Optimization of ultrasonic-stimulated solvent extraction of sinigrin from Indian mustard seed (Brassica Juncea L.) using response surface methodology

Introduction – Sinigrin, a major glucosinolate present in Indian mustard (Brassica juncea L.) seeds as the precursor of the anticancer compound allyl isothiocyanate, shows a wide range of biological activities. It's necessary to optimize the extraction methods and conditions, in order to improve the extraction productivity and save raw material. Objective – To systemically investigate and optimize the most important factors affected the productivity of sinigrin in the process of extraction using response surface methodology. Methodology – The ranges of three main factors including the ethanol concentration, extraction time and extraction temperature were selected by the one‐factor‐at‐a‐time method. The conditions of ultrasonic‐stimulated extraction of sinigrin from defatted Indian mustard seed powder were optimized by Box‐Behnken design to obtain the maximum productivity. Result – The predicted productivity (3.81%) was obtained using 57% ethanol concentration at 81°C for 60 min, with the coefficient of the model R² > 0.96 (n = 17). The actual productivity (3.84 ± 0.02%) of sinigrin under the optimized condition was increased by 70.67% compared with the result of conventional extraction. Meanwhile, HPLC, UV and IR were applied to examine if there is a difference between the ultrasonic‐stimulated solvent extraction and conventional extraction, and the improvement of productivity of sinigrin depended on the destruction of cell wall caused by the elimination of outer pectinous material was explained by SEM and composition content analysis. Conclusion – The ultrasonic‐stimulated solvent extraction was suggested to be a promising method to improve the productivity of sinigrin. And the results demonstrated that sinigrin productivity may be related to pectinous materials existed in the seeds. Copyright © 2010 John Wiley & Sons, Ltd.     

The role of glucosinolates and their hydrolysis products in oviposition and host-plant finding by cabbage webworm, Hellula undalis

The cabbage webworm, Hellula undalis (Fabricius) (Lepidoptera: Pyralidae), a tropical pest on crucifers (Brassicaceae), differentiated among host‐plant species for oviposition in laboratory and field tests. White mustard, Sinapis alba (L.) var. Selinda, was the preferred host‐plant, followed by Brassica juncea (L.) Czern. et. Coss var. Canadian brown mustard, and pak‐choi, Brassica campestris L. ssp. chinensis var. Joi Choi, Black Behi and Bai Tsai. Glucosinolates (GS), secondary plant compounds characteristic to the Cruciferae plant family, and their breakdown products were analyzed by using HPLC and GC‐MS‐techniques. Species differed in GS composition and concentration. Content of GS was highest in S. alba with progressively lower contents detected in B. juncea and B. chinensis. The aromatic GS, 4‐hydroxybenzyl‐GS and benzyl‐GS, were detected in S. alba. In B. juncea the alkenyl GS, allyl‐GS, dominated, whereas in varieties of B. chinensis indolyl and alkenyl GS predominated. Oviposition of H. undalis females on the non‐host‐plant Vigna unguiculata ssp. sesquipedalis (L.) Fruwirth was stimulated by application of GS extracts from the crucifer species; the extract from S. alba was preferred, followed by extracts from B. juncea and B. chinensis. Hydrolysis of GS in the plant extract from B. chinensis causes loss of the oviposition stimulatory effect of the extract. Application of the GS, allyl‐GS, and benzyl‐GS also stimulated oviposition by H. undalis. Significantly more eggs were laid on leaves treated with the aromatic GS, benzyl‐GS, than with the alkenyl GS, allyl‐GS. Host‐plant odor attracted H. undalis females but not males, in behavioral assays conducted in a Y‐tube olfactometer. Low concentrations of the GS hydrolysis product, allyl‐isothiocyanate, induced anemotaxis of females, but a high concentration of allyl‐isothiocyanate was repellent. Oviposition by H. undalis females was not stimulated by host‐plant volatiles. Females laid eggs on inserted traps and the walls of the Y‐tube regardless of presence or absence of host‐plant odor. The relevance of these results in the context of crucifer‐insect interactions is discussed.     

Agrobacterium tumefaciens-mediated transformation to alter ethylene and cytokinin biosynthesis in broccoli

Broccoli (Brassica oleracea var. italica) deteriorates rapidly following harvest. Postharvest treatment of broccoli with 6-benzylaminopurine delays senescence, whilst exogenous ethylene has been shown to accelerate this process following harvest. To alter ethylene biosynthesis, broccoli was transformed, using Agrobacterium tumefaciens-mediated transformation, with an antisense ACC oxidase gene from broccoli driven by the asparagine synthetase promoter from asparagus. In addition, broccoli was transformed with the chimeric gene construct SAG12-IPT to alter cytokinin biosynthesis during harvest-induced senescence. Transformation was achieved using both hypocotyl and cotyledonary petiole explants. The presence of an antisense ACC oxidase gene enhanced transformation efficiency, but Ag⁺ incorporated into the medium did not. The transgenic nature of these plants was confirmed by PCR and Southern analyses.     

Identification of AFLP fragments linked to seed coat colour in Brassica juncea and conversion to a SCAR marker for rapid selection

A Brassica juncea mapping population was generated and scored for seed coat colour. A combination of bulked segregant analysis and AFLP methodology was employed to identify markers linked to seed coat colour in B. juncea. AFLP analysis using 16 primer combinations revealed seven AFLP markers polymorphic between the parents and the bulks. Individual plants from the segregating population were analysed, and three AFLP markers were identified as being tightly linked to the seed coat colour trait and specific for brown-seeded individuals. Since AFLP markers are not adapted for large-scale application in plant breeding, our objective was to develop a fast, cheap and reliable PCR-based assay. Towards this goal, we employed PCR-walking technology to isolate sequences adjacent to the linked AFLP marker. Based on the sequence information of the cloned flanking sequence of marker AFLP8, primers were designed. Amplification using the locus-specific primers generated bands at 0.5 kb and 1.2 kb with the yellow-seeded parent and a 1.1-kb band with the brown-seeded parent. Thus, the dominant AFLP marker (AFLP8) was converted into a simple codominant SCAR (Sequence Characterized Amplified Region) marker and designated as SCM08. Scoring of this marker in a segregating population easily distinguished yellow- and brown-seeded B. juncea and also differentiated between homozygous (BB) and heterozygous (Bb) brown-seeded individuals. Thus, this marker will be useful for the development of yellow seed B. juncea cultivars and facilitate the map-based cloning of genes responsible for seed coat colour trait. Received: 2 October 1999 / Accepted: 11 November 1999     

影响茎用芥菜愈伤组织诱导和植株再生的因素

茎用芥菜子叶培养在MS 0.25 mg·L-1NAA 0.5 mg·L-16-BA 0.25 mg·L-12,4-D培养基上,可获得较高质量的愈伤组织.愈伤组织培养在MS 1～1 mg·L-16-BA 0.2 mg·L-1NAA培养基上分化频率为8.3%,而在加有羧苄青霉素和头孢霉素的培养基上,最高分化频率可达52.4%.将获得的再生植株转移到1/2MS 0.1 mg·L-1 NAA的生根培养基中,可获得完整植株.     

混合小麦亲本与新麦草不对称体细胞杂交体系的建立

以小麦品种济南177悬浮细胞系来源的原生质体与同品系胚性愈伤组织制备的原生质体混合后作为受体;以经过380μＷ/ｃｍ²紫外线照射1min、2min的新麦草原生质体分别作为供体,用PEG法诱导融合。组合Ⅰ(176+cha9+新麦草UV 1min)获得16个再生克隆。经过形态学、同工酶、染色体和RAPD分析,确定其全部为属间体细胞杂种。其中的5个克隆再生杂种植株。用7对小麦SSR引物对杂种克隆的叶绿体基因组进行了分析;组合Ⅱ(176+cha9+新麦草UV 2min)只获得3个克隆,且逐渐褐化死亡。表明以小麦济南177的两种培养细胞混合作受体的融合体系有利于杂种的获得及再生;紫外线对融合产物的生长发育有明显的剂量效应。     

Effect of Salinity on Zinc uptake by Brassica juncea

Salinity is a major worldwide problem that affects agricultural soils and limits the reclamation of contaminated sites. Despite the large number of research papers published about salt tolerance in Brassica juncea L., there are very few accounts concerning the influence of salinity on the uptake of trace metals. In this study, B. juncea plants divided through soil sets comprising 0, 900 and 1800 mg Zn kg^?1, were treated with solutions containing 0, 60 and 120 mmol L^?1 of NaCl, with the purpose of observing the effect of salt on Zn uptake, and some physiological responses throughout the 90 days experiment. Increasing concentrations of NaCl and Zn produced a decline in the ecophysiological and biochemical properties of the plants, with observable synergistic effects on parameters like shoot dry weight, leaf area, or photochemical efficiency. Nevertheless, plants treated with 60 mmol L^?1 of NaCl accumulated striking harvestable amounts of Zn per plant that largely exceed those reported for Thlaspi caerulescens. It was concluded that salinity could play an important role on the uptake of Zn by B. juncea. The potential mechanisms behind these results are discussed, as well as the implications for phytoremediation of Zn on saline and non-saline soils.     

Development of Plantlets from Leaf Protoplast Culture in Brassica juncea var. tsatsi

Protoplast of two mustard cultivars: Brassica juncea var. tsatsi cv. “Quxian Jiaoercai” and “Bangbangcai”, were isolated by enzymolysis from leaf grown in vitro. Protoplasts were suspended in liquid medium and semi-solidified medium with 0.35% low melting point agarose which formed a thin layer floating on the surface of the liquid medium. The first division appeared after 48h in the culture. One week after the original culture, a diluted medium with gradual dicrease of mannitol concentrations (6%→4%→zero) was then added to the culture three times respectively at one week's interval. In this culture method cell division and formation of microcalli were achieved. During the liquid culture of protoplasts, shaking at 20 rpm from time to time was beneficial in the formation of cell colonies and microcalli. Cell colonies developed into calli of approx 0.5—1mm in diameter one month after culture. The plating efficiency, which defined as the percentage of microcatli to numbers of protoplasts, was 0.2%—1%. Shoot regeneration occured when leaf protoplast-derived calli of “Quxian Jiaoercai” were transferred onto the modified MS medium supplemented with BAP 2.0mg/L, KT 1.0mg/L and NAA 0.2mg/L, and those of -'Bangbangcai" were transferred onto the modified MS medium supplemented with BAP 2.0mg/L. Individual shoot was rooted on a rooting medium supplemented with NAA 0.2 or 0.4 mg/L.     

芥菜型油菜抗虫转基因植株及其后代株系的研究

带有１ ～２ ｍ ｍ 子叶柄的芥菜型油菜子叶经农杆菌感染后,培养在附加１０ ～２０ ｍｇ／ Ｌ卡那霉素的 Ｍ Ｓ 选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含３０ ～５０ ｍ ｇ／ Ｌ 卡那霉素的选择培养基上继代培养,再转移到含２０ ｍｇ／ Ｌ 卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫晶体蛋白基因为探针,进行 Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 分子杂交,得到阳性结果。 Ｐ Ｃ Ｒ 分析也证明外源基因整合到油菜基因组并稳定传递到后代。转基因植株的抗虫性和卡那霉素抗性在自交后代中得到保持,筛选得到纯合的转基因植株后代株系