Melissopalynological Determination of Pollen Density and Botanical Origin of Autumn Honeys of Kullu Hills, Himachal Pradesh, India

In present investigations (field and laboratory studies were done as a part of Ph.D work during 2009 to 2012) twelve autumn honey samples were procured from domesticated Indian hive bee, A. cerana from different areas of Kullu hills of Himachal Pradesh so as to identify important pollen and nectar sources of this region (Table 1, Fig 1). Collection sites (and elevation in meters) are shown in Table 1. All honey samples collected were raw and unprocessed. The honey samples were subjected to Qualitative and Quantitative analysis. For Qualitative and Quantitative analysis, the methodology recommended by the International Commission for Bee Botany (ICBB) was followed (Louveaux et al., 1978) modified by (Iwama and Melhem, 1979). Identification of different pollen types was done with the help of standard works (Erdtman, 1960; Nair, 1964; Nair, 1985) and pollen slide collection of Sociobiology and Behavioral Ecology Research Lab, Department of Biosciences, Himachal Pradesh University, Shimla. For the preparation of pollen slides of honey, 10 gm of honey was dissolved in 20 ml of hot distilled water at 40^oC. This solution was poured equally into different centrifuge tubes and centrifuged at 2500-3000 rpm for 10 min. The supernatant liquid was drained off with a fine pipette. The sediment was dispersed again and transferred into another centrifuge tube, centrifuged again for five minutes and sediment was separated. It was then acetolysed by adding sulphuric acid and acetic anhydride in ratio 1:9. The tube was then placed in a water bath for ten minutes at 70^oC and centrifuged after incubation for five minutes. The centrifuge tube was filled with distilled water and a drop of strong detergent (teepol) was added. It was again centrifuged for five minutes and a drop of glycerine and water mixture (1:1) was added to the sediment. Then, this sediment solution was transferred to the slides which were placed in an oven (40-45^oC) to get surplus water evaporated. The pollen grains were mounted in glycerine gelatin. Reference slides were prepared from the identified honey plants using Acetolysis Method and Glycerine Jelly Method (Louveaux et al., 1978).
 

 

       
Quantitative analysis is done with the help of a haemocytometer (Suryanarayana et al., 1981; Sharma, 1989). Pollen spectra, absolute pollen count as well as percentage of pollen types were calculated and formulated on the basis of these percentages (Sharma and Nair, 1965). The honey samples were categorized as rich, poor and extremely poor as per total number of pollen grains present in 10 gm of honey i.e., 1,00,000, 20,000 to 1,00,000 and below 20,000 respectively (Maurizio, 1975). The frequencies of pollen grains were recorded according to the system adopted by Louveaux et al., 1978: ‘Predominant pollen’ (>45% of the pollen grains counted); ‘Secondary pollen’ (16-45%); ‘Important minor pollen’ (3-15%) and ‘Minor pollen’ (<3%). Honey sample was termed as ‘Unifloral honey’ if it was having 45% or more grains of a single pollen type and honey sample’ with several pollen types in considerable percentage was termed as ‘Multifloral honey’ (Chaturvedi, 1983; Sharma, 1989; Iwama and Melhem, 1979).

During this study, 12 autumn honey samples produced in the Kullu hills were analyzed (Table 1, Fig 1). Fifty pollen types belonging to 25 different families were recognized in the quantitative analysis (Table 2 and 3; Fig 2 and 3-14). Five of the 12 samples analyzed were considered to be unifloral; Prunus cerasoides in Bhunter; Eriobotrya japonica in Garsa; Prinsepia utilis in Mohal; Plectranthus sp. in Patlikuhl and Solidago sp. in Bhekli were present as predominant sporomorphs. While, 17 pollen types were secondary sources and 38 were important minor and minor pollen sources. Cassia sp., Taraxacum officinale Callistemon citrinus, Salvia splendens, Eriobotrya japonica, Fagopyrum sp., Impatiens sp., Prunus sp., Salvia officinalis, Medicago sp., Rubus ellipticus, Aesculus indica, Brassica sp., Pyrus sp., Plectranthus coesta and Plectranthus gerardiana were the secondary pollen sources.Whereas, Salvia sp., Adhatoda vasica, Cannabis sativa, Phoenix sylvestris, Zea mays, Coriandrum sativum, Indigofera pulchella, Tilia sp., Taraxacum officinale, Aesculus indica,Rumex nepalensis, Prunus sp., Sesamum indicum, Vitis vinifera, Polygonum sp., Ageratum conyzoides, Medicago sp., Aster sp., Brassica  sp., Rubus sp., Ocimum sp., Foeniculum vulgare, Ocimum basilicum, Senecio sp., Caesalpinia sp., Rhus sp., Luffa cylindrica, Elaeagnus sp., Juglans regia, Bidens pilosa, Sonchus  asper, Solidago sp., Prinsepia utilis, Chenopodium album, Vitex negundo, Pyrus sp., Lonicera sp. and Viola odorata were important minor and minor pollen types. The most widely represented families were Lamiaceae (Salvia sp., Salvia splendens, Salvia officinalis, Plectranthus coesta , Plectranthus gerardiana, Plectranthus  sp., Ocimum basilicum, Ocimum sp.); Asteraceae (Ageratum conyzoides, Aster sp., Bidens pilosa, Solidago sp., Sonchus asper, Taraxacum officinale, Senecio sp.); Rosaceae (Eriobotrya  japonica, Prinsepia utilis, Prunus cerasoides, Prunus sp., Pyrus sp., Rubus sp., Rubus ellipticus); Fabaceae  (Caesalpinia sp., Cassia sp., Indigofera pulchella, Medicago sp.); Polygonaceae (Polygonum sp., Fagopyrum sp., Rumex nepalensis); Apiaceae (Coriandrum sativum, Foeniculum vulgare), whereas, Acanthaceae, Amaranthaceae, Anacardiaceae, Arecaceae, Balsaminaceae, Brassicaceae, Cannabinaceae, Caprifoliaceae, Cucurbitaceae, Elaeagnaceae, Juglandaceae, Myrtaceae, Pedaliaceae, Poaceae, Sapindaceae, Tiliaceae, Verbenaceae, Violaceae and Vitaceae are represented by one member each.
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

       
Microscopic analysis showed that out of 50 morphological pollen types, 44 from were nectariferous and 6 from nectarless species as Cannabis sativa, Juglans regia, Rumex nepalensis, Polygonum sp. and Zea mays.
       
Melissopalynological analysis of autumn honeys indicates that Kullu hills have tremendous potential for the development of bee colonies and for sustainable beekeeping because of the diversity of nectar and pollen sources. The presence vast array of predominant, secondary, important and important minor pollen types in honey samples indicates that all samples were of botanical and geographical origin. The honey samples, rich in both pollen concentration and pollen diversity display a vivid landscape of the bee forage plants growing vicinity of beehive. This was also a clear indication of the wide range of collection of pollen grains from many flowering plants (Ebenezer and Olugbenga, 2010).
       
Fifty pollen types belonging to twenty five families were recorded in this study. Thirty three were identified to species level and 17 upto family level. The identified species belong to varying genera of native trees, herbs, shrubs and trees. There were pollen of varying shapes, sizes and morphological features. The pollen spectra of these honeys showed that honeybee’s foraged considerable distance in search nectar and pollen which is needed for their survival and production of honey. The presence of these large number of pollen types also indicated that the honey samples were pure and not adulterated.
       
Pollen analytical data of the samples was given in Table 2 and 3. The predominant pollen types were Prunus cerasoides, Eriobotrya japonica, Prinsepia utilis, Plectranthus sp. and Solidago sp.  The dominant wild trees/plants were Adhatoda vasica, Aesculus indica, Aster sp., Ageratum conyzoides, Bidens pilosa, Cannabis sativa, Cassia sp., Caesalpinia sp., Elaeagnus sp., Fagopyrum sp., Impatiens sp.,  Indigofera  pulchella,  Lonicera sp., Phoenix sylvestris, Plectranthus coesta, Plectranthus gerardiana, Polygonum sp., Prinsepia utilis, Prunus cerasoides, Rhus sp., Rubus ellipticus, Rumex nepalensis, Salvia officinalis, Senecio sp., Solidago sp., Sonchus asper, Taraxacum officinale, Tilia sp., Viola odorata and Vitex negundo. Cultivated plants were represented by Brassica sp., Coriandrum sativum, Chenopodium sp., Eriobotrya  japonica, Juglans regia, Foeniculum vulgare, Luffa cylindrica, Medicago sp., Ocimum basilicum, Pyrus sp., Prunus sp., Sesamum indicum,Vitis vinifera and Zea mays. Ornamental plants include Callistemon citrinus and Salvia splendens.

From the above observations it is clear that the study area exhibits diversified flora and unlimited potential for bee forage. Moreover, the diverse pollen spectrum indicated that the honey samples are of botanical origin and are adulterated. This study also gives insights into the vegetation type of the respective locations in which honeys were produced as well as provides additional information on the major botanical florals foraged by honeybees in the studied region. Thus, identification of melliferous pollen taxons also helps in establishment of organized apicultural industry and for scientific development. It also helps beekeepers to formulate seasonal bee management programmes especially for migratory beekeeping.
               
Present melissopalynological studies revealed that honeys from Kullu fall under the category of Group I (<20,000 pollen grains), Group II (20,000-100,000) and to Group III (100,000-500,000) formed by International Commission for Plant Bee Relationships. i.e. honeys having absolute pollen count from 10,000 to 5,00,000 per 10 gm of honey.