Exploration of Root-associated Bacteria from the Medicinal Plant Platycodon grandiflorum.

The present study was performed to investigate root-associated bacteria from Platycodon grandiflorum, a medicinal plant commonly grown in East Asia. Isolates were obtained from the rhizosphere or root interior with various culture media, and phylogenetic analyses were performed based on their 16S rDNA sequences. In consideration of practical applications, traits related to plant growth promotion and niche adaptation were assessed in several endophytic strains with fewer biosafety concerns. The effects of a bacterial inoculation on seedling and mature plant growth were evaluated. Seventeen genera that encompassed more than 30 bacterial lineages were successfully retrieved from the roots, the majority of which have not been reported as P. grandiflorum-associated bacteria, particularly for non-negligible Proteobacteria. Although nitrogen-fixing or phosphate-solubilizing and indole acetic acid-producing activities were recorded in all of the strains selected, these strains were beneficial or detrimental to plant growth as evidenced by their influence on the length of seedlings and biomass of mature plants. Among the 4 endophytic Rhizobium species tested in the present study, the potentially novel Rhizobium sp. BF-E16, which was more compatible with the non-leguminous medicinal plant P. grandiflorum, was identified. Other than plant growth-promoting traits, characteristics such as plant constituent-hydrolyzing activities need to be taken into consideration and their roles clarified when investigating plant growth-promoting rhizobacteria.

[Effects of Platycodon grandiflorum/pepper intercropping on root growth, yield and quality of Platycodon grandiflorum].

In order to investigate effects of Platycodon grandiflorum and pepper intercropping on root growth, yield and quality of P. grandiflorum, field experiments were conducted in the soils of continuously cultivated P. grandiflorum for three years. The cultivation model was designed as monoculture and intercropping. The monoculture of P. grandiflorum was denoted as CK and the intercrop association of P. grandiflorum/pepper was arranged as follow: in intercrops every two rows of pepper was planted between every three, four and five rows of P. grandiflorum, respectively, and denoted as JC32, JC42 and JC52. Results showed that taproot length and diameter of P. grandiflorum in intercropping association of JC32 was higher than those of P. grandiflorum in monoculture association. This fact suggested that P. grandiflorum intercropped with pepper facilitated its root growth. Compared with monoculture association, the number of lateral root in intercropping association was significantly decreased and the location of lateral root at taproot also altered. This fact suggested that P. grandiflorum intercropped with pepper enhanced appearance quality of P. grandiflorum root. Total root yield and taproot yield of P. grandiflorum in JC42 and JC52 intercropping associations were increased by 4.88%, 8.91% and 14.23%, 12.92%, respectively, compared with monoculture, while root rot incidence decreased significantly. Compared with JC52 intercropping association, JC42 intercropping association significantly increased total saponin and protein contents of P. grandiflorum, decreased root rot incidence, but did not affect taproot yield significantly. Considering root yield and quality, when P. grandiflorum planted in the soil having continuously cultivated P. grandiflorum for three years, the optimal cultivation model was every two rows of pepper was planted between four rows P. grandiflorum.

[In vitro autotetraploid induction and analysis on DNA methylation diversity of Platycodon grandiflorum].

In order to investigate the epigenetic variations between diploid and autotetraploid of Platycodon grandiflorus. The diploid buds of P. grandiflorus were soaked in the mixture of different concentration colchicines and 0.002 g•mL ⁻¹ dimethyl sulphoxide (DMSO)．The identification of autotetraploid plants were based on morphological characteristics, chromosome number and flow cytometry. And then the level and pattern of DNA methylation explored by using the technology of methylation sensitive amplified polymorphism (MSAP)．The result demonstrated that the buds soaked in 0.2% colchicines and 0.002 g•mL ⁻¹ DMSO solution for 12 h was ideal conditions to induce autotetraploid of P. grandiflorus, with induction rate of 32.0%.The diploid and tetraploid plants existed distinctly differences in morphological indexes.Totally,1 586 bands were amplified by 20 pairs of selective primers, of which 764 and 822 bands were detected in diploid and autotetraploid respectively. The total methylation ratio,full methylation ratio and hemimethylated ratio were 91.25%,61.25% and 30.65% in diploid of P. grandiflorus,respectively.However,the total methylation ratio,full methylation ratio and hemimethylated ratio of autotetraploid of P. grandiflorus were 86.13%,54.38% and 31.75%, respectively. Compared with diploid, the genomic DNA total methylate ratio and full methylation ratio of autotetration plants decreased by 6.02% and 7.14%.But the hemimethylated ratio of autotetraploid was higher than that of diploid, which more than 1.6%. All this results indicated that DNA methylation patterns have adjusted during the polyploidy process．.

[Effect of different nitrogen forms and ratio on growth and active ingredient content of Platycodon grandiflorum].

To providing evidence about nitrogen adequate application of Platycodon grandiflorum, the pot culture experiment was conducted to study the effect of nitrogen on the growth, physiological metabolism and the quality of P. grandiflorum. The activity of NR, GS and SOD, POD and CAT were determined. And the nitrate and ammonium nitrogen content, photosynthetic characteristics, active components of P. grandiflorum were determined. The results showed that the nitrate nitrogen content and P. biomass reached its maximum value, when NH4(+)-N/NO3(-) -N was 0: 100, the activity of NR. The activity of GS was the highest at the NH4(+) -N/NO3(-) -N ratio of 25:75 and ammonium nitrogen content was the highest at 75:25. The activity of SOD decreased and then increased with the increasing of NO3(-) -N. At the NH4(+) -N/NO3(-) -N ratio of 25: 75, the activity of CAT had its maximum value and the content of MDA had the minimum value. At the same time, the content of platycodon D was the highest at this treatment. The studies had shown that different nitrogen forms and ratio had a significant effect on the characteristics of photosynthetic physiology, nitrogen metabolism and resistance adjustment, growth and the quality of P. grandiflorum. The NH4(+) -N/NO3(-) -N ratio of 25: 75 was a suitable ratio of nitrogen forms for the growth of P. Grandiflorum and accumulating the content of platycodon D.

[Preliminary study on the effect of climate factors on pollen fertility in Platycodon grandiflorum].

OBJECTIVE: To have a better utilization of male sterile lines in heterozygotic breeding of Platycodon grandiflorum and provide theoretical basis for Platycodon grandiflorum hyboridization. METHODS: The pollen viability was detected by the means of aceto carmine dyeing, and the correlation analysis between climate factors of each anther development stage and pollen viability was estimated by Pearson coefficients. RESULTS: Pollen viability variation range of male-sterile line GP1BC1-12 was 0% - 27%. That of male-sterile line GP12BC4-10 and chifeng germplasm was respectively 1.3% - 17.9% and 75.9% - 98.5%. Further linear regression analysis between climate factors of each anther development stage and pollen viability indicated that the degree of sensitivity varied with different germplasm of Platycodon grandiflorum. Among three germplasm, male sterile line GP12BC4-10 was the most stable one to the climate factors, and the male-sterile line GP1BC1-12 was the most sensitive one. CONCLUSION: Temperature and solar irradiation are the most important climate factors to affect pollen viability in Platycodon grandflorum, and microspore mother cells stage (MMC) is its sensetive stage.

[The performance of agronomic traits in F1 crossing combinations derived from male sterile line of Platycodon grandiflorum].

OBJECTIVE: To determine the possibility of heterosis utilization by male sterility in Platycodon grandiflorum. METHODS: The analysis of variance, heterosis over standard and correlation on 12 agronomic in 18 crossing combinations were carried out. RESULTS: Except the dry weight of upground part of plant, the length of main root and the diameter of middle part of the main root, all other agricultural traits were different significantly among the 18 F1 combinations. The diameter of middle part of the main root had the highest coefficient of variation and the ratio of fresh weight of main root and total root had the lowest coefficient of variation,which was 85.42% and 3.66%, respectively. For the traits related to the yield of root,average heterosis over standard in fresh main root and total root was 42.35% and 45.50%, respectively; For the traits related to the form of root, the diameter of up part and middle part of main root had a middle heterosis over standard, which was 12.38% and 29.97%, respectively. The shoot growth was significantly related to its root growth in combination (0.651). The number of nodes on main stem was significantly related to five out of six traits of root in hybrids. CONCLUSION: Variation of agricultural traits in crossing combination is rich, heterosis of traits in root is evident,and F1 hybrids with good yield and the forms could be selected from these combinations. The Platycodon grandiflorum of crossing combinations have reasonable morphological characteristics, and the number of nodes of main stem could be taken as the selected index. All show us that the heterosis by the male-sterility could be utilized in the breeding of Platycodon grandiflorum.

Effect of plant age on endophytic bacterial diversity of balloon flower (Platycodon grandiflorum) root and their antimicrobial activities.

Balloon flower (Platycodon grandiflorum) is widely cultivated vegetable and used as a remedy for asthma in East Asia. Experiments were conducted to isolate endophytic bacteria from 1-, 3-, and 6-year-old balloon flower roots and to analyze the enzymatic, antifungal, and anti-human pathogenic activities of the potential endophytic biocontrol agents obtained. Total 120 bacterial colonies were isolated from the interior of all balloon flower roots samples. Phylogenetic analysis based on 16S rRNA gene sequences showed that the population of 'low G + C gram-positive bacteria' (LGCGPB) gradually increased 60.0-80.0% from 1 to 6 years balloon flower sample. On the other hand, maximum hydrolytic enzyme activity showing endophytic bacteria was under LGCGPB, among the bacterial strains, Bacillus sp. (BF1-1 and BF3-8), Bacillus sp. (BF1-2 and BF3-5), and Bacillus sp. (BF1-3, BF3-6, and BF6-4) showed maximum enzyme activities. Besides, Bacillus licheniformis (BF3-5 and BF6-6) and Bacillus pumilus (BF6-1) showed maximum antifungal activity against Phytophthora capsici, Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum. Moreover, Bacillus licheniformis was found in 3 and 6 years balloon flower roots, but Bacillus pumilus was found only in 6 years sample. It is presumed that older balloon flower plants invite more potential antifungal endophytes for there protection from plant diseases. In addition, Bacillus sp. (BF1-2 and BF3-5) showed maximum anti-human pathogenic activity. So, plant age is presumed to influence diversity of balloon flower endophytic bacteria.

[Effects of applying controlled-release compound fertilizer on Platycodon grandiflorum growth].

A pot experiment was conducted in 2008 to study the effects of applying controlled-release compound fertilizer (N:P2O5:K2O = 14:14:14) on the growth of Platycodon grandiflorum in the medicinal herbal farm of Shandong Agricultural University. Comparing with the application of common compound fertilizer (N:P2O5: K2O=15: 15: 15), applying equivalent amount of the controlled-release fertilizer increased the leaf chlorophyll content, root volume, root activity, and root diameter of P. grandiflorum at the late growth stage, but decreased the root length. When the N application rate was 0.24 and 0.32 g x kg(-1) soil, applying the controlled-release compound fertilizer increased the root yield by 26.78% and 22.50%, and the root soluble sugar, protein, and total saponin contents by 9.77% and 6.99%, 11.38% and 2.20%, and 8.85% and 5.47%, respectively, compared with applying the common compound fertilizer. More nitrogen application made the root soluble sugar content decreased but the total saponin content increased. Under our experimental condition, applying the controlled-release compound fertilizer with an application rate of 0.24 g N x kg(-1) soil could obtain the best effect for P. grandiflorum.

[Effect of seed soaking with aluminum on seed germination and seedling physiology of Platycodon grandiflorum].

In order to study the effect of seed soaking with different aluminum solution on seed germination and seedling physiological characteristics of Platycondon grandiflorum, two P. grandiflorum varieties'seed (the white flower and the purple flower) were soaked in Al3+ solution with different concentrations (0, 10, 100, 250, 500, 750 and 1000 mg x L) for 24 h, then germinated in illumination incubator. Results showed that the aluminum toxicity on the trends of the germination rate, germination index and vigor index was positive associated with its concentration, and the Al tolerance of the purple was slightly greater than that of the white. There were some relationships between the physiological indices, which were the leakage rate of electrolyte, the malonaldehyde (MDA) content, the activities of peroxidase (POD) and superoxide dismutase (SOD) , the free praline(Pro) and the soluble sugar contents, with the concentrations of Al. It was suggested that there was Al tolerance difference between the two P. grandiflorum varieties: the purple flower was greater than the white.

[Heterosis analysis of the main agronomic characters of Platycodon grandiflorum].

OBJECTIVE: Breeding the good varieties characters of Platycodon grandiflorum. METHODS: 20 combination, derived from 2 x 10 incomplete diallel cross with 2 male sterile lines and 10 four-generation inbred lines, analysis the 13 agronomic characters. RESULTS: The agronomic characters of F1 has obvious heterosis. Dry plant weight and fresh root weight have the largest heterosis. Their middle parent heterosis (MPH), high parent heterosis (HPH) and heterosis over CK (CH) were 96.07%, 62.62%, 116.21%, 108.99%, and 111.98%, 113.47%, respectively. CONCLUSION: The F1 hybrids derived from the crossing of male sterile lines and in-bred lines have obvious heterosis. It applied the hybrid heterosis breeding is an effective way for the new variety of Platycodon grandiflorum.

[Dynamic research of density on Platycodon grandiflorum dry material accumulation and platycodin D content in Platycodon grandiflorum].

OBJECTIVE: To study the dynamic changes of dry material accumulation and platycodin D content in Platycodon grandiflorum in different planting densities. METHOD: Five different planting densities M1 (4 cm x 25 cm), M2 (6 cm x 25 cm), M3 (8 cm x 25 cm), M4 (10 cm x 25 cm) and M5 (12 cm x 25 cm) were designed in the plot experiment. The individual and colony biomass accumulation, dry material distribution, root yield and platycodin D content were measured in different stage. RESULT: In a certain density range the individual biomass in P. grandiflorum obviously declined with increasing density with the exception of biomass M2 > biomass M3. On the contrary, the colony biomass increased with the increasing density. Dry material accumulation in each organ in P. grandiflorum in different planting densities showed significance (P<0.05). The dry material distribution in organs in the different planting densities showed significance (P<0.05), and the dry material distribution in flower and fruit reached the minimal level in M2, in the same planting density the distribution in root reached the maximal; The dry material in stem, flower and fruit obviously declined with the increasing density, while the dry material in leaf increased. The individual root output increased with the increasing density, and it reached the highest in M2. The colony root yield increased with the increasing density. The platycodin D content in P. grandiflorum reached the highest in M2. CONCLUSION: The result showed that a suitable planting density is very important to P. grandiflorum dry material accumulation and distribution, root yield, platycodin D content and colony yield.

[Effects of top pruning on fruiting characters of Platycodon grandiflorum].

OBJECTIVE: To investigate the effects of top pruning on fruiting characters of Platycodon grandiflorum, and find the suitable stage, in which seed growth and development furtherly. METHOD: One-year old seedlings were chosen and planted in field. Plant height, branching number, fruit number per plant, 1000 grains weight were measured during growth and development period, respectively. RESULT: The treatment of top pruning postponed in turn the flowering date, lowered the plant heights and the fruit number per plant, increased the branching number and influenced significantly on 1000 grains weights. CONCLUSION: The suitable stage of top pruning for producing seeds was from June 20th to July 5th.

[Observation of pollen tube germination of Platycodon grandiflorum by fluorescence microscopy].

OBJECTIVE: To determine the self-compatibility of Platycodon grandiflorum and the location of microspore germination on stigma. METHOD: The microspore germination of self-pollination, self-plant-pollination and cross-pollination and the pollination microspore germination in and outside of stigma were observed with fluorescence microscopy. RESULT: Most pollens of self-pollination, self-plant-pollination and cross-pollination can germinate on the stigma, and after 24 hours, pollen tube entered into the ovary successfully. Pollinated on the outer-surface of stigma, microspores could not germinate, but on the inner-surface of the stigma when it dehisced most microspores can germinate. CONCLUSION: The compatibility of self-plant-pollination of Platycodon grandiflorum is high. The microspore germination loci is on the inner-surface of the dehisced stigma.

[Studies on germination characteristics of Platycodon grandiflorum seed].

OBJECTIVE: To find out the optimal condition for the germination by studying the effects of light, temperature, seed soaking and cultivation condition on Platycodon grandiflourm, and offer the basis for standardized culture of P. grandflorum. METHOD: The rate of water absorption of the seed was determined. The germination of P. grandiflorum seed was tested under following conditions: treatments under different temperature (10,15,20,25,30 degrees C), light treatments (light and dark), seed soaking time (12,24,36 h) and different germination cultivation conditions (on filter paper, between filter paper, between gauze,on gauze). RESULT: The water absorption of the seeds reached the maximal 120% within 36 h at 25 degrees C. The time of seeds soaking had limited effect on the germination of the seed, while temperature and light condition showed significant effects on the germination. CONCLUSION: The optimal condition for the germination of the seed of P. grandiflorum is as follow:seed soaking for 12 h, and 25 degrees C underlight.

[Influence of exogenous substance on germination of Platycodon grandiflorum seeds].

OBJECTIVE: To study of exogenous substances on the germination rate of Platycodon grandiflorum and offer the basis for the standardized culture of P. grandiflorum. METHOD: At 25 degrees C, under darkness and lightness, do pretreatmeats on seeds by using GA3, H2O2, KMnO4, PEG at different concentrations. RESULT: The results indicated that the pretreatments with GA3 at a concentrating of 50-250 mg x L(-1) and 1%-2% of H2O2 could increase germination of P. grandiflorum seed at a certain degree. Contrariwise 0.1%-0.5% of KMnO4 and 15%-35% of PEG inhibited the germination. CONCLUSION: Pretreatment with 50-250 mg x L(-1)GA3 and H2O2 at a low cencontration could increase the seed germination of P. grandiflorum.

[Study on the stigma/pollen vigor and self-compatibility of Platycodon grandiflorum].

OBJECTIVE: To provide the basal data for the breeding and cultivation of Platycodon grandiflorum. METHOD: The field investigation and pollination by bagging were carried out. TTC(2,3,5-triphenyl tetrazolium chloride) solution was used to test the pollen vigor. RESULT: The stigma life-span of P. grandiflorum was about 9 days, however the optimal time for pollination is 4-6 days after the petals opening, with the stigma was splitting lightly or significantly. When the petals opened, the anther began scattering pollen, and finished in the same day. The pollen vigor was about 81.4% at the beginning, and decreases to 27.6% three days later. The pollen vigor still remains 64.4% three days later, when the flower was kept in the desiccant. The natural fructification rate of self-flower-pollination was 4.8%. The fructification rate and compatible index was about 62.7% and 54.6, respectively, when the self-plant-pollination performed by hand. They decreased to 12.8% and 6.5 when the pollination was implemented during the flowering period. CONCLUSION: Low fructification percentage of self-flower-pollination attributes to the difference of maturing period of pistil and stamen, as well as the short pollen life-span of P. grandiflorum. The compatibility of self-plant-pollination is high during flowering period when pollination performed by hand. The life-span of the pollen can be prolonged significantly when keeping in the dry environment.

[Induction and identification of autotetraploid plant of Platycodon grandiflorum].

The methods and technique of inducing polyploid plants of Platycodon grandiflorum were done. Many autotetraploid plant lines were induced by colchicine in vitro successfully. The identification of main agronomic characteristics of these clonal lines was conduced in the fields. The results showed the giant type occurred in tetraploid plants comparing with that in diploid plants. It may show prospective way in developing new varieties with high yield and high content of useful compounds.

[Ultrastructural changes after space flight in Platycodon grandiflorum (Jacq.) DC].

OBJECTIVE: To substantiate the effect of space conditions on medicinal plants. METHOD: The seeds of platycodon grandiflorumg were set up in retrievable satellites. After returning to earth, the ultrastructural changes of leaves were studied. RESULT: Under light microscope, the leaf tissue structure of the flight group and the earth controlled group were alike and under electron microscope, some differences could be found in the nucleus and choloroplasts between the two groups. CONCLUSION: Space environment has some effect on Platycodon grandiflorum, and the effect needs to be further explored.