FLOWERING PATTERN, THE STRUCTURE OF NECTARY SURFACE AND NECTAR SECRETION IN TWO VARIETIES OF OCIMUM BASILICUM L

The studies conducted in the years 2003 2004 covered two varieties of Ocimum basilicum L.: var. purpurascens Benth. and var. lactucaefolium I. The flower lifetime, daily flowering pattern, morphology of pollen grains and the abundance of nectar secretion of flowers were compared. The surface of the nectaries of O. basilicum var. lactucaefolium was observed using scanning electron microscopy (SEM). The automorphic nectaries of basil formed the following parts: a flat part from the side of the upper lip and a convex part with three protrusions on the opposite side. On the top of two outer protrusions, stomatal areas were located, responsible for nectar secretion. The stomata occurred at the level of other epidermal cells. On the average, nectar contained 42% of sugars. The sugar yield of flowers of var. lactucaefolium was 36% higher than this feature in var. purpurascens. In terms of their size, the pollen grains were rated as medium sized. Their viability ranged 88 96%. The shape of the pollen grains was described as oblate and suboblate.


INTRODUCTION
The genus Ocimum numbers 150 plant species occurring in the natural environment in wild state, primarily in the tropical and subtropical zone of Africa and Asia (S z w e y k o w s c y, 2003). Basil belongs to the family Labiatae which includes a numerous group of melliferous plants recommended for cultivation in order to improve bee pollen benefits (J a b ł o ń s k i , 1997b, 2000; Ma s i e r o w s k a i K r z y s i a k , 2000; B o ż e k , 2003).
Plants from the genus Ocimum provide food attractants to insects in the form of pollen and nectar. Flowers of this species were visited in large numbers by insects, in particular honey bees (R a j u , 1989 (2001) showed that honeys from labiate plants inhibited most strongly the growth of staphylococcus. Due to its favourable effect on human organism, showing anti-inflammatory, antibacterial and sedative activity, basil has found application in the pharmaceutical, food and cosmetics industries (M a r t y n i a k -P r z y b y s z e w s k a , 2000; P o db i e l k o w s k i i S u d n i k -W ó j c i k o w s k a , 2003). Basil herb is used in phytotherapy, homeopathy and aromatherapy (K o h l m ü n z e r, 1993; C z y g a n , 1997; T h o p p i l et al. 1998; N d g o u n g a i O u a m b a , 1997).
The aim of the studies conducted was to compare ecological features of flowers, daily flowering pattern, nectar secretion and the morphology of pollen grains of two basil varieties. The morphology and structure of the secretory epidermis of the nectaries, as well as insect visitation of flowers of O. basilicum var. lactucafolium, were also determined.

MATERIAL AND METHODS
The studies conducted in the years 2003--2004 covered two varieties of Ocimum basilicum L.: var. purpurascens Benth. and var. lactucaefolium I. The taxa studied grew in the Botanical Garden of the Maria Curie-Skłodowska Univeristy in Lublin, Poland. The lifetime of flowers was estimated from the time of opening of lobels in the corolla until the wilting stage. The daily pattern of flower blooming was observed on five plants of each variety. Readings were made every hour between 7 00 and 19 00 on three sunny days. The visitation of flowers of var. lactucaefolium by pollinating insects was recorded at 2-hour intervals between 7 00 and 19 00 . The abundance of nectar secretion of basil was examined using the pipette method. Nectar was collected three times in four samples at full bloom stage of the plants. One sample was the amount of nectar from the whole lifetime of 8 -10 flowers. The sugar yield was calculated from the weight of nectar and the percentage content of sugars in it, determined with an Abbe refractometer. The surface of the nectaries of O. basilicum var. lactucaefolium was observed using scanning electron microscopy (SEM). Morphometric measurements of the secretory tissue and flower elements were also made. The morphology of pollen grains was characterised, by determining the length of the equatorial (E) and polar (P) axis for 200 grains. To determine the pollen viability, the pollen was stained with acetocarmine. 400 grains were analysed in the successive view fields of a light microscope.

RESULTS
The plants of Ocimum basilicum var. lactucaefolium burst into bloom in the second decade of June. The variety purpurascens produced the first flowers several days later compared to the former variety. Buds of the studied varieties burst into bloom from early morning hours (7 00 ) throughout the whole day until 17 00 . The largest number of blooming flowers on the plants of the both taxa was found at 11 00 (32% -36%). The daily flowering pattern of the analysed varieties was similar. The intensity of formation of new flowers was the greatest between 9 00 and 13 00 . The flower lifetime was 1.5 day (var. purpurascens) or 2 days (var. lactucaefolium) (Fig. 1). The observed varieties bloomed for a period of about three months.
The flowers of the studied basil varieties differed in their size and the colour of the perianth. The flowers of O. basilicum var. lactucaefolium formed a shorter calyx (by 34%) and a longer corolla (by 22%) than these elements in the other variety (Fig. 2). Shining trichomes present on their surface and white (var. lactucaefolium) or purple-tinged colour (var. purpurascens) of the corolla (Figs. 5-8), as well as an intensive scent were signallers for insects. A short corolla tube (5 mm) provided easy access for insects to the nectar secreted (Tab. 2, Fig. 8). Numerous protective hairs growing on the surface of the epidermis covering the outgrowths of the filaments of two shorter stamens protected the nectar against evaporation (Fig. 9).
In the structure of the automorphic nectaries in the flowers of the studied basil, two parts were distinguished at the base of the ovary of the pistil: a flat part from the side of the upper lip and a convex part with three protrusions from the side of the lower lip. The convex fragments of the secretory tissue were situated between the parts of the four-parted ovary . The glandular tissue from the side of the upper lip accounted for, on the average, 35% of the height of the protruding part (100%=794 μm). The nectaries in the flowers of var. purpurascens had a larger size, lower protrusions and a thicker tier of cells in the flat part, compared to these parameters in the other variety (Fig. 3).
Nectar secretion in the basil flowers was observed from the bursting bud stage throughout their whole lifetime. The stomata forming clusters, that is, the stomatal areas on the two outer protrusions, were responsible for nectar secretion onto the nectary surface       The studied basil varieties secreted nectar abundantly. Ten flowers of var. lactucaefolium secreted about 9 mg of nectar. The weight of nectar in the flowers of var. purpurascens was found lower by 31% compared to the former variety. The concentration of sugars in nectar ranged 43 -46 %. When comparing the sugar content in this solution, it was found that the sugar yield of the flowers of var. lactucaefolium was 36% higher than the weight of sugar in the nectar of the other variety (Tab. 1).
Insects also collected pollen from the basil flowers. The high pollen viability ranging 88 -96% evidences its good nutritional value for insects. The six-colpate white pollen grains of basil developed a reticulate sculpture of the exine. A comparison of the length of the equatorial and polar axis of the grains shows that the flowers of var. purpurascens produced smaller pollen grains compared to the pollen of the other variety. In terms of the size, the pollen grains of basil were classified as medium-sized. Based on the shape index (P/E), these grains were classified as oblate in the flowers of var. lactucaefolium and suboblate in the anthers of var. purpurascens (Tab. 2).
The main insects visiting the basil flowers were bees and bumblebees. Honey bees accounted, on the average, for 52% of insects recorded. The share of bumblebees in flower visitation was 31%. Visitations of other insects accounted for 17% of all pollinators recorded (Fig. 4).

DISCUSSION
The lifetime of basil flowers depends on the variety and climatic conditions. In the varieties studied, this period was 1.5 -2 days.  Chwil, 2003). We r y s z k o -C h m i e l e w s k a (2000), taking into account the shape and number of protrusions in the nectaries, distinguished their three groups in the family Lamiaceae: irregular with three protrusions, symmetrical with one protrusion and regular with a symmetrically divided tissue.
The nectar in the flowers of the studied basil was secreted by the stomata located on two outer convexities of the nectary. Their number on a single protrusion was 13-19. In several earlier described Labiate taxa, this parameter ranged between 4 and 18 (We r y s z k o -C h m i e l e w s k a , 2000). Most of the modified stomata in the secretory epidermis of the analysed varieties of basil occurred at the level of other cells. A similar position of the stomata was found by Dafni et al. (1988) in the nectaries of Pholomis viscosa, Rosmarinus officinalis and Salvia fruticosa, whereas in Coridothymus capitatus and Melissa officinalis these cells were elevated over the epidermis surface or depressed as in Salvia hiersohymitana.
Nectar secretion in the flowers of the analysed basil continued throughout their whole lifetime. An identical secretion pattern was noted in Ocimum kilimandscharicum and O. gratissimum, with the maximum amount of nectar at 10.00 in the former taxon and at 12.00 in the latter (S a j j a n a r i in. 2005a).
The concentration of sugars in the studied basil was 45%, on the average, and their weight from ten flowers ranged 2.6-3.5 mg. This value was higher than the sugar yield of: O. gratissimum, Dracocephalum moldavica, Nepeta melissiflora, Salvia glutinosa and S. Przewalski, but it was lower than the yield of O. basilicum cv. Genovese, O. kilimandscharicum (M a s i e r o w s k a and K r z y s i a k , 2000; C h w i l , 2003; S a j j a n a r et al., 2005a; We r y s z k o -C h m i el e w s k a et al., 2005). The hairs present in the epidermisco vering the outgrowths of the stamens protected the nectar against evaporation in the flowers of the observed varieties of basil. According to B a n a s z a k (1987), these trichomes also made it impossible for insects unable to cross pollinate flowers to use the nectar.
From the flowers of the studied basil and basil described in literature, insects collected pollen and formed white pellets (R a j u , 1989). In terms of the size, the Due to its high sugar yield and a long flowering period, basil may be considered as a good source of nectar for honey bees. On the other hand, the small number of sterile grains evidences their good nutritional value for insects, a high efficiency of cross pollination and the possibility to create new forms in breeding works.