Journal of Science, Engineering and Technology 6:160-168(2018) Southern Leyte State University, Sogod, Southern Leyte, Philippines

Morphological Events on the Development of Flowers, Fruits, and Seeds of Calamansi (XCitrofortunella microcarpa Bunge)

Nikiann G. Mapalo Alma P. Rosillo-Magno Mindoro State College of Agriculture and Technology Victoria, Oriental Mindoro, Philippines

Abstract The development of flowers, fruits, and seeds of calamansi, from initiation to commercial maturity, is described in terms of time intervals. This study provides the morphological ontology of calamansi fruit, a prerequisite for crop breeding program. Sections of the flowers, fruits and seeds were stained with 1% safranin solution and examined under a dissecting microscope. The developmental phases can be grouped into 9, 3 and 4 in floral, fruit and seed, respectively, with distinct morphological events. The time for the flower to reach anthesis from bud formation was 17 to 23 days. The development of the fruit took 78 to 84 days from anthesis to commercial maturity. The development of the fruit from flower bud formation to commercial maturity took 94 to 101 days. During this period, the ovule development became evident on the 7th day from the flower bud formation. On the 59th day, the zygotic embryo became visible. On the 73rd day, the somatic embryo became distinct, and the endosperm tissue became gummy. On the 94th to 101th day, when the fruit reached commercial maturity, the somatic embryos occupy the whole sac and the peel became smooth and shiny. The results imply that cross-pollination can be accomplished before 17 to 23 days from the flower bud formation. An Immature fruit at 59 to 73 days from flower formation is an appropriate fruit-age to obtain the zygote, the nucellus and the endosperm tissues for breeding through plant tissue culture.

Keywords: Anthesis; Calamondin; Embryo; Endosperm; Nucellar tissue

Introduction This shows that calamansi is an emerging important crop. Interest in calamansi is strong in the Korean market (Garcia, 2017). Calamansi (XCitrofortunella microcarpa The plant, which is popular for its acidic Bunge), known as calamondin or Philippine fruit juice with distinct aroma and taste lime belonging to the Rutaceae family, is an (Nilankeeta, 2019), is processed into a indigenous plant of the Philippines. Calamansi beverage, concentrate, and seasoning that is is included in the 12 major fruit crops of the exported to foreign countries like the United Philippines that are cultivated and exported States of America, Hongkong, United Arab (PSA, 2017a). Although fresh calamansi’s Emirates and Saudi Arabia (Espino and contribution to the national revenue generated Espino, 2014). In 2017, the production of less than 0.001 percent share to the country’s calamansi declined from 182.6 thousand export earnings from agricultural products metric tons to 118.2 thousand metric tons (totaled to US$5.28 billion), there is a (https://www.statista.com/statistics/752695/ph 24.68% growth rate in demand (PSA, 2017b).

*Correspondence: [email protected] ISSN 2545-9732 Mapalo and Rosillo-Magno JSET Vol.6, 2018 ilippines-calamansi-production/). The decline randomly. The tags contained information was caused by the decrease in the land area, about the dates of tagging the bud initials, the decrease in the number of bearing trees, commencement of full bloom, and the date smaller fruits produced due to aphids, extreme of anthesis. The development of the tagged heat and delayed fruiting/harvesting in some flower buds was monitored until it reached farms (PSA, 2017a). The improvement commercial fruit maturity or when the fruit peel of a crop stand that is tolerant to biotic became smooth, shiny and light green. (Baniqued, 1998) and abiotic stresses, with improved fruit quality, i.e. seedlessness, requires wider sources of genetic variability Collection of Flowers and Fruits (Khan, 2007). Plant tissue culture provides many options in increasing the genetic variations from the existing variety. There Five flowers were collected every day prior are two methods of increasing genetic to anthesis for flower development. Five variation as a possible source of elite fruits were also collected every week from characteristics: somatic embryogenesis and fruit set until commercial maturation for fruit somatic hybridization. These methods created development. The collected samples were tools for genetic transfer and improvement of placed inside a plastic with labels. These the plant. Furthermore, before performing an were used as experimental materials in in vitro method, one must know the temporal monitoring various phases of morphological morphological development of flowers, fruits, developments during flower and fruit and seeds of calamansi to know the suitable development. growth stage to harvest fruits. Knowledge in the development of seeds for the isolation of zygotic embryo as a source material for Light Microscopy breeding is also important. However, reports on the temporal development of calamansi flowers and seeds are limited. The information Flowers and fruits were characterized based about the approximate time that the different on their distinct morphological events. The floral parts, fruits and seeds develop is still classification of the different organogenic lacking. Hence, the present study aimed to stages was patterned from the report of describe the temporal development of the Dornelas et al. (2007). Fruit characterization floral and fruit parts of calamansi to provide a was based on the report of Tadeo et al. basis on the time to harvest calamansi fruits (2008), who described the phases of fruit for endosperm and nucellar tissue culture growth and the development of citrus fruit. using the in vitro system. These were categorized according to distinct embryo developments. Cross and longitudinal Materials and Methods sections of flowers and fruits were done using a sharp blade to identify their anatomical Plant Material development. The sections were then stained with safranin solution (20 mg safranin: 20 ml The study was conducted from November 6, tap water) for 3 min to increase the contrasts 2017 to February 16, 2018. The calamansi of different parts of the sections. After which, trees used in the experiment were 10 years old the stained specimens were viewed under a bud grafted plants grown in Canaan, Victoria, dissecting stereo microscope (Swift SM101 Oriental Mindoro. Five trees were selected. At stereo microscope). Pictures of the different the middle portion of each tree, 50 flower bud growth stages of flowers and fruits were taken initials exposed to full sunlight were tagged using a digital camera (Sony Cyber-shot).

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Table 1. Stages of floral development in calamansi

Data are means of the five fruits ± standard deviation

Results and Discussion During Stage 4, the stamen primordia became visible 5 days after bud initiation (Fig. Floral structure and morphology 1D), with an average size of 1.90 mm. As it grew, the filaments that bear the anther grew Table 1 and Fig. 1 show the different vertically as the petal expanded (Fig. 1I). developmental stages of the calamansi flower The anthers, about 20 to 22, were yellow and from initiation to anthesis. During Stage 1, oval. The anther started to dehisce in the 3rd the floral meristems were produced by the week of floral development. At Stage 5, carpel inflorescence meristem, following a pattern of primordia became visible in the 6th to the 7th cymose and racemose panicles (Fig.2). The day after floral initiation, with an average bud flowers were borne singly in the leaf axils or size of 2.93mm (Fig. 1E). By Stage 6, or in clusters like in Citrus australasica, in Citrus about 8 days after bud initiation, the locules australis (Clarke and Prakash, 2001) and in formed inside the carpel (Fig. 1F), followed Citrus maxima (Hoque, 2015). Stage 1 was by the emergence of ovule primordia (Stage initially detected in the inflorescence meristem 7, Fig. 1G) that developed on the placenta as a bulge with an average size of 0.19 mm in each locule. At this stage, the ovule is during the 2nd day of development (Fig. 1A). plump fluid-filled, soft and transparent, which Formation of the flower bud was preceded by occurred from 2nd until 4th week after floral sepal primordia (Stage 2), also known as a initiation. The size of the flower at this point button, with an average size of 0.44mm. The was about 3.45mm. sepals took three days to open (Fig. 1B). On Stage 8 (10 days after bud initiation), the It had a dark green color that turned light ovary, stigma, and style became visible (Fig. green as it reached 3.45mm on the 7th day of 1H).At the beginning of Stage 9 (17 to 23 days flower development. As the developing sepal after floral emergence), the floral parts were primordia elongated, it curved inward towards fully developed (Fig. 1I) in which the petal also the center of the flower. Five petal primordia started to fall as the flower reached 13.71mm simultaneously developed at the center of in size. the flower bud on the 4th day (Stage 3) of The white flowers of calamansi are bisexual, development (Fig. 2C). They continuously similar to pummelo (Hoque, 2015), but the elongated until 17 to 23 days. The five petals anthesis of the pummelo is longer (28 to of calamansi flower were ovate and white. 32 days; Hoque, 2015) compared to the

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Figure 1. The calamansi flower at various stages of development. A to C, 1-4 DAFF; D to H, 5-10 DAFF; I, 17-23 DAFF. Abbreviations: (pe) petal, (se) sepal, (sti) stigma, (sty) style, (an) anther, (ova) ovary, (ov) ovule, (lo) locule. DAFF=Days After Flower Formation. Magnification: 40X (A-E), 20X (F-I) calamansi. This is because the period of from the floral bud initiation or 78 to 84 days anthesis varies among the species, with the from the fruit set. climatic conditions of the location, and the position of flower in the shoot (Spiegel-Roy The Phase I (cell division) stage of the fruit & Goldschmidt, 1996). Knowing the time of development was observed 24 to 38 days from anthesis of the specific crop is very important the floral bud initiation, with the fruit attaining to breeders, as selfing and crossing must be a size between 2.38 to 7.17mm. The juice done before anthesis. vesicles were not too plump and dry (Fig. 3B). Although the peel was thick, the separations of the exocarp and mesocarp were not visible Fruit Development yet. The Phase II stage took place during the succeeding 45 to 87 days and constituted Table 2 and Fig. 3 show the different stages a fast period of increase in fruit size (Fig. of calamansi fruit development. The fruits 3E), between 10.22 to 22.45 mm in length attained commercial maturity at 94 to 101 days and 11.08 to 23.97 mm in diameter, mostly

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Figure 2. Cymose (A) and racemose (B) inflorescence in calamansi tree

Table 2. Phases of fruit development in calamansi.

The data are means of the five fruits ± standard deviation by cell enlargement and water accumulation commercial maturity. During this stage, (Tadeo, 2008). This event also synchronized the juice vesicles were transparent, slightly with the increase of exocarp (0.54 to 0.84mm) plump and contained trace amounts of juice and mesocarp (0.40 to 0.73 mm) until 66 (Fig. 3F). The boundary between the exocarp days, then both tissues decreased (0.63 to and mesocarp were already visible. The 0.47mm in exocarp and 0.55 to 0.32 mm in exocarp, which was about 10% thicker than mesocarp) from 73 days until the fruit reached the mesocarp, comprised of cuticle-covered

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Figure 3. Fruit development in calamansi from Phase I (A, 24th -38th day), Phase II (C, 45th -87th day) and to Phase III (E, 94th -101 day). Longitudinal section of calamansi fruit in Phase I (B) and a cross-section of fruit in Phase II (D) and Phase III (F). Abbreviations: (ca) carpel, (cal) calyx, (car) carpel wall, (ce) central core, (ex) exocarp, (ju) juice vesicles, (lo) locule, (me) mesocarp, (pe) pedicel, (ped) pedicel extremity, (pl) placenta, (re) remains of style, (se) seed

Table 3. Stages of ovule development in calamansi

Data are means of five calamansi flower or fruits ± standard deviations epidermis, while parenchyma cells comprised took 94 to 101 days to reach commercial the outermost tissue layers (Bain, 2008). The maturity. During the cease growth, juice exocarp or flavedo of the the calamansi fruit vesicles were yellow to orange and fully plump was green, while the mesocarp or albedo was compared to the previous stage (Fig. 3J). white, and both are spongy in texture. At this stage, as the fruit matured, its peel became shiny and thinner. At Phase III (final stage, Fig. 3I), the fruit

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Figure 4. Ovule development of the calamansi. The ovule was evident (A, 7th day from flower bud formation). The zygotic embryo became visible (B, 59th day) followed by somatic embryo (C, 73rd days). Mature seed (D, 94th day). Abbreviations: (ov) ovule (nu), nucellar tissue, (zy) zygotic embryo, (en) endosperm, (em) embryonic axis, (so) somatic embryo, (co) cotyledon of somatic embryo. Magnification:20X

Ovule Development stage was about 19.92mm. At this stage the endosperm became gummy. Finally in Stage 4 which took 94 days, the seeds were already Table 3 and Fig. 4 show the different stages hard and each part was fully developed and of ovule development. In Stage I (Fig. 4A), or visible (Fig. 4D). The nucellar tissue had about 7th days of floral development, with bud degenerated. The fruit size during this stage a size of 8.98mm, the ovule developed, which was about 23.84mm. was plump, transparent and soft in texture. In Stage 2, the zygotic embryo which This study obtained similar observations emerged within 59 days (Fig. 4B) was green with Avenido et al. (1991), wherein and smaller than the somatic embryo (Fig 4C). they used 31 to 52 days-old immature During this stage, the fruit size was 14.14mm. fruit (after anthesis) or 58-65 days after In Stage 3, the somatic embryo emerged pollination (Dimaculangan et al., 1994) for the within 73 days (Fig. 4C), which evolved from establishment of in vitro culture of calamansi nucellar tissue that is genetically identical to plants from nucellus and endosperm tissue. the parent plant (Hamilton et al., 2007). It Although the results are comparable, there is was white and bigger than the zygotic embryo still a need to find the most suitable stage of as it developed. The fruit size during this endosperm development for in vitro response

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(Gmitter et al., 1990). Dornelas, M., Rodriguez, A., & Gouve, C. (2007). Floral development in Conclusion the tribe Cedreleae (Meliaceae, Subfamily Swietenioideae): Cedrela The time of calamansi flower initiation to and Toona. Retrieved from anthesis took 17 to 23 days to develop. This www.aob.oxfordjournals.org.on implies that selfing or cross-pollination can be done before this time. For in vitro culture, Dimaculangan , J.G., Avenido, R.A., the zygotic embryo, endosperm, and nucellus Patena, L.F. & Barba, R.C. (1994). tissue can be isolated from 59 to 73 days-old Cytological investigation of endosperm immature fruit from flower initiation. explant and long-term maintenance of endosperm-derived calli of calamansi (XCitrofortunella mitis J. Ingram and H. Acknowledgment Moore). In: Transactions of the National Academy of Science and Technology. The authors extend their gratitude to MinSCAT Metro Manila: National Academy of for the research grant. Science and Technology, Bicutan, Taguig.

Espino, R. C., & Espino, M. C. (2014). References Cited The Status of the fruit industry in the Philippines. Retrieved 2017 from Avenido, R.A., Zamora, A. B., Patena, L.F. http://www.fftc.agnet.org & Barba, R.C. (1991). Callus induction and plant regeneration in calamansi Garcia, CRA. Nov 20, 2017. Calamansi is hot (XCitrogortunella mitis J. Ingram & HE in Seoul, but can local suppliers deliver? Moore). Philippine Agriculturist. Business World. Retrieved 2018 from https://www.bworldonline.com/calamansi- Bain, J.M. (2008). Morphological, anatomical, hot-seoul-can-local-suppliers-deliver/ and physiological changes in the developing fruit of Valencia orange Gmitter, F.G. Jr, Ling, X.B. & Deng, X.X. (Citrus sinensis L. Osbeck). Australian (1990). Induction of triploid citrus plants Journal of Botany, 6, 1–25. from endosperm calli in vitro. Theoretical and Applied Genetics, 80(60), 785-790. Baniqued, C.A. (1998). Status of disease management of citrus in the Philippines. Hamilton, K., Ashmore, S., Drew, R. & Focus in the Cordillera. In: Proceedings Pritchard, H. (2007). Seed morphology of a regional workshop on disease and ultrastructure in Citrus garrawayi management of banana and citrus through (Rutaceae) in relation to germinability. the use of disease-free planting materials. Australian Journal of Botany, 55(6), Davao City: Philippines. Editors: AB 618-627. Retrieved 2018 from Molina, VN Roa, J Bay-Peterson, AT http://www.publish.csiro.au/bt Carpio and JEA Joven. Hoque, M.A. (2015). Floral biology of Clarke, K. & Prakash, N. (2001). Floral indigenous pummelo genotypes. morphology and embryology of two Bangladesh Journal of Agricultural Australian species of Citrus (Rutaceae). Research, 40(2), 177-188. Australian Journal of Botany, 49, 199-207.

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Khan, I.A. (2007). Citrus genetics, breeding, and biotechnology. UK & USA: CAB International. Nilankeeta. (2019). [Blog] How to use calamansi for beautiful skin? What are the benefits and side effects? Retrieved from https://www.beautyglimpse.com/calamans i-calamondin- use- beautiful-skin/

Philippine Statistics Authority (PSA), April-June 2017a. Major Fruit Crops Quarterly Bulletin. Retrieved from https://psa.gov.ph/sites/default/files/Fruitcr ops 0.pdf

Philippine Statistics Authority (PSA). September 2017b. Agricultural Exports and Imports, Report No. 2017-4. Agricultural Indicators System.

Tadeo, F.R., Cercos,´ M., Colmenero-Flores, J.M., Iglesias, D.J., Naranjo, M.A., R´ıos, G., Carrera, E., Ruiz-Rivero, O., Lliso, I., Morillon, R., & Talon, M. (2008). Molecular physiology of development and quality of citrus. Advances in Botanical Research, 49, 147-223

The Statistics Portal. Calamansi production in the Philippines from 2011 to 2016. Retrieved from https://www.statista.com/statistics/752695 /philippines-calamansi-production/

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