Role of Entomophily and Artificial Pollination in Enhancing Quality and Yield of Seed Onion (Allium cepa L.) in Indian Himalayas

The pollination studies on onions were conducted at the Experimental Farm, Hawalbagh, ICAR-Vivekananda Parvatiya Krishi Anusandhan Sansthan (VPKAS), Almora, Uttarakhand, India (29°38′01″ N and 79°37′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 m² field (14.0x14.3 m) on 12^th December, 2020 (50^th standard week) at a spacing of 50×50 cm. Fertilization, irrigation, earthing up, weeding and pest management were followed as per the agronomic practices recommended for VL Piaz-3.

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.

To assess the diversity of insects visiting onion flowers during the flowering period, the fields were monitored regularly for sixty-seven 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 flower-visiting 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.

In order to assess the density of flower-visiting insects per unit area, in-situ counts of the insects visiting the umbels were recorded. In this method of sampling, three plots of 1 m² 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.

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.

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 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.

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 number of flowers per umbel, average of 25 umbels), average number of seeds formed per umbel, weight of seeds obtained from each umbel, test weight of 1000 seeds in different treatments and yield per hectare (extrapolated values obtained from seed yield per 1 m² area) in different treatments. A total of thirty umbels were bagged per treatment (each umbel was considered as a separate replication) and the data were recorded separately for each umbel under each treatment. For calculation of the test weight, seeds of all thirty umbels under each treatment were pooled and 1000 seeds randomly were counted and weighed. Moreover, to estimate the seed yield per unit area, the number of umbels per 1 m² (36.66±11.43 umbels per 1 m², average of 12 plots) was counted and the seed yield per umbel under different treatments was multiplied and the values were extrapolated as seed yield (in kg) per 1 hectare area.

All the field experiments were set up in a completely randomized block design (CRBD) with nine treatments and thirty replications each. The quantitative yield data was analysed through the calculation of the average values with Microsoft Office Excel 2019 (Microsoft corp., USA) and the ANOVA was assessed at p<0.05 level of significance. The F-values, SE(m) and CV (coefficient of variation) values were calculated with SPSS software to compare the means of density of insect flower visitors, and post-hoc Tukey's-B test was conducted to analyse the statistically significant data of the pollination behaviour and foraging activity of A. c. indica. Furthermore, the post-hoc Duncan's multiple range test (DMRT) was conducted with SPSS software for WINDOWS version 16.0 (SPSS Inc, Chicago) for the comparison of statistically significant yield data in different treatments.

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.

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 flower-visiting 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 and diurnal coleopterans that visited the flowers actively throughout the day and foraged on pollen and nectar source of onion flowers.

Fig. 1

Fig. 2

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).

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.

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 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.

Fig. 3