ROLE OF ENTOMOPHILY AND ARTIFICIAL POLLINATION IN ENHANCING QUALITY AND YIELD OF SEED ONION ( ALLIUM CEPA L.) IN INDIAN HIMALAYAS

Onion (Allium cepa L.) is a highly cross pollinated crop that needs insect pollination (entomophily) for optimum seed set. In the present study, more than 120 insect species were noted to visit onion flowers, out of which fifty-nine species were collected through in-situ sampling technique and identified up to species level. The Indian bee ( Apis cerana indica ) was the most abundant insect pollinator visiting onion flowers in the study location. The pollination behaviour and foraging activity of A. c. indica were assessed, and the pollen foragers were observed to be swift flyers visiting a significantly higher number of flowers per minute during the peak flowering period and spending less time per flower (3 seconds) to collect nectar from deep seated nectaries of the flowers. The seed yield enhancement assessed through entomophily and artificial pollination methods showed that the open pollinated flowers recorded the highest yield statistically, followed by A. c. indica and A. mellifera pollinated flowers. However in artificial pollination treatments, sponge puff pollinated flowers recorded significantly high seed yield per hectare and percentage seed set per umbel, followed by camel brush, hand gloves (cloth) and hand gloves (rubber). In conclusion, all the cross pollination treatments were statistically significant on the closed pollination treatment concerning to the entire yield parameters calculated.


INTRODUCTION
The onion (Allium cepa L.) is the third most important vegetable crop cultivated in the world after the potato and tomato (Semida et al., 2020).India stands first in onion cultivation and second in its production after China (Parimalarangan, 2020).Production in India Pollination studies in Onion (Allium cepa L.) during 2017-18 was estimated to be 22.42 million tons from 1.28 million hectare area (Barla & Upasani, 2019;Mahmadajaruddin & Mamani, 2020).Although the supply of onions for various culinary purposes is met with sufficient quantities, the demand for seeds is always high and both the public and private sectors seldom meet the increasing demands (Panacci et al., 2020;Meghana et al., 2021).The onion flowers are strongly protandrous and highly cross pollinated in nature (Baswana, 1984;Yucel & Duman, 2005), and due to presence of sticky pollen, pollination through insects inevitably produces high quality seeds (Gjorgji & Rukie, 1997;Chandel et al., 2004).More than 267 insect species belonging to seven insect orders are known to pollinate the onion during its entire flowering period (Bohart et al., 1970;Walker et al., 2011).Among all the pollinators recorded, honey bees are reported to assist in 60-95% of pollination (Kumar et al., 1985;Chandel et al., 2004) and the bee pollinated umbels are reported to have a yield up to ten to eleven times higher than the closed control (Munawar & Muzaffar, 1999).However, the use of bees or other insects in controlled pollination for producing pure line hybrids is almost impossible due to the difficulty in having control over the pollination activity of insects.Mayer & Lunden (2001) and Yucel & Duman (2005) tested several other cross pollination methods for onion hybrid seed production with little success and concluded that, due to sticky pollen, the pollination in onion has to be facilitated by insects (either solitary bees or honey bees) for optimum seed set.Therefore, in the present study, such cross pollination techniques as hand pollination, brush pollination and controlled insect pollination were practised and their efficiency was analysed both qualitatively and quantitatively.Additionally, the pollinator diversity of onions in Uttarakhand, Indian Himalayan conditions was analysed, and the pollinator density per unit area in onion crop was calculated.The Indian bee (Apis cerana indica), the most abundant pollinator of onion in Alpine and Humid subtropical climatic zone of India, was selected for understanding its pollination behaviour and pollination efficiency in onion.Moreover, the quantitative data including seed yield per umbel, test weight of 1000 seeds, seed yield per unit area and per cent fruit set under different pollination conditions were also assessed.The results of the current study to assess the yield enhancement through entomophily and artificial pollination in onion would form a strong scientific base for both researchers and farmers of Uttarakhand, Himalayas for utilizing the native bee species for attaining higher seed yield of onion.

Study site and experimental materials
The pollination studies on onions were conducted at the Experimental Farm, Hawalbagh, ICAR-Vivekananda Parvatiya Krishi Anusandhan Sansthan (VPKAS), Almora, Uttarakhand, India (29º38´01˝ N and 79º37´49˝ E, altitude 1250 amsl) in India's Alpine and Humid subtropical climatic zone.Onion bulbs of the VL Piaz-3 composite variety were directly sown in a 200 m 2 field (14.0x14.3m) on 12 th December, 2020 (50 th standard week) at a spacing of 50x50 cm.Fertilization, irrigation, earthing up, weeding and pest management were followed as per the agronomic practices recommended for VL Piaz-3.

Floral biology of onion
To study the floral biology of onion variety VL Piaz-3, flower colour, number of flowers per umbel, required time for umbel formation, first flowering and last flowering from the date of sowing were recorded.The total number of umbels per plant and total number of umbels per square metre were counted in order to obtain the quantitative yield data.The data was recorded from a total of twenty-five plants selected randomly from the field and the values were averaged and presented as mean±SD values.

Pollinator diversity
To assess the diversity of insects visiting onion flowers during the flowering period, the fields were monitored regularly for sixtyseven days (from initial flowering to seed set) through traversing zigzag manner three times a day (10.00,13.00 and 16.00 h IST UTC+05:30).The traversing distance was up to 15 m in one direction and a total distance of 75 m (5 transect lines) was covered for the entire field once in a span of seven minutes.All the flowervisiting insects were counted and recorded in a field register.One representative specimen of each insect visitor was caught with a hand net and a repository of onion-flower visiting insects was prepared.For the characterization of coleopterans, the specimens collected from the fields were compared with the type specimen of the white grub beetles preserved in a Himalayan white grub species compendium at ICAR-VPKAS, Almora, Uttarakhand, India.However the technical bulletin on butterflies of Almora prepared by Stanley et al. (2018) was referred to for the identification of Lepidopterans.Furthermore, adult dipterans were identified through the pictorial, linear and dichotomous taxonomic keys designed by Buck et al. (2009) and Marshall et al. (2017).The hymenopterans were morphologically characterized through the literatures of Goulet & Huber (1993) and Seltmann (2004) with pictorial, interactive and dichotomous keys.

Pollinator density per unit area
In order to assess the density of flowervisiting insects per unit area, in-situ counts of the insects visiting the umbels were recorded.In this method of sampling, three plots of 1 m 2 area were demarcated in the field and the insects visiting the onion umbels in one minute time were counted.The insects moving out of the marked area and returning back within a span of one minute were counted as fresh entry.The sampling was conducted three times a day (10.00,13.00 and 16.00 h IST) for five consecutive days during the peak flowering period (>50% flowering).The five-day values of three time frames of pollinators' visit into the demarcated area for a one-minute duration were averaged and presented graphically.The ANOVA was assessed at p<0.05 significance level, while F-values, SE(m) and CV (coefficient of variation) values were calculated through SPSS software to compare the means.The insects thus collected were classified into various groups based on the orders and families they belonged to.

Experimental design
After initial assessment of pollinator density and diversity, three major insect pollinators' viz., A. c. indica, Apis mellifera and Bombus haemorrhoidalis were selected for assessing their pollination behaviour and foraging rate in onion.Additionally, such traditional artificial pollination methods as hand pollination with rubber and cloth gloves and both brush and sponge puff pollination were included in the experiment.Moreover, one positive control (open pollination) and one negative control (close pollination) treatments were also added in the study to assess the best pollination method to enhance the yield of onion seeds in a hybrid seed-production system.A total of thirty umbels were bagged separately per each treatment with a plastic mesh of 500 micron diameter prior to flower opening, and immediately afterwards the flowers were pollinated through different methods of pollination as described in Tab. 1 and covered with plastic mesh to avoid contamination.
Pollination behaviour and foraging rate of A. c. indica Based on the results of pollinator density; the Indian bee (A.c. indica) was identified as the most efficient onion pollinator in the mid-Himalayas of Uttarakhand, India.In order to study the pollination behaviour of A. c. indica in the onion crop, such foraging activity of bees as number of flowers visited per minute, number of umbels visited per minute and time spent per flower by a single bee and a total number of pollen and nectar foragers visiting onion flowers at three different time intervals (10.00, 13.00 and 16.00 h IST) of a given day were estimated.For the present study, three flowering time-frames viz., pre-peak flowering (<25%), peak flowering (>50%) and post-peak Pollination studies in Onion (Allium cepa L.) Table 1.Entomophily and artificial pollination methods followed for pollinating onion flowers

Treatments
Procedure of pollination Treatments The pollens were collected from un-bagged umbel through the cloth hand gloves and gently rubbed over the bagged umbel and covered with plastic mesh after pollination.Umbels were gently rubbed for 3 minutes T5-Hand gloves (Rubber) The pollens were collected from un-bagged umbel through the rubber hand gloves and gently rubbed over the bagged umbel and covered with plastic mesh after pollination.Umbels were gently rubbed for 3 minutes T6-Sponge puff The pollens were collected from un-bagged umbel through the puff and gently rubbed over the bagged umbel and covered with plastic mesh after pollination.
Umbels were gently rubbed for 3 minutes T7-Camel Brush The pollens were collected from un-bagged umbel through the camel brush and gently sprinkled over the bagged umbel and covered with plastic mesh after pollination.Pollens were sprinkled for 3 minutes flowering (<25%) were selected and the pollinator behaviour was observed for three consecutive days in three time-frames to obtain an averaged data.The ANOVA was calculated at p<0.05 significance level and F-values were calculated through post-hoc Tukey's-B test to analyse the statistically significant data.

Fruit set and yield parameter studies
To estimate the yield enhancement of onions through the entomophily and artificial pollination methods described above, the following yield data parameters were recorded: percentage of fruit set with respect to average number of flowers per umbel (842±132.51 The umbels were not covered and allowed for free cross pollination by all the pollinating agents T9-Control (Close) The umbels were covered with plastic mesh and only self-pollination was allowed

RESULTS
Floral biology of onion composite variety "VL Piaz-3" Onion composite variety VL Piaz-3 is highly cross pollinated with strongly protandrous flowers, wherein anther dehisces occurs well before the stigma becomes receptive.The colour of the VL Piaz-3 flowers varied from whitish to light bluish in colour with six perianth segments arranged in two whorls.The numbers of stamens were six and arranged in two whorls.Anthers were bilocular and the ovary was superior.The colour of pollens varied from dark greenish to light purplish colour.The additional details of floral biology are furnished in Tab. 2.
Pollinator diversity and abundance at three time frames of the day The pollinator diversity of onion was recorded for sixty-seven flowering days, and through visual observations, more than 120 insect species were recorded to visit onion flowers during the entire flowering period.Fifty-five of these insect species belonging to four insect orders (Hymenoptera, Diptera, Lepidoptera, and Coleoptera) were considered as pollinators, because they spent ample time foraging on the umbels (>45 seconds) and traces of pollens were observed on their bodies.These fifty-five flowervisiting insects were collected through the in-situ sampling technique (Fig. 1) and identified up to species level using taxonomic keys.The hymenopterans were the most dominant flower visitors with nineteen known species, followed by lepidopterans with seventeen species, dipterans with fourteen species and coleopterans with five species respectively (details of insects up to species level are furnished in Tab. 3).The pollinator abundance study in the time frames 10.00, 13.00 and 16.00 h IST showed that (Fig. 2) syrphids and A. c. indica were the most abundant flower-visiting insects in onion during all three time frames, followed by megachilids.
Moreover, the density of flower-visiting insects was statistically superior during the afternoon hours (13.00 h) followed by density in the morning hours (10.00 h).However, the density of insects declined drastically in the evening hours (16.00 h), except for those of moths and megachilids, which were found to visit onion flowers abundantly in the later part of the day.
The other insects in Fig. 2 were hemipterans  Pollination studies in Onion (Allium cepa L.) and diurnal coleopterans that visited the flowers actively throughout the day and foraged on pollen and nectar source of onion flowers.

Pollination behaviour and foraging rate of A. c. indica
Based on the pollinator density results, A. c. indica was identified as the most abundant onion pollinator in the study location.The studies were conducted to understand the pollination behaviour and foraging activity of A. c. indica, and the results showed that (Tab.4) the pollen foragers visited a significantly higher number of flowers during the peak flowering period in comparison to the pre-and post-peak flowering periods.Wherein during the pre-and post-peak flowering periods (<25% flowering), the numbers of flowers per umbel were less, so, the pollen foragers visited more umbels and fewer flowers and spent more time in search of flowers.However, during the peak flowering period (>50% flowering) there were more flowers per umbel and pollen foragers spent more time on a single umbel collecting pollens and thus visited a significantly lower number of umbels during this period.A similar trend was observed in case of nectar feeders; due to profuse flowering during the peak flowering period and deep seated nectaries in onion, the nectar feeders spent more time on a single flower in order to collect nectar and thus visited fewer flowers and umbels per unit time.However, during the pre-and post-peak flowering periods, there were fewer flowers per umbels and thus the bees spent more time in search of flowers and visited a significantly higher number of flowers and umbels per unit time.Moreover, the studies conducted to assess the time spent by individual pollen and nectar foragers (Tab.5) showed that the pollen foragers spent statistically on par time per each flower during the pre-and post-peak flowering periods, while they swiftly collected the pollen from flowers and spent significantly less amount of time per flower during the peak-flowering period.However in contrast, nectar foragers were recorded spending significantly more time per flower during the peak flowering period to collect nectar from deep seated nectaries in the flowers and significantly less time spent per flower during the pre-and post-peak flowering periods because of fewer flowers per umbel and more time spent searching for nectar bearing flowers.The number of pollen and nectar foragers visiting the onion flowers at three time intervals and three flowering periods were recorded (Tab.6).The number of pollen foragers was noted to be significantly lower in the morning hours (10.00 h), increased drastically throughout the day and was statistically significant in the evening hours (16.00 h).Pollination studies in Onion (Allium cepa L.) Table 6.
Pollen and nectar foragers of A. c. indica visiting onion flowers at three time intervals in 1 m 2 area in a span of one minute during three flowering periods (averages from 3 flowering days)  Similar results were recorded during all the flowering periods for the pollen foragers.
Highly contrasting results were recorded for nectar foragers, while statistically significant numbers of nectar foragers of A. c. indica were observed to visit onion flowers during morning hours (10.00 h) and then drastically dropped in the evening hours (16.00 h) during all the three flowering periods.Moreover, the number of pollen foragers during the peak and post-peak flowering periods enhanced significantly in the afternoon as well as in the evening hours due to higher anther dehisces from 12.00 h to 16.00 h and the availability of ample pollen during this period in onions.

Fruit set and seed yield
The quantitative seed yield data obtained through the entomophily and artificial pollination methods in onion was analyzed and presented in Tab. 7. Open pollinated onion umbels were recorded to attract the maximum number of insect flower visitors which resulted in a higher percentage of fruit set, followed by A. c. indica and A. mellifera pollinated umbels, which also set a greater percentage of seeds per umbel in comparison to other treatments (Fig. 3).The average number of seeds per umbel Pollination studies in Onion (Allium cepa L.) was highest in open pollinated umbels, followed by A. c. indica and A. mellifera pollinated umbels.These results followed the trend of percentage of seed set per umbel pollinated by different methods.Moreover, the results of the parameters seed weight per umbel and test weight of 1000 seeds also showed similar trends, wherein, the open pollinated umbels recorded statistically significant values, followed by A. c. indica and A. mellifera, which were also statistically significant to other pollination treatments.Finally, the seed yield per hectare area showed a significantly superior yield in open pollinated treatments, followed by A. c. indica, A. mellifera and B. haemorrhoidalis pollinated umbels.In case of artificial pollination treatments, sponge puff pollinated flowers recorded significantly superior seed yield per hectare and percentage seed set per umbel, followed by camel brush, hand gloves (cloth) and hand gloves (rubber).In conclusion, all the cross pollination treatments were statistically significant during the close pollination treatment with respect to the entire yield parameters calculated.
The floral biology of onion is a varietal character, but the number of stamens, ovaries and florets, the colour of flowers and pollens, days for flowering, seed set and harvesting usually lie in a fixed range (Devi et al., 2015;Saleh at al., 2021).
Recently Walker et al. (2011), Devi et al. (2015) and Saleh et al. (2021) reviewed the floral biology of onion and importance of insect pollinators in improving seed yield of onion.Walker et al. (2011) reported a total of 18,407 individual insects belonging to 264 species and twenty-eight families of seven insect orders visiting the onion flowers during the entire flowering period, whereas Bohart et al. (1970) showed the presence of 267 species of insect pollinators visiting onion flowers.Our studies formed a close concurrence with the previous studies, in which through visual observations we recorded more than 120 species of insects from five orders visiting onion flowers.Out of which fifty-nine species were collected from fields and identified up to species level with the help of taxonomic keys.Among all the insect pollinators visiting onion flowers, bees had an important role in efficient pollination and optimum seed set.Jadhav et al. (1981), Kumar et al. (1985) and Chandel et al. (2004) showed that 60-95% of onion pollination is assisted by honey bees of family Apidae.Moreover, Saeed et al. (2008) and Saeed & Masood (2008) reported four bee species and four wasp species and twelve true fly species to be the major insect pollinators of onion.However, Chandel et al. (2004), in the sub-temperate climates of India's Himachal Pradesh (altitude >1200 m) recorded two honey bee species (Apis dorsata and A. c. indica) and three syrphid species (Episyrphus balteatus, Metasyrphus conferator and M. corollae) to be important onion pollinators in the hilly terrains of the Indian Himalayas.These results were in close concurrence with our study carried out to assess the population density of onion pollinators in Uttarakhand, Himalayas, in which we recorded A. c. indica as the most dominant pollinator, followed by A. mellifera, syrphids, B. haemorrhoidalis, non-Apis bees, wasps and butterflies.
Based on the previous studies carried out by Rao & Suryanarayana (1989), Chaudhary & Sihag (2003) and Chandel et al. (2004) and the results of our studies, we selected the Indian bee (A.c. indica) to study its pollination behaviour and pollination efficiency in onions.Our results showed that immediately after anthesis and flower opening, both the pollen and nectar foragers of A. c. indica visited the onion flowers in large numbers and assisted in cross pollination during the their food-collecting process, thus indicating how rich onion flowers are in nectar and pollen source (Yucel & Duman, 2005;Saeed et al., 2008).
Although onion is a cross pollinated crop and insects play an important role in pollination, the exploitation of insects or domesticated bees in hybrid seed production system is still in infant stages in the temperate and sub-temperate areas of Indian Himalayas (Pratap & Pratap, 1997;Chandel et al., 2004).Moreover, the production of pure lines of breeder seeds is a very cumbersome procedure due to unavailability of suitable crossing techniques in onions ( Van der Meer & Van Bennekom, 1968;Mayer & Lunden, 2001;Yucel & Duman, 2005).Therefore, several crossing methods were tested and it was noted that, only controlled pollination by insects helps in efficient pollination and optimum seed set.Similar crossing studies conducted by Matherson et al. (1996) and Sajjad et al. (2008) concluded that, honey bees were the most efficient onion pollinators and assisted in the production of high quality seeds.Quantitative yield analysis studies showed that the insectpollinated umbels recorded superior yield data in comparison to other mechanical methods.Gjorgji & Rukie (1997)  As a result of this study, a total of 120 species of visiting insects were observed to visit onion flowers for collecting pollen and nectar, out of which Indian bee (A.c. indica) was identified as the most abundant and efficient pollinator of onion flowers in the Himalayas of Uttarakhand, India.The open pollinated flowers yielded the highest seed yield among all the treatments, and yield enhancement of more than 1400% was recorded over the control plots.However, several drawbacks still remain on how to produce pure hybrid lines of onion seeds through controlled insect pollination.These aspects require further research and field experimentations.
One individual bee, actively foraging in onion fields was collected through a test tube and released on to a pre-marked umbel and bagged with plastic mesh of 500 micron diameters to prevent escape.One bee per umbel was released every day from initiation of flowering up to seed set.Duration of release 1

Fig. 1 .
Fig. 1.Diversity of insect pollinators of onion in Himalayas of Uttarakhand, India

Fig. 2 .
Fig. 2. Insect pollinator density obtained through in-situ method of sampling in onion crop at three time frames of the day during peak flowering period (*F-values calculated at 5% level of significance)

Fig. 3 .
Fig. 3. Assessment of per cent fruit set with respect to total number of flowers per umbel under different pollination treatments.

Table 2 .
Floral biology of onion composite variety "VL Piaz-3" Floral Character Details Time of flower openingFrom 6.20 h to 17.45 h (Throughout the day) AnthesisEarly morning hours from 6.25 to 7.25 h

Table 3 .
List of insects visiting onion flowers under Himalayan conditions of Uttarakhand, India

Table 4 .
Foraging rate of pollen and nectar foragers of A. c. indica at three time intervals in a day during three flowering periods * post-hoc Tukey's-B test conducted at 5% level of significance

Table 5 .
Time spent in seconds by individual pollen and nectar forager bee of A. c. indica on onion flowers during three time frames and three flowering periods * post-hoc Tukey's-B test conducted at 5% level of significance

Table 7 .
Quantitative seed yield data of onion obtained through entomophily and artificial pollination methods Munawar & Muzaffar (1999)weight of onion seeds pollinated by honey bees was 10.57% higher than those pollinated through hand pollination.Similar results were recorded in our study, in which A. c. indica and A. mellifera pollinated seeds recorded the highest test weight after open pollinated seeds accounting to 11.37% weight increase over closed control.Moreover,Munawar & Muzaffar (1999)recorded a 10-11 time yield increase of onion seeds pollinated through insects over closed control plots where no pollination was allowed.Our results had close concordance, in which 1423.50% yield increase was recorded in open pollinated umbels followed by A. c. indica and A. mellifera recording 345.16% and 238.71% yield increase respectively over closed umbels where, no cross pollination by insects was allowed.