Genetic Transformation of Watermelon with Pumpkin DNA by Low Energy Ion Beam-mediated Introduction

The No.601 watermelon (citrullus lanatus) seeds were treated with 25 keV N+ implantation at the dosage of 7.8 ×1016 ions/cm2. After treatment, watermelon seeds were incubated with 380μg/μl pumpkin (Cucubita, maxima Duch) DNA solution at 35 ℃ for 5 hours. By two-generations of selection and resistance screening at seedling stage, one transformed material was selected out, whose rind color is similar to that of the donor pumpkin and whose size of seeds is between that of the donor and the receptor. Using AFLP (amplified fragment length polymorphism) technique, two polymorphic DNA fragments were amplified. This primarily testified that the donor DNA fragments/gene were introduced into the receptor cell and integrated into the genomic DNA of the receptor.

Preliminary Studies on Base Substitutions and Repair of DNA Mismatch Damage Stimulated by Low Energy N^+ Ion Beam Implantation in Escherichia coli

Ever since the low energy N+ ion beam has been accepted that the mutation effects of ionizing radiation are attributed mainly to direct or indirect damage to DNA. Evidences based on naked DNA irradiation in support of a mutation spectrum appears to be consistent, but direct proof of such results in vivo are limited. Using mutS, dam and/or dcm defective Eschericha coli imitator strains, an preliminary experimental system on induction of in vivo mutation spectra of low energy N+ ion beam has been established in this study. It was observed that the mutation rates of rifampicin resistance induced by N+ implantation were quite high, ranging from 9.2 x 10~8 to 4.9× 10~5 at the dosage of 5.2×1014 ions/cm2. Strains all had more than 90-fold higher mutation rate than its spontaneous mutation rate determined by this method. It reveals that base substitutions involve in induction of mutation of low energy nitrogen ion beam implantation. The mutation rates of mutator strains were nearly 500-fold (GM2929), 400-fold (GM5864) and 6-fold larger than that of AB1157. The GM2929 and GM5864 both lose the ability of repair DNA mismatch damage by virtue of both dam and dcm pathways defective (GM2929) or failing to assemble the repair complex (GM5864) respectively. It may explain the both strains had a similar higher mutation rate than GM124 did. It indicated that DNA cytosine methylase might play an important role in mismatch repair of DNA damage induced by N+ implantation. The further related research were also discussed.

Bioeffects of Low Energy Ion Beam Implantation:DNA Damage,Mutation and Gene Transter

Low-energy ion beam implantation （10 - 200 keV） has been proved to have a wide range of biological effects and is broadly used in the breeding of crops and micro-organisms.To understand its mechanisms better and facilitate its applications, the developments in the bioeffects of low energy ion beam implantation in the past twenty years are summarized in this paper.

Impact of Low-Energy Ion Beam Implantation on the Expression of Ty1-copia-like Retrotransposons in Wheat(Triticum aestivum)

Retrotransposon-like elements are major constituents of most eukaryotic genomes. For example, they account for roughly 90% of the wheat （Triticum aestivum） genome, Previous study on a wheat strain treated by low-energy N^＋ ions indicated the variations in AFLP （Amplified Fragment Length Polymorphism ） markers, One such variation was caused by the re-activation of Tyl-copia-like retrotransposons, implying that the mutagenic effects of lowenergy ions might work through elevated activation of retrotransposons, In this paper an expression profile of Tyl-copia-like retrotransposons in wheat treated by low-energy N^＋ ions is reported, The reverse transcriptase （RT） domains of these retrotransposons were amplified by reverse-transcriptional polymerase chain reaction （RT-PCR） and sequentially cloned, 42 and 65 clones were obtained from the treated （CL） and control materials （CK）, respectively, Sequence analysis of each clone was performed by software. Phylogeny and classification were calculated responding to the sequences of the RT domains. All the results show that there is much difference in the RT domain between the control sample and the treated sample, Especially, the RT domains from the treated group encode significantly more functional ORF （open reading frames） than those from the control sample, This observation suggests that the treated sample has higher activation of retrotransposons, possibly as a consequence of low-energy ion beam irradiation, It also suggests that retrotransposons in the two groups impact the host gene expression in two different ways and carry out different functions in wheat cells.

Mutation Breeding of an Organic Phosphorus-Solubilizing Bacterium B3 by Low Energy Ion Beam Implantation

A preliminary study on the mutation breeding of an organic phosphorus-solubilizing bacterium B3 by low-energy N^＋ ion beam was conducted. Analysis of dosage vs. survival rate and mutation rate showed that the dosage of 3×10^15 ions/cm^2 was optimal for the mutation. Some parameters for determining the efficient phosphorus content were studied to simplify the protocols for screening. Ultimately one reliable mutant was screened out under plating and flask-culturing screening conditions. Importantly, a novel phosphorus-dissolving mechanism is reported for the first time in terms of bio-surfactant production, which indicates that bio-surfactant might play a vital role in phosphorus-dissolving for some phosphobacteria.

Evidence for Base Substitutions and Repair of DNA Mismatch Damage Induced by Low Energy N^+ Ion Beam Implantation in E. coli

Ever since the low energy N + ion beam has been accepted, the mutations of ionizing radiation are attributable mainly to avoidance of DNA damages repair. Evidences based on in vivo proof results are limited. Using the E.coli wild type and mutator strains, the mutant frequencies suggest that base substitutions in rpoB gene are induced by the N + implantation. A highly conserved region is selected to get the direct evidence for base substitutions by sequence of the high fidelity PCR amplification products in mutants. Most of the mutants (90.9%, 40/44) have at least one base substitution in the amplification region. The evidences for CG to TA (55%, 22/40), AT to GC (20%, 8/40) and TA to CG (5%, 2/40) transitions are identified. The transversions are AT to TA (15%, 6/40) and GC to CG (5%, 2/40). It is suggested that DNA cytosine methylase might play an important role in mismatch repair of DNA damage induced by N + implantation by analysis of the mutant frequencies of mutator strains.

Analysis of a Partial Male-Sterile Mutant of Arabidopsis thaliana Isolated from a Low-Energy Argon Ion Beam Mutagenized Pool

A screen for Arabidopsis fertility mutants, mutagenized by low-energy argon ion beam, yielded two partial male-sterile mutants tc243-1 and tc243-2 which have similar phenotypes. tc243-2 was investigated in detail. The segregation ratio of the mutant phenotypes in the M2 pools suggested that mutation behaved as single Mendelian recessive mutations, tc243 showed a series of mutant phenotypes, among which partial male-sterile was its striking mutant characteristic. Phenotype analysis indicates that there are four factors leading to male sterility, a. Floral organs normally develop inside the closed bud, but the anther filaments do not elongate sufficiently to position the locules above the stigma at anthesis, b. The anther locules do not dehisce at the time of flower opening （although limited dehiscence occurs later）, c. Pollens of mutant plants develop into several types of pollens at the trinucleated stage. as determined by staining with DAPI （4＇,6-diamidino-2-phenylindole）. which shows a variable size. shape and number of nucleus. d. The viability of pollens is lower than that of the wild type on the germination test in vivo and vitro.

A Mutant of Bacillus Subtilis with High-Producing Surfactin by Ion Beam Implantation

In order to generate a mutant of Bacillus subtilis with enhanced surface activity through low energy nitrogen ion beam implantation, the effects of energy and dose of ions implanted were studied. The morphological changes in the bacteria were observed by scanning electron microscope （SEM）. The optimum condition of ions implantation, 20 keV of energy and 2.6 × 10^15N^＋/cm^2 in dose, was determined. A mutant, B.s-E-8 was obtained, whose surface activity of 50-fold and 100-fold diluted cell-free Landy medium was as 5.6-fold and 17.4-fold as the wild strain. The microbial growth and biosurfactant production of both the mutant and the wild strain were compared. After purified by ultrafiltration and SOURCE 15PHE, the biosurfactant was determined to be a complex of surfactin family through analysis of electrospray ionization mass spectrum （ESI/MS） and there was an interesting finding that after the ion beam implantation the intensities of the components were different from the wild type strain.

Simple General Purpose Ion Beam Deceleration System Using a Single Electrode Lens

Ion beam deceleration properties of a newly developed low-energy ion beam implantation system were studied. The objective of this system was to produce general purpose low-energy (5 to 15 keV) implantations with high current beam of hundreds of μA level, providing the most wide implantation area possible and allowing continuously magnetic scanning of the beam over the sample(s). This paper describes the developed system installed in the high-current ion implanter at the Laboratory of Accelerators and Radiation Technologies of the Nuclear and Technological Cam-pus, Sacavém, Portugal (CTN).

Anomalous Phenomena in E<18 keV Hydrogen Ion Beam Implantation Experiments on Pd and Ti

Implantation experiments of low energy（1 keV【E【18 keV）hydrogen ion beamson hydrogen loaded metals are performed with high beam density(Jmax1.2 mA/cm2)and lowbeam density(Jmin0.02 mA/cm2).Palladium and titanium foils（plates）are bombarded withproton and deuteron beams in order to compare the atomic and nuclear interactions between dif-ferent ion beams.X-ray and charged particles are measured,and neutron and gamma doses arealso monitored during the implantation experiments.An anomalous peak in X-spectra,whoseenergy is about four times the beam energy,is observed during the high beam density experi-ment.The peak moves from about 40 keV to 62 keV and FWHM reduces rapidly,while thebeam energy and intensity increase.Another wide peak with over twice the beam energy is mea-sured in experiment with low beam density.It is located between 16 keV and 30 keV,and itspeak energy increases with the increase of implantation dose（implantation time）.Some anoma-lous intensities of neutrons correlated with a charged particle peak（2.93,3.85 MeV）are alsoobserved in the deuteron-palladium experiment.The highest neutron intensity reaches about8×104 n/s,while the beam energy and intensity are about 15 keV and 1.0 mA,respectively.

Molecule modification and mass deposition induced by the implantation of low energy Fe^+ ion beams into amino acids

Fe+ ion beams with the energy of 110 keV were implanted into films of L(+)-cysteine (HSCH2CH(NH2)COOH). One of the single crystals grown in hydrochloric acid solution with the implanted samples through slow evaporation was structurally characterized by the X-ray crystallography. The crystal is monoclinic, space group C2, with a = 1.8534(4) nm, b=0.5234(1) nm, c = 0.7212(1) nm, β=103.722°, V = 0.67965(3) nm3, Z = 4, F(000) = 144.0, Dclac = 1.763 g·cm-3, μ (MoKa) = 1.06 mm-1, T = 293(2) K.R = 0.0379, wR = 0.0835 for 660 observed reflections (I】2σ(I)). The structural formula of the crystal compound is (CH2CH(NH2)NO2)ClFe (Mr = 180.38u). Products of heavy ion beam irradiation were purified and it was directly confirmed that the implanted Fe+ ions had been deposited in the novel molecules. The same doses of Fe+ ion beams of the same energy were implanted into films of L(+)-cysteine hydrochloride monohydrate. FTIR spectroscopy of the implanted samples proved that some of the original molecules were

A Mutant Strain of a Surfactant-Producing Bacterium with Increased Emulsification Activity

As reported in this paper, a strain of oil-degrading bacterium Sp - 5 - 3 was determined to belong to Enterobacteriaceae, which would be useful for microbial enhanced oil recovery (MEOR). The aim of our study was to generate a mutant using low energy N+ beam implantation. With 10 keV of energy and 5.2× 1014 N+/cm2 of dose - the optimum condition, a mutant, S-34, was obtained, which had nearly a 5-fold higher surface and a 13-fold higher of emulsifica-tion activity than the wild type. The surface activity was measured by two methods, namely, a surface tension measuring instrument and a recording of the repulsive circle of the oil film; the emulsification activity was scaled through measuring the separating time of the oil-fermentation mixture. The metabolic acid was determined as methane by means of gas chromatography.

Development of a SCAR Marker for Discrimination of a Thai Jasmine Rice (<i>Oryza sativa</i>L. cv. KDML105) Mutant, BKOS6, and Associated with Purple Color Trait in Thai Jasmine Rice-Related Varieties

The potential of SCAR marker for discrimination of a Thai jasmine rice (Oryza sativa L. cv. KDML 105) mutant, BKOS6, obtained from ion-beam-induced mutation, was evaluated. The improved rice variety, BKOS6, exhibited many remarkable characteristics which fitted the multiple cropping system characteristics of progressive agriculture including photoperiod insensitivity, early flowering, short in stature, and purple pigment accumulation in pericarp. The BKOS6 rice grain extract has already been proved that it exhibited higher antioxidant properties than the KDML 105 and other tested rice grain extracts. In this study, the BKOS6 specific SCAR marker was developed by HAT-RAPD analysis of rice genomic DNA. The marker was successfully used to identify BKOS6 variety and its hybrid varieties containing purple pigment accumulation in plant tissues. Moreover, it was found that this marker could be used to detect other purple pigmented rice varieties that genetically related to Thai jasmine rice. Recently, a wide variety of anthocyanin-based foods are believed to provide significant potential health benefits, and become more attractive. KDML 105 is also a Thai premier fragrant rice variety which is one of the main varieties of country’s rice export. Thus this molecular marker could be useful for commercial and breeding purposes of BKOS6 mutant and other developed varieties from KDML 105 which contain anthocyanin accumulation.

建立低能离子束介导小麦转基因方法并获得转GUS基因植株

研究了注入离子种类、能量、剂量等参数对于低能离子束介导的遗传转化的影响，建立了适于小麦成熟胚转化的组培条件和筛选程序。以携带ＧＵＳ基因的质粒为供体，进行了报告基因转化研究。分子生物学证据表明ＧＵＳ基因已整合到小麦基因组中。３个小麦品种的抗性愈伤转化率分别为 ９．５％、 １０．８％、 １１．２％，再生植株转化率分别为 １．４％、 ３．４％、 １７％。首次证明了离子束介导小麦遗传转化是可行的，为离子束介导小麦遗传转化体系的建立打下了基础。

低能离子束介导的遗传转化研究进展

低能离子束注入对细胞的刻蚀作用提供了外源遗传物质进入细胞的途径。离子束介导的遗传转化技术已在水稻、小麦、烟草、棉花等多种植物上取得成功。对离子束介导遗传转化的原理和最新进展进行了评述。

低能离子束刻蚀番茄果皮的α透射能谱研究

用２０ｋｅＶ 的Ａｒ＋ 、Ｎ＋ 离子分别以（５００ － ２５００） ×２ ．６ ×１０１３ｉｏｎｓ／ｃｍ ２ 、（５００ － ４０００） ×２ ．６ ×１０１ ３ｉｏｎｓ／ｃｍ ２ 的剂量对番茄果皮进行刻蚀，刻蚀前后的样品厚度用α透射能谱进行测量，借助Ｔｒｉｍ９７ 计算程序对刻蚀厚度进行标定。结果表明： 刻蚀厚度同剂量的关系曲线呈增、减、增的变化趋势。对质量数不同的Ｎ＋ 、Ａｒ ＋ 离子，其刻蚀厚度和增、减、增的剂量范围不同。Ａｒ ＋ 离子刻蚀番茄果皮的计算结果表明刻蚀深度超过５０μｍ 。

氮离子注入蛹虫草选育高效富硒菌株的研究

为了更好地开发蛹虫草资源,对蛹虫草菌株富集微量元素硒的培养条件进行了优化,选择最佳参数的低能离子束注入蛹虫草,通过石墨炉原子吸收光谱法检测注入前后菌丝体中硒元素的含量。结果表明:蛹虫草发酵富硒的最优培养条件为蛋白胨质量浓度3 g/100 mL、葡萄糖质量浓度3 g/100 mL、亚硒酸钠质量浓度为8?g/mL、pH值为7,此时,富硒率为21.58%。离子束最佳注入参数为:注入离子为N+,注入能量10 keV,注入剂量1.82×1015 ions/cm2,选育出富集微量元素硒较高的10株菌株,最高富硒率为30.97%,比出发菌株提高了近42%。

低能离子束介导水稻遗传转化的研究

为研究影响低能离子束介导水稻遗传转化效率的各种因素 ,以粳稻 0 2 42 8、花培 94 鉴 0 9、籼稻明恢 63这 3个水稻品种为实验材料 ,分析了离子的种类、能量、剂量、剂量率等参数对遗传转化的影响 ,建立了有利于遗传转化的组织培养条件和筛选程序。分子生物学检测证明 ,外源GUS报告基因已经整合到水稻基因组中 ,3个水稻品种获得抗性愈伤组织的转化率分别为 1 1 %、1 1 4%和 7 1 % ,获得再生试管苗的转化率分别为 1 5 2 %、1 87%和 1 1 3 %。为应用离子束介导法进行其它对农杆菌不敏感的禾谷类作物转基因提供了方法学的参照 ,并为深入研究离子束与生物体相互作用机制打下了基础。

低能离子束表面改性对聚碳酸酯(PC)润湿性能的影响

本文利用氩氧混合低能离子束对双酚A型聚碳酸酯(PC)透明材料进行表面改性,主要研究了改性前后表面润湿性的变化。结果表明,改性后的PC表面润湿性能得到了明显的改善,经XPS分析发现,改性后的PC表面生成了大量的含氧极性基团,使得表面含氧量有了很大的提高。原子力显微镜(AFM)的测试结果表明,改性后的PC表面粗糙度先减小后增大。

低能重离子生物学研究进展

相对于高能离子,低能离子与物质相互作用较少受到人们的关注,低能离子与生物体相互作用的研究则更少。离子注入生物效应的发现开拓了低能离子束技术在生物学中应用的新领域,形成了一个新的交叉学科——低能重离子生物学。本文介绍了这门新学科的基础和应用研究进展与展望。