Comparative Study of Ovule Development between Wild ( Passiﬂora foetida L.) and Cultivated ( P. edulis Sims) Species of Passiﬂora L. Provide Insights into Its Differential Developmental Patterns

: The ovules inside the ovary of a plant are the precursors of seeds and they are important for the perpetuation of the plants. The genus Passiﬂora L., produce fruits with numerous seeds and they have economic and medicinal value. The edible portion of the Passiﬂora are the seeds surrounded by pulp. Being the edible parts of a fruit, it is important to investigate the early development of ovules in Passiﬂora that lead to the formation of seeds after pollination. Wild relatives of the domesticated crops are increasingly being investigated for possible genetic resources that can be used for crop improvement programs. The present study was designed to investigate the comparative ovule development between a wild ( Passiﬂora foetida L.) and a cultivated ( Passiﬂora edulis Sims) species of Passiﬂora with an aim that it may provide important information about the common and diverging regulatory mechanisms during ovule development between the wild and the cultivated species. We also investigated the pollen morphology between the wild and cultivated species using light and scanning electron microscopy. Our results show that wild type P. foetida ovule growth is faster when compared with that of cultivated P. edulis. Furthermore, wild species harbour ovules of large size (0.14 mm 2 ) but less in number (6) as compared to cultivated ones which show smaller size (0.05 mm 2 ) of ovules but relatively more in number (21). The differences in ovary wall thickness were also stark between the two species. The ovary wall thickness was 0.10 mm in the wild type whereas it was 0.74 mm in cultivated species. Notable differences were also observed in diameter where the wild type (2.45 mm) reported smaller diameter than cultivated species (3.25 mm). We observed little difference in the pollen morphology between the two species.


Introduction
The genus Passiflora is highly diverse and comprises around 550 known species, among which only 60 are edible [1]. The species of the genus are commonly known as the passion flowers [2,3]. Passiflora plants are generally shrubs or herbs and may produce bisexual or unisexual flowers [4,5]. Passion flowers are widely spread from tropical forests to high mountains in the Andes in the Neotropical region [6]. The plants are found in abundance in Brazil, Asia and Africa [1,7]. The Passiflora plants found in India are both naturalized and cultivated. They are predominantly found in the Western and Eastern Ghats and morphological analysis is important for taxonomic studies [70], we further compared the pollen morphology of the two species using light and scanning electron microscopy.

Plant Acclimatisation and Growth
For comparative study of ovule development in Passiflora, two species namely P. foetida and P. edulis were chosen. The wild species of P. foetida was collected from the Central University of Kerala Kasaragod premises whereas the cultivated P. edulis saplings was collected from a nursery in Kuniya (Kasaragod, Kerala, India). The plants were potted and acclimatised in the polyhouse at Central University of Kerala (Kasaragod, Kerala, India; 12.3929 • N, 75.0917 • E). The maximum and minimum temperature of the region varies between 29.2 to 33.4 • C and 19.7 to 25 • C respectively.

Tissue Harvesting
The development of ovules inside the ovary progresses simultaneously along with the ovary development before pollination and fruit development after pollination. To track the development of ovules, floral buds of various stages are collected from the acclimatized plants. Floral buds of stages 6, 7 and 8 ( Figure 1) harvested 15 days, 10 days and 5 days before pollination respectively from each species were analyzed for both light microscopy and scanning electron microscopy. economic and medicinal importance. This study provides a comparative overview of two species of Passiflora i.e., Passiflora foetida L. and Passiflora edulis Sims. Since pollen morphological analysis is important for taxonomic studies [70], we further compared the pollen morphology of the two species using light and scanning electron microscopy.

Plant Acclimatisation and Growth
For comparative study of ovule development in Passiflora, two species namely P. foetida and P. edulis were chosen. The wild species of P. foetida was collected from the Central University of Kerala Kasaragod premises whereas the cultivated P. edulis saplings was collected from a nursery in Kuniya (Kasaragod, Kerala, India). The plants were potted and acclimatised in the polyhouse at Central University of Kerala (Kasaragod, Kerala, India; 12.3929° N, 75.0917° E). The maximum and minimum temperature of the region varies between 29.2 to 33.4 °C and 19.7 to 25 °C respectively.

Tissue Harvesting
The development of ovules inside the ovary progresses simultaneously along with the ovary development before pollination and fruit development after pollination. To track the development of ovules, floral buds of various stages are collected from the acclimatized plants. Floral buds of stages 6, 7 and 8 ( Figure 1 harvested 15 days, 10 days and 5 days before pollination respectively from each species were analyzed for both light microscopy and scanning electron microscopy.  Three floral buds of each P. foetida and P. edulis were used for the analysis of ovary and ovule morphology. Six ovules were sampled at each developmental stage. The open flowers from both the species were collected to analyze the pollen grains. The buds were collected early in the morning and the histological sectioning was done immediately after collection.

Histology and Staining
For primary comparison of ovule development between both species, histological study is done by using light microscopy [71]. All collected flower buds were kept in formaldehyde-alcohol-acetic acid fixative in the ratio 2:17:1 [72]. The ovaries were separated carefully from the floral bud and the free hand histological sections were prepared. After preparing the histological sections, each section was placed in the safranin O stain for 5-6 s and excess stain was removed by washing followed by dehydration using a series of ethanol gradients (10%, 20%, 30%, 50% and 70%) [73]. For the pollen analysis, dilute staining is preferred as the pollen gets stained faster. A single drop of safranin O stain was mixed with a drop of water and the pollen was dusted on the slide. The slides were observed under a Magnus light microscope (Magnus MLX-DX, Olympus Pvt. Ltd., Noida, Uttar Pradesh, India) and images were captured using Scope Photo software (Scope Photo Ver. 3.0).

Scanning Electron Microscopy
Scanning electron microscopy can be performed using acetolysis and non-acetolysis methods [74]. The scanning electron microscopy analysis was done using a non-acetolysis method [75]. The buds of different developmental stages were collected as described above and ovaries were separated from the bud and passed through the series of alcohol gradients for dehydration [76] and fixed by acetone [77]. The histological sections of the ovary were placed in the carbon conducting tape in the metal stubs and gold sputter coating was done using a sputter coater (SC7620, QUORUM Technologies, Lewes, UK). For pollen morphology, anthers were collected from the open flowers, and the pollen were dusted into the carbon tape fixed in the metal stub, and sputter coating was done [78]. The samples were then placed in the scanning electron microscope (VEGA3 SBH, TESCAN, Czech Republic), and images were taken.

Measurement of Ovary and Ovule Characteristics
The image analysis software ImageJ was used to measure ovary and ovule parameters [79]. Ovary wall thickness, perimeter, equatorial and polar diameter, ovule size (area), number of ovules per placenta and total number of ovules of both species were considered for the comparative analysis [80]. Image files of histological sections were opened in ImageJ. Scale was set with a scale bar from the image as reference. Straight line tool was used to calculate ovary thickness and diameter. For the measuring ovary perimeter and ovule area, a freehand line tool was used.

Morphological Differences between P. foetida and P. edulis
In the present study, two species of Passiflora L., namely P. foetida and P. edulis were used for comparative investigation of ovule development and pollen morphology. Morphologically, the two species show considerable differences in their various characters. The P. foetida is a creeping vine, the flowers are small with white petals and dark pink corona, the three-lobed leaves are hairy and alternate. The tendrils are produced by the stem from the base of the leaves. The stems are thin and contain sticky hairs. The small, edible fruits are green when unripe and yellow when ripe. The size of fruits between the wild and the cultivated varies drastically. The wild P. foetida bears very small fruits as compared to cultivated P. edulis ( Figure 2). The fruits are partially enclosed by the bracts that dry when the fruits mature. On the other hand, P. edulis flowers are large and showy with white petals and violet corona, the leaves are alternate 'V' shaped and the stem is woody and have tendrils. The tendrils have a purple shade when young. The large, round fruits are green when unripe and can be yellow or purple when ripe ( Figure 2). The nature of the pulp is similar in both species. Figure 3 represents morphological differences between plants of P. foetida and P. edulis. Three lobed leaves are present in both species while leaf size was smaller in P. foetida ( Figure 4). We can easily distinguish between the two species from the size and shape of the leaves. The leaves in P. edulis have deeper incisions at the lobes. that dry when the fruits mature. On the other hand, P. edulis flowers are large and showy with white petals and violet corona, the leaves are alternate 'V' shaped and the stem is woody and have tendrils. The tendrils have a purple shade when young. The large, round fruits are green when unripe and can be yellow or purple when ripe ( Figure 2). The nature of the pulp is similar in both species. Figure 3 represents morphological differences between plants of P. foetida and P. edulis. Three lobed leaves are present in both species while leaf size was smaller in P. foetida ( Figure 4). We can easily distinguish between the two species from the size and shape of the leaves. The leaves in P. edulis have deeper incisions at the lobes.   that dry when the fruits mature. On the other hand, P. edulis flowers are large and showy with white petals and violet corona, the leaves are alternate 'V' shaped and the stem is woody and have tendrils. The tendrils have a purple shade when young. The large, round fruits are green when unripe and can be yellow or purple when ripe ( Figure 2). The nature of the pulp is similar in both species. Figure 3 represents morphological differences between plants of P. foetida and P. edulis. Three lobed leaves are present in both species while leaf size was smaller in P. foetida (Figure 4). We can easily distinguish between the two species from the size and shape of the leaves. The leaves in P. edulis have deeper incisions at the lobes.

Floral Characterization of Two Species
The flowers of P. foetida are covered by three reticulated green bracts. The veins of the bracts end with glandular structures that release adhesive ooze. The flowers are white and are borne singly. The flower has five petals and five sepals. They also have five stamens and an ovary topped with three style tipped with prominent stigmas. On the other hand, P. edulis flowers are larger in size and lack the presence of reticulate green bract. The flowers are hence non-glandular. The flowers have five white petals, five green sepals and a showy corona. The base of the flower has five stamens, an ovary and a branched style. Figure 5 shows floral differences between wild and cultivated species of Passiflora.

Floral Characterization of Two Species
The flowers of P. foetida are covered by three reticulated green bracts. The veins of the bracts end with glandular structures that release adhesive ooze. The flowers are white and are borne singly. The flower has five petals and five sepals. They also have five stamens and an ovary topped with three style tipped with prominent stigmas. On the other hand, P. edulis flowers are larger in size and lack the presence of reticulate green bract. The flowers are hence non-glandular. The flowers have five white petals, five green sepals and a showy corona. The base of the flower has five stamens, an ovary and a branched style. Figure 5 shows floral differences between wild and cultivated species of Passiflora.

Floral Characterization of Two Species
The flowers of P. foetida are covered by three reticulated green bracts. The veins of the bracts end with glandular structures that release adhesive ooze. The flowers are white and are borne singly. The flower has five petals and five sepals. They also have five stamens and an ovary topped with three style tipped with prominent stigmas. On the other hand, P. edulis flowers are larger in size and lack the presence of reticulate green bract. The flowers are hence non-glandular. The flowers have five white petals, five green sepals and a showy corona. The base of the flower has five stamens, an ovary and a branched style. Figure 5 shows floral differences between wild and cultivated species of Passiflora.

Ovule Development in Wild and Cultivated Species of Passiflora
Floral buds were arranged in descending order of their sizes to isolate the ovaries for light and scanning electron microscopy analysis (Figure 1). To investigate the morphological differences in ovary, measurements were taken using ImageJ ( Figure 6). Thickness of the ovary wall was less in wild type P. foetida (0.10 ± 0.012 mm) when compared to its cultivated counterpart P. edulis (0.74 ± 0.177 mm). Measurements of the ovary perimeter indicate P. foetida (4.89 ± 0.10 mm) is smaller than P. edulis (8.78 ± 1.78 mm). Length differences in equatorial and polar diameter are negligible, but variation is observed between the two species ( Figure 6). The development of ovules in both the species start with small finger-like projections in the ovary walls. The wild and cultivated species show differences in the development of the ovules. The wild species P. foetida shows a very symmetric and definite distribution of ovules in its ovary. We observed that, P. foetida ovules are larger in size but less in number as compared to cultivated species P. edulis which has smaller ovules but more in number (Figures 7a-c and 8a-c).

Ovule Development in Wild and Cultivated Species of Passiflora
Floral buds were arranged in descending order of their sizes to isolate the ovaries for light and scanning electron microscopy analysis (Figure 1). To investigate the morphological differences in ovary, measurements were taken using ImageJ ( Figure 6). Thickness of the ovary wall was less in wild type P. foetida (0.10 ± 0.012 mm) when compared to its cultivated counterpart P. edulis (0.74 ± 0.177 mm). Measurements of the ovary perimeter indicate P. foetida (4.89 ± 0.10 mm) is smaller than P. edulis (8.78 ± 1.78 mm). Length differences in equatorial and polar diameter are negligible, but variation is observed between the two species ( Figure 6). The development of ovules in both the species start with small finger-like projections in the ovary walls. The wild and cultivated species show differences in the development of the ovules. The wild species P. foetida shows a very symmetric and definite distribution of ovules in its ovary. We observed that, P. foetida ovules are larger in size but less in number as compared to cultivated species P. edulis which has smaller ovules but more in number (Figures 7a-c and 8a-c).       Ovule size was calculated as area in ImageJ (Figure 9), which led to the findings that mean ovule area in P. foetida (0.150 ± 0.04 mm 2 ) was markedly greater than P. edulis (0.053 ± 0.016 mm 2 ). On the other hand, cultivated P. edulis shows a higher number of ovules which vary from fruit to fruit are however smaller in size (Figures 7d-f and 8d-f). Ovule count per placenta for P. foetida and P. edulis is two and seven respectively (Figure 9). The differences in sizes of the ovules may be because of faster development of ovules in the P. foetida in contrast to P. edulis which shows a very slow growth rate of its ovules. The larger ovule size of the wild species (P. foetida) is not reflected in its fruit size. J. Zool. Bot. Gard. 2021, 2, FOR PEER REVIEW 9 Ovule size was calculated as area in ImageJ (Figure 9), which led to the findings that mean ovule area in P. foetida (0.150 ± 0.04 mm 2 ) was markedly greater than P. edulis (0.053 ± 0.016 mm 2 ). On the other hand, cultivated P. edulis shows a higher number of ovules which vary from fruit to fruit are however smaller in size (Figures 7d-f and 8d-f). Ovule count per placenta for P. foetida and P. edulis is two and seven respectively (Figure 9). The differences in sizes of the ovules may be because of faster development of ovules in the P. foetida in contrast to P. edulis which shows a very slow growth rate of its ovules. The larger ovule size of the wild species (P. foetida) is not reflected in its fruit size.

Pollen Morphology
The pollen grains of P. foetida and P. edulis showed distinct ornamentation ( Figure  10a-d). However, pollen grains of both species were oblate, spherical in shape with six colpi. The colpi were fused at the ends, hence forming syncolpate apertures. Reticulate exine is observed in both species. The similarity in characters explains the evolutionary conserved nature of the pollens between the two species.

Pollen Morphology
The pollen grains of P. foetida and P. edulis showed distinct ornamentation (Figure 10a-d). However, pollen grains of both species were oblate, spherical in shape with six colpi. The colpi were fused at the ends, hence forming syncolpate apertures. Reticulate exine is observed in both species. The similarity in characters explains the evolutionary conserved nature of the pollens between the two species.

Differences between Wild and Cultivated Species of Passiflora
The present study dealt with two species belonging to the genus Passiflora L. namely, P. foetida L. and P. edulis Sims. The P. foetida is a wild plant that grows abundantly in various terrains [4]. Both species show considerable morphological differences in the leaf shape, flower morphology, the habit of the plant and the fruit size (Figures 1-5). P. foetida has solid stem, tendrils, trifoliate leaves, eye-catching flowers and carries hairs all over the plant body. Leaves are single, alternate and hairy. Flowers have white colour outside and dark pink to violet colour inside. Each bud is covered by three highly reticulated green bracts and they cover the unopened buds and fruits completely [81]. On the other hand, P. edulis has perennial woody habit, tendrils, evenly serrated bracts, shorter corona than the calyx and bright purple, fleshy berry, ovoid or globose fruit. Its leaves are alternate and either horseshoe or 'V' shaped. The flowers are large and showy and the petals are generally pale green and oblong. The corona is composed of two series of filiform filaments [82].

Ovule Development in P. foetida and P. edulis
The ovule development begins with the primordial initial, which later develops to form the ovule [34]. The ovule, developed within the ovary, is protected by integuments and has a stalk called funiculus that attaches the ovule to the ovarian placenta [83,84].

Differences between Wild and Cultivated Species of Passiflora
The present study dealt with two species belonging to the genus Passiflora L. namely, P. foetida L. and P. edulis Sims. The P. foetida is a wild plant that grows abundantly in various terrains [4]. Both species show considerable morphological differences in the leaf shape, flower morphology, the habit of the plant and the fruit size (Figures 1-5). P. foetida has solid stem, tendrils, trifoliate leaves, eye-catching flowers and carries hairs all over the plant body. Leaves are single, alternate and hairy. Flowers have white colour outside and dark pink to violet colour inside. Each bud is covered by three highly reticulated green bracts and they cover the unopened buds and fruits completely [81]. On the other hand, P. edulis has perennial woody habit, tendrils, evenly serrated bracts, shorter corona than the calyx and bright purple, fleshy berry, ovoid or globose fruit. Its leaves are alternate and either horseshoe or 'V' shaped. The flowers are large and showy and the petals are generally pale green and oblong. The corona is composed of two series of filiform filaments [82].

Ovule Development in P. foetida and P. edulis
The ovule development begins with the primordial initial, which later develops to form the ovule [34]. The ovule, developed within the ovary, is protected by integuments and has a stalk called funiculus that attaches the ovule to the ovarian placenta [83,84]. When it attains maturity, the ovule curves inwards and becomes anatropous [34]. When the ovary matures, it becomes the fruit, and the ovules develop into seeds [85]. As observed by Padhye and Deshpande [86], the ovules were anatropic and bitegmic. The studies conducted by various researchers on the ovules of the different plants suggest that the ovule arises as a finger-like projection [87]. The initials of the integuments also arise from the top portion of these projections [34,86]. The results show that the finger-like projections soon develop into ovules covered by two integuments.

Ovule Size and Number between P. foetida and P. edulis
We observed differences in the number and size of the ovules between wild P. foetida and cultivated P. edulis species. The rate of development of ovules was different in both the species and P. foetida had less number of ovules in comparison to P. edulis. The ovary diameter and ovary wall thickness was also different between the two species. The ovary diameter and ovary wall thickness in P. foetida was considerably lower when compared with P. edulis. Differences in the ovule number and size might be due tradeoffs for size and number between the two species [88]. However, it is important to note that the P. edulis which is a cultivated species might have been domesticated for its larger fruit and more number of the seeds inside it. It was surprising to observe bigger ovules inside a tiny fruit in P. foetida when compared with P. edulis. Another reason that explains increased size of the fruits in P. edulis may be due to the number of seeds inside the growing ovary. It has been reported from various species that the number of ovules inside the ovary directly affects the fruit size [44,[89][90][91].

Pollen Morphology
The pollen characters help to identify and distinguish between the different species and help explain the phylogenetic relationships [92]. The pollen of P. foetida and P. edulis had an oblate, spherical shape and were hexacolporate. The hexacolporate structure of the pollen of P. foetida was also observed earlier by Padhye and Deshpande [86]. Our study did not notice much difference in the pollens between the wild (P. foetida) and cultivated (P. edulis) at the morphological level. However, study of pollens is important for further studies focusing on phylogenetic aspects of the family Passifloraceae.

Conclusions
This study is a preliminary investigation of differences in the development of ovules between the wild (P. foetida) and cultivated (P. edulis) species of Passiflora L. Our study provides an important insight on the differential developmental patterns between wild P. foetida and cultivated P. edulis species. The ovule development study shows that the number and size of ovules vary between the two species and this variation may prove to be associated with the mature size and yield related traits in the passion flowers besides various genes and other regulators. Further studies in the future must consider analysing the genetic basis of this variation. The proteome analysis from different species especially between wild and cultivated species of Passiflora at different developmental stages of their fruits will give important insights on proteomic regulation of fruit and ovule development. There are more than 500 species of Passiflora and many of them are edible and orphan crops in various parts of the world. They provide important genetic stocks for the crop improvement programs aimed at improvement of the P. edulis and other domesticated species. Availability of the crop wild relatives is an asset for the crop improvement programs and investigation of the genetic and molecular mechanisms that govern differences in the desirable traits is an important step towards this end. Future studies must be designed to explain the genetic and molecular mechanisms behind the differences in the ovule size, ovule number, and ovary size and ovule density between the wild and cultivated species. The genes from the wild species could be incorporated into commercially beneficial species that mature at a slow rate and the commercial value of such plants can be improved in future.
Author Contributions: A.V. designed and conducted study, participated in data collection, analysis and drafted the manuscript. A.N. analysed the data and prepared the manuscript. A.M.R. participated in the design of the study, data collection and analysis. T.A. analysed and consolidated the data. A.K.K. analysed the data and prepared the draft of the manuscript. N.K. helped in data analysis and visualization. A.K. participated in the design of the study, analysis of data and preparation of manuscript. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement:
The data presented in this study are available on request from corresponding author.