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Honeybees foraging response in genetically diversified opium poppy
Bioresource Technology 97 (2006) 1578–1581
Short Communication
Honeybees foraging response in genetically diversified opium poppy H.K. Srivastava a, Dwijendra Singh b
b,*
a Genetics and Plant Breeding Division, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226 015, India Entomology Division, Crop Protection Area, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226 015, India
Received 1 September 2004; received in revised form 12 July 2005; accepted 14 July 2005 Available online 16 September 2005
Abstract Studies were carried out on honeybees foraging on plant flowers. Results showed significantly higher foraging response of honeybees (Apis mellifera) in genetically divergent narcotic plant opium poppy (Papaver somniferum). Of the 18 mutants and two locally adapted cultivars of diverse genotypes screened, eight revealed significantly greater foraging response manifesting honeybeeÕs preference towards specific plant morphotypes. The number of flower bloom did not correspond to number of foraging bees in both mutant and cultivar plant types of opium poppy. The genotype specific foraging response of honeybees could be attributed to physico-chemical properties of opium poppy flowers. This could have implications for the development of opium alkaloid fortified honeys for novel pharmaceuticals and isolation of natural spray compounds to attract honeybee pollinators for promoting crossing and sustainable hybridity in crops. 2005 Elsevier Ltd. All rights reserved. Keywords: Honeybee; Apis mellifera; Foraging; Genetically diversified plant; Papaver somniferum
1. Introduction Honeybees play a great role in cross pollinated crops for higher agricultural productivity and are source of nutritious and natural diet materials for human consumption since ancient times (Crane, 1980). It is estimated that about 30–80% productivity of agricultural crops increases during bee foraging for nectars from flowers. Honey is also used as drug vehicle in several ayurvedic medicines. In some countries, honey is the first diet of human baby because of its natural occurrence and unique medicinal properties, known in traditional systems of medicine. Export of honey and related products earn sizable amount of money (Singh, 2000). Evidently, bee-keepers require potential flowering pastures plant species that attract maximum number of bees and their *
Corresponding author. E-mail address: [email protected] (D. Singh).
0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.07.013
high foraging activities for higher production of honey with different quality and fragrance for the supply in the market as already available apple, cherry, berry, pear, clove and litchy types. Therefore, development and identification of new plant type for high honeybee foraging is in great demand to enhance the agricultural productivity along with availability of new type of honey all over the world. In present study, out of the 18 diverse mutants and two locally adapted cultivars of genotypes of opium poppy, we identified eight significantly potential attractant plant morphotypes for high foraging of honeybees. The aim of this work was to study if flowers emanating from genetically diverse opium poppy plants could lure foraging honeybees (Apis mellifera). 2. Methods Eighteen M6 generation mutant lines from two locally adapted cultivars ‘‘Sanchita (S)’’ and ‘‘Vivek (V)’’
H.K. Srivastava, D. Singh / Bioresource Technology 97 (2006) 1578–1581
after the dry seeds (12% moisture) of each one initially treated with six distinct mutagen treatments—physical (G, gamma-rays, 15 kR using the 60CO gamma cell dose rate 6.5 kR/min) and chemicals (E, ethyl methane sulfonate, 0.4%; N, sodium azide, 0.001 M; and ethidium bromide, 0.2%) together with synergistically combined (gamma rays, 5 kR + ethyl methane sulfonate, 0.4%) were developed. These mutants were identified and characterized in M2 generation. Genotypic purity of the mutants was maintained in each subsequent generation(s) from M2 to M6. ‘‘Sanchita’’ is a morphine rich chemovariety and has been earlier developed by line breeding selection method, while ‘‘Vivek’’ is a big capsulated mutant variety recovered in gamma-rays (5 kR) irradiated material (M1 generation) of the popular opium poppy variety ‘‘Shweta’’ (Satpute, 2000). The field experiment was conducted by sowing seeds of opium poppy; 18 mutants were characterized as high seed yielder (SN-1), morphine rich (SN-2), codeine rich
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serrated leaf (SG-35-I), codeine rich normal leaf (SG35-II), codeine rich (SGE-48), codeine rich high seed yielder (SGE-9), vertically ridged capsule (VG-5), downey mildew disease resistant (VG-46), dwarf plant type (VE-1), high seed yielder (SGE-14), early maturing type (VG-20), morphine rich (SE-1), latexless non-waxy capsule (VG-26), fringed petalled (VG-27), trichommed serrated leaf (VN-35-1), trichommed normal leaf (VN35-II), thebaine rich (SGE-29), bold capsulated type (VN-23) and two locally adapted cultivars ‘‘Sanchita’’ and ‘‘Vivek’’ (Table 1) in sandy loam soil in the plot size 7 · 5 m, plant to plant distance 10 cm and 30 cm apart, and replicated three times in randomized block design at the instituteÕs research farm in November, 2001 at Lucknow (26.5N, 80.5E and 120 m altitude), India. All standard agronomic practices were followed including N, P, and K nutrition by fertilizers (Satpute, 2000). The dry and mature capsules of the plant were harvested without lancing during April 2002. The number
Table 1 Foraging response of honeybees (Apis mellifera) to different mutants and genotypes of opium poppy (Papaver somniferum L.) Symbol
Genotype/ treatment
a b c
SN-1 SN-2 SG-35-I
d
SG-35-II
e f
SGE-48 SGE-9
g
VG-5
h
VG-46
i j k l m n o
VE-1 SGE-14 VG-20 SE-1 SANCHITA VIVEK VG-26
p q
VG-27 VN-35-I
r
VN-35-II
s t – – –
SGE-29 VN-23 SEm± LSD5% LSD1%
Brief descriptor
High seed yielder Morphine rich Codeine rich serrated leaf Codeine rich normal leaf Codeine rich Codeine rich high seed yielder Vertically ridged capsule Downey mildew disease resistant Dwarf plant type High seed yielder Early maturing type Morphine rich Fringed leaf Large capsule Latexless non-waxy capsule Fringed petalled Trichommed serrated leaf Trichommed normal leaf Thebaine rich Bold capsuled type – – –
Mean+ number of flower of opium poppy and honeybees per plot on three sampling date March 04, 2002
March 06, 2002
March 08, 2002
Flower
Flower
Flower
Honeybee
Honeybee
15.0 (1.17) 19.6 (1.27) 11.0 (1.02)
1.3 (2.00) 2.0 (6.66) 3.6 (19.00)
12.3 (0.95) 19.3 (1.27) 11.0 (1.01)
2.0 (04.66) 5.0 (45.66) 10.0 (112.66)*
18.3 (1.22)
3.6 (17.00)
23.0 (1.35)
20.3 (1.29) 15.0 (1.17)
3.0 (09.66) 2.6 (08.00)
15.3 (1.16)
Honeybee
11.6 (0.99) 12.6 (1.08) 10.3 (1.00)
3.6 (20.33) 1.6 (05.66) 6.6 (54.00)
8.6 (95.33)**
11.0 (1.34)**
4.6 (23.33)
20.0 (1.28) 15.0 (1.16)
9.6 (187.00)* 3.0 (09.66)
15.0 (1.16) 11.6 (1.01)
5.0 (25.66) 4.0 (18.66)
1.3 (05.33)
18.3 (1.15)
4.6 (22.66)
12.6 (1.06)
4.3 (19.00)
18.0 (1.20)
5.0 (51.00)
19.3 (1.18)
2.6 (08.66)
14.3 (1.15)
4.6 (24.66)
07.6 29.0 18.3 20.3 13.0 18.6 15.0
2.0 8.0 6.0 4.0 6.0 2.3 5.6
08.0 22.3 14.6 19.3 26.0 14.3 16.6
05.3 17.6 07.0 18.3 19.6 05.3 10.3
3.0 4.0 3.6 3.3 5.0 2.3 7.3
(0.82) (1.44)* (1.24) (1.29) (1.30) (1.18) (1.16)
(12.00) (72.66) (31.33) (16.66) (48.66) (08.33) (45.66)
(0.76) (1.33) (1.14) (1.26) (1.39)* (1.15) (1.17)
5.0 9.3 7.3 7.3 13.6 2.6 13.6
(39.00) (88.66)** (67.33)** (63.33) (204.33)* (10.00) (203.66)*
(0.69) (1.23) (0.81) (1.25) (1.24) (0.47) (0.99)
(11.66) (16.66) (15.00) (15.33) (25.66) (09.00) (65.33)
16.0 (1.15) 08.3 (0.76)
4.0 (24.66) 1.6 (05.66)
14.0 (1.13) 7.6 (0.84)
6.3 (41.66) 2.0 (06.00)
11.0 (1.02) 05.3 (0.67)
7.6 (79.00) 4.0 (22.00)
11.3 (1.04)
2.3 (06.33)
9.3 (0.96)
5.6 (41.66)
08.0 (0.86)
2.6 (10.66)
15.3 (1.18) 14.0 (1.13) 0.10 0.25 NS
3.6 (15.00) 4.6 (24.66) 18.58 NS NS
15.0 (1.17) 13.3 (1.11) 0.10 0.27 0.35
6.6 (48.66) 9.0 (91.66)** 41.06 107.09 138.93
09.3 (0.95) 07.0 (0.82) 0.12 0.32 0.42
1.6 (05.66) 2.6 (10.00) 19.15 NS NS
Figures in parentheses for number of flower is transformed in log values and for honeybees in v2, SEm = Standard error of mean and LSD = least standard deviation. * Significant at 5% (P < 0.05). ** Significant at 1% (P < 0.01). + Mean of three replications.
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H.K. Srivastava, D. Singh / Bioresource Technology 97 (2006) 1578–1581
of flowers was counted visibly plot wise for each genotype followed by counting the population of honeybees landed on flowers and collecting the nectar and pollen by visual sampling method (Visual sampling method is one of the standard common method of sampling by which the arthropods density is estimated in natural conditions through eyes for counting the number of insects.). Naturally occurring wild population constituted the bee source for the present experimentation. The data on number of flowers and honeybees associated with the mutants and other genotypes were recorded on three sampling dates, viz., March 04, 06 and 08, 2002 during 7.30–8.30 AM. The data of number of flowers and honeybees were subjected to statistical analysis by working out least standard deviations (LSD) at significance level of P < 0.05 and P < 0.01, respectively (Table 1).
3. Results and discussion Among twenty genetically divergent lines, flower variability of eight genotypes; Sanchita, VG-26, SGE48, SG-35-1 at P < 0.05 and SG-35-II, VN-23, SGE-14, VG-20 at P < 0.01 significantly attracted naturally occurring wild population of honeybees for their foraging visits with regard to nectar and pollen collection over other 12 divergent lines of opium poppy. This study revealed that development of new plant varieties based on suitable flower traits could be of greater significance in increasing the yield potential of plants as well as for increased foraging response to beesÕ vis-a`-vis higher honey production in both quantity and quality (poppy type). SGE-14 was the only mutant genotype that significantly bloomed (P < 0.05) with higher mean number of flowers over other genotypes as sampled. However, none of the genetic variant of opium poppy was found suitable for the honeybee foraging response on the first sampling date (Table 1). In contrast, the numbers of flowers were recorded to be significantly higher at P < 0.01 level of critical difference in the plots of ‘‘Sanchita’’ variety on second sampling date. Further, the numbers of honeybees visiting flowers were significantly higher in eight genotypes for honeybees foraging responses during second sampling date (Table 1). Non-significant observation of bee visit in relation to diverse genotypes of opium poppy first sampling date and third sampling date could be attributed to the age of plant maturity for the biosynthesis of semio-chemicals in nectar/flow of nectar being maximum on the second sampling date. Therefore, only the observations taken on second sampling being only optimum blooming and nectar day showed the significant numbers of foraging bees on different genotypes (Table 1). The numbers of flowers in the third sampling were found to bloom much markedly in the mutant line SG-35-II both at P < 0.01 and P < 0.05 level of critical differences. However, none of the opium poppy geno-
typic variant with diverse flower traits or plant morphological traits showed significant differences in flower suitability for the foraging of A. mellifera on the third sampling date (Table 1). It was noted that the floral behavior of opium poppy was highly specific and bleaking type depending upon intensity of sunlight. Therefore, low number of blooming as compared to number of plant shown per plot was observed during the observational period of 1 h in the morning (7.30–8.30 AM). The visits of foraging bees were only confined to those opened flowers that were freshly open and bear fresh pollen. Usually, bees visited only those flowers that contained petals. However, in opium poppy the floral petals were caudices thus, exposing the crown within few hours of flower opening, making its floral biology typical and irregular (Srivastava, 2002a,b; Satpute, 2000). The results demonstrated that the number of flower was not at all associated with the number of honeybees visiting on different genotypes (Table 1). However, the genetically governed traits of flower along with its specific aromas and flavors (having spectrum of both major and minor secondary metabolites) could have lured greater number of foraging honeybees. Flowering medicinal plants may be valuable source of nectar and pollen for producing honey of specific flavor, fragrance and medicinal principle. Youngken (1950, 1956) and Free (1993) studied bee activity on more than 300 drug plants. However, apparently no systematic efforts have been endeavored so far to identify the narcotic drug plant in relation to bee visits as biological signals for plant germplasm diversity. The demand of drug plants are high and of continuous nature for their utilization as natural products in various new formulations of pharmaceuticals and nutraceuticals for human health care. Opium poppy, Papaver somniferum (Family: Papaveraceae), a self-pollinated species is famous for its latex biosynthesis with natural opium alkaloids used in pharmaceuticals (Srivastava, 2002a,b). P. somniferum is the natural and unique source of major opium alkaloids—morphine and codeine (Budavari et al., 1996) besides thebaine, papaverine, and narcotine (Srivastava, 2002a,b) used abundantly by major pharmaceutical industries at global level. Evidently, the opium alkaloids do possess strong analgesic (morphine), anti-tussive (codeine), and anti-spasmodic (papaverine) activities. Morphine itself has wider applications as the most potent painkiller influencing the central nervous system. The opium type honey specifically obtained from foraging bees through divergent poppy cultivars could lead to new medicated product. New avenue has emerged after the discovery of a specific ‘‘promoter gene’’ in nectary (the nectar producing plant organ) of genetically engineered Petunias for a cure against a highly contagious ‘‘Parovirus’’ (causative for severe diarrhea and vomiting) in dogs (Anonymous, 1999). The idea is to develop medicated honey or honey
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capsule as potential vaccine for curing the disease. The vaccine is likely to be produced by expressing genes under the control of the promoter gene. Specific proteins are produced in the nectar, which is then collected by visiting honeybees and concentrated into honey. The high concentration of sugars in honey stabilizes the protein structure making purification relatively easy (Anonymous, 1999). The difference in foraging responses of honeybees in eight diverse mutants/genotypes over twenty studied could be attributed to the genepool variability in respect of flavoring and aromatic compounds of pollen and/or nectar together with floral traits differences for color, shape and size of anther, stigma, pollen sac, pollen number, stigmatic rays, petal and petal fringing. To produce hybrid seeds, a pollination control system is required to prevent unwanted self-pollination. In crop species with hermaphrodite flowers, this can be a major benefit. The identification of suitable physico-chemical traits for the foraging of higher number of honeybees may be a potentially desirable natureÕs tool to enhance the yield productivity of flowering crop plants in general and can be used as suitable pasture for honeybees (Roubik, 2002; Westerkamp and Gottsberger, 2000; Buchmann and Nabhan, 1996; Ingram et al., 1996). The active principle(s) responsible for foraging high number of bees in these genetically divergent plants, may be derived from flowers (anther and pollen) and can be used to spray on the flowers to lure more number of bees on other self-pollinating flowering plants to create conditions for hybrid vigor to enhance yield out of the 18 mutants and two locally adapted cultivars of diverse genotypes of opium poppy (Srivastava, 2000, 2002a,b). Researchers have identified key volatiles (like s-linalool as scent, flavor and fragrant) from basil, strawberries and other edible plants attracting both human and non-human grazers (Brown, 2002a,b; Kessler and Baldwin, 2001). The result is supportive of the thesis that opium alkaloids or other compounds of pharmaceutical interest may be present as secondary metabolites in the pollen and nectar and can affect the foraging behaviour of bees. However, the specific empirical association between bee foraging vis-a`-vis medicinal and aromatic constituents from diverse floral and anther traits of narcotic or scent producing plants at molecular level is not yet known. A gene ‘‘for’’ governing honeybees foraging behavior and its variant for diverse bee activities has been discovered (Pennisi, 2002). This gene codes for a cell-signalling molecule called a cyclic GMP-dependent protein kinase.
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Acknowledgements We thank Dr. Peter Day of AgBiotech Center, Rutgers University for his critical review of the manuscript and Mr. S.K. Mishra for statistical analysis.
References Anonymous, 1999. Business and regulatory news: a spoon full of honey. Nat. Biotechnol. 17, 837–838. Brown, K., 2002a. Floral evolution: a compromise on floral traits. Science 298, 45–46. Brown, K., 2002b. Something to sniff at: unbottling floral scent. Science 296, 2327–2329. Buchmann, S.L., Nabhan, G.P., 1996. The Forgotten Pollinators. Island Press, Shearwater Books, Washington, DC, Covelo, CA, pp. 320. Budavari, S., OÕNeil, M.J., Smith, A., Heckelman, P.E., Kinneary, J.F., 1996. The Merck Index. An Encyclopedia of Chemical, Drugs and Biologicals, 12th ed. Merck Research Laboratories, Division of Merck and Co., Inc., NJ. Crane, E., 1980. A Book of Honey, first ed. International Bee Research Association, Oxford University Press, Walton Street, Oxford, pp. 198. Free, J.B., 1993. Insect Pollination of Crops. Academic Press, San Diego, London, pp. 684. Ingram, M., Nabhan, G.P., Buchmann, S.L., 1996. Impending pollination crisis threatens biodiversity and agriculture. Tropinet 7, 1. Kessler, A., Baldwin, I.T., 2001. Defensive function of herbivoreinduced plant volatile emissions in nature. Science 291, 2141–2144. Pennisi, E., 2002. One gene determines bee social status. Science 296, 636. Roubik, D.W., 2002. The value of bees to coffee harvest. Nature 417, 708. Satpute, G.K., 2000. Ph.D. Thesis. Mutation breeding for genetic enhancement of desirable alkaloids in opium poppy (Papaver somniferum L.). University of Lucknow, Lucknow, India, 178 pp. Singh, D., 2000. A focus on honeybees in the tropics. Curr. Sci. 79 (9), 1155–1157. Srivastava, H.K., 2000. Nuclear control and mitochondrial transcript processing with relevance to cytoplasmic male sterility in higher plants. Curr. Sci. 79, 176–186. Srivastava, H.K., 2002a. Biotechnology and genetic engineering. In: Govil, J.N., Anand Kumar, P., Singh, V.K. (Eds.), Series Recent Progress in Medicinal Plants, vol. 4. SCI Tech Pub., USA, pp. 455– 474. Srivastava, H.K., 2002b. Nuclear and cytoplasmic diversity in manifestation of disease control and genepool conservation for sustainable crop productivity. J. Sustain. Agric. 21, 47–72. Westerkamp, C., Gottsberger, G., 2000. Diversity pays in crop pollination. Crop Sci. 40, 1209–1222. Youngken Jr. H.W., 1950. Drug plant gardens and apiculture. Iowa State Apiarist Report 1949, 115–122. Youngken Jr., H.W., 1956. The value of medicinal plants to bee keeping. Glean. Bee Cult. 84, 16–18.
Short Communication
Honeybees foraging response in genetically diversified opium poppy H.K. Srivastava a, Dwijendra Singh b
b,*
a Genetics and Plant Breeding Division, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226 015, India Entomology Division, Crop Protection Area, Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow 226 015, India
Received 1 September 2004; received in revised form 12 July 2005; accepted 14 July 2005 Available online 16 September 2005
Abstract Studies were carried out on honeybees foraging on plant flowers. Results showed significantly higher foraging response of honeybees (Apis mellifera) in genetically divergent narcotic plant opium poppy (Papaver somniferum). Of the 18 mutants and two locally adapted cultivars of diverse genotypes screened, eight revealed significantly greater foraging response manifesting honeybeeÕs preference towards specific plant morphotypes. The number of flower bloom did not correspond to number of foraging bees in both mutant and cultivar plant types of opium poppy. The genotype specific foraging response of honeybees could be attributed to physico-chemical properties of opium poppy flowers. This could have implications for the development of opium alkaloid fortified honeys for novel pharmaceuticals and isolation of natural spray compounds to attract honeybee pollinators for promoting crossing and sustainable hybridity in crops. 2005 Elsevier Ltd. All rights reserved. Keywords: Honeybee; Apis mellifera; Foraging; Genetically diversified plant; Papaver somniferum
1. Introduction Honeybees play a great role in cross pollinated crops for higher agricultural productivity and are source of nutritious and natural diet materials for human consumption since ancient times (Crane, 1980). It is estimated that about 30–80% productivity of agricultural crops increases during bee foraging for nectars from flowers. Honey is also used as drug vehicle in several ayurvedic medicines. In some countries, honey is the first diet of human baby because of its natural occurrence and unique medicinal properties, known in traditional systems of medicine. Export of honey and related products earn sizable amount of money (Singh, 2000). Evidently, bee-keepers require potential flowering pastures plant species that attract maximum number of bees and their *
Corresponding author. E-mail address: [email protected] (D. Singh).
0960-8524/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.07.013
high foraging activities for higher production of honey with different quality and fragrance for the supply in the market as already available apple, cherry, berry, pear, clove and litchy types. Therefore, development and identification of new plant type for high honeybee foraging is in great demand to enhance the agricultural productivity along with availability of new type of honey all over the world. In present study, out of the 18 diverse mutants and two locally adapted cultivars of genotypes of opium poppy, we identified eight significantly potential attractant plant morphotypes for high foraging of honeybees. The aim of this work was to study if flowers emanating from genetically diverse opium poppy plants could lure foraging honeybees (Apis mellifera). 2. Methods Eighteen M6 generation mutant lines from two locally adapted cultivars ‘‘Sanchita (S)’’ and ‘‘Vivek (V)’’
H.K. Srivastava, D. Singh / Bioresource Technology 97 (2006) 1578–1581
after the dry seeds (12% moisture) of each one initially treated with six distinct mutagen treatments—physical (G, gamma-rays, 15 kR using the 60CO gamma cell dose rate 6.5 kR/min) and chemicals (E, ethyl methane sulfonate, 0.4%; N, sodium azide, 0.001 M; and ethidium bromide, 0.2%) together with synergistically combined (gamma rays, 5 kR + ethyl methane sulfonate, 0.4%) were developed. These mutants were identified and characterized in M2 generation. Genotypic purity of the mutants was maintained in each subsequent generation(s) from M2 to M6. ‘‘Sanchita’’ is a morphine rich chemovariety and has been earlier developed by line breeding selection method, while ‘‘Vivek’’ is a big capsulated mutant variety recovered in gamma-rays (5 kR) irradiated material (M1 generation) of the popular opium poppy variety ‘‘Shweta’’ (Satpute, 2000). The field experiment was conducted by sowing seeds of opium poppy; 18 mutants were characterized as high seed yielder (SN-1), morphine rich (SN-2), codeine rich
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serrated leaf (SG-35-I), codeine rich normal leaf (SG35-II), codeine rich (SGE-48), codeine rich high seed yielder (SGE-9), vertically ridged capsule (VG-5), downey mildew disease resistant (VG-46), dwarf plant type (VE-1), high seed yielder (SGE-14), early maturing type (VG-20), morphine rich (SE-1), latexless non-waxy capsule (VG-26), fringed petalled (VG-27), trichommed serrated leaf (VN-35-1), trichommed normal leaf (VN35-II), thebaine rich (SGE-29), bold capsulated type (VN-23) and two locally adapted cultivars ‘‘Sanchita’’ and ‘‘Vivek’’ (Table 1) in sandy loam soil in the plot size 7 · 5 m, plant to plant distance 10 cm and 30 cm apart, and replicated three times in randomized block design at the instituteÕs research farm in November, 2001 at Lucknow (26.5N, 80.5E and 120 m altitude), India. All standard agronomic practices were followed including N, P, and K nutrition by fertilizers (Satpute, 2000). The dry and mature capsules of the plant were harvested without lancing during April 2002. The number
Table 1 Foraging response of honeybees (Apis mellifera) to different mutants and genotypes of opium poppy (Papaver somniferum L.) Symbol
Genotype/ treatment
a b c
SN-1 SN-2 SG-35-I
d
SG-35-II
e f
SGE-48 SGE-9
g
VG-5
h
VG-46
i j k l m n o
VE-1 SGE-14 VG-20 SE-1 SANCHITA VIVEK VG-26
p q
VG-27 VN-35-I
r
VN-35-II
s t – – –
SGE-29 VN-23 SEm± LSD5% LSD1%
Brief descriptor
High seed yielder Morphine rich Codeine rich serrated leaf Codeine rich normal leaf Codeine rich Codeine rich high seed yielder Vertically ridged capsule Downey mildew disease resistant Dwarf plant type High seed yielder Early maturing type Morphine rich Fringed leaf Large capsule Latexless non-waxy capsule Fringed petalled Trichommed serrated leaf Trichommed normal leaf Thebaine rich Bold capsuled type – – –
Mean+ number of flower of opium poppy and honeybees per plot on three sampling date March 04, 2002
March 06, 2002
March 08, 2002
Flower
Flower
Flower
Honeybee
Honeybee
15.0 (1.17) 19.6 (1.27) 11.0 (1.02)
1.3 (2.00) 2.0 (6.66) 3.6 (19.00)
12.3 (0.95) 19.3 (1.27) 11.0 (1.01)
2.0 (04.66) 5.0 (45.66) 10.0 (112.66)*
18.3 (1.22)
3.6 (17.00)
23.0 (1.35)
20.3 (1.29) 15.0 (1.17)
3.0 (09.66) 2.6 (08.00)
15.3 (1.16)
Honeybee
11.6 (0.99) 12.6 (1.08) 10.3 (1.00)
3.6 (20.33) 1.6 (05.66) 6.6 (54.00)
8.6 (95.33)**
11.0 (1.34)**
4.6 (23.33)
20.0 (1.28) 15.0 (1.16)
9.6 (187.00)* 3.0 (09.66)
15.0 (1.16) 11.6 (1.01)
5.0 (25.66) 4.0 (18.66)
1.3 (05.33)
18.3 (1.15)
4.6 (22.66)
12.6 (1.06)
4.3 (19.00)
18.0 (1.20)
5.0 (51.00)
19.3 (1.18)
2.6 (08.66)
14.3 (1.15)
4.6 (24.66)
07.6 29.0 18.3 20.3 13.0 18.6 15.0
2.0 8.0 6.0 4.0 6.0 2.3 5.6
08.0 22.3 14.6 19.3 26.0 14.3 16.6
05.3 17.6 07.0 18.3 19.6 05.3 10.3
3.0 4.0 3.6 3.3 5.0 2.3 7.3
(0.82) (1.44)* (1.24) (1.29) (1.30) (1.18) (1.16)
(12.00) (72.66) (31.33) (16.66) (48.66) (08.33) (45.66)
(0.76) (1.33) (1.14) (1.26) (1.39)* (1.15) (1.17)
5.0 9.3 7.3 7.3 13.6 2.6 13.6
(39.00) (88.66)** (67.33)** (63.33) (204.33)* (10.00) (203.66)*
(0.69) (1.23) (0.81) (1.25) (1.24) (0.47) (0.99)
(11.66) (16.66) (15.00) (15.33) (25.66) (09.00) (65.33)
16.0 (1.15) 08.3 (0.76)
4.0 (24.66) 1.6 (05.66)
14.0 (1.13) 7.6 (0.84)
6.3 (41.66) 2.0 (06.00)
11.0 (1.02) 05.3 (0.67)
7.6 (79.00) 4.0 (22.00)
11.3 (1.04)
2.3 (06.33)
9.3 (0.96)
5.6 (41.66)
08.0 (0.86)
2.6 (10.66)
15.3 (1.18) 14.0 (1.13) 0.10 0.25 NS
3.6 (15.00) 4.6 (24.66) 18.58 NS NS
15.0 (1.17) 13.3 (1.11) 0.10 0.27 0.35
6.6 (48.66) 9.0 (91.66)** 41.06 107.09 138.93
09.3 (0.95) 07.0 (0.82) 0.12 0.32 0.42
1.6 (05.66) 2.6 (10.00) 19.15 NS NS
Figures in parentheses for number of flower is transformed in log values and for honeybees in v2, SEm = Standard error of mean and LSD = least standard deviation. * Significant at 5% (P < 0.05). ** Significant at 1% (P < 0.01). + Mean of three replications.
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of flowers was counted visibly plot wise for each genotype followed by counting the population of honeybees landed on flowers and collecting the nectar and pollen by visual sampling method (Visual sampling method is one of the standard common method of sampling by which the arthropods density is estimated in natural conditions through eyes for counting the number of insects.). Naturally occurring wild population constituted the bee source for the present experimentation. The data on number of flowers and honeybees associated with the mutants and other genotypes were recorded on three sampling dates, viz., March 04, 06 and 08, 2002 during 7.30–8.30 AM. The data of number of flowers and honeybees were subjected to statistical analysis by working out least standard deviations (LSD) at significance level of P < 0.05 and P < 0.01, respectively (Table 1).
3. Results and discussion Among twenty genetically divergent lines, flower variability of eight genotypes; Sanchita, VG-26, SGE48, SG-35-1 at P < 0.05 and SG-35-II, VN-23, SGE-14, VG-20 at P < 0.01 significantly attracted naturally occurring wild population of honeybees for their foraging visits with regard to nectar and pollen collection over other 12 divergent lines of opium poppy. This study revealed that development of new plant varieties based on suitable flower traits could be of greater significance in increasing the yield potential of plants as well as for increased foraging response to beesÕ vis-a`-vis higher honey production in both quantity and quality (poppy type). SGE-14 was the only mutant genotype that significantly bloomed (P < 0.05) with higher mean number of flowers over other genotypes as sampled. However, none of the genetic variant of opium poppy was found suitable for the honeybee foraging response on the first sampling date (Table 1). In contrast, the numbers of flowers were recorded to be significantly higher at P < 0.01 level of critical difference in the plots of ‘‘Sanchita’’ variety on second sampling date. Further, the numbers of honeybees visiting flowers were significantly higher in eight genotypes for honeybees foraging responses during second sampling date (Table 1). Non-significant observation of bee visit in relation to diverse genotypes of opium poppy first sampling date and third sampling date could be attributed to the age of plant maturity for the biosynthesis of semio-chemicals in nectar/flow of nectar being maximum on the second sampling date. Therefore, only the observations taken on second sampling being only optimum blooming and nectar day showed the significant numbers of foraging bees on different genotypes (Table 1). The numbers of flowers in the third sampling were found to bloom much markedly in the mutant line SG-35-II both at P < 0.01 and P < 0.05 level of critical differences. However, none of the opium poppy geno-
typic variant with diverse flower traits or plant morphological traits showed significant differences in flower suitability for the foraging of A. mellifera on the third sampling date (Table 1). It was noted that the floral behavior of opium poppy was highly specific and bleaking type depending upon intensity of sunlight. Therefore, low number of blooming as compared to number of plant shown per plot was observed during the observational period of 1 h in the morning (7.30–8.30 AM). The visits of foraging bees were only confined to those opened flowers that were freshly open and bear fresh pollen. Usually, bees visited only those flowers that contained petals. However, in opium poppy the floral petals were caudices thus, exposing the crown within few hours of flower opening, making its floral biology typical and irregular (Srivastava, 2002a,b; Satpute, 2000). The results demonstrated that the number of flower was not at all associated with the number of honeybees visiting on different genotypes (Table 1). However, the genetically governed traits of flower along with its specific aromas and flavors (having spectrum of both major and minor secondary metabolites) could have lured greater number of foraging honeybees. Flowering medicinal plants may be valuable source of nectar and pollen for producing honey of specific flavor, fragrance and medicinal principle. Youngken (1950, 1956) and Free (1993) studied bee activity on more than 300 drug plants. However, apparently no systematic efforts have been endeavored so far to identify the narcotic drug plant in relation to bee visits as biological signals for plant germplasm diversity. The demand of drug plants are high and of continuous nature for their utilization as natural products in various new formulations of pharmaceuticals and nutraceuticals for human health care. Opium poppy, Papaver somniferum (Family: Papaveraceae), a self-pollinated species is famous for its latex biosynthesis with natural opium alkaloids used in pharmaceuticals (Srivastava, 2002a,b). P. somniferum is the natural and unique source of major opium alkaloids—morphine and codeine (Budavari et al., 1996) besides thebaine, papaverine, and narcotine (Srivastava, 2002a,b) used abundantly by major pharmaceutical industries at global level. Evidently, the opium alkaloids do possess strong analgesic (morphine), anti-tussive (codeine), and anti-spasmodic (papaverine) activities. Morphine itself has wider applications as the most potent painkiller influencing the central nervous system. The opium type honey specifically obtained from foraging bees through divergent poppy cultivars could lead to new medicated product. New avenue has emerged after the discovery of a specific ‘‘promoter gene’’ in nectary (the nectar producing plant organ) of genetically engineered Petunias for a cure against a highly contagious ‘‘Parovirus’’ (causative for severe diarrhea and vomiting) in dogs (Anonymous, 1999). The idea is to develop medicated honey or honey
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capsule as potential vaccine for curing the disease. The vaccine is likely to be produced by expressing genes under the control of the promoter gene. Specific proteins are produced in the nectar, which is then collected by visiting honeybees and concentrated into honey. The high concentration of sugars in honey stabilizes the protein structure making purification relatively easy (Anonymous, 1999). The difference in foraging responses of honeybees in eight diverse mutants/genotypes over twenty studied could be attributed to the genepool variability in respect of flavoring and aromatic compounds of pollen and/or nectar together with floral traits differences for color, shape and size of anther, stigma, pollen sac, pollen number, stigmatic rays, petal and petal fringing. To produce hybrid seeds, a pollination control system is required to prevent unwanted self-pollination. In crop species with hermaphrodite flowers, this can be a major benefit. The identification of suitable physico-chemical traits for the foraging of higher number of honeybees may be a potentially desirable natureÕs tool to enhance the yield productivity of flowering crop plants in general and can be used as suitable pasture for honeybees (Roubik, 2002; Westerkamp and Gottsberger, 2000; Buchmann and Nabhan, 1996; Ingram et al., 1996). The active principle(s) responsible for foraging high number of bees in these genetically divergent plants, may be derived from flowers (anther and pollen) and can be used to spray on the flowers to lure more number of bees on other self-pollinating flowering plants to create conditions for hybrid vigor to enhance yield out of the 18 mutants and two locally adapted cultivars of diverse genotypes of opium poppy (Srivastava, 2000, 2002a,b). Researchers have identified key volatiles (like s-linalool as scent, flavor and fragrant) from basil, strawberries and other edible plants attracting both human and non-human grazers (Brown, 2002a,b; Kessler and Baldwin, 2001). The result is supportive of the thesis that opium alkaloids or other compounds of pharmaceutical interest may be present as secondary metabolites in the pollen and nectar and can affect the foraging behaviour of bees. However, the specific empirical association between bee foraging vis-a`-vis medicinal and aromatic constituents from diverse floral and anther traits of narcotic or scent producing plants at molecular level is not yet known. A gene ‘‘for’’ governing honeybees foraging behavior and its variant for diverse bee activities has been discovered (Pennisi, 2002). This gene codes for a cell-signalling molecule called a cyclic GMP-dependent protein kinase.
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Acknowledgements We thank Dr. Peter Day of AgBiotech Center, Rutgers University for his critical review of the manuscript and Mr. S.K. Mishra for statistical analysis.
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