ComparisonoftheCold_r_省略_ourLigustrumCul

Comparison of the Cold-resistance Capabilities of Four Ligustrum CultivarsHAO Ming-zhuo ，HAN Ming-hui ，PENG Fang-ren *，LIANG You-wangCollege of Forest Resources and Environment ，Nanjing Forestry University ，Nanjing ，Jiangsu 210037，ChinaAbstract ［Objective ］To compare the cold-resistance capabilities of four Ligustrum cultivars．［Method ］Based on semilethal low temperature testand low temperature stress test in phytotron ，cold-resistance capabilities of two-year-old seedlings of 4Ligustrum cultivars were compared at leaf-ex-pansion and shoot-growing stage．［Result ］At leaf-expansion and shoot-growing stage ，the semilethal low temperatures of Ligustrum ˑVicaryi Hort．，Ligustrum lucidum Ait．，Ligustrum japonicum Thunb．‘Howardii ’and Ligustrum lucidum Ait．‘Excelsum Superbum ’were －6．34，－5．69，－4．55and －3．27ħ，respectively．SOD activity ，soluble protein content ，soluble sugar content and MDA content in leaves under low temperature stress were all higher than those under normal temperature．As the low temperature stress enhanced ，soluble protein content in leaves firstly en-hanced and then decreased．Among the 4cultivars ，soluble sugar content in 3cultivars firstly increased and then decreased ；and MDA content con-tinuously enhanced．［Conclusion ］The cold-resistance capabilities of Ligustrum ˑVicaryi Hort．and Ligustrum lucidum Ait．were higher than other cultivars ；Ligustrum japonicum Thunb．‘Howardii ’was middling ；and Ligustrum lucidum Ait．‘Excelsum Superbum ’was the worst．Key words Ligustrum ；Cultivars ；Cold-resistance capability ；Semilethal low temperatureReceived ：August 24，2012Accepted ：December 20，2012Supported by the Science and Technology Support Project of Jiangsu Province （BE2010311），and the Science and Technology Develop-ment Program of Northern Jiangsu Province （SBN200910136，SBE200870251）．*Corresponding author．E-mail ：frpeng@njfu．com．cnPlants of genus Ligustrum in family Oleaceae have been widely applied in landscape greening and the production of Kuding tea．Researches have shown that the roots ，leaves and fruits of most Ligustrum plants have the functions of clearing heat ，detoxification ，analgesia ，hemostasis ，relieving cough ，bacteriostasis and so on．More and more attention has been paid to its medicinal value and health function ［1－2］．In recent years ，new Ligustrum cultivars have been introduced in China ，such as Ligustrum japonicum Thunb．‘Howardii ’，Ligustrum ˑVicaryi Hort．，and Ligustrum lucidum Ait．‘Excelsum Super-bum ’．These cultivars are very popular and their cultivation ar-ea and range have increased each year．At present ，they have been introduced into northern Jiangsu and Anhui Prov-inces ［3－5］．However ，some new varieties of Ligustrum are ex-tended relatively late ，so the growth habitat and cold resistance mechanism are still unclear．Some reports are found on the cold resistance of Ligustrum vicaryi ，Ligustrum quihoui and so on at home and abroad ［6－13］．But few are about the cold resist-ance mechanism of Ligustrum japonicum ‘Howardii ’and Ligus-trum lucidum ‘Excelsum Superbum ’．Plant freeze injury is commonly seen in winter and spring．When the there is late spring cold and other natural disasters in plant germination stage ，leaf-expansion and shoot-growing stage ，freeze injury ofplant often becomes more serious．This phenomenon also cause the attention of many experts and scholars ［14－16］．In thisresearch ，cold-resistance capabilities of Ligustrum japonicum Thunb．‘Howardii ’，Ligustrum ˑVicaryi Hort．，Ligustrum lucid-um Ait．，and Ligustrum lucidum Ait．‘Excelsum Superbum ’were compared ，aiming at providing references for the cultiva-tion management and regionalized extension of new Ligustrum cultivars．1Materials and methods1．1Materials1．1．1Plant materials．Two-year-old cutting seedlings of Ligu-strum japonicum Thunb．‘Howardii ’，Ligustrum ˑVicaryi Hort．，and Ligustrum lucidum Ait．‘Excelsum Superbum ’were introduced from Hangzhou Base of Zhejiang Senhe Group inMarch 2011；and two-year-old seedlings of Ligustrum lucidum Ait．were introduced from Tangquan Town of Nanjing City．1．2Methods1．2．1Pretreatment of plant materials．Test site was located in Nanjing Forestry University．After seedling introduction ，they were planted in the round plastic pots ，which were 28cm in di-ameter and 32cm in height．There was a small hole in the pot bottom with 10kg soil in each pot．Soil used was the mixture of organic matter under forest and yellowish-brown loam soil．Itsphysicochemical properties were as follows ：1．883ˑ106mg /kg organic matter ，0．27ˑ106mg /kg total nitrogen ，921mg /kg to-tal phosphorus ，1．34ˑ106mg /kg total potassium ，134．8mmol /kg CEC ，and pH 5．5．After 30d of transplantation ，seedlings at leaf-expansion stage were put in phytotron at 5ħfor 7d．The condition was as follows ：12h illumination eachday ，30μmol /（m 2·s ）light intensity ，and 75%－80%relative humidity．Proper irrigation should be ensured during the cultiva-tion in order to maintain the soil moisture in pot．1．2．2Imitated freeze injury test in phytotron under low tem-perature stress treatment．As for each Ligustrum cultivar ，healthy plants in 6pots were put in phytotron at 0，－7and －14ħand the greenhouse at 25ħ．After treated by 48h ，Medicinal Plant 2012，3（12）：111－115Germplasm Resources and Cultivationmorphological appearance of plants with freeze injury was ob-served．Then，50g leaves of each Ligustrum cultivar were ran-domly collected and immediately stored in ultra low temperature refrigerator at－75ħ．The soluble sugar content，soluble pro-tein content，SOD activity，MDA content in leaves were detec-ted［17－18］．1．2．3Determination of semilethal low temperature［19－20］．Based on pre-cooling test，10standard plants were selected for each Ligustrum cultivar．10－15normal leaves were collected randomly from each standard plant．After well mixed，the leav-es were divided into6groups and stored in refrigerators at the designed temperature for1h．After thawing for24h，the rela-tive conductivity was detected．According to the Logistic equa-tion，semilethal low temperatures of four Ligustrum cultivars were calculated．The designed temperatures were0，－5，－10，－15，－20and－25ħ．2Results and analyses2．1Morphological appearance of Ligustrum cultivars with freeze injury Table1reported that Ligustrum lucidum Ait．and LigustrumˑVicaryi Hort．had relatively good cold-resistance capability．Their freeze injury was relatively not severe；their growth status was relatively good；and their survival rate under low temperature stress at－14ħwas relatively high．Ligustrum japonicum Thunb．‘Howardii’showed middle cold-resistance capability and grew relatively good at25and0ħtreatments．However，plants of Ligustrum japonicum Thunb．‘Howardii’had slight freeze injury at－7and－14ħtreat-ments；some leaves turned yellow and crimped and some even died．Ligustrum japonicum Thunb．‘Howardii’had the poorest cold-resistance capability under low temperature treatment．Leaves began to be withered under the environment of0ħ；and survival rate of plants was extremely low under low temper-ature stress of－14ħ．2．2Changes of nutritional component contents in leaves of different Ligustrum cultivars under low temperature stress The soluble protein content in plant is closely related to the cold re-sistance．Under normal conditions，soluble protein content en-hances in the plants under low temperature stress，which ad-justs the cold resistance of plant［21］．Fig．1illustrated that un-der normal temperature，Ligustrum lucidum Ait．‘Excelsum Su-perbum’had higher content of soluble protein than other three cultivars．As the temperature fell，soluble protein contents in leaves of different cultivars firstly enhanced and then reduced．Among them，soluble protein content in Ligustrum lucidum Ait．‘Excelsum Superbum’began to fall at－7ħ，while those in the rest three cultivars began to fall at－14ħ．Under low tem-perature stress，the increasing extent of soluble protein content in leaves varied；and they were in the order of Ligustrum lucid-um Ait．（96．2%）＞LigustrumˑVicaryi Hort．（69．4%）＞Li-gustrum japonicum Thunb．‘Howardii’（55．0%）＞Ligustrum lucidum Ait．‘Excelsum Superbum’（13．7%）．Table1Morphological appearance of Ligustrum cultivars under low temperature stressPlant name Tempera-ture∥ħSurvivalrate∥%FreezinggradeGrowth statusLigustrum lucidum Ait．25100．01Normal，deep green leaves0100．01Normal，deep green leaves－7100．01Normal，relatively small amount of leaves，blackish green leaves－1483．32Relatively poor，relatively small amount of leaves，dry leaf edge LigustrumˑVicaryi25100．01Normal，many leaves，deep green leaves，relatively few yellow leaves Hort．0100．01Normal，many leaves，deep green leaves，relatively few yellow leaves －7100．01Normal，many leaves，deep green leaves，30%of yellow leaves－1483．32Relatively poor，curled leaves，light green leaves，half yellow leaves Ligustrum lucidum Ait．25100．01Normal，many leaves，deep green leaves‘Excelsum Superbum’083．32Relatively poor，curled leaves，slight yellow leaves，wilting－766．73Drying，withered leaves，rare，slight yellow leaves，wilting－1416．74slight yellow and dry leaves，mostly dyingLigustrum japonicum25100．01Normal，many leaves，deep green old leaves，golden yellow new leaves Thunb．‘Howardii’0100．01Normal，many leaves，more golden yellow leaves－7100．02Relatively poor，curled leaves，dry and withered leaves，deep yellow leaves－1466．72Relatively poor，curled leaves，dry and withered leaves，wiltingNote：Freezing grade1indicated no freeze injury；2was slight freeze injury；3was middle freeze injury；and4was severe freeze injury．The soluble sugar content could enhance the osmotic con-centration of plant cells，reduce the water potential，increase the water retention capacity，decrease the freezing point，and protect the cell cytoplasm colloids from solidification．Soluble sugar content showed positive correlation with the cold resist-ance of most plants．When under cold environment，plants en-hanced their soluble sugar content to defense against the cold［15］．Fig．2indicated that with the decrease of temperature，soluble sugar content in Ligustrum lucidum Ait．leaves en-hanced，and those in LigustrumˑVicaryi Hort．，Ligustrum luci-dum Ait．‘Excelsum Superbum’and Ligustrum japonicumThunb．‘Howardii’enhanced firstly and then reduced．Soluble sugar content in leaved under－14ħcold temperature treat-ment was significantly lower than that in－7ħtreatment．The decreasing extents of both Ligustrum japonicum Thunb．‘Howardii’and Ligustrum lucidum Ait．‘Excelsum Superbum’were significantly higher than that of LigustrumˑVicaryi Hort．；the three decreasing extents were all higher than those at25ħtreatment．2．3Changes of SOD activity and MDA content in Ligustrum cultivars under low temperature stress SOD has close correla-tion with the stress resistance of plants．Under low temperature211Medicinal Plant2012Fig．1Changes of soluble protein content in Ligustrum culti-vars under low temperaturestressFig．2Changes of soluble sugar content in Ligustrum cultivars under low temperature stressstress ，SOD has super oxidation disproportionation reaction with oxygen and free radicals in order to ensure the stability of membrane ［21］．Fig．3illustrated that with the decrease of tem-perature ，SOD activities in the leaves of Ligustrum lucidum Ait．and Ligustrum japonicum Thunb．‘Howardii ’firstly enhanced and then maintained stable ；SOD activity in Ligustrum lucidum Ait．‘Excelsum Superbum ’enhanced firstly and then reduced．Under normal temperature ，SOD activity of Ligustrum lucidum Ait．‘Excelsum Superbum ’was significantly lower than those of the rest three cultivars ；but it increased by 59．7%from 25ħto －7ħ，and reduced by 21．2%from －7ħto －14ħ，which was obviously different from other cultivars．Besides ，SOD activities of all the Ligustrum cultivars at －7and －14ħtreatments were higher than those in 25ħtreatment．MDA is a product of membrane lipid peroxidation induced by free radicals ，which has certain inhibitory effects on SOD ac-tivity．MDA content also has close correlation with the cold re-sistance of plants ［22］．Fig．4indicated that as the treatment temperature fell ，MDA contents in Ligustrum lucidum Ait．，Ligustrum lucidum Ait．‘Excelsum Superbum ’and Ligustrum japonicum Thunb．‘Howardii ’enhanced by 88．7%，70．9%and 74．0%，respectively．MDA content in Ligustrum ˑVicaryi Hort．firstly enhanced and then decreased ；and it enhanced by 94．4%from 25ħto －7ħ，but reduced by 5．3%from －7to－14ħ．Under normal temperature ，MDA contents in Ligus-trum lucidum Ait．，Ligustrum ˑVicaryi Hort．and Ligustrum ja-ponicum Thunb．‘Howardii ’were higher than that in Ligustrum lucidum Ait．‘Excelsum Superbum ’．Fig．3Changes of SOD activity in Ligustrum cultivars underlow temperaturestressFig．4Changes of MDA content in Ligustrum cultivars underlow temperaturestressFig．5Change laws of relative conductivity of the leaves of Li-gustrum cultivars under low temperature stress311HAO Ming-zhuo et al．Comparison of the Cold-resistance Capabilities of Four Ligustrum Cultivars2．4Determination of semilethal low temperature Relative conductivity of leaves is an important physiological index com-monly used to evaluate the injury degree of plants under low temperature stress．Low temperature stress enhances the per-meability of cell membrane．Plants have poor coldness resist-ance show a rapid increase of membrane permeability，which is expressed in the sharp rising of conductivity［21］．Fig．5indica-ted that from0to－10ħtreatments，the four Ligustrum culti-vars showed slowly increasing of relative conductivity；from －10and－15ħtreatments，relative conductivity enhanced sharply；from－15to－25ħtreatments，relative conductivity enhanced gradually by100%，showing a slowly increasing tendency．The relative conductivity of the leaves of four Ligus-trum cultivars showed the change of"S"curve with the de-crease of treatment temperature．At the same time，the testshowed that the relative conductivity of Ligustrum lucidum Ait．‘Excelsum Superbum’was significantly higher than other culti-vates between0and－10ħ．Research results showed that under low temperature stress treatment，Logistic equation could better reflect the changes of plant organs with temperature［20］．According to the results of equation and test，coefficients K，A and B were ob-tained．Thus，the conductivity-derived Logistic equations of Li-gustrum lucidum Ait．，LigustrumˑVicaryi Hort．，Ligustrum lu-cidum Ait．‘Excelsum Superbum’and Ligustrum japonicum Thunb．‘Howardii’were obtained under low temperature stress［20］．According to the equation coefficients，the semilethal low temperatures of the four cultivars were－6．34，－5．69，－4．55and－3．27ħ，respectively．Table2Logistic equation analyses of semilethal low temperatures of different Ligustrum cultivarsCultivars Coefficient A Coefficient B Coefficient KLT50Semilethallow temperature∥ħF value test P value testLigustrum lucidum Ait．3．44123355960．2171396．9－5．6935．340．0040 LigustrumˑVicaryi Hort．3．76836040110．2091198．0－6．3432．180．0048 Ligustrum japonicum2．72298852610．2203293．9－4．5527．010．0065 Thunb．‘Howardii’Ligustrum lucidum Ait．1．86468101330．1906594．0－3．2754．390．0018‘Excelsum Superbum’Note：Logistic equation was Y=K/（1+Ae－BX），where Y was conductivity，X was temperature，K，A and B were the equation coefficients．3Conclusions and discussionsUnder low temperature stress，the soluble protein content，soluble sugar content，SOD activity，MDA content and relative conductivity in leaves of Ligustrum cultivars were researched．Ac-cording to the morphological appearance of plants，Ligustrum luci-dum Ait．and LigustrumˑVicaryi Hort．showed relatively strong cold-resistance capability，followed with Ligustrum japonicum Thunb．‘Howardii’．And Ligustrum lucidum Ait．‘Excelsum Su-perbum’had the poorest cold-resistance capability．Xiang Yan-jun et al．［21］found out that with the decrease of temperature from9to－7ħ，soluble protein contents in the leaves of5types of vine plants showed an ascendant trend．However，results of this research indicated that with the de-crease of temperature，soluble protein contents in the leaves of 4Ligustrum cultivars firstly enhanced and then decreased，which was similar to the research results of Feng Xian-bin et al．［23］．Results also showed that under low temperature stress，the increasing extent of soluble protein content in plant leaves was closely related to the cold-resistance capability．Li-gustrum cultivars with relatively great increasing extent had strong physiological adjustment ability under low temperature stress，and had relatively strong cold-resistance capability．Be-sides，with the decrease of temperature，decrease of soluble protein content in plant in early stage would lead to poor cold-resistance capability．In this research，Ligustrum lucidum Ait．‘Excelsum Superbum’had the poorest cold-resistance capa-bility and its soluble protein content decreased greatly at －7ħ；while the soluble protein contents in other3cultivars started at－14ħ．This preliminarily proved this conclusion．Research results showed that under low temperaturestress，soluble sugar contents in Ligustrum cultivars enhanced significantly．However，with the further decrease of tempera-ture，soluble sugar content sometimes reduced．Gao Wen-fang et al．［11］researched on the cold tolerance mechanism of5 types of colorful vegetations；research results showed that the soluble sugar content in Ligustrum vicaryi leaves firstly en-hanced and then reduced，which was consistent with the results of LigustrumˑVicaryi Hort．，Ligustrum lucidum Ait．‘Excelsum Superbum’，and Ligustrum japonicum Thunb．‘Howardii’in this research．Reduction of soluble sugar content might be caused by the following reason：leaf chlorophyll was destroyed under low temperature；photosynthesis mechanism was re-stricted；and synthesis of soluble sugar content was greatly af-fected［24］．Besides，as the temperature decreased，soluble sugar content in plant reduced in early stage and the reduction extent was great，which indicated that the cold-resistance capa-bility of this plant was poor．Xie Xiao-jin et al．argued that the decreasing extent of soluble sugar content was closely related to the cold-resistance capability under low temperature treat-ment［25］．Results showed that the SOD activity in plant firstly en-hanced and then reduced as the temperature fell［23］．Change of SOD activity was complex under low temperature stress．Some reduced firstly and then enhanced；some enhanced and then maintained stable；and some increased and then decreased．It was noteworthy that SOD activity of Ligustrum lucidum Ait．‘Excelsum Superbum’was relatively low under normal temper-ature，but enhanced under low temperature，showing the in-crease and decrease with relatively great extent．SOD activity of Ligustrum lucidum Ait．‘Excelsum Superbum’was quite411Medicinal Plant2012sensitive to temperature change，which might be one manifes-tation of its poor cold-resistance capability．Under adversity stress，plant cells produced a large amount of free radicals，induced or accelerated the cell mem-brane lipid peroxidation，and further destroyed the cell mem-brane．As the final product of membrane lipid peroxidation，MDA was an important index reflecting the damage degree of cell membrane［21］．Research results showed that MDA content in plant leaves enhanced as the low temperature stress in-creased［11］．Test results showed that as the temperature de-creased，MDA content in the leaves of4Ligustrum cultivars was not always increasing．For instance，LigustrumˑVicaryi Hort．firstly enhanced and then reduced，which might because that plant could not stand extreme low temperature，so that cell death appeared；cell membrane was completely destroyed；membrane lipid oxidation reaction basically stopped；cell inclu-sion flew away；and MDA content reduced［26］．Research results also indicated that there were differences in the semilethal low temperature of plants in different seasons．Xie Xiao-jin and Hao Ri-min researched on the cold resistance of12types of evergreen broad-leaved trees；and the semilethal low temperature was detected in different months［27］．Results showed that the semilethal low temperatures of12trees from October2005to March2006were all higher than－10ħ，those from December2005to January2006were mostly lower than－10ħ；and there were significant differences in the cold-resistance capability of seedlings in different phenophases．An-alyses of Logistic equation showed that at leaf-expansion and shoot-growing stage，the semilethal low temperatures of Ligus-trumˑVicaryi Hort．，Ligustrum lucidum Ait．，Ligustrum japoni-cum Thunb．‘Howardii’and Ligustrum lucidum Ait．‘Excelsum Superbum’were－6．34，－5．69，－4．55and－3．27ħ，re-spectively，indicating that the cold-resistance capabilities of4 Ligustrum cultivars were poor and introduction should be care-fully considered．References［1］LI XM，LI XQ，ZHAG DZ．Chemical composition，pharmacological action and clinical application research situation of Ligustrum［J］．Academic Journal of Guangdong College of Pharmacy，1997，13（2）：44－47．（in Chinese）．［2］TU YH，GAO NN．Survey in study on chemical constituents and pharmacologic action of Ligustrum［J］．Lishizhen Medicine and Ma-teria 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