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BIO CHEMICAL AND MOLECULAR BASIS OF POLLEN GERMINATION
- 1. BIO CHEMICALAND MOLECULAR BASIS OF POLLEN GERMINATION Seminar-II SST-681 1 WELCOME
- 2. 2 Introduction Pollination and methods to detect pollen Pollen and its cytochemistry Stage and steps Biochemical and molecular key factors for pollen germination Case studies Conclusion
- 3. Pollen- Powdery substance consisting of pollen grains which are male micro gametophytes (2n) of seed plants, which produce the male gametes (sperm cells) Transmit nuclear genetic material Tiny grains with greater importance in plant life cycle Study of pollens / spores is commonlycalled Palynology Moisture level is < 20 % Size - 10 -100 µm Shape- Round, oval, disc, filamentous Texture- From smooth to spiky Colour- White, cream, yellow or orange INTRODUCTION 3 Pollen production - Stigma - Pollen - Pistil interaction - Fertilization - Seed set Franchi et al., 2011
- 4. History 1760’s (J.G. Kolreuter) - Pollen morphology 1820’s (Amici) - Pollen germination in stigmatic tissue of Portulaca 1830’s (Lister and Julius)-studied structure 1840’s (John Lindley)- Pollen characters 1970’s (Yang et al.) - Development of pollen tube 1980’s (Zec and Xu) - Development of male organs in rice sexual reproduction 1990’s (Tian et al.) - Role of calcium during pollen tube growth 4
- 6. Pollination Pollination- Processof transfer of pollen from the male anther to the female stigma Abiotic pollination Biotic pollination Self Pollination Cross pollination Pollen grains adhering to a compatible stigma respond to signals from the mother plant and become active, undergoing cytoplasmic reorganization and activation of stored RNA and protein to produce a pollen tube originating from the vegetative cell. 6
- 7. Pollen morphology 7 Crop Size (in µm) Colour Shape Maize 80-125 (Speed- 0.2m/s) Pale yellow Egg Sunflower 30-32 Orange/yellow/ white cream Elongated ball Paddy 39-40 Yellow/pale white Round Green gram 28-30 Red/pink Aborted pollen- Blue/green Round
- 8. Detection methods of pollen Acetocarmine- Presence of cytoplasm Alexander’s stain test- Differentiate aborted and non aborted pollen DAPI (4,6-diamidino-2-phenylindole) staining- Viability Aniline blue- Detects callose wall and pollen tube IKI (Iodine potassium Iodide)-Viability and starch content NBT (p-nitro blue tetrazolium) - Viability MTT (2, 5-diphenyletetrazolium bromide) - Viability TTC (2, 3, 5-triphenyletetrazolium chloride)- Study living tissues In vitro pollen germination Test-germination Enzyme Assay Method- Viability Tetrazolium Test- Viability FDA- Viability and germination In-Vivo Pollen Germination and Pollen Tube Growth 8 (Rathod et al., 2018)
- 9. Pollen formation Pollen Germination How pollen forms and germinates 9
- 10. Schematic representation of a rice flower 10 (Moon and Jung, 2020)
- 12. It is complex in nature Composed of several layers. Intine: Innermost, cellulose wall. Exine: Outer layer Chemically resistant biopolymer sporopollenin Further divided into sexine and nexine Aperture Region on the surface where exine deposition is reduced or absent Regulates the rate of water entry Site of exit for pollen tubes during germination (Shi et al. 2010) 12 Pollen wall
- 13. Cytochemistry of mature pollen Pollenkit/ Tryphine/Pollencoat Oily substance Contains carotenoids Mixture of lipids, waxes, flavonoids and proteins Derived from degeneration of the tapetum, Exine Composed-Sporopollenin Highly resistant biopolymer-fatty acids,carotenoids,phenolics Outside exine layer the wall that contains lipid, protein and other material deposited from the tapetum of the anther. Intine Consists of cellulose, pectins and hemicellulose and callose (Nepi et al. 1999) 13
- 14. Reserves in Mature Pollen Grains: Carbohydrates- Synthesis of pollen tube wall Sterol esters, free fatty acids triglycerides & fatty alcohols-Pollen coat development Glycerophospholipids - intosol, glycerol-Plasma lemma 14
- 15. • Adhesion of pollen to the stigma • Pollen-hydration • Pollen polarization and germination: preparing for pollen tube growth • Pollen tube invasion: Growing into style Stages 15
- 16. Pollen Germination Hydration causes changes in water content, which trigger pollen germination Consequently, vegetative cell germinates to produce pollen tube Compatible pollination - Pollen grain germinates and extrudes pollen tube upon landing on stigmatic surface Incompatible pollen - Arrested at stigma for pollen tube elongation (Yu-jin kim et al., 2018) 16
- 17. Ca & K signaling Essential for pollen tube growth Accumulation at pollen tip Pollen tube emerges after hydration and calcium influx takes place at the pollen tube tip. If there is no gradient in the pollen grains, there is no rupture after hydration, and germination is inhibited.
- 18. Model of cellular responses to compatible and incompatible pollen 18 Compatible pollen Incompatible pollen
- 19. Structure of the pistil, pollination events and components 19 (Wheeler et al., 2001)
- 20. Pollen tube journey and signalling events involved in tube reception 20 (Dresselhaus and Tong, 2013)
- 21. Model showing male female cross talk and Pollen tube journey SR1 EA1 . GABA, D-serine ES4 EC1 repulsion signals 21 (Dresselhaus and Tong, 2013)
- 22. Biochemical and molecular key factors in pollen germination Protein synthesis begins Pollen-specific genes-Lepro,OsMS188 In maize - 43% & 52–70% genes In rice –genes in late-maturing pollen Proteomic analysis-membranne proteins in cell polarity Biochemical analysis of mRNAs, ribosomes, and tRNAs (Kim et al. 2018) 22
- 23. CASE STUDIES 23
- 24. Objective: In the anther, competence of pollen to germinate & to produce pollen tubes and in the pistil, competence to support pollen germination & tube growth was observed Material and methods: Plant material- RLD and C24 strains TEM, Fluorescence Cont.. NAAS-11.76 IF-5.611 24 (Kandasamy et al., 1994)
- 25. Path of pollen tube growth through a mature pistil of Arabidopsis Cont.. 25 (Kandasamy et al., 1994)
- 26. TEM of the pollen-papillar cell interface Cont.. Typhine 26 (Kandasamy et al., 1994)
- 27. TEM analysis of pollen tube growth through the papillar cell wall and the transmitting tissue of the stigma Cont.. Adhesion zone 27 (Kandasamy et al., 1994)
- 28. TEM analysis of pollen tube growth through the papillar cell wall and the transmitting tissue of the stigma, 30 minutes after pollination Cont.. 28 (Kandasamy et al., 1994)
- 29. Microspore competence in developing anthers Cont.. 29 (Kandasamy et al., 1994)
- 30. Inference: Cytological changes were evident that within 5 minutes after pollen capture Establishment of polarity within pollen grain and pollen tube emergence occurred within 15 minutes after pollination 30
- 31. Objective: Role of a Shaker K+ channel OsAKT1.2 in rice pollen germination and growth Material and methods: Plant materials and growth conditions Gene expression analysis In vitro pollen analysis-Alexander & DAPI staining Aniline blue staining for observation of pollen germination in vivo Cont.. NAAS rating-8.83 IF-3.013 31 (Fan Yang et al., 2020)
- 32. Tissue expression patterns K+ channel genes Cont.. 32 Fan Yang et al., 2014
- 33. Observation of mature pollen grains Cont.. Pollen germination in vitro 33 Fan Yang et al., 2014
- 34. K+ is an essential cation for pollen germination and tube growth Reported that plasma membrane localized K+ channel OsAKT1.2 is required for pollen germination and tube growth 34 Inference:
- 35. Objective: Pollen’s germinating potential and length of growing pollen tubes at two temperatures: 18°C and 4°C. Material and methods: Pollen material Pollen germination assay Observations made on length of pollen tube Observations made on length of style and anther Cont.. NASS-7.42 IF-1.490 35 (Padureanu and Patras 2020)
- 36. Pollen germination (%) in Galanthus nivalis after 1.5–120 hours from inoculation Cont.. 36 (Padureanu and Patras 2020)
- 37. Length of pollen tube (μm) in Galanthus nivalis after 1.5 -120 h from inoculation Cont.. 37 (Padureanu and Patras 2020)
- 38. Pollen germination was optimum (> 90 %) on nutritive mediums containing 15 % and 20 % sucrose, at both studied temperatures 18 °C and 4 °C after 24 h. Our results suggest that in natural conditions, the pollen tubes which may fertilize the ovules are those formed on a stigmatic liquid with 10–25 % sucrose at 18 °C and 10–15 % sucrose at 4 °C. 38 Inference:
- 39. Objective: Role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Material and methods: • Plant growth and in vitro pollen germination • Immunolocalization of pollen tubes Cont.. 2020 NAAS-10.18 IF-4.556 39 (Kim et al., 2020)
- 40. The effect of PME and Polyphenon 60 treatments on rice pollen germination and tube growth. Cont.. 40 (Kim et al., 2020)
- 41. Immunodetection of pectins upon pollen germination Cont.. 41 (Kim et al., 2020)
- 42. Immunodetection of pectins during pollen tube development Cont.. 42 (Kim et al., 2020)
- 43. Evidence showing the essentiality of HGA status during the germination and elongation of pollen tubes, which is primarily governed by the finetuning of PME and PMEI activities. 43 Inference:
- 44. Objective: Growth dynamic of rice pollen tube & growth rate of pollen tube in stigma and ovary was investigated with a fluorescence microscopy. Material and methods: Plant materials-Yangdao 6 and YW-2S Observation of pollen grain germination in vitro in potato medium Observation of pollen tube growth in pistil under a fluorescence microscope. Examination of seed setting when stigma is removed at different times after pollination. Cont.. NASS-8.37 IF-2.680 44 (Shi-quiang et al., 2008)
- 45. Germination of pollen grains in the potato culture medium at 30°C at 0, 2, 4, 6, 8, 10 min Cont.. 45 (Shi-quiang et al.,2008)
- 46. Growth of rice pollen tubes observed with a fluorescence microscope at 2, 5, 10, 20, 30, 40, 45, 50, 60 minutes after pollination Cont.. 46 (Shi-quiang et al.,2008)
- 47. Number of fertilized florets and seed setting rate Cont.. Comparison on the pollen tube growth between wheat and rice 47 (Shi-quiang et al.,2008)
- 48. Pollen tubes growing in the whole pistil at 40 min after pollination Seed setting rate was quite low when stigma was removed at 10–15 min. after pollination, increased at 20 - 50 min after pollination Over 60% seed set when it removed at 50 min after pollination and finally tended to be stable 48 Inference:
- 49. Objective: Biological role of LePro1 during pollen development Materials and methods: Plant materials- cv. Money maker RNA gel blot analysis Pollen germination and morphological analysis Cont.. NASS-8.78 IF-2.870 49 Yu et al., (2014) ,2014
- 50. Temporal and spatial expression of Lepro1 during pollen development Cont.. 50 Yu et al., (2014)
- 51. RNA gel blot analysis of the Lepro1 transcript in pollen grains of wild type Immunoblot analysis of total soluble proteins extracted from pollen grains of wild type Cont.. 51 Yu et al., (2014)
- 52. Cont.. Ambient temperature SEM and low temperature SEM images of pollen Invitro germinated wildtype and antisense pollen 52 Yu et al., (2014)
- 53. Comparison of seed-setting among LePro1 sense, antisense and wild type plants Seeds Cont.. Comparison of invivo germination of wild type and antisense pollen. 53 Yu et al., (2014)
- 54. Using antisense RNA, successfully knocked down the expression of LePro1 in tomato plants Two antisense lines, A2 and A3 showing significant down-regulation of LePro1 in pollen resulting in poor pollen germination and abnormal pollen tube growth 54 Inference:
- 55. Objective: Study the role of OsMS188 in tapetum and cell wall of pollen Material and methods: •Plant materials-cv.nippanbare •TUNEL assay-Spikelets were fixed in FAA solution •RT-PCR and qRT-PCR NASS-9.51 IF-3.840 55 (Han et al., 2021)
- 56. Gene structure and the editing site of OsMS188 & Alexander staining of spikelets, mature anthers 56 (Han et al., 2021)
- 57. Electron micrographs of anthers and pollen grains 57 (Han et al., 2021)
- 58. TUNEL analysis in the anthers of WT and osms188 58 (Han et al., 2021)
- 59. Overall, OsMS188 plays multiple roles during anther development, including tapetum development, pollen wall formation and anther surface formation 59 Inference:
- 60. Composition and localisation of different cell wall polymers or proteins based on bio chemical analysis will helps to study the pollen biology and germination Genes like OsAKT1.2 & OsMS188 in rice and Lepro1 in tomato will helps in studying the pollen germination and pollen tube development Better molecular understanding of pollen tube initiation and guidance will help to overcome hybridization barriers between genotypes and even between species to improve the gene pool for breeding Gene regulatory network established may facilitate future investigations CONCLUSION 60
- 61. THANK YOU 61
Editor's Notes
- Submitted By: G.ADHITHYA PALB-6091 II Ph.D DSST.
- Schematic representation of a rice flower. (A) Flower before anthesis; (B) flower at anthesis; (C) Transverse image of an anther at the mature stage; (D) transverse image of an anther at anthesis. Rapid pollen swelling is the driving force behind the rupture of the anther wall. Red arrows indicate the pressure caused by swollen mature pollen grains; (E) pollen before landing on the stigma; (F) pollen after landing on the stigma. The pollen coat is mobilized on the stigma to form a “pollen foot” in rice. Numerous membranous inclusions appear on the stigma. The illustration of structural changes that occur at the point of adhesion between pollen and stigma is based on observations from Arabidopsis. Cu & Pe—cuticle and proteinaceous pellicle; CV—central vacuole; CW—cell wall; En—endothecium; Ex—exine; FL—foot layer; In—intine; GP—germination pore; MP—mature pollen; PC—pollen coat; PT—pollen tube; SG—starch granule; Se—septum; Sg—stigma; SP—swollen pollen; St—stomium; Vs—vesicle. 2. Pollen Swelling Is a Key Event During Anther Dehiscence in Rice Anther dehiscence is an essential process for the release of mature pollen for pollination and fertilization. Three anther tissues, the endothecium, septum and stomium, play important roles during anther dehiscence in both Arabidopsis and rice (Figure 1C,D) [12,13]. Secondary wall thickening of the endothecium generates the tensile force necessary to rupture the stomium during anther wall dehydration [14–16]. The septum is located between the vascular bundles and two adjacent anther locules [13]. The stomium comprises a single layer of specialized epidermal cells, which has been weakened by the action of hydrolytic enzymes and is the final breakage site for anther dehiscence [13,14,17]. Several genes regulating anther dehiscence have been identified, including auxin response factor17 (ARF17), MYB26, MYB108, NAC secondary wall-promoting factor1 (NST1), NAC secondary wall-promoting factor2 (NST2), FT-interacting protein 7 (OsFTIP7), OsYUCCA4 and homeobox1 (OSH1) [18–21]. Auxin negatively regulates endothecium lignification and jasmonic acid biosynthesis [22,23]. In addition, irregular xylem1 (IRX1), receptor-like protein kinase 2 (RPK2), teosinte branched1, cycloidea, PCF (TCP24), Arabidopsis histidine-containing phosphotransfer factor 4 (AHP4), secondary wall thickening-associated F-box 1 (SAF1), cystathionine β-synthase domain-containing protein (CBSX2), anther dehiscence repressor (ADR) and SUMO E3 ligase1 (SIZ1) are reported to be involved in endothecium thickening, and mutations in these genes result in non-dehiscent anthers [12,16,24–29]. MYB21, MYB24 and jasmonate resistant 1 (OsJAR1) function in jasmonic acid-mediated anther dehiscence [30,31]. Jasmonic acid controls stomium breakage during anther dehiscence. All of these genes are involved in the biomechanical changes that occur in the anther walls to elicit successful pollen release. In rice, the pollen itself plays important roles in anther dehiscence [32]. The rapid swelling of pollen grains drives the septum and stomium to rupture (Figure 1A,B) [32]. Increased pollen pressure results in the locule to bulge, resulting in the rupture of the septum, which has already been weakened by the action of hydrolytic enzymes (Figure 1D) [13,17]. Pollen pressure combined with
- Structure of the pistil, pollination events, and components identified as playing a role in pollination. The left-hand side of this figure shows a generalized basic pistil structure (indicated in green, labelled in red). The basic steps in the pollination process are indicated in blue. The boxes indicate some of the components (genes, gene products and mutants) identified as being important in pollination.
- The cartoon shows a generalized flower, with the major stages of pollen interactions with the pistil. Pollen grains (yellow) are shed from the anthers and land on a suitable stigma. They adhere, hydrate, and germinate on the stigma (Phase I). Pollen tubes (yellow) invade the stigma (Phase II) and travel during Phase III, using tip growth, through the pistil tissue transmitting tract (TT, pale pink), which secretes the extracellular matrix (ECM). During the final stages, it negotiates various layers of tissues comprising the ovary (pale green) in Phase IV and is guided to the micro-pyle, helped by signals from the synergid cells (Phase V). It enters the female gametophyte (green) and interacts with the egg apparatus, which comprises the egg (turquoise) and two synergid cells (orange) in most angiosperms. Here, growth is arrested and the pollen tube tip bursts. This releases the two sperm cells, which can perform double fertilization with the egg cell and central cell (dark pink). Sperm cell nuclei are indicated in red. To the right, the colored boxes indicate the various phases (Phases I–V) of the pollen tube pathway, with some of the female and male components identified as being involved in regulating pistil–pollen interactions at particular stages
- Ovular pollen tube guidance: small highly diffusible signaling molecules (e.g. GABA, D-serine, and NO; drawn as blue dots) are generated by ovule tissues, especially the tips of the integuments, to trigger exit of pollen tubes from ovary tissues towards the micropylar region of the ovule. Integument tips express corresponding biosynthesis genes like SR1 and POP2 (indicated in blue).(B) Micropylar guidance (ovary tissue removed): less diffusable polymorphic peptides/small proteins (LUREs, EA1; orange dots) are secreted mainly by the synergid cells to guide pollen tubes inside the female gametophyte.(C) Pollen tube perception includes induction of synergid cell death, pollen tube burst (stimulated by ES4; green dots), and sperm delivery at the female gamete boundary. Sperm cells are activated by EC1 (yellow dots) and repulsion signaling is initiated (unknown repulsion signals, small violet dots). Note that receptive synergid and pollen tube cell nuclei degenerate.(D) Prevention of polyspermy: successful fertilization of egg and central cell induces death of the second synergid cell (indicated by arrows to skull and crossbones), which removes the micropylar pollen tube guidance cues. Repulsion signaling superimposes remaining pollen tube attraction and burst molecules, thus preventing attraction of secondary pollen tubes and sperm release. APs, antipodal cells; CC, central cell; dSY, degenerated synergid cell; EC, egg cell; II, inner integument; OI, outer integument; SY, synergid cell; Z, zygote.
- Biochemical analysis of mRNAs, ribosomes, and tRNAs required for germination are synthesized during pollen maturation and persist in the pollen grain until they are utilized for translation during the germination process. Protein synthesis begins rapidly once pollen germination is initiated. Single ribosomes, mRNAs, ribosomes and tRNAs aggregate into polysomes within 2 minutes Pollen transcriptomes contain fewer expressed genes than other plant tissues, but comprise large numbers of genes that are pollen-specific or upregulated in pollen compared with sporophytic tissues. In maize, 43% of genes were highly expressed in mature pollen and 52–70% of genes were expressed in other reproductive and vegetative tissues. In rice, genes expressed in late-maturing pollen are transcribed or translated for use during pollen germination and tube growth. Proteomic analysis has validated that PM proteins were involved in signal transduction, transport, cell wall remodeling and metabolism, and membrane trafficking. Quantitative proteomic analysis of sterol rich PM microdomains, which play a key role in directing cell polarity in the pollen aperture and at the tip of germinating pollen tubes. Transcriptomic and proteomic studies have found that transcripts implicated in cell-wall metabolism, signaling, and cytoskeletal dynamics are synthesized during late pollen development and are stored until pollen germination.
- (A) Real-time quantitative RT-PCR detection of the expression of eleven K+ channel genes in rice pollen. (B) The tissue expression patterns of OsAKT1.2, OsAKT1, OsKAT3, OsGORK and OsSKOR were detected by semi-quantitative RT-PCR. Total RNA was isolated from root, stem, leaf, seed, pistil, uninucleate microspore (UNM), bicellular pollen (BCP), tricellular pollen (TCP), mature pollen grain (MPG) and germinated pollen grain (GPG).
- (A) Alexander staining of wildtype (WT), osakt1.2 and osakt1.2a pollen grains and the percentage of viable pollen grains. (B) DAPI staining of WT, osakt1.2 and osakt1.2a pollen grains and the percentage of tricellular pollen grains. Scale bar, 50 μm.
- The present research studies the pollen biology of snowdrop (Galanthus nivalis L.), from the point of view of pollen’s germinating potential and length growth of pollen tubes at two temperatures: 18 °C and 4 °C. The germination was conducted “in vitro” on nutritive mediums containing different concentrations of sucrose (0, 5, 10, 15, 20, 25, 40, 50, 70, 100%). The pollen germination was optimum (more than 90 %) on nutritive mediums containing 15 % and 20 % sucrose, at both studied temperatures, after 24 h. In parallel with germination potential, the adaptation of male gametophytes to the two temperatures was analyzed. The longest pollen tubes were formed on 10–25% sucrose mediums at 18 °C and on 10–15% sucrose mediums at 4 °C, and they were maintained also after 120 h since pollen inoculation. For these concentrations, the pollen tube length was similar at both temperatures. In conclusion, 18 °C is suitable for long tubes formation using a largest range of sucrose concentration than 4 °C. In fact, in most plant species, low temperatures not only inhibit pollen tube growth, but also induce flowers abortion. Present results bring new evidences that the snowdrop’s male gametophyte is genetically settled to have a normal development at low temperatures. The optimal germination rate and pollen tube growth at 4 °C highlight the vernal character of the pollen of G. nivalis.
- TheeffectofPMEandPolyphenon60treatmentsonricepollengerminationandtubegrowth. In vitropollengerminationofricepollenwereanalyzedontheliquidgerminationmediawithout(A,D) and with exogenous PME (B,C,E–G) and Polyphenon 60 (H–M). The concentration was indicated in eachimage. Redarrowsindicatepollengrainswhichruptureearlywithoutformingintactpollentubes. Bars = 200 µm (A–C,H–J) and 50 µm (D–G,K–M). (N,O) The percentage of germinated pollen grain with an intact tube was presented. Red arrow marks pollen grains with burst or disintegrated tubes. Three independent experiments + SD (n > 100).
- Pollen grains were probed with the monoclonal antibodies LM19 and LM20. LM19 is used to indicate pectins with a low level of methylesterificationwhereasLM20bindstopectinswithahighlevelofmethylesterification. (A)Control: LM19-labelingsignalsuponpollengermination. (B)LM19-labelingsignalsofPME-treatedpollengrain. (C)LM19-labelingsignalsofthePolyphenon60-treatedpollengrain. (D)Control: LM20-labelingsignals uponpollengermination. (E)LM20-labelingsignalsofthePME-treatedpollengrain. (F)LM20-labeling signals of the Polyphenon 60-treated pollen grain. Moderate concentration of PME (2 U/mL) was added onto the germination medium. (G) Quantification analysis of LM19 labeling. (H) Quantification analysis of LM20 labeling. FI, fluorescence intensity. All error bars represent SD of at least twenty independent experiments. Student’s t test: ** p < 0.01. Scale bars = 5 µm.
- Pollen tubes were probed with the monoclonal antibodies LM19 and LM20. LM19 is used to indicate pectins with a low level of methylesterificationwhereasLM20bindstopectinswithahighlevelofmethylesterification. (A)Control: fluorescent signals of dimethyl-esterified pectin. (B) Fluorescent signals of dimethyl-esterified pectin of the PME-treated pollen tube. (C) Fluorescent signals of the dimethyl-esterified pectin of Polyphenon 60-treated pollen tube. (D) Control: fluorescent signals of methyl-esterified pectin. (E) Fluorescent signals of methyl-esterified pectin of the PME-treated pollen tube. (F) Fluorescent signalsofmethyl-esterifiedpectinofthePolyphenon60-treatedpollentube. Moderateconcentrationof PME (2·U/mL) was added onto the germination medium. (G) Quantification analysis of fluorescent signals of dimethyl-esterified pectin on the pollen tube tip and shank. (H) Quantification analysis of fluorescent signals of the methyl-esterified pectin on the pollen tube tip and shank. FI, fluorescence intensity. All error bars represent SD of at least twenty independent experiments. Student’s t test: ** p < 0.01. Scale bars = 5 µm.
- A, Pollen grains at 0 min after cultured in the potato medium; B, Most pollen grains germinated 2 min later; C, The length of most pollen tubes up to the diameter of pollen grain 4 min later; D, The length of pollen tubes exceeded twice of the diameter of pollen grain 6 min later; E, The tips of pollen tubes had already enlarged 8 min later; F, The tips of pollen tubes had broken 10 min later.
- A, Pollen grain germinated at 2 min after pollination; B, Pollen tube was longer than the diameter of pollen grain 5 min later; C, Most pollen tubes grew into the style 10–15 min later; D, Pollen tubes growing in the ovary 25 min later; E, In ovary the tip of pollen tube enlarged 30 min later; F, A pollen tube got through the micropyle 40 min later; G, Lots of pollen tubes abnormally enlarged and stopped growing 40 min later; H, Pollen grains were shriveled and their color under the fluorescence microscope changed 50 min later; I, Pollen tubes growing in the whole pistil at 40 min afterpollination.