Comparative reproduction mechanisms of three species of Ocimum L . ( Lamiaceae )

Ocimum species have a combination of reproductive system which varies with the locality and cultivar. We have studied here the reproductive mechanisms of five variants of three Ocimum species in Nigeria, namely: Ocimum canum Sims., O. basilicum L., and O. americanum L. Flowers from each variant were subjected to open and bagged pollination treatments of hand self-pollination, spontaneous selfpollination and emasculation. All open treatments of the five Ocimum variants produced more fruit and seed than the corresponding bagged treatments. The two O. canum variants and O. basilicum ‘b1’ produced high fruit and seed set in the open and bagged treatments of spontaneous self-pollination. Ocimum basilicum ‘b2’ and O. americanum produced higher fruit and seed set in the self-pollination open treatment but significantly lower fruit and seed set in the bagged treatment. Fewer fruit and seeds were produced in the emasculated open treatments but none in the emasculated bagged treatments of the five Ocimum variants. The floral foragers comprising of bees, wasps and butterflies visited the Ocimum species to collect pollen or nectar in the open treatments. The two O. canum variants and O. basilicum ‘b1’ variant reproduced mainly through autogamy but O. basilicum ‘b2’ and O. americanum showed mixed reproduction of autogamy and outcrosssing. Insect visitation to the flowers enhanced pollination resulting in higher fruit and seed set in all the Ocimum species studied.


Introduction
The genus Ocimum L. (basil) belongs to the family Lamiaceae and comprises approximately 30 species of herbs and subshrubs distributed across tropical regions of Asia, Africa, Central and South America, with Africa as the primary center of diversity of the genus [1].In Nigeria Ocimum is represented by four species, namely Ocimum basilicum L., O. canum Sims., O. gratissimum L., and O. suave Willd.[2].The genus Ocimum is an important essential oil crop with around 100 tons of essential oil being produced annually throughout the world, about half of this is produced from O. basilicum and close relatives [3].Ocimum species are also very important in indigenous systems of medicine [4][5][6][7][8].In addition to these uses, Ocimum is widely cultivated as a potherb for culinary uses [9] and for the fresh herb market for which export of Ocimum from Israel alone is worth 4 million US dollars per year.Floral nectar and pollen are offered as a reward for the biotic pollinating agents [10].The pollen and nectar of members of the genus Ocimum are known to support certain potential pollinators of several wild plants species when the latter are not in bloom [11].Insect and other organisms play major role in boosting agricultural production by significantly increasing the yields of crops, vegetables, fruit and seeds by visiting flowers to aid pollination processes.Self-incompatible and cross-pollinated crops require pollinating service of efficient pollinators.Self-pollinated crops also benefit from insect pollination which increases yield up to 30%.Lack of pollinators causes decline in fruit and seed production [12].There are various reports on the pollination system in species of the family Lamiaceae.According to Darrah [13] and Khosla [14] species of Lamiaceae are primarily crossbreeding and have floral structures suitable for pollination by bees.Raju [11] reported that O. americanum and O. basilicum are predominantly autogamous.A study performed by Almeida et al. [15] revealed that O. officinalis L. is predominantly autogamous but also able to reproduce by cross-fertilization.The floral structure of O. basilicum is adapted to pollinator-mediated out-crossing [16].The pollination mechanisms and plant-insect interactions of the Lamiaceae species vary greatly with the locality and cultivar concerned, ideally pollination investigations are necessary in each locality where the species are grown.Also due to the extensive uses and economic importance of the species in the Ocimum genus there is need to have better understanding of the reproductive mechanism in this taxon.This study compared the mode of pollination in three Ocimum species: (O.  ) and one variant of O. americanum L. The Ocimum variants used here were selected from germplasm collections of the genetic unit of the Department of Botany, Obafemi Awolowo University, Ile-Ife.Ocimum canum 'c 1 ' is a tetraploid with chromosome number of 2n = 52 and O. canum 'c 2 ' is diploid with chromosome number of 2n = 24.Ocimum basilicum 'b 1 ' is tetraploid with chromosome number of 2n =52 and O. basilicum 'b 2 ' is a hexaploid with chromosome number of 2n = 72.Ocimum americanum is a tetraploid with chromosome number of 2n = 52.The seeds of each Ocimum variants were raised separately on moistened filter paper in 100 × 15 mm Petri-dishes at room temperature in the laboratory.At the four leaves stage, seedlings were transferred into small plastics cups containing topsoil and were kept in the screen house for the seedlings to be established.Three weeks after germination, seedlings were transplanted one plant per bucket into 7-liters plastic bucket filled with topsoil using five replicates per variant in each treatment.

Flower morphology
For each variant, the anthers color was determined before anther dehiscence and the number of male and female reproductive structures per flower were also determined by observing the flower bloom each day.The length of twenty stamens and carpel were measured separately on each of the Ocimum variants and compared.In each of the variants, the time of opening and closing of flowers and anther dehiscence were observed three times a week between 6 a.m. and 7 p.m. over a period of 2 months.The arrangements of the floral parts immediately after anthesis and after pollination were also observed.

Pollination experiment
At anthesis time, five potted plants of each Ocimum variant were transferred into a screen house made of wired net of mesh size 0.5 mm to exclude the activity of external pollinators.Also five potted plants of each Ocimum variant were left in the open.On each of the Ocimum variants in the open and screen house (bagged), the following pollination treatments were investigated on 200 flowers per treatment.The treatments included: (i) emasculation of flower buds before anthesis and anther dehiscence occurs; (ii) hand self-pollination of stigma by touching it with pollen from its anther immediately after anther dehiscence; (iii) spontaneous self-pollination in which flower buds were untouched.In each of the treatments flowers were examined every day for fruit sets.After fructification, the number of fruit and seeds produced from each treatment in the open and screen house were recorded for each Ocimum variant.Statistical analysis was performed using system analysis software (SAS) version 9.2 (SAS Institute, Cary, NC, USA).Data obtained from floral measurements, fruit and seed sets were subjected to one-way analysis of variance and their means were compared using Duncan's multiple range test (DMRT).

Insect visitor behavior
Observations on the behavior of different foragers were recorded.The observations were carried out five times a week during the day from 6 a.m. to 6 p.m.The foraging period, the part of the plant visited and the type of reward collected by different visitors were recorded through close observations.The time spent on a foraging visit was also recorded.The visiting insects were captured with hand net and identified using identification keys [17,18].The type of pollen grains picked up by the floral visitors were assessed through body washings and examined under a light microscope and compared with pollen of the Ocimum variants.

Flower morphology
In each of the Ocimum species the terminal inflorescence consist of flowers arranged in whorls and oriented horizontally to the axis.Each of the three Ocimum species has white petal, green sepal four filaments with white anther, purple-pigmented style and white style in O. basilicum 'b 1 ' .The flower of each of the Ocimum species opens by the separation of the upper and lower corolla lips.Immediately the style bends down but the stigma remains closed for some time.Later the stigma forms a bi-forked shape in the five Ocimum variants.Anther dehiscence occurred before anthesis in O. canum 'c 1 ' and in two variants of O. basilicum.In O. canum 'c 2 ' and O. americanum anthesis occurred before anther dehiscence.Anthesis occurred from 7 a.m. to 2 a.m. in all the Ocimum variants.The style is significantly longer than the filament in the five Ocimum variants studied (Tab.1).

Pollination experiment
The average number and percentage fruit and seed sets produced for each treatment on each Ocimum variant in the open treatments and bagged treatments were recorded (Tab.2).In the three Ocimum species all the open treatments produced higher fruit and seed sets than their corresponding bagged treatments.In the spontaneous selfpollination of all the five Ocimum variants the open treatments produced significantly higher number of fruit and seed set than in the bagged treatments except in fruit set of O. basilicum 'b 1 ' which showed no significant difference (Tab.2).Fruit and seed sets of spontaneous self-pollinated were low in bagged treatments of O. basilicum 'b 2 ' and O. americanum but significantly higher in the open treatment (Tab.2).In all three Ocimum species fruit and seed sets were low in the emasculated open treatment and no seed production was recorded for the emasculated bagged treatment, revealing the necessity of pollination (Tab.2).

Insect visitor behavior
All insects observed visited each of the Ocimum variant studied.Insect visits were observed as early as 7 a.m. to 6 p.m.The frequency of insects' visits increased as soon as the flowers started opening between 7 a.m. and 2 p.m.The bees observed visiting the Ocimum species are shown in (Tab.3), and include: Apis mellifera Linn., Amegilla cingulata Fabricius, Dactylurina staudingeri Gribido, Lasioglossum Curtis sp., and Xylocopa calens Lepeletier.The wasps observed visiting the Ocimum species include: Vespula Thomsom sp., Pseudomasaris Ashmead sp., and Polybioides tabidum Fabricius.Other insects observed on the Ocimum species were the butterflies, dragonflies, hoverflies, and the housefly.The bees and wasps visited the flowers of the Ocimum species to collect either nectar or pollen grains while Musca domestica L. and the dragonflies were observed visiting leaves and racemes so they cannot be considered as flower visitors.The butterflies visited the flower to collect nectar.The Ocimum pollen grains were observed on the insects most often but sometimes they carried pollen grains of other plants along with that of the Ocimum species.All bees, with the exception of Apis mellifera regularly visited studied Ocimum species.All wasps: Vespula sp. and Polybioides tabidum visited the plants very regularly except Pseudomasaris which visits were rare.The dragonflies and Musca domestica were also rare.The butterflies, however, were frequent in their visit.On all the bees, pollen grains were observed on their legs and abdomen but Dactylurina staudingeri carried pollen on legs only.
The wasps also carried pollen on their legs and abdomen, except Polybioides tabidum which carried pollen on mouthparts.The butterflies carried few pollen grains but the Tab. 3 The insect visitors observed on the Ocimum species studied.dragonflies and Musca domestica were not observed to carry any pollen.Compared to other floral visitors, the bees were observed to carry more pollen grains due to their hairy body.The insects contacted pollen grains when they tried to feed on the nectar or pollen.The time spent on each flower visit by the floral visitors is indicated in Tab. 3. Insect individuals that feed on nectar alone spent less time on a flower than those that feed on pollen.

Discussion
The seed set observed in the emasculated open treatments and lack of seed production in the emasculated bagged treatments of all three studied Ocimum species indicated that the insects that visited the flowers in the open treatments enhanced fruit and seed production.Also the production of higher fruit and seed sets in the open treatments when compared to the corresponding bagged treatments showed that insect visit enhanced fruit and seed production.Insects such as bees, wasp and hoverflies have been reported to play important functional roles as pollinators which facilitate fruit production in Ocimum species [19].The production of high fruit and seed sets in the spontaneous self-pollination bagged treatments of the two O. canum variants and O. basilicum 'b 1 ' indicated that their pollination occurred through autogamy.Autogamy in O. canum has been reported to be optional as it could also reproduce by cross-fertilization [20].It has also been reported that O. canum is predominantly allogamous due to formation of smaller number of fruits from manual selfing and spontaneous selfing [21].The low fruit and seed in the bagged spontaneous self-pollination treatment but significantly higher in the open treatment of O. basilicum 'b 2 ' and O. americanum indicated that these two species showed out-crossing features and more flowers were pollinated by insects in the open treatment.Ocimum basilicum and O. selloi Benth have been reported to exhibits mixed reproduction system via out-crossing and autogamy [16].A combination of protandrous condition (a mechanism promoting out-crossing), annual habit, herbaceous nature, more conspicuous floral parts and bee pollination in the Basilicum group which includes three studied Ocimum species have been considered as advanced characteristics of the genus [22].The mixed mating system indicated a great reproductive versatility of these species which might have contributed to the high genetic variability in the genus, through intraspecific and interspecific hybridization.According to Darrah [13] the genus Ocimum is characterized by a great variability of both morphological features and chemical composition because cross-pollination has created a large number of species and subspecies.This might also be responsible for the variability in the chromosome number of the variants of O. canum and O. basilicum used in this study.This study showed that successful pollination is marked by the corolla abscission (dropping) in all the three Ocimum species.Corolla abscission was observed to occur after successful pollination in other species of the family Lamiaceae [23] and was also directly related to pollination efficiency [24,25].The corolla of the non-pollinated flowers remains intact for 4-5 days which still attract insects to effect pollination.The withering of corolla in Ocimum species is common in plants with many flowers that pollinators failed to visit [25].According to Whitten [24] and Cruden [25] variation in flower longevity, marked by abscission (dropping) of the corolla, indicated an association with pollinating insects visit.This is also confirmed in the five variants of Ocimum species studied here.In the case of already pollinated flowers, post-pollination abscission increases the probability of visits to the remaining (non-pollinated) flowers on the plant.
Result of anther dehiscence indicated that the five Ocimum variants varied in their period of anther dehiscence.In O. canum 'c 1 ' and the two variants of O. basilicum anther dehisces before anthesis while in O. canum 'c 2 ' , O. americanum anther dehisces occurred after anthesis takes place.Anther dehiscence has been reported to occur before anthesis in O. canum, O. basilicum, O. americanum, and O. officinalis [11,15,21,26].Pollination in O. basilicum and O. americanum has been reported to occur mainly in post-anthesis which coincides with period of intense insect visitation which favored out-crossing because the insect carried pollen from flower to flower [14,16,27].The period of anthesis coincided with the peak of insect visit in this study which might be due to the peak of nectar production and availability of pollen.According to Kulloli et al. [28] nectar secretion occurred 2 hours before flower opened and increased soon after the flower opened which continued till early morning in members of the Lamiaceae.This also coincided with the time of maximum pollinator activity.
Insects foraging for both pollen and nectar of studied plants could affect pollination.Ocimum species have been reported to rely on insects for pollination [11].Bees foraging on pollen grains spent more time using their legs to rub against the anther and stigma in a bid to harvest pollen.But those foraging on nectar spent lesser time on flower but their landing position on the lower lip of the petal made their ventral surface to contact the stigma and anther which enhanced pollination.Longer visits were reported to correlate positively with pollination efficiency because it allowed more pollen collection from flowers [29,30].Other factors such as larger body size, navigation pattern might also effect pollination [31].Insects that spent less time on a flower were able to visit and navigate more flowers.This might increase the number of flowers pollinated during a single visit to an inflorescence.However, the tendency of pollinators to visit several flowers on a single plant increases opportunity of geitonogamy and self-pollination rate [32][33][34][35].The repeated visits by various insects to our study plants to collect nectar and pollen suggests that they produced enough reward to the visitors which may enhance pollination.Nectar production is the most common floral reward furnished by animal pollinated plants to their mutualistic partners [36].

Conclusion
canum 'c 1 ' and O. canum 'c 2 '), O. basilicum (O.basilicum 'b 1 ' and O. basilicum 'b 2 ') and O. americanum, type of floral visitors associated with these Ocimum species, and the effect of insects on fruit and seed production.
study was conducted in the Department of Botany, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria, located at N07°31 146', E 004° 31 583', 287 m a.s.l., between April and August 2013.The Ocimum species used in this study included two variants of O. canum Sims.(O.canum 'c 1 ' and O. canum 'c 2 ') two variants of O. basilicum L. (O.basilicum 'b 1 'and O. basilicum 'b 2 ' The two O. canum variants and O. basilicum 'b 1 ' are primarily adapted to reproduction by autogamy while O. basilicum 'b 2 ' and O. americanum exhibited mixed mating system via out-crossing and autogamy.Insect pollination in the studied plants enhanced fruit and seed production. 1ab.1Comparative mean length of stamen and style (mm) in each Ocimum variant.Means in the same column followed by the same letter are not significantly different at 0.05 probability.Result of pollination experiment on each Ocimum variant.