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In vitro pollen germination of Betula utilis, a typical tree line species in Himalayas
South African Journal of Botany 131 (2020) 214221
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In vitro pollen germination of Betula utilis, a typical tree line species in Himalayas Mohammad Saleem Wania,*, Maroof Hamidb, Younas Rasheed Tantraya, Raghbir Chand Guptaa, A.H. Munshic, Vijay Singha a
Department of Botany, Punjabi University, Patiala, Punjab 147002, India Centre for Biodiversity & Taxonomy, Department of Botany, University of Kashmir, Srinagar 190006, J & K, India c Department of Botany, University of Kashmir, Jammu & Kashmir 190006, India b
A R T I C L E
I N F O
Article History: Received 2 April 2019 Revised 18 November 2019 Accepted 16 February 2020 Available online xxx Edited by D Honys Keywords: Betula utilis Pollen grains Pollen germination Pollen tube growth
A B S T R A C T
The study was conducted to determine the optimum medium for in vitro pollen germination for B. utilis based on germination percentages and length of the pollen tube. Studies about pollen germination and tube growth of B. utilis are limited. Therefore, this study aimed to investigate the effects of sucrose (1, 3, 5, 10 and 15%), boric acid (25, 50, 100 and 200 ppm), calcium nitrate (50, 100, 200, 300 and 400 ppm), magnesium sulfate (50, 100, 200 and 300 ppm) and potassium nitrate (50, 100 and 200 ppm) and time of incubation on pollen germination and tube growth. Pollen germination rates and tube growth were recorded periodically at 2, 4, 6, and 10 h. The length varied significantly with different concentrations and incubation time. The germination rate and pollen tube length varied significantly with different concentrations and incubation time. Pollen germination was maximal (98 § 6.1%) when the germination medium contained 10% sucrose solution supplemented with 100 ppm H3BO3, 300 ppm Ca(NO3)2, 200 ppm MgSO4 and 100 ppm KNO3, after 6 h of incubation. In addition, results revealed that pollen grains collected immediately after anther dehiscence showed the best germination ability but gradually decreased with time. © 2020 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Betula utilis, commonly known as Himalayan birch or Bhojpatra belongs to the family Betulaceae and is native to Himalayan region. This species is an important member of tree lines and forms a large portion of the fragile ecosystem of Himalaya. It is a deciduous tree attaining a height of 1520 m and grows in altitudinal range from 2800 to 4511 m. It plays an important role in Himalayan ecology and its bark and roots provide niches to many insects especially beetles. Aromatic and medicinal plants have been used for therapeutic, religious, cosmetic, nutritional, and beautification purposes since ancient times and mankind of all civilizations and culture are familiar with their usage (Abramovic et al., 2018; Senkal et al., 2019). The curing properties of Betula bark and bark extracts have been acknowledged for a long time in traditional medicine in different parts of the world. Different Betula species find mention in several pharmacopoeias (Menkovic et al., 2011; Shikov et al., 2014) including the Russian, French, European, Deutsches Pharmacopoeias, the Ayuevedic Pharmacopoeia of India and Pharmacopoeia Jugoslavica. Several old texts as well as modern books additionally contain monographs that describe the botanical, chemical, as well as pharmacological properties and uses of Betula species (Rastogi et al., 2015). * Corresponding author. E-mail address: [email protected] (M.S. Wani). https://doi.org/10.1016/j.sajb.2020.02.025 0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.
The plant also contains several commercially important compounds like, acetyloleanolic acid, betulin, betulinic acid, lupeol, lupenone, methylebetulonate, methyl betulate, oleanolic acid, sitosterol and karachic acid (Selvam, 2008; Khan, 1975). In the recent past, it is noted that natural populations of this species are declining in several regions of Himalaya and is placed in endangered species category (Siwach et al., 2013). In a genetic breeding program, information on pollen viability is a main factor for artificial hybridization (Soares et al., 2008). Understanding the viability and capacity of pollen germination and pollen tube growth are essential for investigation of reproductive biology and genetic selection and breeding of plants (Salles et al., 2006). Pollen germination potential has been defined as the capacity of viable pollen to germinate under an appropriate environment (Fortescue and Turner, 2004). Pollen germination and pollen tube growth are also essential study material for biochemical, biotechnological, ecological, environmental, evolutionary, molecular biological, morphological and physiological studies (Ottavio et al., 2012). Thus, determination of pollen viability is crucial in tree breeding. An essential study includes investigating chemical changes during germination of pollen and growth of pollen tubes through the use of direct methods such as the induction of in vitro germination (Sorkheh et al., 2018). In vitro pollen germination contributes a novel approach and strategy to hasten the genetic improvement of tree breeding. It is a very advantageous and effective method for studying many basic and
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applied aspects of pollen biology (Shekari et al., 2016). This technique is the best alternative for selecting viable and potential pollen that can be utilized for cross-pollination (Sakhanokho and Rajasekaran, 2010). The conditions needed for in vitro pollen germination differ across species. Pollen germination and pollen tube growth can be enhanced by many internal and external factors (Schueler et al., 2005; Kremer and Jemric, 2006). The external factors which affect the germination of pollen include incubation time, most favorable temperature, and composition of medium (Lin et al., 2017). Organic and inorganic substances like sucrose, boric acid, calcium nitrate, potassium nitrate, and magnesium sulfate exert an effect on the in vitro pollen germination (Parton et al., 2002; Kopp et al., 2002). Undoutedly, the quality of pollen, the optimum concentrations of the media and environmental conditions have special effects on germination of pollen and tube growth (Sakhanokho and Rajasekaran, 2010). A variety of methods and media with different components have been recommended by previous researchers (Liu et al., 2013; Shekari et al., 2016; Lin et al., 2017). Little information is available regarding the effect of different minerals on pollen germination of Betula. However, the effect of sucrose, boric acid, and calcium ions on pollen germination was investigated by Shen et al. (2010). Wu et al. (2012) studied pollen germination and preservation attributes under different storage conditions. Metabolomic analysis during pollen germination and tube growth was studied by Fragallah et al. (2018). In spite of these examples, a more detailed study about the effect of sucrose, boric acid, magnesium sulfate, calcium nitrate, potassium nitrate concentrations and optimal incubation time is not yet reported. Therefore, present work was designed to investigate the effect of sucrose, boric acid, magnesium sulfate, calcium nitrate, potassium nitrate and time of incubation on germination and pollen tube growth of B. utilis pollen grains. Our report is expected to be useful to plant breeders, geneticists, or gene bank curators who need pollen viability tests for B. utilis pollen. 2. Materials and methods 2.1. Description of the study area This study was carried out in 2017 on a population growing in Gulmarg, Jammu and Kashmir. The climate of the study area is continental temperate type, with annual average precipitation about 1049 mm yr1, and most of the precipitation falls during winter in the form of snow. July is the warmest month of the year with temperature rising to an average 20 °C, whereas January is the coldest month with temperature going down to 6 °C (http://www.imd.gov.in/ pages/main.php). 2.2. Collection of pollen samples Male catkins were collected during the pollination period from the end of February to mid-March 2017. The catkins were collected from 10 randomly selected mature wild trees of B. utilis, when the majority of male cones had dehisced anthers. The catkins were collected in the morning between 7:00 and 9:00 a.m. The male catkins were hung upside down in open plastic bags, labeled separately. Then, fresh pollen grains were shed, poured into 1.5 mL centrifuge tubes, and sealed with ParafilmÒ . The tubes were put into silica gel and kept at 4 °C for overnight. 2.3. In vitro pollen germination In vitro pollen germination was carried out following the Lagera et al. (2017) method to know the impact of various supplements like sucrose, boric acid, and other nutrient salts. For the germination test, the glass staining blocks were inoculated in a growth chamber. The relative humidity and temperature conditions set for inoculation
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were 70% and 25 °C, respectively, as previously described (Fragallah et al., 2018). The tests were designed to achieve three goals. First, the effects of various sucrose concentrations on the germination rate of pollen were determined. Second, the effects of different boric acid concentrations on the growth of the pollen tube were checked. Third, the effects of different culture media in various concentrations and combinations (Bhattacharya et al., 1997) were used to determine the germination rate of pollens. This test included five different concentration treatments and three repetitions. 2.4. Pollen tube growth measurements Slides were observed under an Olympus microscope at low magnification (10x and 40x) to monitor the germination rate and pollen tube length following the system of Brewbaker and Kwack (1963). The lengths of the pollen tubes were measured directly by an ocular micrometer. Development of pollen tube from a pollen grain indicates pollen germination, whereas lengthening of pollen tube illustrates pollen tube growth. All the germinated pollen grains do not show tube growth. Hence, both parameters were investigated. A pollen grain was considered germinated when pollen tube length was at least equivalent to or exceeded the grain diameter (Hebbar et al., 2018) and was calculated from the formula below. Moreover, the mean pollen tube growth was measured from randomly selected 10 pollen tubes at an interval of 2, 4, 6 and 10 h. Percentage pollen germination ¼
Number of pollen grains germinated 100 Total number of pollen grains observed
2.5. Data analyses All statistical analyses were performed in R version 3.5.1 (R Core Team, 2019). To test how the in vitro germination of B. utilis pollen grains was effected by using different concentrations of culture media and incubation time, a two-way ANOVA test was performed with ‘concentration of culture media’, ‘incubation time’ and their interactions as fixed effects, and the germination of pollen grains as a response variable. Similar procedures were applied for mean tube length. Prior to all analyses, data were subjected to Levene’s test for checking homogeneity of variance using car- package (Fox and Weisberg, 2018). 3. Results A two-way ANOVA test revealed a significant interaction between culture media and time for the in vitro germination and mean tube length of the pollen grains of B. utilis (Table S3-S10 in supplementary material). Table 1 shows mean values (§ standard deviation (SD)) of the effect of sucrose concentration on pollen germination and tube growth after 2, 4, 6 and 10 h of incubation. The pollen germination along with tube development decreased in lower concentrations as well as in higher concentrations of sucrose. The effect was significant with pollen germination and tube growth being highest at sucrose concentrations of 10%. Compared to the former, sucrose concentrations of 5 and 15% had reduced pollen germination. Fig. 2 shows mean values (§ SD) of the effect of boric acid concentrations on pollen germination and tube growth after 2, 4, 6 and 10 h of incubation. Boric acid significantly promoted the germination of pollen and tube growth. The promotion of germination was most notable at a boric acid concentration of 100 ppm. The pollen germination and tube development generally were lower with boric acid at 25, 50, and 200 ppm. Boric acid (100 ppm) in combination with 10%
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M.S. Wani et al. / South African Journal of Botany 131 (2020) 214221 Table 1 Effect of sucrose on in vitro pollen germination. Conc.%
After 2 h.
After 4 h.
After 6 h.
Sucrose Germination (%) Mean tube Germination (%) Mean tube Germination (%) Mean tube length length (mm) length (mm) (mm) 1 3 5 10 15
21.1 24.5 23.5
11 § 0.4 15 § 0.8 9 § 0.5
26.4 35.5 29.5
14 § 1.7 29 § 3.5 14 § 2.3
28.3 40.8 33.3
24 § 2.1 30 § 3.2 25 § 3.6
After 10 h. Germination (%) Mean tube length (mm) 26.1 27.8 31.4
11 § 2.6 27 § 3.1 12 § 3.4
*Mean § SD.
sucrose showed significantly higher pollen germination and tube elongation (Fig. 2). Other nutrients also had variable effects on pollen germination and tube growth that were concentration-dependent. For example, 300 ppm of calcium nitrate was favorable for pollen germination in
combination with 10% sucrose and 100 ppm boric acid. On the other hand, less pollen germination and tube elongation were observed at 50, 100, 200 and 400 ppm of calcium nitrate (Fig. 3). It was observed that maximum germination occurred at 100 ppm of potassium nitrate (Fig. 4) and 200 ppm of magnesium sulfate (Fig. 5). The
Table 2 Effect of sucrose (10%), H3BO3 (100 ppm), Ca(NO3)2 (300 ppm), MgSO4 (200 ppm) and KNO3 (100 ppm) on in vitro pollen germination. After 2 h.
After 4 h.
After 6 h.
After 10 h.
Germination (%) Mean tube Germination (%) Mean tube Germination (%) Mean tube Germination (%) Mean tube length (mm) length (mm) length (mm) length (mm) 49.3
59 § 4.6
56.3
65 § 5.1
98.1
98 § 6.1
70.8
*Mean § SD.
Fig. 1. . Germinated pollen grains in the medium under in vitro condition.
74 § 5.2
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Fig. 2. Effect of concentration of culture media (Sucrose 10%+ H3BO3) and incubation time on pollen germination and pollen tube length of B. utilis (a-b) germination, pollen tube lenght at 2 h; (c-d) germination, pollen tube lenght at 4 h; (e-f) germination, pollen tube lenght at 6 h; (g-h) germination, pollen tube lenght at 10 h. H3BO3 (Conc).
germination rate reached as high as 98% along with 98 mm long pollen tube developed in the 6th hour, when potassium nitrate was used in 100 ppm supplemented by 10% sucrose solution, 100 ppm boric acid, 300 ppm calcium nitrate and 200 ppm magnesium sulfate (Table 2). It was found during the study that pollen grains began to germinate immediately after being placed in the media. After 1 h, germination rates increased and reached the maximum at 6 h, with a significant decrease at 10 h. On the other hand, pollen tube growth starts to elongate at 2 h and dramatically increased over time and reached the maximum growth at 6 h of incubation. 4. Discussion Pollen grains of B. utilis are triporate (Fig 1) and round (Perveen and Qaiser, 1999) with a smooth surface. During germination one, two or three pollen tubes emerges, but only one grows and increases in length.
Pollen germination and pollen tube growth responded differently to exogenously applied chemicals. Apart from moisture, they usually require a sugar source, boron, and calcium for good germination and tube growth. The media preparation for pollen germination differs as per the plant species and many internal and external factors (Dane et al., 2004). Besides, there are various other factors that have been noted to influence the in vitro germination of pollen grains. For instance, humidity, genotypic differences, vigor and physiological phase of the plant and the age of the flower and ingredients of the substrate used for germination have an effect on pollen germination (Shivanna and Johri, 1985). Nambudiri and Thomas (1974) reported that some nutrients (e.g., boric acid, calcium nitrate, magnesium sulfate, and potassium nitrate) influenced pollen germination. In this study, various combinations of culture media were tested for the provision of optimum germination conditions required by the pollen of B. utilis. However, to study the effects of different medium concentrations on pollen
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Fig. 3. Effect of concentration of culture media (Sucrose 10%+ H3BO3 100ppm + Ca (NO3)2) and incubation time on pollen germination and pollen tube length of B. utilis (a-b) germination, pollen tube lenght at 2 h; (c-d) germination, pollen tube lenght at 4 h; (e-f) germination, pollen tube lenght at 6 h; (g-h) germination, pollen tube length at 10 h.
germination and tube growth, in vitro pollen germination was performed in different concentrations of sucrose separately and in combination with varying calcium, magnesium, potassium and boric acid concentration. An appropriate concentration of sucrose favors both germination and pollen tube development, whereas a high concentration of sucrose can suppress both processes (Lin et al., 2017). The addition of sucrose in a culture medium is essential as it serves as the main supplement for the germination of the pollen and the formation of the pollen tube walls. Besides, sucrose can act as an energy pool in the form of carbon that is needed throughout germination for active metabolism and membrane transport (Fei and Nelson, 2003). During normal growth and development, sucrose has dual functionality; for example, it nourishes the pollen during germination and in the development of a pollen tube; further, it maintains the osmotic pressure of the external environment
(Huang et al., 2004). The differences in pollen germination within the same culture medium with different concentrations of sucrose could, therefore, be endorsed to a difference in osmotic pressure of the culture medium (Youmbi et al., 2015). Therefore the proper concentration of sucrose is essential to maintain a well-balanced osmotic pressure in a system to deliver an environment that will be helpful for pollen germination (Zhang et al., 2004). Principally, pollen germination and tube growth increased with an increase of the sucrose concentration. Here the maximum germination and tube growth of pollen of B. utilis were obtained in media of 10% sucrose concentrations. The effects of boric acid on the germination and growth of Pistacia vera L. and Areca catechu L pollen were reported by Acar et al. (2010) and Liu et al. (2013). During this study, the results revealed that boric acid promoted pollen germination and tube growth. Boric acid in a
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Fig. 4. Effect of concentration of culture media (Sucrose 10%+ H3BO3 100ppm + Ca (NO3)2 300ppm + KNO3) and incubation time on pollen germination and pollen tube length of B. utilis (a-b) germination, pollen tube lenght at 2 h; (c-d) germination, pollen tube lenght at 4 h; (e-f) germination, pollen tube length at 6 h; (g-h) germination, pollen tube length at 10 h.
concentration of 100 ppm significantly increased pollen germination rates and tube growth compared with that in the boric acid-deficient media. Pollen germination and tube growth were hindered at 25, 50 and 200 ppm concentration. Sucrose jointly with boric acid improves pollen germination along with tube development because boron makes a complex with sugar and this sugar-borate complex is known to be capable of better translocation than non-borate, non-ionised sugar molecules (Sidhu and Malik, 1986). Stanley and Linskens (1974) reported that sucrose improves the osmotic balance between the pollen and the germination medium and provides energy to the development of the pollen tube. Boron is supposed to promote pollen germination by affecting H+-ATPase movement, which commences pollen germination and tube growth (Obermeyer and Blatt, 1995). It is required at a concentration of 100 ppm for most of the species investigated (Brewbaker and Majumder, 1961). Exclusion of boric acid from the culture medium generally leads to tube bursting (Acar et al., 2010; Holdaway-Clarke and Hepler, 2003).
Calcium is another inorganic substance with a prominent effect on pollen tube growth (Bendnarska, 1989). Cytosolic free Ca2+ is an important secondary messenger in the signaling networks regulating pollen tube elongation and reorientation (Steinhorst and Kudla, 2013). In vivo pollen tube growth usually relies on external calcium stores in the pistil, and external Ca2+ enhances the elongation of pollen tube in vitro (Brewbaker and Kwack, 1963; Franklin-Tong, 1999). Application of Ca2+ channel blockers or Ca2+ionophores hinders pollen tube growth, indicating the requirement for fine-tuning of cytosolic Ca2+concentration in the elongation of pollen tubes (Obermeyer and Weisenseel, 1991; Picton and Steer 1983; Pierson et al., 1994). rdenas et al. (2008) there is a close interaction According to Ca between intracellular Ca2+ and the cytoskeleton in the pollen tube apex. Actin polymerization participates in the pollen tube growth process, Qian and Xiang (2019) reported that Ca2+regulates actin filament assembly in the tip region of the pollen tube.
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Fig. 5. Effect of concentration of culture media (Sucrose 10%+ H3BO3 100ppm + Ca(NO3)2 300ppm + MgSO4) and incubation time on pollen germination and pollen tube length of B. utilis(a-b) germination, pollen tube lenght at 2 h; (c-d) germination, pollen tube lenght at 4 h; (e-f) germination, pollen tube lenght at 6 h; (g-h) germination, pollen tube length at 10 h.
It is also well-known that K+ is essential for both pollen germination and tube growth (Brewbaker and Kwack, 1963; Weisenseel and Jaffe, 1976). Both Ca2+and K+are mutually dependent upon each other, since the inward K+ channels are highly regulated by Ca2+ as noticed in Arabidopsis (Fan et al., 2001). Moore and Jung (1974) brought up that NO3and Mg2+ improved the tube growth in the case of in vitro pollen germination of Saccharum sp. Osmotic potential for the swelling of pollen grains controlled by KNO3 was noted in barley (Matsui et al., 2000). In our study, we have also found that the germination rate and the growth of the pollen tube were highly sensitive to incubation time. Incubation time has played a fundamental role in both pollen germination and pollen tube formation. An increasing trend in pollen germination has been witnessed with a peak at 6 h. There might be two reasons to clarify such a reduction in the pollen germination and pollen tube formation: (1) plasmolysis of pollen and pollen tube may
occur because of low supplements in the medium after utilization by the developing pollen; (2) continuous germination and growth of pollen tubes demanded water that might evaporate during the prolonged incubation resulted in deformed pollen and pollen tube.
5. Conclusion This research has demonstrated that various factors (culture medium and incubation time) significantly influenced pollen germination and pollen tube formation of Betula pollen. We concluded that a medium containing sucrose (10%), H3BO3 (100 ppm), Ca(NO3)2 (300 ppm), MgSO4 (200 ppm) and KNO3 (100 ppm) is the standout medium for in vitro Betula pollen germination and growth. Likewise, the pollen could be incubated for 6 h in order to achieve a higher germination rate and uniform pollen tubes. The results given here are
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the first observation on pollen germination of B. utilis that will help its reproduction and artificial pollination studies. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements The authors are highly thankful to the Head, Department of Botany, Punjabi University, Patiala for providing necessary lab facilities during the work. This work was supported by the Department of Biotechnology, IPLS-DBT [reference number: BT/PR 4548/INF/22/146/2012]. Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2020.02.025. References Abramovic, H., Abram, V., Cuk, A., Ceh, B., Smole-Mozina, S., Vidmar, M., Pavlovic, M., Ulrih, N.P., 2018. Antioxidative and antibacterial properties of organically grown thyme (Thymus sp.) and basil (Ocimum basilicum L.). Turkish Journal of Agriculture and Forestry 42, 185–194. Acar, I., Ak, B.E., Sarpkaya, K., 2010. Effect of boron and gibberellic acid on in vitro pollen germination of pistachio (Pistaci avera L.). African Journal of Biotechnology 9, 5126–5130. Bendnarska, K., 1989. The effect of exogenous Ca2+ ions on pollen grain germination and pollen tube growth. Sexual Plant Reproduction 2, 53–58. Bhattacharya, A., Mondal, S., Mandal, S., 1997. In vitro pollen germination of delonix regia (Boj.). Science and Culture 63, 143–144. Brewbaker, J., Kwack, B.H., 1963. The essential role of calcium ion in pollen germination and pollen tube growth. American Journal of Botany 50, 859–865. Brewbaker, J., Majumder, S.K., 1961. Cultural studies of pollen population effect and self-incompatibility inhibition. American Journal of Botany 48, 457–464. C ardenas, L., Lovy-Wheeler, A., Kunkel, J.G., Hepler, P.K., 2008. Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization. Plant Physiology 146, 1611–1621. € 2004. In vitro pollen germination of some plant species in Dane, F., Olgun, G., Dalgi¸c , O, basic culture medium. Journal of Cell and Molecular Biology 3, 71–76. Fan, L., Wang, Y., Wang, H., Wu, W., 2001. In vitro Arabidopsis pollen germination a characterization of inward potassium currents in Arabidopsis pollen grain protoplasts. Journal of Experimental Botany 52, 1603–1614. Fei, S., Nelson, E., 2003. Estimation of pollen viability, shedding pattern, and longevity of creeping bentgrass on artificial media. Crop Science 43, 2177–2181. Fortescue, J., Turner, D., 2004. Pollen fertility in Musa: viability in cultivars grown in Southern Australia. Australian. Journal of Agricultural Research 55, 1085–1091. Fox, J., Weisberg, S., 2018. An R Companion to Applied Regression, third ed. Sage. in press https://CRAN.R-project.org/package=car. Fragallah, S.A.D.A., Wang, P., Li, N., Chen, Y., Lin, S., 2018. Metabolomic analysis of pollen grains with different germination abilities from two clones of chinese fir (Cunninghamia lanceolata (Lamb) hook). Molecules 23, 3162. Franklin-Tong, V.E., 1999. Signaling and the modulation of pollen tube growth. The Plant Cell 11, 727–738. Hebbar, K.B., Rose, H.M., Nair, A.R., Kannan, S., Niral, V., Arivalagan, M., Gupta, A., Samsudeen, K., Chandran, K.P., Chowdappa, P., Prasad, P.V., 2018. Differences in in vitro pollen germination and pollen tube growth of coconut (Cocos nucifera L.) cultivars in response to high temperature stress. Environmental and Experimental Botany 153, 35–44. Holdaway-Clarke, T.L., Hepler, P.K., 2003. Control of pollen tube growth: role of ion gradients and fluxes. New Phytologist 159, 539–563. Huang, J., Cao, Q.F., Meng, Y.P., 2004. Effect of culture medium components on in vitro germination of pumpkin pollen. China Cucurbits Veg 3, 6–7. Khan, M.A., 1975. Karachic acid: a new triterpenoid from Betula utilis. Phytochemistry 14, 789–791. Kopp, R.F., Maynard, C.A., Rocha de Niella, P., Smart, L.B., Abrahamson, L.P., 2002. Collection and storage of pollen from Salix (Salicaceae). American Journal of Botany 89, 248–252. Kremer, D., Jemric, T., 2006. Pollen germination and pollen tube growth in Fraxinus pennsylvanicus. Biologia 61, 79–83. Lagera, A.J., Balinado, L.O., Baldomero, J.R., Rotairo, H.F.I., Tero, N.L., Maghinay, M.S., Baluyo, I.F., Reyes, M.R., Galve, R., Sibao, S.A., Rufino, J.V., 2017. Varying sugars and sugar concentrations influence in vitro pollen germination and pollen tube growth of Cassia alata l. Journal of Young Investigators 33. Lin, Y., Wang, Y., Iqbal, A., Shi, P., Li, J., Yang, Y., Lei, X., 2017. Optimization of culture medium and temperature for the in vitro germination of oil palm pollen. Scientia Horticulturae 220, 134–138.
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Effects of mineral elements on pollen germination and pollen tube growth of Cunninghamia lanceolata (Lamb.) Hook. China Forestry Science and Technology 24, 57–60. Shikov, A.N., Pozharitskaya, O.N., Makarov, V.G., Wagner, H., Verpoorte, R., Heinrich, M., 2014. Medicinal plants of the Russian Pharmacopoeia; their history and applications. Journal of Ethnopharmacology 153, 481–536. Shivanna, K.R., Johri, B.M., 1985. The Angiosperm Pollen: Structure and Function. Wiley Eastern. Sidhu, R.J.K., Malik, C.P., 1986. Metabolic role of boron in germinating pollen and growing pollen tubes. Springer -Verlag, New York, pp. 373–378. Siwach, M., Siwach, P., Solanki, P., Gill, A.R., 2013. Biodiversity conservation of Himalayan medicinal plants in India: a retrospective analysis for a better vision. International Journal of Biodiversity and Conservation 5, 529–540. Soares, T.L., Silva, S., Costa, M., Santos-Serejo, J., Souza, A.d.S., Lino, L.S.M., Souza, E., Jesus, O., 2008. In vitro germination and viability of pollen grains of banana diploids. Crop Breeding and Applied Biotechnology 8, 111–118. sz, J., Ercisli, S., Koubouris, G.C., Sorkheh, K., Azimkhani, R., Mehri, N., Chaleshtori, M.H., Hala 2018. Interactive effects of temperature and genotype on almond (Prunus dulcis L.) pollen germination and tube length. Scientia Horticulturae 227, 162–168. Stanley, R.G., Linskens, H.F., 1974. Pollen: Biology Biochemistry Management. Springer Science & Business Media. Steinhorst, L., Kudla, J., 2013. Calcium-a central regulator of pollen germination and tube growth. Biochimica et Biophysica Acta-Molecular Cell Research 1833, 1573–1581. Weisenseel, M.H., Jaffe, L.F., 1976. The major growth current through lily pollen tubes enter as K+ and leaves as H+. Planta 133, 1–7. Wu, Y., Liu, J., Xiao, F., Li, Y., Zhu, X., Xu, H., Huang, B., 2012. Study on pollen germination and preservation characteristic of Cunninghamia lanceolata. Jiangxi For. 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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
In vitro pollen germination of Betula utilis, a typical tree line species in Himalayas Mohammad Saleem Wania,*, Maroof Hamidb, Younas Rasheed Tantraya, Raghbir Chand Guptaa, A.H. Munshic, Vijay Singha a
Department of Botany, Punjabi University, Patiala, Punjab 147002, India Centre for Biodiversity & Taxonomy, Department of Botany, University of Kashmir, Srinagar 190006, J & K, India c Department of Botany, University of Kashmir, Jammu & Kashmir 190006, India b
A R T I C L E
I N F O
Article History: Received 2 April 2019 Revised 18 November 2019 Accepted 16 February 2020 Available online xxx Edited by D Honys Keywords: Betula utilis Pollen grains Pollen germination Pollen tube growth
A B S T R A C T
The study was conducted to determine the optimum medium for in vitro pollen germination for B. utilis based on germination percentages and length of the pollen tube. Studies about pollen germination and tube growth of B. utilis are limited. Therefore, this study aimed to investigate the effects of sucrose (1, 3, 5, 10 and 15%), boric acid (25, 50, 100 and 200 ppm), calcium nitrate (50, 100, 200, 300 and 400 ppm), magnesium sulfate (50, 100, 200 and 300 ppm) and potassium nitrate (50, 100 and 200 ppm) and time of incubation on pollen germination and tube growth. Pollen germination rates and tube growth were recorded periodically at 2, 4, 6, and 10 h. The length varied significantly with different concentrations and incubation time. The germination rate and pollen tube length varied significantly with different concentrations and incubation time. Pollen germination was maximal (98 § 6.1%) when the germination medium contained 10% sucrose solution supplemented with 100 ppm H3BO3, 300 ppm Ca(NO3)2, 200 ppm MgSO4 and 100 ppm KNO3, after 6 h of incubation. In addition, results revealed that pollen grains collected immediately after anther dehiscence showed the best germination ability but gradually decreased with time. © 2020 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Betula utilis, commonly known as Himalayan birch or Bhojpatra belongs to the family Betulaceae and is native to Himalayan region. This species is an important member of tree lines and forms a large portion of the fragile ecosystem of Himalaya. It is a deciduous tree attaining a height of 1520 m and grows in altitudinal range from 2800 to 4511 m. It plays an important role in Himalayan ecology and its bark and roots provide niches to many insects especially beetles. Aromatic and medicinal plants have been used for therapeutic, religious, cosmetic, nutritional, and beautification purposes since ancient times and mankind of all civilizations and culture are familiar with their usage (Abramovic et al., 2018; Senkal et al., 2019). The curing properties of Betula bark and bark extracts have been acknowledged for a long time in traditional medicine in different parts of the world. Different Betula species find mention in several pharmacopoeias (Menkovic et al., 2011; Shikov et al., 2014) including the Russian, French, European, Deutsches Pharmacopoeias, the Ayuevedic Pharmacopoeia of India and Pharmacopoeia Jugoslavica. Several old texts as well as modern books additionally contain monographs that describe the botanical, chemical, as well as pharmacological properties and uses of Betula species (Rastogi et al., 2015). * Corresponding author. E-mail address: [email protected] (M.S. Wani). https://doi.org/10.1016/j.sajb.2020.02.025 0254-6299/© 2020 SAAB. Published by Elsevier B.V. All rights reserved.
The plant also contains several commercially important compounds like, acetyloleanolic acid, betulin, betulinic acid, lupeol, lupenone, methylebetulonate, methyl betulate, oleanolic acid, sitosterol and karachic acid (Selvam, 2008; Khan, 1975). In the recent past, it is noted that natural populations of this species are declining in several regions of Himalaya and is placed in endangered species category (Siwach et al., 2013). In a genetic breeding program, information on pollen viability is a main factor for artificial hybridization (Soares et al., 2008). Understanding the viability and capacity of pollen germination and pollen tube growth are essential for investigation of reproductive biology and genetic selection and breeding of plants (Salles et al., 2006). Pollen germination potential has been defined as the capacity of viable pollen to germinate under an appropriate environment (Fortescue and Turner, 2004). Pollen germination and pollen tube growth are also essential study material for biochemical, biotechnological, ecological, environmental, evolutionary, molecular biological, morphological and physiological studies (Ottavio et al., 2012). Thus, determination of pollen viability is crucial in tree breeding. An essential study includes investigating chemical changes during germination of pollen and growth of pollen tubes through the use of direct methods such as the induction of in vitro germination (Sorkheh et al., 2018). In vitro pollen germination contributes a novel approach and strategy to hasten the genetic improvement of tree breeding. It is a very advantageous and effective method for studying many basic and
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applied aspects of pollen biology (Shekari et al., 2016). This technique is the best alternative for selecting viable and potential pollen that can be utilized for cross-pollination (Sakhanokho and Rajasekaran, 2010). The conditions needed for in vitro pollen germination differ across species. Pollen germination and pollen tube growth can be enhanced by many internal and external factors (Schueler et al., 2005; Kremer and Jemric, 2006). The external factors which affect the germination of pollen include incubation time, most favorable temperature, and composition of medium (Lin et al., 2017). Organic and inorganic substances like sucrose, boric acid, calcium nitrate, potassium nitrate, and magnesium sulfate exert an effect on the in vitro pollen germination (Parton et al., 2002; Kopp et al., 2002). Undoutedly, the quality of pollen, the optimum concentrations of the media and environmental conditions have special effects on germination of pollen and tube growth (Sakhanokho and Rajasekaran, 2010). A variety of methods and media with different components have been recommended by previous researchers (Liu et al., 2013; Shekari et al., 2016; Lin et al., 2017). Little information is available regarding the effect of different minerals on pollen germination of Betula. However, the effect of sucrose, boric acid, and calcium ions on pollen germination was investigated by Shen et al. (2010). Wu et al. (2012) studied pollen germination and preservation attributes under different storage conditions. Metabolomic analysis during pollen germination and tube growth was studied by Fragallah et al. (2018). In spite of these examples, a more detailed study about the effect of sucrose, boric acid, magnesium sulfate, calcium nitrate, potassium nitrate concentrations and optimal incubation time is not yet reported. Therefore, present work was designed to investigate the effect of sucrose, boric acid, magnesium sulfate, calcium nitrate, potassium nitrate and time of incubation on germination and pollen tube growth of B. utilis pollen grains. Our report is expected to be useful to plant breeders, geneticists, or gene bank curators who need pollen viability tests for B. utilis pollen. 2. Materials and methods 2.1. Description of the study area This study was carried out in 2017 on a population growing in Gulmarg, Jammu and Kashmir. The climate of the study area is continental temperate type, with annual average precipitation about 1049 mm yr1, and most of the precipitation falls during winter in the form of snow. July is the warmest month of the year with temperature rising to an average 20 °C, whereas January is the coldest month with temperature going down to 6 °C (http://www.imd.gov.in/ pages/main.php). 2.2. Collection of pollen samples Male catkins were collected during the pollination period from the end of February to mid-March 2017. The catkins were collected from 10 randomly selected mature wild trees of B. utilis, when the majority of male cones had dehisced anthers. The catkins were collected in the morning between 7:00 and 9:00 a.m. The male catkins were hung upside down in open plastic bags, labeled separately. Then, fresh pollen grains were shed, poured into 1.5 mL centrifuge tubes, and sealed with ParafilmÒ . The tubes were put into silica gel and kept at 4 °C for overnight. 2.3. In vitro pollen germination In vitro pollen germination was carried out following the Lagera et al. (2017) method to know the impact of various supplements like sucrose, boric acid, and other nutrient salts. For the germination test, the glass staining blocks were inoculated in a growth chamber. The relative humidity and temperature conditions set for inoculation
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were 70% and 25 °C, respectively, as previously described (Fragallah et al., 2018). The tests were designed to achieve three goals. First, the effects of various sucrose concentrations on the germination rate of pollen were determined. Second, the effects of different boric acid concentrations on the growth of the pollen tube were checked. Third, the effects of different culture media in various concentrations and combinations (Bhattacharya et al., 1997) were used to determine the germination rate of pollens. This test included five different concentration treatments and three repetitions. 2.4. Pollen tube growth measurements Slides were observed under an Olympus microscope at low magnification (10x and 40x) to monitor the germination rate and pollen tube length following the system of Brewbaker and Kwack (1963). The lengths of the pollen tubes were measured directly by an ocular micrometer. Development of pollen tube from a pollen grain indicates pollen germination, whereas lengthening of pollen tube illustrates pollen tube growth. All the germinated pollen grains do not show tube growth. Hence, both parameters were investigated. A pollen grain was considered germinated when pollen tube length was at least equivalent to or exceeded the grain diameter (Hebbar et al., 2018) and was calculated from the formula below. Moreover, the mean pollen tube growth was measured from randomly selected 10 pollen tubes at an interval of 2, 4, 6 and 10 h. Percentage pollen germination ¼
Number of pollen grains germinated 100 Total number of pollen grains observed
2.5. Data analyses All statistical analyses were performed in R version 3.5.1 (R Core Team, 2019). To test how the in vitro germination of B. utilis pollen grains was effected by using different concentrations of culture media and incubation time, a two-way ANOVA test was performed with ‘concentration of culture media’, ‘incubation time’ and their interactions as fixed effects, and the germination of pollen grains as a response variable. Similar procedures were applied for mean tube length. Prior to all analyses, data were subjected to Levene’s test for checking homogeneity of variance using car- package (Fox and Weisberg, 2018). 3. Results A two-way ANOVA test revealed a significant interaction between culture media and time for the in vitro germination and mean tube length of the pollen grains of B. utilis (Table S3-S10 in supplementary material). Table 1 shows mean values (§ standard deviation (SD)) of the effect of sucrose concentration on pollen germination and tube growth after 2, 4, 6 and 10 h of incubation. The pollen germination along with tube development decreased in lower concentrations as well as in higher concentrations of sucrose. The effect was significant with pollen germination and tube growth being highest at sucrose concentrations of 10%. Compared to the former, sucrose concentrations of 5 and 15% had reduced pollen germination. Fig. 2 shows mean values (§ SD) of the effect of boric acid concentrations on pollen germination and tube growth after 2, 4, 6 and 10 h of incubation. Boric acid significantly promoted the germination of pollen and tube growth. The promotion of germination was most notable at a boric acid concentration of 100 ppm. The pollen germination and tube development generally were lower with boric acid at 25, 50, and 200 ppm. Boric acid (100 ppm) in combination with 10%
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M.S. Wani et al. / South African Journal of Botany 131 (2020) 214221 Table 1 Effect of sucrose on in vitro pollen germination. Conc.%
After 2 h.
After 4 h.
After 6 h.
Sucrose Germination (%) Mean tube Germination (%) Mean tube Germination (%) Mean tube length length (mm) length (mm) (mm) 1 3 5 10 15
21.1 24.5 23.5
11 § 0.4 15 § 0.8 9 § 0.5
26.4 35.5 29.5
14 § 1.7 29 § 3.5 14 § 2.3
28.3 40.8 33.3
24 § 2.1 30 § 3.2 25 § 3.6
After 10 h. Germination (%) Mean tube length (mm) 26.1 27.8 31.4
11 § 2.6 27 § 3.1 12 § 3.4
*Mean § SD.
sucrose showed significantly higher pollen germination and tube elongation (Fig. 2). Other nutrients also had variable effects on pollen germination and tube growth that were concentration-dependent. For example, 300 ppm of calcium nitrate was favorable for pollen germination in
combination with 10% sucrose and 100 ppm boric acid. On the other hand, less pollen germination and tube elongation were observed at 50, 100, 200 and 400 ppm of calcium nitrate (Fig. 3). It was observed that maximum germination occurred at 100 ppm of potassium nitrate (Fig. 4) and 200 ppm of magnesium sulfate (Fig. 5). The
Table 2 Effect of sucrose (10%), H3BO3 (100 ppm), Ca(NO3)2 (300 ppm), MgSO4 (200 ppm) and KNO3 (100 ppm) on in vitro pollen germination. After 2 h.
After 4 h.
After 6 h.
After 10 h.
Germination (%) Mean tube Germination (%) Mean tube Germination (%) Mean tube Germination (%) Mean tube length (mm) length (mm) length (mm) length (mm) 49.3
59 § 4.6
56.3
65 § 5.1
98.1
98 § 6.1
70.8
*Mean § SD.
Fig. 1. . Germinated pollen grains in the medium under in vitro condition.
74 § 5.2
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Fig. 2. Effect of concentration of culture media (Sucrose 10%+ H3BO3) and incubation time on pollen germination and pollen tube length of B. utilis (a-b) germination, pollen tube lenght at 2 h; (c-d) germination, pollen tube lenght at 4 h; (e-f) germination, pollen tube lenght at 6 h; (g-h) germination, pollen tube lenght at 10 h. H3BO3 (Conc).
germination rate reached as high as 98% along with 98 mm long pollen tube developed in the 6th hour, when potassium nitrate was used in 100 ppm supplemented by 10% sucrose solution, 100 ppm boric acid, 300 ppm calcium nitrate and 200 ppm magnesium sulfate (Table 2). It was found during the study that pollen grains began to germinate immediately after being placed in the media. After 1 h, germination rates increased and reached the maximum at 6 h, with a significant decrease at 10 h. On the other hand, pollen tube growth starts to elongate at 2 h and dramatically increased over time and reached the maximum growth at 6 h of incubation. 4. Discussion Pollen grains of B. utilis are triporate (Fig 1) and round (Perveen and Qaiser, 1999) with a smooth surface. During germination one, two or three pollen tubes emerges, but only one grows and increases in length.
Pollen germination and pollen tube growth responded differently to exogenously applied chemicals. Apart from moisture, they usually require a sugar source, boron, and calcium for good germination and tube growth. The media preparation for pollen germination differs as per the plant species and many internal and external factors (Dane et al., 2004). Besides, there are various other factors that have been noted to influence the in vitro germination of pollen grains. For instance, humidity, genotypic differences, vigor and physiological phase of the plant and the age of the flower and ingredients of the substrate used for germination have an effect on pollen germination (Shivanna and Johri, 1985). Nambudiri and Thomas (1974) reported that some nutrients (e.g., boric acid, calcium nitrate, magnesium sulfate, and potassium nitrate) influenced pollen germination. In this study, various combinations of culture media were tested for the provision of optimum germination conditions required by the pollen of B. utilis. However, to study the effects of different medium concentrations on pollen
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Fig. 3. Effect of concentration of culture media (Sucrose 10%+ H3BO3 100ppm + Ca (NO3)2) and incubation time on pollen germination and pollen tube length of B. utilis (a-b) germination, pollen tube lenght at 2 h; (c-d) germination, pollen tube lenght at 4 h; (e-f) germination, pollen tube lenght at 6 h; (g-h) germination, pollen tube length at 10 h.
germination and tube growth, in vitro pollen germination was performed in different concentrations of sucrose separately and in combination with varying calcium, magnesium, potassium and boric acid concentration. An appropriate concentration of sucrose favors both germination and pollen tube development, whereas a high concentration of sucrose can suppress both processes (Lin et al., 2017). The addition of sucrose in a culture medium is essential as it serves as the main supplement for the germination of the pollen and the formation of the pollen tube walls. Besides, sucrose can act as an energy pool in the form of carbon that is needed throughout germination for active metabolism and membrane transport (Fei and Nelson, 2003). During normal growth and development, sucrose has dual functionality; for example, it nourishes the pollen during germination and in the development of a pollen tube; further, it maintains the osmotic pressure of the external environment
(Huang et al., 2004). The differences in pollen germination within the same culture medium with different concentrations of sucrose could, therefore, be endorsed to a difference in osmotic pressure of the culture medium (Youmbi et al., 2015). Therefore the proper concentration of sucrose is essential to maintain a well-balanced osmotic pressure in a system to deliver an environment that will be helpful for pollen germination (Zhang et al., 2004). Principally, pollen germination and tube growth increased with an increase of the sucrose concentration. Here the maximum germination and tube growth of pollen of B. utilis were obtained in media of 10% sucrose concentrations. The effects of boric acid on the germination and growth of Pistacia vera L. and Areca catechu L pollen were reported by Acar et al. (2010) and Liu et al. (2013). During this study, the results revealed that boric acid promoted pollen germination and tube growth. Boric acid in a
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Fig. 4. Effect of concentration of culture media (Sucrose 10%+ H3BO3 100ppm + Ca (NO3)2 300ppm + KNO3) and incubation time on pollen germination and pollen tube length of B. utilis (a-b) germination, pollen tube lenght at 2 h; (c-d) germination, pollen tube lenght at 4 h; (e-f) germination, pollen tube length at 6 h; (g-h) germination, pollen tube length at 10 h.
concentration of 100 ppm significantly increased pollen germination rates and tube growth compared with that in the boric acid-deficient media. Pollen germination and tube growth were hindered at 25, 50 and 200 ppm concentration. Sucrose jointly with boric acid improves pollen germination along with tube development because boron makes a complex with sugar and this sugar-borate complex is known to be capable of better translocation than non-borate, non-ionised sugar molecules (Sidhu and Malik, 1986). Stanley and Linskens (1974) reported that sucrose improves the osmotic balance between the pollen and the germination medium and provides energy to the development of the pollen tube. Boron is supposed to promote pollen germination by affecting H+-ATPase movement, which commences pollen germination and tube growth (Obermeyer and Blatt, 1995). It is required at a concentration of 100 ppm for most of the species investigated (Brewbaker and Majumder, 1961). Exclusion of boric acid from the culture medium generally leads to tube bursting (Acar et al., 2010; Holdaway-Clarke and Hepler, 2003).
Calcium is another inorganic substance with a prominent effect on pollen tube growth (Bendnarska, 1989). Cytosolic free Ca2+ is an important secondary messenger in the signaling networks regulating pollen tube elongation and reorientation (Steinhorst and Kudla, 2013). In vivo pollen tube growth usually relies on external calcium stores in the pistil, and external Ca2+ enhances the elongation of pollen tube in vitro (Brewbaker and Kwack, 1963; Franklin-Tong, 1999). Application of Ca2+ channel blockers or Ca2+ionophores hinders pollen tube growth, indicating the requirement for fine-tuning of cytosolic Ca2+concentration in the elongation of pollen tubes (Obermeyer and Weisenseel, 1991; Picton and Steer 1983; Pierson et al., 1994). rdenas et al. (2008) there is a close interaction According to Ca between intracellular Ca2+ and the cytoskeleton in the pollen tube apex. Actin polymerization participates in the pollen tube growth process, Qian and Xiang (2019) reported that Ca2+regulates actin filament assembly in the tip region of the pollen tube.
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Fig. 5. Effect of concentration of culture media (Sucrose 10%+ H3BO3 100ppm + Ca(NO3)2 300ppm + MgSO4) and incubation time on pollen germination and pollen tube length of B. utilis(a-b) germination, pollen tube lenght at 2 h; (c-d) germination, pollen tube lenght at 4 h; (e-f) germination, pollen tube lenght at 6 h; (g-h) germination, pollen tube length at 10 h.
It is also well-known that K+ is essential for both pollen germination and tube growth (Brewbaker and Kwack, 1963; Weisenseel and Jaffe, 1976). Both Ca2+and K+are mutually dependent upon each other, since the inward K+ channels are highly regulated by Ca2+ as noticed in Arabidopsis (Fan et al., 2001). Moore and Jung (1974) brought up that NO3and Mg2+ improved the tube growth in the case of in vitro pollen germination of Saccharum sp. Osmotic potential for the swelling of pollen grains controlled by KNO3 was noted in barley (Matsui et al., 2000). In our study, we have also found that the germination rate and the growth of the pollen tube were highly sensitive to incubation time. Incubation time has played a fundamental role in both pollen germination and pollen tube formation. An increasing trend in pollen germination has been witnessed with a peak at 6 h. There might be two reasons to clarify such a reduction in the pollen germination and pollen tube formation: (1) plasmolysis of pollen and pollen tube may
occur because of low supplements in the medium after utilization by the developing pollen; (2) continuous germination and growth of pollen tubes demanded water that might evaporate during the prolonged incubation resulted in deformed pollen and pollen tube.
5. Conclusion This research has demonstrated that various factors (culture medium and incubation time) significantly influenced pollen germination and pollen tube formation of Betula pollen. We concluded that a medium containing sucrose (10%), H3BO3 (100 ppm), Ca(NO3)2 (300 ppm), MgSO4 (200 ppm) and KNO3 (100 ppm) is the standout medium for in vitro Betula pollen germination and growth. Likewise, the pollen could be incubated for 6 h in order to achieve a higher germination rate and uniform pollen tubes. The results given here are
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the first observation on pollen germination of B. utilis that will help its reproduction and artificial pollination studies. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements The authors are highly thankful to the Head, Department of Botany, Punjabi University, Patiala for providing necessary lab facilities during the work. This work was supported by the Department of Biotechnology, IPLS-DBT [reference number: BT/PR 4548/INF/22/146/2012]. Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2020.02.025. References Abramovic, H., Abram, V., Cuk, A., Ceh, B., Smole-Mozina, S., Vidmar, M., Pavlovic, M., Ulrih, N.P., 2018. Antioxidative and antibacterial properties of organically grown thyme (Thymus sp.) and basil (Ocimum basilicum L.). Turkish Journal of Agriculture and Forestry 42, 185–194. Acar, I., Ak, B.E., Sarpkaya, K., 2010. Effect of boron and gibberellic acid on in vitro pollen germination of pistachio (Pistaci avera L.). African Journal of Biotechnology 9, 5126–5130. Bendnarska, K., 1989. The effect of exogenous Ca2+ ions on pollen grain germination and pollen tube growth. Sexual Plant Reproduction 2, 53–58. Bhattacharya, A., Mondal, S., Mandal, S., 1997. In vitro pollen germination of delonix regia (Boj.). Science and Culture 63, 143–144. Brewbaker, J., Kwack, B.H., 1963. The essential role of calcium ion in pollen germination and pollen tube growth. American Journal of Botany 50, 859–865. Brewbaker, J., Majumder, S.K., 1961. Cultural studies of pollen population effect and self-incompatibility inhibition. American Journal of Botany 48, 457–464. C ardenas, L., Lovy-Wheeler, A., Kunkel, J.G., Hepler, P.K., 2008. Pollen tube growth oscillations and intracellular calcium levels are reversibly modulated by actin polymerization. Plant Physiology 146, 1611–1621. € 2004. In vitro pollen germination of some plant species in Dane, F., Olgun, G., Dalgi¸c , O, basic culture medium. Journal of Cell and Molecular Biology 3, 71–76. Fan, L., Wang, Y., Wang, H., Wu, W., 2001. In vitro Arabidopsis pollen germination a characterization of inward potassium currents in Arabidopsis pollen grain protoplasts. Journal of Experimental Botany 52, 1603–1614. Fei, S., Nelson, E., 2003. Estimation of pollen viability, shedding pattern, and longevity of creeping bentgrass on artificial media. Crop Science 43, 2177–2181. Fortescue, J., Turner, D., 2004. Pollen fertility in Musa: viability in cultivars grown in Southern Australia. Australian. Journal of Agricultural Research 55, 1085–1091. Fox, J., Weisberg, S., 2018. An R Companion to Applied Regression, third ed. Sage. in press https://CRAN.R-project.org/package=car. Fragallah, S.A.D.A., Wang, P., Li, N., Chen, Y., Lin, S., 2018. Metabolomic analysis of pollen grains with different germination abilities from two clones of chinese fir (Cunninghamia lanceolata (Lamb) hook). Molecules 23, 3162. Franklin-Tong, V.E., 1999. Signaling and the modulation of pollen tube growth. The Plant Cell 11, 727–738. Hebbar, K.B., Rose, H.M., Nair, A.R., Kannan, S., Niral, V., Arivalagan, M., Gupta, A., Samsudeen, K., Chandran, K.P., Chowdappa, P., Prasad, P.V., 2018. Differences in in vitro pollen germination and pollen tube growth of coconut (Cocos nucifera L.) cultivars in response to high temperature stress. Environmental and Experimental Botany 153, 35–44. Holdaway-Clarke, T.L., Hepler, P.K., 2003. Control of pollen tube growth: role of ion gradients and fluxes. New Phytologist 159, 539–563. Huang, J., Cao, Q.F., Meng, Y.P., 2004. Effect of culture medium components on in vitro germination of pumpkin pollen. China Cucurbits Veg 3, 6–7. Khan, M.A., 1975. Karachic acid: a new triterpenoid from Betula utilis. Phytochemistry 14, 789–791. Kopp, R.F., Maynard, C.A., Rocha de Niella, P., Smart, L.B., Abrahamson, L.P., 2002. Collection and storage of pollen from Salix (Salicaceae). American Journal of Botany 89, 248–252. Kremer, D., Jemric, T., 2006. Pollen germination and pollen tube growth in Fraxinus pennsylvanicus. Biologia 61, 79–83. Lagera, A.J., Balinado, L.O., Baldomero, J.R., Rotairo, H.F.I., Tero, N.L., Maghinay, M.S., Baluyo, I.F., Reyes, M.R., Galve, R., Sibao, S.A., Rufino, J.V., 2017. Varying sugars and sugar concentrations influence in vitro pollen germination and pollen tube growth of Cassia alata l. Journal of Young Investigators 33. Lin, Y., Wang, Y., Iqbal, A., Shi, P., Li, J., Yang, Y., Lei, X., 2017. Optimization of culture medium and temperature for the in vitro germination of oil palm pollen. Scientia Horticulturae 220, 134–138.
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