Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 768-777 ISSN: 2319-7706 Volume 5 Number 1(2016) pp. 768-777 Journal homepage: http://www.ijcmas.com Original Research Article http://dx.doi.org/10.20546/ijcmas.2016.501.078 Studies on in vitro pollen germination of Mitragyna parvifolia (Roxb.) Korth. Krishnendu Dey, Subrata Mondal and Sudhendu Mandal* Department of Botany, Visva-Bharati, Santiniketan-731235, India *Corresponding author A B S T R A C T K e y w o r d s In vitro pollen germination test was performed on Mitragyna parvifolia (Roxb.)Korth. belonging to the family Rubiaceae to study the effect of Pollen different nutrients like sucrose, boric acid at various concentration germination, separately and in combinations and salts like Calcium nitrate, Magnesium Pollen tube sulphate and Potassium nitrate. The flowers open in the early evening 19.00 growth, to 20.30 hrs. Anther dehiscence takes place before flower opening. Mitragyna parviflora Maximum 96% pollen germination along with 1105µm pollen tube development was observed in 10% sucrose solution supplemented with Article Info 100ppm boric acid and among the salts maximum 90% pollen germination along with 910µm pollen tube was observed in 50ppm Calcium nitrate Accepted: solution and in case of Magnesium sulphate 85% pollen germination along 22 December 2015 with 728µm pollen tube was observed in 500ppm while in case of Potassium Available Online: 10 January 2016 nitrate 65% pollen germination along with 897µm pollen tube was observed in 500ppm solution. Introduction Pollen grains are reduced, non-motile, pollen vigour during storage; genetics and microscopic male gametophytes which, pollen-stigma interaction; crop improvement upon pollination, produce pollen tubes that and breeding programmes; gene bank grow through the pistil for effective maintenance; incompatibility and fertility fertilization and seed set. Pollen fertility and studies; evaluation of pollen germ inability viability have a paramount importance in after exposure to certain conditions, and plant reproduction. So pollen fertility, evaluation of dispersal and gene flow viability, and its longevity are basic aspect (Stanley and Linkens 1974, Heslop- for the improvement of plant before going to Harrison et al. 1984, Heslop-Harrison 1992, successful breeding programme. Pollen Dafni 1992, Mulugeta et al. 1994,Shivanna viability is critical for the study of following and Rangaswamy 1993, Shivanna and aspects of pollination biology: monitoring Heslop-Harrison 1981). Viability has been 768 Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 768-777 defined as having the capacity to live, grow, sucrose and boric acid at various germinate or develop (Lincoln et al. 1982). concentrations separately and in The term viability has also been used to combinations and salts of Calcium, describe pollen grains capable of Magnesium and Potassium on in vitro pollen germinating on the stigma (Morse 1987, germination of Mitragyna parvifolia (Roxb.) Preston 1991, Vaughton and Ramsey 1991, Korth..,an economically and medicinally Niesenbaum 1992).germination in vitro important plant belonging to the family (Shchori et al. 1992, Beardsell et al. 1993, Rubiaceae popularly knowen as Guli- Lindgren et al. 1995). Kadam. Pollen grains are simple structure of plant Materials and Methods cells and pollen tube formation is a good and appropriate model of growth and For the study of in vitro pollen germination, development (Taylor and Hepler, 1997). newly opened flowers were collected in the Thus pollen germination and pollen tube evening (19.00-20.30 hours) and transferred growth are important research material for to polythene bags. In vitro pollen morphological, physiological, germination was studied to know the effect biotechnological, ecological, environmental, of nutrients like sucrose and boric acid at evolutional, biochemical and molecular different concentrations individually as well biological studies (Ottavio et al., 1992). as in combinations and salts of Calcium Pollen tube elongation is a lively process in nitrate, Magnesium sulphate, Potassium which pollen tubes navigates and respond to nitrate. The fresh pollen samples were female tissues to accomplish their mission of shown on several grooved slides containing delivering the sperm cells for fertilization. solution of different concentrations Pollen tube extents exclusively at the cell separately or in combination. Slides were apex via an extreme form of polar growth, then kept in Petri dishes lined with moist known as tip growth, producing uniformly filter paper and examined under the set cylindrical cells (Cheung, 2001). Pollen microscope, at different time intervals to tubes are excellent system for the study of record the germination percentage and polarized tip growth, cell movement, cell to pollen tube length following the method of cell communication, cell to cell recognition Shivanna and Rangaswamy (1993). A pollen and signalling in plants. In recent years, grain was considered as germinated if pollen pollen germination and pollen tube tube at least as long as pollen grain diameter development are used as materials for (Stanley and Liskens 1974). determining the importance of cytoskeleton in cell growth and differentiation (Ma et al., Results and Discussion 2000). Pollens normally germinate on stigma and the required environment for in Studies on in vitro pollen germination at vitro pollen germination is related to genetic different time intervals after anthesis composition and also the quality of nutrient indicated that 92% germinating pollen with reserves of pollen (Baker and Baker, 1979). a mean of 1014µm long pollen tube During the past few years pollen tube development was observed in 10% sucrose growth in vitro becomes a popular model solution (Table-1). Individually, 100 ppm system for cell biology studies in plant cell boric acid showed 90% germination along (Moutinho et al., 2001). The present with 871 µm long pollen tube (Table-2). The investigation is aimed to study the effect of maximum 96% pollen germination along 769 Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 768-777 with 1105 µm long pollen tube developed and amino acids are supplied by the style to after 3 hours in 10% sucrose solution nourish the growing pollen tubes. Boron is supplemented with 100 ppm boric acid (Fig- also provided by stigmas and styles and 1 and Table-3). The maximum 90% pollen facilitates sugar uptake and play a vital role germination along with 910µm long pollen in pectin production in the pollen tubes tube developed after 3 hours in 50ppm (Richards, 1986). Boric acid is known to be Calcium Nitrate solution while 85% pollen crucial for pollen germination and tube germination along with 728 µm long pollen growth and it is required at concentration of tube development was observed in 500ppm 100 ppm for most species (Brewbaker and Magnesium Sulphate solution and 65% Majumder, 1961). pollen germination along with 897 µm long pollen tube developed in 500ppm Environmental factors and especially Potassium Nitrate solution (Table-4). desiccation risks are considered a main Though the effect of either sucrose or boric selective force leading to better protection of acid individually showed good results, but the pollen grain and from the evolutionary sucrose in combination with boric acid ecology view-point, the possible relation promoted pollen germination as well as tube between pollen longevity and pollination development (Table-1, Table-2, Table-3), chances,pollen competition, and breeding because boron makes a complex with sugar system is noteworthy. Even if pollen is and this sugar-borate complex is known to delivered successfully into the proper be capable of better translocation than non- receptive stigma, there is no guarantee that it borate, non-ionized sugar molecules (Gauch is still viable and one may point out that and Dugger, 1953; Sidhu and Malik, 1986. pollen longevity on the vector body even at In vitro germination measures pollen the right location to meet the stigma may germinability under the specific conditions also be a crucial factor in pollination of the medium and temperature conditions efficiency (Dafni and Firmage 2000). reveals the state of the reserves, the condition of the membranes and the sub The pronounced effect of sucrose and boric sequent rate of reserve conversion (Heslop- acid on germinating pollen might be Harrison et al. 1984). reflected with the views of Johri and Vasil (1961). The induced role of Calcium and Shivanna and Johri (1989) stated that the boron on in vitro pollen germination was externally supplied sucrose maintains the reported by Brewbaker and Kwack (1964). osmotic pressure and acts as a substrate for The role of boron in flowering and fruiting pollen metabolism. The role of boron has process has been established (Brown et al., been confirmed in germinating pollen and 1994) and its deficiency resulted in low growing pollen tubes in vascular plants pollen viability, poor pollen germination and (Lewis, 1980; Sidhu and Malik, 1986). The reduced pollen tube growth (Nyomora and studies of Stanley and Loewus (1964) Brown, 1997). Boron takes part in pollen indicated that boron is directly involved in germination and style tube formation and pectin synthesis and thus indirectly involved therefore has a vital function in fertilization in development of pollen tube membrane. of flowering crops. Boron added in the form Scott (1960) suggested that boron could of boric acid, is also essential for the in vitro exert a protective effect in preventing culturing of pollen from most species and it excessive polymerization of sugars at sites is also reported that elimination of boric acid of sugar metabolism. In nature water, sugar from the culture medium often leads to tube 770 Int.J.Curr.Microbiol.App.Sci (2016) 5(1): 768-777 bursting (Holdaway-Clarke and Hepler, wall of pollen tubes in Picea meyeri. Acar et 2003; Acar et al., 2010).Wang et al., (2003) al. (2010) also reported the stimulatory studied the effect of boron on the effect of boron on in vitro pollen localization of pectins and callose in the germination of Pistacia vera.