Somoclonal Variation in Salinity Tolerance for Creeping Bentgrass

Eddie Zhang*, Jinpeng Xing, and Thomas GianfagnaDepartment of Plant Biology and Pathology, Rutgers University

INTRODUCTIONTurfgrasses are being subjected to rising amounts of salinity stress resulting from increasedsalinization of agricultural areas, and the use of non-potable water sources as a method ofturfgrass irrigation. Improving salt tolerance of turfgrasses is important for conservation offresh water. Plant tissue culture techniques are recently employed to improve the geneticvariability of plant tolerance to environmental stresses. The objective of this study was todetermine whether in vitro selection of somoclonal variation could be used for improvingsalt tolerance for creeping bentgrass (Agrostis palustris), a cool-season turfgrass used widelyon golf courses. This information is important for determining the feasibility of irrigatingturfgrasses with non-potable water in a high salt environment. This study will also select salttolerant plants that could be used as breeding materials.

RESULTS AND DISCUSSIONCallus growth rate decreased with the concentration of NaCl in the culture medium. After 28days of culture, surface area of the control calli increased 48%. Similarly, the calli exposed to0.5% NaCl concentration showed a surface area increase of 48%. However, calli in 0.75%and 1.0% NaCl increased only 35% and 43% in size, respectively. At a high level of salinity,surface area increases were only 20% at 1.25% NaCl and 13% for 1.5% NaCl.(Table 1)

NaClconcentration%

Avg. (%)Two weeks

Avg. (%)Four weeks

0 30.4 ± 1.18 47.8 ± 1.23

0.5 41.5 ± 0.94 48.4 ± 1.33

0.75 31.5 ± 1.26 35.2 ± 1.21

1.0 34.4 ± 1.32 42.9 ± 1.44

1.25 21.6 ± 0.76 20.4 ± 0.81

1.5 9.1 ± 0.88 13.2 ± 0.97

Table 1: The growth rates of calli in MS medium containing varying solutions of NaCl(0%, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%) in two and four weeks

Despite showing a significant decrease in growth rate, a large number of calli in the 1.25% -1.5% salinity range continued to grow after 28 days. Calli survived at 1.25% - 1.5% salinityrange maintained higher survival rate when transferred to 2.0% NaCl, while those calligrown previously at in 0%, 0.5%, 0.75%, 1.0% NaCl lost viability quickly when transferredto 2.0% NaCl medium. Surviving calli from the culture at 1.5% NaCl medium weretransferred to regeneration medium and regenerated into plants (Fig 1).

MATERIALS AND METHODSSeeds of creeping bentgrass (cv. Penncross) were sterilized and calli were generated in MSmedium containing varying solutions of NaCl (0%, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%). Thegrowth rates of calli were evaluated using surface area measurements taken with a digitalimage analysis program. Plants were regenerated from calli that survived high salt treatmentsto evaluate for their tolerant to higher level of salinity. Salinity tolerance of 1.5%-NaClregenerated plants was compared against those regenerated from calli with 0% NaCl. Bothgroups of plants were sprayed weekly with 100 mL of 0% NaCl solution or 1.5% NaClsolution. Prior to each salt treatment, leaf samples were taken from the plants and rinsedunder running deionized water in order to remove salt particles from leaves. Samples weresubsequently tested for electrolyte leakage (EL%), leaf relative water content, andchlorophyll content.

CONCLUSIONSOur results suggest that callus selected through increasing concentrations of saltgenerated bentgrass plants with improved tolerance to high salinity. The studydemonstrated that it may be feasible to produce salinity tolerant creeping bentgrassthrough somoclonal selection. Field test is needed to confirm the controlled-environment study.

A B

Fig1. A. Surviving calli from the culture at 1.5% NaCl medium were transferredto regeneration medium and regenerated into plants. B. Regenerated plants.

Salinity tolerance of 1.5%-NaCl regenerated plants and the plants regenerated fromcalli with 0% NaCl were treated weekly with 100 mL of 0% NaCl solution or 1.5%NaCl solution. After 5 weeks treatment, plants treated with salt and with waterexhibited different growth rate and morphology (Fig 2).

Leaf samples were taken from non-NaCl and NaCl-selected plants treated with wateror 1.5% NaCl, electrolyte leakage (EL%), leaf relative water content, and chlorophyllcontent were measured. The initial pretreatment values indicated no differences in EL,realtive water content, and chlorophyll content between NaCl-selected and non-NaCl-selected plants. However, NaCl-selected plants had lower EL% than non-NaCl-selectedplants when both were treated with 1.5% NaCl (Fig 3). Leaf relative water content ofnon-NaCl-selected plants were less than that of NaCl-selected plants after 21 d of salttreatment (Fig 4). Leaf chlorophyll content of NaCl-selected plants were higher thanthat of non-NaCl-selected plants after 14 d of salt treatment (Fig 5).

Fig 2. 1. Non-NaCl-selected plants treated with water. 2. NaCl-selectedplants treated with water. 3. Non-NaCl-selected plants treated with 1.5%NaCl . 4. NaCl-selected plants treated with 1.5% NaCl .

Electrolyte Leakage

813182328333843

0 7 14 21 35

Days of Treatment

Elec

troly

te L

eaka

ge

Fig 3. Leaf electrolyte leakage

Relative Water Content

75

80

85

90

95

100

0 7 14 21 28 35

Days of Treatment

Rela

tive

Wat

er C

onte

nt

Fig 4. Leaf relative water content.

Chlorophyll Content

0.81

1.21.41.61.8

2

0 7 14 21 28 35

Days of Treatment

Chlo

roph

yll

Cont

ent

Fig 5. Leaf chlorophyll content