根癌农杆菌介导乙肝病毒表面抗原基因转化樱桃番茄体系优化

以樱桃番茄为试材,建立了樱桃番茄高频再生体系,并对农杆菌介导乙肝病毒表面抗原(HBsAg)基因转化樱桃番茄的条件进行了优化研究。结果表明:11d苗龄的子叶外植体在MS+6-BA 3.0mg/L+IAA 0.2mg/L的培养基上预培养2d后,用OD值为0.6的农杆菌侵染15min,可使樱桃番茄植株达到最佳的遗传转化效果。     

根癌农杆菌介导的西瓜遗传转化研究

利用带有内含子的GUS基因的瞬时表达,研究影响根癌农杆菌介导的西瓜遗传转化的若干因素。结果表 明:西瓜子叶块外植体对潮霉素较为敏感,15 mg/L是适宜的筛选浓度,可明显抑制非转化组织的生长;脱菌过程中 采用500 mg/L的头孢霉素,对外植体生长影响较小;农杆菌菌株EHA105对西瓜子叶块的侵染能力较强;预培养有 利于转化;共培养3-4 d有利于提高转化频率并避免了农杆菌的过度生长;OD600值为0.3的菌液侵染10min效果最 佳;共培养培养基中添加乙酰丁香酮可提高转化频率。     

小西葫芦黄花叶病毒外壳蛋白基因导入西瓜的遗传转化

研究了西瓜品种ZKM的再生体系和农杆菌介导小西葫芦黄花叶病毒(Zucchini yellowmosaic virus,ZYMV)的外壳蛋白(cp)基因导入西瓜的遗传转化体系。结果表明,质量分数0.6%琼脂是适宜种子萌发的基本培养基,切取5d龄的西瓜无菌苗子叶为外植体,接种在MS+BA2.0mg/L诱导培养基上,诱导不定芽效率最高。选用75mg/L卡那霉素筛选西瓜抗性芽较为合适,外植体经预培养2～3d,菌液浓度以OD600nm值为0.4,共侵染10min有利于提高西瓜转化的效率。经PCR检测,ZYMVcp基因已经导入西瓜植株,转化频率为0.15%。     

The culture of isolated microspores of ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo regeneration

The culture of isolated microspores of kale (Brassica oleracea var. acephala) was studied including the importance of genotype to embryo regeneration, medium composition chiefly the sucrose concentration and the use of colchicine, simultaneously medium renovation. It was initiated using 29 different genotypes as donor plants. Embryos were induced from six of the kale genotypes and these corresponded to the more out-bred genotypes. Embryogenesis was achieved using four different combinations of culture media: (a) microspores initially cultured in NLN medium supplemented with 13% (w/v) sucrose (NLN-13) for 48 h, followed by transfer to fresh NLN-13 medium; (b) microspores cultured for 48 h in NLN-13 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium; (c) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-16 medium; (d) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium. The embryos obtained from four of the genotypes developed into plantlets and these regenerated plants have been successfully transplanted to soil.     

根癌农杆菌介导的韭菜基因转化体系的建立

 以GUS 基因为报告基因, 建立了农杆菌介导的韭菜基因转化体系。研究结果表明, 以预培养4～6 d 的根尖为转化受体, 用OD₆₀₀0. 6 ～0. 8 的LBA4404 浸染2～5 min , 共培养2 d , 然后在MS + NAA1 mg/L + BA 2 mg/L + Km 25 mg/L + Timentin 400 mg/L + As 100 mg/L 培养基上培养40 d , 后有愈伤组织产生,转入光下培养20 d 后有少量绿色不定芽产生。经X-Gluc 染色、PCR 分析和Southern blot 检验, GUS 基因已经整合到韭菜基因组染色体上。     

Changes in kale (Brassica oleracea L. var. acephala) carotenoid and chlorophyll pigment concentrations during leaf ontogeny

There has been recent market interest in “baby” salad greens. However, little information exists on the nutritional differences between immature “baby” greens and produce traditionally sold at the fully mature stage. Kale (Brassica oleracea L. var. acephala D.C.) contains high levels of lutein and β-carotene, which possess important human health properties. Kale was grown in a controlled environment and pigments were measured in young (<1 week), immature (1–2 weeks), mature (2–3 weeks), fully developed (3–4 weeks) and senescing (>4 weeks) leaves using high-performance liquid chromatography (HPLC). Significant differences were observed for all pigments during leaf development. Pigment accumulation followed a quadratic trend, with maximums occurring between the 1st and 3rd week of leaf age. The highest concentrations of lutein measured 15.1 mg/100 g fresh mass and occurred in 1–2 week old leaves. The remaining pigments reached maximum levels at 2–3 weeks (β-carotene at 11.6 mg/100 g; chlorophyll a at 251.4 mg/100 g; and chlorophyll b at 56.9 mg/100 g fresh mass). Mature fully expanded kale leaves accumulated higher carotenoid concentrations than immature or “baby” leaves, with senescent leaves having the lowest carotenoid concentrations. Harvesting kale leaves at a mature stage of development resulted in maximum carotenoid values. Cultural management practices that increase carotenoid concentrations would be expected to improve nutritional quality for fresh markets.     

Efficient embryo induction in cucumber ovary culture and homozygous identification of the regenetants using SSR markers

Several factors, i.e. the duration of thermal shock pretreatment at 35 °C, the concentrations of TDZ and the silver nitrate were investigated for their effects on embryo formation in a variety of cucumber (Cucumis sativus L) ovary culture. The results showed that a thermal shock for 3 days at 35 °C at the start of the culture resulted in higher frequency of embryo formation than 2 or 4 days. TDZ had a positive effect on the embryo formation. Highest embryo formation frequency (72.7%) was recorded by adding 0.04 mg/L TDZ into the induction medium. The results found that addition of AgNO₃ to induction medium had no significant effect on frequency of embryo formation but shortened embryo sprouting period and improved number of embryos formed in each ovary slice. All the experiment materials responded well to ovary culture, and there were no difference among genotypes used in this study. Among the forty regenerated plants obtained, two were identified as haploid plants (2n = x = 7), five were tetraploid plants (2n = 4x = 28), and the rest were diploid plants. Microsatellite markers (SSR) were used to analyze the homozygosity of the diploid plants, the putative chromosome-doubled haploids. Of the 33 diploid plants, 17 (51.5%) were identified as double haploids. Based on the above results, we have established a useful protocol for production of cucumber doubled haploids with ovary culture.     

Phytochemical composition and antioxidant activity of high-lycopene tomato (Solanum lycopersicum L.) cultivars grown in Southern Italy

In this study, the antioxidant components and of six high-lycopene (Lyco 1, Lyco 2, HLY 02, HLY 13, HLY 18 and Kalvert) and one ordinary (Donald) tomato cultivars (cvs) grown simultaneously in an open-field of the Southern Italy were investigated. Lycopene, β-carotene, lutein, total phenols, flavonoids, ascorbic acid (AsA), dehydroascorbic acid (DHA) and total vitamin C (AsA + DHA) contents, as well as hydrophilic and lipophilic antioxidant activities (HAA and LAA) were determined. Significant differences were detected among tomato cvs in all studied antioxidant components, as well as in the antioxidant activity of their hydrophilic and lipophilic fractions. High-lycopene tomato cvs showed higher lycopene, β-carotene, HAA and LAA when compared to cv Donald. Cv HLY 18 showed the highest lycopene and β-carotene content with 232.9 mg/kg fresh weight (fw) and 19.4 mg/kg fw, respectively. Except for Kalvert, high-lycopene tomato cvs also obtained higher total vitamin C levels, with cv HLY 13 top ranking with an average of 352.8 mg/kg fw. LAA ranged from 133.5 μM Trolox/100 g fw in cv Donald to 540.1 μM Trolox/100 g fw in cv Lyco 2 and was significantly correlated to lycopene (r = 0.53; p < 0.01) and β-carotene (r = 0.56; p < 0.01) contents. A variation between 2.7- and 4.0-fold was found in LAA of high-lycopene tomato cvs compared to Donald. HAA was significantly correlated to the amount of DHA (r = 0.61; p < 0.01) and total vitamin C (r = 0.60; p < 0.01). Although these data require confirmation over a longer period of time, this investigation suggests a promising use of the high-lycopene tomato cvs for the production of tomatoes with higher nutritional quality.     

Regeneration and transformation system in Mirabilis jalapa

Protocols for in vitro regeneration and production of in vitro-propagated plants and a transformation system were developed for Mirabilis jalapa (Nyctaginaceae). Among the types of explants and the different media tested, consistent shoot regeneration was obtained only from nodal segments grown in a regeneration medium consisting of Murshashige and Skoog medium supplemented with 2 mg l⁻¹ 6-benzyladenine, 2 mg l⁻¹ zeatin and 1 mg l⁻¹ indole acetic acid. Regeneration efficiency was dependent on the type of plant – white or pink flowers – used as the source of explants. Stable transformation was obtained following inoculation of nodal segments with Agrobacterium tumefasciens strain EHA105, which harbours the binary plasmid pAD1339 containing both nptII and gus genes under the control of the 35S promoter. Transformation was confirmed by PCR and Southern blot analysis of genomic DNA from mature regenerated plants. β-Glucuronidase (GUS) activity was observed only in tissues regenerated from in vitro-grown plants and not in tissues originating from greenhouse-grown plants. GUS expression was not uniform in regenerated leaves and showed a chimera pattern.     

Agrobacterium × plant factors influencing transformation of ‘Joseph's coat’ (Amaranthus tricolor L.)

A protocol is developed for Agrobacterium-mediated genetic transformation of Amaranthus tricolor via explant co-cultivation with Agrobacterium rhizogenes. Bacteria-plant specific factors which influenced transformation were optimized. Of the two Agrobacterium strains employed, LBA9402 was more infectious compared to A4. Bacterial suspensions grown overnight with 100 μM acetosyringone and experiencing O.D.₆₆₀ = 0.6 followed by dilution to a density of 10⁹ cells ml⁻¹ were the most effective. Explants from garden-grown plants were more responsive than those from in vitro cultures; stem internodes being better than leaves. Immersion of the pre-pricked explants in bacterial suspension resulted in a markedly higher transformation frequency compared to the direct injection method. The infection of internode explants with the LBA9402 strain followed by co-cultivation on growth regulator-free MS medium (MS0) for 5 days resulted in emergence of hairy roots up to a maximum frequency of 97.22%. Roots were individually cultured in MS0, but fortified with bactericidal antibiotic (500 μg ml⁻¹ cefotaxime). Rhizoclones showing prolific growth were renewed through successive subcultures in MS0. Opine gene expression was revealed by positive agropine and mannopine synthesis in all selected transformed rhizoclones. Shoot regeneration from root clones, capable of auxin-independent growth and opine proficiency, was stimulated in MS augmented with 2.0 mg l⁻¹ zeatin. pRi TL–DNA rolB and pRi TR–DNA man2 ORF were detected in leaf tissues of regenerated plants from selected hairy root clones through PCR amplification. The implication of such findings is discussed on the possibility of conferring protection to crop amaranths against biotic stress challenges, particularly due to insects, viruses or fungal pathogens.     

In vitro propagation of Lychnis senno Siebold et Zucc., a rare plant with potential ornamental value

Lychnis senno is a rare and valued ornamental plant. Seed propagation is not efficient because of the low germination rate. To grow commercially L. senno in China, a protocol for in vitro germination and propagation of this species was developed. Various germination rates were obtained by treating seeds with GA₃ during 1–6 months storage period. The highest germination rate reached 19.4% when seeds were treated with 250 mg/l GA₃ and stored for 5 months at 4 °C. Axillary shoot proliferation was induced in the nodal segments of the seedlings on medium containing specific concentrations of BA and NAA [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497]. Maximum number of shoots was developed on a medium supplemented with 5 mg/l BA and 0.5 mg/l NAA, while the higher shoots were observed on a medium supplemented with 0.5 mg/l BA and 0.05 mg/l NAA. Rooting was induced in 91.7% of the regenerated explants on a half-strength MS medium supplemented with 0.5 mg/l NAA. The plantlets grew well and flowered after transfer to the greenhouse. The chromosome numbers of seedlings and propagated plants were also determined to be 2n = 2x = 24.     

Improvement of caper (Capparis spinosa L.) propagation using in vitro culture and gamma irradiation

Some of the factors influencing the propagation of caper (Capparis spinosa L.) plants in vitro and germination of the seed were studied. The number of adventitious shoots emerging from caper stems cultured in vitro increased from 2.2 shoots per explant when the growth medium contained 2 mg/L of gibberellic acid (GA3) to 5.5 when the growth medium contained 2 mg/L zeatin riboside (ZR) and 1 mg/L naphthalene acetic acid (NAA). The best medium for callus formation from leaf and stem parts contained the growth regulators 1 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L NAA and the best medium for plant regeneration contained 1 mg/L kinetin and 0.1 mg/L indole-3-acetic acid (IAA). The effect of gamma irradiation on the growth of caper shoots in vitro was also studied. A 10 Gy dose of gamma irradiation stimulated growth of shoots up to 200% and increased shoot rooting percentage from 75 to 100%.     

Stoloniferous shoot protoplast,an efficient cell system in potato for somatic cell genetic manipulations

The present study reports that protoplasts isolated from stoloniferous shoots (SS) of potato represent an efficient system for somatic cell genetic manipulations. SS were established from single-node cuttings on MS medium supplemented with either 0.1 or 0.2 M sucrose (Suc), and protoplasts were isolated and cultured within the alginate strip, following an improved method. SS induced by 0.1 M Suc yielded 8–22 × 10⁵ protoplasts g⁻¹ fresh mass, with a high morphogenic competence. However, 0.2 M Suc-induced SS yielded protoplasts that contained large amounts of starch grains, resulting in their high degree of fragility, delayed cell division and poor morphogenic competence. For symmetric somatic hybridization (electrofusion) between Solanum tuberosum Gp. Tuberosum androgenic (di)haploid (2n = 2x = 24) ‘C-13’ and diploid (2n = 2x = 24) wild species S. pinnatisectum, protoplasts isolated from 0.1 M Suc-induced SS were also found to be most responsive. Out of several putative somatic hybrids, there were two tetraploids and five diploids, with 48 and 24 chromosomes, respectively at all the three shoot layers (L₁–L₃). This precluded the occurrence of mixoploidy vis-à-vis chimaerism in regenerants, as common in somatic fusion involving mesophyll protoplasts of S. pinnatisectum. Nuclear microsatellite analyses based on the two single-locus nSSR loci (STM0037 and STM2030) confirmed that one of the tetraploids was a true nuclear hybrid (heterokaryon), while the other a homokaryon of the Tuberosum parent ‘C-13’. The use of 0.2 M Suc-induced SS protoplasts for fundamental studies on tissue- and/or cell type-specific transient gene expression underlying tuberization has been discussed.     

High frequency plant regeneration from microspore-derived embryos of ornamental kale (Brassica oleracea L. var. acephala)

The aim of this work was to study the effect of solid medium, developmental stage, embryonic age, cold treatment and additives to the medium on plant regeneration from microspore-derived embryos in four F₁ hybrids of ornamental kale (Brassica oleracea L. var. acephala). The results showed that all of the cultivars responded best when the embryos were cultured in solidified B5 medium with 1% agar. Optimal regeneration was gained when cotyledonary embryos were cultured for 25 days. Cold treatment significantly improved plant regeneration with a frequency of up to 79.0% under 4 °C for 2 d or 5 d. The addition of 3.0 or 5.0 mg/L silver nitrate (AgNO₃) increased the frequency of plant regeneration. In the Zhouyehongxin cultivar, the frequency of plantlet development reached 84.4%. The addition of activated charcoal reduced embryo hyperhydricity.     

The genotypic effects on the chemical composition and antioxidant activity of sea buckthorn (Hippophae rhamnoides L.) berries grown in Turkey

In this study, chemical composition of berries of 10 sea buckthorn (Hippophae rhamnoides L.) genotypes in Turkey was investigated. The total phenolic content of the berries ranged from 21.31 mg gallic acid equivalents (GAE) per g dry weight basis to 55.38 mg GAE per g. The highest antioxidant activity was 93.54% (similar to the standard BHT at 200 mg/L) and the lowest was 80.38%. There was no correlation (R = 0.688) between the total phenolic content and the antioxidant activity. The major fatty acids in berries were palmitoleic acid (35.48%), followed by palmitic acid (28.13%), oleic acid (22.89%) and linoleic acid (3.96%). Total soluble solid content of sea buckthorn genotypes varied from 10.15 to 14.80%, titratable acidity varied from 2.64 to 4.54%, the pH varied from 2.63 to 2.98 and Vitamin C varied from 19 to 121 mg/100 mL. The average content of minerals in the sea buckthorn berries of different genotypes was 20,800 ppm N, 7100 ppm P, 7260 ppm K, 1960 ppm Ca, 1465 ppm Mg, 32 ppm Zn, 24 ppm Cu, 22 ppm Mn and 7 ppm Fe.     

橙色大白菜遗传转化体系的初步建立

以06J28橙色大白菜子叶段为外植体,通过根癌农杆菌介导CMS7311-orf224基因,探讨了潮霉素、头孢霉素、预培养时间、农杆菌浓度、感菌时间和共培养时间等因素对橙色大白菜遗传转化的影响.试验结果表明,子叶段预培养2~3 d后,在OD600值为0.3~0.5的农杆菌EHA-105菌液中侵染5 min,再共培养2~3 d,在培养基中加入5 mg/L Hyg (抗性筛选)和500 mg/L Cef(脱菌)可得到转化植株.试验初步建立了橙色大白菜遗传转化体系,为大白菜种质资源创新奠定了基础.     

甜菜转基因体系的相关影响因素研究

以16种基因型的二倍体甜菜（Beta vulgarisl L．）为材料,建立并优化了甜菜的植株再生体系;对影响甜菜转基因的相关因素进行了比较分析,确定了抗生素标记基因的适宜筛选浓度为200mg／L;菌液适宜浸染浓度OD600为0．3;浸染最适时间为10min;外植体与农杆菌适宜共培养时间为4d;添加乙酰丁香酮的适宜浓度为100μmol／L。研究结果的获得,为完善甜菜的遗传工程以及甜菜基因组学的相关研究奠定基础。     

Improving the micropropagation efficiency of hybrid Dendrobium orchids with chitosan

The appropriate chitosan types and concentrations for enhancing the in vitro micropropagation of Dendrobium ‘Eiskul’ were studied using 70, 80 and 90% N-deacetylated polymeric (P-70, P-80 and P-90) and oligomeric (O-70, O-80 and O-90) forms of crab (Portunus pelagicus) chitosan. For the initial protocorm-like body (PLB) multiplication, the application of 10 mg/L of P-70 or 20 mg/L of P-90 was optimal, although 10 mg/L of P-80 and O-70 were also effective, and attained maximal PLB replication rates without increasing the detectable levels of somaclonal variation. However, during PLB-shoot induction, 10 or 20 mg/L of O-80 was the most appropriate chitosan and also induced further PLB formation. For plantlet regeneration, the addition of 10 mg/L of O-80 or P-80 gave the best quantity and quality, respectively, of plantlets. Finally, 20 mg/L of P-70 chitosan as a supplement during exflasking enhanced both the survival rate and the growth of the plantlets at one month after exflasking. Together, these data reveal a potentially beneficial and applicable protocol for commercial orchid micropropagation.     

Somatic embryogenesis and Agrobacterium-mediated transformation of Japanese apricot (Prunus mume) using immature cotyledons

This study describes a successful method of somatic embryogenesis and genetic transformation using immature cotyledons of Prunus mume. Immature cotyledons from four different developmental stages of eight different P. mume cultivars were used for the experiments to optimize somatic embryogenesis and genetic transformation protocols. Somatic embryogenesis was induced when the explants were cultured on somatic embryo inducing medium consisting of MS basic medium supplemented with 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 μM 6-benzyladenine (BA). They were cultured for 30 days and then transferred to somatic embryo propagation medium containing 0.1 μM α-naphthaleneacetic acid (NAA) and 5 μM BA. It appeared that the developmental stage of the immature cotyledons used as explants was the most important factor for somatic embryogenesis; higher frequencies of somatic embryogenesis were observed when the immature cotyledons were less than 5 mm in length regardless of cultivars. For genetic transformation, the immature cotyledons were inoculated with Agrobacterium tumefaciens EHA101 harbouring a binary plasmid vector with neomycin phosphotransferase II and an intron-interrupted β-glucuronidase gene under the control of cauliflower mosaic virus 35S promoter, and three transgenic plant lines were obtained from inoculated “Sirakaga” immature cotyledons. Transgenic somatic embryos and shoots were selected using 25 mg l⁻¹ kanamycin. Integration of transgenes in the genome of GUS-positive putative transgenic shoots was confirmed by PCR and Southern blot analyses.