Influence of Plant Growth Regulators on Pollen Germination and Growth of Xinjiang Apricots

[Objective] This study aimed to investigate the influence of different plant growth regulators on apricot pollen germination and pollen tube growth. [Method] Pollens of six kinds of Xinjiang apricots were cultured in solid media supplemented with five plant growth regulators （GA3 , NAA, 2, 4-D, 6-BA, IAA）. Then the rate of pollen germination and the length of pollen tube were respectively measured. [Result] In a certain concentration range, GA3 most significantly promoted the pollen germination and the pollen tube growth of Shushanggan, Kalayulvke, Dayoujia, Yiliakeyulvke and Kabakehuanna; NNA had the strongest improvement function on Kumaiti’s pollen germination and pollen tube growth. [Conclusion] All the five plant growth regulators promoted the pollen germination and the pollen tube growth of apricots at low concentration but inhibited them at high concentration.

BRASSICA DE-EXINED POLLEN AS A NEW EXPERIMENTAL SYSTEM FOR STUDYING POLLEN GERMINATION

Exine deprived pollen only coated with inline has been isolated and artificially germinated in Brassica L. In view of the lack of exine and germ furrows, the de-exined pollen is an interesting experimental system for studying pollen germination. This report focuses on the microscopic and ultrastructural changes in the aspects of polarization, predetermination of the germination site and new wall synthesis during the course of isolation and germination of the de-exined pollen. It was shown that the de-exined pollen was already in an active state and had established its polarity and germination sites prior to exine detachment. The germination sites were still localized at the region of the previous germ furrows even after exine detachment. The new wall deposited at the germination sites appeared to have an important morphogenetic role in setting a limit to the size of pollen tube diameter . These results support our assumption that the de-exined pollen may have an extensive application in the research of pollen biology.

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcll is male sterile as a result of the failure ofpollen germination. We show that the bcll mutant allele cannot be transmitted by male gametophytes and no homozygous bcll mutants were obtained. Analysis of pollen developmental stages indicates that the bcll mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting （VPS） in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.

Influence of Fungicides on Almond Pollen Germination and Fruit Set

The almond （Prunus dulcis） is a self-incompatible species that requires various orchard management techniques to encourage pollination and achieve a good fruit set. Fungicides are commonly applied to almond flowers to control fungaI infections, such as ＂blossom blight＂ and ＇brown rot＂ that damage the flowers and developing fruits. However there is evidence that the application of some of these products may adversely affect pollination and fruit set. The effects of the fungicides captan, chlorothalonyl, propiconazole, mancozeb and dichloran on fruit set were studied in an orchard using ＂Nonpareil＂ and ＂Carmel＂ almond trees. The effects on in vitro pollen germination and pollen tube elongation were studied using ＂Carmel＂ pollen. With respect to Non Pareil fruit set, all fungicidal treatments were statistically similar to the control （11.5%） but being propiconazole （13.1%） and chlorotalonil （5.6%） different between them. Fruit set for ＂Carmel＂ was significantly lower than the control （26.1%） with all fungicidal treatments. After 24 hours, in vitro ＂Carmel＂ pollen germination was significantly higher in the control （90.5%） compared with the fungicidal treatments, with the exception of chlorothalonyl （70.2%）. Pollen tube growth in the control was four times greater than in the fungicide treatments at 24 hours, none of which were significantly different from the other treatments. It can be concluded that the application of certain fungicides has a clearly detrimental effect on fruit set in ＂Nonpareil＂ and ＂Carmel＂ almond trees and on ＂Carmel＂ pollen activity.

Pollen Germination and Pollen Tube Elongation of Tomato （Lycopersicum esculentum L.） Regulated by Cell Wall Invertase through Sucrose Hydrolysis

Sucrose is a sugar required for pollen germination and pollen tube elongation. It is little known on the regulation mechanism. As such, this research was conducted to reveal mechanism pathway of the sugar in regulating pollen germination and pollen tube elongation by cell wall invertase. The pollen grains, respectively originated from wild type and transgenic tomato plants, which had been silenced their inhibitor gene （INVINH1） of the cell wall invertase were used in this study. The pollen grains were cultured in media containing glucose, fi＇uctose or sucrose. Results of the study showed that percentage of the pollen germination derived from transgenic plants was much higher than that from wild type plants. Moreover, pollen tube elongation was longer in transgenic plants compared with wild type plants. Interestingly, these results were observed in medium containing sucrose only, but not in glucose and fructose. This result suggests that cell wall invertase activity regulates pollen germination and pollen tube elongation through sucrose hydrolysis.

Screening for High-Temperature Tolerant Cotton Cultivars by Testing In Vitro Pollen Germination,Pollen Tube Growth and Boll Retention

With radical global climate change and global warming, high temperature stress has become one of major factors exerting a major Influence on crop production, In the cotton （Gossyplum hirsutum L.）-growlng areas of China, especially in the Yangtze River valley, unexpected periodic episodes of extreme heat stress usually occur In July and August, the peak time of cotton flowering and boll loading, resulting In lower boll set and lint yield. Breeding programs for screening high temperature-tolerant cotton germplasm and cultlvars are urgent In order to stabilize yield in the current and future warmer weather conditions. In the present study, 14 cotton cultivars were quantified for in vitro pollen germination and pollen tube growth in response to temperatures ranging from 10 to 50 ℃ at 5 ℃ intervals. Different cotton genotypes varied In their in vitro pollen germination and pollen tube length responses to the different temperatures. Maximum pollen germination and pollen tube length ranged from 25.2% to 56.2% and from 414 to 682 pro, respectively. The average cardinal temperatures （Tmin,, Topt, and Tmax） also varied among the 14 cultivars and were 11.8, 27.3, and 42.7 ℃ for pollen germination and 11.8, 27.8, and 44.1 ℃ for maximum pollen tube length. Variations In boll retention and boll numbers per plant in field experiments were found for the 14 cotton cultivars and the boll retention and boll retained per plant on 20 August varied considerably In different years according to weather conditions. Boll retention on 20 August was highly correlated with maximum pollen germination （R^2 = 0.84） and pollen tube length （R^2=0.64）. A screening method based on principle component analysis of the combination of pollen characterlatics In an in vitro experiment and boll retention testing In the field environment was used In the present study and, as a result, the 14 cotton cultlvars could be classified as tolerant, moderately tolerant, moderately susceptible and susceptible to high temperature.

Higher-Ordered Actin Structures Remodeled by Arabidopsis ACTIN-DEPOLYMERIZING FACTOR5 Are Important for Pollen Germination and Pollen Tube Growth

Dynamics of the actin cytoskeleton are essential for pollen germination and pollen tube growth. ACTIN- DEPOLYMERIZlNG FACTORs （ADFs） typically contribute to actin turnover by severing/depolymerizing actin filaments. Recently, we demonstrated that Arabidopsis subclass III ADFs （ADF5 and ADF9） evolved F-actin-bundling function from conserved F-actin-depolymerizing function. However, little is known about the physiological function, the evolutional significance, and the actin-bundling mechanism of these neo- functionalized ADFs. Here, we report that loss of ADF5 function caused delayed pollen germination, retarded pollen tube growth, and increased sensitive to latrunculin B （LatB） treatment by affecting the generation and maintenance of actin bundles. Examination of actin filament dynamics in living cells revealed that the bundling frequency was significantly decreased in adf5 pollen tubes, consistent with its biochem- ical functions. Further biochemical and genetic complementation analyses demonstrated that both the N- and C-terminal actin-binding domains of ADF5 are required for its physiological and biochemical functions. Interestingly, while both are atypical actin-bundling ADFs, ADF5, but not ADF9, plays an important role in mature pollen physiological activities. Taken together, our results suggest that ADF5 has evolved the function of bundling actin filaments and plays an important role in the formation, organization, and maintenance of actin bundles during pollen germination and pollen tube growth.

Arabidopsis AtVPS15 Plays Essential Roles in Pollen Germination Possibly by Interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells. The knowledge about the function of its homologue in plants remains limited. Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis, plays an essential role in pollen germination. Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants. Reciprocal crosses between atvps15 mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes. DAPI staining, Alexander＇s stain and scanning electron microscopic analysis showed that atvps15 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen, whereas in vitro germination experiments revealed that germination of the pollen grains was defective. GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPS15 was mainly expressed in pollen grains. Finally, DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34. These results suggest that AtVPS15 is very important for pollen germination, possibly through modulation of the activity of PI3-kinase.

Stigmatic exudate in the Annonaceae:Pollinator reward,pollen germination medium or extragynoecial compitum?

Although ＂dry-type＂ stigmas are widely re- garded as ancestral in angiosperms, the early-divergent family Annonaceae has copious stigmatic exudate. We evaluate three putative functions for this exudate： as a nutritive reward for pollinators; as a pollen germination medium; and as an extragynoecial compitum that enables pollen tube growth between carpels. Stigmatic exudate is fructose dominated （72.2%）, but with high levels of glucose and sucrose; the dominance of hexose sugars and the diversity of amino acids observed, including many that are essential for insects, support a nutritive role for pollinators. Sugar concentration in pre-receptive flowers is high （28.2%）, falling during the peak period of stigmatic receptivity （17.4%）, and then rising again toward the end of the pistillate phase （32.9%）. Pollen germination was highest in sugar concentrations 〈2%. Sugar concentrations during the peak pistillate phase therefore provide optimal osmolarity for pollen hydration and germination; subsequent changes in sugar concentration during anthesis reinforce protogyny （in which carpels mature before stamens）, enabling the retention of concentrated exudate into the staminate phase as a pollinator food reward without the possibility of pollen germination. Intercarpellary growth of pollen tubes was confirmed： the exudate therefore also functions as a suprastylar extragynoecial compitum, overcoming the limitations of apocarpy.

Arabidopsis Synaptotagmin 2 Participates in Pollen Germination and Tube Growth and Is Delivered to Plasma Membrane via Conventional Secretion

Arabidopsis synaptotagmin 2 （SYT2） has been reported to participate in an unconventional secretory pathway in somatic cells. Our results showed that SYT2 was expressed mainly in the pollen ofArabidopsis thaliana. The pollen of syt2 T-DNA and RNA interference mutant lines exhibited reduced total germination and impeded pollen tube growth. Analysis of the expression of SYT2-GFP fusion protein in the pollen tube indicates that SYT2 was localized to distinct, patchy compartments but could co-localize with the Golgi markers, BODIPY TR C5 ceramide and GmManl-mCherry. However, SYT2-DsRed-E5 was localized to the plasma membrane in Arabidopsis suspension cells, in addition to the Golgi apparatus. The localization of SYT2 at the plasma membrane was further supported by immunofluorescence staining in pollen tubes. Moreover, brefeldin A treatment inhibited the transport of SYT2 to the plasma membrane and caused SYT2 to aggregate and form enlarged compartments. Truncation of the SYT2-C2AB domains also resulted in retention of SYT2 in the Golgi apparatus. An in vitro phospholipid-binding assay showed that SYT2-C2AB domains bind to the phospholipid membrane in a calcium-dependent manner. Take together, our results indicated that SYT2 was required for pollen germination and pollen tube growth, and was involved in conventional exocytosis.

Ultrastructural Changes of Vegetative Cells in Amaryllis Pollen during Its Germination

Structural and organizational changes of the vegetative cell in Amaryllis vittata Ait. during such dynamic processes of pollen as hydration, activation and germination have been examined with electron microscopy. The mature pollen grain is composed of such organelles as plastids, mitochondria,endoplasmic reticulum, dictyosomes and lipid bodies which are in their resting state. Microfilaments appear as aggregates. After pollen activation, however,the organelles undergo great changes in number and shape: the lamellae of plastids and the cristae of mitochondria increase conspicuously in number, the cisternae of the endoplasmic reticulum become narrower; the dictyosomes produce vesicles actively the lipid bodies become degraded and the microfilament aggregates disperse. Cortical microtubules and spiny vesicles appear in the cytoplasm after germination of the pollen tube. No apparent structural changes of the organelles were noticed any longer during this period and microfilaments are distributed throughout the entire pollen tube as a three-dimensional network.

Pollen Grain Germination and Pollen Tube Growth in Pistil of Rice

The germination of pollen grain in vitro and the growth of pollen tube in the pistil of rice were observed with a microscope. The stigma was removed at different time points after pollination to study its effect on seed setting rate. The rice pollen grain started to germinate at 2 min after pollination and the pollen tube penetrated stigma into style in 5-10 min, 30 min later the end of pollen tube reached the bottom of ovary, and only some pollen tubes arrived at embryo sac at 40 min after pollination. Meanwhile, a small amount of callose began to deposit in the pollen tubes, a great deal of callose was observed at 50 min after pollination, whereas the pollen grain began to shrink. The growing rates of pollen tube in the rice stigma, style and ovary were 1500, 5000, and 5400 μm/h, respectively. The seed setting rate was quite low when the stigma was removed at about 10-15 min after pollination, gradually increased when it removed at 20 min to 50 min after pollination, and over 60% when it removed at 50 min after pollination and finally tended to be stable.

Pollen Viability in Three Xinjiang Hawthorn Species

[Objective] The aim was to explore the most efficient medium for pollen germination and suitable pollen storage temperature for three different hawthorn species in Xinjiang, and to lay foundation for the development and utilization of wild hawthorn resources. [Method] A randomized block design was adopted to select the most suitable concentrations of sucrose and boric acid in the medium for pollen germination under in vitro culture. The vitality of the pollens stored at different tem- peratures for six mouths was measured once every 10 d using the optimum medi- um selected in above steps. [Result] The most efficient medium for Altai hawthorn pollen germination was 1% agar ＋ 0.05% boric acid ＋ 15% sucrose, and its germi- nation rate in this medium was 30.8%; the most suitable medium for crabapple hawthorn pollen germination was 1% agar ＋ 0.01% boric acid ＋ 15% sucrose, and its germination rate in this medium was 58.7%; and the highest germination rate of Junggar hawthorn pollen was found in the medium containing 1% agar ＋ 0.01% boric acid ＋ 20% sucrose, and the germination rate reached 67.2%. There were extremely significant differences （P〈0.01） in germination rate between these treat- ments and others in each species. The pollen vitality of Altai hawthorn, crabapple hawthorn and Junggar hawthorn were lost completely after they were stored at room temperature for 40, 60 and 70 d; at 4-5 ℃ for 90, 130, and 140 d; or at -18 ℃ for 130, 160 and 170 d. Therefore, -18 ℃ was found the most efficient storage temperature for the three hawthorn species＇ pollen among the three different stor- age temperatures. [Conclusion] A certain concentration of sucrose and boric acid can promote pollen germination of the three hawthorn species. Under different stor- age temperatures, the pollen vitality of the three hawthorn species can maintain for a longer period at -18 ℃ than at 4-5 ℃, and for the shortest period at room tem- perature.

Study on in vitro Culture and Germination of Three Cultivars of Lagerstroemia indica L.

[Objective] The research aimed at investigating the in vitro culture and germination of three cultivars of Lagerstroemia indica L.[Method] The pollen of three L.indica cultivars Hongdiefeiwu,Cuipanjinwei and Zizhuayinwei in full flowering stage was collected as materials to study the effects of in vitro culture on pollen viability and germination.[Result] The optimum medium for L.indica pollen germination was 150 g/L sucrose ＋ 60 mg/L boric acid ＋ 20 mg/L CaCl2＋20 mg/L MgSO4 ＋100 mg/L KNO3,in which,the concentrations of sucrose and boric acid could significantly influence the pollen germination.In addition,the vitality of pollen at long filament differed from that at short filament,wherein,the vitality of pollen at short filament of Zizhuayinwei and Hongdiefeiwu was higher than that at their long filament,while Cuipanjinwei showed the opposite result.Within 2-6 h after pollination self,pollen germinated to form pollen tube,which grew fast firstly and then slowly.[Conclusion] The study provided theoretical reference for breeding new L.indica cultivar.

Decreased Pollen Viability and Thicken Pollen Intine in Antisense Silenced Brassica campestris Mutant of BcMF19

Brassica campestris male fertility 19 （BcMF19; GenBank accession number GQ902048.1）, a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, which is learned from the previous in situ hybridization study. In the present study, we constructed antisense-silenced plants of BcMF19 using B. campestris ssp. chinensis to validate this prediction. The morphology of the pistils, long anthers, and short anthers was significantly affected in 35sbcmf19 compared with the control samples. 4＇-6-Diamidino-2-phenylindole staining revealed that two generative nuclei and one large vegetative nucleus were not affected in the mutant compared with control. Statistical analysis of Alexander＇s staining results showed that 96% of the control pollen grains had vitality, whereas only 86% of the mutant pollen grains did. Under scanning electron microscopy, the mutant demonstrated numerous abnormal pollen grains and resembled dried persimmon. The frequency of normal pollen grains was approximately 18%. Under transmission electron microscopy, the pollen intine during the binucleate and mature pollen stages in 35sbcmf19 exhibited abnormal thickening, especially at the germinal furrows, compared with control. In vitro pollen germination test showed that the tips of the mutant pollen tubes transformed into globular alveoli and stopped growing compared with control. On the other hand, in vivo pollen germination test suggested that BcMF19 affected the pollen tube extension in the pistil. These findings indicate that BcMF19 is essential to the pollen development and pollen tube extension orB. campestris ssp. chinensis.

Influence of Storage Time on Pollen Traits in Actinidia eriantha

We examined the influence of storage time on germinability and tube growth of freeze stored pollens collected from 25 wild male plants in Actinidia eriantha variety. Pollens were stored in freezer at - 20°C for six months and one year periods to determine changing at germinability in time. In vitro germination was conducted in certain cultural medium defined for Actinidia genus. The results showed that the germination percentages and tube lengths of genotypes decreased at the end of storage period. MH22, MH45, MH47, MH56, MH67, MH70, MH71, MH72, MH74, MH55 and MH61 genotypes were evaluated as vigor genotypes, because they maintained their viability and germination capability displaying statistically insignificant decreasing although their tube lengths significantly decreased except MH67. This investigation provided to determine some robust wild male germplasm resources in A. eriantha in point of durability of pollens against long term conservation for using at future pollination and breeding programs.

Observation of Flowering Process of Grape(Vitis vinifera L.)—Insight into the Starting of Pollination of Flower Hiding in Calyptras(Cap)

It is generally agreed that many Vitis vinifera L.cultivars are self-fertile,where self-pollination often occurs before capfall in a process called cleistogamy.Therefore,it is difficult to identify the right time to remove stamens before self-pollination during the cross-breeding of grape.For this paper,we observed the process of grape flowering and measured the pollen viability and stigma receptivity of grape flowers of‘Shine Muscat’in order to identify the starting time of self-pollination before capfall and to provide useful information for improving the efficiency of cross-breeding.The results demonstrate that the anther is not cracked during the visible clusters and separated clusters stages.Meanwhile,in the separated floral buds,flowering begins,and full bloom stages,the pollen viability is 60.7%,73.2%and 80.3%,respectively;however,at the berry set stage,pollen viability drops to zero.The top of the mature stigma is composed of a layer of nearly cylindrical papillary cells,and the stigma receptivity for pollen changes with the development of flowers:in particular,no reaction was observed in the visible clusters stage;weak positive reaction at the separated clusters stage;strong positive reaction at the separated floral buds,flowering begins,and full bloom stages;and no reaction at the berry set stage.In the separated floral buds stage,pollen tubes were seen germinating in the style.In the flowering begins stage,more pollen tubes were observed at the entry of the ovary.During the full bloom stage,most pollen tubes elongated into the ovary base and some entered the pearl hole.At the berry set stage,newborn endosperm nucleus could be seen in the ovule.From the above,we can conclude that the initiation time of closed fertilization for‘Shine Muscat’grape can be judged as the separated floral buds stage,and it is best to discard the stamen before the separated floral buds stage when conducting cross-breeding.

Production of Partial New-typed Brassica Napus by Introgression of Genomic Components from B. rapa and B. carinata

A breeding strategy for widening the germplasm of Brassica napus was proposed by introgression of the A^r subgenome of B. rapa （A^rA^r） and C^c of B. carinata （B^cB^cC^cC^c） into natural B. napus （AnAncncn）. The progenies with 38 chromosomes that were derived from the self-pollinated seeds of pentaploid hybrids （A^rA^nB^cC^cC^n） were used for further research. Some of the partial new-typed B. napus showed normal meiotic behavior, high portion of germinated pollen and normal embryological development. This indicates that the selected new-typed B. napus had a balanced genetic base. Molecular analysis showed that about 50% of the genome in the new-typed B. napus was replaced by A^r and C^c subgenome from B. rapa and B. carinata. Considering the genetic diversity among different lines of new-typed B. napus, it was deduced that the introgression of the genomic components from B, rapa and B. carinata could widen the genetic diversity of rapeseed.

Plant fertility defects induced by the enhanced expression of microRNA167

The plant hormone auxin plays a critical role in regulating plant growth and development. Recent advances have been made in the understanding of auxin response pathways, primarily by the characterization of auxin response mutants in Arabidopsis. In addition, microRNAs （miRNAs） have been shown to be critical regulators of genes important for normal plant development and physiology. However, little is known about possible interactions between miRNAs and hormonal signaling during normal development. Here we show that an Arabidopsis microRNA, miR167, which has a complementary sequence to a portion of the A UXINRESPONSE FACTOR6 （ARF6） and ARF8 mRNAs, can cause transcript degradation for ARF8, but not for ARF6. We report phenotypic characterizations of 35S：：MIR167b transgenic lines, and show that severe 35S：：MIR167b transgenic lines had phenotypes similar to those of an arf6 arf8 double mutant. The transgenic phenotypes suggest that miR167 may repress ARF6 at the level of translation. We demonstrate that the transgenic plants are defective in all four whods of floral organs. In the transgenic flowers, filaments were abnormally short, anthers could not properly release pollen, and pollen grains did not germinate. Our results provide an important link between the miRNA-mediated regulatory pathway of gene expression and the auxin signaling network promoting plant reproductive development.

Methods for Improving Hybrid Seed-setting Rate of a Potato Cultivar Linshu 17

[Objective] This study aimed to explore methods for improving hybrid seed-setting rate of a potato cultivar Linshu 17. [Method] L0527-4 was taken as male parent and Linshu 17 was taken as female parent to breed a new potato cul- tivar, so as to take advantage of the excellent characters of Linshu 17. [Result] The optimum pollination time of Linshu 17 was 10：00-12：00 am or after 04：00 pm. Spraying 10 mg/L of 2, 4-D, 50 mg/L of gibberellin or 20 mg/L of 6-BA ＋ 50 mg/L of gibberellin once every 7 days since the 3^rd day after hybridization all could im- prove the hybrid seed-setting rate of Linshu 17. [Conclusion] The hybrid seed-setting rate-improving effect of 2, 4-D sprayed on the 3^rd d after hybridization was best for Linshu 17. This study will provide technical references for other varieties to improve the hybrid seed-setting rate.