Brassicaceae,a Plant Family Well Suited for Modern Biotechnology

Abstract

A survey of recent progress in various biotechnological areas, on various Brassica crops, is presented. Wide hybridization and subsequent culture of embryos, ovaries and ovules can facilitate sexual gene transfer between species. The protoplast fusion technique extends the possibilities of transferring genes between species, even from different genera and tribes, it being independent of sexual incompatibility barriers. Furthermore, somatic hybridization provides novel possibilities for modifying the organelle composition of plants. Genetic engineering for crop improvements and fundamental studies in plant biology involve isolation of genes, studies of gene expression and gene regulation. In order to change or improve agronomically desirable traits, or to investigate further specific gene functions, methods for transfer of cloned genes into plants have developed. Furthermore, cryopreservation would enable conservation of genetic material, including genetically modified plants deriving from various biotechnological manipulations.     

STE20L4促进拟南芥下胚轴伸长的机制研究

下胚轴伸长是高等植物进行正常生命活动的保障,下胚轴的伸长协助植株破土而出并由异养转变为自养生长。本研究通过对一系列拟南芥突变体材料进行下胚轴观察,鉴定到一个具有短下胚轴的突变体ste20l4,并对其调控下胚轴伸长的分子机制进行初步探索。结果表明：STE20L4编码与酵母中STE20同源的丝裂原活化蛋白激酶,在MAPK级联信号通路中作为MAP4K激活下游的MAP3K。基因型鉴定和半定量结果显示,ste20l4-1、ste20l4-2均为功能缺失的突变体。通过表型观察,ste20l4突变体无论在长日照（16 h光照/8 h黑暗）、短日照（8 h光照/16 h黑暗）及黑暗（24 h黑暗）条件下与野生型相比均呈现出短的下胚轴。细胞学结果显示,突变体ste20l4短的下胚轴是由于其细胞长度的变短而不是细胞数目的变化导致的。植物激素赤霉素可促进下胚轴的伸长,对其处理的敏感性可作为是否参与GA信号转导途径的判断方法。在不同梯度浓度的GA处理（0、0.5、1、2、5 µmol/L）下,ste20l4突变体与野生型相比下胚轴伸长作用不明显。多效唑（PAC）是内源GA合成的抑制剂,随着不同梯度浓度的PAC处理（0、0.01、0.02、0.05、0.1、0.2、0.5 µmol/L）,ste20l4突变体下胚轴伸长的抑制作用相对于野生型不明显。上述生理学结果表明,STE20L4参与到GA信号转导通路中调控拟南芥下胚轴的生长发育。将STE20L4蛋白与荧光标签融合后转染烟草或拟南芥原生质体观察其亚细胞定位,结果表明STE20L4在细胞膜和细胞质中均有表达。对野生型、ste20l4突变体中进行一系列GA下游与下胚轴伸长相关基因的RT-qPCR检测,结果表明XTH17、EXP2、PRE1、PIF4、YUC2和SAUR19基因的转录水平在ste20l4突变体中明显下调,即STE20L4可能通过间接调控这些下游基因的表达参与下胚轴的伸长。综上,STE20L4参与GA信号转导通路,且通过促进GA下游下胚轴伸长相关基因的表达调控下胚轴的伸长。本研究初步阐述了STE20L4调控植物下胚轴伸长的分子机制,为进一步研究MAPK与GA互作调控植物生长发育提供参考。     

甘蓝型油菜‘南盐油1号’不同外植体再生体系的比较研究

以甘蓝型油菜‘南盐油1号’下胚轴和具柄子叶为材料,通过植物组织培养方法,探讨不同预培养和分化培养的交叉组合对油菜植株再生的影响。结果表明:不同浓度的2,4-D、6-BA和AgNO3组合对外植体的分化有不同程度的影响,但分化出的芽苗均能生根,并生长成为植株。下胚轴在预培养(MS+0.5 mg.L-12,4-D+1.0 mg.L-16-BA)和分化培养(MS+3.0 mg.L-16-BA+0.1 mg.L-1NAA+5.0 mg.L-1AgNO3)条件下的分化率最高,达到95.23%;具柄子叶在预培养(MS+0.5 mg.L-12,4-D+1.0 mg.L-16-BA)和分化培养(MS+4.5 mg.L-16-BA+0.1 mg.L-1NAA+5.0 mg.L-1AgNO3)下的分化率最高,达到89.52%。上述结果表明,下胚轴很可能更适合作为‘南盐油1号’油菜在组织培养研究中的外植体。     

壮丰安浸种对地膜甜菜幼苗生长和产量效应的研究

用壮丰安1：800倍液浸甜菜种球，使地膜甜菜幼苗的下胚轴缩短，增加苗期干物质积累和叶柄干物质积累比率；最终使甜菜的根颈缩短，青头变小，含糖率提高1．6个百分点，产糖量与对CK无显著差异。     

Effect of Sodium Chloride Salinity on Seedling Emergence in Chickpea

Although laboratory (Petri dish) germination as an estimate of seed viability is a standard practice, it may not give an accurate prediction of seedling emergence in the field, especially when saline irrigation water is used. Experiments were conducted to investigate seedling emergence in two chickpea cultivars (ILC 482 and Barka local) in response to varied salinity levels and sowing depths. Seeds were sown in potted soil at a depth of 2, 4 or 6 cm. The salinity treatments were 4.6, 8.4 and 12.2 dS m^–1. Tap water (0.8 dS m^–1) served as the control. Depth of sowing had a significant effect on seedling emergence. Seeds sown 6 cm deep showed the lowest seedling emergence. Similarly, salinity had an adverse effect on seedling emergence. The lowest seedling emergence percentages were obtained at the highest salinity treatment (12.2 dS m^–1). The interaction between salinity treatment and seeding depth was significant. Hypocotyl injury was implicated as a possible cause of poor seedling emergence in chickpea under saline water irrigation and was less severe when pre-germinated seeds were used. ILC 482 appeared to be more tolerant to salinity than Barka local, suggesting that breeding programmes involving regional exchange of germplasm may be helpful.     

小盐芥下胚轴再生体系的建立

以小盐芥下胚轴为外植体,研究了不同激素与不同浓度组合对愈伤诱导和不定芽分化的影响。结果表明,MS 6-BA 2,4-D组合能诱导出愈伤,诱导率为100%。MS 6-BA NAA组合既能诱导出愈伤又能分化不定芽,愈伤诱导率为100%,激素不同浓度不定芽分化率不同,最佳分化培养基为MS 2.5mg/L 6-BA 0.1mg/L NAA,不定芽分化率为43%。生根培养基为1/2MS,生根率为74%。     

Plant regeneration in sweet potato (Ipomoea batatas L., Convolvulaceae)

The application of new techniques for improvement of sweet potato crops, particularly including the exploitation of somaclonal variation, gene transfer by genetic transformation and somatic hybridization, requires the control of plant regeneration from tissue cultures. Shoots can easily be regenerated from explants of stems, petioles, leaves and roots, while callus cultures do not produce any shoots. The potential of somatic embryogenesis and plant regeneration via embryogenesis was evaluated for 10 cultivars of sweet potato. Protocols for plant regeneration from cultured protoplasts have also been developed. Since mesophyll was resistant to enzyme digestion, fragments of stems and petioles, callus and cell suspensions were used as source of protoplasts of sweet potato. Series of transfers of protoplast-derived calluses, particularly those which had been obtained from in vitro plants, to media containing a high level of zeatin resulted in successful formation of shoots in only two sweet potato cultivars. In addition, the embryogenic potential was irreversibly lost through protoplast culture, since protoplasts isolated from embryogenic cell suspensions developed into non-embryogenic callus. Consequently, an alternative protocol is being successfully developed to improve plant regeneration from cultured protoplasts of sweet potato, involving first root formation from which shoots can then be regenerated. Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gene specificity is maintained in transient expression assays with protoplasts derived from different tissues of barley

Summary In some cereal species that are still recalcitrant to stable transformation and regeneration, transient expression in isolated protoplasts is a useful tool for the study of gene expression and regulation. We have successfully applied these techniques to barley protoplasts derived from developing endosperm, aleurone, leaves and roots in order to characterize functionally cis-acting motives in two gene promoters, corresponding to trypsin inhibitor BTI-CMe and to sucrose synthase Ss1. Gene specificity is maintained in transient expression assays with protoplasts isolated from these different barley tissues and the pattern of expression parallels the mRNA levels observed for the corresponding genes in the same tissues.Abbreviations CaMV35S 35S-cauliflower mosaic virus promoter - GUS -glucuronidase - MU 4-methyl umbelliferone - NOS nopaline synthase gene - PEG polyethyleneglycol     

Transformation of modified cowpea trypsin inhibitor gene and anti-bacterial peptide gene in Brassica pekinensis protoplasts mediated by Agrobacterium tumefaciens

Summary A transformation system for Chinese cabbage protoplasts was developed using Agrobacterium tumefaciens strain LBA4404 (harbouring the plasmid pBinΩSCK and the plasmid pMOG 411 respectively). The plasmid pBinΩSCK contains a 415 bp insert derived from the Cowpea trypsin proteinase inhibitor gene and The plasmid pMOG 411 contains a 870 bp fragment which codes an anti-bacterial peptide gene. Freshly isolated protoplasts of Chinese cabbage (Brassica campestris L.ssp.pekinensis) lines were pre-treated at 4 ^∘C for 1 h, then incubated at 25 ^∘C for 2–3 days in the dark. 3 drops of A. tumefaciens solution in log-phase were added to 10 ml protoplasts and cocultivated for 48 h at 25 ^∘C. Some kanamycin-resistant plants and a number of kanamycin-resistant calli were obtained. Southern blot hybridization analysis demonstrated the presence of the CpTI gene and the anti-bacterial peptide gene in the Chinese cabbage genome. Northern blot analysis of the kanamycin-resistant plantlets and calli confirmed the accumulation of the CpTI and the anti-bacterial peptide mRNAs.