Effects of Ion Implantation on in Vitro Pollen Germination and Cellular Organization of Pollen Tube in Pinus thunbergii Parl. (Japanese Black Pine)

Low-energy ion implantation, as a new technology to produce mutation in plant breeding, has been widely applied in agriculture in China. But so far there is a little understanding of the underlying mechanisms responsible for its biological effects at the cellular level. Here we report the biological effects of a nitrogen ion beams of 30 keV on the pollen grains of Pinus thunbergii Parl. In general, ion implantation inhibited pollen germination. The dose-response curve presented a particular saddle-like pattern. Ion implantation also changed the dimension of the elongated tubes and significantly induced tip swelling. Confocal microscopy indicated that the pollen tube tips in P. thunbergii contained an enriched network of microtubules. Ion implantation led to the disruption of microtubules especially in swollen tips. Treatment with colchicine demonstrated that tip swelling was caused by the disruption of microtubules in the tip, indicating a unique role for microtubules in maintaining the tip integrality of the pollen tube in conifer. Our results suggest that ion implantation induce the disruption of microtubule organization in pollen and pollen tubes and subsequently cause morphological abnormalities in the pollen tubes. This study may provide a clue for further investigation on the interaction between low-energy ion beams and pollen tube growth.

Performance of Weibull function as a diameter distribution model for Pinus thunbergii stands in the eastern coast of South Korea

This study was carried out to determine the performance of percentile-based Weibull diameter distribution model for Pinus thunbergii stands thriving along the eastern coast of South Korea. The parameter recovery technique was used to estimate the three parameters of the Weibull model. The analysis demonstrated satisfactory results based on the following test statistics for the principal percentile models： fit index （FI） range from 0.501（minimum diameter） to 0.932 （50th diameter percentiles） and root mean square error （RMSE） range from 0.112 （quadratic mean diameter） to 3.572 （minimum diameter）. The developed model was further evaluated by determining the mean bias （E） in trees per ha （TPH） for each diameter class, and the results showed highest over-prediction in the 20 cm, and under-prediction in the x6 cm and 24 cm diameter classes. The goodness of fit tested by Kolmogorov- Smirnov （KS） test showed no significant differences （P〉0.05） between the observed and predicted diameter distributions for almost all plots. Using site index and aboveground biomass （AGB） models developed for P. thunbergii in South Korea, a model to predict the AGB per ha for each diameter class and subsequently the total AGB of the stand was created. An application guide was also created, which will serve as a decision-support tool for forest managers in quantifying the future total AGB in P. thunbergii stands located in the eastern coast of South Korea and, subsequently, the quantification of potential carbon stocks aside from being a vital input in designing efficient management and protection strategies for these stands.

Ion Implantation Hampers Pollen Tube Growth and Disrupts Actin Cytoskeleton Organization in Pollen Tubes of Pinus thunbergii

Pollen grains of Pinus thunbergii Parl. （Japanese black pine） were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implantation significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for continuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam implantation and is involved in mediating certain subsequent cytological changes.

Characterization of 14 anonymous nuclear loci in Pinus thunbergii and their cross-species transferability

We characterized 14 anonymous nuclear loci from Pinus thunbergii Parl., an important pine species native to Japan. One hundred and twenty-six single nucleotide polymorphisms （SNPs） were identified from these loci, giving a frequency of 1 SNP per 51 bp. Nucleotide di- versity （0） ranged from 1.06 × 10^-3 to 11.87 × 10^-3, with all average of 4.99 × 10^-3. Only one locus （mK45） deviated significantly from the Hardy-Weinberg equilibrium. Thirteen of 14 loci were applicable in other pine species. These loci will be useful for nucleotide variation studies and will provide material for SNP-based marker development in P. thun- bergii and related species.

Evaluation of somatic embryo production during embryogenic tissue proliferation stage using morphology,maternal genotype,proliferation rate and tissue age of Pinus thunbergii Parl

To determine the optimal embryogenic capacity(somatic embryo production)of the selected elite nematode-resistant genotypes of Pinus thunbergii,variables such as embryogenic tissue(ET)morphology,maternal genotype,proliferation rate and tissue age were analyzed.ET morphology and histological evaluation of the proliferation stage showed a decrease in filamentous clump and protuberant surfaces and a decline in the acetocarmine-staining area,which indicates a decrease in somatic embryo production(SEP).Variations in cell physiology during the prolifera-tion stage showed that SEP was positively correlated with soluble sugars and proteins,but negatively correlated with starch,peroxidase,and superoxidase.In addition,SEP was significantly(p<0.001)affected by maternal genotype,tis-sue age and proliferation rate.Moreover,SEP was positively correlated with proliferation rate(r=0.98,p<0.001),but negatively correlated with tissue age(r=−0.95,p<0.001).In general,the results suggest that SEP could be assessed in ET proliferation stages by the apparent cell morphology,histology,proliferation rate and tissue age,which provides novel insights for evaluating the ET maturation capacity(number of somatic embryos)during the proliferation stage of P.thunbergii somatic embryogenesis.

Adaptation of pine wood nematode,Bursaphelenchus xylophilus,early in its interaction with two Pinus species that differ in resistance

Pine wilt disease(PWD)is one of the most devastating diseases of Pinu s spp.and is caused by the pine wood nematode(PWN),Bursaphelenchus xylophilus(Steiner&Buhrer)Nickle.To study adaptation of PWN to survive in hosts that differ in resistance,we examined the self-regulatory characteristics of PWN at the biological and molecular levels early in the interaction.Two-year-old susceptible Pinus thunbergii and resistant Pinus taeda were selected for this experiment,and changes in PWNs after inoculation were assessed.q RT-PCR was used to detect changes in genes related to PWN pathogenicity and detoxification.The results showed that the migration and reproductive abilities of PWNs in P.thunbergii were stronger than those of PWNs in P.taeda.After 7 d,the number of nematodes in P.thunbergii was approximately 3.2-fold higher than in P.taeda.After 15 d,the number of nematodes in P.thunbergii was approximately twofold higher than that in P.taeda.Because PWN can adjust its sex ratio after infection,we compared the sex ratio of uninoculated PWNs,to that in the two pine species.In P.thunbergii,the female to male ratio first decreased and then stabilized over time;in P.taeda first decreased and then increased.Relative fat accumulation in PWNs increased significantly after the PWNs entered the tree body;the accumulation rate in P.thunbergii was higher than in P.taeda at 7 d,but lower after 15 d.Scanning electron microscopy(SEM)showed significantly more bacteria on the surface of PWNs in P.taeda compared with PWNs in susceptible P.thunbergii.At 12 h after inoculation,the expression of genes related to cell-wall degradation(Bx Beta1-4 and Bxpel1),effectors(Bx CDP1,Bx Sap B1),and active oxygen metabolism(Bxy-ctl-1 and Bx GST3)was 2–6×higher in the resistant pine than in the susceptible one.In contrast,in PWNs,the expression of autophagy-related genes Bx ATG1 and Bx ATG16 was 1.5–2 times higher in P.thunbergii than in P.taeda.These results indicate that the interaction between PWNs and pine trees with different resistance levels elicits a series of physiological and molecular adaptations that affect nematode reproduction and virulence.This study will help elucidate the adaptive mechanisms of PWN in different pine trees.

Electrical wave transmission in healthy black pine seedlings

Non-injurious local stimuli, such as a cold shock, and injurious stimuli, such as local burning, punctures or chemicals, were applied to study electrical wave transmission in black pine (Pinus thunbergii) seedlings. The results showed that non-injurious stimuli evoked the action potential (AP) transmission and injurious stimulation induced both AP transmission and the more complex variation (VP) transmission in the seedlings. The causes of these phenomena were discussed. Key words Black pine - Pinus thunbergii - Action potential (AP) - Variation potential (VP) - Electrical wave transmission CLC number S791.256 Document code A Foundation item: This paper was supported by the National Nature Science Foundation of China (grant No. 39670613).Biography: GUO Jian (1971-), male, lecturer in Haikou Bureau of Forestry. Hainan, P. R. China.Responsible editor: Song Funan

Effect of pine wood nematode invasion on pine community functions in the Pinghu region, Zhejiang Province, eastern China

In order to investigate the effect of invastion by pine wood nematode(PWN), this study analyzed several functional indices, i.e., the increment in DBH and stand volume and biomass, in the damaged stands with various mixed percentages of Pinus massoniana and P. thunbergii and with different levels of damage. According to the results of rate of change in increment of DBH and stand volume, the forest ecosystem resistance against PWN increased with a reduction in the mixed ratio of pine. The resistance was highest with a mixed percentage of 50%. The invasion of PWN changed the corresponding relationship of increment between DBH and stand volume(pure stands > 7:3 conifer and broadleaf > 6:4 conifer and broadleaf > 5:5 conifer and broadleaf) among the P. thunbergii stands when there is no damage, but for P. massoniana stands this phenomenon did not occur. For the increment rate of DBH and stand volume, this significant change in P. thunbergii forest indicates that the resistance of pure P. thunbergii forest was higher than that of P. massoniana. The invasion of PWN accelerates the succession from pure stands to mixed stands and then to the broadleaf evergreen stands.

松梢螟危害黑松诱发次生冻害观察

通过对日照地区近10年气候资料的观察分析,发现黑松(Pinus thunbergii)受松梢螟(Dioryctria rubella Hampson)危害,松梢枯死后,枯死部位下针叶束萌发新芽,簇生枝端,由于这些新梢木质化较差,很难越冬,第2年全部冻死,形成次生冻害。促进黑松健康生长,必须加强松梢螟的科学防控。

黑松针叶束促萌硬枝扦插繁殖技术

选择当年生黑松(Pinus thunbergii Parl.)枝条做插穗,通过抹顶芽,促进插穗顶端针叶束萌发新芽,产生生长素刺激插穗基部产生愈合组织生根,插条生根率达91.2%,大田移栽育苗成活率89.7%,解决了生产中黑松不能硬枝扦插繁殖的问题。