Impaginato 549 Adv. Hort. Sci., 2018 32(4): 549-556 DOI: 10.13128/ahs-23504 Flower development and pollen vitality of Moringa oleifera Lam. grown in a humid temperate climatic condition S. Radice 1, E. Giordani 2 (*) 1 Department of Plant Physiology, FAyCAUM - CONICET, Machado 914, University of Morón, Argentina. 2 D i p a r t i m e n t o d i S c i e n z e d e l l e P r o d u z i o n i A g r o a l i m e n t a r i e dell’Ambiente, Viale delle Idee, 30, 50019 Sesto Fiorentino (FI), Italy. Key words: fertility, flower anatomy, microsporogenesis. Abstract: Moringa oleifera is a tropical tree cultivated in many countries. This species has acquired a great importance in human nutrition and it was recently indicated as a “novel food” by the European Commission. Recently, moringa plants have been introduced in humid temperate climatic areas, among which Moreno (Buenos Aires Province - Argentina). In such area, the cultivation is possible for the production of leaves, but plants need protection during winter time in order to overcome damages due to low temperatures and hence to pro- duce capsules and seeds. The main objective of this research was to study flower morphology and anatomy of M. oleifera, as well as microsporogenesis and viability of pollen grains of plants cultivated in Moreno in comparison with those produced in a humid sub-tropical climatic area of Argentina (San Miguel de Tucumán). Flowers grown in the temperate environment resulted similar for morphological parameters to those observed in the sub-tropical environment. Nevertheless, pollen grain fertility depended directly on air temperature and it was negatively affected by the lower temperatures registered in the temperate site. According to the observed results, pollen viability increases with mean monthly temperatures above 16°C. 1. Introduction Moringa oleifera Lam. (moringa) is a multipurpose small to medium- sized, evergreen or deciduous tree, native to northern India, Pakistan and Nepal. It has a spreading open crown with drooping, fragile branches, feathery foliage with tripinnate leaves, and a thick corky whitish bark (Marcu, 2005). M. oleifera is utilised as animal fodder and employed in human nutrition due to its healthy properties (Fuglie, 1999; Palada and Chang, 2003; Ganatra et al., 2012; Paula et al., 2017), as well as in the production of fuel (Foidl et al., 2001), water sanitation (Wilson, 1992; Lekgau, 2009; Padilla et al., 2012). Moringa leaves are considered a “novel food” by the European Commission, so confirming their valuable properties in terms of energy, nutrients, proteins and minerals, as report- ed by several authors (Atawodi et al., 2010; Tende et al., 2011; Yameogo (*) Corresponding author: edgardo.giordani@unifi.it Citation: RADICE S., GIORDANI E., 2018 - Flower develop- ment and pollen vitality of Moringa oleifera Lam. grown in a humid temperate climatic condition. - Adv. Hort. Sci., 32(4): 549-556 Copyright: © 2018 Radice S., Giordani E. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 26 June 2018 Accepted for publication 28 September 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(4): 549-556 550 et al., 2011; Gopalakrishnan et al., 2016; Vats and Gupta, 2017). Araujo et al. (2016) highlighted also the importance of M. oleifera in regions character- ized by desertification and water deficit. Moringa cultivation is expanding all over the world, including in climatic areas, which differ notice- ably from those of its tropical origin. Recently some experiments showed the feasibility of cultivating moringa in the humid temperate climatic conditions of Buenos Aires Province (Argentina) for leaf produc- tion. Leaf extracts from trees grown in that condi- tions showed higher phenol content and antioxidant activity than those obtained from plants cultivated in typical tropical climates (Arena and Radice, 2016). Nevertheless, flower differentiation, anthesis and fertility resulted negatively altered and the produc- tion of pods and seeds, both of them important source of nutrients, was very low. The main objective of this research was to study the effect of air temper- ature on flower morphology and anatomy of M. oleifera, as well as on the microsporogenesis and pollen grain viability, observed on trees cultivated at Moreno (Buenos Aires Province) in comparison with those grown in San Miguel de Tucumán (Argentina), the first characterized by a humid temperate climate and the latter by a humid subtropical environment. 2. Materials and Methods Plant material All plants were obtained from the same seed lot. Homogeneous seedlings (n= 10) were grown in soil and in open air and cultivated in San Miguel de Tucumán (26° 49’59.00” S, 65° 13’00” W, elevation 456 m asl), while another similar set of seedlings (n=10) was planted in Moreno (34° 39’ 0” S, 58° 47’ 0” W, elevation 14 m asl) in 25 l plastic pots under a glasshouse from April to September. Successively pots were placed in open air. Moreno has a humid temperate climate, with an average temperature of 23.4°C in January and 10.0°C in winter time (June); San Miguel de Tucumán, has humid subtropical cli- mate (19.4°C the average annual temperature) with a hot and long summer and mild and dry winter. The precipitation pattern is monsoonal with an average of 997 mm (climate-data.org). A set of monthly air temperature parameters is reported in figure 1. Flower morphology Flowers (n = 100) on different phenological stages were observed on both groups of plants, and samples were collected monthly for further observations from September to December. Button flower collected were used fresh and fixed in FAA (formaldehyde, 100 ml; ethyl alcohol, 500 ml; acetic acid, 50 ml; distilled water, 350 ml). Light microscopy Button flowers (n= 10) were immediately frozen to -25°C and embedded in a medium consisting of p o l y e t h y l e n e g l y c o l a n d p o l y v i n y l a l c o h o l . Successively they were cut frozen by microtome inside the cryostat. Histologic slices were cut at 5 to 10 µm. A set of ten button flowers were dehydrated in an ethanol series and embedded in Spurr’s resin. Thin sections (75-90 nm thick) were stained with uranyl acetate and lead citrate. Sections were observed with a Leica DM 2500 microscope. Fluorescent microscopy Flowers in anthesis phase (n= 50) fixed in FAA were shaved with distilled water and softened with NaOH (8N) as described by Martin (1959). Then, they were stained with aniline blue to study pollen tube growth. Squash material was observed by a Leica microscope (DM 2500) using fluorescence with exci- tation filter BP: 450-490. Scanning electron microscopy (SEM) Button flowers fixed in FAA (n= 10) were dehy- drated in an ethanol series and critical point-dried with liquid CO2 was employed. Then it was sputter- coated with gold-palladium (40% gold and 60% palla- dium) for 3 minutes. Samples were observed with Philips XL30 SEM. Fig. 1 - Mean (A) and average minimum (B) air monthly tempe- ratures (°C) in San Miguel de Tucumán and Moreno loca- tions. Radice and Giordani - Moringa in temperate climate 551 Pollen viability Pollen viability was performed with fluorescent microscopy according to Radice and Arena (2016) on fresh anthers taken from button flowers of two local- ities (Moreno and San Miguel de Tucumán). Pollen evaluation was expressed in percentage. To deter- mine the statistical significance of the hypothesis the chi-squared test (χ2) was used. 3. Results Flower development Flower development starts with the appearance of the flower on a clustered inflorescence (Fig. 2). These first buds are green reddish and about 1mm long (Fig. 2A). They grow up to about 10mm and turn to white greenish (Fig. 2A). Anthesis takes place sequentially among the flowers of the inflorescence (Fig. 2B). At anthesis, flower shows a zygomorphy symmetry. The larger transversal petal is bent upwards, while the others are reflexed downwards together with the sepals (Fig. 2C). Anthers are yellow and no dehiscent; flowers have odour and nectar in this phase. As the flower develops, anthers change colour to dark yellow (Fig. 2C) and finally to brown (Fig. 2D). It was observed that 1-3 anthers were not developed in flowers collected from Moreno field. Pistil, which at the time of the anthesis is below the anthers, contin- ues to grow until it protrudes several millimetres above the androecium (Fig. 2C). Finally, petals wither and fall, while the ovary enlarges and turns to red- dish colour regardless its fertilisation (Fig. 2E). Flower structure Moringa oleifera plants grown in Moreno and San Miguel de Tucumán experimental fields developed flowers with average values of ≈5 petals, ≈6 sepals, ≈6 stamens, ≈5 staminoides and 22 ovules (data not showed). In the observed flowers, the unique largest petal (referred as “primordium petal”) stands right; the others are folded (Fig. 2C). Female part of the flower shows the complete pistil with the style and a hairy ovary (Fig. 3A). Ovary is tricarpelar and ovules are located in parietal placentation (Fig. 3B). Stigma is just a hole (Fig. 3C). Some nectarostomata sur- round the gynophore (Fig. 4). Glandular hairs, with the function of expelling the nectar, are present on the nectarostomata surface (Fig. 4A). Nectarostoma- ta produces nectar in sub epidermal cells, that accu- mulate it and transfer it through the intercellular spaces (Fig. 4B). Frozen section of a button flower just before the anthesis phase allows to appreciate the state of the Fig. 2 - Flower development of Moringa oleifera Lam. A, from but- ton flower to anthesis. B, beginning of anthesis. C, anthe- sis; D, flower senescence; E, pod formation. Bar = 1 cm. Fig. 3 - Pistil of Moringa oleifera Lam. A, external view of the pistil; B, internal view of the ovary with ovules; C, detail of the stigma. Bars = A-C, 1mm. Adv. Hort. Sci., 2018 32(4): 549-556 552 structures and their normal coloration (Fig. 5). In fact, it is possible to see anthers with pollen grains already formed wrapped in a yellow substance similar to sporopollenin (Fig. 5B). Pistil shows developing ovules attached to the carpelar wall (Fig. 5A). Finally, all internal organs are enveloped by sepals and petals (Fig. 5A). Flowers studied by SEM showed there is no defined stigmata structure. Pistil is coronate by a smooth cell structure as a continuation of the style (Fig. 6A). The internal cavity of the ovary is covered with hairs (Fig. 6C) and ovules adhere to their walls on the connection of two carpels. Ovules appear to be campylotropous (Fig. 6D). Microsporogenesis Button flowers from 1mm to 10 mm (Fig. 2A) have been used to observe different steps of pollen grain formation. Different stages of pollen grain differenti- ation were observed: microsporocytes mother cells, tetrads, microsporocytes and mature pollen (Fig. 7). Development was very fast and it accomplished in less than one week. M o r i n g a f l o w e r s h a v e m o n o t h e c a l a n t h e r s . Button flowers shorter than 5 mm contains inside the just formed pollen sac microspore mother cells (Fig. 7A). These cells show a big and visible nucleus and a very dense cytoplasm indicating a high activity. Fig. 4 - Nectary of Moringa oleifera Lam. (light micrographs). A, longitudinal section of the nectary; B, detail of the nectar cumulated in the intercellular spaces (arrows). Bars = A, 100 µm; B, 10 µm. Fig. 5 - Flower in pre anthesis phase of Moringa oleifera Lam. (frozen section). A, Longitudinal section of flower with petals (pe), anthers (an) and ovary with ovules (arrows); B, mature pollen grain surrounded by sporopollenin. Bars = A, 10 cm; B, 10 µm. Fig. 6 - SEM micrographs of Moringa oleifera Lam pistil. A-B, style and stigma; B, detail of the stigma with pollen grain; C, ovarian cavity with ovules; D, detail of an ovule; E-F, pollen grains. Bars = A, 50 µm; B, E, 20 µm; C, 500 µm; D, 100 µm; F, 10 µm. Radice and Giordani - Moringa in temperate climate 553 During this stage, the last inner layer of the anther wall corresponds to tapetal cells that are formed by large and binucleate cells (Fig. 7A). As the buds lengthen, more advanced stages of microsporogenesis are observed. In fact, it was observed the tetrad (Fig. 7B) and then the release of microsporocites (Fig. 7C). At this point, tapetum degrades (Fig. 7C). The young microspores show a central nucleus and a vacuolated cytoplasm. The last stage shows mature pollen grains and free orbicules (Fig. 7D). When mature pollen grains are formed, tapetum disappears completely. Mature pollen grains measure about 20 µm. It is possible to observe the exine of the grains well formed and the cytoplasm of the vegetative cell with a lot of amylo- plasts (Fig. 7D). Pollen viability It was possible to differentiate green, red and orange yellowish pollen grains corresponding to viable, non-viable and sub viable pollen grains respectively. Pollen viability among flowers at different dates (Table 1) showed great variations on flowers collect- ed in Moreno; on the contrary, no differences were observed between flowers collected from San Miguel de Tucumán throughout the study period (Table 2). Moreno flowers showed a very low percentage of Table 1 - Viability of pollen grains collected on different months and different locality Values with different letters between the same column are signifi- cant different. Tukey (p≤0.05). v i a b l e p o l l e n g r a i n s r e s p e c t t o S a n M i g u e l d e Tucumán flowers during September to November. Although the number of pollen grains per anther in Source Month Viable Non Viable Sub-viable Moreno September 10 c 79 a 11 a Moreno October 28 c 53 a 19 a Moreno November 48 b 32 b 20 a Moreno December 74 a 3 c 23 a S.M.Tucumán September 68 a 22 b 10 a S.M.Tucumán October 63 a 25 b 12 a S.M.Tucumán November 70 a 22 b 8 a S.M.Tucumán December 69 a 28 b 13 a Table 2 - Viability of pollen grains collected on September from anthers of the same flower Values are expressed on percentage. Values with different letters between the same column are significant different. Tukey (p≤0.05). Source Anther Viable Non Viable Sub-viable Moreno 1-1 10 a 84 a 6 a Moreno 1-2 7 a 91 a 2 a Moreno 1-3 10 a 82 a 8 a Fig.. 7 - Microsporogenesis of Moringa oleifera Lam (light micrographs). microsporangium with conspicuous microspore mother cells (mmc) and tapethal cells (tc); B, tetrads (td); C, microporocytes free (m) and tapetal cells (tc) in metabolization phase; D, mature pollen grains (arrows) with amyloplast (a) in the cytoplasm and orbicules (circles). Bars = A 10 µm; B-D, 20 µm. 554 Adv. Hort. Sci., 2018 32(4): 549-556 the flowers was not evaluated, it was observed that flowers collected from San Miguel de Tucumán had more amount of pollen grain in each anther. Some anthers of Moreno flowers contained immature pollen and a viscous substance. Additionally, accord- ing to the harvesting period of the flowers, some anthers developed only dead pollen grains. According to these results, a more detailed analy- sis of pollen viability was performed between pollen grains derived from different anthers of the same flower and between anthers from different flowers collected from Moreno trial. Viability of pollen grains of different anthers collected from the same flower was not statistically different (Table 2), while viability of pollen from different flowers of different trees col- lected on the same date resulted significantly differ- ent between flowers (Table 3). Viable pollen grains were contained on great proportion in some flowers but scarce in others, with a random distribution among trees. Pollination During anthesis, pistils both with or without ger- minated pollen were observed. When pollination occurs, pollen grains fall freely into the stigma cavity and then germinate (Fig. 6B). In effect, pistils treated by Martin technique showed that a mass of pollen grains is housed in the cavity and that many pollen tubes germinated (Fig. 8A) and later on reached the ovary and fertilized the ovules (Fig. 8B). Pollination was rarely observed on flowers collect- ed from Moreno during spring time (September to November) but it was very frequent in summer time. On the contrary, it was observed that flowers collect- ed on San Miguel de Tucumán were profusely polli- nated in both periods and fruit production was con- tinuous throughout the year. Fruit production on Moreno plants started in summer until May, while it resulted continuous in San Miguel de Tucumán. 4. Discussion and Conclusions Flower morphology and anatomical structure observed in flowers collected from Moreno field and San Miguel de Tucumán did not show significant dif- f e r e n c e w i t h t h o s e d e s c r i b e d i n l i t e r a t u r e (Ramachandran et al., 1980). Furthermore, flowers from the two localities studied in Argentina have a normal external and internal development. Frozen sections allowed to see the internal anato- my with its natural colorations and to show more clearly the presence of orbicules, that, in the tradi- tional cuts, appear very confused because of the dif- ferent colorations applied. On the other hand, flow- ers studied by SEM allowed clarifying some concepts. In fact, it was clearly showed how pollen germination begins even in the absence of any connection to any structure of the stigma or style. Bhattacharya and Mandal (2004) found that some extra proteins and esterases contribute towards the stigmatic receptivi- ty; furthermore, the occurrence of intraovarian tri- chomes, which is not widespread in the angiosperms, could facilitate the growth of the pollen tubes. This assessment made by Dickison (1993) is based on that the trichomes functionally resemble obturators. Pollen grain formation seems to be very variable depending on the geographical location and the time of year. In fact, Muhl et al. (2011) showed that low temperature regime induces flowering but provokes low pollen viability and this statement would explain the results obtained in Moreno spring flowers, thus confirming that pollen grain viability is affected by air temperature. In fact, the lowest values of pollen Fig. 8 - Fertilization of Moringa oleifera Lam (fluorescent light micrographs). A, pollen tube growing toward the style (arrows); B, ovules (ov) and pollen tube crossing the micropile and entering the embryo sac (arrows). Bars = A-B, 100 µm. Table 3 - Viability of pollen grains from different flowers of the same plant Source Flower Viable Non viable Sub-viable Moreno 1 A 7 b 78 ab 15 a Moreno 1 B 6 b 82 a 12 a Moreno 1 C 18 a 67 b 15 a Moreno 1 D 2 b 95 a 3 b Moreno 1 E 15 a 72 b 13 a Values are expressed on percentage. Values with different letters between the same column are significant different. Tukey (p≤0.05). 555 Radice and Giordani - Moringa in temperate climate grain viability were observed on September and October when mean temperatures were lower than 16°C. Additionally, the higher percentage of pollen viability observed in San Miguel de Tucuman is con- sistent with the air temperatures registered in that location, which resulted about 4/5°C higher than those observed in Moreno (Fig. 1). The difference of the quality of pollen grains col- lected in spring and summer could be related to the amount of orbicules or Ubisch bodies. Studies on Ubisch body formation in Brachypodium support the evidence that they are formed in the tapetum and are involved in exine synthesis (Sharma et al., 2014). Actually, during spring time, anthers with mature pollen grains brings many orbicules. This fact sug- gests that when the exine was not well formed the quantity of dead pollen was important. On the other hand, pollen viability seems to have an influence on the efficiency of pollination. As directly observed, all plants were very visited by insects during blooming on both experimental loca- tions, but pollination was rarely observed in Moreno flowers, while it was very frequent in flowers collect- ed in San Miguel de Tucumán. Although tropical climates are those considered ideal for M. oleifera according to Muhl et al. (2011), good results obtained with San Miguel de Tucumán flowers confirm that sub-tropical climates are also suitable for this species. Taking into account repro- ductive functions, and namely the microsporogene- sis, Moreno environment seems to be just below the threshold of good temperature regime for M. oleifera during spring time despite the climatological predic- tions made by Falasca and Bernabé (2008). In conclusion, the reported results demonstrate the possibility of moringa cultivation unusual non tropical climates. Flowers of the trees grown in Moreno field were normal from the morphological point of view only in some periods of the year and the quality of their development was related to air t e m p e r a t u r e s . P l a n t s g r o w n o n S a n M i g u e l d e Tucumán have an un-interrupted production of flow- ers despite not being in a tropical climate. On the other hand, moringa cultivated in marginal areas, as the locality of Moreno can be considered, could offer important advantages. Acknowledgements The authors thank Ing. Oscar Dantur for the col- lection of plant material in San Miguel de Tucumán and Mrs. Isabel Farías for her assistance with the his- tology. This research was supported by IFUND - University of Florence project. References ARAúJO M., SANTOS C., COSTA M., MOUTINHO-PEREIRA J., CORREIA C., DIAS M.C., 2016 - Plasticity of young Moringa oleifera L. plants to face water deficit and UVB radiation challenges. - J. Photochem. Photobiol., B. Biol., 162: 278-285. 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