Cold stress is a major factor limiting the growth of warm-season turfgrass species. Cold tolerance in warm-season turfgrass species could be improved through in vitro selection for somaclonal variations. The objectives of this study were to establish an effective in vitro culture protocol for generating plants from calli using mature seeds of seashore paspalum (Paspalum vaginatum) and to determine whether in vitro cold selection of somaclonal variations would lead to improved cold tolerance in seashore paspalum. The optimal concentrations of supplemental compounds in the culture medium for callus induction, embryogenic callus formation, and plant regeneration were determined. The supplemental compounds included 2,4–dichlorophenoxy acetic acid (2,4-D), 6-benzylaminopurine (6-BA), kinetin (KT), naphthalene-1-acetic acid (NAA), CuSO4, and acidic hydrolysis casein (AHC). The highest rates of callus induction (97.50%), embryogenic callus formation (66.88%), and regeneration (55.94%) were obtained with the supplemental compounds of 3.0 mg·L−1 2,4-D and 10.0 mg·L−1 CuSO4 for callus induction; with 3.0 mg·L−1 2,4-D, 15 mg·L−1 CuSO4, and 1.0 g·L−1 AHC for embryogenic callus formation; and with 8.0 mg·L−1 6-BA, 0.2 mg·L−1 KT, 0.5 mg·L−1 NAA, and 10 mg·L−1 CuSO4 for plant regeneration. Embryogenic calli were subjected to 2 or 6 °C treatment for 90 days for in vitro cold selection of somaclonal variation. Plants regenerated from calli surviving cold treatment (cold-selected) for 45 or 60 days were then exposed to low temperatures [15/10 or 5/3 °C (day/night)]. Plant variants derived from cold-selected calli exhibited significant improvement in their tolerance to low temperature of either 15/10 or 5/3 °C (day/night), as manifested by higher turf quality, leaf chlorophyll content, and membrane stability as well as lower levels of lipid peroxidation compared with the control plants. This study demonstrated the feasibility of in vitro selection for cold tolerance in seashore paspalum. The cold-tolerant variants could be useful germplasm for breeding programs and further molecular characterization of cold tolerance mechanisms.
June Liu, Zhimin Yang, Weiling Li, Jingjin Yu, and Bingru Huang
Lili Zhuang, Mengxian Liu, Xiuyun Yuan, Zhimin Yang, and Bingru Huang
Aquaporin (AQP) proteins serve important roles in regulating water movement across cellular membranes and affect plant responses to drought stress. The objective of this study was to characterize and examine functions of an AQP gene FaPIP2;1, isolated from a drought-tolerant perennial grass species tall fescue (Festuca arundinacea), for involvement in leaf dehydration status during water stress by overexpressing the gene in arabidopsis (Arabidopsis thaliana). FaPIP2;1 had characteristic transmembrane domains and Asn–Pro–Ala motifs and was similar to PIP2;1 in rice (Oryza sativa) and maize (Zea mays). Quantitative real-time reverse transcriptase polymerase chain reaction analysis showed that FaPIP2;1 was upregulated during moderate water stress (hydroponic culture, osmotic potential (ΨS) at −0.47 and −0.78 MPa) and the transcript level decreased as ΨS further decreased. Transgenic arabidopsis plants overexpressing FaPIP2;1 showed greater number of leaves per plant and improved survival rate compared with the wild type (WT) during drought stress. Transgenic plants also maintained higher leaf relative water content (RWC), chlorophyll content (Chl), net photosynthetic rate (Pn), and lower leaf electrolyte leakage (EL) than the WT. However, there was no difference in root length between the transgenic and WT plants following drought stress. The results demonstrated that overexpressing FaPIP2;1 could improve plant tolerance to drought stress by enhancing leaf water status, Chl, and photosynthetic rate, as well as maintaining improved cellular membrane stability relative to the WT plants. FaPIP2;1 may be used as a candidate gene for genetic modification of perennial grasses to develop new drought-tolerant germplasm and cultivars.
Jingjin Yu, Mengxian Liu, Zhimin Yang, and Bingru Huang
Drought stress is one of the most important abiotic stresses limiting plant growth, while high recuperative capacity of plants from drought damages is critical for plant survival in periods of drought stress and rewatering. The objective of our study was to determine physiological and growth factors in association with drought tolerance and recuperative capacity of cool-season kentucky bluegrass (Poa pratensis cv. Excursion II) and warm-season zoysigrass (Zoysia matrella cv. Diomand), which were grown in controlled environment chambers and maintained well watered (control) or subjected to drought stress and subsequently rewatering. Compared with kentucky bluegrass, zoysiagrass maintained higher leaf hydration level during drought stress, as shown by greater relative water content (RWC), improved osmotic adjustment (OA), increased leaf thickness, and more extensive root system at deeper soil layers. Turf quality (TQ) and photosynthesis recovered to a greater level and sooner in response to rewatering for zoysiagrass, compared with kentucky bluegrass, which could be due to more rapid reopening of stomata [higher stomatal conductance (g S)] and leaf rehydration (higher RWC). The aforementioned physiological factors associated with leaf dehydration tolerance during drought and rapid resumption in turf growth and photosynthesis in zoysiagrass could be useful traits for improving drought tolerance in turfgrasses.
Zhimin Yang, Lixin Xu, Jingjin Yu, Michelle DaCosta, and Bingru Huang
Carbohydrate metabolism is important for plant adaptation to drought stress. The objective of this study was to examine major forms of carbohydrates associated with superior drought tolerance and post-drought recovery in kentucky bluegrass (Poa pratensis) by comparing responses of different forms of carbohydrates with drought stress and re-watering in two cultivars contrasting in drought tolerance. Plants of drought-tolerant ‘Midnight’ and drought-sensitive ‘Brilliant’ were maintained well watered or subjected to drought stress for 10 days by withholding irrigation, and drought-stressed plants were re-watered for 3 days. Physiological analysis (turf quality, relative water content, and electrolyte leakage) confirmed the genetic variability of the two cultivars in drought tolerance. The two cultivars exhibited differential responses to drought stress and re-watering for the content of water-soluble sugars (sucrose, fructose, and glucose) and storage carbohydrates (starch and fructan), and ‘Midnight’ maintained higher sucrose content at 10 days of drought stress and more fructan at 3 days of re-watering. The greater accumulation of sucrose in ‘Midnight’ under drought stress corresponded with higher activities of two sucrose-synthesizing enzymes (sucrose phosphate synthase and sucrose synthase) but was not related to the sucrose-degrading enzyme activity (acid invertase). These results suggested that increased sucrose accumulation resulting from the maintenance of active sucrose synthesis could be associated with superior turf performance during drought stress, whereas increased fructan accumulation could contribute to rapid re-growth and post-drought recovery on re-watering in kentucky bluegrass.
Zhimin Yang, Jingjin Yu, Emily Merewitz, and Bingru Huang
Abscisic acid (ABA) and glycine betaine (GB) may regulate plant responses to drought or salinity stress. The objectives of this controlled-environment study were to determine whether foliar application of ABA or GB improves turf quality under drought or salinity and whether improved stress responses were associated changes in antioxidant metabolism in two C3 turfgrass species, creeping bentgrass (Agrostis stolonifera) and kentucky bluegrass (Poa pratensis). Physiological parameters evaluated included turf quality, leaf relative water content, membrane electrolyte leakage (EL), membrane lipid peroxidation [expressed as malondialdehyde (MDA) content], and activity of superoxide dismutase (SOD), peroxidase (POD), and ascorbate peroxidase (APX). Abscisic acid and GB were both effective in mitigating physiological damage resulting from drought or salinity for both grass species, but effects were more pronounced on kentucky bluegrass. The most notable effects of ABA or GB application were the suppression of EL and MDA accumulation and an increase in APX, POD, and SOD activities after prolonged periods of drought (21 days) or salinity stress (35 days). These results suggest foliar application of ABA or GB may alleviate physiological damage by drought or salinity stress in turfgrass and the maintenance of membrane stability and active antioxidant metabolism could contribute to the positive effects in the stress mitigation effects.