藏红花瓣状体的诱导与花柱-柱头状物的定向分化

藏红花 (ＣｒｏｃｕｓｓａｔｉｖｕｓＬ .)又名番红花、西红花 ;藏药中称为苟日苟木。它是鸢尾科草本植物 ,原产于欧洲、地中海地区。我国长期以来把它作为珍贵的中藏药。其药用部位是柱头 ,有效成分主要是藏红花素、藏红花酸、藏红花醛和藏红花苦素。丁葆祖等于 1979年首次从藏红花的球茎获得完整的植株[1 ] 。Ｓａｎｏ等人 1987年在离体条件下 ,诱导花柱 柱头状物再生获得成功[2 ] ,此后国内外不断有相关报道[3～ 7] 。但试验大多集中在由外植体直接或由愈伤组织间接诱导柱头状物 ,且花柱 柱头状物的频率低和数量少。我们试图先由藏红花…     

藏红花瓣状体的诱导与花柱-柱头状物的定向分化

藏红花(Crocus sativus L.)又名番红花、西红花;藏药中称为苟日苟木.它是鸢尾科草本植物,原产于欧洲、地中海地区.我国长期以来把它作为珍贵的中藏药.其药用部位是柱头,有效成分主要是藏红花素、藏红花酸、藏红花醛和藏红花苦素.丁葆祖等于1979年首次从藏红花的球茎获得完整的植株[1].Sano等人1987年在离体条件下,诱导花柱-柱头状物再生获得成功[2],此后国内外不断有相关报道[3～7].但试验大多集中在由外植体直接或由愈伤组织间接诱导柱头状物,且花柱-柱头状物的频率低和数量少.我们试图先由藏红花花瓣诱导瓣状体,再定向分化花柱-柱头状物,来提高花柱-柱头状物的诱导频率和数量.从而为以后的大规模生产奠定基础.     

番红花雄蕊柱头状物的离体再生

以番红花（Crocus sativus L.)雄蕊为材料诱导培养出花柱柱状结构,诱导率可达30％,起源于花丝基部。影响雄蕊柱头状物诱导的主要因素为外植体的发育期和生长素NAA的使用浓度。幼嫩浅黄色雄蕊适于诱导柱头状物。温度和光照在不同激素水平下对雄蕊柱头状物诱导的影响不同。紫外检测表明,由雄蕊诱导出的柱头状物含有番红花甙、番红花醛和番红花苦甙。其含量明显高于由药柱诱导出的柱头状结构。在诱导花魔王、子房、花瓣的柱头状物的过程中,观察到成药逆转现象。     

离体培养下番红花花柱—柱头状物再生的研究

番红花(Crocus sativus L.)幼花被和幼花柱可以诱导花柱-柱头状物的再生,长度为24mm 的花芽上分离的花被具有最高的诱导频率(37.5%);花被外植体附加 K5 mg/L 和NAA 4mg/L 有利于再生花柱-柱头状物,频率达到35.3%;附加6-BAP 7 mg/L 和 NAA9mg/L 则有利于柱头状物的大量形成。     

莴苣授粉前后柱头与花柱中钙的分布变化

莴苣柱头呈两裂片状,有接受花粉的接受面和非接受面之分。授粉前后,柱头接受面乳突细胞的细胞壁中贮存丰富的细小钙颗粒,而非接受面的表皮细胞壁中的钙颗粒很少。在花柱组织中钙的分布具有明显特征,在同一水平上,钙主要分布在引导组织的质外体中,如细胞壁和细胞间隙中,而在引导组织外的薄壁组织中钙主要分布在的细胞内液炮和细胞壁中以及维管束导管内。在花柱不同水平上,花柱中的钙呈现出梯度分布,顶端各组织中的钙颗粒较少,基部各组织中的钙颗粒较多。授粉后1h花柱基部组织的钙颗粒增多,钙梯度分布现象增强。花柱引导组织中的钙梯度分布很可能是吸引花粉管向下生长的原因。讨论了莴苣花柱引导组织中钙梯度分布特征和花粉管在其体内生长的关系。     

不同葡萄品种柱头、花柱发育与种子形成的关系

对‘京秀’、‘香妃’(二倍体)和‘黑奥林’、‘巨峰’(四倍体)葡萄品种的柱头、花柱发生发育以及花粉管在雌蕊中生长与坐果和果实中种子形成的关系进行了研究。结果显示:‘京秀’、‘香妃’的坐果率和果实中的种子数明显高于‘黑奥林’和‘巨峰’,而‘巨峰’葡萄的坐果率和种子数最低。开花期‘京秀’和‘香妃’的花柱直径略小于‘黑奥林’和‘巨峰’,但花柱中部引导组织的直径和引导组织所占花柱的比例却大于‘黑奥林’和‘巨峰’;半实心花柱的比例也高于‘黑奥林’和‘巨峰’,说明花柱中引导组织的结构和发育状况可能与坐果和果实中种子形成有关;人工授粉24 h后,在‘京秀’和‘香妃’雌蕊中花粉管的生长速度快于‘黑奥林’和‘巨峰’,授粉后48 h‘京秀’和‘香妃’雌蕊中到达各部位的花粉管数要多于‘黑奥林’和‘巨峰’,授粉后72 h各品种到达子房各部位的花粉管数开始减少。表明‘京秀’和‘香妃’花柱中引导组织较为发达,而且半实心花柱比例也较高,有利于花粉管在雌蕊中的生长和完成授粉受精,使‘京秀’和‘香妃’的坐果率和果实中种子的形成都较高。     

莴苣授粉前后柱头与花柱中钙的分布变化

莴苣柱头呈两裂片状,有接受花粉的接受面和非接受面之分。授粉前后,柱头接受面乳突细胞的细胞壁中贮存丰富的细小钙颗粒,而非接受面的表皮细胞壁中的钙颗：粒很少。在花柱组织中钙的分布具有明显特征,在同一水平上,钙主要分布在引导组织的质外体中,如细胞壁和细胞间隙中,而在引导组织外的薄壁组织中钙主要分布在的细胞内液泡和细胞壁中以及维管束导管内。在花柱不同水平上,花柱中的钙呈现出梯度分布,顶端各组织中的钙颗粒较少,基部各组织中的钙颗粒较多。授粉后1h花柱基部组织的钙颗粒增多,钙梯度分布现象增强。花柱引导组织中的钙梯度分布很可能是吸引花粉管向下生长的原因。讨论了莴苣花柱引导组织中钙梯度分布特征和花粉管在其体内生长的关系。     

In Vitro Production of Stigma-like Structures from Stigma Explants of Crocus sativus L.

Stigma-like structures (TC stigmas) were produced in tissuecultures from stigma explants of Crocus sativus under definedconditions. MS medium supplemented with NAA (10 mg dm^–3)+BA (1.0 mg dm^–3) induced the optimum response. NAA wasfound to be an important addendum to achieve a good response.The TC stigma regeneration response as a function of explantage showed significant differences (except between stage 1 andstage 4). A culture temperature of 20 °C seems to be betterthan 25 °C with reference to all parameters. Crocin andpicrocrocin pigments, responsible for colour and bitter taste,respectively, were extracted, identified and quantified fromthe TC stigmas. Safranal was not detected in fresh samples. Key words: Crocus sativus, stigma-like structures, organ regeneration, crocin, picrocrocin, safranal     

西红花的离体成花

1植物名称西红花(Crocus sativus). 2材料类别球茎的幼嫩顶芽,长度在20～30 mm. 3培养条件诱导培养基分别为:(1)MS NAA 5mg·L-1(单位下同) 6-BA 5;(2)MS NAA 5 6-BA7;(3)MS KT 5 NAA 4;(4)MS 2,4-D 2 KT0.5;(5)MS 6-BA 2 2,4-D 0.1;(6)MS 6-BA2 IBA 1;(7)B5 KT 5 IAA 4 2,4-D 1 Pro 0.5;(8)B5 KT 5 IAA 4 2,4-D 1 Pro 0.5.以上培养基均添加琼脂0.8%;(1)～(5)中加蔗糖4%,(6)中3%,(7)中6%,(8)中4%;pH 5.8.培养温度(20±2)℃,黑暗培养.     

Variation in Saffron (Crocus sativus L.) accessions and Crocus wild species by RAPD analysis

Crocus, a genus of Iridaceae is mostly grown in areas with Mediterranean environment as the best region. Saffron (C. sativus) is a perennial plant and is cultivated as an industrial crop in several regions of Iran. In this research, five accessions of cultivated Saffron from five areas in Khorasan, and Esfahan including Gonabad, Ferdows, Torbat-e-Heyidariye, Estahbanat, Gopayegan were used. Other nine species of saffron were grown naturally in Iran; so we collected two wild species (C. caspius and C. speciosus) from north of Iran (Gilan Province). RAPD markers were used to classify these species and to find their relationships. In the results of this study, the cluster analysis showed two distinct groups. Also, the maximum similarity was seen between C. caspius and C. speciosus (0.82) and the minimum was between Estahbanat, Ferdows accessions and C. speciosus (0.33). Finally, this method as a convenience procedure could be used to separate different accessions and species of Crocus as well.     

Callus Induction from Explants of Crocus sativus

Saffron calli were induced from ovary explants on Murashige-Skoog (MS) medium supplemented with beyzyladenine (BA) and naphthalene acetic acid (NAA) as growth factors. MS medium with 5 mg l^?1 BA and 10 mg l^?1 NAA was selected for calli induction and undifferentiated calli growth, while MS medium with 1 mg l^?1 BA and 1 mg l^?1 NAA was the most appropriate for stigma differentiation. On this medium, stigma-like structures measuring 0.5–1.5 cm were obtained. Initially they were colourless, but yellow pigmentation, due to the presence of crocin, progressively increased with calli growth. Extracts of stigma-like structures were analysed by HPLC and the presence of saffron secondary metabolites was demonstrated. In addition, calli also showed yellow pigmentation.     

藏红花花瓣和雄蕊挥发油化学成分GC-MS分析及比较

采用水蒸气蒸馏法从藏红花花瓣和雄蕊中提取挥发油,用GC-MS技术结合计算机检索对其二者化学成分进行分离和鉴定,用色谱峰面积归一化法计算各组分的相对含量.花瓣中共鉴定出16种化合物,主要成分为正二十六烷(11.60％)、正十五烷(11.31％)、棕榈酸甲酯(10.82％)、油酸甲酯(10.35％)、2,4-二叔丁基苯酚(9.63％)、亚油酸甲酯(7.18％)、藏红花醛(5.66％);雄蕊中共鉴定出20种化合物,主要成分为油酸甲酯(30.83％)、亚油酸甲酯(24.12％)、环已醇(16.80％)、硬脂酸甲酯(12.88％)、棕榈酸甲酯(8.97％)、花生酸甲酯(1.18％)、苯并噻唑(1.01％).     

Production of crocin using Crocus sativus callus by two-stage culture system

Saffron callus was grown in a two-stage culture on B5 medium supplemented with casein hydrolysate (300 mg l^–1) at 22 °C in dark with naphthalene acetic acid (2 mg l^–1) and 6-benzyladenine (1 mg l^–1) to give maximum biomass (16 g dry wt l^–1), and with indole 3-acetic acid (2 mg l^–1) and 6-benzyladenine (0.5 mg l^–1) for crocin formation. The maximum crocin production (0.43 g l^–1) was achieved by this two-stage culture method, which was three times that by a one-stage method.     

Dehydration and viability of saffron crocus (Crocus sativus)

Saffron pollen reaches the shedding stage with a high water content which averages 58% under 18-20°C and RH 55-70%. Because of the precocious incision of anther stomium and the apertures of pollen exine, when the flower opens the pollen is soon exposed to dehydration. As a result, it goes from the highly hydrated stage to 15% water within 2 h from flower opening, and to the minimum 7% within 10 h. The transition to dry state results in a progressive loss of the plasmamembrane integrity, but this is restored after gradual rehydration. On average, 50% of the total pollen grains shows capacity for semi-vivo germination regardless of the dehydration degree. Pollen loses the competence for germination within 3 weeks from shedding. The results are discussed in comparison with fertile Crocus species.     

Germination and viability of the pollen of Crocus sativus L

Abstract

Viability and germinability tests were carried out on the pollen of Crocus sativus L., a sterile triploid commonly known as saffron. Pollen taken from dehiscent anthers was examined by means of vital staining and cytochemical techniques in order to detect its viability; germination in vitro was evaluated. From the results obtained it is evident that saffron pollen is viable at a high percentage (65%) but germinates at a very low percentage. Moreover in vitro germination is very slow, and is accompained by numerous morphological anomalies. The low germination of the pollen is due to the triploidy of C. sativus.