LI Ling-li,HE Xin-ru,SUN Bing,FEI Yong-jun

College of Horticulture and Gardening,Yangtze University,Jingzhou 434025,PRC

Abstract To identify suitable grass species for growth in the Jianghan Plain,the micromorphologies of the upper and lower epidermis of the leaves of 14 bermudagrass varieties of Cynodon dactylon were investigated,and the number,length,and width of long and short cells and stomata were measured.The results showed that the anticlinal wall of epidermal cells looked like waves and the stoma was distributed in the upper and lower epidermis.However,the various indicators of the leaf epidermis of different species of Cd were significantly different.The length and width of long cells and the length of short cells of Cd Mohawk,Cd Suncity,and wild type Cd were relatively large,which may improve their ability to adapt to the pastoral soil and make them suitable for the rainy environment of the Jianghan Plain.On the contrary,the results of all indicators of Cd Xinnong 1,Cd hulled,and Cd unhulled ranked at the bottom,which was not suitable for pastoral soil habitats,while the wild type Cd could adapt to both the pastoral soil and the vegetation concrete habitat.Moreover,the long cells and short cells of Cd Kashi were tightly arranged,and the stoma in Cd Pyramid and Cd hulled were more dense,which may improve their ability to adapt to an arid environment.

Key words Cynodon dactylon;Epidermal cell;Micromorphology

1.Introduction

Poaceae,the name of the family of plants known as grasses,is one of the most ecologically and economically important plant families[1].Cynodon dactylon(Cd) belongs to theCynodonofChloridoideaesubfamily Poaceae[2].And it is one of the three largest warm-season turfgrass species in the world,native to southeastern Africa[3-4]and widely distributed in tropical,subtropical,and temperate coastal areas.Cd not only has a profound cultural history in South Asia,but also colonizes six continents and islands in Antarctica[5-6].In China,Cd is mainly distributed in the Yellow River Basin and the area to the south of it[7-8],and is also distributed in the northwest plateau areas,such as Xinjiang and Tibet[9].The strong reproductive ability of Cd in different environments,and its resistance to drought,barrenness,trampling,and soil consolidation,has made it an invasive weed[10].At present,Cd is one of the principle turf species used in urban greening,vegetation slope protection,sports fields and golf courses,as well as for soil protection and restoration[11-12].

The practicability and importance of micromorphological features for systematic studies of Poaceae plants have been confirmed by many researchers[13-14].The anatomical structure of leaves is widely used to describe the characteristics of different populations of Poaceae[15-19].As the main organ of plant photosynthesis and respiration,leaves are more easily exposed to the air than any other plant organ[20],and are sensitive to changes in environmental factors such as moisture,temperature,and light.Therefore,the leaves will show different external morphology and anatomical structures as the plant adapts to the changing environment[21-22].

In this study,we investigated the micromorphology of the leaf epidermis of 14 species of Cd,including the length,width,and number of long cells,short cells,and stomata,and then compared and analyzed the differences between the various species.Our study provided a research basis for finding suitable grass species for growth in the Jianghan Plain and understanding the ecological adaptability of Cd.Additionally,we provided a research basis for the development and utilization of bermudagrass resources in subtropical regions.

2.Materials and Methods

2.1.Overview of the test site

The test site was located in the Botanical Garden of the College of Horticulture and Gardening,Yangtze University,located in the hinterland of the Hubei Jianghan Plain at an altitude of 35 m,110°15′E～144°05'E,29°26'N～31°37'N.The region is characterized by a subtropical humid monsoon climate with sufficient light energy,and an annual frost-free period of 242～263 d,an annual average temperature of 15.9～16.6°C,an annual rainfall of 1 100～1 300 mm,and 1 800～2 000 h of annual sunshine.In this region,the total annual solar radiation of the city is 104～110 kcal/cm2,the solar radiation accounts for about 75%of the annual solar radiation in this region,and the accumulated temperature of≥10°C in this area accounts for 80% of the whole year’s amount.The soil is yellow loam,fluvo-aquic soil,and paddy soil,which are suitable for the growth of a variety of turfgrasses.

2.2.Materials

The tested materials were as follows:Cynodon dactylon(L.) Pers.cv.040459 (CdP 040459),Cynodon dactylon(L.) Pers.cv.hulled (CdPH),Cynodon dactylon(L.) Pers.cv.Nanjing (CdPNJ),Cynodon dactylon(L.) Pers.cv.Suncity,(CdPSC),040459/680Cynodon dactylon(L.) Pers.cv.040459/680 (Cd680),Cynodon dactylon(L.) Pers.cv.Xinnong No.1(CdXN1),Cynodon dactylon(L.)Pers.cv.Kashi (Kashi),Cynodon dactylon(L.) Pers.cv.Mohawk (Mohawk),Cynodon dactylon(L.) Pers.cv.Unhulled (CdPU),Cynodon dactylon(L.)Pers.cv.Pyramid (Pyramid),Cynodon dactylonL (L.) Pers.cv.Jackpot (Jackpot),Cynodon dactylon(L.) Pers.cv.Sahara (Sahara),Cynodon dactylon(L.) Pers.cv.Heaven (Heaven),Cynodon dactylon(L.) Pers.wild type (CdWT).All plant materials were planted in the pastoral soil of the Botanical Garden of Yangtze University.The recipe of the pastoral soil was as follows:the texture was clay loam;the alkali hydrolyzable nitrogen concentration was 65 mg/kg;the available phosphorus concentration was 8.6 mg/kg;the available potassium concentration was 113 mg/kg;the organic matter concentration was 14.48 g/kg,and the soil pH value was 7.9.

2.3.Methods

The plant preparation method was performed as previously described in WANG Z Aet al[23].Briefly,collected the middle part of the third leaf with healthy and mature Cd from top to bottom.Only the middle part of the leaf was kept and was subsequently divided into 2～3 cm pieces.All leaf pieces were fixed with FAA stationary liquids for 24 h at room temperature and then soaked in water to remove the fixative.A new double-faced blade,with the blade perpendicular to the leaf surface,was used to evenly scrape off the epidermis and part of the mesophyll,leaving only epidermal tissue.Then,it was transferred to a mixture of 1% pectinase and cellulase and dissociated for 30 min at room temperature,and then it was moved to a petri dish and washed with distilled water.The distilled water was replaced and transferred to the surface dish,followed by 4% alum staining for 5～10 min,tap water rinsing for 10 min,distilled water rinsing once,0.5% hematosin staining for 3～5 min,and tap water rinsing for 30 s.Finally,the product was placed on a glass slide,and evenly scraped the remaining mesophyll tissue to mount the slide for observation.Slices were observed with an Olympus BH-2 photographic biomicroscope (Japan),and a typical field of view was selected for imaging under the magnification conditions of eyepiece ×10,objective lens ×40,eyepiece ×10 and objective lens×100,respectively.Using the JNO-EC XS-212-103 optical microscope (China),30 complete long cells,short cells,and stomata were randomly observed,and their lengths and widths were measured.The number of long cells,short cells,and stomata per unit area was recorded in 30 randomly selected complete fields of view.

2.4.Data processing

In this study,SPSS software was used to process the data,andP＜0.05 was considered as a significant difference.Excel was used for image production.

3.Results and Analysis

3.1.Epidermal cell structure of leaf of Cd

The micromorphological structures of the epidermis of different varieties of Cd were similar(Fig.1 and 2),it showed that the epidermal cells were closely arranged and rectangular in shape,and could be divided into long cells and short cells.

Fig.1 Micromorphological characteristics of leaf upper epidermis of Cynodon dacylon L.on rural area soil

Fig.2 Micromorphological characteristics of leaf lower epidermis of Cynodon dacylon L.on rural area soil

The cells above the veins were all short cells,and the leaf vein intervein were alternately arranged with long cells and short cells.All the cell wall was serrate,and the anticlinal wall of the epidermis with sinuous thickening.The upper and lower epidermis contained stomata,which were composed of 2 guard cells and a pair of subsidiary cells.Among them,the guard cells were long and narrow dumbbell-shaped,and the subsidiary cells were long prismatic,and the long axis of the stomatal apparatus was parallel to the long axis of the leaf.The stoma was distributed almost equidistant in the cell row,but only between the veins.

3.2.Comparison of long cells in leaf epidermis of different species of Cd

The length of long cells varied from 22.47 to 48.47 μm (Fig.3 and 4).The longest cells were observed in Kashi,and the shortest cells were observed in Cd PU,with the former being 2.2 times longer that of the latter,showing significant differences.The width of long cells varied from 10.62 to 15.5 μm,among which Heaven was the widest and CdXN1 was the narrowest.In the lower epidermis,the Mohawk had the longest long cell length at 45.08 μm,and Heaven was the shortest at 33.54 μm.Among them,the length of long cells showed no significant differences among the 8 varieties of Jackpot,Sahara,CdWT,CdP040459,Pyramid,CdPSC,Cd680,and Heaven.

Fig.3 Comparisons between the length of long cells on the leaf epidermis of Bermudagrass varieties

Fig.4 Comparisons between the width of long cells on the leaf epidermis of Bermudagrass varieties

Fig.5 Comparisons between the number of long cells on the leaf epidermis of Bermudagrass varieties

Under the 10× eyepiece and the 100× objective lens,the number of upper epidermal long cells varied from 20.1 to 38.7 (Fig.5),with the greatest number observed in CdPSC and the least number observed in Kashi.The number of long cells in the lower epidermis ranged from 20.7 to 48.8,and the size order was different from that in the upper epidermis.The greatest number of long cells in the lower epidermis was observed in Kashi,and the least number of long cells was observed in CdXN1,a very significant difference of 2.4 times.Additionally,there were significant differences between Kashi and other Cd breeds.

3.3.Comparison of short cells in leaf epidermis of different species of Cd

In the upper epidermis,the short cell length of Pyramid was the longest at 17.07 μm (Fig.6 and 7),and CdXN1 was the shortest at 10.81 μm.There were no significant differences among the 5 species of Sahara,CdPSC,Mohawk,CdPNJ,and Jackpot.The width of the short cells ranged from 6.82 to 10.88 μm,with the widest short cells observed in CdWT,and the narrowest short cells observed in CdPU.In the lower epidermis,the short cell length of Mohawk was the longest at 15.91 μm,and the short cell length of CdPH was the shortest at 10.91 μm.There were no significant differences among the 4 species of Mohawk,CdPSC,Sahara and CdWT,and there were no significant differences between the three species of Kashi,CdPU,and CdPH.The width of the short cells ranged from 8.20 to 11.32 μm,and the descending order was CdWT,Sahara,Mohawk,CdPNJ,CdXN1,CdPSC,Pyramid,CdP040459,Heaven,Cd680,Jackpot,Kashi,CdPH,and CdPU.

Under the 10× eyepiece and the 100× objective lens,we observed the greatest number of short cells in the upper epidermis of CdPU at 45.3 (Fig.8),while the least number of short cells was observed in CdPH at 15.0.The number of short cells in the former was three times that of the latter,and the number of short cells was extremely significant differences between CdPU and CdPH.The number of short cells in the lower epidermis ranged from 23.5 to 49.2,with the greatest number observed in Kashi and and the least observed in CdXN1.There was also a significant difference between Kashi and other Cd breeds.

Fig.6 Comparisons between the length of short cells on the leaf epidermis of Bermudagrass varieties

Fig.7 Comparisons between the width of short cells on the leaf epidermis of Bermudagrass varieties

Fig.8 Comparisons between the number of short cells on the leaf epidermis of Bermudagrass varieties

3.4.Comparison of the epidermal stoma of different species of Cd

The stoma length in the upper epidermis of Kashi was the longest at 19.69 μm (Fig.9 and 10),and the stoma length of CdXN1 was the shortest at 15.24 μm.The stoma width varied from 5.55 to 6.61 μm,with the widest observed in Kashi and the narrowest observed in CdWT.In the lower epidermis,the stoma length varied from 14.97 μm to 21.55 μm,with the longest observed in Kashi and the shortest observed in Pyramid.There was no significant difference in the stoma length among the 4 species of CdP040459,Sahara,CdWT,and Mohawk.Stomata width in the lower epidermis ranged from 5.10 μm to 7.30 μm,and the descending order was CdPH,CdXN1,CdP040459,CdPSC,CdWT,Heaven,CdPNJ,Pyramid,Mohawk,Sahara,Jackpot,CdPU,Cd680,and Kashi.

Under the eyepiece of 10× and the objective lens of 100×,we observed the greatest number of stoma in the upper epidermis of CdPH at 11.1 (Fig.11),and the least number in CdPU at 6.5,a 1.7 fold difference.The number of stoma in the lower epidermis was 5.2 to 9.2,with the greatest number observed in Pyramid and the lowest number observed in Mohawk.The number of former was 1.8 times that of the latter,which was a significant difference.

4.Discussion

Fig.9 Comparisons between the length of stoma on the leaf epidermis of Bermudagrass varieties

Fig.10 Comparisons between the width of stoma on the leaf epidermis of Bermudagrass varieties

The micromorphological characteristics of leaf epidermis refer to the microscopic morphology of plant leaf epidermis,including the shape,length,width,and stomata types of long cells and short cells[24-25].The thickness of the cuticle,and the size and number of stomata of leaves are directly related to water loss of the plants[26].Thus,according to these characteristics,the degree of adaptation to adverse conditinos,such as excess moisture and drought,can be judged.By observing the upper and lower epidermis of the leaf from the different breeds of Cd in the pastoral soil,we observed similar micromorphological features in the leaf epidermis of different breeds of Cd.Also,the anticlinal wall of epidermal cells looked like waves,these specific structures alleviate the deformation effect caused by water loss to a certain extent and increase cell rigidity[27].However,there were significant differences in the length,width,and number of upper and lower epidermal cells and stoma.Studies have shown that the epidermal cells are tightly arranged with small intercellular spaces,which are better suited to adapt to an arid environment[28].Among the different breeds of Cd,we observed the greatest number of long cells and short cells in the lower epidermis of Kashi,as well as the largest stomatal width in the upper epidermis and the longest stomatal length in the upper or lower epidermis,indicating that Kashi displays strong drought resistance.In addition,the larger epidermal cells have increased water storage capacity[28].The length and width of long cells and the length of short cells in the lower epidermis of Mohawk,and the short cell width of the upper and lower epidermis of CdWT were the largest,followed by those of Sahara.These results suggested that Mohawk,CdWT,and Sahara could better regulate the water content in plant,and thus,might be suitable for the rainy environment of the Jianghan Plain.The indicators of CdXN1,CdPH and CdPU ranked at the bottom,suggesting that they could not adapt well to the pastoral soil and would not be suitable for growth in the Jianghan Plain.This result was contrary to the results that the epidermal cells of Jackpot,CdPSC,and Heaven were large and could enhance their ability to regulate water,whereas it was consistent with the conclusion that,under vegetation concrete habitat,the width of short cell of upper epidermal cells in the CdWT was the widest[29].However,the indicators of CdPH were low in both pastoral soil and vegetation concrete,indicating that it could not adapt to the environment,while CdWT could adapt to both habitats at the same time.

Gas exchange in the plant epidermis occur at the stoma,which provides the main control over water movement in the plant.The size,density,shape and related auxiliary cells location of stoma all affect plant gas exchange and water passages inside leaves[30-32].The structure and density of stoma reflect the adaptability of plants to the environment,as increased stoma density results in increased drought resistance[33].Moreover,increased stoma density accelerates the gas exchange between the body and the outside,and improves the efficiency of photosynthesis and transpiration.This accelerates the transport of water and achieves the effect of cooling the leaves[28].In many plants that live under water stress,stoma can sink under the epidermis to reduce water loss[34].In this study,we found stoma distributed in the upper and lower epidermis of different breeds of Cd[35],and observed that the stoma of Paphiopedilum was only distributed on the lower epidermis.Moreover,we also observed an increased number of stoma in the upper and lower epidermis of CdPSC,Pyramid,and CdPH,with Pyramid and CdPH having more stoma in the lower epidermis,but less stoma in the upper epidermis,suggesting these breeds may be well suited to adapt to drought.However,this conclusion was contrary to the conclusion that the density of stoma increased in the lower epidermis and decreased in the upper epidermis in CdWT on vegetation concrete[29].These results indicated that the response mechanism of plants to drought was the disparity in different habitats.

5.Conclusion

A comparison of the micromorphology of the leaf epidermis of 14 species,and the conclusions are drawn as follows:

1) The leaf epidermis of different species of Cd had similar micromorphological structures.The epidermal cells were with wave anticlinal wall and the stoma was distributed in the upper and lower epidermis.However,the various incdicators of the leaf epidermis of different species of Cd were significantly different.

2) Cd Mohawk,Cd Suncity,and wild type Cd could adapt better to the pastoral soil and were suitable for the rainy environment of the Jianghan Plain.On the contrary,Cd Xinnong 1,Cd hulled,and Cd unhulled were not suitable for pastoral soil habitats,while the wild type Cd could adapt to both the pastoral soil and the vegetation concrete habitat.

3) Kashi,Pyramid,and Cd hulled could better adapt to arid environments.