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American-Eurasian Journal of Sustainable Agriculture, 6(4): 344-348, 2012 ISSN 1995-0748
美国-欧亚可持续农业杂志， 6（4）： 344-348， 2012 ISSN 1995-0748

ORIGINAL ARTICLE
原文

In Vitro Screening of Potato Cultivars for Salinity Tolerance
马铃薯品种耐盐性的体外筛选

C. Sudhersan, S. Jibi Manuel, J. Ashkanani and A. Al-Ajeel
C. Sudhersan、S. Jibi Manuel、J. Ashkanani 和 A. Al-Ajeel

Biotechnology Department, Food Resources and Biological Sciences Division Kuwait Institute for Scientific Research P.O. Box 24885, Safat 13109, Kuwait
科威特科学研究所食品资源和生物科学司生物技术部 P.O. Box 24885， Safat 13109， Kuwait.

C. Sudhersan, S. Jibi Manuel, J. Ashkanani and A. Al-Ajeel: In Vitro Screening of Potato Cultivars for Salinity Tolerance
C. SudhersanS.Jibi Manuel、J. Ashkanani 和 A. Al-Ajeel：马铃薯品种耐盐性的体外筛选

Corresponding Author: C. Sudhersan, Biotechnology Department, Food Resources and Biological Sciences Division
通讯作者： C. Sudhersan， 生物技术部， 食品资源与生物科学部

Kuwait Institute for Scientific Research P.O. Box 24885, Safat 13109, Kuwait
科威特科学研究所 P.O. Box 24885， Safat 13109， Kuwait.

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Am.-Eurasian J. SustainAgric.6(4): 344-348, 2012

The main objective of this study was to select salt tolerant potato cultivars for the utilization of saline soil and brackish water irrigation in Kuwait for the commercial potato production. The details of the study are presented in this report
本研究的主要目的是选择耐盐马铃薯品种，利用科威特的盐碱地和咸水灌溉进行商业马铃薯生产。本报告详细介绍了该研究的细节

Materials and Methods
材料与方法

Disease-free certified seed potato tubers of cultivars Ajiba, Almera, Arnova, Atlas, Bellini, Diamant, Fontane, Lola, Matador, Timate, Safrane, Santae and Spunta obtained from Agrico Co., Holland, Anabelle, Desiree and Nicola obtained from Moracco in the form of tissue cultured minitubers, certified tubers of Charlotte and Rembrandt obtained from British Potato Council (BPC), United Kingdom and Costanera, Maria Tropica-1, MF-1, MF-II, Primavera, Tacna and Unica obtained from the International Potato Research Center (CIP), Lima, Peru were used for the study
从荷兰农业公司获得的无病认证马铃薯种薯块茎，从莫拉科获得的无病认证马铃薯块茎，从英国马铃薯理事会（BPC），英国和科斯塔内拉获得的夏洛特和伦勃朗的认证块茎，从英国马铃薯理事会（BPC），英国和科斯塔内拉获得的夏洛特和伦勃朗的认证块茎， 该研究使用了从秘鲁利马国际马铃薯研究中心（CIP）获得的Maria Tropica-1、MF-1、MF-II、Primavera、Tacna和Unica.

Surface sterilized seed potatoes of all the 25 cultivars were maintained at room temperature under total darkness for sprouting. The shoot buds were removed from the sprouted tubers using a sterile surgical knife and treated with 20% commercial Chlorox® containing approximately 1% sodium hypochlorite with a drop of Tween 20 for 15 min. After 3-4 times washing in sterile distilled water, the shoot buds were treated with 70% ethyl alcohol for one minute and washed three times in sterile distilled water. The shoot meristems with two undeveloped leaves were isolated from the surface sterilized shoot buds under the laminar hood using a dissection microscope for the in vitro culture.
所有25个品种的表面灭菌种薯在室温下在完全黑暗下保持发芽。用无菌手术刀将芽芽从发芽的块茎中取出，并用含有约1%次氯酸钠的20%商业氯氧®处理，滴Tween 20 15分钟后，在无菌蒸馏水中洗涤3-4次后，芽芽用70%乙醇处理一分钟，并在无菌蒸馏水中洗涤3次。使用解剖显微镜从层流罩下的表面灭菌芽中分离出具有两片未发育叶子的枝条分生组织进行体外培养。

Murashige and Skoog (1962) shoot multiplication media obtained from Hi-Mdia was used as the culture initiation media. The pH of the media was adjusted to 5.6 prior to the addition of Gelrite. The media was dispensed in 25x125 mm culture tubes and autoclaved at 121。C for 15 min. Low concentration (0.1 mg/l) of benzyl adenine (BA) was added to the media for the meristem culture initiation. All cultures were incubated in a culture room containing 1000lux light intensity with 16 h photoperiod at 25。C for plantlet development. Large numbers of plantlets from 25 cultivars were produced in vitro through standard stem nodal multiplication method (Sudhersan and Hussain, 2002; Sudhersan, 2004).
Murashige 和 Skoog （1962） 使用从 Hi-Mdia 获得的射击繁殖培养基作为培养起始培养基。在加入凝胶体之前，将培养基的pH值调节至5.6。将培养基分装在25x125 mm培养管中，并在121.C 15 min将低浓度（0.1 mg/l）的苄基腺嘌呤（BA）加入培养基中，用于分生组织培养起始。所有培养物均在光强度为1000lux的培养室中孵育，光周期为16 h，光周期为25.C 用于小植株发育通过标准茎节点繁殖方法在体外生产了来自 25 个品种的大量小植株（Sudhersan 和 Hussain，2002 年;Sudhersan，2004年）。

For salt tolerance screening experiments, isolated stem nodal explants of 25 different potato cultivars were grown on MS medium supplemented with NaCl concentrations of 0,750, 1000, 2000, 3000 and 4000 ppm for 90 days to test their tolerance or sensitivity to high salt media. Shoots were cultivated on the same medium without NaCl was used as control. In all experiments each treatment had 20 replicates and the experiment was repeated twice. Growth and morphological changes due to the salt stress in culture were observed and recorded periodically.
在耐盐筛选实验中，将25个不同马铃薯品种的分离茎节点外植体在补充有0,750、1000、2000、3000和4000 ppm浓度NaCl的MS培养基上生长90天，以测试其对高盐培养基的耐受性或敏感性。在未使用NaCl作为对照的相同培养基上培养芽。在所有实验中，每个处理有20次重复，实验重复两次。定期观察和记录培养物中盐胁迫引起的生长和形态变化。

Results:
结果：

High salinity in the culture media greatly affected the survival, growth and rooting of the potato cultivars that were experimented. In the control culture media all cultivars showed normal growth and development on both shoot and root. Plantlets of all cultivars reached 12 cm in lengths after 30 days. In the salt treatments shoot elongation and root production decreased with the increase in salt concentration in culture (Table 1). In culture media with 750 ppm NaCl, plantlets were developed normally from the nodal cuttings of all the 25 cultivars similar to the control. The leaves and stems were succulent in all other treatments higher than 1000 ppm in majority of the cultivars tested. Mature and expanded leaves produced by the plantlets in high salt media showed burns in their mesophyll tissue and subsequently died. Salt burns were noticed on the older leaves first and then the younger ones. Shoot tip injury was also noticed. The symptoms appeared on the plantlets at high salinity in vitro were: succulent nature of stem and leaves, yellowish colour of stem and leaves, stunted growth, poor root development, shoot tip dying, and burns on mature leaf tissues.
培养基中的高盐度极大地影响了试验马铃薯品种的存活、生长和生根。在对照培养基中，所有品种的枝条和根部均表现出正常的生长和发育。所有品种的植株在30天后达到12厘米长。在盐处理中，随着培养物中盐浓度的增加，枝条伸长率和根系产量降低（表1）。在含有750 ppm NaCl的培养基中，与对照相似的所有25个品种的节点插条正常发育小植株。在大多数测试品种中，叶子和茎在所有其他处理中都是多汁的，高于 1000 ppm。小植株在高盐培养基中产生的成熟和膨化的叶子在其叶肉组织中显示烧伤并随后死亡。首先在较老的叶子上注意到盐灼伤，然后在较年轻的叶子上注意到盐灼伤。还注意到了芽尖损伤。体外高盐度小植株出现的症状有：茎叶多汁、茎叶发黄、生长迟缓、根系发育不良、芽尖死亡、成熟叶组织烧伤等。

Among the 25 potato cultivars screened for salt tolerance in vitro, 12 cultivars showed tolerance up to 750 ppm NaCl, 6 cultivars showed tolerance up to 1000 ppm NaCl and 7 cultivars showed tolerance up to 2000 ppm NaCl (Table 2; Figs. 1-3)). Based on the salt tolerance level, the potato cultivars were grouped in to three categories (Table 3) such as 1. Highly sensitive (tolerance level 750 ppm), 2. Sensitive (tolerance level 1000 ppm) and 3. Tolerant (tolerance level 2000 ppm).
在体外筛选的25个马铃薯品种中，12个品种的耐盐性高达750 ppm NaCl，6个品种的耐受性高达1000 ppm NaCl，7个品种的耐受性高达2000 ppm NaCl（表2;图 1-3））。根据耐盐性水平，将马铃薯品种分为三类（表3），如1.高灵敏度（公差等级 750 ppm），2.灵敏度（公差等级 1000 ppm）和 3.耐受性（公差水平 2000 ppm）。

Table 1: Effect of NaCl on potato cultivar Ajiba in vitro
表1：NaCl对马铃薯品种Ajiba体外的影响

(±) Standard error; data from 20 replicates; experiment was repeated twice; data collected after 20 days.
(±)标准误差;来自 20 次重复的数据;重复实验两次;20 天后收集的数据。

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Table 2: In vitro screened potato cultivars and their level of salinity tolerance
表2：体外筛选马铃薯品种及其耐盐性水平

Table 3: Grouping of potato cultivars based on NaCl tolerance
表3：基于NaCl耐受性的马铃薯品种分组

1 2

3

Fig. 1-3: Effect of NaCl on potato plantlet growth in vitro. Fig.1. Potato cultivar MF-2; Fig. 2. Potato cultivar Ajiba; Fig.3. Potato cultivar Armada
图1-3：NaCl对马铃薯小植株体外生长的影响。图1 马铃薯品种MF-2;图2.马铃薯品种Ajiba;图3 马铃薯品种Armada.

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Discussion:
讨论：

Potato is sensitive to salinity. Fresh water irrigation is necessary to get good yield. The yield decreases when the salinity increases in the soil. Desalination of the soil is an expensive process for potato production. Therefore, selection of salt tolerant cultivars is necessary for the economic production. Screening many potato cultivars in the field is difficult and time consuming. Therefore, tissue culture method was used to screen 25 potato cultivars to identify the salt tolerant cultivars.
马铃薯对盐度敏感。淡水灌溉是获得高产的必要条件。当土壤中的盐分增加时，产量会降低。土壤淡化是马铃薯生产的一个昂贵的过程。因此，选择耐盐品种对于经济生产是必要的。在田间筛选许多马铃薯品种既困难又耗时。因此，采用组织培养法筛选了25个马铃薯品种，鉴定了耐盐品种。

High salinity causes hyperosmotic stress and ion disequilibrium that produce secondary effects in crops (Zhu, 2001). Saline stress induces several alterations on growth, cell division and metabolic activities (Wincov, 1993). Similarly, higher concentrations of salt in the culture media greatly affected the shoot and root initiation and growth of potato plantlets in vitro. Shoot elongation and root elongation decreased corresponding to the increase of NaCl concentration in the culture media. Among the cultivars screened for the salinity tolerance seven cultivars showed tolerance up to 2000 ppm NaCl. Majority of the potato cultivars screened in this study were sensitive to salinity. However, a few sensitive cultivars maintained in high salt media for longer time showed tolerance to high salinity. This is due to the physiological changes occurred in the plant cells to adapt them selves to the salt stress in vitro. Osmotic stress due to the salinity in the culture environment induces a specific cellular response (Csonka, 1989; Lucht and Bremer, 1994; Luyten et al., 1995) in the plant that is under stress to produce substances named osmoprotectants (Bohnert et al.,1995; Bohnert and Jensen, 1996). Similarly, potato plantlets of a few cultivars that developed tolerance to certain level of salinity during the subculture process may be produced osmoprotectants to adopt them selves to the saline environment in culture. From this result it is clear that stress tolerant plants could be developed through tissue culture methods.
高盐度会导致高渗胁迫和离子不平衡，从而对作物产生次生效应（Zhu，2001）。盐水应激诱导生长，细胞分裂和代谢活动的几种改变（Wincov，1993）。同样，培养基中较高的盐浓度对马铃薯小植株的枝条和根系萌生和生长有很大影响，体外枝条伸长率和根系伸长率降低，与培养基中NaCl浓度的增加相对应。在筛选耐盐度的品种中，有7个品种的耐受性高达2000 ppm NaCl。本研究中筛选的大多数马铃薯品种对盐度敏感。然而，一些在高盐培养基中保存较长时间的敏感品种表现出对高盐度的耐受性。这是由于植物细胞中发生的生理变化，使它们自身适应体外盐胁迫，由于培养环境中的盐度引起的渗透胁迫诱导特定的细胞反应（Csonka1989;Lucht 和 Bremer1994;Luyten等人，1995）在植物中应激产生名为渗透保护剂的物质（Bohnert等人，1995;Bohnert和Jensen1996）类似地，在传代培养过程中对一定盐度产生耐受性的少数品种的马铃薯小植株可以生产渗透保护剂，以使其在培养中适应盐碱环境。从这一结果可以清楚地看出，可以通过组织培养方法开发耐胁迫植物。

Genotypes within many crop species showed considerable differences in growth response to saline conditions (Jones and Qualset, 1984). Similar growth difference to salinity was noticed in different potato cultivars tested in the present study. Cultivars Arnova, Bellini, Costanera, Desiree, MF I, Primavera and Timate showed better shoot growth in the saline media containing 2000 ppm NaCl (4770 ppm MS basal salts + 2000 ppm NaCl= 6770 ppm TDS). These cultivars can be used for the potato production in Kuwait
许多作物物种的基因型在对盐碱条件的生长响应方面表现出相当大的差异（Jones和Qualset，1984年）。在本研究中测试的不同马铃薯品种中，观察到与盐度相似的生长差异。栽培品种Arnova、Bellini、Costanera、Desiree、MF I、Primavera和Timate在含有2000 ppm NaCl（4770 ppm MS基础盐+ 2000 ppm NaCl = 6770 ppm TDS）的盐水培养基中表现出更好的芽生长。这些品种可用于科威特的马铃薯生产.

Conclusion:
结论：

Tissue culture system can be used as a tool for the screening of stress tolerant genotypes from the existing cultivars. It is an easiest and less time consuming method for testing the salinity tolerance among the different potato cultivars. From this study it is clear that most of the potato cultivars are highly sensitive to salinity and some cultivars developed physiological adaptation to tolerate certain level of salinity from the environment. Among the 25 potato cultivars tested for salinity stress tolerance in vitro, seven cultivars showed tolerance up to 6770 ppm TDS salinity in culture. Further study is required for the field confirmation of these cultivars towards their respective level of salinity tolerance prior to recommending these salt tolerant cultivars to the farmers.
组织培养系统可用作从现有品种中筛选抗逆基因型的工具。这是测试不同马铃薯品种之间耐盐性的一种最简单且耗时较少的方法。从这项研究中可以清楚地看出，大多数马铃薯品种对盐度高度敏感，一些品种发展出生理适应能力，可以耐受环境中一定程度的盐度。在25个马铃薯品种的盐度胁迫耐受性测试中，有7个品种在培养物中显示出高达6770 ppm TDS盐度的耐受性。在向农民推荐这些耐盐品种之前，需要进一步研究这些品种的田间确认其各自的耐盐水平。

Acknowledgement
确认

This research was financed by the Kuwait Foundation for the Advancement of Sciences (KFAS) grant No. 2004-1207-03 and hereby the support is acknowledged. The support and encouragement of the Kuwait Institute for Scientific Research (KISR) management is also acknowledged. The authors thank British Potato Council (BPC), UK and International Potato Research Centre (CIP) Lima, Peru for providing potato cultivars for our experiments.
这项研究由科威特科学促进基金会（KFAS）资助，第2004-1207-03号赠款，特此感谢科威特科学研究所（KISR）管理层的支持和鼓励。作者感谢英国马铃薯理事会（BPC）、英国和秘鲁利马国际马铃薯研究中心（CIP）为我们的实验提供马铃薯品种。

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