本地早蜜橘胚性愈伤组织的离体诱导

柑橘胚性愈伤组织诱导技术在柑橘种质资源离体保存、理化诱变、原生质体融合、外源基因转化等柑橘种质保存及新种质创制过程中有着重要的基础研究地位。目前随着柑橘分子生物育种技术的快速发展,胚性愈伤组织作为柑橘理化诱变、原生质体融合、外源基因转化的理想起始材料,其在柑橘种质创新研究中的基础应用作用更为明显。柑橘胚性愈伤组织目前主要可通过败育的成熟胚珠、未成熟胚珠、实生幼苗胚轴茎段、子叶以及花药培养等途径来诱导获得。     

绿色荧光蛋白基因转化柑橘胚性愈伤组织与高效再生

采用根癌农杆菌介导法对13份柑橘胚性愈伤组织进行绿色荧光蛋白基因(gfp)的转化,短时间内获得了温州蜜柑、橘橙杂种GWZ等11份柑橘材料的82个转化细胞系,其中佛罗斯特脐橙和日辉橘最易转化,分别获得28个和25个转化细胞系。选取3份材料的不同转化细胞系进行gfp的PCR分析,均获得了预期的gfp片段。研究表明,GFP标记可以在转化早期快速检测并分离转化子,及时剔除逃逸体,获得纯合的转化系;高水平的gfp表达不影响转化细胞增殖、生长和分化。这些带有GFP标记的转化细胞系为柑橘的有关基础研究提供了良好的试材,目前正用于柑橘体细胞杂交和不同倍性愈伤组织的社会化控制现象与机理等研究。     

二甲戊乐灵在枣树多倍体诱变中的应用初探

以‘月光’枣为试材,研究除草剂二甲戊乐灵在枣树上的多倍体诱变效果,包括二甲戊乐灵浓度、作用时间、施用时期对愈伤组织形成、芽再生及多倍体诱变的影响等。结果表明:二甲戊乐灵明显抑制了枣愈伤组织生长和芽再生,且随着浓度(0.01%～0.1%)的升高,出愈率和单枝截面出芽数均呈逐渐下降趋势;作用时间(12～48 h)对愈伤组织生长及芽再生能力影响的差异不明显;随着施用时期(6～10 d)的推移,单枝截面出芽数增多,截枝处理10 d后施用二甲戊乐灵的出愈率、单枝截面出芽数均最高;优化后的条件下,即二甲戊乐灵浓度0.025%～0.05%,作用时间24～48 h,施用时期为愈伤组织诱导(施用4.0 mg/L TDZ+2.0 mg/L AgNO_3,12～14 h)6～10 d后,获得了80个再生芽,成功诱变出3个四倍体再生芽;与秋水仙素相比,二甲戊乐灵诱导的出芽时期晚10 d左右,出芽总数较低,但其诱导的芽变异率为3.57%,较秋水仙素诱导的高659.57%。     

不同生长调节因子对耐寒柑橘愈伤组织诱导效应研究

以耐寒柑橘子叶为试验材料,研究了不同浓度的2,4-D、NAA、6-BA与KT对柑橘子叶愈伤组织诱导的影响作用。结果表明:2,4-D、NAA、6-BA与KT对柑橘子叶愈伤组织诱导具有重要效应,其综合贡献率分别为86.4%、93.9%、93.9%与94.5%。4种生长调节因子对柑橘子叶愈伤组织诱导的膨大率、膨大速度、出愈率、出愈速度的效应大小分别为2,4-DNAA6-BAKT、KT2,4-DNAA6-BA、2,4-DNAA6-BAKT与2,4-DKTNAA6-BA。综合考虑测量指标效应,对于柑橘子叶愈伤组织诱导的最适生长调节因子组合配比为2,4-D 1.0mg/L+NAA 2.0mg/L+6-BA 0.5mg/L+KT 0.5mg/L。     

红掌愈伤组织和再生团块发育过程中糖和蛋白质的组织化学研究

以红掌叶片诱导出的4类愈伤组织和气生根根段诱导的再生团块为试材,研究糖和蛋白质在愈伤组织衰老与再生团块发生发育过程中的变化。经石蜡切片PAS和汞溴酚兰组织化学染色观察发现,糖主要以颗粒状存在于愈伤组织细胞中,并有明显的围绕核的现象,蛋白质主要集中于细胞核及其周围。糖和蛋白质的含量在黄绿愈伤组织中有明显的从外向内减少的梯度变化;深绿愈伤组织中梯度不太明显。糖在金黄色愈伤组织中含量很低,没有梯度;在褐色愈伤组织中散乱分布。蛋白质含量变化在金黄色愈伤组织中由外层细胞向内层细胞有一定的梯度但不明显;在褐色愈伤组织中没有梯度。刚形成的再生团块的糖和蛋白质含量在由外向内的细胞中有明显的梯度变化,在再生团块膨大生长过程中两者含量少,没有梯度变化。分化期再生团块中的糖主要集中在分裂旺盛的分生细胞团和维管组织周围,蛋白质则集中于分生细胞团的细胞中。在各类愈伤组织和再生团块中均出现各种特化细胞。糖的含量及其梯度变化是愈伤组织和再生团块分化的重要内在条件,蛋白质含量以及核形态是否完整是愈伤组织和再生团块分化的关键。     

长期继代培养的柳橙胚愈伤组织再分化能力和遗传性研究

长期继代培养的愈伤组织,不仅是建立细胞无性繁殖系和作为种质资源保存的一种材料,也是细胞突变育种的理想试材。这主要是材料一致,容易处理,不受季节和植物发育阶段的限制,任何时候都可以进行试验,给研究工作提供了很多方便,避免了对种子和接穗作辐射诱变和化学诱变时容易形成的大量嵌合体,提高了诱变频率。 但是,除必须提高长期继代培养愈伤组织的增殖能力外,还要解决愈伤组织再生植株的分化率,以及植株的染色体倍性和其它遗传性状是否发生变化的问题。本试验试图通过对长期继代培养的柳橙(Citrus sinensis Osbeck)胚的愈伤组织进行研究,以对上述问题进行探讨。     

不同培养方法对长春花愈伤组织生长及吲哚生物碱积累的影响

分别对4种不同愈伤组织采用固体培养和悬浮培养,并对其生长鲜重和吲哚生物总碱的含量进行了分析测定,实验结果表明,EMS诱变的长春花愈伤组织生长和吲哚生物总碱积累适于固体培养,而对于秋水仙碱诱变的四倍体长春花愈伤组织、叶愈伤组织和子房愈伤组织更适于悬浮培养,但前者优势表现更为突出.     

中国柑橘育种60年回顾与展望

过去的60年,中国柑橘遗传改良与品种选育研究取得长足进展。据统计,在中国重庆、武汉等地建立的柑橘种质资源迁地保存圃、愈伤组织库分别保存了1 700多份芸香科材料和100多个柑橘品种的胚性愈伤组织。经调查,发掘到道县野橘、莽山野柑、红河大翼橙等多个野生种及迷你野生柑橘——单胚山金柑;发掘的‘资阳香橙’已作为砧木应用于产业。累计选育柑橘新品种122个,包含121个接穗品种和1个砧木品种,涉及宽皮橘、橙、柚等主要柑橘类型。上述接穗品种88.5%来自芽变和实生选种,余下的11.5%来自人工创制的变异,包括杂交、诱变和细胞融合等途径产生的变异;改良的性状主要是无籽、熟期和色泽等。在育种技术方面,组学技术全面应用于柑橘遗传改良,完成了甜橙等柑橘主要类型的基因组测序,发掘到柑橘重要农艺性状的相关基因,如控制多胚的基因CitRWP等;建立起柑橘遗传转化和基因编辑技术体系,为柑橘基因组设计育种奠定了坚实基础。     

苹果离体叶片再生体系两步培养法的研究

通过对苹果离体叶片两步再生法的研究,建立了乔纳金苹果试管苗的叶片再生体系。试验表明,2, 4-D能促进愈伤组织的形成和增殖,影响外植体脱分化和再分化的趋向。BA影响外植体形成愈伤组织的能力,对不定芽再生具有显著影响。IAA对愈伤组织的形成几乎无影响,但对再生不定芽的生长影响较大。诱导愈伤组织的培养基为MS+BA 1 0mg·L-1 +IAA 0 2mg·L-1 +2, 4 -D 0 8mg·L-1时诱导率可达100%;分化不定芽的培养基为MS+BA4 0mg·L-1 +IAA0 2mg·L-1时最高分化率也达 100%。暗培养 1月左右即可得到再分化的不定芽。     

影响洋葱愈伤组织及胚性愈伤组织诱导的因素研究

以洋葱雄性不育系种子(10-8F)为外植体,研究了蔗糖浓度、暗处理及取材时间对洋葱愈伤组织及胚性愈伤组织诱导的影响,以期优化胚性愈伤组织的形成条件,为转基因提供良好受体并为建立高频再生体系奠定基础.结果表明:40 g/L蔗糖及光培养最适合愈伤组织的诱导;50 g/L蔗糖及暗培养最适合胚性愈伤组织的诱导,最适宜的取材时间为45 d.     

柑橘愈伤组织RNAi沉默CCD1基因对其类胡萝卜素积累的影响

类胡萝卜素裂解双加氧酶(CCD)可催化类胡萝卜素在特定位点氧化裂解生成多种生物活性化合物,其活性差异一定程度上决定了植物体内类胡萝卜素含量和组成变化。为了探讨柑橘中CCD1蛋白对类胡萝卜素积累的影响,从红肉脐橙(Citrus sinensis L. Osbeck)果实中克隆了CCD1基因,将其特异片段通过重组反应连接到pHGRV载体中,构建CCD1的RNA干扰载体pHGRV-CCD1。利用农杆菌介导的方法将其转入‘国庆1号’蜜柑、‘伏令夏橙’和‘马叙’葡萄柚愈伤组织中,经PCR鉴定分别获得6、4、4个转基因干扰系。3种柑橘材料转基因干扰系中CCD1基因的表达水平均较野生型显著下调,其中‘马叙’葡萄柚转基因干扰系Rm2的表达水平仅为野生型的8.4%,干扰效果最佳。CCD1转基因干扰系愈伤组织外观多数呈现与野生型对照相近的色泽,仅Rm2系可见较为明显的颜色变化;综合分析3种柑橘材料转基因前后类胡萝卜素含量变化,发现CCD1基因抑制表达后,紫黄质、9–顺式–紫黄质的含量均较野生型显著增加,表明其可能是CCD1在柑橘愈伤组织体内优先选择的裂解底物。     

依据多倍体形态特征快速高效发掘柑橘四倍体

基于柑橘多胚性品种存在珠心细胞自然加倍的特点,建立了基于种子催芽、生长室实生播种、幼苗"观根辨叶看油胞"形态初选和流式细胞仪倍性鉴定的发掘柑橘四倍体的快速方法。采用该方法,分别从无酸甜橙、红江橙、贡柑、年橘、新会柑、滑皮金柑的432、506、1 356、1 870、2 749和685株实生苗中,发掘出疑似四倍体5、6、5、17、45、7株;通过倍性检测,从上述6个品种分别获得四倍体4、5、5、14、39、5株,形态初选准确率分别为80.0%、83.3%、100.0%、82.4%、86.7%和71.4%。与之前的四倍体发掘方法相比,本方法四倍体初选准确率高,并且从种子播种到获得四倍体植株耗时不超过40 d。SSR分子鉴定表明,所鉴定的四倍体均来自其二倍体品种珠心细胞自然加倍形成的双二倍体,为柑橘三倍体无核育种提供了珍贵的四倍体亲本资源。     

Rootstock influence on the incidence of rind breakdown in ‘Navelate’ sweet orange

Summary

The influence of three rootstocks, Carrizo citrange (Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.), Cleopatra mandarin (Citrus reshni Hort. ex Tanaka) and sour orange (Citrus aurantium L.) on the incidence and severity of rind breakdown in ‘Navelate’ sweet oranges was studied over seven years in four orchards. The proportion of affected fruit was greater in trees on Carrizo citrange (60% on an average) followed by Cleopatra mandarin (38%) and then on sour orange (9%). There were also significant differences in the severity of the disorder among rootstocks; more than 35% and 20% of fruit from trees on Carrizo citrange and Cleopatra mandarin, respectively, were rejected for commercialization, whereas only 4% of fruit on sour orange was rejected. Cryo-scanning electron microscopy (SEM) of fruit peduncles showed that average diameter of xylem vessel elements of peduncles of fruits on Carrizo citrange rootstock were 6% and 17% larger than those on Cleopatra mandarin and sour orange, respectively. Defoliation of fruit-bearing branches reduced water loss from fruit, maintained fruit peel water potential at a higher value than that of fruit on undefoliated branches and reduced the proportion and severity of affected fruit. It is concluded that fruit-tree water relationships are related to rind breakdown in ‘Navelate’ oranges and low fruit water content might be responsible for the disorder.     

Somaclonal variation with early juice-sac granulation obtained by inter-specific protoplast fusion in citrus

Summary

Six plants with an early juice–sac granulation trait derived from inter-specific protoplast fusions between embryogenic calli of ‘Bonanza’ navel orange (Citrus sinensis [L.] Osbeck) and mesophyll protoplasts of ‘Dahongpao Red’ tangerine (C. reticulata Blanco) were analysed by flow cytometry and by using molecular markers, including simple sequence repeats (SSR) and restriction fragment length polymorphism (RFLP). The results indicated that all six plants were diploids and had inherited their nuclear DNA from the embryogenic callus parent ‘Bonanza’ navel orange. However, an analysis of morphological and fruit characteristics, and measurements of components of the cell walls in the juice-sacs, showed that they were not true-to-type for ‘Bonanza’ navel orange, especially for fruit traits such as juice-sac granulation and navel structure. These results confirmed that these plants were not hybrids, and were more likely to be somaclonal variants that arose during the regeneration of the navel orange protoplasts. These plants will provide material for studying the mechanism of granulation in juice-sacs, a common phenomenon during the storage of pummelo and other citrus fruits.     

世界柑橘品种改良的进展

 柑橘鲜食品种的发展趋势是容易剥皮、无核、有香味和风味浓。过去30年, 通过芽变选种、杂交育种等途径, 全世界培育出了上百个柑橘品种。芽变选种获得的新品种主要来自如温州蜜柑、脐橙和克里曼丁橘, 它们分别形成了品种群, 不同品种采收期可长达4～6个月; 杂交育种途径培育出70多个品种(系) , 主要利用了单胚性品种克里曼丁橘、胞质雄性不育的温州蜜柑以及外观色泽好的红橘等作为杂交亲本; 新的砧木品种(系) 主要来自中国的枳与酸橘等品种的杂交; 加工品种的选育相对落后。生物技术, 特别是胚抢救及细胞融合等技术大大加快了柑橘育种步伐。     

利用转座子显示技术鉴别柑橘芽变品种

在依据柑橘全基因组信息的基础上,针对转座子序列开发了一种适合鉴别柑橘芽变品种的分子技术。以43个柑橘芽变品种和7个普通柑橘品种为材料,采用转座子显示技术(transposon display,TD)对转座子进行多态性分析。结果表明:5个DNA转座子(DTM63、DTM58、Helitron1、Spem和mPing2)和4个反转录转座子(Line1、LTR-Gypsy3、LTR-Gypsy5和Tcs2)可以较好地鉴别柑橘不同芽变品种。罗伯逊脐橙及其早熟芽变品种罗脐35号能被DTM63、Spem和mPing2鉴别;m Ping2能鉴别罗伯逊脐橙/罗脐35号、暗柳/红暗柳、普通椪柑/黔阳无核椪柑,以及红肉琯溪蜜柚/橙皮琯溪蜜柚这4对芽变品种。DNA转座子DTH3在甜橙芽变品种中多态性高,可以鉴别不同的甜橙芽变品种。因此,TD技术是一种值得关注的能有效鉴别柑橘芽变品种的分子标记技术。     

柑橘芽变选种以及芽变性状形成机理研究进展

综述了柑橘芽变选种的成果。近年来,我国选育了崀丰、长红、新世纪、奉晚等脐橙品种和华柑2号椪柑、岩溪晚芦、光明早温州蜜柑等宽皮柑橘品种,还有锦橙101、红肉琯溪蜜柚等其他柑橘品种。介绍了RAPD、AFLP、ISSR、MSAP和基于反转录转座子的分子标记在柑橘芽变品种鉴别中的应用。阐述了部分柑橘芽变性状形成的细胞学机理和分子机理,指出目前对芽变的研究主要集中在反转录转座子的插入、DNA甲基化、基因结构和表达差异等方面。结合果树芽变研究的现状,对今后进行芽变研究提出了展望。     

柑橘新品种——粤引红脐橙的选育

粤引红脐橙品种树形呈自然圆头形,树势中等,成枝力强,枝梢较开张。成花能力强,与枳、枳橙、温州蜜柑和甜橙嫁接表现亲和。果个中等,单果质量180g左右,近球形;闭脐或露脐;果皮橙色,果肉紫红色。可溶性固形物含量15.0%,可滴定酸含量0.876%,维生素C594.0mg/L,总糖116g/L,固酸比17.12:1,肉质脆嫩化渣,风味清甜,有独特芳香味,无核,品质优。成熟期为翌年1月份,耐贮,留树挂果可贮至2月份,常温下常规贮藏,能保存至3月底,品质风味优良。     

Application of 2,4-dichlorophenoxypropionic acid 2-ethylhexyl ester reduces mature fruit abscission in Citrus navel cultivars

Summary

2,4-dichlorophenoxyacetic acid (2,4-D) isopropyl ester has been used extensively, since the 1950’s, to reduce mature fruit abscission in Citrus navel cultivars. However, this synthetic auxin is no longer registered for this purpose in the European Union (EU). 2,4-dichlorophenoxypropionic acid (2,4-DP) 2-ethylhexyl ester has been registered in the EU for use in citrus growing and can be an effective replacement for 2,4-D to reduce pre-harvest fruit abscission. Use of the 2-ethylhexyl ester of 2,4-DP sprayed before mature fruit abscission significantly reduced fruit drop in sweet orange (Citrus sinensis L. Osbeck) ‘Washington navel’ and ‘Navelate’. The magnitude of the response depended on the concentration applied. At 15 mg l^–1, the percentage of abscised fruit was reduced by 50–75% compared to untreated trees, depending on the variety and the orchard. Increasing the concentration applied to 50 mg l^–1 did not improve this response. The response to this auxin was as effective as that obtained with 2,4-D applied on the same day at the same concentration (15 mg l^–1). 2, 4-DP treatment had no effect on skin colour or on the internal and external characteristics of the fruit at harvest.     

Evaluation of ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid for tolerance to Xanthomonas axonopodis pv. citri and Xylella fastidiosa

Asiatic citrus canker (ACC), caused by Xanthomonas axonopodis Starr & Garces pv. citri (Hasse) Vauterin et al., and citrus variegated chlorosis (CVC), caused by Xylella fastidiosa Wells et al., are considered the main diseases affecting sweet orange scion varieties in Brazil. Among commercial varieties, mandarins and tangerines are recognized as tolerant to these pathogens. We report herein the production of ‘Hamlin’ sweet orange (Citrus sinensis L. Osbeck) + ‘Montenegrina’ mandarin (Citrus deliciosa Ten.) allotetraploid somatic hybrid plants by protoplast fusion with improved disease tolerance that could be used as a donor of resistance genes in interploid hybridisation. Somatic hybridisation was confirmed by leaf morphology, flow cytometry and RAPD analyses. The somatic hybrid was propagated by grafting and cultivated in a screenhouse for tolerance assays. For X. axonopodis pv. citri assays, buds were collected from both ‘Hamlin’ sweet orange and the somatic hybrid and grafted onto ‘Cleopatra’ mandarin (Citrus reshni hort. ex Tanaka). As a negative control, buds from ‘Mexerica Tardia’ mandarin (C. deliciosa) were collected and grafted onto ‘Cleopatra’ mandarin. Two-month old plants with at least one young vegetative flush were individually spray-inoculated with a 10⁶ CFU mL⁻¹X. axonopodis pv. citri suspension and incubated in a growth chamber, at 27 °C, under 16-h photoperiod. The somatic hybrid showed a statistically significant reduction in susceptibility to ACC 30 days after inoculation. Compared to ‘Hamlin’ sweet orange, disease severity was reduced by 70%, with similar tolerance to that of the mandarin negative control. For X. fastidiosa assays, buds were collected from the somatic hybrid and its parental plants and grafted onto ‘Rangpur’ lime (Citrus limonia Osbeck). The developed plants were needle-inoculated with a X. fastidiosa suspension (8.7 × 10¹⁰ CFU mL⁻¹) into the new growth flush stem. Bacterial population was quantified both at 4 (at the inoculation point) and 8 months (50 cm above the inoculation point) after inoculation. The first evaluation detected X. fastidiosa in 63% of ‘Hamlin’ sweet orange and ‘Hamlin’ + ‘Montenegrina’ mandarin samples. In the second evaluation, X. fastidiosa was detected in 47.4% of ‘Hamlin’ sweet orange and 10.5% of ‘Hamlin’ + ‘Montenegrina’ somatic hybrid samples, suggesting that bacterial movement was restricted in the somatic hybrid. X. fastidiosa was not detected in both evaluations in samples collected from leaves of ‘Montenegrina’ mandarin. These results indicate that the ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid has potential for improved disease tolerance that should enhance its value regarding future use in citrus breeding programs.