Embryogenic cell suspension culture and plant regeneration in zoysiagrass (Zoysia japonica Steud)

팽왼주안,Liebao Han,Chunhui Qi,Deying Li 한국잔디학회 2009 Weed & Turfgrass Science Vol.23 No.2

Zoysiagrass (Zoysia japonica Steud) is a warm season turfgrass species widely used for sports field and golf courses. Many cultivars are propagated through vegetative methods. This study was conducted to develop an optimum culture medium and culture conditions for embryogenic callus induction and plant regeneration, and to establish a cell suspension culture system for use in zoysiagrass breeding and propagation. The results indicated that adding Cu++ at 2.5 mg L-1 to the induction medium was optimum for callus induction. Increasing the numbers of sub-culture cycles improved the quality of calli. The optimum dosage for cell suspension culture ranged from 2.5 to 10 mL. The embryogenic callus suspension used in this study had a plant regeneration rate of 58%. Zoysiagrass (Zoysia japonica Steud) is a warm season turfgrass species widely used for sports field and golf courses. Many cultivars are propagated through vegetative methods. This study was conducted to develop an optimum culture medium and culture conditions for embryogenic callus induction and plant regeneration, and to establish a cell suspension culture system for use in zoysiagrass breeding and propagation. The results indicated that adding Cu++ at 2.5 mg L-1 to the induction medium was optimum for callus induction. Increasing the numbers of sub-culture cycles improved the quality of calli. The optimum dosage for cell suspension culture ranged from 2.5 to 10 mL. The embryogenic callus suspension used in this study had a plant regeneration rate of 58%.

Effect of localized reduction of gibberellins in different tobacco organs on drought stress tolerance and recovery

Tianxiu Zhong,Huiming Zeng,Liebao Han,Lijuan Zhang,Shanshan Sun 한국식물생명공학회 2014 Plant biotechnology reports Vol.8 No.5

Drought resistance is increased in plants by theabsence of the hormone gibberellic acid (GA) or by a lackof GA sensitivity. We studied the effects of tissue-specificreduction in GA levels on drought tolerance, on recoveryfrom drought stress, and on primary and secondary growthusing transgenic tobacco plants expressing the GA-inactivatinggene PtGA2ox1 (GA 2-oxidase) specifically inleaves, stems, or roots. Localized reduction of bioactiveGA1 levels was achieved by tissue-specific expression ofthe PtGA2ox1 gene in leaves using the rbcs promoter (LDplants), in roots using the TobRB7 promoter (RD plants),and in stems using the LMX5 promoter (SD plants). Inresponse to drought stress, all transgenic tobacco plantsexhibited reduced primary and secondary growth andincreased drought tolerance with a corresponding reductionin malondialdehyde levels, higher relative water content,increased proline and sugar content, and elevated peroxidase,superoxide dismutase, and catalase activities relativeto wild-type plants. The highest level of drought toleranceand the most rapid recovery from stress was achieved bylocalized reduction of GA1 in the roots of the RD transgenicplants. In addition, although the total bioactive GA1content in RD and LD plants was essentially identical, theheights of LD plants were significantly greater and droughttolerance was significantly less than in RD plants. It ispossible that the site of gibberellin-related gene expressionplays an important role in the balance between growth anddrought tolerance.

Endophytes from Natural Festuca spp. in Southwest China and Their Compatibility with Tall Fescue Cultivars

Yaoyao Wang,Yongji Du,Liebao Han,Deying Li 한국잔디학회 2009 Weed & Turfgrass Science Vol.23 No.1

Investigating endophyte distribution, naturally occurring in native grasses, is important for understanding endophyte-grass associations and using the beneficial effects of endophytes in cultivated plants. The goal of this study was to investigate endophytes from natural Festuca spp. in Yunnan, Guizhou, and Sichuan Provinces of China, and to study the compatibility between the endophytes and turf type tall fescue (Festuca arundinacea) which is widely used for lawn and athletic fields in that region. Of 628 accessions in F. ovina, 421 had endophytes identified in leaf sheath from on-site microscopic examination. From Festuca spp, three isolates were obtained from the seeds and ninety isolates were obtained from seedlings established from the collected seeds. The isolates from F. ovina and F. stapfii were tentatively identified as Neotyphodium typhinum and Neotyphodiumstarii, respectively. We tested compatibility of the two fungal species with seven tall fescue cultivars, Little Hero, Sub Boy, Eldorado, Arid III, Millennium, Crossfire, and Fawn. N. typhinum or N. starii did not infect ‘Fawn’with either seed injection or seed soaking method. The highest infection rate byboth N. typhinum and N. starii was in ‘Sun Boy’ and ‘Eldorado’. There were significant interaction effects between tall fescue cultivar and type of endophyte on infection. Investigating endophyte distribution, naturally occurring in native grasses, is important for understanding endophyte-grass associations and using the beneficial effects of endophytes in cultivated plants. The goal of this study was to investigate endophytes from natural Festuca spp. in Yunnan, Guizhou, and Sichuan Provinces of China, and to study the compatibility between the endophytes and turf type tall fescue (Festuca arundinacea) which is widely used for lawn and athletic fields in that region. Of 628 accessions in F. ovina, 421 had endophytes identified in leaf sheath from on-site microscopic examination. From Festuca spp, three isolates were obtained from the seeds and ninety isolates were obtained from seedlings established from the collected seeds. The isolates from F. ovina and F. stapfii were tentatively identified as Neotyphodium typhinum and Neotyphodiumstarii, respectively. We tested compatibility of the two fungal species with seven tall fescue cultivars, Little Hero, Sub Boy, Eldorado, Arid III, Millennium, Crossfire, and Fawn. N. typhinum or N. starii did not infect ‘Fawn’with either seed injection or seed soaking method. The highest infection rate byboth N. typhinum and N. starii was in ‘Sun Boy’ and ‘Eldorado’. There were significant interaction effects between tall fescue cultivar and type of endophyte on infection.

E ffects of exogenous spermidine and spermine on antioxidant metabolism associated with cold-induced leaf senescence in Zoysiagrass (Zoysia japonica Steud.)

Xinbo Sun,Lijuan Xie,Liebao Han 한국원예학회 2019 Horticulture, Environment, and Biotechnology Vol.60 No.3

Zoysiagrass ( Zoysia japonica Steud.) leaves lose their green colour as they undergo cold acclimation (CA) in late fall inmany regions, and delaying leaf senescence can extend their green period. The objective of this study was to investigatewhether exogenous spermidine (Spd) and spermine (Spm) can delay leaf senescence by enhancing reactive oxygen species(ROS)-scavenging antioxidant enzyme activity. Mature zoysiagrass (cv. Zenith) plants were treated with Spm (1 mM) or Spd(1 mM) or water (control). The treated plants were grown under normal conditions or subjected to cold acclimation CA for35 days. CA treatment increased leaf electrolyte leakage (EL) and malondialdehyde (MDA) level, decreased chlorophyll (Chl)content, and induced leaf senescence. Foliar application of Spd and Spm reduced the decline in Chl content and reduced ELand MDA levels. CA treatment increased the ROS level and reduced antioxidant enzyme activity. Application of Spm andSpd reduced ROS accumulation and enhanced the activities of superoxide dismutase, peroxidase, catalase, and ascorbateperoxidase under CA conditions. The results of this study indicated that exogenous Spd and Spm at 1 mM can delay CAinducedleaf senescence through enhancement of ROS-scavenging antioxidant activity in zoysiagrass.

한국들잔디 배아세포의 부유배양과 식물체 재생

팽왼주안,한리바우,퀴충휘,리다이엥,박태윤,Fang, Wenjuan,Han, Liebao,Qi, Chunhui,Li, Deying,Park, Tae-Yun Turfgrass Society of Korea 2009 한국잔디학회지 Vol.23 No.2

한국들잔디는 운동경기장이나 골프 코스 등에 폭넓게 쓰이는 난지형 잔디이다. 많은 품종은 영양체로 번식한다. 이 연구는 배아세포 유도와 식물체 재생을 위한 적정 배양액 개발과 배양조건을 연구하여, 한국잔디의 육종과 번식을 위한 세포배양 체계 확보를 목적으로 하였다. 그 결과 $Cu^{++}\;2.5mgL^{-1}$를 유도 배양액에 첨가하는 것이 캘러스 유도를 위한 최적 농도 조건 이였음을 보여주었다. Sub-culture의 횟수의 증가는 캘러스의 질을 향상시켰다. 세포 부유배양을 위한 최적 용량은 2.5에서 10mL 사이였으며 본 연구에서 58%의 식물체 재생산비율을 보였다. Zoysiagrass (Zoysia japonica Steud) is a warm season turfgrass species widely used for sports field and golf courses. Many cultivars are propagated through vegetative methods. This study was conducted to develop an optimum culture medium and culture conditions for embryogenic callus induction and plant regeneration, and to establish a cell suspension culture system for use in zoysiagrass breeding and propagation. The results indicated that adding $Cu^{++}$ at 2.5 mg $L^{-1}$ to the induction medium was optimum for callus induction. Increasing the numbers of sub-culture cycles improved the quality of calli. The optimum dosage for cell suspension culture ranged from 2.5 to 10 mL. The embryogenic callus suspension used in this study had a plant regeneration rate of 58%.

Protective Effect of Exogenous Spermidine on Ion and Polyamine Metabolism in Kentucky bluegrass under Salinity Stress

Xuehua Puyang,Mengying An,Lixin Xu,Liebao Han,Xunzhong Zhang 한국원예학회 2016 Horticulture, Environment, and Biotechnology Vol.57 No.1

Spermidine (Spd) acts as a plant protectant under salinity stress. The objective of this study was to determine whether exogenous Spd pre-treatment could improve the salinity tolerance of Kentucky bluegrass (Poa pratensis L.). Exogenous Spd was applied before the grass was exposed to 200 mM sodium chloride (NaCl) for 28 d in the growth chamber. Salinity stress decreased chlorophyll (Chl) content, K+/Na+ratio and K+, Ca2+, and Mg2+ contents, and increased electrolyte leakage (EL), proline, Na+, putrescine (Put), Spd and Spermine (Spm) levels and the activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (SAMDC) and polyamine oxidase (PAO). Pre-treatment with 1 mM Spd effectively alleviated the decline of Chl content, K+/Na+ratio and K+, Ca2+, and Mg2+ contents under salinity stress conditions. Spd pre-treatment also reduced EL, Na+ content, Put levels and the activities of ADC and PAO, but increased proline content, endogenous Spd and Spm levels and the activities of ODC and SAMDC upon salinity stress. These results indicate that exogenous Spd pre-treatment could enhance salinity tolerance by increasing proline levels and regulating ion and polyamine metabolism.

Heterologous expression of the Arabidopsis DREB1A/CBF3 gene enhances drought and freezing tolerance in transgenic Lolium perenne plants

Li, Xue,Cheng, Xiaoxia,Liu, Jun,Zeng, Huiming,Han, Liebao,Tang, Wei The Korean Society of Plant Biotechnology 2011 Plant biotechnology reports Vol.5 No.1

The dehydration-responsive element binding proteins (DREB1)/C-repeat (CRT) binding factors (CBF) function as transcription factors and play an important role in agricultural biotechnology and molecular biology studies of drought and freezing stress tolerance. We generated transgenic Lolium perenne plants containing the PCR-cloned Arabidopsis DREB1A/CBF3 gene (AtDREB1A/CBF3) to study the function of this gene construct in drought and freezing tolerance in a species of turfgrass. Compared to the control, AtDREB1A/CBF3 transgenic L. perenne plants showed enhanced drought and freezing stress tolerance. The activities of the enzymes superoxide dismutase (SOD) and peroxidase (POD) were higher in transgenic plants than in the non-transgenic plant control. These results demonstrate that the expression of the AtDREB1A/CBF3 gene in transgenic L. perenne plants enhanced drought and freezing tolerance and that the increased stress tolerance was associated with the increased activities of antioxidant enzymes. These results are relevant to stress biology and biotechnology studies of turfgrass.

Antioxidant enzyme activity and microRNA are associated with growth of Poa pratensis callus under salt stress

Luo Hongsong,Zhou Zhixiang,Song Guilong,Yao Hongxiang,Han Liebao 한국식물생명공학회 2020 Plant biotechnology reports Vol.14 No.4

Kentucky bluegrass (Poa pratensis L.) is an important species of turfgrass that is commonly planted on golf courses and landscapes all over the world. It is sensitive to salt stress; however, details relating to its molecular mechanisms of salt resistance are not available. We, therefore, analyzed the changes in growth, antioxidant enzyme activity, and microRNA expression in the callus 1 week after treatment with 200 mM NaCl for 12 h, 24 h, 48 h, 96 h, and 144 h. The results demonstrated that callus growth declined and thiobarbituric acid-reactive substance production and cell membrane permeability increased. Treatment with salt increased ascorbate peroxidase, glutathione reductase, superoxide dismutase, catalase, peroxidase, and polyphenol oxidase activity. Changes in the expression levels of microRNAs were observed under salt treatment. The expression of miR162, miR173, miR391, miR408, miR773, and miR857 increased by 70% after 24 h of salt treatment, after which it declined to a level similar to that of the control. The expression level of miR775 and miR827 decreased by 20% after 24 h, and then further decreased by 80% after 144 h. The expression level of miR841 increased by 50% after 24 h of salt treatment, and then stabilized. In contrast, salt treatment increased the expression of the auxin response factors ARF6, ARF8, ARF10, and ARF16 in the callus from 12 to 144 h of salt treatment, during which the expression increased twofold. Gene expression analysis indicated that salt-responsive gene families were regulated by microRNAs in the callus under salinity stress. The activity of antioxidant enzymes is also changing. MiR841 is considered to be a positive regulator of antioxidant enzyme biosynthesis. The present investigation elucidates the manner in which P. pratensis responds to salt stress in the callus, and could be used to inform further studies on the molecular mechanisms of stress tolerance.

Gene expression involved in dark-induced leaf senescence in zoysiagrass (Zoysia japonica)

Xiaoxia Cheng,Xiaomei Dai,Huiming Zeng,Yunxia Li,Wei Tang,Liebao Han 한국식물생명공학회 2009 Plant biotechnology reports Vol.3 No.4

Zoysiagrass (Zoysia japonica) is one of the important turfgrass species. Extending green period of zoysiagrass via delaying leaf senescence will make this species have more potential in the turfgrass industry. In this study, we found that zoysiagrass seedlings treated with GA3 could delay the leaf senescence induced by darkness. To study expression of genes responsive to staying green in zoysiagrass, suppression subtractive hybridization (SSH) was used to identify differentially expressed genes between non-GA3-treated and GA3-treated seedlings subjected to darkness. A total of 307 ESTs were generated, of which 226 ESTs clustered into 54 contigs and 81 were singlets. Differentially expressed genes selected by subtractions were classified into six categories according to their putative functions generated by BLAST analysis. Expression of five selected genes, Met, SAM, V-ATPase, Cry (Cryptochrome gene), and An (diphthine synthase gene) were examined by RT-PCR and Real-time PCR. Both RT-PCR and Real-time PCR results demonstrated that the differential expressions of these genes were attributable to delaying senescence by exogenously applied gibberellic acid. This is the first genome-wide study of senescence in a species of turfgrass. Zoysiagrass (Zoysia japonica) is one of the important turfgrass species. Extending green period of zoysiagrass via delaying leaf senescence will make this species have more potential in the turfgrass industry. In this study, we found that zoysiagrass seedlings treated with GA3 could delay the leaf senescence induced by darkness. To study expression of genes responsive to staying green in zoysiagrass, suppression subtractive hybridization (SSH) was used to identify differentially expressed genes between non-GA3-treated and GA3-treated seedlings subjected to darkness. A total of 307 ESTs were generated, of which 226 ESTs clustered into 54 contigs and 81 were singlets. Differentially expressed genes selected by subtractions were classified into six categories according to their putative functions generated by BLAST analysis. Expression of five selected genes, Met, SAM, V-ATPase, Cry (Cryptochrome gene), and An (diphthine synthase gene) were examined by RT-PCR and Real-time PCR. Both RT-PCR and Real-time PCR results demonstrated that the differential expressions of these genes were attributable to delaying senescence by exogenously applied gibberellic acid. This is the first genome-wide study of senescence in a species of turfgrass.

Endophytes from Natural Festuca spp. in Southwest China and Their Compatibility with Tall Fescue Cultivars

왕야오야오,두용지,한리바우,리다이엥,Wang, Yaoyao,Du, Yongji,Han, Liebao,Li, Deying Turfgrass Society of Korea 2009 한국잔디학회지 Vol.23 No.1

자생화본과 식물에서 자연적으로 생기는 엔도파이트 분포의 연구는 엔도파이트 공생에 대한 이해와 재배식물에서의 엔도파이트의 잇점에 대하여 중요하다. 본 연구는 중국 유난, 구이죠우, 시촨지역에서 자생하는 페스큐로부터 엔도파이트를 조사하고, 그 지역에서 널리 식재되는 잔디형 페스큐와의 융화에 대하여 연구하는데 목표를 두었다. 현장의 현미경 관찰 결과628개체의 sheep fescue 중 421개체가 엽초조직에서 엔도파이트가 동정되었다. 페스큐종에서 3개는 종자에서, 90개의 엔도파이트 분리체가 수집된 종자의 유묘에서 수집되었다. sheep fescue와 stapfii 페스큐의 엔도파이트는 Neotyphodium typhinum와 Neotyphodium starii로 각각 동정되었다. Little Hero, Sub Boy, Eldorado, Arid III, Millennium, Crossfire, 그리고 Fawn 톨페스큐 7품종에서 위 2종의 균류의 융화성을 조사한 결과, N. typhinum 나 N. typhinum and N. starii는 'Sun Boy'와 'Eldorado'에서 가장 높은 접종율을 보였다. 톨페스큐 품종과 엔도파이트 타입과의 접종에서 매우 높은 유의성의 교호 효과를 보였다. Investigating endophyte distribution, naturally occurring in native grasses, is important for understanding endophyte-grass associations and using the beneficial effects of endophytes in cultivated plants. The goal of this study was to investigate endophytes from natural Festuca spp. in Yunnan, Guizhou, and Sichuan Provinces of China, and to study the compatibility between the endophytes and turf type tall fescue (Festuca arundinacea) which is widely used for lawn and athletic fields in that region. Of 628 accessions in F. ovina, 421 had endophytes identified in leaf sheath from on-site microscopic examination. From Festuca spp, three isolates were obtained from the seeds and ninety isolates were obtained from seedlings established from the collected seeds. The isolates from F. ovina and F. stapfii were tentatively identified as Neotyphodium typhinum and Neotyphodiumstarii, respectively. We tested compatibility of the two fungal species with seven tall fescue cultivars, Little Hero, Sub Boy, Eldorado, Arid III, Millennium, Crossfire, and Fawn. N. typhinum or N. starii did not infect 'Fawn' with either seed injection or seed soaking method. The highest infection rate byboth N. typhinum and N. starii was in 'Sun Boy' and 'Eldorado'. There were significant interaction effects between tall fescue cultivar and type of endophyte on infection.