Morphological Characterization of a New Biotype Moringa of Saudi Arabia

Dari total 13 spesies Moringa, di Saudi Arabia hanya terdapat dua spesies, yaitu M. peregrina dan M. oleifera. Kedua spesies ini memiliki kandungan nutrisi dan bahan obat yang tinggi serta mampu bertahan pada iklim kering. Biotipe Moringa baru yang berbeda dengan kedua spesies ini telah teramati di daerah Al Bahah, Saudi Arabia. Pada penelitian ini, karakterisasi dari biotipe baru ini telah dilakukan dengan mengamati 22 karakter kualitatif dan kuantitatif dari daun, biji dan bunga. Semua karakter ini kemudian dibandingkan dengan M. peregrina dan M. oleifera untuk mengetahui hubungan biotipe baru dengan kedua spesies. Hasil pengamatan pada karakter kualitatif menunjukan bahwa biotipe baru ini memiliki 8 karakter (tepi anak daun, warna bunga, bentuk kelopak dan mahkota bunga, perlekatan kelopak bunga, warna dan orientasi kepala sari, dan bentuk dasar bunga) yang sama dengan kedua spesies, satu karakter (distribusi warna merah pada perhiasan bunga) dengan M. peregrina saja, tiga karakter (tipe susunan anak daun, kulit biji dan sayap biji) dengan M. oleifera saja. Untuk karakterkarakter kuantitatif, hasil pengukuran menunjukan semua karakter pada biotipe baru berada pada posisi antara M. peregrina and M. oleifera. Berdasarkan hasil ini, kami menyimpulkan bahwa biotipe baru dari Moringa ini adalah hibrid hasil persilangan M. peregrina dan M. oleifera.


INTRODUCTION
Moringa is the only genus of the Moringaceae family.With just 13 species, the genus is one of the most morphologically varied groups of angiosperms.It ranges from very small tuberous shrubs to vast bottle trees and from radial to bilateral floral symmetry (Olson, 2002).All the species are originated from India and Africa (Amaglo, 2010), but then introduced into many tropical and subtropical countries as food, medicinal or ornamental plants.In Saudi Arabia, Migahid (1978) reported that only two species of Moringa are present i.e.M. oleifera and M. peregrina.Due to its edible leaves and high quality seed oil, M. oleifera is widely cultivated in the country.For M. peregrina, the species is not as famous as the former and is naturally distributed in South and North Hijaz.
Due to their conventional trunk and fibrous roots, M. oleifera and M. peregrina are classified within slender trees class of Moringaceae (Olson, 2002).Both species are tree with irregular flower, hairy ovary and smaller pollen grains.According to Olson (2002), the main morphological differences between M. oleifera and M. peregrina are that the former has winged seeds and deciduous  leaf axes whereas the latter has unwinged seeds and their leaf axes are persistent.
In this study we aim to characterize a new biotype Moringa observed in Al Bahah Region, Saudi Arabia.The new biotype has both M. oleifera and M. peregrina characteristic in its leaflets and seeds.Its leaflets shapes are round, elliptic or oblanceolate, which are combination between leaflets shapes of M. oleifera (round) and M. peregrina (linear, elliptic or oblanceolate).In addition, while having similar size to those of M. peregrina, its seeds bear wings like those of M. oleifera.
As both M. oleifera and M. peregrina are known as the source of nutritious leaf and fruit vegetables, high-quality seed oil and pharmacologically active compounds (Tsaknis, 1998;Saleem and Meinwald, 2000;Jahn, 2001;Padayachee and Baijnath, 2012), detection of genetic diversity is of great value for the improvement of nutritional and medicinal value of these plants.Morphological variation is one indication of genetic variation and may serve as a basis for more detailed molecular studies.Thus in the present study, we morphologically characterized the new biotype Moringa by measuring its leaf, flower and seed traits and comparing them with of M. oleifera and M. peregrina to determine its relation with both species.

Sample preparation and traits measurement
All leafs, seeds and flowers of the new biotype, M. oleifera and M. peregrina were collected from Al Bahah Region, Saudi Arabia, where the new biotype was observed (19°45'14.0"N41°27'28.2"E).
For data presentation simplification, all measured traits were divided into qualitative and quantitative traits (Table 1).These traits are key morphological characteristics of Moringa which can be used to differentiate between species (Olson, 2002;Mgendi et al., 2011).For leaf traits measurement, a total of 12 individuals of the new biotype, 11 individuals of M. peregrina and 8 individuals of M. oleifera were examined in this study.From each individual, one leaf which fully exposed to sun light was selected and photographed.From these photographs, leaflet length and width, leaf area and petiole length were measured using ImageJ software (Schneider et al., 2012).Specific leaf area (SLA) of leaves was measured by dividing leaf area by its dried weight (mm 2 /mg).To get constant dried weight of the leaves, the samples were put in the oven at 70° C for 24 hours.For seed traits measurement, a total of 38, 45 and 37 seeds were randomly selected from the new biotype, M. peregrina, and M. oleifera, respectively.Similar to the leaves, seed surface area was measured using ImageJ software.As not all examined individuals produced flower during the study, minimum of 2 flowers were examined for flower traits measurement.

Data analysis
ANOVA one way was used to compare all quantitative traits between the biotype, M. peregrina and M. oleifera.The analyses were performed using PASW Statistic 18, release version 18.0.0(SPSS Inc., 2009, www.spss.com).

RESULTS AND DISCUSSION
Most of the qualitative traits of the new biotype were similar to M. peregrina and M. oleifera.These similarities were observed for leaflets margin, flower color, petals and sepals shape, sepal fusion, anther color and orientation, and receptacle shape (Table 2 and Figure 1).All three studied samples had entire leaflet margin, white flower, unequal sepals and petals, fussed sepals, orange and centrally-pointed anther, and long tubular receptacle.Three traits of the new biotype namely pinnate type, seed cover and seed wing resembled M. oleifera (2 or 3-pinnate leaf, membranous seed cover, and winged seed), while only one trait i.e. distribution of red pigmentation on perianth resembled M. peregrina.Furthermore, leaflet shapes of the new biotype (round, elliptic or oblanceolate) were the only trait observed to have mixed characteristic of both M. peregrina and M. oleifera (Table 2 and Figure 1).
The examination of qualitative morphological traits revealed that the new biotype shared eight characters (leaflets margin, flower color, petals and sepals shape, sepal fusion, anther color and orientation, and receptacle shape) with both M. peregrina and M. oleifera, one character (distribution of red pigmentation on perianth) with M. peregrina only, three characters (pinnate type, seed cover and seed wing) with M. oleifera, and none of unshared character.For quantitative traits, all measured characters of the new biotype showed intermediate position between M. peregrina and M. oleifera.Based on these results, we suggested that the new biotype is a hybrid between M. peregrina and M. oleifera.Previous studies on plant hybrid identification and characterization showed that most hybrids occupied intermediate position between their parental species, and were additive in their characters which reflected the contribution of both parental species characters (e.g.Takamiya et al., 1999;Horandl & Greilhuber, 2002;Rieseberg et al., 2003;Lin et al., 2010;Hoyo & Tsuyuzaki, 2013;Hodac et al., 2014).Further studies using codominant and highly polymorphic molecular markers are needed to test our conclusion.For this purpose, we suggest to use simple sequence repeat (SSR) and single nucleotide polymorphism (SNP).With simple cross-amplification, common loci can be selected and hybridization between involved Moringa species could be analyzed.

CONCLUSION
Using 22 qualitative and quantitative morphological markers, the present study was able to characterize the new biotype Moringa of Saudi Arabia.Due to the high number of shared character with both M. peregrina and M. oleifera, we suggested that the new biotype is a hybrid between the two species.The results of the present study may serve as a basis for future studies on genetic diversity of Moringa in Saudi Arabia.

Figure 2 .
Figure 1 Comparison of leaf, seed and flower of M. peregrina, M. oleifera and the new biotype.Black lines on photos are1 cm scale

Figure 3 .
Figure 3.Comparison of leaf area, specific leaf area (SLA) and petiole length between Moringa peregrina, the new biotype and Moringa oleifera.Bars with line above them are mean values and their standard deviation and n is number of sample.Different colors of bars indicate statistically significant difference (p < 0.0001).

Figure 4 .
Figure 4. Comparison of seeds weight and weight to surface area ratio between Moringa peregrina, the new biotype and Moringa oleifera.Bars with line above them are mean values and their standard deviation and n is number of sample.Different colors of bars indicate statistically significant difference (p < 0.0001).

Table 1 .
List of leaf, seed and flower traits examined to characterize the new biotype.

Table 2 .
Comparison of leaf, seed and flower characteristics between M. peregrina, the new biotype and M. oleifera.