with inferior ovary bearing a single ovule. Corolla (group of petals) tube consists of five fused petals with epipetalous five stamens and bicarpellary syncarpous inferior ovary with single ovule bearing bilobed stigma constitutes the gynoecium. Anthers are linear, dehiscing longitudinally and enclosing style. Bilobed stigma extrose with anthers positioned below, releasing their pollen on the outside of the flower.
2.9.1. Gas chromatography–mass spectrometry GC–MS analysis was carried out with the help of QP2010 (Shimadzu 3
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
Fig. 3. Florets of wild marigold (A) Ray Floret (B) Disc Floret.
morphological description has been studied for plants like Arabidopsis thaliana (L.) Heynh (Schneit et al., 1995) and Stevia rebaudiana (Bertoni) (Yadav et al., 2014). Floral developmental stages were studied for use as visual reference for understanding the pollination behavior of the plant and distinctions among different developmental stages (Fig. 5) are as under: Stage-1: Flower head was found to be immature, small and elongated in this stage and bract tightly enclosing 5–8 immature florets. All the floral parts are green including stigma and anthers. Average size of the Flower head in this stage was 3.5 mm and diameter was 0.91 mm, while stigma and anthers were indistinct in this stage. Stage-2: Flower head size increased almost double as compared with stage-1 (6.5 mm) and average diameter of bud was 1.41 mm. Florets are inside the closed flower head and average size of pistil at this stage was observed to be 2.5 mm. Pollen production starts in stage-2. Pollen grains are shed in the cavity between anthers and stigma as stigma is also surrounded by anthers at this early stage. As the stigma grows it takes out pollen grains out of this cavity along with its slow movement. Pollen viability test suggested 22.6 per cent viability pointing towards
3.2. Floral induction and development The flower head is initially greenish in colour and turns yellowish at maturity. Flower heads are characterized by the presence of glands which contributes to essential oil production. The arrangement of flowers in wild marigold is panicle-like. Plants produce many clusters of 45–80 cylindrical flower heads. Each head is 10−15 mm long surrounded by 4–5 involucre bracts. Each head generally contains about 3 ray florets and 3–5 disc florets (Fig. 3A and B). Color of corolla is white, Achenes are brown in colour 09−12 mm long and the pappus consisting of 1–4 tiny scales and awns 1−3 mm long. Test weight of 1000 achenes varies from 0.58 g to 0.68 g. Anthers are small, elongated, five in number and are fused (Fig. 4A). Stigma is bi-lobed (Fig. 4B) and style is surrounded by anthers. Similar type of observations was also made for head, ray florets and disc florets (Wang and Chen, 2006), essential oil (Singh et al., 2003) and floral examination (Bandana, 2017). Wild marigold floral development stages can be differentiated into 11 distinct stages, each having distinct morphological features which are described stage wise accordingly. Earlier such type of
Fig. 4. Floral development stages of Tagetes minuta (A) anthers (B) pistil. 4
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
Fig. 5. Variations at different flower developmental stages in (A) Bud/Flower head length (including stigma) (B) Bud/Flower head diameter, (C) Pistil length. Vertical bars represent standard error.
Fig. 7. Variations at different developmental stages in (A) Pollen count/anther (B) Pollen viability, Vertical bars represent standard error.
immature stage of pollen grains production. Pollen shape was spherical tricolpate and reticulately ornamental on the surface. Pollen size ranged from 0.025 mm to 0.031 mm. Stage-3: Average length and diameter of the flower head during this stage was 8.38 mm and 1.76 mm, respectively. Pistil length was observed to be 3.37 mm and pollen viability was 32.65 per cent at this particular stage. Stage-4: In this stage, average flower head length was found to be 9.27 mm and diameter was 2.04 mm, whereas average length of pistil was 3.52 mm. Pollen viability is 33.47 per cent at this stage. Oil glands were observed to be present on the flower head from this stage (Fig. 6A). These specialized structures contribute to essential oil production. Oil contains different biomolecules and have sweet smell. This helps to attract insects for ensuring cross pollination and seed set. Plants species have evolved this mechanism as strategies to strengthen fitness of the population. Stage-5: Flower head become ready to open at this stage and florets
still remains inside. Average length and diameter of flower head was observed to be 11.28 mm and 2.21 mm, respectively. Pistil length at this stage was 3.65 mm. pollen viability was observed to be 45.05 per cent at this stage. A curvation in stigma lobes was observed during this stage. This stage represents the initiation of stigma receptivity. Stage-6: Flower head distinguished by one prominent white petal (one petal stage) emerging from the flower head. Average flower head length at this stage was 12.12 mm and diameter was 2.34 mm. Stigma length enlarge to 4.57 mm at this stage. Stage-6 recorded highest pollen viability (67.19 per cent) as well as highest stage for pollen availability (Fig. 7A and B). Stage-7: This stage was distinguished by two prominent white petals emerging from flower head (two petal stage). The average length of flower head was 12.98 mm and diameter was 2.34 mm. At stage-6 the stigma becomes “curved” and continues till last stage. Pistil growth is highest at stage-7 with average pistil length 5.22 mm (Figs. 5C and 4 B)
Fig. 6. Oil glands (A) Immature flower head (Stage-4) (B) Mature flower heads. 5
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
At this stage bilobed stigma is clearly visible outside the floret, which is curved and average length of floret including stigma is 12.98 mm. It is the stage of highest stigma receptivity which is very important stage for pollination. Pollen availability further declines and the pollen viability is reduced to 40.67 per cent. Stage-8: Flower head is distinguished by three prominent petals (three petal stage) and is characterized by maximum growth in size of floral bud and diameter. The average length of flower bud is 13.20 mm and bud diameter is 2.36 mm. Pistil length enlarge to 5.18 mm at this stage. Pollen viability is 37.94 per cent at this stage. Ovary starts turning light black at this stage. This indicates the stage of initiation of seed development process. Stage-9: Corolla and stigma show senescence and dry up. Stigma turns to pale brown colour and corolla gets fully dried up and turn dark brown but calyx is still light green. The average length of flower head was 12.18 mm and diameter reduce to was 1.85 mm. Pistil length was 4.77 mm at this stage. Stage-10: Whole flower head starts drying; corolla and stigma turn dark brown and seeds attain maturity. The average length /of flower head was 10.92 mm and diameter was reduced to 1.71 mm. stigma length is 4.98 mm at this stage. Stage-11: At this stage seed is ready for dispersal. During this stage fragrance in form of sweet smell is still prevalent which attracts insects. Insects in turn help in seed dispersal.
were considerably variable in the different parts of the plant. The (Z)-βocimene was significantly higher (46.42 %) in inflorescence compared with its content in foliage. On the contrary, dihydrotagetone content was significantly higher (73.91 %) in foliage as compared to its content in inflorescence. The limonene content was also recorded to be higher (1.83 %) in foliage while lower in inflorescence. These observations suggest that dihydrotagetone content decreases and (Z)-β-ocimene content increases when plant enters reproductive phase. Prakasa rao et al., 1999 showed similar findings that essential oil in leaf contains more dihydrotagetone and less (Z)-β-ocimene as compared to oil in inflorescence. Similar to (Z)-β-ocimene, (Z)-tagetone, (Z)-ocimenone and (E)-ocimenone contents were higher in inflorescence (6.43 %, 4.67 % and 17.17 %, respectively). 3.6. Variation of essential oil composition at each floral development stages Analysis of essential oil components at each floral development stages (Table 2) revealed considerable variations. The dihydrotagetone content was highest at stages 1–5 (vegetative phases) while (E)-ocimenone content was significantly lower in all these stages. The (Z)-βocimene content was significantly higher at satges 6–10 (reproductive phases) while limonene content was significantly lower in these stages. 4. Conclusion Eleven floral developmental stages of wild marigold each having distinct characteristic features were identified on the basis of size of bud development including corolla growth, anthesis, length of stigma, pollen production and viability which gave an insight regarding the breeding behavior of wild marigold. Pollen availability starts from stage-2 and continues till stage-8. Stage-5 represents the initiation of stigma receptivity. Stigma growth is highest in stage-7 indicating highest receptivity during this stage. Total seed production was higher in open field conditions as these conditions favors cross pollination. Seed weight was also higher in cross pollinated condition as compare to self-pollinated condition. Despite poor seed set in self-pollinated condition germination percentage was not effected much. Significant variations in essential oil composition of inflorescence and foliage were recorded. (Z)-β-ocimene content was highest in inflorescence, while Dihydrotagetone content was highest in foliage. These variations in essential oil composition are possibly due to change of crop from vegetative phase to reproductive phase. Self-pollination in a species results in homozygous genotypes and maintains homogeneity of populations, while cross pollination leads to heterozygous individuals and promotes heterogeneity in population. The floral biology of wild marigold suggests often cross pollinated breeding behaviour.
3.3. Seed set At maturity when wild marigold seeds are ready for dispersal, total seed formation was manually counted by dissecting out the seeds from floret. Seed set percentage was higher (94.43 %) in open field condition than that of self -pollinated conditions (45.57 %) (Fig. 8). Open field conditions favors cross pollination resulting in higher seed set percentage than the self-pollinated conditions. Results suggest open field conditions favor good seed set in wild marigold. 3.4. Seed viability and germination test Seed viability test suggest 86 % viabilty in open pollinated and 83 % viabilty in self pollinated condition. There was no significant difference between seed germination percentage for both open pollinated and self pollinated conditions and it was comparable, 81.40 ± 2.06 for open pollinated and 79.80 ± 1.46 for self-pollination under polyhouse conditions. 3.5. Variation of essential oil composition in inflorescence and foliage Essential oil composition of wild marigold analysed by GC and GC–MS led to the identification of six major compounds which contributed maximum to the total volume (Table 1). In the present study, the major components of Tagetes essential oil
Authors contribution All authors contributed equally. Disclosure statements No potential conflict of interest was reported by the authors. Declaration of Competing Interest Authors declare that they don’t have any conflict of interest Acknowledgement We thankfully acknowledge the support of Dr. Sanjay Kumar, Director, CSIR-Institute of Himalayan Bioresource Technology, Palampur (HP, India) for providing the facilities for the study and Council of Scientific and Industrial Research, New Delhi (India) for providing financial assistance under the Project HCP-0007 entitled
Fig. 8. Percent seed set in open field and self pollinated conditions. 6
Industrial Crops & Products xxx (xxxx) xxxx
A. Kumar, et al.
Table 1 Variations of essential oil composition of wild marigold in foliage and inflorescence. Chemical compounds (%) Plant Part
(Z)-β-ocimene
Dihydrotagetone
Limonene
(Z)-tagetone
(Z)- ocimenone
(E)-ocimenone
Foliage Inflorescence RIa RI Lit.b
5.59 ± 3.50 52.01 ± 3.23 1041 1050
84.85 ± 5.04 10.94 ± 0.62 1057 1054
3.30 ± 0.11 1.47 ± 0.29 1034 1031
1.87 ± 0.62 8.30 ± 0.36 1157 1153
0.09 ± 0.09 4.76 ± 0.69 1234 1231
0.14 ± 0.14 17.31 ± 3.97 1243 1239
RIa (retention indices), RI Lit.b (retention indices from literature). Table 2 Variations of essential oil composition of wild marigold at different floral development stages. Chemical compounds (%) Stages
(Z)-β-ocimene
Dihydrotagetone
Limonene
(Z)-tagetone
(Z)- ocimenone
(E)-ocimenone
Stage-1 Stage-2 Stage-3 Stage-4 Stage-5 Stage-6 Stage-7 Stage-8 Stage-9 Stage-10
0.29 ± 0.15 0.56 ± 0.23 0.95 ± 0.47 0.65 ± 0.12 2.66 ± 0.42 24.27 ± 2.23 53.70 ± 0.25 52.88 ± 0.55 46.18 ± 1.87 38.7 ± 0.52
53.92 ± 1.06 51.26 ± 2.16 60.56 ± 2.56 72.97 ± 2.22 82.07 ± 1.22 27.60 ± 1.14 7.20 ± 0.01 10.65 ± 0.88 7.08 ± 0.78 7.31 ± 0.84
4.36 ± 0.32 5.03 ± 0.97 6.72 ± 0.38 5.24 ± 0.59 3.13 ± 0.18 3.83 ± 0.19 2.44 ± 0.01 1.70 ± 0.33 1.61 ± 0.11 1.98 ± 0.06
1.13 ± 0.58 2.58 ± 0.56 5.06 ± 1.04 1.02 ± 0.57 1.83 ± 0.36 13.10 ± 2.05 6.67 ± 0.02 8.74 ± 0.18 7.77 ± 0.08 7.94 ± 1.43
2.71 ± 0.29 3.41 ± 1.09 4.30 ± 0.74 1.25 ± 0.43 0.13 ± 0.06 7.38 ± 0.88 5.45 ± 0.27 4.49 ± 0.55 9.0 ± 1.64 7.94 ± 1.43
0.75 ± 0.40 0.81 ± 0.44 1.18 ± 0.16 0.25 ± 0.25 0.20 ± 0.10 9.97 ± 0.60 11.73 ± 0.66 15.87 ± 1.74 20.33 ± 0.87 18.86 ± 0.06
Data are arithmetic means ± S.E., n = 3.
“CSIR Aroma-Mission”
activities of Tagetes minuta, Lippia javanica and Foeniculum vulgare essential oils from the Eastern Cape Province of South Africa. J. Essent. Oil. Bear Pl. 7 (1), 68–78. Prakasa Rao, E.V.S., Syamasundar, K.V., Gopinath, C.T., Ramesh, S., 1999. Agronomical and chemical studies on Tagetes minuta grown in a red soil of a semiarid tropical region in India. J. Essent. Oil Res. 11, 259–261. Schneit, K., Hülskamp, M., Pruitt, R.E., 1995. Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. Plant J. 7, 731–749. Scrivanti, L.R., Zunino, M.P., Zygadlo, J.A., 2003. Tagetes minuta and Schinusareira essential oils as allelopathic agents. Biochem. Syst. Ecol. 31 (6), 563–572. Shahzadi, I., Hassan, A., Khan, U.W., Shah, M.M., 2010. Evaluating biological activities of the seed extracts from Tagetes minuta L. found in Northern Pakistan. J. Med. Plants Res. 4 (20), 2108–2112. Singh, V., Singh, B., Kaul, V.K., 2003. Domestication of wild marigold (Tagetes minuta L.) as a potential economic crop in western Himalaya and north Indian plains. Econ. Bot. 57, 535–544. Stein, S.E., 2005. Mass Spectral Database and Software, Version3.02. National Institute of Standards and Technology (NIST), Gaithersburg, MD. Thappa, R., Agarwal, S., Kalia, N., Kapoor, R., 1993. Changes in chemical composition of Tagetes minuta oil at various stages of flowering and fruiting. J. Essent. Oil Res. 5, 375–379. Vasudevan, P., Suman, K., Satyawati, S., 1997. Tagetes: a multipurpose plant. Bioresour. Technol. 62 (29), 33. Wang, C.M., Chen, C.H., 2006. Tagetes minuta L. (Asteraceae), a newly naturalized plant in Taiwan. Taiwania. 51 (1), 32–35. Wanzala, W., 2009. Ethnobotanicals for Management of the Brown Ear Tick, Rhipicephalus Appendiculatus in Western Kenya. Ponsen & Looijen, Wageningen, pp. 231. Wanzala, W., Ogoma, S.B., 2013. Chemical composition and mosquito repellency of essential oil of Tagetes minuta from the Southern slopes of Mount Elgon in Western Kenya. J. Essent. Oil. Bear Pl. 16 (2), 216–232. Weaver, D.K., Wells, C.D., Dankel, F.V., Bertsch, W., Sing, S.E., Sirharan, S., 1994. Insecticidal activity of floral, foliar and root extracts of Tagetes minuta (Asterales: asteraceae) against adult Mexican bean weavils (Coleoptera: bruchidae). J. Econ. Entomo. 87, 1718–1725. Yadav, A.K., Singh, S., Rajeev, 2014. Self-incompatibility evidenced through scanning electron microscopy and pollination behaviour in Stevia rebaudiana. Indian J. Agr. Sci. 84 (1), 93–100. Zygadlo, J.A., Maestr, D.M., Ariza Espinar, L., 1993. The volatile oil of Tagetes Argentina Cabrera. J. Essent. Oil Res. 5 (1), 85–86.
References Adams, P.R., 1995. Identification of essential oil components by Gas Chromatography/ Mass Spectroscopy, 4th ed. Allured Publishing Corporation, Suite A Carol Stream, IL, USA, pp. 60188–62403 336 Gundersen Drive. Adekunle, O.K., Acharya, R., Singh, B., 2007. Toxicity of pure compounds isolated from Tagetes minuta oil to Meloidogyne incognita, Australas. Plant Dis. Notes. 2, 101–104. Arora, K., Batish, D.R., Singh, P.H., Kohli, R.K., 2015. Allelopathic potential of the essential oil of wild marigold (Tagetes minuta L.) against some invasive weeds. J. Agri. Environ. Sci. 3, 56–60. Bahare, S., Marco, V., Maria, F., Bezerra, M.B., Joara, N.P.C., Antonio Linkoln, A.B.L., Henrique Douglas, M.C., Sara, V., Dorota, K., Hubert, A., Mehdi, S., Nathália, C.C.L., Zubaida, Y., Miquel, M., Marcello, I., Simone, C., Javad, S.R., 2018. Tagetes spp. essential oils and other extracts: chemical characterization and biological activity. Molecules 23, 2847. Bandana, K., 2017. Studies on morphological, essential oil and molecular diversity in Tagetes minuta L. in Himachal Pradesh. PhD Thesis. YSPUHF Nauni, Solan (H.P) India. Ester, R.C., Griselda, B., Alfredo, F.S., Gustavo, A.V., María, F.Z., 2008. Chemical composition of essential oil from Tagetes minuta L. leaves and flower. J. Arg. Chem. Soc. 96, 1–2. Gil, A., Ghersa, C.M., Leicach, S., 2000. Essential oil yield and composition of Tagetes minuta accessions from Argentina. Biochem. Syst. Ecol. 28, 261–274. Grange, M., Ahmed, S., 1988. Handbook of Plants With Pest-control Properties. John Wiley & Sons (Wiley-Interscience), Inc., New York, pp. 470. Kamenetsky, R., 1994. Life cycle, flower initiation and propagation of the desert geophyte Allium rothii. Int. J. Plant Sc. 155, 597–605. Kéïta, S., Vincent, C., Schmit, J.P., Ramaswamy, S., Bélanger, A., 2000. Effect of various essential oils on Callosobruchus maculatus (F.) (Coleoptera: bruchidae). J. Stored Prod. Res. 36, 355–364. Kyarimpa, C., Omolo, I.N., Kabasa, J.D., Nagawa, C.B., Wasswa, J., Kikawa, C.R., 2015. Evaluation of anti-oxidant properties in essential oil and solvent extracts from Tagetes minuta. Afr. J. Pure Appl. Chem. 9 (5), 98–104. Mahmoud, G.L., 2013. Biological effects, antioxidant and anticancer activities of marigold and basil essential oils. J. Med. Plants Res. 7 (10), 561–572. Muyima, O.N., Nziweni, S., Mabinya, L.V., 2013. Antimicrobial and antioxidative
7