生物科学中英文对照外文翻译文献

1、 外文翻译资料中英文资料外文翻译文献译文标题：传统意大利榛子的体外繁殖用于当地遗传资源库的稳定和保存译文：关键词：欧洲榛，榛属，传统种质，体外繁殖摘要：在地中海盆地，榛子（欧洲榛）是非常重要的一种作物。体外繁殖能够有效的稳定当地遗传资源库。为了提高榛子微组织繁殖实验记录的精确性，各种不同的研究已经在进行。这些研究通常以重要的品种为材料，然而，微组织繁殖实验记录应用在这些幼小品种上比起传统方法通常会产生相反的结果，这种技术在幼小品种上很少取得成功。本实验的目的是为重要品种微组织繁殖的操作积累相关的知识和信息。实验过程中需要设计不同成分的培养基，灭菌时间和培养时间都要进行详细的讨论。传统意大利品种

2、植株茎芽中的N6-异戊烯腺嘌呤的作用是改善这种状态。生根阶段是榛属微组织繁殖应用于大型商业生产的关键步骤。欧洲榛在欧洲特别是生物地理分布区地中海盆地代表一种重要的经济类林木。榛子主要产于土耳其，意大利，美国和西班牙（分别是每年55,000, 110,000, 25,000, 18,000+吨），其次是法国，希腊，葡萄牙。大约 90%的产品被去皮并且以树芯的形式卖出，然而剩余的 10%则作为树苗消费。极好的营养成分和营养制品的特性也使该物种产生很高的利润。此外，在一些特有的栽培地区，传统和文化身份严重受榛子产量的影响，文化身份常常会促进贫瘠土地的回收和利用。即使这样，在一些地区，这种林业作物仍然

3、不是重要的农业资源，然而，就当地足够维持的生产式系统和作为宝贵的食物的传统而言，它却是一种有趣的收入来源。世界第二大生产商意大利说一些传统的品种主要种植在 Campania ,Latium, Piedmont,在西西里岛有大量的属典型种。近几年，一些主要品种由于质量和传统特性获得了欧洲质量印模。此外，这些品种还被引进其他国家特定的果园中以增大他们的生长范围。没有经过检验的物质可能会传播疾病，也可能会导致原因不明的物质的出现。微组织繁殖法等生物技术的应用会促进健康的合乎本性的物质的产生(Nas et al.,2004)，并且提高这种林木的经济价值。育种计划可以通过样本或者新品种的快速分配来加快进

4、程。 外文翻译资料榛子成熟组织体外快繁的一个主要障碍是高程度的内源性污染 (Diaz-Sala etal.,1998)，这使得培养体系的建立艰难又费时。此外，一些培养基成分被用于促进一些主要榛属品种(Andres et al.,2002;Damiano etal.,2005;Messeguer and Mele,1987;Yu and Reed,1993茎) 芽数目的增多和长度的增长。然而，当标准化的实验设计应用于当地数目较少的品种时，实验结果是相反的，通过观察，经的生长存在一定的困难(Bacchetta et al.,2005)。以营养成分能够保证籽苗和体外抽枝合适的生长状况的理念为基础，N

5、as and Read (2004)最近提出了一种新的培养基成分结构。然而，在植物的体细胞和种子细胞之间存在着生理差别。此外，种子内必需营养素化合物的浓度对于体细胞则太高甚至有毒。在目前的工作中，这种状况有了轻微的改善，我们通过利用不同物种种子中大量元素和微量元素的差异来使榛子无机培养基的成分最优化，使得其中遗物中能够很好的适应体外的生长状况。微组织繁殖法是多因素共同作用的结果，其中包括组织型和激素要求，外植体原始细胞培养阶段生理因素的影响和N6-异戊烯腺嘌呤对促进茎数目增多的影响。这些实验目的正是一些工业机构和科研机构支持的SCRIGNO 实验计划的目标，这项实验主要是为了估计当地传统型和典

6、型物种的农业生物多样性，同时，它还支持了 AGRI GENRES 068 Safenut EU 计划中遗传资源库的鉴定、保存及利用的内容。实验材料和试验方法榛属培养基的进展状况。桃枣作为参考物种(Rugini and Verma, 1983)，两物种无机营养素的比例作为调节 MS 培养基的调节因子(Murashige and Skoog, 1962)。培养基中刚开始培养的是种子，因此，此营养素成分必须尽可能的接近体细胞组织对营养素的要求。通过原生质发射光谱法来估计无机组分的应用于意大利中部果园搜集的（100g）杏仁和榛子的嫩叶和裸露种子样本。这种新型培养基的研究过程如下：含盐 MS 培养基中阴

7、阳离子的测定；榛子和杏仁种子中无机元素浓度比例的测定；校正系数用于计算阴离子和阳离子数量和测定新的盐浓度；KH PO 中钾的含量少于磷的含量时，KI 的量不发发生变化。新的营24养成分构成改良培养基，通过改良培养基我们可以进行HM 诱导，辅以含有维生素的 MS 培养基，加入200mgL 的肌醇，0.4 mg L 赤霉素，0.05 mg L 吲哚乙酸 (IBA) ，3% 的蔗-1-1-1 外文翻译资料糖，BA (0.5 mgL )。-1外植体的来源。组织培养首先要收集盆栽植物茎部分离部分（1cm 长）。这些生长 2 年的植物分别来自六个意大利品种的根出条。这些根出条于 2003 年 2 月被种植

8、于含有 1:1:1粘土，沙，泥炭混合物的直径为 30 厘米的塑料容器。这些被称为母体植株的盆栽植物在自然光的照射下，并定期施肥，修剪，进行杀菌处理。灭菌过程。外植体在自来水中用消毒剂于抗菌性药皂冲洗一小时，将植物漂洗干净。灭菌结束后将植株放入 70乙醇中浸泡 5 秒。重新切割，外植体表面用 0.05次氯酸钠的消毒 10 分钟，滴加几滴 Tween-20，然后将植体在无菌蒸馏水冲洗三次。10 分钟次氯酸钠和几滴 Tween-20只用于在冬季采集外植体。接种到培养基之前，每个外植体基底两端分别翻新，有利于营养物质的吸收。体外培养的建立和生根。单节外植体取自于植物的两个不同的生理阶段：快速增长和休眠

9、期接种于 HM 诱导培养基。在继代培养时，高压灭菌后的 HM 培养基中加入加入 BA(1.5mg L ) 和 N6-异戊烯腺嘌呤 (1 mgL )以促进其增值。-1-1生根刺激：1）茎的基部浸入 1 mgL 的 IBA 溶液 20s，然后将外植体在没有激素的 HM-1培养基中培养 20 天。或者 2）在含有 1/3 HM 培养基营养素成分与 2 mg L 的IBA 的培养基-1中培养一个月。不同的学者认为，中等浓度的矿物质浓度可以提高生根率。培养液的 PH 调到 5.7，然后加入 0.7% (w/v)的琼脂，121条件下高压灭菌 20 分钟。外植体培养环境设置为：室温 251，16 小时的光周

10、期，70% 到 80%的相对湿度。每个塑料瓶中分装 30 毫升的培养基。实验设计。每个榛子品种的一百个辅助芽进行培养在含有 30 毫升培养基的单独的试管中。每一个品种随机抽取十个重复，记录芽的数量、新叶以及茎白天的重量。通过观察估计小植株的形状。将实验重复一次。用外植体培养过程中记录的外植体数，平均芽长和腋芽数进行统计分析。统计分析采用 SPSS 完成（13.0，SPSS 软件，芝加哥）：描述性分析和普通线性分析。结果与讨论榛子校正培养基。表 1 包含榛子和杏仁的坚果和树叶中大量元素和微量元素的浓度。差别主要体现在硼，钡，锰，铜，硅，锶的浓度上，表明榛子具有相对大的元素浓度。另一方 外文翻译资

11、料面，榛子所含铁和锌浓度较低。 Mobdilene 并未在坚果和叶片中发现。其中大量元素，钙，镁的含量在榛属中低于李属。新型培养基HM 与传统培养基的不同之处，见表 2。比起 MS培养基，HM 培养基中含有少量的的氯化钙、硫酸镁、硫酸钾和磷酸二氢钾，这是 MS 和NRM 中没有的成分。另一方面，在HM 中锌浓度减少到 MS 和 NRM 中锌含量的 1/4。即使实验表明，葡萄糖和果糖对于茎的伸长具有促进作用，蔗糖仍被作为为碳源；记录含有乳糖的培养基中芽的形状与生长习性（数据未显示）。关于榛子，Yu and Reed报道过类似的结果，蔗糖作为替代碳源可以促进树种茎的伸长和提高繁殖率被Ding et

12、 al.(1985 年) 和 Marino etal.(1993 年)报道过。 外文翻译资料接种阶段。榛快繁的局限性主要是外植体微组织繁殖能力的降低和植物材料的污染比较严重。Damiano et al.在 2005 年报道说大约 95%的外植体都会受到污染，掩盖了体外快繁的优势。高压灭菌比起直接处理材料会减少 20%到 30%的内生菌污染，可以提高母体植株的利用率。 外文翻译资料图 1图 2芽消毒产品也比较敏感，出现组织坏死的情况。这种现象很普遍，特别是在外植体的生长过程中。乙汞硫代水杨酸钠比次氯酸钠效果更好，因为它渗入细胞却不会引起组织坏死（数据未显示）。此外，根据研究发现，对于榛子的休眠品

13、种，离体的芽是最合适的外植体；单芽比单节外植体产生的正常植株比例高。Messeguer and Mele(1987)就西班牙 cv. Negret 报道过同样的解释。接种前去除小叶，是的组织在灭菌后更容易坏死（比有芽时高出 25%）。Damiano ed al （2005）报道说，体外培养榛子的主要困难之一是接种后芽坏死。另一方面，低温处理提高了休眠材料体外形态发生的能力。由于低温可以 外文翻译资料降低内生菌污染程度（表 3），所以在灭菌之前榛子小枝要在 5条件下保存三周，在灭菌过程，诱导 50%而不是 30的不育材料。培养体系的建立。六个榛子品种对新型培养基的反应见表4。HM 培养基上的外植

14、体长出了绿叶，在基部产生了相对较少的愈伤组织。MS 培养基上的外植体基本全部萎黄，在它们的基部几乎没有愈伤组织产生。所有的品种芽和辅芽的长度在两种培养基上的生长状况类似。这是培养基与基因型共同作用的结果，当培养基有利于生长时，芽的长度和每芽芽数会增加。此外，由于培养基对芽湿重及干重的积极影响，小植株的质量会得到提高。Nas and Read(2004)也报道说芽没有发生增殖。 然而，培养基会影响潜在的增值率，长枝更适合做继代培养。外植体生理阶段影响芽的生长长度。Tonda Giffoni and Tonda Romana研究表明，春季和冬季收集的外植体的差异及其在诱导培养基 HM 上的生长差异

15、（图 1）。另一方面，两种外植体在 Mortarella，Ghirara，Avelana Speciable 和 Napoletanedda 培养基上表现出类似的根伸长状况。基本上，春季收集的外植体长出的芽可以区分出不同的品种。各种植物生长调节剂对榛子组织的不同影响也被记录。榛子组织生长旺盛时测定出高浓度的吲哚-乙酸和细胞分裂素，这些激素促进了植物的发育。季节和基因型的相互作用可以对芽的生长产生显著的影响。增殖阶段。由于低繁殖率，榛子体细胞在体外组织培养仍然受限。春季采摘的材料在分别加有锌(1 mg L-1)和 BA (1.5 mg L-1)的培养基上生长状况有所不同（图 2）。双向的变量分析

16、表明品种不同，培养基不同，生长状况也会不同。通过t 检验，不同品种不同培养基会出现显著的差异 P0.05。另一方面，N6-异戊烯腺嘌呤可以更好的促进茎的增值。相反的，BA 有更有利于促进芽的伸长。两种细胞激肽对于茎的生长有类似的影响。榛子品种的特点是不同水平的影响植物生长调节的内生细胞肽激素。Andres etal.(2002)研究表明2iP/zeatin 比例影响影响榛的组织形态发生能力，该比例作为鉴别体外培养竞争力的指标。无论如何设置条件，没有侧芽和不定芽发生。生根阶段。将根浸在 IBA 溶液中进行根的诱导。基本不同品种的生长状况都表明含有 IBA (2 mg L-1)的生长体系都很差。3

17、0%的 cv. Tonda romana每个外植体长出 2.10.7 长度的不定根，然而只有 20%的 cv. Tonda Giffoni 每个外植体长出 1.50.6 长度的根。更低比例的（10%）生根的茎从 cvs. Mortarella 和 Ghirara 中获得。在 cvs. Napoletanedda 和 Avellana 外文翻译资料Speciale 中没有根系形成（见表5）。结论微组织培养是应用于榛子商业化生产的一种重要技术。当地品种或者选择的一些杂种对于体外培养表现出相反的反应。正如 Nas 和 Read（2004）所说，决定快繁成功的一个关键是优化的培养基使用，及其与基因型的

18、适合。从相同的假设开始，培养基的研究有效的提高了优良品种的质量与生产水平。此外，这种快捷的实验方法作用到作为模式植物榛子，表明它可以应用于大规模的植物品种培养基生产。在离体繁殖的成功还取决于外植体的生理阶段。春季采集的茎段处的芽以及单独的芽都保证了体外繁殖的客观条件，实验表明他们可以用于大规模的商业化生产。由于只有很少的增值率，因此进一步的研究是通过刺激多芽/外植体的比例来达到的增殖阶段。另一方面，榛子体外繁殖的关键阶段生根。不同的内部因素（生理阶段）和外部因素（矿物和植物激素的浓度，物理参数）在很大程度上影响跟的产生。需要进一步的研究体外繁殖的有效和可靠的技术，通过这种无性繁殖达到保存榛子遗

19、传资源库的目的。 外文翻译资料In vitro propagation of traditional Italian Hazelnut Cultivars as a tool for the Valorization andConservation of Local Genetic ResourseAbstractThe hazelnut(corylus avellana)is one of the most important crops in the Mediterranean basin. Theavailablility of efficient and reliable in vit

20、ro propagation could valorize the local geneticresources.Different studies have been carried out for the definition of an effcient hazelnutmicropropagation protocol. These have usually been performed on the most important cultivars,but the application of the micropropagation protocol to the minor on

21、es has produced contradictoryresults and the technique sometimes had less success than the traditional one. The aim of this workwas to gather knowledge and additional information on the in vitro performance of some minorcultivars in comparison with the most used for micropropagation .A revised proce

22、dure for thespecific medium formulation is suggested. The sterilization and culture establishment phases arediscussed in detail. The role of zeatin and 6-benzylamminopurine(BA) in shoot proliferation in theItalian traditional cultivars to improve this phase.The rooting stage proves to be one of the

23、mostcrucial steps in achieving a large-scale commercial application of hazelnut micropagation.Corylus avellana L.(Brtulaceae)represents an ecomomically important crop in the EuropeanCommunity ,particularly in the biogeographic Mediterranean basin .Hazelnuts are producedprincipally in Turkey ,Italy,t

24、he United States,and Spain (55,000,110,000, 25,000,18,000+tons,respectively,per year)followed by France,Greece, and Portugal. Approximately 90%of production is shelled and sold as kernels, whereas the remaining 10% gose to freshconsumption.Interest in this species is also the result of its excellent

25、 nutritional and nutraceuticalproperties(Phillipset al.2005: Sovakumar and Bacchetta, 2005).Moreover,in the typical cultivationareas, traditions and cultural identity are strongly tied to hazelnut production,whereas the latteralso contributes to asuitable use and recovery of marginal land .Even if,

26、in some regions, this cropis not the major agricultural resource ,it nevertheless represents an interesting source of income forthe local sustainable production system and a precious food for traditional local use. Italy, theworlds second largest producer, boasts several traditional cultivars, which

27、 are mainly cultivated inCampania ,Latium, Piedmont, and Sicily with a large number of local genotypes (Bacchetta etal.,2005).In the last few years, some of the major cultivars (Tonda Romana from Latium, Tonda diGiffoni from Campania,and Tonda delle Langhe from Piedmont) obtained the EuropeanCommuni

28、ty quality stamp for their quality and traditional peculiarity. Morever, these cultivars areoften introduced into other countries to increase their range of “vigorous mother plants”selectedin orchards. Without certified materials, it is possible to spread diseases widely (Scortichini,2002)or reprodu

29、ce materials of unknown origin. The use of biotechnologies such as micropropagationpromots the production of healthy and true-to-type materials (Nas et al.,2004), improving theeconomic value of the crop. Using micropropagation, the breeding program could be acceleratedby a rapid distribution of stan

30、dard or new cultivars. 外文翻译资料One of the main limitations of hazelnut in vitro propagation from mature tissues is the highdegree of endogenous contamination (Diaz-Sala et al.,1998), which makes the establishment of theculture a very laborious and timeconsuming phase.Moreover, several media formulatio

31、ns have been proposed for optimizing shootmultiplication and elongation in the main hazelnut cultivars (Andres et al.,2002;Damiano etal.,2005;Messeguer and Mele,1987;Yu and Reed,1993). However, when the standardized protocolwas applied to local and minor cultivars, the result were contradictory and

32、some difficulties inshoot growth were observed (Bacchetta et al.,2005). Nas and Read (2004) recently proposed anovel method for medium formulation based on the concept that nut nutritional reserves are ableto guarantee suitable conditions for seedlings as well as for in vitro shoots. However, there

33、arephysiological differences between seed tissues and the somatic parts of plants. Moreover, theconcentration of essential nutritional compounds present in seeds can be too high or toxic forsomatic tissues.In the present work, this approach was slightly revised and the mineral hazelnut mediumformila

34、tion was optimized by using the differences in seed macro- and micro- elements of twospecies, one of them well adapted to in vitro conditions.Because the success of micropropagation is the result of a combination of many fators,including tissue type and hormone requirment, the effect of the physiolo

35、gical stages of the initialexplants on culture establishment and the influence of zeatin or 6-benzylamminopurine (BA)concentrations in stimulating shoot proliferation were evaluated.These goals met the objectives of research project SCRIGNO, supported by the Ministry ofIndustry and Scientific Resear

36、ch, aimed at evaluating local agrobiodiversity for “typical,traditional”products and meet those of the AGRI GEN RES 068 Safenut EU program covering thecharacterization, conservation, and utilization of genetic resources.Materials and MethodsDevelopment of the hazelnut-specific medium. Prunus dulcis

37、was chosen as the referencespecies (Rugini and Verma, 1983) and the ratio of the mineral nut material between the twospecies was used as Murashige and Skoog medium (MS) mineral composition correction factor(Murashige and Skoog, 1962). This approach took account of the importance of seed compositiona

38、s a starting point for formulation of the culture medium composition as closely as possible withthe demands of somatic tissue growth.Mineral composition (macro- and micro-elements) was estimated with inductively coupledplasma-atomic emission spectrometry VARIAN S.p.A (VISTA MPX assail configuration)

39、 appliedto the fresh leaves and raw seeds of both almond ( Pruns dulcis) and hazelnut ( Corylus avellana)samples (100g) collected in orchards located near Viterbo (Central Italy). The procedure fordeveloping the novel medium was as follows: determination of anions and cations of MS saltmedium; evalu

40、ation of the ratio between the concentrations of mineral elements found in hazelnutand almond seeds (factors of correction); use of the correction factor for calculating the cation andanion amounts and for estimating the new concentrations of salts; the quantity of KI wasunchanged while the quantity

41、 of potassium in KH PO was calculated after phosphorous (Rugini,241984). The novel formulation was a modified MS medium, which we term HM induction,supplemented with vitamins MS, 200mg L myo-inositol as suggested by Nas and Read (2004),-1gibberellicA (GA3) 0.4 mg L ,indole-3-butirrc acid (IBA) 0.05

42、mg L , 3% sucrose,and BA (0.5-1-13mg L ).-1 外文翻译资料Explant source. Tissue culture was initiated using uninodal shoot explants (1 cm in length)gathered from potted plants. The plants were 2 years old and were obtained from rooted suckers ofsix Italian cultivars in an ex situ hazelnut plant collection

43、located in Vico Matrino. The suckerswere potted in Feb.2003 and were grown in 30-cm-diameter plastic pots containing clay, sand, andpeat in a 1:1:1ratio. The potted plants, used as“mother plants,”were maintained in a healthy stateunder natural light and were periodically fertilized, pruned, and trea

44、ted with fungicides.Sterilization procedure. Primary explants were washed in tap water (1h), cleaned with adisinfectant, antibacterial soap, and then rinsed again with tap water. The sterilization wasperformed by immersion in 70% ethanol for 5s. After the recut of basal ends, the explants weresurfac

45、e-sterilized with 0.05% Na Merthiolate for 10 min, a few drops of Tween-20 were added,and then the explants were rinsed in sterile, distilled water three times. Sodium hypochlorite for 10min and a few drops of Tween-20 were only used for explants collected during winter. Beforeinoculation onto the c

46、ulture medium, the basal ends of each explant were renovated to favor theabsorption of nutrients.Establishment of in vitro culture and rooting. The uninodal explants were collected in the twodifferent physiological phase of the plants: rapid growth and the dormant phase and inoculatedonto the HM ind

47、uction medium. In the second subculture, BA(1.5 mg L ) or zeatin (1 mg L )-1-1was added after autoclaving to HM to promote proliferation.Rooting was stimulated with: 1) shoot basal end immersion in an IBA solution (1 mg L ) for-120s and 20-d culture on the HM medium without hormones; or 2) 1-month c

48、ulture on the one-thirdHM mineral content supplemented with IBA(2 mg L ). As suggested by different authors, a-1reduction of the medium mineral concentration can increase the rooting percentage.The media were adjusted to pH5.7 before adding 0.7% (w/v) agar and autoclaved at 121 for20 min. The explan

49、ts were cultured in a growth conditioned chamber at 251 with a 16-hphotoperiod (32molm s coolwhite fluorescent illumination) and 70% to 80% relative-2 -1humidity. Thirty milliliters of medium were distributed into glass test tubes (12 cm length, 3 cmdiameter) with plastic lids.Experimental design. O

50、ne hundred uninodal auxiliary buds of each hazelnut cultivar werecultured in separate glass test tubes containing 30 mL of medium. Ten replications (test tubes) ofeach cultivar were randomly applied bud (node) number, and fresh and day weight of shoots wererecorded. The appearance of plantlets was v

51、isually evaluated. The experiment was repeated once.Data for the explants in a culture vessel were divided by the number of explant, and the meanshoot length and auxiliary bud numbers were used for statistical analysis. The statistical analysiswas completed using SPSS (13.0; SPSS, Chicago): descript

52、ive analysis and general linear modelmultivariate at P0.05.Results and DiscussionHazelnut revised medium. Table 1 contains the concentrations of macro- and micro- elementsin both hazelnut and almond nuts and leaves. Differences were found for B, Ba, Mn, Cu, Si, andSr concentrations, which showed hig

53、her values in hazelnuts than in almonds. On the other hand,Fe and Zn displayed lower concentrations in hazelnut. Mobdilene was not found in either thekernel or leaves of either. Among macroelements, Ca and Mg were consistently lower in Corylusavellana than in Prunus dulci. The novel medium HM, devel

54、oped on the basis of these differences,is shown in Table 2. With respect to MS, the HM medium shows reduced concentrations of CaCl ,2MgSO , and KH2PO4 and includes K2SO4, which is missing in both MS and NRM. On other4 外文翻译资料hand, Zn concentration in HM was reduced to 1:4 compared with MS and NRM med

55、ia. Sucrosewas chosen as the carbon source , even if precious experiments showed the postive effect onhazelnut shoot elongation of both glucose and fructose; shoots with a good general appearance andgrowth habit were also observed in a medium supplemented with lactose (data not shown).Analogous resu

56、lts were reported by Yu and Reed in hazelnut, but an alternative carbon source tosucrose was reported to improve growth and the multiplication rate in this and other tree speciesby Ding et al.(1985) and Marino et al.(1993).Inoculation phase. The ongoing limitations of hazel micropropagation are attr

57、ibuted not onlyto the reduced specific morphogenetic capacity of explants, but also to the endocontaminationfound in plant materials. Damiano et al.(2005) reported that 95% of explants could be infected,limiting the advantage of in vitro culture. The availability of “mother plants” cared for in pots

58、facilitated sterilization because of the reduced endo contamination of the initial explants, thepercentage of contamination decreased by 20% to 30% compared with the material collecteddirectly in the field.Buds also seemed particularly sensitive to the sterilizing products and displayed necrosis oft

59、issues. This phenomenon was evident especially in actively growing explants. In this case,Na-merthiolate proved more effective than Na-hypochlorite, because it is able to penetrate cellswithout causing necrosis (data not shown). Moreover, according to our results, isolated buds werethe most suitable

60、 explants for in vitro establishiment of hazelnut dormant cultivars; the percentageof healthy responsive explants was higher in single buds than in uninodal explants. The sameconsiderations were reported by Messeguer and Mele(1987) for the in vitro establishment of theSpanish cv. Negret. The removal