Cytological Observations and In Vitro Germination of Nipa ......of Nipa (Nypa fruticans Wurmb.,...

Keywords boron microspores microsporocyte Nypa fruticans pollen staminate rachilla sucrose

Cytological Observations and In Vitro Germination of Nipa (Nypa fruticans Wurmb Arecaceae) Pollen

Corresponding Author jamantiquillaupeduph sectFormerly Affiliated with DBSES CSM UP Mindanao

1Department of Biological Sciences and Environmental Studies (DBSES) College of Science and Mathematics (CSM) University of the Philippines (UP)

Mindanao Mintal Davao City 8022 Philippines2Davao Doctorrsquos College Gen Malvar St Davao City 8000 Philippines

The staminate rachillae from laterals of nipa (Nypa fruticans Wurmb) at different stages of inflorescence development were sampled for cytological observations using squash technique with 2 acetocarmine as the stain Results showed that the dividing microsporocytes exhibited a tetrad in tetragonal shape enclosed in thick callose walls at the end of meiosis II The microspore products consisted not only of uninucleate but also of binucleate and trinucleate pollen during anthesis Meiotic stages were interpreted as highly normal which suggests high pollen viability In vitro germination observed every 4-h intervals for 24 h showed that sucrose level at 15 attained significantly highest germination at 53 The germinated pollen developed a knob protrusion instead of an elongated pollen tube There was a significant difference in pollen knob length at 15 sucrose measuring 96 microm the longest observed compared to the control With significant interaction between sucrose and boron levels 10 and 15 sucrose levels achieved higher in vitro germination when combined with 001 and 0005 boron respectively Indeed sucrose and boric acid consisting of the liquid medium can initiate nipa pollen germination confirming earlier results with lower concentrations

Philippine Journal of Science149 (3-a) 719-730 October 2020ISSN 0031 - 7683Date Received 07 Apr 2020

Junaldo A Mantiquilla1 Keren Mnhz B Chispa1 Salsabel A Ladjahassan1 and Reynaldo G Abad2sect

INTRODUCTIONLocally known as rdquonipardquo Nypa fruticans Wurmb belongs to the family Arecaceae It is monotypic ndash being the only known species of its genus Nypa ndash under subfamily Nypoideae which is isolated from other palms (Chan 2003 Uhl and Dransfield 1987) owing to its distinctive characteristics

This palm adapts to tidal estuarine habitats which is a unique trait in the family (Dowe 2010) Its distribution ranges from Sri Lanka the Ganges Delta the Malay

Peninsula and archipelago and Pacific Islands from northern Australia to the Solomons Philippines and the Ryukyus in Japan ndash making tropical conditions essential for its growth (Corner 1966 Janick and Paull 2008 Tomascik et al 1997 Tomlinson 1961 1971 Uhl 1972) It tolerates a wide range of environmental conditions in the intertidal zone from brackish water and extends upstream to permanent freshwater areas where seeds can be carried and deposited by water level fluctuations (Chan 2003 Cushion et al 2010 Joshi et al 2006) This is a general indication of a strong influence of freshwater where it can also withstand waterlogged substrates extreme floods and high concentrations of salt The saltwater tides are vital in

719

dispersing seeds and depositing silt (Janick and Paull 2008 Joshi et al 2006 MacNae 1968 Tomascik et al 1997)

Nypa fruticans is characterized by a rhizomatous habit of its dichotomously branching stem that is 30 cm wide to 15 cm thick and often remains underground (Dowe 2010 Janick and Paull 2008) Each of branching stem holds less than of a dozen upright pinnate leaves that can usually reach a length of 5ndash9 m and numerous lanceolate leaflets that are 12ndash15 m long (Janick and Paull 2008) Monoecious flowers are borne in a 1ndash2-m long spadix-like inflorescence that is axillary The laterals bear the terminal catkin-like rachillae where the staminate flowers are produced and the terminal globose head structure of the main axis consists of pistillate flowers that are larger and positioned below the staminate rachillae after anthesis (Chan 2003 Dowe 2010 Janick and Paull 2008)

Staminate rachillae which measure 6ndash9 cm long support the short dense spikes of staminate flowers that are yellow-orange (Dassanayake and Clayton 2000 Dowe 2010) The staminate flowers are arranged in rows usually 21 rows per spike Each flower is composed of perianth parts and a large stalk at its center which gives forth to longitudinally positioned anthers Perianth parts are arranged in two whorls The androecium forms from three stamens that fused into a column of about 4ndash6 cm in length and no pistillodes are present There are three ndash sometimes four ndash anthers that reach a length of 2 mm each containing four sporangia with conniving half-anthers near the tip (Stone 1970 Uhl 1972 Dassanayake and Clayton 2000 Dowe 2010)

The pollen of extant N fruticans is unique among the palm family and has little chance of being mistaken for pollen of other taxa except for the fossil nipa pollen (Germeraad et al 1968 Tschudy and Van Loenen 1970 Kedves 1980) Its pollen grain is classified according to aperture as zonasulcate with oblate spheroidal to subprolate shape The sulcus furrow in pollen surface is 120ndash180 microm in width the meridian is ring-like while the membrane is psilate Ornamentation is echinate while the interspinal area is perforate or some reticulate (Chumchim and Khunwasi 2011) Completely zonasulcate aperture in Arecaceae is known only in the Calamoideae and the Nypoideae The spiny exine along with zonasulcate aperture and large size makes the pollen of nipa highly distinctive (Harley and Baker 2001)

Good fruit-set and high crop yield depend largely on viable pollen which is often determined by germination (Opute 1975 Lwin 2010) Besides being used for measuring male fertility to monitor stored pollen (Oliveira et al 2001 Soares et al 2013) pollen viability generally indicates the ability of the pollen grain to perform its function by delivering the sperm cells to the embryo sac

following compatible pollination (Shivanna et al 1991 Wang et al 2004) One approach to estimate pollen viability is by employing in vitro germination assay This method is used to germinate a small sample of pollen in an appropriate culture medium to simulate the stigma environment It provides a balanced environment for the development of the pollen tubes by observing the percentage of pollen grains that produced them after a certain period The composition sucrose concentration and pH of the medium are among the factors that affect pollen germination (De Franca et al 2009)

In the study conducted by Mortazavi et al (2010) on the in vitro germination of the date palm the germinated pollen count was highest at the medium containing 15 sucrose 002 calcium nitrate and 0005 boric acid Sugar is utilized as an energy source for the synthesis of cell wall materials such as pectins cellulose and callose The sucrose in the medium maintains osmotic pressure and provides nutrition to germinating pollen (Nair 1985 Soni et al 2010) During in vitro germination boron is added in the form of boric acid for most species as it directly involves in pectin synthesis and thus indirectly with the development of pollen tube membrane (Holdaway-Clarke and Hepler 2003 Stanley and Loewus 1964) It affects H+-ATPase activity that initiates pollen germination and tube growth (Obermeyer and Blatt 1996) ndash its absence in the culture medium often leads to pollen tube bursting (Acar et al 2010) Sucrose in combination with boric acid promotes pollen germination as well as tube development since boron makes a complex with sugar This sugar-borate complex is capable of translocation of sugar (Gauch and Dugger 1953)

An initial study conducted on in vitro germination of nipa showed no pollen tube growth but pollen broke open by the aperture thus releasing its content This was observed across 2ndash10 sucrose and germination time (1ndash4 h) by germinating pollen grains using oil palm and date palm culture media It is composed of 50 mL distilled water 10 g sucrose and 001 g boric acid (Mantiquilla et al 2018) Given that the germination medium can dramatically affect pollen metabolism (Taylor and Hepler 1997) finding the optimal medium has to be explored further

This study was conducted to characterize the pollen development of N fruticans Wurmb cytologically through acetocarmine squash technique and describe nipa pollen during in vitro germination This study aims to

1 assess which phase in the inflorescence development where cell division occurs in staminate rachilla

2 analyze meiosis of N fruticans during microsporogenesis

3 estimate optimum levels of sucrose and boron concentration for in vitro germination of pollen and

Mantiquilla et al Cytology of Pollen Development of Nypa fruticans Wurmb

Philippine Journal of ScienceVol 149 No 3-a October 2020

720

4 evaluate in vitro pollen germination every 4-h interval for 24 h

MATERIALS AND METHODS

SamplingThe study was conducted from June 2015 to April 2017 Samples were collected from the semi-wild stands in Bago Aplaya Davao City Philippines (Figure 1) for field observations of the male phase Staminate rachillae were obtained from different regions and stages of the inflorescence to identify where cell division occurs The male flowers were fixed in a freshly prepared Farmerrsquos fixative solution (3 parts absolute ethyl alcohol to 1 part 100 glacial acetic acid) for 24 h at 4 degC After this the materials were transferred and maintained in 70 ethyl alcohol at 4 degC until further use

For pollen collection the harvesting of staminate rachillae from nipa inflorescence was conducted at dawn between 500 to 600 AM before insect activities affect dehisced pollen Pollen samples were collected by tapping and brushing off from the spike and were kept oven-dried at 40 degC (Mantiquilla et al 2018) The experiment was conducted at the Plant Science Laboratory UP Mindanao Mintal Davao City

Acetocarmine Squash TechniqueThe anthers were gently scraped and tapped using a bent needle with a drop of 2 acetocarmine The prepared

slides were then passed over a flame and repeated tapping was made as needed to improve the spread of fixed cells Samples were magnified either at HPO (400x) or OIO (1000x) for documentation

Pollen GerminationThe liquid culture medium for in vitro germination test was based on the modified Brewbaker and Kwack medium for date palm consisting of 15 of sucrose 05 g of boric acid (H3BO3) 03 g of calcium nitrate [Ca(NO3)24H2O] 02 g of magnesium sulfate (MgSO4) and 01 g of potassium nitrate (KNO3) dissolved in 1 L of distilled water (Zaid and de Wet 2002) The pH level of the medium was recorded

This stock solution was added with the following levels of sucrose (0 5 10 15 and 20) and boron (0 0005 and 001) as treatments Ten (10) mg of pollen grains were added to a Petri dish containing 10 mL of the germination medium observed in 4-h intervals for 24 h The emergence of pollen knob was considered as an indicator of pollen germination

Three random microscopic fields under high power objective (400x) were used to measure pollen grains under photomicroscope in three slides as replicates Percentage germination was obtained using this formula [adapted from Lwin (2010) and Mortazavi et al (2010)]

Germination=

Number of germinated pollen grains per field of view

percentage () Total number of pollen grains per field of view

Figure 1 Map of the sampling site (arrowhead) in Bago Aplaya Davao City (7deg221 N 125deg3203 E) in southern Philippines (Source Google Maps)



721

Pollen MeasurementThe mean pollen diameter was measured from 63 random samples using the Motic photomicroscope software excluding the exine layer (Figure 2) It was determined by measuring the length parallel to the aperture of the pollen for uniformity For the germinated pollen with a knob it was measured at the point of emergence up to its tip

RESULTS AND DISCUSSION

Cytological ObservationThe male spikes or staminate rachillae varied from white to yellow and so the anthers do which also corresponds to their development stages Yellow anthers (Figure 3A) were observed to contain mature pollen grains while tetrads dividing cells and pollen mother cells (PMCs) were observed in white anthers In addition microsporocytes contained within the anthers of an individual staminate flower were of the same developmental stage Prior to male anthesis it was found out that meiosis had already started due to the presence of tetrads This is contrary to what Bhowmick and Bose (2011) reported that it is usually during anthesis where the meiotic cell division of the PMCs are observed Mature pollen grains are generally observed in the upper laterals during the male phase Since nipa follows a basipetal development dividing microsporocytes are expected in the middle laterals while PMCs are abundant in lower laterals of the inflorescence

PMCs or microsporocytes were spheroid or ovoid when they are about to undergo meiosis Figure 5 shows the stages that occur during meiosis I Particularly at prophase I the chromosomes at the leptotene stage appear to be exceptionally long and thin (Figure 4A) It is in this phase where chromosomes start to contract and continually do so throughout the entire prophase I (Griffiths et al 2012) Figure 4B shows the pachytene stage Chromosomes appeared to stain deeper and the individuality of some of the paired chromosomes can be distinguished The

Figure 2 Screenshot of Nypa fruticans Wurmb (Arecaceae) pollen grain measured using Motic photomicroscope software (magnification 400x)

Figure 3 The mature male inflorescence of nipa (Nypa fruticans Wurmb Arecaceae) A) staminate rachilla (male spike) B) longitudinally positioned anthers C) pollen inside the anther (40x)

Experimental Design and Statistical AnalysisThe experiment was laid out in a factorial completely randomized design The data was analyzed using DSAASTAT Ver 1101 (Onofri 2006) Least significant difference was employed to compare treatment means



722

nucleolus was still prominent at this stage with some chromosomes attached to it The attached chromosome can likely be a satellite chromosome (Schulz-Schaeffer 1980) The pairing of some of the chromosomes is seen during diplonema where they appeared denser and contracted in Figure 4C

While nucleolus can be observed as very prominent and deeply stained at prophase I it disappeared at the end of this stage At metaphase I most of the chromosomes in the equatorial plate without the nuclear membrane were rod-shaped (Figure 4D) Chromosomes moving to opposite poles ensued at anaphase I where the separation of homologs in all the cells examined showed no anomalies (Figure 4E) This is then followed by the first telophasic transformation in which two groups of chromosomes organize to form two nuclei at the end of the first meiotic division Based on the appearance of a dyad in Figure 4F the plate formation of nipa follows conventional cytokinesis called a successive type This means that cell plate in the meiocytersquos midzone grows alongside a centrifugal cell wall expansion which is typical among

monocotyledonous PMCs (De Storme and Geelen 2013)

At prophase II two groups of chromosomes were observed to have their respective nuclear envelopes broken down (Figure 5A) and then migrated at the equatorial plate during metaphase II (Figure 5B) At anaphase II four groups of chromosomes migrate towards opposite poles (Figure 5C) Each of these groups organized to form tetrad or four nuclei stage PMCs at telophase II (Figure 5D) For nipa the resulting tetrad of haploid microspores at the end of microsporogenesis is classified as tetragonal (Figure 5D) This shape is one of the results of the successive type of cytokinesis where every nuclear division is always accompanied by cell plate formation (De Storme and Geelen 2013)

The microspore tetrad is enclosed in a callose wall separating them from each other and from the other surrounding cells (Figures 6A and 6B) It becomes a thick wall due to the continuous deposition of callose in between the primary cell wall and cell membrane during meiotic divisions However it is temporary and may have

Figure 4 Meiosis I of Nypa fruticans microsporogenesis stained with 2 acetocarmine (magnification 1000x) with prophase I substages [A) leptotene B) pachytene C) diplotene] and the succeeding stages [D) metaphase I E) anaphase I and F) telophase I] Legend ch ndash chromosome nu ndash nucleolus chn ndash chromosomes attached to nucleolus h ndash homolog



723

many different roles It is believed that the formation of a temporary callose wall prevents cell cohesion and fusion prior to microspores release into the locular space upon its degradation (Waterkeyn 1962) It also functions as a molecular filter that protects the developing microspores from the influence of the surrounding diploid tissues (Heslop-Harrison and Mackenzie 1967) As a physical

barrier it prevents the premature swelling and bursting of the microspores The callose wall also acts as a mold wherein the primexine provides a guide for the formation of the exine pattern on the mature pollen grain from the pollen wall (Waterkeyn and Beinfait 1970 Stanley and Linskens 1974 Zhang et al 2002)

Figure 5 Meiosis II of Nypa fruticans microsporogenesis stained with 2 acetocarmine (magnification 1000x) A) prophase II B) metaphase II C) anaphase II and D) telophase II (chromosomes under karyokinesis arrowheads)



724

Each tetrad will eventually give rise to an immature pollen In Figure 6C the four microspores dissociated from the surrounding callose matrix through the enzyme callase that degrades the callose and releases the microspores into the locular space (Dong et al 2005) The free microspore becomes more spherical in shape with thick even cell wall According to Perera (2003) the spherical shape of the cells is due to vacuolation and the dense cytoplasm The cells then developed into uninucleate pollen with clear cytoplasm and prominent nucleus (Figure 6D) then it becomes binucleate pollen with one cell slightly bigger (vegetative cell) than the other (generative cell) (Figure 6E) and finally into trinucleate pollen with two cells smaller than the other (Figure 6F)

It was observed that binucleate pollen cells were more frequent compared to other types of pollen cells ie nearly 60 of the total number of cells observed (Table 1) These cells were observed during the anthesis of the laterals in the middle region of the inflorescence The duration of the male phase takes much longer than female receptivity ensuring pollen source for cross-pollination along with high viability of nipa pollen (Mantiquilla et al 2018) Trinucleate pollens germinate and elongate at a faster rate but have shorter viability as compared to binucleate pollens that germinate slower but have higher viability (Devrnja 2012) They are also more adapted in conditions where temperature slightly increase and seeds are dispersed via wind and water accelerating the female development and the whole reproductive process

Table 1 Percent pollen type undergoing microgametogenesis observed during the anthesis of staminate rachillae from the middle laterals

Pollen cell typePercent per random field

Mean percent Total count1 2 3 4

Uninucleate 191 200 80 200 168 15

Binucleate 571 650 600 560 595 54

Trinucleate 238 150 320 240 237 22

Figure 6 Developing pollen of Nypa fruticans A) tetrad stained with 2 acetocarmine (B) tetrad enclosed in two callose walls (magnification 1000x) C) dissociating microspores D) uninucleate pollen E) binucleate pollen F) trinucleate pollen (magnification 400x) Legend cw ndash thick callose wall nu ndash nucleus vc ndash vegetative cell gc ndash generative cell



725

(Sanzol and Herrero 2001) Binucleate pollen on the other hand can survive longer and can still be available for the pollinating insects during female anthesis (Gottsberger 1989) Since nipa is outcrossing by protandry and entomophily (Mantiquilla et al 2016) the presence of both binucleate and trinucleate pollens at anthesis can be an adaptive strategy for a higher chance of fertilization to occur (Lora et al 2009)

In Vitro Germination TestFaiz et al (2011) described nipa pollen as yellow and circular with zonasulcate aperture and spiky projections of uniform length that comprise the exine layer According to Henny (1977) for pollen germination to occur it is expected to develop a pollen tube where its length is the same or greater than its diameter However initial observations conducted by Mantiquilla et al (2018) through in vitro pollen germination showed that nipa pollen did not produce an elongated pollen tube Instead it split open to release its content using the liquid culture media of oil palm (Elaeis guineensis Jacq) and date palm (Phoenix dactylifera) Again the results of this follow-up study described it as a knob that emerged by the pollen aperture Albeit not elongated the presence of the granular substance and a clear zone at its extreme tip make this knob pollen tube-like as shown in Figure 7

Statistical results showed that using 15 sucrose attained a significant difference to a high of 53 germination compared to control (no sucrose) (Table 2) However this level of sucrose did not vary with 5 and 10 by obtaining nearly 50 and 49 germination respectively In nature water sugar and amino acids are supplied by the

style to nourish the growing pollen tube (Richards 1986) The sticky substances of the stigma also supplied sugar to the growing pollen tube Sugar can regulate the osmotic potential in pollen tube growth It also serves as a source of nutrients and energy At suitable sucrose concentration the balance between the internal and external osmotic pressures of pollen can be maintained thereby preserving the normal vitality of pollen (Zhang and Huang 2009)

The presence of boron attained mean germination between 38ndash41 Nevertheless it significantly increased germination from 60 to as high as 69 by adding 0005 and 001 depending on sucrose concentration in the medium In nature boron is provided by the stigma and style It facilitates sugar uptake and has a role in pectin production in the pollen tube It can also promote pollen germination and help the pollen tube rapidly enter the ovary (Richards 1986) Stanley and Loewus (1964) indicated that boron is directly involved in pectin synthesis and thus indirectly involved in the development of the pollen tube membrane It affects H+-ATPase activity that initiates pollen germination and tube growth (Obermeyer and Blatt 1996)

As shown in Table 2 sucrose at 10 added with 001 boron significantly increased germination to 60 This was increased further to 69 when 15 sucrose was combined with 0005 boron With these significant effects between the interaction of sucrose and boron sucrose clearly played a primary role in germination as enhanced by boron It appears that 10ndash15 sucrose favored higher germination At the lower limit it needs 001 boron while at the upper limit with 0005

Figure 7 Germinated pollen of Nypa fruticans Wurmb (Arecaceae) under 15 sucrose at different time intervals a) 0 h b) 4 h c) 8 h d) 12 h e) 16 h f) 20 h and g) 24 h Legend ap ndash zonasulcate aperture kn ndash knob protrusion (magnification 400x)



726

Table 4 Germination pollen diameter and knob length of Nypa fruticans Wurmb (Arecaceae) measured at regular 4-h intervals

Germinated pollen measurementTime intervals (h)z

0 4 8 12 16 20 24

Germination () 2630a 2870a 3204b 4944bc 3648cd 4074d 6000c

Pollen diameter (microm) 2361a 2275b 2202c 2195c 2199c 2198c 2217c

Knob length (microm) 60c 59c 67bc 89a 80b 86ab 112a

zTreatments having similar letter(s) within a row are not significantly different at α = 005 Knob length and germination rate values were transformed using modified square root transformation

Table 3 Pollen diameter (microm) and knob length (microm) under different levels of sucrose measured at regular 4-h intervals

Sucrose ()Pollen measurementz

Pollen diameter (microm) Knob length (microm)

0 2204c 00b

5 2261ab 87a

10 2262a 71a

5 2234ab 96a

20 2216bc 63a

zTreatments having similar letter(s) within a column are not significantly different at α = 005 Knob length and percent germination values were transformed using modified square root transformation

Table 2 Mean of in vitro germination of nipa pollen () under different levels of sucrose and boron z

Sucrose () Boron () Germination ()

Mean

0 0 00e 00C

0005 00e

001 00e

5 0 4484a-e 4960AB

0005 5476a

001 4921a-e

10 0 5040a-c 4881AB

0005 3571de

001 6032ab

15 0 4563b-e 5330A

0005 6865a

001 4563b-e

20 0 5357a-e 4378BC

0005 4405b-e

001 3373c-e

zTreatments having similar letter(s) within a column are not significantly different at α = 005 Germination rate values were transformed using modified square root transformation

Pollen in 15 sucrose medium produced the longest knob at 96 microm long (Table 3) Figure 7 showed the changes in the appearance of the germinating pollen at regular 4-h intervals As mentioned the pollen tube did not elongate as expected during germination but initially a knob would break out by the aperture

The initial diameter of pollen was measured at 24 microm but the trend was decreasing (Table 4) Pollen diameter decreased as the time elapsed during germination likely due to the movement of the protoplasmic activities upon knob formation The emergence of a pollen tube causes the decrease in pollen diameter since the pollen cytoplasm vegetative nucleus and sperm cells are transported within this structure during the elongation process (Ray et al 1997 Palanivelu and Preuss 2000 Johnson and Preuss

2002 Kim et al 2003) However the decline was quick as it only happened in the first 4 h during germination No significant differences were observed in pollen diameter beyond four hours as the knob protrusion started to increase at this point (Table 4) This appears to be the combined effects of sucrose and boron concentrations

Sucrose in combination with boric acid promotes pollen germination as well as tube development since boron makes a complex with sugar This sugar-borate complex is capable of translocating of sugar (Gauch and Dugger 1953) Scott (1960) also suggested that boron could exert a protective effect in preventing excessive polymerization of sugars at sites of sugar metabolism

Despite boronrsquos important role in pollen germination using it in an assay may have some setbacks The presence of boron can change the pH of the culture medium De Franca et al (2009) noted that the pH of the medium can be another variable affecting pollen germination In this study the pH of the medium decreased with the addition of boron At 5 and 10 sucrose without boron the pH was measured between 7ndash75 An addition of 0005 boric acid however resulted in dropping of pH to 65 at 001 boric acid pH dropped further to 63 Increasing sucrose to 15 and 20 further slid pH down to 54 and to as low as 53 for a higher level of boric acid After 24 h the sucrose in the media was fermenting by then emitting a rancid odor The effect of this needs to be explored in testing pollen germination in the future



727

SUMMARY AND CONCLUSIONStaminate rachillae or male spikes of N fruticans were collected for cytological observations of microsporogenesis Using squash technique with 2 acetocarmine as stain the dividing microspores were abundant from the staminate rachillae of middle laterals during inflorescence development Mature pollen grains in the upper laterals and microsporocytes (PMCs) in the lower laterals confirmed the basipetal development of nipa

Meiosis exhibited successive cytokinesis with the tetrad formed a tetragonal shape The microspores were enclosed with thick callose walls consisting not only of uninucleate but binucleate and trinucleate cells as well sampled from the middle laterals of the staminate rachillae undergoing anthesis This highly normal meiosis I and II suggest the high viability of nipa pollen

In vitro germination of nipa pollen was conducted in a liquid medium of different levels of sucrose (0 5 10 15 and 20) and boron (0 0005 and 001) as treatments At the same time changes in germinating pollen were observed every 4-h intervals for 24 h

At 15 sucrose a significantly higher 53 germination was obtained over the control It also attained the longest knob length at 96 microm However the significant interaction between sucrose and boron levels indicated that 10 sucrose likewise improved pollen germination and knob length when combined with 001 boron while 15 sucrose achieved a significant increase in germination and knob length when combined with 0005 boron Indeed different levels of sucrose with varying boron concentrations allowed pollen germination with observed knob protrusion This was least expected than an elongated pollen tube confirming the knob formation of initial pollen germination from the previous testing

ACKNOWLEDGMENTThe authors would like to thank the Nipa Research Project of the Commission on Higher Educations Zonal Research Center and UP Mindanaos Office of the Vice Chancellor for Academic Affairs for the funding support

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boron and gibberellic acid on in vitro pollen germina-tion of pistachio (Pistacia vera L) J Biotechnol 9 5126ndash5130

BHOWMICK G BOSE S 2011 Analytical techniques in biotechnology a complete laboratory manual Tata

McGraw Hill Education Private Limited New Delhi

CHAN E 2003 Handy Pocket Guide to Tropical Plants Periplus Editions (HK) Ltd Singapore

CHUMCHIM N KHUNWASI C 2011 Pollen morphol-ogy of true mangrove species in Thailand Department of Botany Faculty of Science Chulalongkorn Univer-sity Bangkok p 1ndash8

CORNER EJH 1966 The Natural History of Palms Berkeley CA University of California Press

CUSHION E WHITEMAN A DIETERLE G 2010 Bioenergy development issues and impacts for poverty and natural resources management Washington DC World Bank Publications

DASSANAYAKE MD CLAYTON WD eds 2000 A Revised Handbook to the Flora of Ceylon Vol 14 National Herbarium Department of Agriculture Per-adeniya Sri Lanka p 76ndash78

DE FRANCA L NASCIMENTO W CARMONA R DE FREITAS R 2009 Viability of eggplant pollen Crop Breed Appl Biotechnology 9 320ndash327

DE STORME N GEELEN D 2013 Cytokinesis in plant male meiosis Plant Signal Behav (Article ID e23394)

DEVRNJA N MILOJEVIĆ J TUBIĆ L ZDRAVKOVIĆ-KORAĆ S CINGEL A ĆALIĆ D 2012 Pollen Mor-phology Viability and Germination of Tanacetum vulgare L HortScience 47 440ndash442

DONG X HONG Z SIVARAMAKRISHNAN M MAHFOUZ M VERMA DPS 2005 Callose synthase (CalS5) is required for exine formation during micro-gametogenesis and for pollen viability in Arabidopsis The Plant Journal 42 315ndash328

DOWE J 2010 Australian palms biogeography ecology and systematics Clayton CSIRO Publishing

FAIZ MR SULONG I ZAINUDIN B KARTINI M 2011 The study on the life pollen of Kelantan delta mangrove forest Univ Mob Telecomm Syst 149p

GAUCH HG DUGGER WM JR 1953The role of bo-ron in the translocation of sucrose Plant Physiol 28 457ndash466

GERMERAAD JH HOPPINGS CA MULLER J 1968 Palynology of tertiary sediments from tropical areas Rev Palaeobotany Palynology 6 189ndash348

GOTTSBERGER G 1989 Comments on flower evolu-tion and beetle pollination in the genera Annona and Rollina (Annonaceae) Plant Syst Evol 167 89ndash94

GOOGLE MAPS 2020 Davao City Philippines Re-trieved on 19 Feb 2020 from httpswwwgooglecommaps



728

GRIFFITHS AJF WESSLER SR CARROLL SB DOE-BLEY J 2012 Introduction to Genetic Analysis 10th ed New York WH Freeman and Company

HARLEY MM BAKER WJ 2001 Pollen aperture mor-phology in Arecaceae application within phylogenetic analysis and a summary of a fossil record of palm like pollen Grana 40 45ndash77

HENNY R 1977 Effect of sucrose level medium com-position and pH on the in vitro germination of pollen from Spathiphyllum floribundum (Linden Andre) Fla State Hortic Soc 90 304ndash306

HESLOP-HARRISON J MACKENZIE A 1967 Auto-radiography of soluble [2-14C] thymidine derivatives during meiosis and microsporogenesis in Lilium an-thers J Cell Sci 2 387ndash400

HOLDAWAY-CLARKE TL HEPLER PK 2003 Control of pollen tube growth role of ion gradients and fluxes New Phytology 159 539ndash563

JANICK J PAULL R eds 2008 Encyclopedia of fruit and nuts CAB International Oxfordshire United Kingdom

JOHNSON MA PREUSS D 2002 Plotting a course multiple signals guide pollen tubes to their targets Dev Cell 2 273ndash281

JOSHI L KANAGARATNAM U ADHURI D 2006 Nypa fruticans ndash useful but forgotten in mangrove reforestation programs World Agroforestry Centre (ICRAF) Indonesia p 1ndash4

KEDVES M 1980 Morphological investigation of recent Palmae pollen grains Bot Acad Sci Hung 26 339ndash373

KIM S MOLLET JC DONG J ZHANG K PARK SY LORD EM 2003 Chemocyanin a small basic protein from the lily stigma induces pollen tube chemotro-pism Proc Natl Acad Sci USA 100 16125ndash16130

LORA J HERRERO M HORMAZA JI 2009 To coex-istence of bicellular and tricellular pollen in Annona cherimola (Annonaceae) implications for pollen evo-lution Amer J Bot 96 802ndash808

LWIN NM 2010 Seed maturity and pollen source influ-ence on Dura X Pisifera (Elaeis guineensis Jacq) Seed Quality [PhD Dissertation Abstract] University Putra Malaysia 123p

MACNAE W 1968 A general account of the fauna and flora of mangrove swamps and forests in the Indo West Pacific region Adv Mar Bio 6 73ndash270

MANTIQUILLA JA ABAD RG BARRO KMG BASI-LIO JAM RIVERO GC SILVOSA CSC 2016 Potential pollinators of nipa palm (Nypa fruticans Wurmb) Asia Life Sciences 25(1) 1ndash22

MANTIQUILLA JA ELUMBA ME ADTOON JA ABAD RG MILLADO CSS RIVERO GC 2018 In vitro germination and viability testing of nipa (Nypa fruticans Wurmb) pollen under different storage condi-tions Philipp J Sci 147(4) 617ndash627

MORTAZAVI SM ARZANI K MOIENI A 2010 Optimizing storage and in vitro germination of date palm (Phoenix dactylifera) pollen Agric Sci Technol 12 181ndash189

NAIR PKK 1985 Essentials of palynology plant breed-ing Monogram Theory Application Genetics Volume II Berlin Germany p 12ndash15

OBERMEYER G BLATT MR 1996 Electrical prop-erties of intact pollen grains of Lilium longiflorum characteristics of the non-germination grain J Exp Bot 46 803ndash813

OLIVEIRA MSP MAUEacuteS MM KALUME MAA 2001 Teste de viabilidade de poacutelen in vivoe in vitro emgenoacutetipos de accedilaizeiro Acta Bot Brasil 15 27ndash33

ONOFRI A 2006 DSAASTAT a new Excelreg VBA macro to perform basic statistical analyses of field trials Retrieved on 18 Apr 2016 from httpcommfacultyfullertoedujreinardexcel_add-inshtm

OPUTE FI 1975 Lipid and sterol composition of the pollen of the West African oil palm Elaeis guineensis Phytochemistry 14 1023ndash1026

PALANIVELU R PREUSS D 2000 Pollen tube targeting and axon guidance parallels in tip growth mechanisms Trends Cell Biol 10 517ndash524

PERERA PIP 2003 Cytological examination of micro-spore development for microspore and anther culture of coconut (Cocos nucifera L) cv Sri Lanka tall Cocos 15 53ndash59

RAY SM PARK SS RAY A 1997 Pollen tube guidance by the female gametophyte Dev 124 2489ndash2498

RICHARDS AJ 1986 Plant Breeding Systems London George Allen Unwin

SANZOL J HERRERO M 2001 The lsquoeffective pollina-tionrsquo period in fruit trees Sci Hort 90 1ndash17

SCHULZ-SCHAEFFER J 1980 Cytogenetics Plants Animals Humans New York Springer-Verlag

SCOTT EG 1960 Effect of supra optimal boron levels on respiration and carbohydrate metabolism of Helianthus annuus Plant Physiol 35 653

SHIVANNA KR LINSKENS HF CRESTI M 1991 Pollen viability and pollen vigor Theory Appl Genet 81 38ndash42



729

SOARES TL DE JESUS ON DE SOUZA EH SEREJO JA OLIVEIRA EJ 2013 Morphology and viability of pollen grains from passion fruit species (Passiflora spp) Acta Bot Brasil 27(4) 779ndash787

SONI JN PATEL MN JHA CV 2010 Effect of different concentration of sucrose during in vitro pollen germi-nation and pollen tube growth in Cleome gynandra L Life Sci Leaflet 8 222ndash225

STANLEY RG LINSKENS HF 1974 Pollen Biology Biochemistry Management New York Springer-Verlag

STANLEY RG LOEWUS FA 1964 Boron and myo-inositol in pollen pectin biosynthesis In Pollen Physi-ology and Fertilization Linkens HF ed North Holland Publ Corp Amsterdam p 128ndash139

STONE BC 1970 The flora of Guam Micronesica 6 1ndash659

TAYLOR PL HEPLER PK 1997 Pollen germination and tube growth Annual Review Plant Physiology Plant Mol Biol 48 461ndash491

TOMASCIK T MAH AJ NONTJI A MOSSA MK 1997 The ecology of the Indonesian seas part two Singapore Berkeley Books Private Ltd

TOMLINSON PB 1961 Anatomy of the Monocotyle-dons II Palmae Oxford

TOMLINSON PB 1971 The Shoot Apex and its Di-chotomous Branching in the Nypa Palm Ann Bot 35 865ndash879

TSCHUDY RH VAN LOENEN SD 1970 Illustrations of plant microfossils from the Yazoo Clay (Jackson Group upper Eocene) Mississippi United States Geol-ogy Survey [Professor Paper 643-E] p 1ndash5

UHL NW 1972 Inflorescence and flower structure in Nypa fruticans (Palmae) Am J Bot 59(7) 729ndash743

UHL NW DRANSFIELD J 1987 Genera Palmarum St Lawrence KS Allen Press

WANG ZY GE Y SCOTT M SPANGENBERG G 2004 Viability and longevity of pollen from transgenic and nontransgenic tall fescue (Festuca arundinacea Poa-ceae) plants Am J Bot 91(4) 523ndash530

WATERKEYN L 1962 Les parois microsporocytaires de nature callosique chez Helleborus et Fadescantia Cellule 62 225ndash255

WATERKEYN L BENFAIT A 1970 On a possible func-tion of the callosic special wall in Ipomoea purpurea (L) Roth Grana 10 13ndash20

ZAID A DE WET PF 2002 Pollination and bunch management In FAO Plant Production and Protec-tion [Paper 156 Rev 1] Date Palm Cultivation Zaid A Arias-Jimenez EJ eds Rome Retrieved on 24 Feb 2020 from httpwwwfaoorg3Y4360Ey4360e0chtmbm12

ZHANG C GUINEL FC MOFFATT BA 2002 A com-parative ultrastructural study of pollen development in Arabidopsis thaliana ecotype Columbia and male-sterile mutant Apt1-3 Protoplasma 219 59ndash71

ZHANG T HUANG M 2009 The effect of sucrose and PEG on the in vitro pollen germination of camellia Northern Hort 1 101ndash102



730

dispersing seeds and depositing silt (Janick and Paull 2008 Joshi et al 2006 MacNae 1968 Tomascik et al 1997)

Nypa fruticans is characterized by a rhizomatous habit of its dichotomously branching stem that is 30 cm wide to 15 cm thick and often remains underground (Dowe 2010 Janick and Paull 2008) Each of branching stem holds less than of a dozen upright pinnate leaves that can usually reach a length of 5ndash9 m and numerous lanceolate leaflets that are 12ndash15 m long (Janick and Paull 2008) Monoecious flowers are borne in a 1ndash2-m long spadix-like inflorescence that is axillary The laterals bear the terminal catkin-like rachillae where the staminate flowers are produced and the terminal globose head structure of the main axis consists of pistillate flowers that are larger and positioned below the staminate rachillae after anthesis (Chan 2003 Dowe 2010 Janick and Paull 2008)

Staminate rachillae which measure 6ndash9 cm long support the short dense spikes of staminate flowers that are yellow-orange (Dassanayake and Clayton 2000 Dowe 2010) The staminate flowers are arranged in rows usually 21 rows per spike Each flower is composed of perianth parts and a large stalk at its center which gives forth to longitudinally positioned anthers Perianth parts are arranged in two whorls The androecium forms from three stamens that fused into a column of about 4ndash6 cm in length and no pistillodes are present There are three ndash sometimes four ndash anthers that reach a length of 2 mm each containing four sporangia with conniving half-anthers near the tip (Stone 1970 Uhl 1972 Dassanayake and Clayton 2000 Dowe 2010)

The pollen of extant N fruticans is unique among the palm family and has little chance of being mistaken for pollen of other taxa except for the fossil nipa pollen (Germeraad et al 1968 Tschudy and Van Loenen 1970 Kedves 1980) Its pollen grain is classified according to aperture as zonasulcate with oblate spheroidal to subprolate shape The sulcus furrow in pollen surface is 120ndash180 microm in width the meridian is ring-like while the membrane is psilate Ornamentation is echinate while the interspinal area is perforate or some reticulate (Chumchim and Khunwasi 2011) Completely zonasulcate aperture in Arecaceae is known only in the Calamoideae and the Nypoideae The spiny exine along with zonasulcate aperture and large size makes the pollen of nipa highly distinctive (Harley and Baker 2001)

Good fruit-set and high crop yield depend largely on viable pollen which is often determined by germination (Opute 1975 Lwin 2010) Besides being used for measuring male fertility to monitor stored pollen (Oliveira et al 2001 Soares et al 2013) pollen viability generally indicates the ability of the pollen grain to perform its function by delivering the sperm cells to the embryo sac

following compatible pollination (Shivanna et al 1991 Wang et al 2004) One approach to estimate pollen viability is by employing in vitro germination assay This method is used to germinate a small sample of pollen in an appropriate culture medium to simulate the stigma environment It provides a balanced environment for the development of the pollen tubes by observing the percentage of pollen grains that produced them after a certain period The composition sucrose concentration and pH of the medium are among the factors that affect pollen germination (De Franca et al 2009)

In the study conducted by Mortazavi et al (2010) on the in vitro germination of the date palm the germinated pollen count was highest at the medium containing 15 sucrose 002 calcium nitrate and 0005 boric acid Sugar is utilized as an energy source for the synthesis of cell wall materials such as pectins cellulose and callose The sucrose in the medium maintains osmotic pressure and provides nutrition to germinating pollen (Nair 1985 Soni et al 2010) During in vitro germination boron is added in the form of boric acid for most species as it directly involves in pectin synthesis and thus indirectly with the development of pollen tube membrane (Holdaway-Clarke and Hepler 2003 Stanley and Loewus 1964) It affects H+-ATPase activity that initiates pollen germination and tube growth (Obermeyer and Blatt 1996) ndash its absence in the culture medium often leads to pollen tube bursting (Acar et al 2010) Sucrose in combination with boric acid promotes pollen germination as well as tube development since boron makes a complex with sugar This sugar-borate complex is capable of translocation of sugar (Gauch and Dugger 1953)

An initial study conducted on in vitro germination of nipa showed no pollen tube growth but pollen broke open by the aperture thus releasing its content This was observed across 2ndash10 sucrose and germination time (1ndash4 h) by germinating pollen grains using oil palm and date palm culture media It is composed of 50 mL distilled water 10 g sucrose and 001 g boric acid (Mantiquilla et al 2018) Given that the germination medium can dramatically affect pollen metabolism (Taylor and Hepler 1997) finding the optimal medium has to be explored further

This study was conducted to characterize the pollen development of N fruticans Wurmb cytologically through acetocarmine squash technique and describe nipa pollen during in vitro germination This study aims to

1 assess which phase in the inflorescence development where cell division occurs in staminate rachilla

2 analyze meiosis of N fruticans during microsporogenesis

3 estimate optimum levels of sucrose and boron concentration for in vitro germination of pollen and



720










Germination=






721










722









723






724






Uninucleate 191 200 80 200 168 15

Binucleate 571 650 600 560 595 54

Trinucleate 238 150 320 240 237 22




725










726



0 4 8 12 16 20 24








0 2204c 00b

5 2261ab 87a

10 2262a 71a

5 2234ab 96a

20 2216bc 63a




Mean

0 0 00e 00C

0005 00e

001 00e

5 0 4484a-e 4960AB

0005 5476a

001 4921a-e

10 0 5040a-c 4881AB

0005 3571de

001 6032ab

15 0 4563b-e 5330A

0005 6865a

001 4563b-e

20 0 5357a-e 4378BC

0005 4405b-e

001 3373c-e









727



























728
































729





















730










Germination=






721










722









723






724






Uninucleate 191 200 80 200 168 15

Binucleate 571 650 600 560 595 54

Trinucleate 238 150 320 240 237 22




725










726



0 4 8 12 16 20 24








0 2204c 00b

5 2261ab 87a

10 2262a 71a

5 2234ab 96a

20 2216bc 63a




Mean

0 0 00e 00C

0005 00e

001 00e

5 0 4484a-e 4960AB

0005 5476a

001 4921a-e

10 0 5040a-c 4881AB

0005 3571de

001 6032ab

15 0 4563b-e 5330A

0005 6865a

001 4563b-e

20 0 5357a-e 4378BC

0005 4405b-e

001 3373c-e









727



























728
































729





















730










722









723






724






Uninucleate 191 200 80 200 168 15

Binucleate 571 650 600 560 595 54

Trinucleate 238 150 320 240 237 22




725










726



0 4 8 12 16 20 24








0 2204c 00b

5 2261ab 87a

10 2262a 71a

5 2234ab 96a

20 2216bc 63a




Mean

0 0 00e 00C

0005 00e

001 00e

5 0 4484a-e 4960AB

0005 5476a

001 4921a-e

10 0 5040a-c 4881AB

0005 3571de

001 6032ab

15 0 4563b-e 5330A

0005 6865a

001 4563b-e

20 0 5357a-e 4378BC

0005 4405b-e

001 3373c-e









727



























728
































729





















730









723






724






Uninucleate 191 200 80 200 168 15

Binucleate 571 650 600 560 595 54

Trinucleate 238 150 320 240 237 22




725










726



0 4 8 12 16 20 24








0 2204c 00b

5 2261ab 87a

10 2262a 71a

5 2234ab 96a

20 2216bc 63a




Mean

0 0 00e 00C

0005 00e

001 00e

5 0 4484a-e 4960AB

0005 5476a

001 4921a-e

10 0 5040a-c 4881AB

0005 3571de

001 6032ab

15 0 4563b-e 5330A

0005 6865a

001 4563b-e

20 0 5357a-e 4378BC

0005 4405b-e

001 3373c-e









727



























728
































729





















730






724






Uninucleate 191 200 80 200 168 15

Binucleate 571 650 600 560 595 54

Trinucleate 238 150 320 240 237 22




725










726



0 4 8 12 16 20 24








0 2204c 00b

5 2261ab 87a

10 2262a 71a

5 2234ab 96a

20 2216bc 63a




Mean

0 0 00e 00C

0005 00e

001 00e

5 0 4484a-e 4960AB

0005 5476a

001 4921a-e

10 0 5040a-c 4881AB

0005 3571de

001 6032ab

15 0 4563b-e 5330A

0005 6865a

001 4563b-e

20 0 5357a-e 4378BC

0005 4405b-e

001 3373c-e









727



























728
































729





















730






Uninucleate 191 200 80 200 168 15

Binucleate 571 650 600 560 595 54

Trinucleate 238 150 320 240 237 22




725










726



0 4 8 12 16 20 24








0 2204c 00b

5 2261ab 87a

10 2262a 71a

5 2234ab 96a

20 2216bc 63a




Mean

0 0 00e 00C

0005 00e

001 00e

5 0 4484a-e 4960AB

0005 5476a

001 4921a-e

10 0 5040a-c 4881AB

0005 3571de

001 6032ab

15 0 4563b-e 5330A

0005 6865a

001 4563b-e

20 0 5357a-e 4378BC

0005 4405b-e

001 3373c-e









727



























728
































729





















730










726



0 4 8 12 16 20 24








0 2204c 00b

5 2261ab 87a

10 2262a 71a

5 2234ab 96a

20 2216bc 63a




Mean

0 0 00e 00C

0005 00e

001 00e

5 0 4484a-e 4960AB

0005 5476a

001 4921a-e

10 0 5040a-c 4881AB

0005 3571de

001 6032ab

15 0 4563b-e 5330A

0005 6865a

001 4563b-e

20 0 5357a-e 4378BC

0005 4405b-e

001 3373c-e









727



























728
































729





















730



0 4 8 12 16 20 24








0 2204c 00b

5 2261ab 87a

10 2262a 71a

5 2234ab 96a

20 2216bc 63a




Mean

0 0 00e 00C

0005 00e

001 00e

5 0 4484a-e 4960AB

0005 5476a

001 4921a-e

10 0 5040a-c 4881AB

0005 3571de

001 6032ab

15 0 4563b-e 5330A

0005 6865a

001 4563b-e

20 0 5357a-e 4378BC

0005 4405b-e

001 3373c-e









727



























728
































729





















730



























728
































729





















730
































729





















730





















730

Cytological Observations and In Vitro Germination of Nipa ......of Nipa (Nypa fruticans Wurmb.,...

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Transcript of Cytological Observations and In Vitro Germination of Nipa ......of Nipa (Nypa fruticans Wurmb.,...